JAML transmembrane and co-signaling domains for chimeric antigen receptors

Abstract

The present disclosure relates to the field of cell therapy, and more specifically, to chimeric antigen receptor (CAR) cell therapy. In one embodiment, the disclosure provides CAR-T constructs comprising transmembrane and / or co-stimulatory cytoplasmic domains of JAML. In one embodiment, the disclosure provides CAR-T cells that co-express two CAR-T constructs, or a bicistronic construct, one of which comprises a JAML transmembrane and / or co-stimulatory domain. In one embodiment, the disclosure provides CAR-T cells that co-express a CAR-T construct and a full length JAML construct. In one embodiment, the disclosure provides a chimeric construct comprising a TGFbeta- receptor with an intracellular JAML domain, or a fragment thereof, and cells expressing the same. In one embodiment, the disclosure provides methods of treating cancer and autoimmune disorders with the constructs and cells of the disclosure.

Description

JAML TRANSMEMBRANE AND CO-SIGNALING DOMAINS FOR CHIMERIC ANTIGEN RECEPTORSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Patent Application No. 63 / 735,847, filed on December 18, 2024 which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 10, 2025, is named K-l 177-WO-PCT_SL.xml and is 3,248 bytes in size.TECHNICAL FIELD

[0003] The present disclosure relates to the field of cell therapy, and more specifically, to chimeric antigen receptor (CAR) cell therapy. In one embodiment, the disclosure provides CAR-T constructs comprising transmembrane and / or co-stimulatory cytoplasmic domains of JAML and cells expressing the same. In one embodiment, the disclosure provides CAR-T cells that coexpress two CAR-T constructs, one of which comprises a JAML transmembrane and / or co¬ stimulatory domain. In one embodiment, the disclosure provides CAR-T cells that co-express a CAR-T construct and a full length JAML const met. In one embodiment, the disclosure provides a chimeric construct comprising a TGF -Receptor-extracellular domain with a TGFp-R or JAMI, transmembrane domain, and an intracellular JAML domain, or a functional fragment thereof, and cells expressing the same.BACKGROUND

[0004] Human cancers are by their nature comprised of normal cells that, have undergone a genetic or epigenetic conversion to become abnormal cancer cells. In doing so, cancer cells begin to express proteins and other antigens that are distinct from those expressed by normal cells. These aberrant tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells.

[0005] Current T cell therapies rely on enriched or modified human I’ cells to target and kill cancer cells in a patient. To increase the ability of T cells to target and kill a particular cancer cell, methods have been developed to engineer T cells to express constructs, which direct T cells to aparticular target cancer cell. Chimeric antigen receptors (CARs), which comprise binding domains capable of interacting with a particular tumor antigen, allow T cells to target and kill cancer cells that express the particular tumor antigen.

[0006] Chimeric antigen receptors are also being explored for autoimmune diseases by targeting immune cells that drive autoimmune responses or by modifying T cells to target specific antigens in the disease pathology. In chronic infections like HIV, CAR-modified T cells are being investigated to find and eliminate infected cells that evade the immune system. Graft-Versus-Host Disease (GVHD) occurs when transplanted immune cells attack the recipient's tissues. CARs are being studied to help control GVHD by designing CAR- 1 cells that selectively target and eliminate cells causing GVHD. In addition, CAR-T therapy is being investigated for treating certain neurological disorders and genetic diseases by modifying the immune system to recognize and address malfunctioning cells. Accordingly, a need exists for improved methods of generating antigen receptor modified T cells for specifically targeting and killing cells in an autologous or allogeneic setting.SUMMARY

[0007] Junctional adhesion molecule-like protein (JAM-L / JAML / AMICA1) is a member of the immunoglobulin superfamily of cell adhesion molecules, and it plays an important role in regulating the adhesion and migration of immune cells, particularly during immune responses and inflammation. It is part of the broader JAM family, which includes other members like JAM-A, JAM-B, and JAM-C, that are involved in the maintenance of tight junctions and the regulation of leukocyte transmigration through endothelial and epithelial barriers. JAML is a cell surface transmembrane protein of monocytes, neutrophils, activated CD8 T cells, and tissue-resident y§ T cells that controls their migration and activation through interaction with CAR, also known as CX ADR (coxsackie and adenovirus receptor), a plasma membrane receptor found on adjacent epithelial and endothelial cells. The interaction between both receptor and ligand mediates the activation of y5 and aPT-cells, a subpopulation of T-cells residing in epithelia and involved in tissue homeostasis and repair. Upon epithelial CAR-binding, JAML induces downstream cell signaling events through PI3-kinase and MAP kinases. It results in proliferation and production of cytokines and growth factors by T-cells that in turn stimulate epithelial tissues repair. It also controls the transmigration of leukocytes within epithelial and endothelial tissues through adhesive interactions with epithelial and endothelial CXADR. JAML expression also plays a vital role in maintaining effective function of CD8 and y5 T cells within tumors.

[0008] The cytoplasmic region of JAM-L is short but significant for interacting with intracellular signaling molecules. It is involved in adaptor protein binding, such as to cortactm or ZO- 1, whichare involved in regulating actin cytoskeleton dynamics and tight junction formation. The cytoplasmic tail of JAM-L contains a PDZ-bindmg motif at its C-terminus. This motif allows JAM-L to interact with PDZ-domain-containing scaffolding proteins, such as Zonula Occludcns-1 (ZO-1). which are involved in the assembly and maintenance of tight junctions. This interaction helps to link JAM-L to the actin cytoskeleton and other junctional proteins, thus contributing to the regulation of tight junction integrity. The cytoplasmic tail may also interact with proteins containing SH2 domains, allowing for further regulation of signaling pathways, especially those related to cell migration, cell-cell adhesion, and immune responses. The cytoplasmic region of JAM-L is involved in transducing signals from the extracellular environment into the cell. This includes the regulation of small GTPases such as Rho, which are involved in cytoskeletal reorganization, cell adhesion, and cell migration. These signaling pathways are particularly important in processes like leukocyte migration, endothelial cell permeability, and immune cell trafficking.

[0009] Disclosed are chimeric antigen receptors (CAR), comprising at least one extracellular target binding domain; a hinge / spacer region; a transmembrane domain; a costimulatory domain; and an activation / signaling domain; wherein the CAR comprises the transmembrane domain and / or the costimulatory domain of J AML, or a fragment thereof. In one embodiment, the extracellular binding domain comprises an antigen-binding domain selected from a Fab, Fab', F(ab')2, Fv, scFv, linear antibodies, single domain antibodies such as sdAb (either VL or VH), cameiid VHH domains, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and multi-specific antibodies. In one embodiment, the hinge / spacer domain comprises an IgG4 hinge, an IgG2 hinge, a CD8a hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In one embodiment, the transmembrane domain comprises a transmembrane domain from JAML, CD3, CD8a, CD28, CD4, or ICOS. In one embodiment, the costimulatory domain comprises a JAML domain. In one embodiment, the costimulatory domain comprises one or more CD28, 4- IBB, 0X40, ICOS, DAP10, DAP12, CD27, MYD88-CD40, or KIR2DS2 costimulatory domains. In one embodiment, the activation / signaling domain comprises a signaling domain of one or more of CD3, CD3e, CD3y, CD35, FcyR, DAP- 12. or IT AM mut signaling domain.

[0010] In one embodiment, the transmembrane domain of the CAR comprises a JAML transmembrane domain, or a fragment thereof. In one embodiment, the transmembrane domain of the CAR comprises a polypeptide of SEQ ID NO:69. In one embodiment, the transmembrane domain of the CAR comprises an amino acid sequence having at least about 75%, 80%. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69.SEQ ID NO: 69: VI1VGIVCATILLLPVLIL1V

[0011] In one embodiment, the co-stimulatory domain of the CAR comprises a JAML intracellular domain, or a fragment thereof. In one embodiment, the co-stimulatory domain of the CAR comprises a polypeptide of SEQ ID NO:70. In one embodiment, the co-sliniulaiory domain of the CAR of the CAR comprises an ammo acid sequence having at least about 75%, 80%. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 70.SEQ ID NO:70:KKTCGNKSSVNSTVLVKNTKKTNPEIKEKPCHFERCEGEKHIYSPUVREVIEEEEPSEKS EATYMTMHP V WPSLRSDRN NSLEKKS GGGMPKTQQAF

[0012] In one embodiment, the disclosure provides that anti-CD19 CAR-T cells comprising a CAR-T construct carrying the transmembrane and / or the co-stimulatory domain of JAMI, had higher juvenile cells (CD45RA+, CCR7+), are less exhausted, exhibit lower tonic signaling, and have lower percentages of Th2 (CD4+, CCR4+, CCR6-, CXCR3-), and T-reg like (CD25high, CD127-) cell than anti-CD19 CAR-T cells comprising a CAR-T construct with the transmembrane and / or co-stimulatory domain of CD28. Accordingly, in one embodiment, the disclosure provides CAR-T cells that have better anti-cancer properties and are advantageous for cancer treatment, including cells that are more juvenile, less exhausted, exhibit lower tonic signaling, and have lower percentages of Th2 and T-reg like cells than earlier generation CD28-based CAR-T cells.

[0013] In one embodiment, the disclosure provides that anti-CD19 CAR-T cells comprising a CAR-T construct carrying the transmembrane and / or the co-stimulatory domain of JAML had comparable cytotoxicity to anti -CD 19 CAR-T cells comprising a CAR-T construct with the transmembrane and / or co-stimulatory domain of CD28.

[0014] In one embodiment, the disclosure provides that anti-CD19 CAR-T cells comprising a CAR-T construct carrying the transmembrane and / or the co-stimulatory domain of JAML were activated following co-culture with antigen-expressing target cells and produced cytokines but at a lower level than anti-CD 19 CAR-T cells comprising a CAR-T construct with the transmembrane and / or co-stimul tory domain of CD28. Accordingly, in one embodiment, the disclosure provides CAR-T cells that are less likely to result in serious adverse events such as high-grade cytokine release syndrome (CRS) and / or neurologic events (NE) post-transplantation than CAR-T cells comprising a CAR-T construct with the transmembrane and / or co-stimulatory domain of CD28.

[0015] In one embodiment, the disclosure provides that anti-CD19 CAR-T cells comprising a CAR-T construct carrying the transmembrane and / or the co-stimulatory domain of JAML proliferated similarly to CD28-based CAR-T cells during co-culture with target cells, although CD8+ CAR-T cells had a greater proliferation capacity than CD4+ CAR-T cells in JAML-based CAR-T cells. Accordingly, in one embodiment, the disclosure provides CAR- 1' cells with a highercytotoxic potential for cancer treatment than anti-CD19 CAR-T cells comprising a CAR-T construct with the transmembrane and / or co-stimulatory domain of CD28.

[0016] In one embodiment, the disclosure provides CAR-T cells that co-cxprcss a CAR-T construct and full length JAMI, having improved T cell activation, T cell survival, T cell extravasation, and I' cell migration toward tumor cells than cells with the CAR-T construct alone. Any CAR-T construct can be used in these cells, including, without limitation, one or more first- generation CAR-T, second generation CAR-T, third generation CAR-T, fourth generation CAR- T, fifth generation CAR-T. bivalent CAR-T, multi-target CAR, Tandem CAR, Fc binding CAR, Conditional CAR, ZipCAR. Inhibitory CAR, Split CAR, Switch CAR, iCasp9 CAR, sMASh CAR, Tet-ON CAR, Adaptor CAR, SynNotch CAR. LINK CAR, SNIP CAR, and Stealth CAR. In one embodiment, the full length JAMI, comprises the sequence of SEQ ID NO: 74. In one embodiment, the full length JAMI, comprises a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 74.SEQ ID NO: 74:MFCPI KLII DPVLLDYSLGLNDLNVSPPEI TVHVGDS Al, MGCVFQSTEDKCIFKIDWTI, S PGEHAKDEYVLYYYSNLSVPIGRFQNRVHLMGDILCNDGSLLLQDVQEADQGTYICEIR LKGESQVFKKAVVLI1VLPEEPKELMVHVGGLIQMGCVFQSTEVKHVTKVEW1FSGRRA KEEIVFRYYHKI, RMSVEYSQSWGHFQNRVNLVGDIFRNDGSIMLQGVRESDGGNYTCS IHLGNLVFKKTIVLHVSPEEPRTLVTPAALRPLVLGGNQLVIIVGTVC ATILLLPVLII, IVK KTCGNKSSVNSTVLVKNTKKTNPEIKEKPCHFERCEGEKHIYSPIIVREVIEEEEPSEKSE ATYMTMHPVWPSLRSDRNNSLEKKSGGGMPKTQQAF

[0017] In one embodiment, the disclosure provides CAR-T cells comprising two CAR-T constructs, one of which has a JAMI, transmembrane and / or costimulatory domain. These cells exhibit increased T cell activation, T cell survival. T cell extravasation, and T cell migration toward tumor cells than cells with one CAR- 1’ const met alone. Any CAR-T construct can be used in these cells, including, without limitation, one or more first-generation CAR-T. second generation CAR-T. third generation CAR-T, fourth generation CAR-T, fifth generation CAR-T, bivalent CAR-T. multi-target CAR. Tandem CAR, Fc binding CAR, Conditional CAR, ZipCAR, Inhibitory CAR, Split CAR, Switch CAR, iCasp9 CAR, sMASh CAR, Tet-ON CAR, Adaptor CAR, SynNotch C / XR, LINK CAR, SNIP CAR, and Stealth CAR. In one example, the two CAR- T constructs are expressed as a bicistronic construct. In one example, one CAR-T construct has a CD28 costimulatory domain or a 4- IBB costimulatory domain and the other has a J AML transmembrane and / or costimulatory domain.

[0018] Dominant-negative TGFp- receptor II has been used in CAR-T cell therapy to enhance the antitumor activity of CAR-T cells by blocking the immunosuppressive effects of TGF'P within thetumor microenvironment, allowing the CAR-T cells to proliferate, infiltrate tumors more effectively, and exert stronger cytotoxic activity against cancer cells, particularly in solid tumors where TGF-bcta is often highly expressed. In one embodiment, the disclosure provides a novel chimeric construct comprising a TGFp-Receptor extracellular domain with the transmembrane domain of the TGF'P-Receptor or the transmembrane domain of J AML and the intracellular domain of J AML, or a fragment thereof. In one embodiment, this construct is co-expressed in T cells comprising a CAR-T construct. The resultant CAR-T cells have improved antitumor efficacy, relative to those without a TGFp-dominant negative receptor and those with a corresponding TGF'P- receptor without an intracellular J AML domain. Any CAR construct can be used in these cells, including, without limitation, any CAR construct of the disclosure one or more first-generation CAR, second generation CAR, third generation CAR. fourth generation CAR, fifth generation CAR, bivalent CAR, multi-target CAR, Tandem CAR, Fc binding CAR, Conditional CAR, ZipCAR, Inhibitory CAR, Split CAR, Switch CAR, iCasp9 CAR, sMASh CAR, Tet-ON CAR, Adaptor CAR, SynNotch CAR, LINK CAR, SNIP CAR, and / or Stealth CAR constructs.

[0019] In one embodiment, the extracellular domain (ECD) of the TGFP-Receptor is selected from the ECD of TGF - RI or TGF PR1I.

[0020] In some embodiments described herein, the chimeric TGF-pRI / JAML construct comprises an extracellular ligand binding domain for binding TGF-3 having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%. or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence of SEQ ID NO: 75.SEQ ID NO: 75:LQCFCHLCTKDNFTCV'rDGLCFVSVl’ETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGS VTTTYCCNQDHCNKIELPTTVKSSPGLGPVEL

[0021] In some embodiments described herein, the chimeric TGF- RII / JAML construct comprises the extracellular ligand binding domain for binding of TGF-P to wild-type TGF-PRII having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%. at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence of SEQ ID NO: 76.SEQ ID NO: 76:TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSIT'SICEKPQE VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPD

[0022] The full length wild-type amino acid (aa) sequence for TGF-pRI is 503 am o acids as shown in SEQ ID NO: 77. The full length TGF-pRI polypeptide of SEQ ID NO: 77 includes a signal peptide (at approximately amino acids 1-33), an extracellular domain (at approximately amino acids 34-126), a transmembrane domain (at approximately amino acids 127-147) and an intracellular domain (at approximately amino acids 148-503). The intracellular domain includes four, key threonine sites located between amino acids 185-204 of the intracellular domain of TGF-PRI and initiates pSMAD signaling.SEQ ID NO: 77:MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVS VTETTDKVIHNSMCIAEIDLlPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTrVKSS PGLGPVELAAVIAGPVCFVCISEMI. MVYICHNRTVIIIHRVPNEEDPSLDRPFISEGTTI. K DLIYDMTTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSRE ERSWFREAEIYQTVMLRHENILGFLAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYT VTVEGMIKLALSTASGLAHLHMEIVGTQGKPAIAURDLKSKNILVKKNGTCCIADLGLA VRHDSATDTIDIAPNIIRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIAR RCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMR ECW Y AN GA ARLT ALRIKKTLS QLS QQEGIKM

[0023] The full length wild-type amino acid sequence for TGF-pRII is 567 amino acids as shown in SEQ ID NO: 78. The full length TGF-pRII polypeptide of SEQ ID NO: 78 includes a signal peptide (at approximately amino acids 1-22), an extracellular domain (at approximately amino acids 33-170). a transmembrane domain (at approximately amino acids 171-201) and an intracellular domain (at approximately amino acids 202-567).SEQ ID NO: 78:MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAAS PKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPI. GVAI SVIIIFYCYRVNRQQKLSSTWETGKTRKEMEFSEHCAIILEDDRSDISSTCANNINHNTEL LPIELDTLVGKGRFAEVYKAKLKQNESEQFETVAVKIFPYEEYASWKTEKDIFSDINLKII ENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAH I HSDHTPCGRPKMPIVHRDI. KSSNILVKNDI. TCCLCDFGLSI. RLDPTLS VDDI. ANSGQV GTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGS KVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCV AERFSELEHLDRLSGRSCSEEKIPEDGSLNTTKK-1177-WO-PCT

[0024] Disclosed are nucleic acids encoding a CAR, J AML, and chimeric TGFbeta- receptor with an intracellular JAML domain disclosed herein. Disclosed is a recombinant vector comprising such nucleic acids. Disclosed is a host cell transduced with such nucleic acids and recombinant vectors. In embodiments the host cell comprises a T cell, a macrophage, an iPSC, an iNKT cell, or a NK cell.

[0025] Disclosed are pharmaceutical compositions comprising such nucleic acids, vectors, T cells, macrophages, iPSCs, iNKT cells and / or NK cells. Disclosed are methods of treating a disease in a patient in need of thereof, comprising administering a T cell, iNKT cell and / or the NK cell or pharmaceutical composition of the disclosure to the patient.

[0026] Disclosed are methods of inducing an immune response in a subject or immunizing a subject against a cancer, the method comprising administering to the subject the T cell, iNKT cell and / or the NK cell or the pharmaceutical composition the patient.

[0027] Disclosed are methods of treating cancer and autoimmune disorders in a subject, the method comprising administering to the subject the I' cell, iNKT cell and / or the NK cell or the pharmaceutical composition the patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGs. 1A and IB illustrate embodiments of anti-CD19 CAR constructs with transmembrane domains of CD28 or JAML and signaling domains selected from the group consisting of a CD3 Zeta, CD28. and JAML co-stimulatory domains.

[0029] FIG. 2 shows viability measurements for CAR-T cells expressing exemplary CAR-T constructs of the disclosure.

[0030] FIG. 3 shows diameter measurements for CAR-T cells expressing exemplary CAR-T constructs of the disclosure.

[0031] FIG. 4 shows cell expansion measurements for CAR-T cells expressing exemplary CAR-T constructs of the disclosure,

[0032] FIG. 5 shows CAR-T construct expression levels in CAR-T cells expressing exemplary CAR-T constructs of the disclosure.

[0033] FIG. 6 shows the percentages of CD4+ and CD8+ CAR-T cells at harvest in manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure,

[0034] FIG. 7 shows the memory phenotype of CAR-T cells at harvest in manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure.

[0035] FIG. 8 shows the activation status of CAR-T cells at harvest in manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure.

[0036] FIGs. 9 A and 9B show the exhaustion signatures of CAR-T cells at harvest in manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure.

[0037] FIG. 10 shows the cellular composition of the CD4+ population in CAR-T cells at harvest in manufactured populations of CAR-T cells expressing exemplary' CAR-T constnicts of the disclosure.

[0038] FIG. 11 shows the activation status of manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure co-cultured with various antigenexpressing target cancer cells.

[0039] FIG. 12 shows the CD4 / CD8 cellular ratio after 4 days of co-culture of manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure with various antigen-expressing target cancer cells.

[0040] FIGs. 13A and 13B show the cytotoxicity of manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure co-cultured with various antigen¬ expressing target cancer cells.

[0041] FIG. 14 shows the proliferation of CD3, CD4, and CDS subpopulations of manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure co-cultured with various antigen-expressing target cancer cells.

[0042] FIG. 15 show's the CAR-T construct expression in the CD3, CD4. and CD8 subpopulations of manufactured populations of CAR-T cells expressing exemplary' CAR-T constnicts of the disclosure co-cultured with various antigen-expressing target cancer cells.

[0043] FIG. 16 show's cytokine release activity of manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure co-cultured with various antigenexpressing target cancer cells.

[0044] FIG. 17 shows mTOR activity in manufactured populations of CAR-1’ cells expressing exemplary CAR-T constructs of the disclosure co-cultured with Nalm6 CD19 antigen-expressing target cancer cells and CD 19 KO Nalm6 cells.

[0045] FIGs. 18A. 18B. and 18C show transcriptoniic analyses of manufactured populations of CAR-T cells expressing exemplary CAR-T constructs of the disclosure co-cultured with CD 19-expressing Nalm6 target cancer cells.

[0046] FIG. 19 shows lower number of non-activated T cells in JAML-overexpressing CAR-T cells and JAMLAD2 CAR-T cells compared to CAR-only T cells. This indicates better overall CAR-T cell activation when JAML was overexpressed in the CAR-T cells.

[0047] FIG. 20 show's a higher number of live CD8+ T cells and a higher number of CD25+ activated T cells were observed in JAML-overexpressing CAR-T cells and JAMLAD2 CAR-Tcells compared to CAR-only T cells 96 hours after co-culture with antigen-positive (Nalm6, Raji, ST486) versus antigen-negative (K562) ceil lines.

[0048] FIG. 21 shows JAM.-ovcrcxprcssing CAR-T cells and JAMLAD2 CAR-T cells exhibited higher cytotoxic activity compared to CAR-only T cells 96 hours after co-culture with antigenpositive (Nalm6. Raji, ST486).

[0049] FIG. 22 shows JAML-overexpressing CAR-1' cells and JAMLAD2 CAR-T cells showed superior proliferation compared to CAR-only T cells 96 hours after co-culture with antigen¬ positive (Nalm6, Raji, ST486) versus antigen-negative (K562) cell lines.DETAILED DESCRIPTIONTerms

[0050] In order for the present disclosure to be more readily understood, certain terms arc first defined below. Additional definitions for the following terms and other terms are set forth throughout the Specification.

[0051] As used in this Specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0052] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

[0053] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] The term “e.g.,” as used herein, is used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification.

[0055] The terms “or more”, “at least”, “more than”, and the like, e.g., “at least one” are understood to include but not be limited to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71. 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82. 83. 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139. 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150. 200, 300,400, 500, 600, 700, 800, 900, 1000. 2000, 3000, 4000, 5000 or more than the stated value. Also included is any greater number or fraction in between.

[0056] Conversely, the term “no more than"’ includes each value less than the stated value. For example, “no more than 100 nucleotides’" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91. 90, 89, 88. 87, 86, 85, 84, 83. 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72. 71. 70, 69, 68, 67, 66, 65. 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included is any lesser number or fraction in between.

[0057] The terms “plurality”, “at least two”, “two or more”, “at least second”, and the like, are understood to include but not limited to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32. 33, 34, 35, 36, 37, 38, 39, 40. 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55. 56. 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67. 68, 69, 70. 71. 72, 73, 74, 75, 76, 77, 78, 79, 80, 81. 82. 83, 84, 85. 86, 87, 88, 89, 90, 91, 92, 93, 94. 95, 96, 97, 98, 99. 100, 101. 102, 103, 104, 105, 106, 107, 108, 109. 110, 111, 112. 113. 114, 115, 116, 117, 118, 119, 120, 121. 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133. 134.135, 136, 137, 138. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also included is any greater number or fraction in between.

[0058] Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and / or “consisting essentially of” are also provided. The term "consisting of" excludes any element, step, or ingredient not specified in the claim. The term “consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed disclosure.

[0059] Unless specifically stated or evident from context the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of’ can mean within one or more than one standard deviation per the practice in the art. “About” or “comprising essentially of” can mean a range of up to 10% (i.e., ±10%). Thus, “about” can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%,K-I177-WO-PCT 4%, 3%, 2%, 1 %, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “comprising essentially of’ should be assumed to be within an acceptable error range for that particular value or composition.

