And-gated chimeric antigen receptors comprising kinase domains

Abstract

Cells engineered to express AND-gated chimeric antigen receptor (CAR) pairs comprising a CAR comprising a kinase domain and a CAR comprising a substrate for that kinase are provided, as are nucleic acids encoding the CARs, vectors comprising the nucleic acids, and host cells comprising the nucleic acids and vectors.

Description

Attorney Docket No. 48104-713601AND-GATED CHIMERIC ANTIGEN RECEPTORS COMPRISING KINASE DOMAINSCROSS-REFERENCE

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 734,359, filed December 16, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on December 11, 2025, is named 48104-713_601_SL and is 286,252 bytes in size.BACKGROUND

[0003] T cell-expressing chimeric antigen receptors (CAR-T cells) have been shown to effectively treat numerous blood cancers. However, treatment of solid tumors has been limited because the solid tumor markers that could be used to target CAR-T cells are often shared with non-cancerous cells, causing great risk of off-target effects. Accordingly, improved and alternative CAR-T cell platforms that have greater specificity are needed.SUMMARY

[0004] Provided herein are cells comprising: a first chimeric antigen receptor comprising a first extracellular domain and a first intracellular domain, wherein the first extracellular domain comprises a first antigen-binding domain, and wherein the first intracellular domain comprises a first kinase domain; and a second chimeric antigen receptor comprising a second extracellular domain and a second intracellular domain, wherein the second extracellular domain comprises a second antigen-binding domain, wherein the second intracellular domain comprises a tyrosine, and wherein the tyrosine is a substrate for phosphorylation by the first kinase domain.

[0005] Provided herein are cells comprising: a first chimeric antigen receptor comprising a first extracellular domain and a first intracellular domain, wherein the first extracellular domain comprises a first antigen-binding domain, and wherein the first intracellular domain comprises a first kinase domain; a second chimeric antigen receptor comprising a second extracellular domain and a second intracellular domain, wherein the second extracellular domain comprises a second antigen-binding domain; and a PLCyl, wherein the PLCyl is phosphorylated by the first kinaseAttorney Docket No. 48104-713601domain when the first chimeric antigen receptor is in contact with the second chimeric antigen receptor.

[0006] In some embodiments, the first kinase domain comprises a biologically active domain of any one of KIT, DDR1, FGFR1, TIE2, FLT1, ZAP-70, LCK, FYN, SYK, ErbB2, DDR2, PDGFRa, FLT1, ITK, or RLK proteins. In some embodiments, the first kinase domain comprises a biologically active domain of any one of KIT, DDR1, FGFR1, TIE2, FLT1, ZAP-70, LCK, FYN, SYK, or RLK proteins. In some embodiments, the first kinase domain comprises the amino acid sequence of any one of SEQ ID NOs: 27 or 33-45. In some embodiments, the first kinase domain comprises the amino acid sequence of any one of SEQ ID NOs: 33 or 46-49. In some embodiments, the first kinase domain comprises a biologically active domain of FGFR1. In some embodiments, the first kinase domain comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the first kinase domain comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the first kinase domain comprises a biologically active domain of any one of KIT, FGFR1, TIE2, or SYK proteins. In some embodiments, the first kinase domain comprises a biologically active domain of TIE2. In some embodiments, the first kinase domain comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the first kinase domain comprises an amino acid sequence of SEQ ID NO: 49. In some embodiments, the second intracellular domain comprises a domain that recruits PLCyl upon phosphorylation. In some embodiments, the second intracellular domain comprises an intracellular domain of any one of LAT, VEGFR, PDGFRa, FGFR1, or KIT proteins. In some embodiments, the second intracellular domain comprises an amino acid sequence of any one of SEQ ID NOs: 28 or 50-53. In some embodiments, the second intracellular domain comprises an intracellular domain of LAT. In some embodiments, the second intracellular domain comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the second intracellular domain comprises an intracellular domain of LAT and wherein the first kinase domain comprises a biologically active domain of TIE2. In some embodiments, the second intracellular domain comprises an intracellular domain of LAT and the first kinase domain comprises a biologically active domain of FGFR1. In some embodiments, the second intracellular domain comprises an intracellular domain of any one of VEGFR, PDGFRa, FGFR1, or KIT proteins. In some embodiments, the second intracellular domain comprises an intracellular domain of PDGFRa. In some embodiments, the second intracellular domain comprises an amino acid sequence of SEQ ID NO: 53. In some embodiments, the second intracellular domain comprises an intracellular domain of PDGFRa and the first kinase domain comprises a biologically activeAttorney Docket No. 48104-713601domain of TIE2. In some embodiments, the second intracellular domain comprises an intracellular domain of PDGFRa and the first kinase domain comprises a biologically active domain of FGFR1.

[0007] In some embodiments, the second chimeric antigen receptor comprises a second kinase domain.

[0008] In some embodiments, the second kinase domain comprises a kinase regulatory domain. In some embodiments, the kinase regulatory domain is an activator of the first kinase domain. In some embodiments, the kinase regulatory domain is a repressor of the first kinase domain.

[0009] In some embodiments, the first kinase domain and the second kinase domain are activated when dimerized to each other.