[0060] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0061] Units, prefixes, and symbols used herein are provided using their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, Juo, “The Concise Dictionary of Biomedicine and Molecular Biology”, 2nded., (2001), CRC Press; “The Dictionary of Cell & Molecular Biology”, 5thed., (2013), Academic Press; and “The Oxford Dictionary' Of Biochemistry And Molecular Biology", Cammack et al. eds., 2nded, (2006), Oxford University Press, provide those of skill in the art with a general dictionary for many of the terms used in this disclosure.

[0063] ‘ ‘Administering” refers to the physical introduction of an agent to a subject, such as an engineered T cell disclosed herein, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally. vaginally. rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0064] The terms "activated'' and "activation" refer to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In one embodiment, activation may also beassociated with induced cytokine production, and detectable effector functions. The term "activated T cells" refers to, among other things, T cells that are proliferating. Signals generated through the TCR alone may be insufficient for full activation of the T cell and one or more secondary or costimulatory signals may also be required. Thus, T cell activation comprises a primary stimulation signal through the TCR / CD3 complex and one or more secondary costimulatory signals. Costimulation may be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR / CD3 complex.

[0065] The term “agent” may refer to a molecule or entity of any class comprising, or a plurality of molecules or entities, any of which may be. for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, cell (such as a T cell or progenitor of such cells), or organism (for example, a fraction or extract thereof) or component thereof. In some embodiments, an agent may be utilized in isolated or pure form. In some embodiments, an agent may be utilized in a crude or impure form. In some embodiments, an agent may be provided as a population, collection, or library, for example that may be screened to identify or characterize members present therein,

[0066] The term “allogeneic” refers to any material derived from one individual which is then introduced to another individual of the same species, e.g., allogeneic T cell transplantation.

[0067] The term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced. For example, the engineered autologous cell therapy method described herein involves collection of lymphocytes from a patient, which are then engineered to express, e.g., a CAR construct, and then administered back to the same patient.

[0068] The term “antibody” (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen. In general, and antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VII) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VII and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors,K-1177-WO-PCT including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. In general, human antibodies are approximately 150 kD tetrameric agents composed of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. The heavy and light chains are linked or connected to one another by a single disulfide bond; tw'O other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, e.g., on the CH2 domain.

[0069] The term “human antibody” is intended to comprise antibodies having variable and constant domain sequences generated, assembled, or derived from human immunoglobulin sequences, or sequences indistinguishable therefrom. In some embodiments, antibodies (or antibody components) may be considered to be “human” even though their amino acid sequences comprise residues or elements not encoded by human germline immunoglobulin sequences (e.g., variations introduced by in vitro random or site-specific mutagenesis or introduced by in vivo somatic mutation). The term “humanized” is intended to comprise antibodies having a variable domain with a sequence derived from a variable domain of a non-human species e.g., a mouse), modified to be more similar to a human germline encoded sequence. In some embodiments, a “humanized” antibody comprises one or more framework domains having substantially the amino acid sequence of a human framework domain, and one or more complementary determining regions having substantially the amino acid sequence as that of a non-human antibody. In some embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), generally that of a human immunoglobulin constant domain. In some embodiments, a humanized antibodies may comprise a CH1, hinge, CH2, CH3. and, optionally, a CH4 region of a human heavy chain constant domain.

[0070] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), and antigen binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations. Antibodies may also comprise, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, single chain or Tandem diabodies (TandAb®), Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans¬ bodies®, Affibodies®, TrimerX®, MicroProteins, Fynorners®, Centyrins®, and KALBITOR®s.

[0071] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, IgE and IgM, IgG subclasses are also well known to those in the art and include but are not limited to human IgG I, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The term “antibody" includes, by way of example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or nonhuman Abs; wholly synthetic Abs; and single chain Abs. A nonhuman Ab may be humanized by recombinant methods to reduce its immunogenicity in man. Where not expressly stated, and unless the context indicates otherwise, the term “antibody” also includes an antigen binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab.

[0072] An “antigen binding molecule,” “antigen binding portion,” “antigen binding fragment,” or “antibody fragment” or “antigen binding domain” refers to any molecule that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived. An antigen binding molecule can include the antigenic complementarity determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen. In certain embodiments an antigen binding molecule is a chimeric antigen receptor (CAR). In certain embodiments, the antigen binding molecule or domain binds CD 19. In certain embodiments, the antigen binding molecule or domain is an antibody fragment that specifically binds to the antigen, including one or more of the complementarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv).

[0073] In some instances, a CDR is substantially identical to one found in a reference antibody (<?. g., an antibody of the present disclosure) and / or the sequence of a CDR provided in the present disclosure. In some embodiments, a CDR is substantially identical to a reference CDR (e.g., a CDR provided in the present disclosure, for example in Table 4) in that it is either identical in sequence or contains between 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions as compared with the reference CDR. In some embodiments a CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR (e.g., 85-90%. 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments a CDR is substantially identical to a reference CDR in that it shows at least 96%. 96%. 97%. 98%. 99%. or 100%’ sequence identity with the reference CDR. In some embodiments a CDR is substantially identical to a reference CDR in that one amino acid within the CDR is deleted, added, or substituted as compared with the reference CDR while the CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments a CDR is substantially identical to a reference CDR in that 2, 3, 4, or 5 (e.g., 2-5) amino acids within the CDR are deleted, added, or substituted as compared with the reference CDR while the CDR has an amino acid sequence that is otherwise identical to the reference CDR. In various embodiments, an antigen binding fragment binds a same antigen as a reference antibody. In various embodiments, an antigen binding fragment crosscompetes with the reference antibody, for example, binding to substantially the same or identical epitope as the reference antibody.

[0074] An antigen binding fragment may be produced by any means. For example, in some embodiments, an antigen binding fragment may be enzymatically or chemically produced by fragmentation of an intact antibody. In some embodiments, an antigen binding fragment may be recombinantly produced (such as by expression of an engineered nucleic acid sequence). In some embodiments, an antigen binding fragment may be wholly or partially synthetically produced. In some embodiments, an antigen binding fragment may have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino adds or more; in some embodiments at least about 200 amino acids (e.g., 50-100, 50-150. 50-200, or 100-200 amino acids).

[0075] The term “variable region’ or “variable domain’’ is used interchangeably. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementaritydetermining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0076] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody or an antigen -binding molecule thereof.

[0077] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.

[0078] A number of definitions of the CDRs are commonly in use: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular’s AbM antibody modelling software. The contact definition is based on an analysis of the available complex crystal structures.Table 1. CDR NumberingLoop Kabat AbM Chothia Contact LI L24-L34 L24-L34 L24-L34 L30-L36 1.2 L50-L56 L50-L56 L50-L56 L46-L55 1.3 L89-L97 L89-L97 L89-L97 L89-L96 Hl H31-H35B H26-H35B H26— II32..34 H30-H35B (Kabat Numbering)Hl H31--H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering)H2 H50 --H65 H50--H58 H52-H56 H47-H58 H3 H95-H102 H95-H102 H95-H102 H93-H101

[0079] The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen-binding molecule thereof. In certain aspects, the CDRs of an antibody can bedetermined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR.3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0080] In certain aspects, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Moi Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontane A et al., (1990) J Mol Biol 215(1): 175-82; and U. S. Patent No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR- L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-HI loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H3 A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme.

[0081] The terms “constant region” and “constant domain” are interchangeable and have a meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0082] The term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (e), gamma (y) and mu (p), based on the amino acid sequenceof the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3 and IgG4.

[0083] The term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (k) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

[0084] An “antigen” refers to a compound, composition, or substance that may stimulate the production of antibodies or a T cell response in a human or animal, including compositions (such as one that includes a tumor-specific protein) that are injected or absorbed into a human or animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is not substantially found on the surface of other normal (desired) cells and to which a binding domain of a CAR contemplated herein, is designed to bind. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be endogenously expressed, i.e. expressed by genomic DNA, or can be recombinantly expressed. An antigen can be specific to a certain tissue, such as a cancer cell, or it can be broadly expressed. In addition, fragments of larger molecules can act as antigens. In one embodiment, antigens are tumor antigens. In one particular embodiment, the antigen is all or a fragment of CD 19. In certain embodiments, the antigen may include, but is not limited to, 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b- catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD 19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22). CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7 / 8 (cluster of differentiation 44, exons 7 / 8), CAMEL (CTL- recognized antigen on melanoma), CAP-1 (carci noembryonic antigen peptide - 1 ), CASP-8 (caspase-8), CDC27m (cell-division cycle 27 mutated). CDK4 / m (cyclin-dependent kinase 4 mutated). CEA (carcinoembryonic antigen). C-tvpe lectin-like-1 (CLL-1). CT (cancer / testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen melanoma), EGER (epidermal growth factor receptor), EGFRvlll (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40). Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (elongation factor 2 mutated), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), EBP (folate binding protein), fAchR (Fetal acetylcholine receptor), G250 (glycoprotein 250), G / \GE (G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), glypican 3 (GPC3), GnT-V (N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100kD). HAGE (helicose antigen), HER- 2 / neu (human epidermal receptor- 2 / neurological; also known as EGFR2), HLA-A (human leukocyte antigen-A) HPV (human papilloma virus), HSP70-2M (heat shock protein 70 - 2 mutated). HST-2 (human signet ring tumor - 2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxyl esterase), IL-13R-a2 (Interleukin- 13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert domain receptor), i -light chain, LAGE (L antigen), LDLR / FUT (low density lipid receptor / GDP-L-fucose: b-D-galactosidase l a Lfucosyltransferase). LeY (Lewis-Y antibody), LI CAM (LI cell adhesion molecule), MAGE (melanoma antigen), MAGE- Al (Melanoma-associated antigen 1 ), mesothelin, Murine CMV infected cells, MART-l / Melan-A (melanoma antigen recognized by I’ cells-I / Melanoma antigen A), MC1R (melanocortin 1 receptor), Myosin / m (myosin mutated), MUC1 (mucin 1 ), MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural killer group 2, member D) ligands, NY-BR-1 (New York breast differentiation antigen 1 ), NY-ESO-1 (New York esophageal squamous cell carcinoma- 1 ), oncofetal antigen (h5T4), P15 (protein 15), pl 90 minor bcr-abl (protein of 190KD bcr-abl), PML / RARa (promyelocytic leukaemia / retinoic acid receptor a), PRAME (preferentially expressed antigen of melanoma), PSA (prostate-specific antigen), PSCA (Prostate stem cell antigen), PSMA (prostate¬ specific membrane antigen), RAGE (renal antigen), RU 1 or RU2 (renal ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen rejecting tumor 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma XI, -2, -3. -4), TAA (tumor-associated antigen), TAG-72 (Tumor-associated glycoprotein 72), TEL / AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1 ), TPI / rn (triosephosphate isomerase mutated), TRP-1 (tyrosinase related protein 1, or gp75), TRP-2 (tyrosinase related protein 2), TRP-2 / TNT2 (TRP-2 / intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), or WT1 (Wilms' tumor gene).

[0085] In certain embodiments, the antigen is a tumor antigen selected from CD 19, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD 10, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases Erb- B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2). human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (LI CAM), melanoma antigen family A, 1 (MAGE- Al), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin(MSLN), ERBB2. MAGEA3, p53. MARTLGP100, Proteinase3 (PR1), Tyrosinase. Survivin, hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tumor-associated glycoprotein 72 (TAG- 72). vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII. CD99, CD70. ADGRE2, CCR1, LILRB2, FRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, Integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB.

[0086] In some embodiments, the antigen is a tumor antigen selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20. CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudm 18.2, claudin 6, c-Mct, DLL3, DR5. epidermal growth factor receptor (EGER), EGERvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (EBP), folate receptor beta (FRb). follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, TCAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-AI, MAGE-A3, MAGE- A4, Melan A, mesothelia, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof.

[0087] In some embodiments, the antigen is a tumor antigen selected from the group consisting of CD19, CD20, CD22, CD123, CD33, CD3, CD4, CD8, CD38, SLAMF7, BCMA, GD2, GPRC5D, MUC16, FIER2, EGFR, EGFRvIII, CLL-1, CD44v6, folate receptor-a, mesothelia, CD20, CD37, ROR1, carcinoembryonic antigen (CEA), 0-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE- 1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M- CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE- la, p53, prostein, PSMA, Her2 / neu, surviving, telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, 1GE-II, 1GF-I receptor, IL13Ra2, B7-H3 (CD276), EPHA2, GRP78, NKG2D, and CD70.

[0088] In some embodiments, the antigen is a tumor antigen selected from the group consisting of CEA, ERBB2, EGFR, GD2, mesothelia, MUC1, PSMA, GD2, PSMA1, LAP3,ANXA3, Tumor-associated glycoprotein 72 (TAG72), MUC16, 5T4, FRa, MUC28z, NKG2D, HRG10, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), carboxy-anhydrasc-IX (CA-IX), Trop2, claudinl8.2, folate receptor 1 (FOLRl), CXCR2, B7-II3, CD 133, CD24, receptor tyrosine kinase-like orphan receptor 1 -specific (R0R1), EGFR, EGFRvIIL VEGF, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), DLL3, glypican-3, epithelial cell adhesion molecule (EpCAM), GUCY2C (Guanylate Cyclase 2C), doublecortin-like kinase 1 (DCLK1), HER receptors HERE HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigens Ley, Lex, Leb, STEAP1, CD 166, CD24, CD44. E-cadherin, SPARC, and ERBB3.

[0089] In some embodiments, the antigen is a tumor antigen selected from the group consisting of CD19; CD123; CD22; CD30; CD17I; CS-1; C-type lectin-like molecule-1, CD33;epidermal growth factor receptor variant III (EGFRvTII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member; B-cell maturation antigen; Tn antigen ((Tn Ag) or (GalNAca- Ser / Thr));prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2; Mesothelin; Interleukin 11 receptor alpha (IL-1 IRa); prostate stem cell antigen (PSCA); Protease Serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet- derived growth factor receptor beta (PDGFR-beta); Stage- specific embryonic antigen-4 (SSEA- 4); CD20; Folate receptor alpha; Receptor tyrosine -protein kinase ERBB2 (Her2 / neu); Mucin I, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gp100); oncogene polypeptide consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type- A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight-melanoma- associated antigen (HMWMAA); o-acetyl -GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-rel ted (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR): G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta- specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary glanddifferentiation antigen (NY-BR-1); uroplakin 2 (UPK2): Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TOR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY -ESO-1); Cancer / testis antigen 2 (LAGE-la); Melanoma-associated antigen 1 (MAGE- Al); ETS translocation- variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin- binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen- 1 (MAD-CT-1 ); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen- 1. melanoma antigen recognized by T cells 1; Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML- TAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl- transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC- Binding Factor (Zinc Finger Protein) -Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax -5 (PAX5); proacrosin binding protein sp32 (OY-TES 1); lymphocyte- specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the antigen binding domain binds an antigen selected from the group consisting of CD 19, CD22, BCMA. CD20. CD 123, EGFRvIII, and mesothelm.

[0090] In some embodiments, the antigen is selected from alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3 -antigen, carbonic anhydrase EX, CD1, CDla, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CDI23, CD 138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6,CSAp. EGFR, EGP-1, EGP-2. Ep-CAM, EphAl, EphA2, EphA3. EphA4, EphA5, EphA6, EphA7, EphA8, EphA10. EphBl. EphB2, EphB3, EphB4, EphB6. FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-I), la, IL- 2, IL-6, IL-8, insulin growth factor- 1 (IGF-I), KC4- antigen. KS-l-antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF). MAGE, MUC2, MUC3, MUC4. NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, SI 00, TAG, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thornson- Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-1 A-antigen, an angiogenesis marker, an oncogene marker and an oncogene product.

[0091] In one embodiment, the antigen is selected from the group consisting of CD3, NKp46, CD5, CD 19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, GRAGG, SLAMF7, CD319, and 19A24); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); FmsLike Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine -protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr- abl); tyrosinase; ephrin type- A receptor 2 (EphA2); Fucosyl GM I; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker7-related (TEM7R): claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR): G protein coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD 179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51 E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE- la); Melanoma- associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen- 1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53): p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen- 1 (PCT A-l or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl- transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v- myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 IB 1 (CYP1B 1 ); CCCTC-Binding Factor (Zinc Finger Protein)- Like (BORIS or Brother of the Regulator oflmprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation End products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72: Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin -like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope Tag, CD34, LAMP1 TROP2. GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea(CA19.9; Sialyl Lewis Antigen) Fucosyl-GMl. PTK7, gpNMB, CDH1-CD324. DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-lGLll, ALK TCRgamma-delta. NKG2D, CD32 (FCGR2A), CSPG4-HMW-MAA, Timl- / HVCR1, CSF2RA (GM-CSFR-alpha), TGFbctaR2, VEGFR2 / KDR, Lews Ag. TCR-betal chain, TCR-beta2 chain, TCR -gamma chain, TC -delta chain, Leutenizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), Chorionic Gonadotropin Hormone receptor (CGHR), CCR4, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLVl-Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, auto-antibody to desmoglem 3 (Dsg3), autoantibody to desmoglein 1 (Dsgl). HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, claudinl8.2 (CLD1 A2 OR CLDN18A.2)), P- glycoprotein, STEAP1, LIVE NECTIN-4, CRIPTO, GPA33, BST1 / CD157, and low conductance chloride channel and Integrin B7.

[0092] In some embodiments, the antigen is selected from the group consisting of one or more of CD5; CD19; CD123; CD22; CD30: CD171; CS1 (also referred to as CD2 subset 1, CRACC, MPL, SLAMF7, CD 19, and 1 A24); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2): ganglioside GD3 (aNeu.5Ac(2- 8)aNeu5Ac(2-3)bDGaIp(l-4 )bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)): prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38: CD44v6; a glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2): Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Receptor tyrosine -protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP): insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l- 4)bDGlcp(l-l)Cer): transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein- coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE- la); Melanoma- associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen- 1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p.53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-l or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-1AP); ERG (transmembrane protease, serine 2 (TMPRSS2 ) ETS fusion gene); N- Acetyl glucosaminyl- transferase V (NA 17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v- myc avian myelocy tomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 IB 1 (CYP1B 1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY- TES1): lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7): intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2): CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A(CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module- containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope Tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GMl, PTK7, gpNMB. CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGLll, TCRgamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Timl- / HVCR1, CSF2RA (GM-CSFR-alpha), TGFbetaR2, Lews Ag, TCR- betal chain, TCR-beta2 chain, TCR -gamma chain. TCR-delta chain, FITC, Leutenizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), Gonadotropin Hormone receptor (CGHR or GR), CCR4, GD3. SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLVl-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC). auto antibody to desmoglein 3 (Dsg3), auto antibody to desmoglein 1 (Dsgl), HLA. HLA-A. HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB. HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, Claudin18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Livl, Nectin-4, Cripto, gpA33, BST1 / CD157, low conductance chloride channel, and the antigen recognized by I N I antibody.