[0010] In some embodiments, the tyrosine binds to a PLCyl when phosphorylated. In some embodiments, the PLCyl comprises a substrate for the first kinase domain. In some embodiments, the PLCyl comprises a substrate for the second kinase domain.

[0011] In some embodiments, the first chimeric antigen receptor comprises a first transmembrane domain, and wherein the second chimeric antigen receptor comprises a second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain do not promote dimerization to each other in the absence of binding of the cell to both a first antigen that binds to the first antigen-binding domain and a second antigen that binds to the second antigen-binding domain. In some embodiments, the first transmembrane domain and the second transmembrane domain promote dimerization when spatially colocalized. In some embodiments, one or both of the first transmembrane domain and the second transmembrane domain comprise a CD4, CD8, 4 IBB, or CD28 transmembrane domain or variations thereof.

[0012] In some embodiments, one or both of the first intracellular domain and the second intracellular domain comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 4 IBB domain; a human CD28 domain; LAT; SLP-76; and any combination thereof. In some embodiments, the intracellular domain comprises a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

[0013] In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell.

[0014] In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to different antigens. In some embodiments, one of the first binding domain or theAttorney Docket No. 48104-713601second binding domain binds to CD 19, and wherein one of the first binding domain or the second binding domain binds to HER2. In some embodiments, one or both of the first antigen-binding domain and the second antigen-binding domain comprises an antibody, an antibody fragment, or an alternative scaffold-based antigen-binding domain. In some embodiments, the alternative scaffold-based antigen-binding domain comprises a D domain.

[0015] In some embodiments, the cell is selectively activated in an AND-gated manner.

[0016] Provided herein are methods of treating a subject having a cancer, the method comprising: administering to the subject an effective amount of the cells described herein. In some embodiments, cancer cells of the subject comprise a first extracellular antigen that binds to the first antigen-binding domain of the cells described herein and a second extracellular antigen that binds to the second antigen-binding domain of the cells described herein, wherein the first extracellular antigen and the second extracellular antigen are different.

[0017] Provided herein are methods of treating a cancer patient having cancer cells, the method comprising: administering to the cancer patient an effective amount of the cells described herein. In some embodiments, the cancer cells comprise a first extracellular antigen that binds to the first antigen-binding domain of the cells described herein and a second extracellular antigen that binds to the second antigen-binding domain of the cells described herein, wherein the first extracellular antigen and the second extracellular antigen are different. In some embodiments, the cancer cells are killed by the administered cells, and wherein cells without one or more of the first extracellular antigen and the second extracellular antigen are not killed by the administered cells.

[0018] Provided herein are methods for depleting cells of a subject, the method comprising: administering to the subject an effective amount of the cells described herein, wherein the cells of the subject comprise a first extracellular antigen that binds to the first antigen-binding domain of the cells described herein and a second extracellular antigen that binds to the second antigenbinding domain of the cells described herein wherein the first extracellular antigen and the second extracellular antigen are different. In some embodiments, the cells of the subject comprising the first extracellular antigen and the second extracellular antigen are killed by the administered cells, and wherein cells without one or more of the first extracellular antigen and the second extracellular antigen are not killed by the administered cells.

[0019] Provided herein are vectors comprising a nucleic acid sequence encoding a first chimeric antigen receptor described herein and a nucleic acid sequence encoding a second chimeric antigen receptor described herein.Attorney Docket No. 48104-713601

[0020] Provided herein are methods for determining the suitability of a subject for treatment with a cell therapy, the method comprising: (a) obtaining a cancerous sample from the subject; (b) testing the cancerous sample for the presence of a first antigen and a second antigen on a cell in the cancerous sample; and (c) determining that the subject is suitable for treatment with the cellular therapy if both the first antigen and the second antigen are present on the cell; wherein the cellular therapy is administered to the subject if the first antigen and the second antigen are detected, wherein the cell therapy comprises administering the cells described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows AND-gated T cell activation in AND-gated CAR systems disclosed herein. When both CARs in the AND-gated CAR pair are bound to their target antigens (e.g., target antigens on the same target cell), the CAR-containing cell is activated. However, when only one CAR binds to its target antigen, the CAR-containing cell is not activated.

[0022] FIGs. 2A-2C show exemplary CARs for use in AND-gated CAR systems comprising two CARs as disclosed herein. FIG. 2A shows an exemplary first CAR which comprises an extracellular antigen-binding domain and an intracellular kinase domain. FIGs. 2B and 2C show exemplary second CARs that each comprise an extracellular antigen-binding domain and an intracellular PLCyl binding site. FIG. 2C further comprises a regulatory domain for regulating the kinase on the first CAR.

[0023] FIGs.3A-3F show an exemplary process of AND-gated T cell activation. First, a first CAR as shown in FIG. 2A and a second CAR as shown in FIG. 2C (constituting an AND-gated CAR pair) each bind to their respective target antigens, and are brought into proximity as a result (FIG.3 A). When both CARs are in proximity, the kinase of the first CAR (shown at right) is activated by the regulatory domain (FIG. 3B) of the second CAR (shown at left) and then phosphorylates the PLCyl binding site (FIG. 3C). Next, PLCyl binds to the phosphorylated PLCyl binding site (FIG. 3D) where it is also optionally phosphorylated by the kinase on the first CAR (FIG. 3E). The phosphorylated PLCyl then causes downstream activation of the T cell (FIG. 3F).