[0093] In some embodiments, the antigen is a surface protein selected from the group consisting of Alpha (a)-fetoprotein (AFP), interferon-inducible protein absent in melanoma 2 (AIM2), adenocarcinoma antigen recognized by T cells 4 (ART-4), BCMA, B antigen (BAGE), CTL- recognized antigen on melanoma (CAMEL), carcinoembryonic antigen peptide-1 (CAP-1), Caspase 8 (CASP8), Cell division cycle 27 (CDC27), Cyclin-dependent kinase 4 (CDK4), CDK12, Carcinoembryonic antigen (CEA), Calcium-activated chloride channel 2 CLCA2), CFTR, CMV, cancer-testis antigen 83 (CT83), desmin, DLK1, DLL3, EBV, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, EGFR E746-A750del, EGFRVIII, epithelial-specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), Ephrin type-A receptor 2, 3 (EphA2,3). epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), Fibronectin (FN), FGF-5, FGF-6, G antigen 1 (GAGE-1), GAGE-2, GAGaE-3, GAGE-4, GAGE-5, GAGE-6. GAGE-7, N- Acetylglucosaminy transferase V (GnT-V), glycoprotein 100 (GP100), Helicase antigen (HGE), H3.3K27M, oncofetal antigen (h5T4), IP3 KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, Human epidermal receptor 2 / neurological (HER2) / neti), HBV, HERV-E, HIV-1 gag, HM1.24, HMB-45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16 E7, Human telomerase reverse transcriptase (hTERT), V-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), KRAS G12D, KRAS G12V, L antigen lb (LAGElb). LMP2, LILRB2, LGR5, Ly49, Ly108, LI celladhesion molecule (Ll-CAM), melanoma-associated antigen (MAGE), Melanoma antigen Al (MAGE-A1). MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, c-Mct, MTCA / B, muscle-specific actin (MSA), protein mclan-A (melanoma antigen recognized by T lymphocytes MART-1), Mesothelin (MSLN), Mucin 1 (MUC1), MUC2, Mucin 16 (Muc-16). myo-Dl, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2- PK), Neel-2, neurofilament, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO, New York esophageal 1 (NY-ESO- 1), Preferentially expressed antigen of melanoma (PRAME), Prostate-specific antigen (PSA), Prostate-specific membrane antigen (PSMA), Renal antigen (RAGE), Ral-B, an abnormal ras protein, R0R1, SLAMF7 / CS1. sperm protein 17 (Spl7), sarcoma antigen (SAGE), squamous antigen rejecting tumor 1, 2, 3 (SART-1, -2, -3). SOXIO, synovial sarcoma, X breakpoint 2 (SSX-2), Survivin, OVAl, IIE4, DR-70, Total PSA, alpha-Methylacyl-CoA Racemase / AMACR, CA125 / MUC16, ER alpha / NR3Al, ER beta / NR3A2, Thymidine Kinase 1, AG-2, BRCA1. BRCA2, CA15-3 / MUC-1, Caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, Cytokeratin 14, HIN-1 / SCGB3A1. K1-67 / MK167, MKP-3, Nestin. NGF R / TNFRSF16, NM23-H1, PARP, PP4, Serpin El / PAI-1, 14-3-3 beta, 14-3-3 sigma, 14-3-3 zeta, 15-PGDH / HPGD, 5T4, TIM-3, TROP-2, Nectin-4, PD1, PD-L1, CTEA-4, PDGFRalpha. VEGF, TRAG-3, T cell receptor gamma alternate reading frame protein (TARP), TGFbll, Thyroglobulin, an abnormal p53 protein, TP53 (p53), TRAIL, Tyrosinase-related protein 1 or gp75 (TRP1), TRP2, TYRP1, Tyrosinase, tumor-associated glycoprotein 72 (TAG-72), TALLA-1, TLR4, TRBC1, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, Vu24, Wilms’ tumor gene (WIT), CDla, CDlb, CDlc, CD2, CD3, CD4, CDS, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD 12, CD 13, CD 14. CD15 (SSEA-1), CD 16, (FcyRIII), CD 17, CD 18, CD20, CD21, CD22. CD23, CD24, CD25. CD26, CD27, CD28, CD29. CD30. CD31, CD32 (FcyRII), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61 (integrin 03), CD62L, CD63, CD64 (FcyRI), CD66b, CD68, CD69, CD70, CD73, CD74, CD79a (Igu), CD79b (Ig0), CD80, CD83. CD85k (ILT3), CD86. CD88, CD93 (CIRqp), CD94, CD95, CD99. CD103, CD 105 (Endoglin), CD107a, CD 107b, CD114 (G-CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (Syndecan-1), CD141 (BDCA3), CD146, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1.1), CD163, CD183, CD191, CD193 (CCR3), CD 194 (CCR4), CD 195 (CCRS), CD 197 (CCR7), CD203c, CD205 (DEC-205), CD207 (Langerin), CD209 (DC-SIGN), CD223, CD235. CD235a, CD244 (2B4), CD252 (OX40L), CD267, CD268 (BAFF-R), CD273 (B7-DC, PD-L2), CD276 (B7-1I3), CD279 (PDI), CD282 (TLR2), CD284 (TLR4), CD294, CD304 (Neuropilin- 1), CD305, CD314 (NKG2D), CD319 (CRACC), CD326, CD328 (Siglec-7), CD335 (NKp46), HLA-DR, Kappa light chain, Lambdalight chain, Pax-5, BCL-2, Ki-67, MPO, TdT, FMC-7, Pro2PSA, ROMA (HE4+CA-125). OVA1 (multiple proteins), HE4, Fibrin / fibrinogen degradation product (DR-70), AFP-L3, Circulating Tumor Cells (EpCAM, CD45, cytokeratins 8, 18+, 19+), Prostate stem cell antigen (PSCA), u2pl, PAP (prostatic acid phosphatase), PAPA, P-cadherin. placental alkaline phosphatase, PRAIVIE, C3AR, carbonic anhydrase IX (CAIX), chromogranin, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), CS-L CSPG4, cytokeratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CAI 9.9), Lewis Y (LeY), Estrogen receptor (ER), Progesterone receptor (PR), Pro2PSA, cancer antigen-125 (CA-125), CAI 5-3, CA27.29, Free PSA, Thyroglobulin, Nuclear Mitotic Apparatus protein (NuMA / NMP22), A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, Aldo-keto Reductase 1C3 / AKR1C3, alpha 1 B- Glycoprotein, alpha 1 -Microglobulin, AlphaB Crystallin / CRYAB, alpha-Methylacyl-CoA Racemase / AMACR, AMFR / gp78, Annexin A3, Annexin A8 / ANXA8, APC, Apolipoprotein A-I / ApoAl, Apolipoprotein A-II / ApoA2, Apolipoprotein E / ApoE. APRIL / TNFSF13, ASCLl / Mashl, ATBF1 / ZFHX3, Attractin. Aurora A, BAP1, Bel- 2, Bcl-6, beta 2-Microglobulin, beta-1, 3-Glucuronyltransferase 1 / B3GAT1, beta-Catenin, beta-III Tubulin, Bikunin,B-Raf, BRCA1, BRCA2, Brk, C4.4A / LYPD3, CA 15-3 / MUC-l, c-Abl, Cadherin-13, Caldesmon / CALDl, Calponin 1, Calretinin, Carbonic Anhydrase IX / CA9, Catalase, Cathepsin D, Caveolin-1, Caveolin-2, CBFB, CCR1, CCR4, CCR7, CCR9, CEA CAM- 19, CEACAM-20, CEACAM-4, CHD1L, Chitinase 3-like 1. Cholecystokinin-B R / CCKBR, Chorionic Gonadotropin alpha Cham (alpha HCG), Chorionic Gonadotropin alpha / beta (HCG), CKAP4 / p63, Claudin-18, Clusterin, c-Maf, c-Myc, Coactosin- like Protein 1 / CotL1, C0MMD1, Cornulin, Cortactin, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL-8, CXCL9 / MIG, CXCR4, Cyclin Al, Cyclin A2, Cyclin D2, Cyclin D3, CYLD, Cyr61 / CCN1, Cytokeratin 14, Cytokeratin 18, Cytokeratin 19, fetal acetylcholine receptor (AChR), ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2 / ESDN, DC-LAMP, Dkk-1, DLL3, DMBT1. DNMT1, DPPA2. DPPA4, E6. E-Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endoglin / CD105. Endosialin / CD248, Enolase 2 / Neuron-specific Enolase, EpCAM / TROPl, Epsl5, ER alpha / NR3Al, ER beta / NR3A2, ERBB, EGFR / ErbBl, ERBB2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E-FABP, Fascin, FTP3, FCRLA, Fetuin A / AHSG. FGF acidic, FGF basic, FGF R3, FGF R4, Fibrinogen, folate-binding protein (EBP). Fibroblast Activation Protein alpha / EAP, Follistatin-like 1 / FSTL1, F0LR1, F0LR2, F0LR3, F0LR4, FosB / GOS3, FoxMl, Fox()3, FRAT2, FXYD5 / Dysadherin, FcRIII, FITC, FLT3, GABA- A R alpha 1, GADD153, GADD45 alpha, Galectin-3. Galectin-3BP / MAC-2BP, galectin, gangliosides, gross cystic diseasefluid protein (GCDFP- 15), GD2 (ganglioside G2), GD3, GM2, GM3, gamma- Glutamylcyclotransferase / CRF21, Gasl, Gastrin-releasing Peptide R / GRPR, Gastrokine 1, Gclsolin / GSN, glial fibrillary acidic protein (GFAP), GLI-2, Glutathione Peroxidase 3 / GPX3, gpA33, glycopeptides, Glypican 2 (GPC2), Glypican 3, Golgi Glycoprotein 1 / GLG1, gp96 / HSP90Bl, GPR10, GPR110, GPR18, GPR31, GPR87. GPRC5A, GPRC6A, GRP78 / IISPA5, HE4 / WFDC2, Heparanase / HPSE, Hepsin, HGF R / c-MET, H1F-2 alpha / EPASl, IIIN-1 / SCGB3A1, HLA-DR, H0XB13, H0XB7, HSP70 / ITSPA1A, IISP90, Hyaluronidase 1 / HYAL1, ID1, IgE, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-I, IGF-I R, IGF-II, IGFL-3, IGFLR1, IL-1 beta / lL-lF2, 1L-17E / IL-25, IL-2, IL-6, ICAM-1, IgG, IgD, IgE, IgM, Interleukin 13 receptor a2 chain (IL-13Ra). Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), integrins, Integrin B7, IMP Dehydrogenase 1 / IMPDHl, Importin alpha 2 / KPNA2, ING1, Integrin beta 1 / CD29, Integrin beta 3 / CD61, IQGAP1, Isocitrate Dehydrogenase 1 / IDH1, ITIH4, ITM2C, Jagged 1, JNK, JunB, JunD, 0GR1, Olig2, Osteopontin / OPN, Ovastacin, OXGR1 / GPR80 / P2Y15, pl30Cas, p!5INK4b / CDKN2B, pl6INK4a / CDKN2A, pl 8INK4c / CDKN2C, p2l / CIP1 / CDKN1A, p27 / Kip1, P2x5 / P2RX5, PARP, PAUF / ZG16B, PBEF / Visfatin, PDCD4, PDCD5, PDGF R alpha, PDGF R beta, PDZD2, PEA- 15, Pepsinogen A5 / PGA5, Peptidase Inhibitor 16 / PI16, Peroxiredoxin 2, PGCP, PI 3-Kinase p85 alpha, PIWIL2, PKM2, PLK1, PLRPI, PP4, P-Rex 1, PRMT1, Profilin I, Progesterone R B / NR3C3, Progesterone R / NR3C3, Progranulin / PGRN, Prolactin, Prostaglandin E Synthase 2 / PTGES2, PSAP, PSCA, PSMA / FOLHl / NAALADase 1, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PIP beta / zeta / PTPRZ, Rab25, RARRES I, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100AI6, S100A2, S100A4, S100A6, S100A7, S100A9, SIOOB, S100P, SARTL SCUBE3. Secretin R, Serpin A9 / Centerin, Serpin El / PAI-1, Serum Amyloid Al, Serum Amyloid A4. SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostein, SLC5A5, SLC5A8 / SMCT1, SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy- 1 / DkkLl, SOX11, SOX17, SOX2, SPARC, SPARC-like 1 / SPARCLL SPINK 1, Src, six-transmembrane epithelial antigen of the prostate 1 (STEAP1), STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, Survivin, Synaptotagmin-1. Syndecan-1 / CD138, Syntaxin 4, Synuclein-gamma, synaptophysin, Kallikrein 2, Kallikrein 6 / Neurosin, KCC2 / SLC12A5, KL67 / MKI67, K1SS1R / GPR54, KLF10, KLF17, LI CAM, Lactate Dehydrogenase A / LDHA, Lamin Bl, LEF1, Leptin / OB, LIN-28A, LIN-28B, Lipocalin-2 / NGAL, LKB1 / STK1 L LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, LYPD8, MAP2, Matriptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, Melanocortin- 1 R / MC1R, Melanotransferrin / CD228, Melatonin, Mer, Mesothelin, Metadherin, Metastin / KiSSl, Methionine Aminopeptidase, MethionineAminopeptidase 2 / METAP2, MFAP3L, MGMT, MIA, MIF, MINA, Mind Bomb 2 / M1B2, Mindin. MITF, MKK4, MKP-1, MKP-3, MMP-1, MMP-10, MMP-13, MMP-2, MMP-3, MMP- 8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSP R / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM-1 / CD56, NCOA3. NDRG1, NEK2, NELLI, NELL2, Nesfatin-l / Nucieobindin-2, Nestin. NFkB2, NF-L, NG2 / MCSP, NGF R / TNFRSF16, Nicotinamide N- Methyltransferase / NNMT, NKX2.2, NKX3.1, NM23-H1, NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, Tankyrase 1, Tau, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, Tenascin C, TFF1, TGF-beta 1, TGF-beta 1, 2, 3, TGF-beta 1 / 1.2, TGF-beta 2 / 1.2, TGF-beta RI / ALK-5, THRSP, Thymidine Kinase 1, Thymosin beta 10, Thymosin beta 4, Thyroglobulin, TIMP Assay Kits. T1MP-1, T1MP-2, T1MP-3, TIMP-4, TLE1, TLE2, TM4SF1 / L6, TMEFF2 / Tomoregulin-2, TMEM219, TMEM87A, TNF-alpha, TOP2A, TopBPl, t-Plasminogcn Activator / tPA, TRA-l-60(R), TRA-1-85 / CD147, TRAF-4, Transgclin / TAGLN, Trypsin 2 / PRSS2, Tryptase alpha / TPSl, TSPAN1, UBE2S. uPAR, u- Plasminogen Activator / Urokinase. Urotensin-II R, VAP-1 / AOC3, VCAM-1 / CD106. VEGFRl / Flt-1, VEGFR2 / KDR / Flk-1, VEGF / P1GF Heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZl / ZimplO, SGK, CNKSR1 / CNK / KSR or any combination thereof.

[0094] In some embodiments, the tumor antigen is associated with an hematological cancer and is selected from the group consisting of BCMA, CD 19, CD20. CD22, CD.30, CD 123, CD70, CD33, GDI 35. CD44, CD276. CD2, CD3, CD4, CD5, CD7, CD8, CD10. CD37. CD79, CD79a, CD80, CD138, CD47, CRLF2, CD38, CLL-1, NKG2D, CALR, IL1RAP, ILT3, TIM3, CD96, VISTA, CS1, TACI, APRIL, GPRC5D, and CD44v6. In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematologic malignancy. In some embodiments, the tumor antigen associated with glioblastoma is selected from IIER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1 and IL13Ra2, the tumor antigen associated with ovarian cancer is selected from FOLRl, FSIIR, MUCI 6, MUCI, Mesothelia, CA125, EpCAM, EGFR, PDGFRa. Nectin-4. and B7H4, the tumor antigen associated with cervical cancer or head and neck cancer is selected from GD2. MUCI, Mesothelin, I1ER2, and EGFR, the tumor antigen associated with liver cancer is selected from Claudin 18.2, GPC-3, EpCAM, cMET, and AFP, the tumor antigen associated with hematological malignancies is selected from CD 19, CD22. CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70, and the tumor antigen associated with bladder cancer is selected from Nectin- 4 and SLITRK6.

[0095] In some embodiments, the cancer is multiple myeloma and the tumor antigen is BCMA. In some embodiments, the cancer is hepatocellular carcinoma and the tumor antigen is GPC3. InK-II77-WO-PCT some embodiments, the cancer is a lymphoma and the tumor antigen is CD 19. In some embodiments, the cancer is a leukemia and the tumor antigen is CD19. In some embodiments, the cancer is an ovarian cancer and the tumor antigen is selected from one or more of mcsothelin, glycoprotein 72 (TAG72), MUC16, Her2, 5T4, and FRa.

[0096] In some embodiments, the cancer is a breast cancer and the tumor antigen is selected from one or more of MUC28z, NKG2D, IIRG10, and HER2.

[0097] In some embodiments, the cancer is a prostate cancer and the tumor antigen is selected from one or more of prostate stern cell antigen (PSCA) and prostate- specific membrane antigen (PSMA).

[0098] In some embodiments, the cancer is a renal cancer and the tumor antigen is carboxy- anhydrase-IX (CA-IX).

[0099] In some embodiments, the cancer is a gastric cancer and the tumor antigen is selected from one or more of Trop2, claudin!8.2, NKG2D, folate receptor 1 (FOLR1), and HER2.

[0100] In some embodiments, the cancer is a pancreatic cancer and the tumor antigen is selected from one or more of mesothelm, MUC1, CXCR2, B7-H3, CD133, CD24, PSCA, CEA, and Her-2.

[0101] In some embodiments, the cancer is a lung cancer and the tumor antigen is selected from one or more of mesothelia, receptor tyrosine kinase-like orphan receptor 1 -specific (R0R1), EGFRvIIT, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), PSCA, MUC1, and DLL3.

[0102] In some embodiments, the cancer is a liver cancer and the tumor antigen is selected from one or more of MUC1, CEA, glypican-3, and epithelial cell adhesion molecule (EPC AM).

[0103] In some embodiments, the cancer is a colorectal cancer and the tumor antigen is selected from one or more of MUC1, NKG2D, CD133, GUCY2C (Guanylate Cyclase 2C), TAG- 72 Doublecortin-Iike kinase 1 (DCLK1), and CEA.

[0104] In some embodiments, the cancer is glioblastoma, and the tumor antigen is EGFRvIII.

[0105] In some embodiments, the hematological cancer is myelofibrosis and the tumor antigen is CALR.

[0106] In some embodiments, the hematological cancer is myelodysplastic syndromes and the tumor antigen is selected from one or more of CD 123, CD33, and NKG2D.

[0107] In some embodiments, the hematological cancer is acute myeloid leukemia and the tumor antigen is selected from one or more of CD123, CLL-1, IL1RAP, CD33, CD135. CD70, CD44, CD276, ILT3, CD7, CD47, TIM3, CD96, and VISTA.

[0108] In some embodiments, the hematological cancer is acute lymphocytic leukemia and the tumor antigen is selected from one or more of CD19, CD22, CD79a, CD10, CD2, CD3, CD4, CD5, CD7, CD8, CRLF2, and CD38.

[0109] In some embodiments, the hematological cancer is non-Hodgkin's lymphoma and the tumor antigen is selected from one or more of CD 19, CD20, CD22, CD80, CD37, CD79, CD30, CD70, and CD38.

[0110] In some embodiments, the hematological cancer is multiple myeloma and the tumor antigen is selected from one or more of BCMA, CD 19, CD138, CS1, CD38, TACI, APRIL, GPRC5D, and CD44v6.

[0111] A “target” is any molecule bound by a binding domain, antigen binding system, CAR or antigen binding agent, e.g., an antibody.

[0112] " Antigen-specific targeting region" (ASTR) refers to the region of the CAR which targets specific antigens. The targeting regions on the CAR are extracellular. In some embodiments, the antigen-specific targeting regions comprise an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof and each of the targeting regions target a different antigen. The targeting regions may comprise full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen. There are, however, numerous alternatives, such as linked cytokines (which leads to recognition of cells bearing (he cytokine receptor), affibodies. ligand binding domains from naturally occurring receptors, soluble protein / peptide ligand for a receptor (for example on a tumor cell), peptides, and vaccines to prompt an immune response, which may each be used in various embodiments of this disclosure. In fact, almost any molecule that binds a given antigen with high affinity can be used as an antigen-specific targeting region, as will be appreciated by those of skill in the art.

[0113] ‘ ‘Antigen presenting cell” or “APC” refers to cells that process and present antigens to 1’ cells. Exemplary APCs comprise dendritic cells, macrophages, B cells, certain activated epithelial cells, and other cell types capable of TCR stimulation and appropriate T cell costimulation.

[0114] An “anti -tumor effect” refers to a biological effect that can present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression- free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor. An anti -tumor effect can also refer to the prevention of the occurrence of a tumor.

[0115] Two events or entities are “associated” with one another if the presence, level, and / or form of one is correlated with that of the other. For example, an entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a disease, disorder, orcondition, if its presence, level, and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). For example, two or more entities arc physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another (e.g., bind). In additional examples, two or more entities that are physically associated with one another are covalently linked or connected to one another, or non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0116] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff / kf,n, whereas KA is calculated from the quotient of koll / koif. kollrefers to the association rate constant of, e.g., an antibody to an antigen, and kofi refers to the dissociation of, e.g., an antibody to an antigen. The konand koff can be determined by techniques known to one of ordinary skill in the art, such as BIACORE® or KinEx.

[0117] The term " KD" (M) refers to the dissociation equilibrium constant of a particular antibody¬ antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. There is an inverse relationship between KD and binding affinity, therefore the smaller the KD value, the higher, i.e. stronger, the affinity. Thus, the terms “higher affinity” or “stronger affinity” relate to a higher ability to form an interaction and therefore a smaller KD value, and conversely the terms “lower affinity” or “weaker affinity” relate to a lower ability to form an interaction and therefore a larger KD value. In some circumstances, a higher binding affinity (or KD) of a particular molecule (e.g. antibody) to its interactive partner molecule (e.g. antigen X) compared to the binding affinity of the molecule (e.g. antibody) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger KD value (lower, or weaker, affinity) by the smaller KD (higher, or stronger, affinity), for example expressed as 5-fold or 10-fold greater binding affinity, as the case may be.

[0118] The term "kd" (sec - 1 or 1 / s) refers to the dissociation rate constant of a particular antibody¬ antigen interaction, or the dissociation rate constant of an antibody or antibod -binding fragment. Said value is also referred to as the ko value.

[0119] The term "ka" (M-l x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0120] The term " KA" (M-l or 1 / M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody binding fragment. The association equilibrium constant is obtained by dividing the kaby the kd.

[0121] The term “binding” generally refers to a non-covalent association between or among two or more entities. Direct binding involves physical contact between entities or moieties. “Indirect” binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities may be assessed in any of a variety of contexts, e.g., where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system such as a cell).

[0122] The terms “immunospecifically binds,” “imniunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope or immune complex) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIACORF®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the KA when the molecules bind to another antigen. Binding may comprise preferential association of a binding domain, antibody, or a CAR with a target of the binding domain, antibody, or CAR as compared to association of the binding domain, antibody, or CAR with an entity that is not the target (i.e. non-target). In some embodiments, a binding domain, antibody, or CAR selectively binds a target if binding between the binding domain, antibody, or CAR and the target is greater than 2-fold, greater than 5-fold, greater than 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or greater than 100-fold as compared with binding of the binding domain, antibody, or CAR and a non-target. In some embodiments, a binding domain, antibody, or CAR selectively binds a target if the binding affinity is less than about 10 ' M, less than about 100M, less than about 10"' M, less than about 108M, or less than about 10"9M.

[0123] In another embodiment, molecules that specifically bind to an antigen bind with a dissociation constant (Ka) of about 1 x 10'7M. In some embodiments, the antigen binding molecule specifically binds an antigen with “high affinity” when the Kd is about 1 x IO’9M to about 5 x 10"9M. In some embodiments, the antigen binding molecule specifically binds anantigen with “very high affinity” when the Kd is I x IO'10M to about 5 x l(f10M. In one embodiment, the antigen binding molecule has a Kd of IO-9M. In one embodiment, the off-rate is less than about 1 x 10"5. In embodiments, the antigen binding molecule binds CD 19 with a Kd of about 1 x 10iOM to about 5 x 1010M.

[0124] In certain embodiments, provided herein is an antibody or an antigen binding molecule thereof that binds to the target human antigen, e.g., In certain embodiments, the antigen binding molecule binds to CD19 with a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher affinity than to another species of the target antigen as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In a specific embodiment, an antibody or an antigen binding molecule thereof described herein, which binds to a target human antigen, will bind to another species of the target antigen with less than 10%, 15%, or 20% of the binding of the antibody or an antigen binding molecule thereof to the human antigen as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

[0125] A “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor. Examples of cancers that can be treated by the methods of the present di closure include, but are not limited to, cancers of the immune system including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods of the present disclosure can be used to reduce the tumor size of a tumor derived from, for example, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, liver cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, multiple myeloma. Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS). primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T- ccll lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and combinations of said cancers. In one particular embodiment, the cancer is multiple myeloma. The particular cancer can be responsive to chemo- or radiation therapy or the cancer can be refractory. A refractory cancer refers to a cancer that is not amendable to surgical intervention and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time. Cancer further includes relapsed or refractory after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary' mediastinal large B-cell lymphoma after two or more lines of systemic therapy, high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma, and follicular lymphoma.

[0126] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a late-stage cancer. In some embodiments, the cancer is a terminal cancer. In some embodiments, the cancer is in stage II, III or IV. In some embodiments, the cancer is an inoperable tumor and / or is malignant. In some embodiments, the tumor is at least 0.2cm, 0.4cm, 0.6cm, 0.8cm, 1cm, 2 cm, 3cm, 4cm or 5cm in length. In some embodiments, the cancer is relapsed / refractory. In some embodiments, the cancer has been subjected to and / or failed one or more prior therapy (e.g., an immune checkpoint blockage therapy (e.g., a PD-1 antibody), a chemotherapy, a surgery, a cell therapy (e.g., an allogenic NK cell infusion therapy)). In some embodiments, the cancer is a recurrent or refractory cancer. In some embodiments, the cancer is refractory to one or more of irradiation therapy, chemotherapy, or immunotherapy (e.g., checkpoint blockade).

[0127] In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is adrenocortical carcinoma, agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligoden glioma, meningioma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer),ependymoma, esophageal cancer, Ewing’s family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non- melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms’ tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome. In some embodiments, the cancer is a virus -infection- related cancer. In some embodiments, the cancer is a human papillomavirus (I IPV)- related cancer (e.g., HPV-related cervical cancer, e.g., HPV-related head and neck cancer, e.g., HPV related squamous cell carcinoma). In some embodiments, the cancer is human herpes virus 8 (HHV8) related cancer (e.g., Kaposi sarcoma). In some embodiments, the cancer is human T- lymphotrophic virus (I ITLV-1 (-related cancer (e.g., adult T cell leukemia or lymphoma). In some embodiments, the cancer is Epstein-Ban* virus (EBV) related cancer (e.g., Burkitt lymphoma, Hodgkin’s and non-Hodgkin’s lymphoma, stomach cancer). In some embodiments, the cancer is hepatitis B virus (HBV) related cancer (e.g., liver cancer). In some embodiments, the cancer is hepatitis C virus) related cancer (e.g., liver cancer, non-Hodgkin’s lymphoma). In some embodiments, the cancer is a liver cancer, a kidney cancer, an endometrial cancer, a thymicepithelial neoplasma, lung cancer, spindle ceil sarcoma, chondrosarcoma, uterine smooth muscle, colon cancer, or pancreatic cancer.

[0128] “Chernokines’' arc a type of cytokine that mediates cell chemotaxis, or directional movement. Examples of chernokines include, but are not limited to, IL-8, IL- 16, eotaxin, eotaxin-3. macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory' protein la (MIP-la, MIP-la), MIP-ip (MIP-lb), gamma-induced protein 10 (IP-10), and thymus and activation regulated chemokine (TARC or CCL17).

[0129] “Chimeric antigen receptor” or “CAR” refers to a molecule engineered to comprise a binding domain and a means of activating immune cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells, iNKT cells, NK cells or combination thereof) upon antigen binding. CARs arc also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. In some embodiments, a CAR comprises a binding domain, an extracellular domain, a hinge / spaced domain, a transmembrane domain, one or more co¬ stimulatory domains, and an intracellular signaling domain. A T cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR T cell.

[0130] “Extracellular domain" (or “ECD”) refers to a portion of a polypeptide that, when the polypeptide is present in a cell membrane, is understood to reside outside of the cell membrane, in the extracellular space.

[0131] The term "extracellular ligand-binding domain." as used herein, refers to an oligo- or polypeptide that is capable of binding a ligand, e.g., a cell surface molecule. For example, the extracellular lig nd-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer). Examples of cell surface markers that may act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

[0132] The binding domain of the CAR may be followed by a "spacer," or "hinge," which refers to the region that moves the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412- 419). The hinge region in a CAR is generally between the transmembrane (TM) and the binding domain. In certain embodiments, a hinge region is an immunoglobulin hinge region and may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8alpha, CD4, CD28 and CD7, which may be wild-type hinge regions from these molecules or may be altered.

[0133] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell, and facilitates binding of the binding domain to the target antigen. The transmembrane domain may be a JAML transmembrane domain. The transmembrane domain may be a CD3zeta transmembrane domain, however other transmembrane domains that may be employed include those obtained from CD8alpha, CD4, CD28, CD45, CD9, CD 16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and GDI 54. In one embodiment, the transmembrane domain is the transmembrane domain of CD137. In certain embodiments, the transmembrane domain is synthetic in which case it would comprise predominantly hydrophobic residues such as leucine and valine. In certain embodiments, the transmembrane domain comprises the transmembrane region of MHC class I molecules, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7. CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDl la / CDlS), 4- IBB (CD137). B7-H3, CDS. ICAM- 1. 1COS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma. IL7R alpha, ITGA4, VLA1, CD49a. ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD. GDI Id, ITGAE, CD103, ITGAL, GDI la, LFA-1, ITGAM, CD1 lb, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D. NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226). SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CR'fAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl()8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, GDI 9a, or a receptor that specifically binds with CD83. or a fragment thereof. The transmembrane domain may also be a fragment of said proteins.

[0134] The "intracellular signaling domain" or “signaling domain’’ refers to the part of the chimeric antigen receptor protein that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of the cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the terms "intracellular signaling domain" or “signaling domain,” used interchangeably herein, refer to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, inK-1177-WO-PCT many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal. The intracellular signaling domain is also known as the, "signal transduction domain," and is typically derived from portions of the human CD3 or FcRy chains.

[0135] It is known that signals generated through the T cell receptor alone are insufficient for full activation of the T cell and that a secondary, or costimulatory signal is also required. Thus. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen dependent primary activation through the T cell receptor (primary cytoplasmic signaling sequences) and those that act in an antigen independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a primary activation manner may contain signaling motifs which are known as immunoreceptor tvrosine-based activation motif or ITAMs. Examples of IT AM containing primary cytoplasmic signaling sequences that are of particular use in the disclosure include those derived from DAP- 12, CD3gamma, CD3delta, and CD3epsilon.

[0136] As used herein, the term, "costimulatory signaling domain," or "costimulatory domain", refers to the portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. In one embodiment, the disclosure provides a co-stimulatory domain of. I AML. Other examples of such co-stimulatory molecules include CD27, CD28, 4- IBB (CD 137). 0X40 (CD134), CD30. CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 CD28, CD27, 0X40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CDS, CD8alpha, CDSbeta, CD11a, CDllb, CDlle, CD1 Id, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49D, CD49L CD58, CD53, ICAM-1 (CD54). CD69, CD70, CD80 (B7-1), CD82, CD83. CD84. CD86 (B7-2), CD90, CD96, CD100, CD103, CD 122, CD132, CD150 (SLAMF1), CD 160 (BY55), CD 162 (DNAMI), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, I, AT, lymphocyte function- associated antigen-I (LFA-1), LIGHT. NKG2C. NKG2D. NK.p30, NKp44, NKp46, NKp80 (KLRFI), DAP10, DAP12. LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIT, 4-1 BBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40I., TAC1, BLAME, CRACC, CD2F-10, NTB-A, integrin a4, integrin a4pl, integrin a4p7, IA4, ICAM-1, IL-2Ralpha, IL-2Rbeta, lL-2Rganima, IL-4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R,IL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76. Toll-like receptors (TLRs, such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, 1TGAL. 1TGAM, 1TGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGLI, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, BTLA, ikaros. LAG-3, LAHR, CEACAM1, CRT AM, TCLIA, DAP 12, TIM-1, Dectin- 1, PDCD6, PD-1, TIM-4, TSLP, and a ligand that specifically binds CD83, or any combination thereof. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from CD28 and 4- IBB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more costimulatory signaling domains may enhance the efficacy and expansion of T cells expressing CAR receptors. The intracellular signaling and costimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0137] Although scFv-based CARs engineered to contain a signaling domain from CD3 or FcRgamma have been shown to deliver a potent signal for T cell activation and effector function, they are not sufficient to elicit signals that promote T cell survival and expansion in the absence of a concomitant costimulatory signal. Other CARs containing a binding domain, a hinge, a transmembrane and the signaling domain together with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from 4-1BB, CD28, CD137, CD 134 and CD278) may more effectively direct antitumor activity as well as increased cytokine secretion, lytic activity, survival and proliferation in CAR expressing T cells in vitro, and in animal models and cancer patients (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0138] In some embodiments, the CAR of the present disclosure further comprising a leader domain. In certain embodiments, the CAR further comprises one or more spacer sequences between one or more of the domains. In certain embodiments, the spacer sequences is a polypeptide linker.

[0139] A “costimulatory signal" refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to a T cell response, such as, but not limited to, proliferation and / or upregulation or down regulation of key molecules.

[0140] A “costimulatory ligand" includes a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A costimulatory' ligand induces a signal that is in addition to the primary signal provided by a stimulatory molecule, for instance, by binding of a T cell receptor (TCR) / CD3 complex with a major histocompatibility complex (MHC) molecule loaded with peptide. A co-stimulalory ligand can include, but is not limited to, 3 / TR6, 4- IBB ligand,agonist or antibody that binds Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin- like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligand that specifically binds with B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain- related protein B (MICE), 0X40 ligand, PD-L2, or programmed death (PD)-Ll. A co-stimulatory ligand includes, without limitation, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligand that specifically binds with CD83, lymphocyte function- associated antigen- 1 (LFA-1), natural killer cell receptor C (NKG2C), 0X40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0141] A “costimulatory molecule” is a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. A “costimulatory molecule” is a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CDL54, CD16, CD160 (BY55), CD18, CD19, CD 19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a. CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1- la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP- 10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, 1TGA4, ITGA6, 1TGAD, 1TGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, K1RDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18). MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRFI), 0X40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMFl; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VL. A-6, or fragments, truncations, or combinations thereof.

[0142] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with basic side chains(e.g.. lysine, arginine, histidine), acidic side chains (e.g.. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isolcucinc, proline, phenylalanine, methionine), beta-branched side chains e.g.. threonine, valine, isoleucine) and aromatic side chains (e.g.. tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR(s) or within a framework region(s) of an antibody or antigen¬ binding molecule thereof can be replaced with an amino acid residue with a similar side chain. In general, two sequences are generally considered to be “substantially similar” if they contain a conservative amino acid substitution in corresponding positions. For example, certain amino acids are generally classified as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may be considered a conservative substitution. Exemplary amino acid categorizations are summarized in Tables 2 and 3 below:K-I177-WO-PCT Table 2Amino Acid 3 -Letter 1 -Letter Property Property Hydropathy Index Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive -4.5 Asparagine Asn N polar neutral -3.5 Aspartic acid Asp D polar negative -3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu E polar negative -3.5 Glutamine Gin Q polar neutral -3.5Glycine Gly G nonpolar neutral -0.4 Histidine His II polar positive -3.2 Isoleucine He I nonpolar neutral 4.5leucine Leu L nonpolar neutral 3.8Lysine Lys K polar positive -3.9 Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8Proline Pro P nonpolar neutral -1.6Serine Ser S polar neutral -0.8 Threonine Thr T polar neutral -0.7 Tryptophan Trp W nonpolar neutral -0.9 Tyrosine Tyr Y polar neutral -1.3Valine Vai V nonpolar neutral 4.2Table 3Ambiguous Amino Acids 3-Letter 1 -Letter Asparagine or aspartic acid Asx B Glutamine or glutamic acid Glx ZIxjucine or Isoleucine Xie J Unspecified or unknown amino acid Xaa X

[0143] “Combination therapy’’ refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic moieties). In some embodiments, the two or more regimens may be administered simultaneously; in someembodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents arc administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0144] “Corresponding to” may be used to designate the position / identity of a structural element in a molecule or composition through comparison with an appropriate reference molecule or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, for purposes of simplicity, residues in a polypeptide may be designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 100, for example, need not actually be the 100th amino acid in an amino acid chain provided it corresponds to the residue found at position 100 in the reference polypeptide. Various sequence alignment strategies are available, comprising software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSLSearch, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SW1MM, or SWIPE that may be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure.

[0145] An antigen binding molecule, such as an antibody, an antigen binding fragment thereof, CAR or TCR, “cross-competes” with a reference binding molecule, such as an antibody or an antigen binding fragment thereof, if the interaction between an antigen and the first antigen binding molecule blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule to interact with the antigen. Cross competition can be complete, e.g., binding of the antigen binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind the antigen, or it can be partial, e.g., binding of the antigen binding molecule to the antigen reduces the ability of the reference antigen binding molecule to bind the antigen. In certain embodiments, an antigen binding molecule that cross-competes with a reference antigen binding molecule binds the same or an overlapping epitope as the reference antigen bindingmolecule. In other embodiments, the antigen binding molecule that cross-competes with a reference antigen binding molecule binds a different epitope than the reference antigen binding molecule. Numerous types of competitive binding assays can be used to determine if one antigen binding molecule competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin ELX (Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct labeled assay, solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (Morel et al., 1988. Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (Cheung, et al., 1 90, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1 90, Scand. J. Immunol. 32:77-82).

[0146] A “cytokine," refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. A cytokine can be endogenously expressed by a cell or administered to a subject. Cytokines may be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce various responses in the recipient cell. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL- 15, promote immune cell survival and proliferation, and pro- inflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory' cytokines include, but are not limited to, IL- la, IL- lb, IL-6, IL-13, IL- 17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony -stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase -proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0147] By "decrease" or "lower," or "lessen," or "reduce," or “abate" refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., a downstream effect) compared to the response caused by either the vehicle alone (i.e., an active moiety) or a control molecule / cornposition. A "decrease" or "reduced" amount is typically a "statistically significant" amount, and may include an decrease that is 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7. 7.5, 8. 8.5, 9, 9.5, 10. 15, 20, 30 or more times (e.g., 500. 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition.

[0148] The term “domain” refers to a portion of an entity. In some embodiments, a “domain” is associated with a structural and / or functional feature of the entity, e.g., so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the structural and / or functional feature. In some embodiments, a domain may comprise a portion of an entity that, when separated from that (parent) entity and linked or connected with a different (recipient) entity, substantially retains and / or imparts on the recipient entity one or more structural and / or functional features, e.g., that characterized it in the parent entity. In some embodiments, a domain is a portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a structural element (e.g., an amino acid sequence or sequence motif, a-helix character, p-sheet character, coiled-coil character, random coil character, etc.), and / or by a functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0149] The term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., an antigen binding system or antibody) for administration to a subject. Generally, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population. The total amount of a therapeutic composition or agent administered to a subject is determined by one or more medical practitioners and may involve administration of more than one dosage forms.

[0150] The term “dosing regimen” may be used to refer to a set of one or more unit doses that are administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, a dosing regimen comprises a plurality of doses and consecutive doses are separated from one another by time periods of equal length; in some embodiments, a dosing regimen comprises a plurality of doses and consecutive doses are separated from one another by time periods of at least two different lengths. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount differentK-1177-WO-PCT from the first dose amount. In some embodiments, a dosing regimen is periodically adjusted to achieve a desired or beneficial outcome.

[0151] “Effector cell” refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, effector cells may comprise, without limitation, one or more of monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, T-lymphocytes, and B-lymphocytes. Effector cells may be of any organism comprising, without limitation, humans, mice, rats, rabbits, and monkeys.

[0152] “Effector function” refers to a biological result of interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions comprise, without limitation, antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). An effector function may be antigen binding dependent, antigen binding independent, or both. ADCC refers to lysis of antibody-bound target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally understood to involve Fc receptor (FcR)-bearing effector cells recognizing and subsequently killing antibody-coated target cells (e.g., cells that express on their surface antigens to which an antibody is bound). Effector cells that mediate ADCC may comprise immune cells, comprising yet not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, eosinophils.

[0153] An “epitope” refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non¬ contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuteriuni exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g.. site-directed mutagenesis mapping). For X-ray¬ crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giege R el al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350: McPherson A (1990) Eur JBiochern 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biot Chem 251: 6300-6303). Antibodytanligen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW el al.,; U. S. 2004 / 00141 4), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pl 1): 37-60; Bricogne G (1997) Meth Enzymol276A: 361-423, ed Carter CW; Rovers! P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies may be accomplished using any method known to one of skill in the art. See, e.g., Charnpe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0154] “Endogenous” with reference to a gene, protein, and / or nucleic acid refers to the natural presence of that gene, protein, and / or nucleic acid in a cell, such as an immune cell.

[0155] “Exogenous” refers to an introduced agent, such as a nucleic acid, gene, or protein, into a cell, for example from an outside source. A nucleic acid introduced into a cell is exogenous even if it encodes a protein which is naturally found in the cell. Such exogenous introduction of a nucleic acid encoding a protein can be used to increase the expression of the protein over the level that would naturally be found in the cell under similar conditions, e.g., without introduction of the exogenous nucleic acid.

[0156] The term “excipient” refers to an agent that may be comprised in a composition, for example to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, a suitable excipient may comprise, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel. sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, or the like.

[0157] A “fragment” or “portion” of a material or entity as described herein has a structure that comprises a discrete portion of the whole, e.g., of a physical entity or abstract entity. In some embodiments, a fragment lacks one or more moieties found in the whole. In some embodiments, a fragment consists of or comprises a characteristic structural element, domain or moiety found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 19, 20, 25, 30, 35. 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200. 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%. 10%, 15%, 20%. 25%, 30%, 25%. 40%, 45%, 50%. 55%, 60%, 65%. 70%, 75%, 80%. 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). The whole material or entity may in some embodiments be referred to as the “parent” of the fragment.

[0158] The term “fusion polypeptide” or “fusion protein” generally refers to a polypeptide comprising at least two segments. Generally, a polypeptide containing at least two such segments is considered to be a fusion polypeptide if the two segments are moieties that (1) are not comprised in nature in the same peptide, and / or (2) have not previously been linked or connected to oneK-1177-WO-PCT another in a single polypeptide, and / or (3) have been linked or connected to one another through action of the hand of woman / man. In embodiments, a CAR is a fusion protein.

[0159] The term “gene product” or “expression product” generally refers to an RNA transcribed from the gene (pre-and / or post-processing) or a polypeptide (pre- and / or post-modification) encoded by an RNA transcribed from the gene.

[0160] The term “genetically engineered” or “engineered” refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some embodiments, the cell that is modified is a lymphocyte, e.g., a T cell, which can either be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as, e.g., a chimeric antigen receptor (CAR), which is incorporated into the cell's genome. Engineering generally comprises manipulation by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences, that are not linked or connected together in that order in nature, are manipulated by the hand of man to be directly linked or connected to one another in the engineered polynucleotide. In the context of manipulation of cells by techniques of molecular biology, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, somatic hybridization, transfection, transduction, electroporation or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by other protocols). In some embodiments, a binding agent is a modified lymphocyte, e.g., a T cell, may be obtained from a patient or a donor. An engineered cell may be modified to express an exogenous construct, such as, e.g., a chimeric antigen receptor (CAR), which is incorporated into the cell's genome. Progeny of an engineered polynucleotide or binding agent are generally referred to as “engineered” even though the actual manipulation was performed on a prior entity. In some embodiments, “engineered” refers to an entity that has been designed and produced. The term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0161] In some embodiments, the cell that is modified is a lymphocyte, e.g., a T cell, which can either be obtained from a patient or a donor. In some embodiments, the engineered cell(s) are induced pluripotent stem cells (iPSCs). In some cases, said engineered T cells or iPSC as described herewith has been genetically modified to suppress or repress expression of a T-cell receptor (TCR) by inactivation of a gene encoding a TCR component (e.g., a TRAC and / or TRBC), suppress or repress expression of at least one gene controlling MI-IC complex surface presentation, such as B2M and class II major histocompatibility complex transactivator (CIITA),and. optionally, to suppress or repress expression of CD52, and, optionally, to suppress or repress expression of at least one immune checkpoint or receptor thereof, in the cell.

[0162] It will be understood by those of ordinary skill in the art, that the term “immune checkpoints” means a group of molecules expressed by T-cells, NK-cells and antigen presenting cells. These molecules effectively serve as "brakes" to down-modulate or inhibit an immune response. Immune checkpoint molecules include, but are not limited to Programmed Death 1 (PD-1, also known as PDCD1 or CD279, e.g. human PD-1: accession number NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, e.g. human CTLA-4: GenBank accession number AF414120.1), LAG3 (also known as CD223. e.g. human LAG3: accession number NM_002286.5), Tim3 (also known as HAVCR2, e.g. human Tim3: GenBank accession number JX049979.1), BTLA (also known as CD272, e.g. human BTLA: accession number NM_181780.3), BY55 (also known as CD160, e.g. human BY55: GenBank accession number CR541888.1), TIGIT (also known as IVSTM3, e.g. human TIGIT: accession number NM_173799), LAIR1 (also known as CD305, e.g. human LAIR1: GenBank accession number CR542051.1), SIGLEC10 (e.g. human SIGLEC10: GeneBank accession number AY358337.1), 2B4 (also known as CD244, e.g. human 2B4: accession number NM_001166664.1), PPP2CA (Also known as: NEDLBA. PP2Ac, PP2CaIpha. RP-C, e.g. human PPP2CA: NCBI Gene ID 5515), PPP2CB (also known as Also known as: PP2Abeta, e.g. human PPP2CB: NCBI Gene ID 5516), PTPN6 (also known as Also known as: HCP, HCPH, HPTP1C, PTP-1C, SH-PTP1, SHP- 1, SHP-1L, SHP1, e.g. human PTPN6: NCBI Gene ID 5777), PTPN22 (NCBI Gene ID 26191), CD96 (NCBI Gene ID 10225), CRTAM (NCBI Gene ID 56253), SIGLEC7 (NCBI Gene ID 27036), SIGLEC9 (NCBI Gene ID 27180), TNFRSF10B (NCBI Gene ID 8795), TNFRSF10A (NCBI Gene ID 8797), CASP8 (NCBI Gene ID 841 ), C ASP 10 (NCBI Gene ID 843), CASP3 (NCBI Gene ID 836). CASP6 (NCBI Gene ID 839), CASP7 (NCBI Gene ID 840), FADD (NCBI Gene ID 8772), FAS (NCBI Gene ID 355), TGFBRII (NCBI Gene ID 7048), TGFRBRI (NCBI Gene ID 7046), SMAD2 (NCBI Gene ID 4087), SMAD.3 (NCBI Gene ID 4088 ), SMAD4 (NCBI Gene ID 4089), SMAD10, SKI (NCBI Gene ID 6497), SKI (NCBI Gene ID 6498), TGIF1 (NCBI Gene ID 7050), IL1 ORA (NCBI Gene ID 3587). IL1 ORB (NCBI Gene ID 3588). HM0X2 (NCBI Gene ID 163), IL6R (NCBI Gene ID 3570), IL6ST (NCBI Gene ID 3572), EIF2AK4 (NCBI Gene ID 440275), CSK (NCBI Gene ID 1445), PAG1 (NCBI Gene ID 55824), SIT1 (NCBI Gene ID 27240), FOXP3 (NCBI Gene ID 50943), PRDM1 (NCBI Gene ID 639), BATF (NCBI Gene ID 10538). GUCY1A2 (NCBI Gene ID 2977), GUCY1A3 (NCBI Gene ID 2977), and GUCY1B2 (NCBI Gene ID 2974) which directly inhibit immune cells. For example, CTLA-4 is a cell-surface protein expressed on certain CD4 and CDS T-cells; when engaged by its ligands (B7-1 and B7-2) on antigen presenting cells, T-cell activation and effector function are inhibited.In some cases, the engineered T-cells are further genetically modified by inactivating at least one gene encoding a protein involved in the immune checkpoint, such as PD1 and / or C’TLA-4 or any immune- checkpoint proteins referred to herein.

[0163] In some cases, at least two genes encoding immune checkpoint proteins are inactivated, selected from the group consisting of: CTLA4. PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD 160, TIG1T, CD96, CRTAM, LA1R1, SIGLEC7, S1GLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIP, TGIF1, IL1 ORA, IL1 ORB, HM0X2, IL6R, IL6ST. EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCYIA3, GUCY1B2, and GUCY1B3.

[0164] In some embodiments, the disclosure provides engineered immune cells, some cases, the engineered immune cells, (e.g. T-cells, NK cells, macrophages), can be modified to confer resistance to at least one immune suppressive or chemotherapy drug, and optionally to comprise a suicide gene. In some cases, the engineered immune cells, (e.g. T-cells, NK cells, macrophages), can be further modified to confer resistance to at least one immune suppressive drug, such as by inactivating CD52 that is the target of anti-CD52 antibody (e.g, alemtuzumab). To improve cancer therapy and selective engraftment of allogeneic immune cells, drug resistance can be conferred to the engineered immune cells to protect them from the toxic side effects of chemotherapy or immunosuppressive agents. In some cases, the engineered immune cell can be further modified to confer resistance to a chemotherapy drug, such as a purine analogue drug, for example by inactivating DCK.

[0165] Drug resistance of immune cells also permits their enrichment in or ex vivo, as immune cells which express a drug resistance gene, will survive and multiply relative to drug sensitive cells. In some cases, the methods further comprise methods of engineering allogeneic and drug¬ resistant immune cells for immunotherapy comprising: (a) providing an immune cell, e.g. a T-cell; (b) selecting at least one drug; (c) modifying the cell to confer drug resistance to said cell; and (d) expanding said engineered cell in the presence of said drug. When the immune cell is a T-cell, the preceding steps may be combined with a step of modifying the T-cell, by inactivating at least one gene encoding a T-cell receptor (TCR) component, and then sorting the transformed T-cells, which do not express TCR on their cell surface.

[0166] Accordingly, the engineered immune cells can be further modified to confer a resistance to a drag, such as a chemotherapy agent. The resistance to a drug can be conferred to an immune cell by expressing a drug resistance gene. Variant alleles of several genes such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDII2), calcineurin or methylguanine transferase (MGMT) have been identified to confer drug resistance to a cell. Insome cases, the drug resistance gene can be expressed in the cell either by introducing a transgene encoding said gene into the cell or by integrating said drug resistance gene into the genome of the cell by homologous recombination.

[0167] The resistance to a drug can be conferred to an immune cell by inactivating one or more gene(s) responsible for the cell’s sensitivity to the drug (drag sensitizing gene(s)), such as the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (Genbank: M26434.1). For instance, HPRT can be inactivated in engineered immune cells to confer resistance to a cytostatic metabolite, the 6-thioguanine (6TG) which is converted by HPRT to cytotoxic thioguanine nucleotide and which is currently used to treat patients with cancer, in particular leukemias (Hacke et al. (2013) Transplantation Proceedings. 45(5): 2040-2044). Another example is the inactivation of the CD3 normally expressed at the surface of the T-cell, which can confer resistance to anti-CD3 antibodies such as tcplizumab.

[0168] Otherwise, drag resistance can be conferred to the immune cell (e.g. T-cell), by the expression of at least one drug resistance gene. The drug resistance gene refers to a nucleic acid sequence that encodes "resistance" to an agent, such as a chemotherapeutic agent (e.g. methotrexate). In other words, the expression of the drug resistance gene in a cell permits proliferation of the cells in the presence of the agent to a greater extent than the proliferation of a corresponding cell without the drug resistance gene. A drug resistance gene can encode resistance to anti -metabolite, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, their analogs or derivatives, and the like.

[0169] One example of drug resistance gene can also be a mutant or modified form of Dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in the cell and is essential to DNA synthesis. Folate analogs such as methotrexate (MIX) inhibit DHFR and are thus used as anti-neoplastic agents in clinic. Different mutant forms of DHFR which have increased resistance to inhibition by antifolates used in therapy have been described. In some cases, the drug resistance gene can be a nucleic acid sequence encoding a mutant form of human wild type DHFR (GenBank: AAH71996.1) which comprises at least one mutation conferring resistance to an anti-folate treatment, such as methotrexate. In some cases, mutant form of DHFR comprises at least one mutated amino acid at position G1, L22, F31 or F34, for instance at positions L22 or F31.

[0170] As used herein, "antifolate agent" or "folate analogs" refers to a molecule directed to interfere with the folate metabolic pathway at some level. Examples of antifolate agents include, e.g. methotrexate (MTX); aminopterin; trimetrexate (Neutrexin™); edatrexate; NlO-propargyl-5,8-dideazafolic acid (CB3717); ZD1694 (Tumodex), 5, 8 -dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid (DDATHF); 5-deazafolic acid; PT523 (N alpha-(4-arnino-4-K-I177-WO-PCT deoxypteroyl)-N delta-hemiphthaloyl-L-omithine); 10-ethyl- 10- deazaaminopterin (DDATHF, lomatrexol); piritrexim; 10-EDAM; ZD 1694; GW1843; Pemetrexate and PDX (10-propargyl-10-dcazaaminoptcri n).

[0171] Another example of drug resistance gene can also be a mutant or modified form of ionisine-5 ’-monophosphate dehydrogenase 11 (IMPDH2), a rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. The mutant or modified form of IMPDH2 is a IMPDII inhibitor resistance gene. IMPDII inhibitors can be mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF). The mutant IMPDH2 can comprise at least one, for instance two mutations in the MAP binding site of the wild type human IMPDH2 (NP 000875.2) that lead to a significantly increased resistance to IMPDII inhibitor. The mutations can be at positions T333 and / or S351. In some cases, the threonine residue at position 333 can be replaced with an isolcucinc residue and the serine residue at position 351 can be replaced with a tyrosine residue.

[0172] Another drug resistance gene is the mutant form of calcineurin. Calcineurin (PP2B) is an ubiquitously expressed serine / threonine protein phosphatase that is involved in many biological processes and which is central to T-cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; three isoforms) and a regulator}' subunit (CnB; two isoforms). After engagement of the T-cell receptor, calcineurin dephosphorylates the transcription factor NF AT, allowing it to translocate to the nucleus and active key target gene such as 1 L2. FK506 in complex with FKBP12, or cyclosporine A (CsA) in complex with CyPA block NFAT access to calcineurin's active site, preventing its dephosphorylation and thereby inhibiting T-cell activation (Brewin et al. (2009) Blood 114(23): 4792-803). The drug resistance gene can be a nucleic acid sequence encoding a mutant form of calcineurin resistant to calcineurin inhibitor such as FK506 and / or CsA. In some cases, said mutant form can comprise at least one mutated ammo acid of the wild type calcineurin heterodimer at positions: V314, Y341, M347, T351, W352, L354, K360, for instance double mutations at positions T351 and L354 or V314 and Y341. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type human calcineurin heterodimer (GenBank: ACX34092.1).