[0024] FIG. 4 is a Western blot of HEK293 cells expressing (i) proteins comprising an extracellular FLAG epitope and an intracellular domain comprising an activated kinase domain and (ii) proteins comprising an extracellular V5 epitope and an intracellular LAT domain. Results from proteins with various activated kinase domains are shown.

[0025] FIG. 5 is a Western blot of HEK293 cells expressing (i) proteins comprising an extracellular FLAG epitope and an intracellular domain comprising a kinase domain and (ii)Attorney Docket No. 48104-713601proteins comprising an extracellular V5 epitope and an intracellular LAT domain. Results from proteins with various wild-type and catalytically inactive kinase domains are shown.

[0026] FIGs. 6A-6E show exemplary T-cell cytotoxicity data from NALM6 cells cultured with AND-gated CAR-expressing T cells.

[0027] FIG. 7 is a Western blot of HEK293 cells expressing (i) proteins comprising an extracellular FLAG epitope and an intracellular domain comprising an activated kinase domain and (ii) proteins comprising an extracellular V5 epitope and an intracellular recruitment site for PLCyl. Results from proteins with various kinase domains and proteins with various recruitment sites for PLCyl are shown.

[0028] FIGs. 8A-8E show T-cell cytotoxicity data from NALM6 cells cultured with AND-gated CAR-expressing T cells.DETAILED DESCRIPTION

[0029] Logic gating systems that cause T cell activation only in the presence of multiple target antigens have the potential to increase specificity of CAR-T cells. For example, AND-gated CAR systems can be designed with two CARs that bind to different antigens, leading to activation only when both CARs are bound to their target antigens. In these systems, the close proximity of the CARs when both bound to their target antigens causes the intracellular domains of the CARs to interact in a way that enables T cell activation. The compositions and methods disclosed herein can have one or more advantages over prior CAR-T cell systems, such as improving specificity and reducing off-target effects.

[0030] In one aspect, the present disclosure provides AND-gated CAR pairs, wherein a first CAR of the AND-gated CAR pair comprises a kinase domain (e.g., as shown in FIG. 2A). In some embodiments, a second CAR of the AND-gated CAR pair comprises a substrate for the kinase domain of the first CAR. Alternatively or in addition, the second CAR of the AND-gated CAR pair may comprise a binding site for PLCyl (e.g., as shown in FIG. 2B). In some embodiments, the binding site for PLCyl comprises a substrate for a kinase domain.

[0031] In another aspect, the present disclosure provides cells engineered to express a pair of AND-gated CARs comprising (i) a first CAR comprising a kinase domain and (ii) a second CAR comprising a substrate for the kinase, as shown in FIGs. 3 A-3F. When both the first CAR and the second CAR are bound to antigens on the surface of a target cell, the kinase domain of the first CAR may phosphorylate the substrate of the second CAR. In some embodiments, phosphorylation of the substrate on the second CAR may cause recruitment of PLCyl to a PLCyl binding site.Attorney Docket No. 48104-713601PLCyl may bind to the second CAR (e.g., at the phosphorylated PLCyl binding site) and may also be phosphorylated by the kinase domain of the first CAR. Phosphorylated PLCyl may then stimulate activation of the cell comprising the first CAR and the second CAR.

[0032] In another aspect, the present disclosure provides cells engineered to express (i) a pair of AND-gated CARs comprising (a) a first CAR comprising a kinase domain and (b) a second CAR and (ii) PLCyl. When both the first CAR and the second CAR are bound to antigens on the surface of a target cell, the kinase domain of the first CAR may phosphorylate PLCyl. PLCyl may then stimulate activation of the cell comprising the pair of AND-gated CARs.Definitions

[0033] The section headings used herein are for organizational purposes only and are not to be construed as in any way limiting of the subject matter described.

[0034] While various embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments described herein may be employed.

[0035] As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0036] The term “about” as used herein generally refers to within (plus or minus) 10% of a designated value.Cells Engineered to Express CARs

[0037] In one aspect, the present disclosure provides cells engineered to express the CARs disclosed herein. In some embodiments, the cells are engineered to express multiple different CARs simultaneously. In some embodiments, the cells are engineered to express a pair of AND-gated CARs. In some embodiments, the pair of AND-gated CARs comprises a first CAR comprising a kinase domain (e.g., as shown in FIG. 2A). In some embodiments, the pair of AND-gated CARs comprises a second CAR comprising a substrate for the kinase domain (e.g., as shown in FIGs. 2B and 2C). Alternatively or in addition, the second CAR may comprise a PLCyl binding site. In some embodiments, the PLCyl binding site comprises a substrate (e.g., a tyrosine) for the kinase domain of the first CAR. In some embodiments, the cells engineered to express CAR(s) comprise PLCyl. For example, a cell may express a pair of AND-gated CARs, wherein the firstAttorney Docket No. 48104-713601CAR comprises a kinase domain and the second CAR comprises a substrate for the kinase domain. Alternatively or in addition, a cell may express a pair of AND-gated CARs and a PLCyl, wherein the first CAR comprises a kinase and the PLCyl is phosphorylated by the kinase domain when the first CAR is in contact with a second CAR of the AND-gated CAR pair.