[0173] In some cases, said mutant form can comprise at least one mutated amino acid of the wild type calcineurin heterodimer b at positions: V120, N123, L124 or K125, for instance double mutations at positions Id 24 and KI 25. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type human calcineurin heterodimer b polypeptide (GenBank: ACX34095.1).

[0174] Another drug resistance gene is 06-methylguanine methyltransferase (MGMT) encoding human alkyl guanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents, such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an inhibitor of AGT that potentiates nitrosourea toxicity and is co - administered with TMZ to potentiate the cytotoxic effects of this agent. Several mutant forms of MGMT that encode variants of AGT are highly resistant to inactivation by 6-BG, but retain their ability to repair DNA damage (Maze, Kurpad et al. 1999). In some cases, AGT mutant form can comprise a mutated amino acid of the wild type AGT position P140 (UniProtKB: P16455).

[0175] Another drag resistance gene can be multi drag resistance protein 1 (MDR1) gene. This gene encodes a membrane glycoprotein, known as P-gly coprotein (P-GP) involved in the transport of metabolic byproducts across the cell membrane. The P-Gp protein displays broad specificity towards several structurally unrelated chemotherapy agents. Thus, drag resistance can be conferred to cells by the expression of nucleic acid sequence that encodes MDR-1 (NP_000918).

[0176] Drag resistance genes can also be cytotoxic antibiotics, such as blc gene or mcrA gene. Ectopic expression of ble gene or mcrA in an immune cell gives a selective advantage when exposed to the chemotherapeutic agent, respectively the bleomycine or the mitomycin C.

[0177] With respect to the immunosuppressive agents, the present disclosure describes the possible optional steps of: (a) providing an immune cell such as a T-cell, for instance from a cell culture or from a blood sample, or an induced pluripotent stem cell (iPSC); (b) selecting a gene in said cell expressing a target for an immunosuppressive agent; (c) introducing into said cell an endonuclease able to selectively inactivate by DNA cleavage, for instance by double-strand break, said gene encoding a target for said immunosuppressive agent, (d) expanding said cells, optionally in presence of said immunosuppressive agent. In some cases, said method comprises a further step of inactivating a component of the T-cell receptor (TCR).

[0178] An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. In other words, an immunosuppressive agent is a compound which is capable of diminishing the extent and / or voracity of an immune response. As non-limiting examples, an immunosuppressive agent can be a calcineurin inhibitor, a target of rapamycin, an interleukin-2oc-cham blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite. Classical cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act through inactivation of T-cells or by inhibiting the activation of helper cells. The method described herewith allows conferring immunosuppressive resistance to immune cells (e.g. T-cells), for immunotherapy by inactivating the target of the immunosuppressive agent in said cells. As non¬ limiting examples, a target for an immunosuppressive agent can be a receptor for an immunosuppressive agent such as CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member.

[0179] In immunocompetent hosts, allogeneic cells are normally rapidly rejected by the host immune system. It has been demonstrated that allogeneic leukocytes present in nonirradiated blood products will persist for no more than 5 to 6 days (Boni ct al. (2008) Blood 112(12): 4746-54). Thus, to prevent rejection of allogeneic cells, the host’s immune system must be effectively suppressed. Glucocorticoid steroids are widely used therapeutically for immunosuppression. This class of steroid hormones binds to the glucocorticoid receptor (GR) present in the cytosol of T-cells resulting in the translocation into the nucleus and the binding of specific DNA motifs that regulate the expression of a number of genes involved in the immunologic process. Treatment of T-cells with glucocorticoid steroids results in reduced levels of cytokine production leading to T-cell anergy and interfering in T-cell activation. Alemtuzumab, also known as GAMPATH 1-11, is a humanized monoclonal antibody targeting CD52, a 12 amino acid glycosylphosphatidyl-inositol-(GPI) linked glycoprotein (Waldmann and Hale (2005) Philos. Trans. R. Soc. Land. B. BiolSci. 360: 1701-11). CD52is expressed at high levels on T and B lymphocytes and lower levels on monocytes while being absent on granulocytes and bone marrow precursors. Treatment with Alemtuzumab, a humanized monoclonal antibody directed against CD52, has been shown to induce a rapid depletion of circulating lymphocytes and monocytes. It is frequently used in the treatment of T-cell lymphomas and in certain cases as part of a conditioning regimen for transplantation. However, in the case of adoptive immunotherapy the use of immunosuppressive drugs will also have a detrimental effect on the introduced therapeutic immune cells (e.g. T-cells). Therefore, to effectively use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be resistant to the immunosuppressive treatment.

[0180] In some cases, the gene that is specific for an immunosuppressive treatment is CD52, and the immunosuppressive treatment comprises a humanized antibody targeting CD52 antigen. In some cases, the gene that is specific for an immunosuppressive treatment is a glucocorticoid receptor (GR) and the immunosuppressive treatment comprises a corticosteroid such as dexamethasone. In some cases, the gene that is specific for an immunosuppressive treatment is a FKBP family gene member or a variant thereof and the immunosuppressive treatment comprises FK506 also known as Tacrolimus or fujimycin. In some cases, the gene that is specific for an immunosuppressive treatment is a FKBP family gene member such as FKBP12 or a variant thereof. In some cases, the gene that is specific for an immunosuppressive treatment is a cyclophilin family gene member or a variant thereof and the immunosuppressive treatment comprises cyclosporine.

[0181] Cytokine Release Syndrome (CRS) is the most common adverse event of CAR-T cell therapy. CRS is defined as a clinical syndrome that may occur after cell therapy due to the release of cytokines (substances secreted by immune cells) into the body’s blood stream. Il has beenshown that inactivation of Granulocyte-macrophage colony-stimulating factor (GM-CSF) can prevent monocyte-dependent release of key cytokine release syndrome mediators (Sachdeva et al. (2019) J. Biol. Chcm. 294(14) 5430-5437). Thus, in a further embodiment, the engineered immune cells as described herewith have been genetically modified to suppress expression, or cell surface presentation, of GM-CSF.

[0182] In some cases, the engineered immune cell as described herewith is one or more of: TCR negative, B2M negative, CIITA negative, PDCD1 negative, GM-CSF negative, CD52 negative; for instance at least TCR negative or at least TCR negative, B2M-negative and CD52-negative.

[0183] In some cases, to reduce fratricide effect, the engineered immune cell as described herewith does not present at its cell surface the antigen targeted by the tumor-CAR. For example, the engineered immune cell as described herewith can have its CD4 or CD 8 gene inactivated, or its expression inhibited, if the tumor-CAR targets CD4 or CD8, respectively.

[0184] In some embodiments, the cell can be modified to express an exogenous construct, such as, e.g., a chimeric antigen receptor (CAR), which is incorporated into the cell's genome. Engineering generally comprises manipulation by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences, that are not linked or connected together in that order in nature, are manipulated by the hand of man to be directly linked or connected to one another in the engineered polynucleotide. In the context of manipulation of cells by techniques of molecular biology, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, somatic hybridization, transfection, transduction, electroporation or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by other protocols). In some embodiments, a binding agent is a modified lymphocyte, e.g.. a T cell, may be obtained from a patient or a donor. An engineered cell may be modified to express an exogenous construct, such as, e.g., a chimeric antigen receptor (CAR), which is incorporated into the cell's genome. Progeny of an engineered polynucleotide or binding agent are generally referred to as “engineered” even though the actual manipulation was performed on a prior entity. In some embodiments, “engineered” refers to an entity that has been designed and produced. The term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man: and / or (tii) that is distinct from natural substances and other known agents.

[0185] A “T cell receptor” or “TCR” refers to antigen-recognition molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes, a, p, y, and 8, may rearrange leading to highly diverse TCR proteins. In embodiments, a T cell disclosed hereinhas been engineered to reduce, eliminate and / or inhibit the surface expression of the a chain of the TCR receptor.

[0186] The term “heterologous” means from any source other than naturally occurring sequences. For example, a heterologous sequence included as a part of a costimulatory protein is amino acids that do not naturally occur as, i.e., do not align with, the wild type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than that of the wild-type human costimulatory protein-encoding sequence.

[0187] Term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g.. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided polypeptide sequences are known. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences may be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison or alignment of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, such as BLAST (basic local alignment search tool). In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%. 45%. 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95- 100%).

[0188] To calculate percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux el al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison. Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span,” as determined by the algorithm). In certain embodiments, a standard comparison matrix see, Dayhoff el al.. 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250comparison matrix; Henikoff et al.. 1992, Proc. Natl. Acad. Sci. U. S. A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm. Other algorithms are also available for comparison of amino acid or nucleic acid sequences, comprising those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAS T, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.. Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying similar sequences, the programs mentioned above generally provide an indication of the degree of similarity. In some embodiments, two sequences are considered to be substantially similar if at least 50%, at least 55%. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%. at least 85%. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of their corresponding residues are similar and / or identical over a relevant stretch of residues (e.g.. 85-90%’, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35. at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80. at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425. at least 450, at least 475, at least 500 or more residues. Sequences with substantial sequence similarity may be homologs of one another.

[0189] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%. 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0190] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0191] The terms ‘‘improve,” “increase,” “inhibit,” and “reduce” indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may comprise a measurement in certain system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) an agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may comprise a measurement in comparable system known or expected to respond in a comparable way, in presence of the relevant agent or treatment.

[0192] An “immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0193] The term “immunotherapy” refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, NK cells, iNKT cells, and T cell therapies. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T cell transplantation. Examples of T cell therapies are described in U. S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, U. S. Patent No. 5,728,388, and International Publication No. WO 2008 / 081035. In some embodiments, the cells are selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor¬ infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC -derived cell, optionally wherein the cell is autologous or wherein the cell is allogeneic. In some cases, an engineered cell provided herein can be a T-cell that has been genetically modified to suppress or repress expression of T-cell receptors (TCRs) (e.g. endogenous TCRs) at the T-cell surface and,optionally, to suppress or repress expression of at least one gene controlling MHC complex surface presentation such as a B2M gene that encodes a p2m polypeptide and / or a CUT A gene that encodes a CIITA polypeptide, and optionally to suppress or repress expression of a gene encoding a CD52 polypeptide, at the T-cell surface. In some cases, an engineered cell provided herein can be a NK-cell that has been genetically modified to suppress or repress expression of at least one gene controlling MHC complex surface presentation such as a B2M gene that encodes a p2m polypeptide and / or a CIITA gene that encodes a (UTA polypeptide, and optionally to suppress or repress expression of a gene encoding a CD52 polypeptide, at the NK-cell surface. In some cases, an engineered T-cell described herein can be designed such that the CD52 gene, the B2M gene, or both the CD52 gene and the B2M gene are inactivated. In some cases, an engineered T-cell described herein can be designed (e.g., genetically modified) to suppress or repress the expression of at least one immune checkpoint protein or receptor thereof. For example, an engineered T-cell described herein can be designed such that the programmed cell death 1 (PDCD1) gene, the CTLA4 gene, or both the PDCD1 gene and the cytotoxic I'- lymphocyte associated protein 4 (CTLA4) gene are inactivated.

[0194] In some cases, the immune cell, e.g. T-cell or NK-cell, can derive from a stem cell. The stem cells can be adult stem cells, embryonic stem cells, such as non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. In some cases, the immune cells (e.g. T-cells or NK cells) to be engineered are allogenic. By “allogeneic” is meant that the cells originate from a donor, from a cell line, or are produced and / or differentiated from stem cells in view' of being infused into patients having a different haplotype. Such immune cells are generally engineered to be less allor eactive and / or become more persistent with respect to their patient host. More specifically, the method of engineering the allogeneic cells can comprise the step of reducing or inactivating TCR expression into T-cells, or into the stem cells to be derived into T-cells. This can be obtained by different sequence-specific reagents, such as by gene silencing or gene editing techniques by using for instance nucleases, base editing techniques, shRNA and RNAi as non-limited examples. In some cases, the immune cells, e.g. T- cells or NK-cells, to be engineered can originate from a human, w'herein the human is a donor, not the patient

[0195] The T cells of the immunotherapy can come from any other source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population or iPSC, or can be obtained from a subject, for example for transplantation into a second subject after engineering. T cells can be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow', lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites,pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation and / or apheresis. Additional methods of isolating T cells for a T cell therapy are disclosed in U. S. Patent Publication No. 2013 / 0287748, which is herein incorporated by references in its entirety.

[0196] Tire term “in vitro” refers to events occurring in an artificial environment, e.g., in a test lube, reaction vessel, cell culture, etc., rather than within a multi-cellular organism. The term “in vitro cell” refers to any cell which is cultured ex vivo. In particular, an in vitro cell can include a T cell. The term “in vivo” refers to events that occur within a multi-cellular organism, such as a human or a non-human animal.

[0197] The term “isolated” refers to a substance that (1) has been separated from at least some components with which it was associated at an earlier time or with which the substance would otherwise be associated, and / or (2) is present in a composition that comprises a limited or defined amount or concentration of one or more known or unknown contaminants. An isolated substance, in some embodiments, may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of other non-substance components with which the substance was associated at an earlier time, e.g., other components or contaminants with which the substance was previously or otherwise would be associated. In certain instances, a substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of molecules of a same or similar type. For instance, in certain instances, a nucleic acid, DNA, or RNA substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of non-substance nucleic acid, DNA, or RNA molecules. For instance, in certain instances, a polypeptide substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of non-substance polypeptide molecules. In certain embodiments, an amount may be, e.g., an amount measured relative to the amount of a desired substance present in a composition. In certain embodiments, a limited amount may be an amount that is no more than 100% of the amount of substance in a composition, e.g.. no more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the amount of substance in a composition (e.g., 85-90%, 85-95%, 85-100%, 90-95%>, 90-100%, or 95-100%). In certain instances, a composition is pure or substantially pure with respect to a selected substance. In some embodiments, an isolated substance is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure (e.g.. 85-90%. 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). A substance is “pure” if it is substantially free of other components or of contaminants. In some embodiments, a substance may still be considered “isolated” or even “pure,” after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without comprising such carriers or excipients.

[0198] " Li nker" (I.) or "linker domain" or "linker region" refers to an oligo- or polypeptide region from about 1 to 100 ammo acids in length, for example linking together any of the domains / regions of a CAR. and / or scFv, or ever one of more of those polypeptides together. Linkers may be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. Other linkers will be apparent to those of skill in the art and may be used in connection with this disclosure. A linker may be a portion of a multi-element agent that connects different elements to one another. For example, a polypeptide comprises two or more functional or structural domains may comprise a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, or 10 to 100 amino acids in length). In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, and instead provides flexibility to the polypeptide. In another example it may be used to connect to or more polypeptides to be expressed. Other linkers include non-cleavable linkers. A number of linkers are employed to realize the subject invention including “flexible linkers.” Tire latter are rich in glycine. Klein et al., Protein Engineering, Design & Selection Vol. 27, No. 10, pp. 325-330, 2014; Priyanka et al., Protein Sei.. 2013 Feb; 22(2): 153-167.

[0199] In some embodiments, the linker is a synthetic linker. A synthetic linker can have a length of from about 10 amino acids to about 200 amino acids, e.g., from 10 to 25 amino acids, from 25 to 50 amino acids, from 50 to 75 ammo acids, from 75 to 100 amino acids, from 100 to 125 aminoacids, from 125 to 150 amino acids, from 150 to 175 amino acids, or from 175 to 200 amino acids. A synthetic linker can have a length of from 10 to 30 amino acids, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. A synthetic linker can have a length of from 30 to 50 amino acids, e.g., from 30 to 35 amino acids, from 35 to 40 amino acids, from 40 to 45 amino acids, or from 45 to 50 amino acids.

[0200] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), e.g., I, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs.

[0201] The term “lymphocyte” includes natural killer (NK) cells, T cells, iNKT cell, or B cells. NK cells arc a type of cytotoxic (cell toxic) lymphocyte that represent a component of the inherent immune system. NK cells reject tumors and ceils infected by viruses. It works through the process of apoptosis or programmed cell death. They were termed “natural killers” because they do not require activation in order to kill cells. T cells play a role in ceil-mediated-iramunity (no antibody involvement). Its T cell receptors (TOR) differentiate themselves from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the T cell’s maturation. There are six types of T cells, namely: Helper T cells (e.g., CD4+ cells), Cytotoxic T cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T cells or killer T cell). Memory 'I' cells ((i) stem memory TSCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of CD95, IL-2R0, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells): (ii) central memory TCM cells express L-selectin and the CCR7, they secrete IL-2, but not IFNy or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFNy and IL-4), Regulatory I’ cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), Natural Killer T cells (NKT) and Gamma Delta T cells. B-cells, on the other hand, play a role in humoral immunity (with antibody involvement). It makes antibodies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow, where its name is derived from.

[0202] The term “neutralizing” refers to an antigen binding molecule, scFv, antibody, or a fragment thereof, that binds to a ligand and prevents or reduces the biological effect of that ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or a fragment thereof, directly blocking a binding site on the ligand or otherwise alters the ligand's ability to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments,the antigen binding molecule, scFv, antibody, or a fragment thereof prevents the protein to which it is bound from performing a biological function.

[0203] “Nucleic acid” refers to any polymeric chain of nucleotides. A nucleic acid may be DNA, RNA, or a combination thereof. In some embodiments, a nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, a nucleic acid comprises of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10. 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0204] “Operably linked” refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. For example, a control element “operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element.

[0205] A “patient” includes any human who is afflicted with a cancer e.g., prostate cancer). The terms “subject” and “patient” are used interchangeably herein.

[0206] The terms “peptide,” “polypeptide.” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two ammo acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0207] The term “pharmaceutically acceptable’’ refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof. With respect to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient. The term “pharmaceutically acceptable carrier” means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another e.g.. from one organ to another). Each carrier present in a pharmaceutical composition must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient. Some examples of materials which may serve as pharmaceutically acceptable carriers comprise: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxyrnethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; al ginic acid: pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0208] The term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, a pharmaceutical composition may be formulated for administration in solid or liquid form, comprising, without limitation, a form adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patchor spray applied to the skin, lungs, or oral cavity, intravaginally or intrarectally, for example, as a pessay, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0209] The term "proliferation" refers to an increase in cell division, either symmetric or asymmetric division of cells. In some embodiments, "proliferation" refers to the symmetric or asymmetric division of T cells. " Increased proliferation" occurs when there is an increase in the number of cells in a treated sample compared to cells in a non-treated sample.

[0210] The term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, a reference or control is tested, measured, and / or determined substantially simultaneously with the testing, measuring, or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. When sufficient similarities are present to justify reliance on and / or comparison to a selected reference or control.

[0211] “Regulatory T cells” (“Treg”, “Treg cells”, or “Tregs”) refer to a lineage of CD4+ T lymphocytes that participate in controlling certain immune activities, e.g., autoimmunity, allergy, and response to infection. Regulatory T cells may regulate the activities of T cell populations and may also influence certain innate immune system cell types. Tregs may be identified by the expression of the biomarkers CD4, CD25 and Foxp3, and low expression of CD127. Naturally occurring Treg cells normally constitute about 5-10% of the peripheral CD4+ T lymphocytes. However, Treg cells within a tumor microenvironment (i.e., tumor-infiltrating Treg cells). Treg cells may make up as much as 20-30% of the total CD4+ T lymphocyte population.

[0212] The term “sample” generally refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may comprise a cell or an organism, such as a cell population, tissue, or animal (e.g., a human). In some embodiments, a source of interest comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and / or combinations or component(s) thereof. In someembodiments, a biological fluid may comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and / or a transcelluiar fluid. In some embodiments, a biological fluid may comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g,, fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and / or purification of certain components, etc.

[0213] " Single chain variable fragment", "single-chain antibody variable fragments" or "scFv" antibodies refer to forms of antibodies comprising the variable regions of only the heavy and light chains, connected by a linker peptide.

[0214] The term “stage of cancer” refers to a qualitative or quantitative assessment of the level of advancement of a cancer. In some embodiments, criteria used to determine the stage of a cancer may comprise, without limitation, one or more of where the cancer is located in a body, tumor size, whether the cancer has spread to lymph nodes, whether the cancer has spread to one or more different parts of the body, etc. In some embodiments, cancer may be staged using the so-called TNM System, according to which T refers to the size and extent of the main tumor, usually called the primary tumor; N refers to the number of nearby lymph nodes that have cancer; and M refers to whether the cancer has metastasized. In some embodiments, a cancer may be referred to as Stage 0 (abnormal cells are present without having spread to nearby tissue, also called carcinoma in situ, or CIS; CIS is not cancer, though could become cancer), Stage I-III (cancer is present; the higher the number, the larger the tumor and the more it has spread into nearby tissues), or Stage IV (the cancer has spread to distant parts of the body). In some embodiments, a cancer may be assigned to a stage selected from the group consisting of: in situ localized (cancer is limited to the place where it started, with no sign that it has spread); regional (cancer has spread to nearby lymph nodes, tissues, or organs): distant (cancer has spread to distant parts of the body); and unknown (there is not enough information to determine the stage).

[0215] “Stimulation,” refers to a primary response induced by binding of a stimulatory molecule with its cognate ligand, wherein the binding mediates a signal transduction event. A “stimulatoryK-1177-WO-PCT molecule” is a molecule on a T cell. e.g., the T cell receptor (TCR) / CD3 complex, that specifically binds with a cognate stimulatory ligand present on an antigen present cell. A “stimulatory ligand” is a ligand that when present on an antigen presenting cell (e.g., an APC, a dendritic cell, a B-ccll, and the like) can specifically bind with a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands include, but are not limited to, an anti-CD3 antibody (such as 0KT3), an MHC Class I molecule loaded with a peptide, a superagonist anti-CD2 antibody, and a superagonist anti-CD28 antibody.

[0216] The phrase “therapeutic agent" may refer to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropri te population. In some embodiments, the appropriate population may be a population of model organisms or human subjects. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, in accordance with presence or absence of a biomarker, etc. In some embodiments, a therapeutic agent is a substance that may be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by a government agency before it may be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans.

[0217] A “therapeutically effective amount,” “effective dose," “effective amount," or “therapeutically effective dosage" of a therapeutic agent, e.g., engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0218] The terms “transduction” and “transduced” refer to the process whereby foreign DNA is introduced into a cell via viral vector (see Jones et al., “Genetics: principles and analysis.” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, apapovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.

[0219] “Transformation” refers to any process by which exogenous DNA is introduced into a host cell. Transformation may occur under natural or artificial conditions using various methods. Transformation may be achieved using any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. In some embodiments, some transformation methodology is selected based on the host cell being transformed and / or the nucleic acid to be inserted. Methods of transformation may comprise, yet are not limited to, viral infection, electroporation, and lipofection. In some embodiments, a “transformed” cell is stably transformed in that the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, a transformed cell may express introduced nucleic acid.

[0220] “Treatment” or “treating” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, “treatment” or “treating” includes a partial remission. In another embodiment, “treatment” or “treating” includes a complete remission. In some embodiments, treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relev nt disease, disorder, and / or condition.

[0221] The term “vector” refers to a recipient nucleic acid molecule modified to comprise or incorporate a provided nucleic acid sequence. One type of vector is a “plasmid.” which refers to a circular double stranded DNA molecule into which additional DNA may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors comprise sequences that direct expression of insertedgenes to which they are operatively linked. Such vectors may be referred to herein as “expression vectors.” Standard techniques may be used for engineering of vectors, e.g., as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989)), which is incorporated herein by reference. A "vector" can include, but is not limited to, a viral vector, a plasmid, an oligonucleotide, a RNA vector or a linear or circular DNA or RNA molecule which may consist of a chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and / or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g. adeno-associated viruses (AAV)), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g. influenza virus), rhabdovirus (e.g. rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double- stranded DNA viruses including adenovirus, herpesvirus (e.g. Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g. vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lenti virus, and spumavirus.

[0222] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0223] In some cases, the promoter is a constitutive promoter. In some embodiments, the promoter can be selected from the group consisting of the promoters of EFl A. CD52, GAPDH, CMV, hPGK, UBC, SV40, PGK, CAGG, TRAC, TRBC, TRGC, TRDC, B2M, CD5, CS1, CD45, RPBSA, CD4, and CD8; and / or the inducible promoter can be selected from the group consisting of the promoters of PDCD1, CD25, TIM3, TIGIT, CCL1, NR4A3. EGR3, G0S2, IL22, RGS16, FASLG, RDH10, CSF1. GM-CSF, LAG3, CTLA-4, ILK). NUR77, and FOXP3.

[0224] In some cases, the constitutive promoter can be selected from the group consisting of the promoters of EFl A, TRAC, B2M, CD52, CS1, CD45, CD5, and GAPDH. For example, the / - 'constitutive promoter can be a EFl A, TRAC, CD52. or B2M promoter, For example, the constitutive promoter can be an EFl A promoter.

[0225] In some cases, the inducible promoter can be responsive to the CD3 zeta signaling. Examples of inducible promoters useful herewith include the promoter of the Programmed Cell Death Protein 1 (PDCD1) gene, Cluster of Differentiation 25 (CD25) gene, T-cell immunoglobulin and mucin-domain containing-3 (TIM3) gene, T Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) gene, C-C Motif Chemokine Ligand 1 (CCL1) gene, Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) gene, Early Growth Response 3 (EGR3) gene, G0 / G1 Switch 2 (G0S2) gene, Interleukin 22 (IL22) gene, Regulator of G Protein Signaling 16 (RGS16) gene. Fas Ligand (FAS LG) gene, Retinol Dehydrogenase 10 (RDH10) gene, Colony Stimulating Factor 1 (CSF1 ) gene, Colony Stimulating Factor 2 (CSF2, also called GM-CSF) gene, Lymphocyte Activating 3 (LAG3) gene, Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4 or CD152) gene, Interleukin-10 (IL10) gene, Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1 or NUR77) gene, Forkhead Box P3 (FOXP3) gene. The inducible promoter useful herewith can be identical to a promoter already present (i.e. without genetic engineering as described herewith) in the cell’s genome. This would be the case for the promoter of PDCD1 or the promoter of GM-CSF, for example. The inducible promoter useful herewith can also be absent from the cell's genome prior to its introduction in the cell by genetic engineering. The inducible promoter can be added to the cell’s genome as an exogenous polynucleotide or can be an endogenous polynucleotide already present in the cell’s genome independently of the cell’s genetic engineering as described herewith, i.e. without addition to the cell of an exogenous polynucleotide corresponding to this promoter. In some cases, the inducible promoter can be selected from the group consisting of the promoters of PDCD1, CD25. GM-CSF, TIM3, and TIGIT. For example, the inducible promoter can be a PDCD1 promoter. In some cases, the constitutive promoter can be an endogenous TRAC promoter or an exogenous EFl A promoter, and the inducible promoter can be an endogenous PDCD1 promoter. In some cases, tire constitutive promoter can be an exogenous EFl A promoter, and the inducible promoter can be an endogenous PDCD1 promoter.

[0226] The disclosure may employ, unless indicated specifically to the contrary, methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover. DNA Cloning: A Practical Approach, vol. I & II (IRE Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B, Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane. Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1998) Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology, as well as monographs in journals such as Advances in Immunology.AUTOIMMUNE DISEASES

[0227] Certain aspects of the disclosure relate to the treatment of an autoimmune condition and / or use of an autoimmune- associated antigen. The autoimmune disease to be treated or antigen may be an antigen associated with any autoimmune condition known in the art or, for example, diabetes, graft rejection. GVHC, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyosilis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis,respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy ), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type 1 and Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidemiolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto¬ immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B- O blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus. including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes) and autoimmune diabetes. Also contemplated are immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizingvasculitis such as systemic necrotizing vasculitis, and ANCA- associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA). Addison’s disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti -glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis. Behcet’s disease / syndrome, Castleman's syndrome, Goodpasture's syndrome. Reynaud's syndrome, Sjogren's syndrome, Stevens- Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM -mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma- associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (nontransplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis,cirrhosis such as primary biliary cirrhosis and pneumoconiosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune, zoster- associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressier's syndrome, alopecia greata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly), and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing’s syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis. Sampler's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinurn, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch’s cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis obliterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, chronichypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck’s disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil- related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler’s syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthri tides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton’s syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephri tides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renalcortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, graft versus host disease, contact hypersensitivity, asthmatic airway hyperreaction, and endometriosis.NEXT-GENERATION CHIMERIC ANTIGEN RECEPTORS COMPRISING JAMI, TRANSMEMBRANE AND / OR CO-STIMULATORY DOMAINS EXEMPLARY ANTIGEN-BINDING DOMAINS

[0228] Chimeric antigen receptors (CARs) are engineered receptors that may direct or redirect T cells (e.g., donor T cells) to target a selected antigen. A CAR may be engineered to recognize an antigen and, when bound to that antigen (such as CD 19), activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR may target and kill the tumor cell. CARs generally comprise an extracellular binding domain that mediates antigen binding (e.g., an anti-CD19 binding domain), a transmembrane domain that spans, or is understood to span, the cell membrane when the CAR is present at a cell surface or cell membrane, and an intracellular (or cytoplasmic) signaling domain.

[0229] One or more antigen binding domains determine the target(s) of an antigen binding system. A binding domain may comprise a binding domain to any antigen of interest, e.g., an antibody provided by the present disclosure, e.g., a binding motif of the present disclosure. Binding domains are used in chimeric antigen receptors at least in part because they may be engineered to be expressed as part of a single chain along with the other CAR components. See, for example, U. S. Pat. Nos. 7,741,465, and 6,319,494 as well as Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136, Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161: 2791-2797, each of which is incorporated herein by reference with respect to binding domains in CARs. A binding domain or scFv, is a single chain antigen binding fragment comprising a heavy chain variable domain and a light chain variable domain, which heavy chain variable domain and light chain variable domain are linked or connected together. See, for example, U. S. Pat. Nos. 7,741,465, and 6,319,494 as well as Eshhar et al.. Cancer Immunol Immunotherapy (1997) 45: 131-136, each of which is incorporated herein by reference with respect to binding domains. When derived from a parent antibody, a binding domain may retain some of, retain all of, or essentially retain the parent antibody's binding of a target antigen. In some embodiments, a CAR contemplated herein comprises antigen-specificbinding domain that may be a scFv (a murine, human or humanized scFv) that binds an antigen expressed on a cancer ceil. In a certain embodiment, the scFv binds CD19.

[0230] In one embodiment, disclosed herein is a next generation CAR T-cell therapy targeted against one or more cell-surface antigens, for the treatment of B- cell malignancies, such as relapsed or refractory (r / r) B-cell malignancies. The allogeneic chimeric antigen receptor (CAR) T-cell may be engineered from T cells derived from healthy human donors. The autologous chimeric antigen receptor (CAR) T-cell may be engineered from appropriate human cancer patients. These engineered T cells undergo insertion of an engineered retroviral vector encoding a gene for a CAR construct, in certain aspects an anti-CD19 CAR.

[0231] In some embodiments, the CAR of the disclosure comprise an antigen-binding domain that binds CD1. CD19 (also known as Cluster of Differentiation 19, B-lyrnphocyte antigen CD19, B-lymphocytc surface antigen B4, B4, CVID3, Differentiation antigen CD 19) is a protein that is encoded by the CD19 gene in humans. Unless otherwise indicated, it is to be appreciated the references to CDF) in the present disclosure relate to human CD 19. It is found on the surface of B cells. Since CD19 expression is a hallmark of B cells, it may be useful as an antigen, e.g., in recognizing B cells and cancer cells that arise from B cells, e.g., B-cell lymphomas. Anti-CD19 antibodies may bind CD 19 expressed on, e.g., B lymphocytes in peripheral blood and spleen, B cell chronic lymphocytic leukemia (B-CLL) cells, pro lymphocytic leukemia (PEL) cells, hairy cell leukemia (HCL) cells, common acute lymphoblastic leukemia (CALL) cells, pre-B acute lymphoblastic leukemia (pre-B-ALL) cells, and NULL-acute lymphoblastic leukemia (NULL- ALL) cells, to provide a few non limiting examples. An exemplary pharmaceutical product that comprises an antigen binding system that comprises an anti-CD19 binding domain is the pharmaceutical product YESCARTA®. YES CARTA® is a CD19-directed genetically modified autologous T cell immunotherapy (See YESCARTA® FDA-approved package insert, the entirety of which is incorporated herein by reference with respect to methods and compositions relating to immunotherapy). Another exemplary pharmaceutical product that comprises an antigen binding system that comprises an anti-CD19 binding domain is the pharmaceutical product KYMRIAH®.

[0232] Both YESCARTA® and KYMRIAH® comprise antibody binding domains derived from an anti-human CD19 antibody. Many anti-CD19 antibodies are thought to bind an epitope of CD19 encoded in exon 4 of the CD1 gene. Other anti-CD19 binding domains may recognize different epitopes of CD 19, or the same epitope with differential affinities.

[0233] An anti-CD19 binding domain of the present disclosure may comprise antigen-binding sequences as found in an antibody described herein. In some embodiments, an anti-CD19 binding domain of the present disclosure comprises an antigen binding fragment provided herein.

[0234] In various embodiments, an anti-CD 19 binding domain of the present disclosure comprises at least one HCDR provided herein, e.g., at least one HCDR disclosed in Table 4 or Table 5. In various embodiments, an anti-CD19 binding domain of the present disclosure composes two HCDRs provided herein, e.g., at least two HCDRs disclosed in Table 4 or Table 5. In various embodiments, an anti-CD19 binding domain of the present disclosure comprises three HCDRs provided herein, e.g., three HCDRs disclosed in Table 4 or Table 5.

[0235] In various embodiments, an anti-CDl 9 binding domain of the present disclosure comprises at least one LCDR provided herein, e.g., at least one LCDR disclosed in Table 4 or Table 5, In various embodiments, an anti-CDl 9 binding domain of the present disclosure comprises two LCDRs provided herein, e.g., at least two LCDRs disclosed in Table 4 or Table 5. In various embodiments, an anti-CD19 binding domain of the present disclosure comprises three LCDRs provided herein, e.g., three LCDRs disclosed in Tabic 4 or Table 5.

[0236] In various embodiments, an anti-CDl 9 binding domain of the present disclosure comprises at least one HCDR provided herein, e.g., at least one HCDR disclosed in Table 4 or 'fable 5 and at least one LCDR provided herein, e.g., at least one LCDR disclosed in Table 4 or Table 5. In various embodiments, an anti-CD1 binding domain of the present disclosure comprises two HCDRs provided herein, e.g., at least two HCDRs disclosed in Table 4 or 'fable 5, and two LCDRs provided herein, e.g., at least two LCDRs disclosed in Table 4 or Table 5. In various embodiments, an anti-CDl 9 binding domain of the present disclosure comprises three HCDRs provided herein, e.g., three HCDRs disclosed in Table 4 or Table 5, and three LCDRs provided herein, e.g., three LCDRs disclosed in Table 4 or Table 5.

[0237] In various embodiments, an anti-CD 19 binding domain of the present disclosure comprises at least one heavy chain framework region (heavy chain FR) of a heavy chain variable domain disclosed herein, e.g.. at least one heavy chain FR of a heavy chain variable domain disclosed in Table 4 or 'fable 5. In various embodiments, an anti-CD 19 binding domain of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in Table 4 or Table 5. In various embodiments, an anti-CD 19 binding domain of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in Table 4 or Table 5.

[0238] In various embodiments, an anti-CD 19 binding domain of the present disclosure comprises at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in Table 4 or Table 5. In various embodiments, an anti-CDl 9 binding domain of the present disclosure comprises two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variabledomain disclosed in Table 4 or Table 5. In various embodiments, an anti-CDI9 binding domain of the present disclosure comprises three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in Tabic 4 or Table 5.

[0239] In various embodiments, an anti-CD19 binding domain of the present disclosure comprises at least one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in Table 4 or Table 5, and at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in Table 4 or Table 5. In various embodiments, an anti¬ CD 19 binding domain of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in Table 4 or Table 5, and two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in Table 4 or Table 5. In various embodiments, an anti-CD19 binding domain of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in Table 4 or Table 5, and three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in Table 4 or Table 5.

[0240] In van ous embodiments, an anti-CD19 binding domain of the present disclosure comprises one, two, or three FRs that together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity: e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to corresponding FR(s) of a heavy chain variable domain of a heavy chain variable domain disclosed in in Table 4 or Table 5. In various embodiments, an anti¬ CD 19 binding domain of the present disclosure comprises one, two, or three FRs that together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to corresponding FR(s) of a light chain variable domain of a light chain variable domain disclosed in Table 4 or Table 5.

[0241] In various embodiments, an anti- CD 19 binding domain of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity to a heavy chain variable domain disclosed in Table 4 or Table 5 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g.. 85-90%, 85-95%, 85-100%, 90-95%, 90-100%. or 95-100%). In various embodiments, an anti-CD19 binding domain of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity to a heavy chain variable domain disclosed in Table 4 or Table 5 (e.g., at least 75%, at least 80%, al least 90%, at least 95%,or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), which heavy chain variable domains may be same or different.

[0242] In various embodiments, an anti-CD19 binding domain of the present disclosure comprises at least one light chain variable domain having at least 75% sequence identity to a light chain variable domain disclosed in Table 4 or Table 5 (e.g.. at least 75%. at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In various embodiments, an anti-CD19 binding domain of the present disclosure comprises two light chain variable domains each having at least 75% sequence identity to a light chain vanable domain disclosed in Table 4 or Table 5 (e.g., at least 75%, at least 80%, at least 90%. at least 95%, or 100% identity; e.g.. 85-90%, 85-95%, 85-100%, 90-95%, 90-100%. or 95-100%). which light chain variable domains may be same or different.

[0243] In various embodiments, an anti -CD 19 binding domain of the present disclosure comprises at least one heavy chain vanable domain having at least 75% sequence identity to a heavy chain variable domain disclosed in Table 4 or Table 5 (e.g.. at least 75%, at least 80%. at least 90%. at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%. or 95-100%) and at least one light chain variable domain having at least 75% sequence identity to a light chain variable domain disclosed in Table 4 or Table 5 (e.g., at least 75%, at least 80%’, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0244] In various embodiments, an anti-CD19 binding domain of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity to a heavy chain variable domain disclosed in Table 4 or Table 5 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) and two light chain variable domains each having at least 75% sequence identity to a light chain variable domain disclosed in Table 4 or Table 5 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), where, in various embodiments, (i) each of the heavy chain variable domains may be same or different; or (ii) each of the light chain variable domains may be same or different.Table 4: Exemplary anti-CD19 Antibody Sequences (Abl)SEQ ID Description SequenceNO:1 Heavy Chain EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY Variable Domain GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLT1IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY AMD YWGQGTS VTVS S2 CDRH1 IMGT GVSLPDYG(Prot)3 CDRH1 Kabat DYGVS(Prot)4 CDRH1 Chothia GVSLPDY(Prot)5 CDRH2 IMGT IWGSETT(Prot)6 CDRH2 Kabat VIWGSETTYYNSALKS(Prot)7 CDRH2 Chothia WGSET(Prot)8 CDRH3 IMGT AKH Y Y YGG S Y AMDY(Prot)9 CDRH3 Kabat HYYYGGSYAMDY(Prot)10 CDRH3 Chothia HYYYGGSYAMDY(Prot)11 Light Chain DIQMTQTTSSLSASLGDRVTISCRASQDISKYL Variable Domain NWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNITLPYTFGGGTKLEIT12 CDRL1 IMGT RASQDISKYLN(Prot)13 CDRL1 Kabat RASQDISKYLN(Prot)14 CDRL1 Chothia RASQDISKYLN(Prot)15 CDRL2 IMGT HTSRLHS(Pro!)16 CDRL2 Kabat HTSRLHS(Prot)17 CDRL2 Chothia HTSRLHS(Prot)18 CDRL3 IMGT QQGNTLPYT(Prot)19 CDRL3 Kabat QQGNTLPYT(Prot)20 CDRL3 Chothia QQGNTLPYT(Prot)21 scFv DIQMTQTTSSLSASLGDRVTISCRASQDISKYL (Prot) NWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNITLPYTFGGGTKLEIT GSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLS VTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAI YYCAKHYYYGGSYAMDYWGQGTSVTVSS22 Linker GSTSGSGKPGSGEGSTKG(Prot)Table 5: Exemplar}’ anti-CD19 Antibody Sequences (Ab2)SEQ ID Description SequenceNO:23 Heavy QVQLVQSGAEVKKPGSSVKVSCKDSGGTFSS Chain Variable YAISWVRQAPGQGLEWMGGIIPIFGTTNYAQQFQGR Domain VTTTADESTSTAYMELSSLRSEDTAVYYCAREAVAA DWLDPWGQG TLVT VS S24 CDRH1 GGTFSSYAIMGT (Prot)25 CDRH1 SYAISKabat (Prot)CDRI11 GGTFSSYChothia (Pro!)CDR1I2 IIPIFGTTIMGT (Prot)CDRII2 GIIPIFGTTNYAQQFQGKabat (Prot)CDRH2 PIFGChothia (Prot)CDRII3 AREAVAADWLDPIMGT (Prot)CDRH3 EAVAADWLDPKabat (Pro!)CDRII3 AVAADWLDChothia (Prot)Light Chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYL Variable Domain AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD FTLTISRLEPEDFAVYYCQQYGSSRFTFGPGTKVD1K CDRL1 QSVSSSYIMGT (Prot)CDRL1 RASQSVSSSYLAKabat (Prot)CDRL1 SQSVSSSYChothia (Prot)CDRL2 GASIMGT (Prot)CDRL2 GASSRATKabat (Prot)CDRL2 GASChothia (Prot)CDRL3 QQYGSSRFTIMGT (Prot)CDRL3 QQYGSSRFTKabat (Prot)42 CDRL3 YGSSRFChothia (Prot)43 scFv EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYL (Prot) AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD FTLTISRLEPEDFAVYYCQQYGSSRFTFGPGTKVD1KG STSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGSSVK VSCKDSGGTFSSYAISWVRQAPGQGLEWMGGIIPIFG TTNYAQQFQGRVTITADESTSTAYMELSSLRSEDTAV YYCAREAVAADWLDPWGQGTLVTVSS22 Linker GSTSGSGKPGSGEGSTKG(Prot)Table 6: Exemplary anti-CD20 sequencesSEQ ID NO: Description Sequence79 Heavy Chain QVQLVQSGAEVKKPGASVKVSCKASGY Variable Domain TFKEYGISWVRQAPGQGLEWMGWISAYSGHT YYAQKLQGRVTM1TDTSTSTAYMELRSLRSDD I’AVYYCARGPIIYDDWSGFIIWFDPWGQGTLV TVSS80 VH (DNA) CAGGTTCAGCTGGTGCAGTCTGGAGCT GAGGTGA AGAAGCCTGGGGCCTCAGTGAAG GTCTCCTGCAAGGCTrCTGGTTACACCrrTAA AGAATATGGTATCAGCTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGATGGA TCAGCGCTTAC AGTGGTCAC AC AT ACTATGC ACAGAAGCTCCAGGGCAGAGTCACCATGACC ACAGACACATCCACGAGCACAGCCTACATGG AGCTGAGGAGCCTGAGATCTGACGACACGGC GGTGTACTACTGCGCCAGAGGGCCTCACTAC GACGACTGGAGCGGATTTATCATATGGTrCG ACCCATGGGGACAGGGTACA'n’GGTCACCGT CTCCTCA81 CDRH1 1MGT (Prot) GYTFKEYG82 CDRH1 Kabat (Prot) EYGISCDRII1 Chothia(Prot) GYTFKECDRH2 IMGT (Prot) ISAYSGHTCDRII2 Kabat (Prot) WISAYSGHTYYAQKLQ CDRH2 Chothia(Prot) SAYSGCDRH3 IMGT (Prot) ARGPHYDDWSGFIIWFDP CDRH3 Kabat (Prot) GPHYDDWSGFIIWFDPCDRH3 Chothia(Prot) GPHYDDWSGFIIWFDPLight Chain V ariable DIQMTQSPS SLS AS VGDRVTITCRAS QSI Domain SSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPP TFGQGTKVEIK VL (DNA) GACATCCAGATGACCCAGTCTCCATCC TCCCTGTCTGCATCTGTAGGAGACAGAGTCA CC ATC A CTTGCCGGG CA AGTC AGAGC ATT AG CAGCTATTTAAATTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAGCTCCTGATCTATGCTG CATCCAGTTTGCAAAGTGGGGTCCCTTCAAG GTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCrCACCA'ICAGCAGTCTGCAACCIGAAG ATl'TTGCAACTTACTACTGTCAACAGAGTTAC AGGTTTCCTCCTACCTTTGGCCAAGGGACCA AGGTTGAGATCAAA CDRL1 IMGT (Prot) RASQSISSYLNCDRL1 Kabat (Prot) RASQSISSYLNCDRL1 Chothia(Prot) RASQSISSYLNCDRL2 IMGT (Prot) AASSLQSCDRL2 Kabat (Prot) AASSLQSCDRL2 Chothia(Prot) AASSLQSCDRL3 IMGT (Prot) QQSYRFPPT99 CDRL3 Kabat (Prot) QQSYRFPPT100 CDRL3 Chothia(Prot) QQSYRFPPTTable 7: Exemplary anti-GPC3 SequencesSEQ ID NO. Description Sequence101 Signal MLLLVTSLLLCELPHPAFLLIPpeptide AA102 Signal ATGCn'GTTATTAGTGACTT'CTTTACTGCnGTGT peptide GAGCTGCCCCACCCCGCTTFCCTCCTCATCCCg nucleotide103 VII AA QVQLVQSGAEVKKPGASVKVSCKTSGYTFTDY YIITWVRQAPGQGI. EWMGEIYPGSGNTYYAQKFQGRV TMTADTSTSTAYMEESSLRSEDTAVYYCARGNDYDA WFVYWGQGTLVTVSS104 VH CAAGTCCAACTGGTGCAGTCCGGAGCCGAGGT nucleotide CAAGAAGCCCGGAGCCAGCGTGAAAGTC ICATGTAA AACCAGCGGCTACACCTTCACCGACTACTACATCCA CTGGGTCCGACAAGCCCCCGGTCAAGGTTTAGAGTG GATGGGCGAGATCTACCCCGGCTCCGGCAACACCTA CTACGCCCAGAAGlTCCAAGGTCGrGTGACCAl’GAC AGCCGACACCAGCACCTCCACCGCCTACATGGAACT GTCCTCTCTGCGTTCTGAGGACACAGCCGTTTACTAC TGCGCCAGAGGCAACGACTACGACGCTTGGTTCGTG TACTGGGGCCAAGGAACAn’AGTGACCGTGTCCTCC 105 Whitlow GSTSGSGKPGSGEGSTKGAA106 Whitlow GGATCCACATCCGGCAGCGGAAAGCCCGGTA nucleotide GCGGCGAGGGCAGCACCAAAGGA107 VLAA DIVMTQSPDSLAVSEGERVTMNCKSSQSEI NSG TRKNYLAWYQQKPGQPPKLLIYWASIRESGVPDRFSGS GSGTDFTLTISSVQAEDVAVYYCKQSYSLYTFGQGTKL EIK108 VL GACATCGTCATGACCCAGAGCCCCGAn’CTTT nucleotide AGCCGTGTCTTTAGGCGAAAGAGTGACCATGAACTG CAAGTCCAGCCAGTCTTTACTGAATTCCGGCACTCGA AAAAACTATTTAGCTTGGTACCAGCAGAAACCCGGC CAGCCCCCTAAGCTGCTGATCTACTGGGCTAGCATTC GAGAATCCGGCGTGCCCGATCGCTTTAGCGGCAGCG GTAGCGGCACCGACTTTACTTTAACCATCAGCAGCG TGCAAGCTGAGGACGTGGCTGTGTACTATTGCAAGC AGTCCTACTCTTTATACACCTTCGGCCAAGGAACAA AGCTGGAGATCAAGEXEMPLARY UNKERS AND SPACERS

[0245] In certain embodiments, the CARs contemplated herein may comprise linker residues between the various domains, e.g., between VH and VL domains, added for appropriate spacing conformation of the molecule. CARs contemplated herein, may comprise one, two, three, four, or five or more linkers. In some embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids long.

[0246] Illustrative examples of linkers include glycine polymers (G)n; glycine-serine polymers (Gi sSi-sln, where n is an integer of at least one, two, three, four, or five; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CARs described herein. Glycine accesses more phi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Client. 11173-142 (1992)). Other linkers contemplated herein include Whitlow linkers (see Whitlow, Protein Eng. 6(8): 989-95 (1993)). The ordinarily skilled artisan will recognize that design of a CAR in some embodiments may include linkers that are all or partially flexible, such that the linker may include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired CAR structure, In one embodiment, any of the constructs described herein may comprise a “GS” linker. In another embodiment, any of the constructs described herein comprise a “GSG” linker. In an example a glycine-serine linker comprises or consists of the amino acid sequence GS (SEQ ID NO: 45). In an example a glycine-serine linker comprises or consists of the amino acid sequence GGGSGGGS (SEQ ID NO: 46). In another embodiment, the CARs described herein comprise the amino acidsequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g.. 85-90%. 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of SEQ ID NO: 22).