[0038] In some embodiments, the antigen-binding domain of the first CAR and the antigen-binding domain of the second CAR bind to different antigens. In some embodiments, the antigen-binding domain of one of the first CAR or the second CAR binds to CD 19. In some embodiments, the antigen-binding domain of one of the first CAR or the second CAR binds to HER2. In some embodiments, the antigen-binding domain of one of the CARs binds to CD 19 while the antigenbinding domain of the other CAR binds to HER2.

[0039] In some embodiments, the cell is selectively activated in an AND-gated manner (e.g., as shown in FIG. 1). For example, the cell may be activated when each CAR in a pair of AND-gated CARs interacts with their respective target antigen, and the cell may not activated when one or fewer CARs in a pair of AND-gated CARs interacts with their target antigen. When both CARs in a pair of AND-gated CARs are bound to their target antigens, the first CAR and the second CAR may come into close proximity. When the first CAR and the second CAR are in close proximity, a kinase domain on the first CAR may phosphorylate PLCyl. In some embodiments, prior to PLCyl phosphorylation, a kinase domain on the first CAR may phosphorylate a substrate on the second CAR. In some embodiments, the PLCyl may bind to the second CAR prior to PLCyl phosphorylation. The phosphorylated PLCyl may cause activation of the cell.

[0040] In some embodiments, the kinase domain of the first CAR in a pair of AND-gated CARs is regulated by a kinase regulatory domain on an intracellular domain of a second CAR in a pair of AND-gated CARs (e.g., as shown in FIG. 2C). In some embodiments, the kinase regulatory domain activates the kinase domain. In some embodiments, the kinase regulatory domain represses the kinase domain.

[0041] In some embodiments, the first CAR and the second CAR of the pair of AND-gated CARs comprise the same transmembrane domain. In some embodiments, the first CAR and the second CAR of the pair of AND-gated CARs comprise different transmembrane domains. In some embodiments, the transmembrane domain of the first CAR and the transmembrane domain of the second CAR do not promote dimerization to each other in the absence of the both CARs in the AND-gated CAR pair binding to their target antigens.

[0042] In some embodiments, the cell engineered to express the AND-gated CAR pair is capable of killing a target cell e.g., when both CARs in the AND-gated CAR pair interact with their targetAttorney Docket No. 48104-713601antigens). In some embodiments, the cell engineered to express the AND-gated CAR pair is capable of degranulating (e.g., when both CARs in the AND-gated CAR pair interact with their target antigens). In some embodiments, the cell engineered to express the AND-gated CAR pair is capable of secreting a cytokine or cytokines (e.g., when both CARs in the AND-gated CAR pair interact with their target antigens). In some embodiments, killing of the target cell, degranulation, or secreting a cytokine or cytokines is a result of cell activation.

[0043] In some embodiments, the cell engineered to express the AND-gated CAR pair is an immune cell. In some embodiments, the cell engineered to express the AND-gated CAR pair is an immune effector cell. In further embodiments, the cell engineered to express the AND-gated CAR pair is a cytotoxic cell. In further embodiments, the cytotoxic cell is selected from a T cell, NK cell, or a cultured NK cell (e.g., a NK92 cell). Cells that are capable of mediating antibodydependent cellular cytotoxicity (ADCC) are examples of immune effector cells. Other immune effector cells include Natural Killer cells, tumor-infiltrating T lymphocytes (TILs), cytotoxic T lymphocytes, and granulocytic cells such as cells that comprise allergic response mechanisms. Other immune effector cells may include cells of non-hematopoietic origin that are capable of mediating immune functions, for example, endothelial cells, keratinocytes, fibroblasts, osteoclasts, epithelial cells, and other cells. Immune effector cells may also include cells that mediate cytotoxic or cytostatic events, or endocytic, phagocytic, or pinocytotic events, or that effect induction of apoptosis, or that effect microbial immunity or neutralization of microbial infection, or cells that mediate allergic, inflammatory, hypersensitivity and / or autoimmune reactions.

[0044] In some embodiments, the cell engineered to express the AND-gated CAR pair is a T cell. In some embodiments, the cell engineered to express the AND-gated CAR pair is a NK cell. In additional embodiments, the cell engineered to express the AND-gated CAR pair is a B cell. Other immune cells, and / or combinations of different immune cell types may optionally be used. In some embodiments, combinations of cell types (e.g., NK cells and T cells) are advantageous because they act synergistically to treat a disease or condition (e.g., myasthenia gravis). When combinations are used, the various cell types may target the same, different, or overlapping tumor antigenic determinants.