[0247] In certain embodiments, an anti-CD 19 binding domain of the present disclosure comprises a binding domain that comprises a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity to SEQ ID NO: 22 e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain embodiments, an anti-CD 19 binding domain of the present disclosure comprises a binding domain that comprises a linker according to SEQ ID NO: 22.

[0248] The engineered CARs described herein may also comprise an N-terrninal signal peptide or tag at the N-tcrminus of the scFv or antigen binding domain. In one embodiment, a heterologous signal peptide may be used. The antigen binding domain or scFV may be fused to a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the ceil surface. It is understood that, once a polypeptide containing a signal peptide is expressed at the cell surface, the signal peptide is generally proteolytically removed during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, a polypeptide such as the CAR constructs described herein, are generally expressed at the cell surface as a mature protein lacking the signal peptide, whereas the precursor form of the polypeptide includes the signal peptide. Any suitable signal sequence known in the art may be used. Similarly, any known tag sequence known in the art may also be used. In certain embodiments, a binding domain of the present disclosure comprises an anti -CD 19 binding domain that comprises a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a signal sequence having at least 75% sequence identity to SEQ ID NO: 44 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), MALPVTALLLPLALLLHAARP (SEQ ID NO: 44).

[0249] In embodiments, a CAR comprises a scFv that further comprises a variable region linking sequence. A "variable region linking sequence," is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. In one embodiment, the variable region linking sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids long.

[0250] In embodiments, the binding domain of the CAR is followed by one or more "spacer domains," which refers to the region that moves the antigen binding domain away from the effector cell surface to enable proper cell / ccll contact, antigen binding and activation (Patel et al, Gene Therapy, 1999; 6: 412-419). The spacer domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino add sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0251] The binding domain of the CAR may generally be followed by one or more "hinge domains," which plays a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. A CAR generally comprises one or more hinge domains between the binding domain and the transmembrane domain. The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0252] In some embodiments, an antigen binding system of the present disclosure may comprise a hinge that is, is from, or is derived from (e.g., comprises all or a fragment of) an immunoglobulin-like hinge domain. In some embodiments, a hinge domain is from or derived from an immunoglobulin. In some embodiments, a hinge domain is selected from the hinge of IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or a fragment thereof.

[0253] A hinge may be derived from a natural source or from a synthetic source. Hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type I membrane proteins such as CD8a, CD4. CD28 and CD7, which may be wild-type hinge regions from these molecules or may be altered, for example a truncated CD28 hinge domain. A hinge may be derived from a natural source or from a synthetic source. In some embodiments, an Antigen binding system of the present disclosure may comprise a hinge that is, is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8p. GDI la (11 GAL), GDI lb (ITGAM), C’Dllc (ITGAX), GDI Id (ITGAD), GDI 8 (ITGB2). CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), GD66e (CEAGAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD 100 (SEMA4D), CD 103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CDI58B1(KIR2DL2), CD158B2 (KIR2DL3), CD158C (K1R3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2). CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zcta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46). CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (GDI la / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, 1L-7R alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL). SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a I NF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, or Toll ligand receptor, or which is a fragment or combination thereof. In embodiments, the hinge domain comprises a CD28 hinge region. In embodiments the CARs described herein comprise a hinge domain from CD28 having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g.. 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of SEQ ID NO: 47 (IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 47)). In embodiments, the hinge domain comprises a CD28T hinge region. In embodiments the C ARs described herein comprise a hinge domain from CD28 having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of SEQ ID NO: 48 (LDNEKSNGTIIIIVKGKHLCPSPLFPGPSKP (SEQ ID NO: 48)). In embodiments, the hinge domain comprises a CD8a hinge region. In embodiments the CARs described herein comprise a hinge domain from CD8a having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of SEQ ID NO: 49 or 72 FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 49)) ITrPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 72)).

[0254] Polynucleotide and polypeptide sequences of these hinge domains are known. In some embodiments, the polynucleotide encoding a hinge domain comprises a nucleotide sequence at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a nucleotide sequence known. In some embodiments, thepolypeptide sequence of a hinge domain comprises a polypeptide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence.EXEMPLARY TRANSMEMBRANE DOMAINS

[0255] In some embodiments, the next -generation CAR constructs of the disclosure utilize a JAM-L transmembrane domain (or a fragment thereof), instead of CD28 transmembrane domain, standard in earlier generation CARs. In some embodiments, the next-generation CAR construct s of the disclosure further comprise a co-stimulatory domain of JAM-L (or active fragment thereof), instead of standard co-stimulatory domain of CD28.

[0256] In one embodiment, the transmembrane domain of the CAR comprises a JAML transmembrane domain, or a fragment thereof. In one embodiment, the transmembrane of the CAR comprises a polypeptide of SEQ ID NO:69. In one embodiment, the transmembrane domain of the CAR comprises an arnino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69.

[0257] In general, a “transmembrane domain” (e.g., of an antigen binding system) refers to a domain ha ving an attribute of being present in the membrane when present in a molecule at a cell surface or cell membrane (e.g.. spanning a portion or all of a cellular membrane). A costimulatory domain for an antigen binding system of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. It is not required that every amino acid in a transmembrane domain be present in the membrane. For example, in some embodiments, a transmembrane domain is characterized in that a designated stretch or portion of a protein is substantially located in the membrane. Amino acid or nucleic acid sequences may be analyzed using a variety of algorithms to predict protein subcellular localization (e.g., transmembrane localization). The programs psort (PSORT.org) and Prosite (prosite.expasy.org) are exemplary of such programs.

[0258] The type of transmembrane domain comprised in an antigen binding system described herein is not limited to any type. In some embodiments, a transmembrane domain is selected that is naturally associated with a binding domain and / or intracellular domain. In some instances, a transmembrane domain comprises a modification of one or more amino acids (e.g., deletion, insertion, and / or substitution), e.g., to avoid binding of such domains to a transmembrane domain of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. A transmembrane domain may be derived either from a natural or froma synthetic source. Where the source is natural, a domain may be derived from any membrane¬ bound or transmembrane protein. Exemplary transmembrane domains may be derived from (<?.§., may comprise at least a transmembrane domain of) an alpha, beta or zeta chain of a T-ccll receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45. CD4, CD5, CD7. CD8, CD8 alpha, CD8beta, CD9, GDI la, GDI lb, CD11c, GDI Id, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / GD137, activating NK cell receptors, an Immunoglobulin protein, B7-II3, BAFFR, BLAME (SLAMF8), BTLA, GDI 00 (SEMA4D), CD103, GDI 60 (BY55), CD18, CD19, GDI 9a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a. CD49D. CD49f. CD69, CD84, CD96 (Tactile). CDS, CEACAM1, CRT AM, cytokine receptor. DAP-10. DNAM1 (CD226), Fc gamma receptor, GADS. G1TR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-I, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (IGOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2. LAT. LFA-1. LFA-1, a ligand that binds with CD83. LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl-la / CD18), MHC class 1 molecule, NKG2C, NKG2D. NKp30, NKp44, NKp46, NKp80 (KL. RF1). OX-40, PAG / Cbp, programmed death- 1 (PD-1), PSGL1, SETJPLG (CD 162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; GDI 50; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl()8), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6. or a fragment, truncation, or a combination thereof. In some embodiments, a transmembrane domain may be synthetic (and can, e.g., comprise predominantly hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan and valine are comprised at each end of a synthetic transmembrane domain. In some embodiments, a transmembrane domain is directly linked or connected to a cytoplasmic domain. In some embodiments, a short oligo- or polypeptide linker (e.g., between 2 and 10 amino acids in length) may form a linkage between a transmembrane domain and an intracellular domain. In some embodiments, a tinker is a glycine-serine doublet. In embodiments the CARs described herein comprise a TM domain from CD28 having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of SEQ ID NO: 50 (FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 50)). In embodiments the CARs described herein comprise a TM domain from CD8a having the ammo acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of SEQ ID NO: 51 or SEQ ID NO: 73K-1177-WO-PCT (IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 51)). (IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 73)).

[0259] In some embodiments, the next -generation CAR constructs of the disclosure utilize a JAM-L transmembrane domain (or a fragment thereof), instead of CD28 transmembrane domain, standard in a earlier generation CARs. In some embodiments, the next-generation CAR constructs of the disclosure comprise a co-stimulatory domain of JAM-L (or active fragment thereof), instead of standard co-stimulatory domain of CD28.

[0260] In one embodiment, the transmembrane domain of the CAR comprises a JAML transmembrane domain, or a fragment thereof. In one embodiment, the transmembrane of the CAR comprises a polypeptide of SEQ ID N():xxx. In one embodiment, the transmembrane domain of the CAR comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: xxx.

[0261] In one embodiment, the co-stimulatory domain of the CAR comprises a JAML intracellular domain, or a fragment thereof. In one embodiment, the co-stimulator ' domain of the CAR comprises a polypeptide of SEQ ID NO:xxx. In one embodiment, the co-stimulatory domain of the CAR of the CAR comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: xxx

[0262] Polynucleotide and polypeptide sequences of transmembrane domains provided herein are known. In some embodiments, the polynucleotide encoding a transmembrane domain comprises a nucleotide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, at least about 96%, al least about 97%, at least about 98%, al least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a nucleotide sequence known. In some embodiments, the polypeptide sequence of a transmembrane domain comprises a polypeptide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%. 85-100%, 90-95%. 90-100%, or 95-100%) identical to a polypeptide sequence known. Optionally, short spacers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0263] Intracellular signaling domains that may transduce a signal upon binding of an antigen to an immune cell are known, any of which may be comprised in an antigen binding system of the present disclosure. For example, cytoplasmic sequences of a T cell receptor (TCR) are known to initiate signal transduction following TCR binding to an antigen (see, e.g., Brownlie el al., Nature Rev. Immunol. 13:257-269 (2013)).

[0264] In embodiments, CARs contemplated herein comprise an intracellular signaling domain. An "intracellular signaling domain," refers to the part of a CAR that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain. In some embodiments, a signaling domain and / or activation domain comprises a cytoplasmic signaling domain derived from CD3y, CD 33, CD3s, or DAP- 12, or a fragment, truncation, or a combi n tion thereof. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of a human CD3 zeta chain, CD3Q, FcyRIII, FcsRI, DAP10, DAP12, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine -based activation motif (IT AM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon. CD5. CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0265] In some embodiments, the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain derived from a protein selected from the group consisting of CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FceRI, DAP 10, DAP 12, and CD66d. In some embodiments, the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain derived from CD3 zeta. In some embodiments, the intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain derived from a protein selected from the group consisting of MIIC class I molecules, TNF receptor proteins. Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4- 1BB (CD137), B7-II3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFI), NKp44, NKp30, NKp46, CD 19, CD4, CDSalpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLAI, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE. CD 103, ITGAL. CD 1 la, LFA-1. ITGAM. CD1 lb, 1TGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRAN CE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMFL CD150. IPO-3), BLAME (SLAMF8), SELPLG (CDI62), LTBR, LAL, GADS, SLP- 76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83. In some embodiments, the CAR is an inhibitory CAR comprising one or more intracellular inhibitory domains that inhibit an immune response. In some aspects, the one or more intracellular inhibitory domains comprise anICD derived from the PD-1, CTLA4, TIGIT, BTLA, LIR1 (LILRB1), TIM3. KIR3DL1, NKG2A, LAG3, LAIR1, SIRPa, KIR2DL1, KIR2DL2, KIR2DL3, K1R3DL2, KLRG-1, CEACAM1, LIR2, LIR3, LIR5, SI I. EC-2, SIGLEC-10, PECAM-1, CD72, IRTA2, IRTA4, NKIR, TLT1, PCDHGC3, MPZL1, FCGR2B, SIGLEC-6, MPIG6B. SIGLEC-12. LIR8, IRTA1, KIR2DL4, KIR2DL5, SIGLEC-7. or FCRH3.

[0266] The term "effector function" refers to a specialized function of the cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain may be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal.

[0267] In one embodiment, the CARs have a CD3s domain having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI(SEQ ID NO: 52). In embodiments. CD3e domain is encoded by a nucleic acid having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, al least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to:AAGAACCGGAAGGCCAAGGCCAAGCCTGTGACAAGAGGTGCTGGTGCTGGCGGCA GACAGAGAGGCCAGAACAAAGAAAGACCTCCTCCTGTGCCTAATCCTGACTACGAG CCCATCCGGAAGGGCCAGAGAGATCTGTACAGCGGCCTGAACCAGCGGCGGATT(SEQ ID NO: 53).In embodiments, CD3E domain is encoded by a nucleic acid having at least 75% sequence identity io (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to:AAGAACCGCAAAGCAAAGGCAAAACCCGTCACACGAGGAGCGGGCGCAGGGGGAC GACAACGCGGTCAGAATAAGGAACGCCCGCCTCCAGTACCAAATCCAGATTATGAAK-1177-WO-PCT CCAATTCGGAAGGGACAACGCGA'rCTClACTCCGGTCTCAArCAGAGGCGAAT'r (SEQ ID NO: 54).

[0268] In one embodiment, the CARs have a CD3E domain, having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%. at least 95%. or 100% identity: e.g.. 85-90%. 85-95%. 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDI. YSGE (SEQ ID NO: 109). In embodiments, this CD3E domain, is encoded by a nucleic acid having at least 75% sequence identity to (such as, at least 75%. at least 80%, at least 90%, at least 95%, or 100% identity; e.g.. 85-90%, 85-95%, 85-100%, 90-95%, 90-100%. or 95-100%) the nucleic acid having the sequence according to: AAGAACCGCAAAGCAAAGGCAAAACCCGTCACACGAGGAGCGGGCGCAGGGGGAC GACAACGCGGTCAGAATAAGGAACGCCCGCCTCCAGTACCAAATCCAGATTATGAA CCAA1TCGGAAGGGACAACGCGATCTCTACTCCGGTCTC (SEQ ID NO: 110).

[0269] In one embodiment, the CARs have a CD3s domain, having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQKRI(SEQ ID NO: 111). In embodiments, this CD3s domain is encoded by a nucleic acid having at least 75% sequence identity to (such as, at least 75%’, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to: AAGAACCGCAAAGCAAAGGCAAAACCCGTCACACGAGGAGCGGGCGCAGGGGGAC GACAACGCGGTCAGAATAAGGAACGCCCGCCTCCAGTACCAAATCCAGATTATGAA CCAATTCGG / XAGGGACAACGCGATCTCTACTCCGGTCTCAATCAGAAGCGAATT (SEQ ID NO: 112).

[0270] In one embodiment, the CARs have a CD3E domain having the amino acid sequence having at least 75%’ sequence identity to (such as, at least 75%’, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEP1RKGQRDLYNGLNQKRI (SEQ ID NO: 113). In embodiments, this CD3e domain is encoded by a nucleic acid having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleicacid having the sequence according to: AAGAACCGCAAAGCAAAGGCAAAACCCGTCACACGAGGAGCGGGCGCAGGGGGAC GACAACGCGGTCAGAATAAGGAACGCCCGCCTCCAGTACCAAATCCAGATTATGAA CCAATTCGGAAGGGACAACGCGATCTCTACAACGGTCTCAATCAGAAGCGAATT(SEQ ID NO: 114).

[0271] In one embodiment, the CARs have a CD3y domain having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 57). In embodiments, CD3y domain is encoded by a nucleic acid having at least 75%> sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95'%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to:GGACAGGATGGCGTCAGACAGAGCAGAGCCAGCGACAAGCAAACCCTGCTGCCTA ACGACCAGCTGTACCAGCCTCTGAAGGACAGAGAGGACGACCAGTACAGCCATCTG CAGGGCAACCAGCTGCGGAGAAAC (SEQ ID NO: 58).

[0272] In one embodiment, the CARs have a DAP- 12 domain having the amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:YFIXiRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 59). In embodiments, DAP-12 domain is encoded by a nucleic acid having at least 75% sequence identity to (such as, at least 75%. at least 80%, at least 90%. at least 95%, or 100% identity; e.g.. 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to:TACTTCCTGGGCAGACTGGTGCCTAGAGGAAGAGGAGCTGCTGAGGCTGCTACCAG AAAGCAGAGAATCACCGAGACCGAGAGCCCITACCAGGAGCTGCAGGGACAGAGA AGCGACGTGTACAGCGACCTGAACACCCAGAGACCTTACTACAAG (SEQ ID NO: 60).EXEMPLARY CO-STIMULATORY DOMAINS

[0273] It is known that signals generated through the 'ICR alone are insufficient for full activation of the T cell and that a secondary or costimulatory signal may also be required. Thus, T cell activation may be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TOR(e.g., a TCR / CD3 complex) and costimulatory signaling domains that act in an antigen independent manner to provide a secondary or costimulatory signal. In some embodiments, a CAR contemplated herein comprises an intracellular signaling domain that comprises one or more "costimulatory signaling domain" and a "primary signaling domain."

[0274] CARs contemplated herein comprise one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing CAR receptors. As used herein, the term, "costimulatory signaling domain," or "costimulatory domain", refers to an intracellular signaling domain of a costimulatory molecule. In some embodiments, costimulatory molecules may include JAML, CD27, CD28, CD137(4-IBB), 0X40 (CD134), CD30, CD40, PD~1, 1COS (CD278), CTLA4. LFA- 1, CD2, CD7, LIGHT, TRIM, LCK3, SLAM, DAP1O, LAG3, HVEM, and NKD2C, and CD83. In some embodiments, co-stimulatory signaling domains comprise intracellular domains of one or more of JAML, CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1. CD2, CD7, LIGHT, NKD2C, B7-H2 CD28, CD27, 0X40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5. CD7, CDS, CD8alpha, CD8beta, CDl la, CD1 lb, CDlle, CD1 Id, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L(CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CDL50 (SLAMF1), CD 160 (BY55), CD 162 (DNAMI), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278. EAT, lymphocyte function- associated antigen-I (LFA-1), LIGHT, NKG2C, NKG2D, NK.p30, NKp44, NKp46, NKp80 (KLRFI), DAP10, DAP12, LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIT, 4- 1BBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TAC1, BLAME, CRACC, CD2F- 10, NTB-A. integrin a4, integrin a4pi, integrin a4 7, IA4, ICAM-1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha. IL-7Ralpha, lL-9Ralpha, 1L-12R, lL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, ’Foil-like receptors (TLRs, such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGL1, SLAMF6 (NTB-A. LyI08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6. BTLA, ikaros, LAG-3. LMIR, CEACAM1, CRTAM, TCLIA, DAP 12, TIM-1, Dectin- 1, PDCD6, PD-1, TIM-4, TSLP, and a ligand that specifically binds CD83, or any combination thereof.

[0275] In one embodiment, the co-stimulatory domain of the CAR comprises a JAML intracellular domain, or a fragment thereof. In one embodiment, the co-stimulatory domain of the CAR comprises a polypeptide of SEQ ID NO:70. In one embodiment, the co-stimulatory domain of the CAR of the CAR comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 70.

[0276] In some embodiments, the CARs described herein comprise a CD28 costimulatory domain having the amino acid sequence of having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90- 95%, 90-100%, or 95-100%) SEQ ID NO: 61. RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 61).

[0277] In embodiments, the CARs described herein comprise a 4-1BB costimulatory domain having the amino acid sequence of having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%;, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) SEQ ID NO: 77. KRGRKKLLYIFKQPFMRPVQ1TQEEDGCSCRFPEEEEGGCE (SEQ ID NO: 77).

[0278] Components of a CAR may be exchanged or “swapped” using routine techniques of biotechnology for equivalent components. To provide just a few non-limiting and partial examples, a CAR of the present disclosure may comprise a binding domain as provided herein in combination with a hinge provided herein and a costimulatory domain provided herein. In certain examples, a CAR of the present disclosure may comprise a leader sequence as provided herein together with a binding domain as provided herein in combination with a hinge provided herein and s costimulatory domain provided herein.

[0279] In certain aspects, the present disclosure comprises nucleic acids encoding anti-CD19 binding domains provided herein. In certain further aspects, the present disclosure comprises nucleic acids encoding antibodies provided herein, comprising, without limitation, nucleic acids encoding anti-CD19 binding domains. In certain further aspects, the present disclosure comprises nucleic acids encoding antigen binding systems provided herein, comprising without limitation nucleic acids encoding anti-CD19 chimeric antigen receptors.

[0280] In embodiments, an anti-CD19 CAR construct (pKR4656) has an amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%’, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:

[0281] MLLLVTSLLLCELPHI’AFLLIPDIQMTQTTSSLSASLGDRVriSCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLIISGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGS YAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKH LCPSPLFPGPSKI’FWVLVVVGGVLACYSLLVTVAFllFWVRSKRSRLLHSDYMNMTPRR PGPTRKI IYQP YAPPRDFAA YRSRVKFSRS AD AP AYQQGQNQEYNELNLGRREEYD VI DK-1177-WO-PCT KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE1GMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR (SEQ ID NO: 62).

[0282] In embodiments an anti-CD19 CAR (pKR4656) is encoded by a nucleic acid having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to:

[0283] ATGCTGCTGCTGGTGACATCTCTGCTGCTTTGCGAGCTGCCCCACCCTGCCTT CCTGCTTATCCCCGACATTCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCTT AGGAGATAGAGTTACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAATACCTGA ACTGGTATCAGCAGAAGCCXXjACGGCACTGTGAAACTGCTTATTTACCACACCT(X) AGACTGCACAGCGGCGTTCCCAGCAGATTCTCTGGCAGCGGATCTGGAACCGACTA CAGCCTCACCATCTCCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGC AGGGCAACACACTGCCCTACACCTTCGGAGGAGGAACCAAGCTGGAGATCACCGGC TCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGT TAAGCTGCAGGAGAGCGGCCCTGGCCTGGTGGCTCCTAGCCAATCTTTATCTGTGAC CTGCACTGTGTCCGGCGTTAGCCTGCCCGATTATGGCGTTTCCTGGATCAGACAGCC CCCX1AGAAAGGGCCTGGAATGGCTGGGC ITATCI GGGGCAGCGAGACCACATACT ACAACAGCGCCCTGAAGAGCAGACTTACGATTATCAAGGACAACAGCAAGAGCCA GGTTTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCG CTAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGAACA AGCGTTACCGTTAGCAGCGCTGCTGCAATTGAAGTTATGTATCCTCCTCCTTACCTG GACAACGAGAAGAGCAACGGCACCATCATCCACGTTAAGGGCAAGCACCTGTGCCC CAGCCCTCTGTTCCCTGGACCTTCTAAGCCTTTCTGGGTTCTGGTGGTGGTCGGCGG CGTTrTAGCCTG i CAGCC rcTGGTGACTGTGGCCTrcATCATCTTTrGGGTrAGA AGCAAGAGAAGCAGACTGCTCCACAGCGACTACATGAACATGACCCCCAGACGGC CTGGCCCCACCAGAAAGCATTACCAGCCCTACGCTCCTCCCAGAGACTTCGCCGCCT ACAGGAGCAGAGTTAAATTCAGCAGATCCGCCGATGCCCCCGCTTACCAACAGGGA CAAAACCAGCTGTACAATGAGCTCAACCTGGGGAGAAGAGAAGAATACGACGTTCT GGATAAGAGAAGGGGCAGAGATCCCGAAATGGGGGGCAAGCCCAGACGCAAGAAC CCTCAGGAGGGGCTTTACAACGAACTGCAGAAGGATAAGATGGCTGAGGCTTACTC GGAGATTGGGATGAAGGGGGAGAGAAGGCGGGGCAAGGGACACGATGGCTTATAC CAGGGGCTGAGCACCGCCACCAAGGACACATACGACGC. TCrrCATATGCAGGCTCT GCCCCCAAGA (SEQ ID NO: 63).

[0284] In one embodiment, the CAR comprises a signal peptide comprising the sequence MLLLVTSLLLCELPHPAFLL1P (SEQ ID NO: 64).

[0285] In embodiments, an anti-CD19 CAR construct (pKR11916) has an amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:MLLLV'rSLLLCELPHPAFLLlPDIQMTQ'ri'SSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLIISGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGEEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFI, KMNSLQTODTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGT1IHVKGKHLCPSP LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKKTCGNKSSVNSWLVKNTKKTNP EIKEKPCIIFERCEGEKHIYSPIIVREVIEEEEPSEKSEATYMTMHPVA\TSLRSDRNNSLEK KSGGGMPKTQQAFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVI., DKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 65).