[0045] In some embodiments, the cell engineered to express the AND-gated CAR pair is a T cell, and the binding of the AND-gated CAR pair to their target antigen(s) stimulates the T cell to initiate intracellular signaling. In further embodiments, binding of the AND-gated CAR pair to their target antigen(s) stimulates the T cell to produce cytokines and degranulate, leading to the cytotoxic effects on the cell expressing the target antigen(s) on its surface (e.g., a cancer cell). In additionalAttorney Docket No. 48104-713601embodiments, the CAR-containing T cell proliferates in response to binding the target antigen(s). In some embodiments, the activity of the CAR-containing T cell does not result in the T cells exhibiting a phenotype associated with T cell exhaustion.

[0046] In some embodiments, the cell engineered to express the AND-gated CAR pair is engineered such that the CAR-coding sequence site is specifically introduced into a locus of a gene highly expressed in the corresponding host cell. In some embodiments the CAR-coding sequence is introduced into a T cell receptor locus. In further embodiments, the CAR-coding sequence is introduced into the T cell receptor a constant (TRAC) of the cell. Modified cells that lack expression of a functional TCR and / or HLA can routinely be obtained using any suitable means, including for example, siRNA, shRNA, CRISPR, TALEN, and / or ZFN.

[0047] Nucleic acids encoding AND-gated CAR pairs are also provided. In some embodiments, the AND-gated CAR pair binds specifically to target antigen(s) and thus functions to deliver the cell expressing the AND-gated CAR pair to the target cell. In some embodiments, the antigen(s) is associated with cancer. In some embodiments, the cell expressing the AND-gated CAR pair is a T cell, a natural killer (NK) cell, or other immune cell type. In some embodiments, the cell expressing the AND-gated CAR pair (whether T cell, NK cell or other cell type) exhibits an anti-target cell response when the polypeptide binds to its target antigen(s).AND-Gated Chimeric Antigen Receptors (AND-Gated CARs)

[0048] In one aspect, the present invention provides a pair of AND-gated CARs. In some embodiments, a first CAR of the AND-gated CAR pair comprises a kinase domain. In some embodiments, a second CAR of the AND-gated CAR pair comprises a substrate for a kinase (e.g., a kinase of the first CAR). Alternatively or in addition, the second CAR may comprise a binding site for PLCyl. In some embodiments, the binding site for PLCyl comprises a substrate for a kinase domain (e.g., a kinase of the first CAR).

[0049] In some embodiments, each CAR comprises an extracellular antigen-binding domain and a transmembrane domain. In some embodiments, each CAR comprises an extracellular antigenbinding domain, a transmembrane domain, and an intracellular domain. In some embodiments, each CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain wherein the intracellular domain comprises a kinase domain. In some embodiments, a first CAR of a pair of CARs comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain wherein the intracellular domain comprises a substrate for a kinase. In some embodiments, the extracellular antigen-binding domain of eachAttorney Docket No. 48104-713601CAR comprises an antigen-binding domain. In some embodiments, the extracellular antigenbinding domain of each CAR comprises an antigen-binding domain that specifically binds to CD 19 or HER2.

[0050] The CARs disclosed herein may be used alone, in combination with one another (e.g., for AND-gating), or in combination with other various CARs or cellular receptors.Extracellular Domain

[0051] In some embodiments, each CAR comprises an extracellular antigen-binding domain that is specific to an antigen on a target cell. In some embodiments, the target cell is a disease-associated cell. In some embodiments, the target cell is a cancer cell.

[0052] In some embodiments, a first antigen is a first epitope of a tumor antigen associated with a malignant tumor. In some embodiments, a second antigen is a second epitope of a tumor antigen associated with a malignant tumor.

[0053] In some embodiments, an antigen is CD 19. In some embodiments, an antigen is HER2.

[0054] In some embodiments, the extracellular antigen-binding domain is an antibody or antibody fragment. In some embodiments, the antibody fragment is an scFv. In some embodiments, the extracellular antigen-binding domain is an alternative scaffold-based antigen-binding domain. In some embodiments, the alternative scaffold-based antigen-binding domain is a D domain (DD).

[0055] In some embodiments, the CAR comprises a signal peptide, which may be linked to the extracellular antigen-binding domain. In some embodiments, the signal peptide is cleavable, and cleavage of the signal peptide may result in a CAR without a signal peptide.Antigen-binding domains

[0056] In some embodiments, the CAR comprises an extracellular antigen binding domain. In some embodiments, the extracellular antigen-binding domain is sufficient to confer recognition and specific binding to a target of interest.

[0057] The target of interest specifically bound by the antigen-binding domain can be any molecule for which it is desirable for a CAR to bind. In some embodiments, the target of the antigen-binding domain can be an extracellular component, a soluble factor (e.g., an enzyme, hormone, cytokine, growth factor, toxin, venom, pollutant, etc.), or a transmembrane protein (e.g., a cell surface receptor).

[0058] In some embodiment, the antigen-binding domain specifically binds a target of interest on the surface of a target cell. In some embodiments, the antigen-binding domain specifically binds a cell surface receptor.Attorney Docket No. 48104-713601

[0059] In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the antigen-binding domain binds to a tumor-associated antigen. In some embodiments, the antigen-binding domain binds to a tumor-specific antigen.

[0060] In some embodiments, the antigen-binding domain binds a cancer antigen. In some embodiments, the antigen-binding domain binds to a cancer-associated antigen. In some embodiments, the antigen-binding domain binds to a cancer-specific antigen.