[0286] In embodiments an anti-CD19 CAR (pKRl 1916) is encoded by a nucleic acid having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to:ATGCTGCTGCTGGTG A C ATCTCTGCTGCTTTGCG A GCTGCCCC A CCCTGCCTTCCTGC TTATCCCCGACATTCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCTTAGGA GATAGAGTTACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAATACCTGAACTG GTATCAGCAGAAGCCCGACGGCACTGTGAAACTGCTTATTTACCACACCTCCAGAC TGCACAGCGGCGTTCCCAGCAGATTCTCTGGCAGCGGATCTGGAACCGACTACAGC CTCACCATCTCCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGG CAACACACTGCCCTACACCTTCGGAGGAGGAACCAAGCTGGAGATCACCGGCTCCA CCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTTAAG CTGCAGGAGAGCGGCCCTGGCCTGGTGGCTCCTAGCCAATCTTTATCTGTGACCTGC ACTGTGTCCGGCGTTAGCCTGCCCGA'TTATGGCGT'rrCCTGGATCAGACAGCCCCCC AGAAAGGGCCTGGAATGGCTGGGCGTTATCTGGGGCAGCGAGACCACATACTACAA CAGCGCCCTGAAGAGCAGACTTACGATTATCAAGGACAACAGCAAGAGCCAGGTTT TCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCTAAG CACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGAACAAGCGT TACCGTTAGCAGCGC / lXiCTGCAATTGAAGTTATGIATCCTCCTCCTTACCTGGACAA CGAGAAGAGCAACGGCACCATCATCCACGTTAAGGGCAAGCACCTGTGCCCCAGCCCrcTGTICCC'IXiGACCTirTAAGCCTT'rCTGGGl'TCIGGlGG'rGGI'CGGCGGCGTri'TAGC TGTTACAGCCTrCTGGTGACTGTGGCCI CATCATCTTITGGGI AAGAAAAC GTGTGGCAACAAATCTAGCGTTAATTCTACAGTTTTGGTGAAGAATACTAAGAAAA CGAACCCGGAGATCAAGGAGAAACCTTGCCACTTTGAACGCTGCGAAGGCGAAAA GCACATCTACTCACCTAT ATTGTCAGAGAAGTAATTGAAGAGGAGGAGCC TCTG AGAAAAGCGAGGCGACrrACATGACGATGCACCCTGTCHjGCCGTCCTTGCGCTCT GATCGCAATAATTCACTCGAGAAGAAATCTGGAGGAGGAATGC. C AAGACGCAGC AGGCTTTCAGAGTTAAATTCAGCAGATCCGCCGATGCCCCCGCTTACCAACAGGGA CAAAACCAGCTGTACAATGAGCTCAACCTGGGGAGAAGAGAAGAATACGACGTTCT GGA TAAGAGAAGGGGCAGAGA TCCCGAAA TGGGGGGCAAGCCCAGACGCAAGAAC CCTCAGGAGGGGCTl ACAACGAACTGCAGAAGGATAAGATGCiCTGAGGCITACTC GGAGATTGGGATGAAGGGGGAGAGAAGGCGGGGCAAGGGACACGATGGCTTATAC CAGGGGCTGAGCACCGCCACCAAGGACACATACGACGCTCTTCATATGCAGGCTCT GCCCCCAAGA (SEQ ID NO: 66).

[0287] In an embodiment, an anti-CD19 CAR construct (pKRl 1917) has an amino acid sequence having at least 75% sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the amino acid sequence according to:MLIJ. VTSI LCELPIIPAHJJPDIQMTQTTSSIAASLGDRVTISCRASQDISKYI. NWYQQ KPDGTVKLI JYHTSRLHSGVPSRFSGSGSGTDYSETISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP LFPGPSKPVIIVGIVCATILLLPVIJLIVKKTCGNKSSVNSTVLVKNTKKTNPEIKEKPCHF ERCEGEKHIYSPIIVREVIEEEEPSEKSEAI’YMTMHPVWPSLRSDRNNSLEKKSGGGMPK TQQAFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGI. YQGLSTATKDTYDALHMQA LPPR(SEQ ID NO: 67).

[0288] In embodiments an anti-CD19 CAR (pKRl 1917) is encoded by a nucleic acid having at least 750 sequence identity to (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the nucleic acid having the sequence according to:ATGCTGCTGCTGGTGACATCTCTGCTGC'FlTGCGAGCTGCCCCACCCTGCCTrCCTGC TTATCCCCGACATTCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCTTAGGA GATAGAGTTACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAATACCTGAACTG GTATCAGCAGAAGCCCGACGGCACTGTGAAACTGCTTATTTACCACACCTCCAGACTGCACAGCGGCGTTCCCAGCAGATTCTCTGGCAGCGGATCTGGAACCGACTACAGC CTCACCATCTCCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGG CAACACACTGCCCTACACCTTCGGAGGAGGAACCAAGCTGGAGATCACCGGCTCCA CCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTTAAG CTGCAGGAGAGCGGCCCTGGCCTGGTGGCTCCTAGCCAATCTTrATCTGTGACCTGC ACTGTGTCCGGCGn’AGCCTGCCCGATTATGGCGlTTCCTGGATCAGACAGCCCCCC AGAAAGGGCCTGGAATGGCTGGGCGTTATCTGGGGCAGCGAGACCACATACTACAA CAGCGCCCTGAAGAGCAGACTTACGATTATCAAGGACAACAGCAAGAGCCAGGTTT TCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCTAAG CACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGAACAAGCGT TACCGTTAGCAGCGCTGCTGCAATTGAAGTTATGTATCCTCCTCCTTACCTGGACAA CGAGAAGAGCAACGGCACCATCATCCACGTTAAGGGCAAGCACCTGTGCCCCAGCC CTCTGITCCCTGGACCTTCTAAGCCTGTCATCATAGTAGGAATI'GTGTGCGCTACAA TACTTCTCn’GCCCG rC rATATTGATAGTGAAGAAAACGTGTGGCAACAAATCTA GCGTTAATTCTACAGTTTTGGTGAAGAATACTAAGAAAACGAACCCGGAGATCAAG GAGAAACCTTGCCACTTTGAACGCTGCGAAGGCGAAAAGCACATCTACTCACCTAT AA ITGTCAGAGAAG TAATTGAAGAGGAGGAGCCA 1( 1 ’GAGAAAAGCGAGGCGAC I TACATGACGATGCACCCTGTCTGGCCGTCCTTGCGCTCTGATCGCAATAATTCACTC GAGAAGAAATCTGGAGGAGGAATGCCGAAGACGCAGCAGGCTTTCAGAGTTAAAT TCAGCAGATCCGCCGATGCCCCCGCTTACCAACAGGGACAAAACCAGCTGTACAAT GAGCTCAACCTGGGGAGAAGAGAAGAATACGACGTTCTGGATAAGAGAAGGGGCA GAGATCCCGAAATGGGGGGCAAGCCCAGACGCAAGAACCCTCAGGAGGGGCTTTA CAACGAACTGCAGAAGGATAAGATGGCTGAGGCTTACTCGGAGATTGGGATGAAG GGGGAGAGAAGGCGGGGCAAGGGACAC GATGGCT I A TACCAGGGGCTGAGCACCG CCACCAAGGACACATACGACGCTCTrCATATGCAGGCTCTGCCCCCAAGA (SEQ ID NO: 68).

[0289] In some embodiments, the disclosure provides a CAR construct as described in FIG. 1A and FIG. IB. In some embodiments, the construct encodes ...

Claims

CLAIMS1. A chimeric antigen receptor (CAR) comprising:an extracellular antigen-binding domain:a hinge / spacer;a transmembrane domain and / or a costimulatory domain of Junctional Adhesion Molecule-lake (JAML) protein, or a fragment thereof;and an activation domain,2. The Ci AR of claim 1, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:69, or a an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69; and / or the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 70, or a an amino acid sequence having at least about 75%. 80%. 85%, 90%. 91%. 92%, 93%. 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 70.

3. The CAR of any one of claims 1 or 2, wherein the antigen-binding domain binds an antigen selected from the group consisting of CD 19 (cluster of differentiation 19), 707- AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b-catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region- Abelson), CAIX (carbonic anhydrase IX), CD la, CDlb, CDlc, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD1 la, CD1 lb, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16, (FcyRIII), CD17, CD18, CD19, CD20, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32 (FcyRlI), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44v6, CD44v7 / 8, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD52, CD.53, CD54, CD56 (NCAM), CD57, CD61 (integrin 33), CD62L, CD63, CD64 (FcyRI), CD66b, CD68, CD69, CD70. CD73, CD56, CD74, CD79a (Igu), CD79b (Ig[3). CD80, CD85k (1LT3), CD86, CD88, CD93 (CIRqp), CD94, CD95, CD99, CD103, CD105 (Endoglin), CD107a, CD107b, CD114 (G-CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138, CD141 (BDCA3), CD146, CD147, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1.1), CD163, CD171, CD183, CD191, CD193 (CCR3), CD194 (CCR4), CD 195 (OCRS), CD 197 (CCR7), CD203c, CD205 (DEC-205), CD207 (Langerin), CD209 (DC-S1GN), CD223, CD235, CD235a, CD244 (2B4), CD252 (OX40L), CD267, CD268 (BAFF-R), CD273 (B7-DC, PD-L2), CD276 (B7-H3), CD279 (PDI), CD282 (TLR2), CD284 (TLR4), CD294, CD304 (Neuropilin- 1), CD305, CD314 (NKG2D), CD319 (CRACC). CD326,CD328 (Siglec-7), CD335 (NKp46), CAMEL (CTL-recognized antigen on melanoma). CLL1, CD44V6CAP-1 (carcinoembryonic antigen peptide - 1 ), CASP-8 (caspase-8), CDC27m (cell¬ division cycle 'll mutated), CDK4 / m (cyclinc-dcpendcnt kinase 4 mutated), CEA (carcinoembryonic antigen), C-type lectin-like- 1 (CLL-1), CT (cancer / testis (antigen)). Cyp-B (cyclophilin B), DAM (differentiation antigen melanoma), EGFR (epidermal growth factor receptor), EGFRvlll (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40), EGP-I, EGP-2, Ep-CAM, EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphBl, EphB2, EphB3, EphB4, EphB6. Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3. 4), ELF2M (elongation factor 2 mutated), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (Fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE (G antigen), GD2 (disialogangliosidc 2), GD3 (disialogangliosidc 3), glypican 3 (GPC3), GnT-V (N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100kD), HAGE (helicose antigen), HER-2 / neu (human epidermal receptor- 2 / neurological: also known as EGFR2), HLA-A (human leukocyte antigen- A) HPV (human papilloma virus), HSP70-2M (heat shock protein 70 - 2 mutated), HST-2 (human signet ring tumor - 2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxyl esterase), ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), KIAA0205, KDR (kinase insert domain receptor), K-light chain. LAGE (I, antigen), LDLR / FUT (low density lipid receptor / GDP-L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase), LeY (Lewis-Y antibody), LI CAM (LI cell adhesion molecule), LMPl, MAGE (melanoma antigen), MAGE- A1 (Melanoma-associated antigen 1 ), MAGE- A3, MAGE- A4. Melan A, rnesothelin, MMP, Murine CMV infected cells, MART-l / Melan-A (melanoma antigen recognized by T cells-I / Melanoma antigen A), MCI R (melanocortin 1 receptor), Myosm / m (myosin mutated). MLJC1 (mucin 1 ), MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural killer group 2, member D) ligands, NY-BR-1 (New York breast differentiation antigen 1 ), NY-ESO-1 (New York esophageal squamous cell carcinoma- 1 ). oncofetal antigen (h5T'4), P15 (protein 15), pl90 minor bcr-abl (protein of 190KD bcr-abl), Pml / RARa (promyelocytic leukaemia / retinoic acid receptor a), PRAME (preferentially expressed antigen of melanoma), PSA (prostate-specific antigen), PSCA (Prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitous I or 2), SAGE (sarcoma antigen), SART-1 or S ART-3 (squamous antigen rejecting tumor 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma XI, -2, -3, -4), TAA (tumor- associated antigen), TAG-72 (Tumor-associated glycoprotein 72), TEL / AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1 ), TPl / m (triosephosphate isomerase mutated),TRP-l (tyrosinase related protein 1. or gp75), TRP-2 (tyrosinase related protein 2), TRP- 2 / INT2 (TRP-2 / intron 2). VEGF-R2 (vascular endothelial growth factor receptor 2), or WT1 (Wilms' tumor gene).

4. The CAR of any one of claims 1-3, wherein the antigen-binding domain comprises an scFv selected from the group consisting of an anti- CD19-binding scFv, an anti-CD20 binding scFv, and an anti-GPC3 binding scFv.

5. The CAR of any one of claims 1-4, wherein the activation domain comprises a CD3C, CD3s, CD: 3y, CD35, FcvR, DAP- 12, or IT AM mut signaling domain6. The CAR of any one of claims 1-5, wherein the transmembrane domain comprises a J AML, CD3 CD8a. CD28, CD4, or 1COS transmembrane domain, or fragment thereof.

7. The CAR of any one of claims 1-6, wherein the costiniulatory domain comprises a JAML, CD27, CD28, 4- IBB (CD137), 0X40 (CD 134). CD30. CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 CD28, CD27, 0X40 (CD1 4), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CDllb, CDlle, CD lid, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154). CD48, CD49a. CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-1), CD82, CD83, CD84, CD86 ( B7- 2 ), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMF1), CD160 (BY55), CD162 (DNAMI), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278, L. AT, lymphocyte function -associ ted antigcn-I (LFA-1), LIGHT, NKG2C, NKG2D, NK.p30, NKp44, NKp46. NKp80 (KLRFI), DAP10, DAP12, LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIT, 4-1BBL, BAFF. GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, 0X401., TAC1, BLAME, CRACC, CD2F-10, NTB-A, integrin a4, integrin a4pi, integrin a4p»7, IA4, ICAM-1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs, such as TLR2), ITGA4, 1TGA6, 1'IGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGLI, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6. BTLA, ikaros, LAG-3, LMIR, CEACAM1, CRTAM, TCLIA, DAP12, TIM-1, Dectin- 1. PDCD6, PD-1, TIM-4, TSLP, and a ligand that specifically binds CD83, costiniulatory domain, or any combination thereof.

8. The C AR of any one of claims 1-7, where the antigen-binding domain comprises an anti-CD19 binding domain having an heavy chain complementarity determining region 1 (TICDR1 ), an HCDR2, and an IICDR3; and a light chain complementarity determining region 1 (LCDRI), an L. CDR2, and an LCDR3, whereinthe HCDR1 comprises an amino acid sequence according to any one of SEQ ID NOs: 2-4; the IICDR2 comprises an amino acid sequence according to any one of SEQ ID NOs: 5-7; the HCDR3 comprises an amino acid sequence according to any one of SEQ ID NOs: 8-10; the LCDR1 comprises an amino acid sequence according to any one of SEQ ID NOs: 12-14; the LCDR2 comprises an amino acid sequence according to any one of SEQ ID NOs: 15-17; and the LCDR3 comprises an amino acid sequence according to any one of SEQ ID NOs: 18-20, orwherein the HCDR1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-26; the HCDR2 comprises an amino acid sequence according to any one of SEQ ID NOs: 27-29; the HCDR3 comprises an amino acid sequence according to any one of SEQ ID NOs: 30-32; the LCDRI comprises an amino acid sequence according to any one of SEQ ID NOs: 34-36; the LCDR2 comprises an amino acid sequence according to any one of SEQ ID NOs: 37-39; and the LCDR3 comprises an amino acid sequence according to any one of SEQ ID NOs: 40-42.

9. The CAR of any one of claims 1-8, comprising a heavy chain variable domain comprising the IICDR1, the IICDR2, and the IICDR3; and a first light chain variable domain comprising the LCDRI, the LCDR2, and the LCDR3 wherein:the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1; and the light chain variable domain is at least 80% identical to SEQ ID NO: 11.

10. The CAR of any one of claims 1-7, where the antigen-binding domain comprises an anti-CD20 binding domain having an heavy chain complementarity determining region 1 (HCDRI), an HCDR2, and an HCDR3; and a light chain complementarity determining region I (LCDRI), an LCDR2, and an LCDR3, whereinthe IICDR1 comprises an amino acid sequence according to any one of SEQ ID NOs: 81-83; the HCDR2 comprises an amino acid sequence according to any one of SEQ ID NOs: 84-86; the HCDR3 comprises an amino acid sequence according to any one of SEQ ID NOs: 87-89; the LCDRI comprises an amino acid sequence according to any one of SEQ ID NOs: 92-94; the LCDR2 comprises an amino acid sequence according to any one of SEQ IDNOs: 95-97; and the LCDR3 comprises an amino acid sequence according to any one of SEQ ID NOs: 98-100.

11. The CAR of any one of claims 1-7 and 10, comprising a heavy chain variable domain comprising the HCDR1. the HCDR2, and the HCDR3; and a first light chain variable domain comprising the LCDR1, the LCDR2, and the LCDR3 wherein:the heavy chain variable domain is at least 80% identical to SEQ ID NO: 79; and the light chain variable domain is at least 80% identical to SEE ID NO: 90.

12. The CAR of any one of claims 1-7, wherein the antigen-binding domain comprises an anti-GPC3-binding domain comprising a heavy chain variable domain that is at least 80% identical to SEQ ID NO: 103; andthe light chain variable domain is at least 80% identical to SEQ ID NO: 107.

13. The CAR of any one of claims 1 -12, where the transmembrane domain comprises a transmembrane domain selected from the group consisting of JAML, CD8, and CD28 transmembrane domains, or fragments thereof.

14. The CAR of any one of claims 1-1, where the costimulatory domain comprises a costimulatory domain selected from the group consisting of a JAML, CD28, and 4- IBB costimulatory domains.

15. The CAR of any one of claims 1-14, comprising the amino acid sequence according to any one of SEQ ID NOs: 62, 65, or 67.

16. A nucleic acid encoding the CAR of any one of claims 1-15.

17. A recombinant vector comprising the nucleic acid of claim 16.

18. A host cell transduced or transfected with the nucleic acid of claim 16 or the recombinant vector of claim 17.

19. A host cell transduced or transfected with a nucleic acid encoding a full length JAML,, or a functional fragment thereof, and a chimeric antigen receptor, preferably wherein the chimeric antigen receptor is selected from the group consisting of a first-generation CAR-T,second generation CAR-T, third generation CAR-T, fourth generation CAR-T, fifth generation CAR-T, bivalent CAR-T, multi-target CAR, Tandem CAR, Fc binding CAR, Conditional CAR, ZipCAR, Inhibitory CAR, Split CAR, Switch CAR, iCasp9 CAR, sMASh CAR, Tet-ON CAR, Adpator CAR, SynNotch CAR, LINK CAR, SNIP CAR, and Stealth CAR,20. A host cell transduced or transfected with a nucleic acid encoding a chimeric TGFβ-receptor comprising a JAML cytoplasmic domain, a JAML transmembrane domain, or a fragment thereof.

21. The host cell of any one of claims 18 to 20, wherein the host cell comprises a T cell, an iPSC cell, a macrophage, an iNKT cell, or a NK cell.

22. A pharmaceutical composition comprising the T cell, iPSC cell, macrophage, iNKT cell, or NK cell of claim 21.

23. A method of treating disease in a patient in need of thereof, comprising administering the 1’ cell, iPSC cell, macrophage, iNKT cell, or NK cell of claim 21 or the pharmaceutical composition of claim 22 to the patient.

24. A method of inducing an immune response in a subject or immunizing a subject against a cancer, the method comprising administering to the subject the T cell, iPSC cell, macrophage, iNKT cell, or NK cell of claim 21 or the pharmaceutical composition of claim 22 to the patient.

25. The method of claim 23 or 24, wherein the disease is a cancer selected from acute lymphoblastic leukemia (ALL) (including non T cell ALL), acute myeloid leukemia, B cell prolymphocytic leukemia, B cell acute lymphoid leukemia (“BALL”), blastic plasmacytoid dendritic cell neoplasm. Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's Disease, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, monoclonal gammapathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorder (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablaslic lymphoma, plasmacytoid dendritic cellneoplasm, plasmacytomas (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukasc syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B cell lymphoma (PMBC), small cell- or a large cell-follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, I' cell acute lymphoid leukemia (“TALL”), T cell lymphoma, transformed follicular lymphoma, or Waldenstrom macroglobulinemia, Mantle cell lymphoma (MCI.), Transformed follicular lymphoma (TFL), Primary mediastinal B cell lymphoma (PMBCL), Multiple myeloma, Hairy cell lymphoma / leukemia, adrenocortical carcinoma, agnogenic myeloid metaplasia, AIDS- related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing’s family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g.. hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma,lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (c.g., non- melanoma (c.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms’ tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome. In some embodiments, the cancer is a virus-infection-related cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-related cancer (e.g., HPV-related cervical cancer, e.g., HPV-related head and neck cancer, e.g., HPV related squamous cell carcinoma). In some embodiments, the cancer is human herpes vims 8 (HHV8) related cancer (e.g., Kaposi sarcoma). In some embodiments, the cancer is human T-lymphotrophic virus (HTLV-l)-related cancer (e.g., adult T cell leukemia or lymphoma). In some embodiments, the cancer is Epstein-Barr virus (EBV) related cancer (e.g., Burkitt lymphoma, Hodgkin’s and non-Hodgkin’s lymphoma, stomach cancer). In some embodiments, the cancer is hepatitis B virus (HBV) related cancer (e.g., liver cancer). In some embodiments, the cancer is hepatitis C virus) related cancer (e.g., liver cancer, non-Hodgkin’s lymphoma). In some embodiments, the cancer is a liver cancer, a kidney cancer, an endometrial cancer, a thymic epithelial neoplasma, lung cancer, spindle cell sarcoma, chondrosarcoma, uterine smooth muscle, colon cancer, or pancreatic cancer.

26. The method of claim 23 or 24, wherein the disease is an autoimmune disease.

27. A chimeric polypeptide comprising an extracellular domain of a transforming growth factor (TGF)-p-receptor and a cytoplasmic domain of JAML, or a fragment thereof.

28. A polynucleotide encoding the chimeric polypeptide of claim 27.

29. A vector comprising the polynucleotide of claim 28.

30. A host cell comprising the vector of claim 29.

1. The host cell of claim 30, further comprising a CAR.

32. The host cell of claim 31, wherein the CAR is the CAR of any one of claims 1 to 12.

33. The host cell of any one of claims 30 to 32, wherein the cell is selected from the group consisting of T cell, an iPSC cell, a macrophage, an iNKT cell, or a NK cell.

34. A pharmaceutical composition comprising the host cell of any one of claims 30 to 33.

35. A method of treating a disease or condition in a patient in need thereof, comprising administering the host cell of any one of claims 30 to 33 or the pharmaceutical composition of claim 34 to the patient.

36. A method of inducing an immune response in a subject or immunizing a subject against a cancer, the method comprising administering to the subject the host cell of any one of claims 30 to 33 or the pharmaceutical composition of claim 34 to the patient.

37. The method of claim 35 or 36, wherein the disease is a cancer selected from acute lymphoblastic leukemia (ALL.) (including non T cell ALL), acute myeloid leukemia, B cell prolymphocytic leukemia, B cell acute lymphoid leukemia (“BALL”), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's Disease, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, monoclonal gammapathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL.), plasma cell proliferative disorder (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytomas (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B cell lymphoma (PMBC), small cell- or a large cell-follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T cell acute lymphoid leukemia (“TAI ”), T cell lymphoma, transformed follicular lymphoma, or Waldenstrom macroglobulinemia, Mantle cell lymphoma (MCL). Transformed follicularlymphoma (TFL), Primary mediastinal B cell lymphoma (PMBCL), Multiple myeloma, Hairy cell lymphoma / leukemia. adrenocortical carcinoma, agnogenic myeloid metaplasia, AIDS- related cancers (e.g., AIDS-rclatcd lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing’s family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g.. hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non- melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms’ tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associatedK-1177-WO-PCT with phakomatoses. edema (such as that associated with brain tumors), and Meigs’ syndrome. In some embodiments, the cancer is a virus-infection-related cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-related cancer (e.g., HPV-related cervical cancer, c.g., HPV-related head and neck cancer, e.g., HPV related squamous cell carcinoma). In some embodiments, the cancer is human herpes virus 8 (HHV8) related cancer (e.g,, Kaposi sarcoma). In some embodiments, the cancer is human T-lymphotrophic virus (HTLV-1)-related cancer (e.g., adult T cell leukemia or lymphoma). In some embodiments, the cancer is Epstein-Barr virus (EBV) related cancer (e.g., Burkitt lymphoma. Hodgkin’s and non-Hodgkin’s lymphoma, stomach cancer). In some embodiments, the cancer is hepatitis B virus (HBV) related cancer (e.g., liver cancer). In some embodiments, the cancer is hepatitis C virus) related cancer (e.g., liver cancer, non-Hodgkin’s lymphoma). In some embodiments, the cancer is a liver cancer, a kidney cancer, an endometrial cancer, a thymic epithelial neoplasma, lung cancer, spindle cell sarcoma, chondrosarcoma, uterine smooth muscle, colon cancer, or pancreatic cancer.

38. The method of claim 35 or 36, wherein the disease is an autoimmune disease.

39. A method of making a CAR T cell, comprising providing for the expression of the CAR of any one or claims 1 to 15 in a cell.

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