[0061] In some embodiments, the antigen-binding domain binds to CD 19. In some embodiments, the antigen-binding domain binds to HER2.

[0062] In some embodiments, the antigen-binding domain binds an antigen expressed on the surface of an immune effector cell.

[0063] In some embodiments, a target of interest bound by the antigen-binding domain is a human protein. In some embodiments, the antigen-binding domain binds a human protein target of interest and its monkey (e.g., cynomolgous monkey), mouse, rabbit, hamster and / or a rabbit ortholog.

[0064] In another embodiment, the antigen-binding domain binds a peptide tag present on a target of interest. In one embodiment, the antigen-binding domain specifically binds a peptide tag selected from: a hexahistidyl (His6) tag (SEQ ID NO: 58), a myc tag, and a FLAG tag. Other peptide tags are described herein or otherwise available.

[0065] Affinity requirements for a given antigen-binding domain binding event are contingent on a variety of factors including, but not limited to: the composition and complexity of the binding matrix, the valency and density of both the antigen-binding domain and target molecules, and the functional application of the antigen-binding domain. In one embodiment, the antigen-binding domain binds a target of interest with a dissociation constant (KD) of less than or equal to IO-4M, 5* 1(T5M, or KT5M. In an additional embodiment, the antigen-binding domain binds a target of interest with a KD of less than or equal to 5* KT6M, KT6M, 5* KT7M, KT7M, 5* KT8M, or 1(T8M. In additional embodiments, the antigen-binding domain binds a target of interest with a KD less than or equal to 5 x 1 O’9M, 1 O’9M, 5 x 1 O’10M, 1 O’10M, 5 x 10’11M, 10’11M, 5 x 1 O’12M, 1(T12. In several embodiments, the antigen-binding domain has a dissociation constant selected from the group: between 10'4M and 10"5M, between 10'5M and 10'6M, between IO"6M and 10’7M, between 10'7M and IO"8M, between 10'8M and 10'9M, between 10'9M and 10'10M, between 10'10M and 10"11M and between 10'11M and 10'12M.

[0066] In one embodiment the antigen-binding domain binds a target of interest in active form. In one embodiment the antigen-binding domain reversibly binds a target of interest in active formAttorney Docket No. 48104-713601and also releases the bound target in active form. In one embodiment the antigen-binding domain binds a target of interest in the native form. In specific embodiments, the antigen-binding domain bind targets of interest with off-rates or Koff of greater than or equal to IO-10sec-1, 5* IO-9sec-1, 10-9sec-1, 5><10-8sec-1, 10-8sec-1, 5 * 10-7sec-1, 10-7sec-1, 5 * 10-6sec-1, 10-6sec-1, 5 * 10-5sec-1, 10-5sec-1, 5* 10-4sec-1, 10-4sec-1, 5* 10-3sec-1, 10-3sec-1, 5* 10-2sec-1, 10-2sec-1, 5><10-1sec-1, or 10-1sec-1.

[0067] Binding experiments to determine KD and off-rates may be performed in a number of conditions including, but not limited to: (1) pH 6.0, 0.01% Tween 2, (2) pH 6.0, 0.1% gelatin, (3) pH5.0, 0.01% Tween 2, (4) pH 9.0, 0.1% Tween 2, (5) pH 6.0, 15% ethylene glycol, 0.01% Tween 2, (6) pH 5.0, 15% ethylene glycol, 0.01% Tween 2, and (7) pH 9.0, 15% ethylene glycol, 0.01% Tween 2. The buffers in which to make these solutions may routinely be determined by one skilled in the art, and depend largely on the desired pH of the final solution. Low pH solutions (<pH 5.5) may be made, for example, in citrate buffer, glycine-HCl buffer, or in succinic acid buffer. High pH solutions may be made, for example, in Tris-HCl, phosphate buffers, or sodium bicarbonate buffers. A number of conditions may routinely be used by those skilled in the art to determine KD and off-rates for the purpose of determining, for example, optimal pH and / or salt concentrations.

[0068] In one embodiment, the antigen-binding domain specifically binds a target of interest with a Koir ranging from 0.1 to 10'7see'1, 10'2to 10'7see'1, or 0.5 X 1 O'2to 10'7see'1. In a specific embodiment, the antigen-binding domain binds a target of interest with an off rate (Koir) of less than 5 X 10'2see'1, 10'2sec'1, 5 X 10'3sec'1, or 10'3sec1. In an additional embodiment, the antigen-binding domain binds a target of interest with an off rate (Koir) of less than 5 X 10'4sec'1, 10'4sec'1, 5 X 10'5sec'1, or 10'5sec1, 5 XI O'6sec'1, 10'6sec'1, 5 X 10'7sec'1, or 10'7sec'1.

[0069] In one embodiment, the antigen-binding domain specifically binds a target of interest with a Kon ranging from 103to 107M'^ec'1, 103to 106M^sec'1, or 103to 105M^sec'1. In other specific embodiments, the antigen-binding domain binds the target of interest its target of interest with an on rate (Kon) of greater than 103M^sec'1, 5 X 103M^sec'1, 104M^ec'1, or 5 X 104M' ^ec'1. In an additional embodiment, the antigen-binding domain binds a target of interest with a Kon of greater than 105M'^ec'1, 5 X 105M^ec'1, 106M'1sec'1, or 5 X 106M'^ec'1, or 107M'1sec'1.

[0070] In some embodiments, the antigen-binding domain is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding domain is a scFv. In some embodiments, the antigen-binding domain is an alternative scaffold-based antigen-binding domain. In some embodiments, the antigen-binding domain is a D domain.Attorney Docket No. 48104-713601Antibody -Derived Antigen-Binding Domains

[0071] In some embodiments, one or more antigen-binding domains can be derived from an antibody molecule, e.g., one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-domain antibodies e.g., a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) from, e.g., human or cam elid origin. In some embodiments, the antigen-binding domain is derived from the same species in which the CAR will ultimately be used, e.g., for use in humans. It may be beneficial for CAR to comprise a human or a humanized antigen-binding domain.

[0072] In some embodiments, the antigen-binding domain comprises a fragment of an antibody that is sufficient to confer recognition and specific binding to the target antigen. Examples of an antibody fragment include, but are not limited to, an Fab, Fab', F(ab')2, or Fv fragment, an scFv antibody fragment, a linear antibody, a minibody, a BiTE, a Tandab, a diabody ((VL-VH)2 or (VH-VL)2), single domain antibody such as an sdAb (either VL or VH), a camelid VHH domain, and multi-specific antibodies formed from antibody fragments.

[0073] In some embodiments, the antigen-binding domain is a scFv, which can comprise a fusion protein comprising a VL chain and a VH chain of an antibody, wherein the VH and VL are, e.g., linked via a short flexible polypeptide linker, e.g., a linker described herein. scFvs can routinely be prepared according to available methods (see, e.g., Bird etal., Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). In some embodiments, the antigen-binding domain is an scFv that binds to CD 19. In some embodiments, the antigenbinding domain is an scFv that binds to HER2. In some embodiments, the antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the antigenbinding domain comprises an amino acid sequence of SEQ ID NO: 2.Table 1. Antibody-Derived Antigen-Binding DomainsAntigen- SequenceBindingDomainsHER2 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVPS scFv RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGSGKPGSGEG SGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQ GTLVTV (SEQ ID NO: 1)Attorney Docket No. 48104-713601CD19 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH scFv SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTS GSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC AKHYYYGGSYAMDYWGQGTSVTV (SEQ ID NO: 2)

[0074] In some embodiments, the antigen-binding domain is a single domain antigen binding (SDAB) molecule. A SDAB molecule includes molecules containing complementary determining regions that are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules naturally devoid of light chains, single domains derived from conventional 4-chain antibodies, engineered domains and singledomain scaffolds other than those derived from antibodies. SDAB molecules can be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. This term also includes naturally occurring single domain antibody molecules from species other than Camelidae and sharks.

[0075] In some embodiments, the antigen-binding domain comprises a human antibody or a fragment thereof. In some embodiments, the antigen-binding domain comprises a humanized antibody or a fragment thereof.

[0076] Humanization of antibodies can essentially be performed following the method of Winter and co-workers (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; Inti. Appl. Publ. No. WO 91 / 09967; and U. S. Pat. Nos. 4,816,567; 6,331,415;5,225,539; 5,530,101; 5,585,089; and 6,548,640; the contents of which are incorporated herein by reference herein in their entirety). Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5): 489-498; Studnicka et al., Protein Engineering 7(6): 805-814 (1994); and Roguska et al., PNAS 91: 969-973 (1994)) or chain shuffling (U. S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference herein in their entirety.Alternative Scaffold-Based Antigen-Binding Domains

[0077] In some embodiments, the antigen-binding domain is an alternative scaffold-based antigen-binding domain. An alternative scaffold-based antigen-binding domain may be anAttorney Docket No. 48104-713601antigen-binding domain that is derived from, or corresponds to, a non-antibody-based binding scaffold.

[0078] In some embodiments, the disclosure provides a CAR comprising an antigen-binding domain that is an alternative scaffold-based antigen-binding domain. In some embodiments, the disclosure provides a cell comprising a CAR that comprises an antigen-binding domain that is an alternative scaffold-based antigen-binding domain. In further embodiments, an immune effector cell that comprises a CAR comprising an alternative scaffold-based antigen-binding domain is provided.

[0079] In some embodiments, the binding of the alternative scaffold-based antigen-binding domain to the target antigen is mediated by secondary structures of the binding scaffold, such as alpha helices or beta sheets. In some embodiments, the alternative scaffold-based antigen-binding domain is a three-helix bundle-based binding domain. In some embodiments, the alternative scaffold-based antigen-binding domain is a D domain-based binding domain.

[0080] Generally, D Domains are target-binding polypeptides sharing certain sequence and certain structural features of a reference scaffold sequence:MGSWAEFI< QRLAAII< TRLQALGGSEAELAAFEI< EIAAFESELQAYI< GI< GNPEVEALRI< EAAAIRDELQAYRHN (SEQ ID NO: 3). The reference scaffold is a variant of a non-naturally occurring and targetless antiparallel three helical bundle reference polypeptide originally engineered as an exercise in protein folding (see, Walsh et al., PNAS 96: 5486-5491 (1999) incorporated by reference herein in its entirety). In some embodiments, the D domain is an antiparallel three helical bundle. In some embodiments, the D domain consists of 72-75 amino acids.

[0081] The “percent sequence identity” between a reference amino acid sequence and a query amino sequence (i.e., the amino sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference amino acid sequence) is determined by aligning the sequences using the Needleman-Wunsch alignment algorithm as implemented using the “Global Align” BLAST program available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi (with a gap existence penalty of 11 and a gap extension penalty of 1) and comparing the sequences. The number of exact matches, divided by the total number of positions in the alignment (which corresponds with the number of amino acids in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query amino acid sequence and theAttorney Docket No. 48104-713601reference amino acid sequence (i.e., percent sequence identity = (# of exact matches / (total # of positions in alignment)* 100).

[0082] The disclosed DD, particularly those administered to a subject, may be not antigenic with respect to the subject (e.g., human). In some embodiments, the sequence of the DD does not contain a human HLA-DR binding motif or cleavage sites for proteasomes and immune-proteasomes. In particular embodiments, the DD sequence does not contain an antigenic sequence as determined by a computer prediction model version existent on the filing date of this specification. In particular embodiments, the DD sequence does not contain an MHC (class I or class II) binding site sequence as predicted by an algorithm selected from ProPred (see, e.g., Singh, Bioinformatics 17(12): 1236-1237 (2001)), ProPredl (Singh, Bioinformatics 19(8): 1009-14 (2003)), SYFPEITHI (see, e.g., Schuler, Immunoinf. Meth, in Mol. Biol. 409(1): 75-93 (2007)), SMM-align (see, e.g., Nielsen, BMC Bioinformatics 8: 238 (2007)), RANKPEP (see, e.g., Reche, Hum Immunol 63: 701-709. (2004)), or TEPITOPE (see, Sturniolo, Nat Biotechnol 17: 555-561 (1999)), wherein the version of the algorithm and the applied database are in existence on the filing date of this application. In some embodiments, the DD does not contain a sequence that shares characteristics with a high affinity (binding threshold less than 6%) T cell epitope. (Singh, Bioinformatics 17: 1236-1237 (2001)). In some embodiments, the DD does not contain a sequence that shares characteristics with a promiscuous (present in greater than 50% of relevant alleles) T cell epitope (Singh, Bioinformatics 17: 1236-1237 (2001)). In some embodiments, the DD does not contain a sequence that shares characteristics with a high affinity or a promiscuous T cell epitope. In particular embodiments, the DD does not contain the sequence LAAIKTRLQ (SEQ ID NO: 4). Techniques for generating, screening, and identifying affinity matured DD variants comprising a sequence alteration that removes a predicted MHC (class I or class II) binding site sequence are known in the art.

[0083] In some embodiments, the D domain comprises a sequence selected from the group: SEQ ID NO: 5-15.

[0084] In some embodiments the alternative scaffold-based antigen-binding domain is a Z-domain scaffold (Affibody)-based antigen-binding domain. Z-domain scaffold-based binding domains generally consist of 58 amino acid residues in which substitutions of up to 13 positions located in the first and second of three alpha helices, confer binding confer target antigen recognition and binding specificity for the target antigen of interest. In some embodiments, the Z-domain-based antigen-binding domain comprises a sequence selected from SEQ ID NO: 16 andAttorney Docket No. 48104-71360117. Z-domain (Affibody) scaffold-based binding domains are further described in U. S. Pat. No.5,831,012, the entire contents of which are herein incorporated by reference in their entirety.

[0085] Additional examples of alternative scaffold-based antigen-binding domains that display secondary structure-mediated target binding include DARPins, affilins, and armadillo repeatbased binding scaffolds.

[0086] In some embodiments, the alternative scaffold-based antigen-binding domain is a DARPin-based antigen-binding domain. DARPin-based binding domains generally contain 2-3 repeats of the sequence of SEQ ID NO: 18 positioned between N- and C- terminal capping repeats (e.g., the sequence MRGSHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAX33 (SEQ ID NO: 19) and the sequence QDKFGKTAFDISIDNGNEDLAEILQ (SEQ ID NO: 20), respectively, wherein the first Gin corresponds to consensus repeat position X33 of the preceding repeat). Each internal repeat may consist of 27 framework residues and up to 6 substituted nonframework residues that that form a [l-turn followed by two antiparallel helices and a loop that connects to the [l-turn of the next repeat. The collective substitutions and structure of the DARPin may confer target antigen recognition and binding specificity.

[0087] In some embodiments, the binding specificity of the alternative scaffold-based antigenbinding domain to the target antein is mediated by amino acids in exposed loops on the alternative scaffold-based antigen-binding domain. Examples of scaffolds having these binding properties include, adnectins, lipocalins, avimers, knottins, fynomers, atrimers, kunitz domainbased binders, and CTLA4-based binding scaffolds.

[0088] In some embodiments, the alternative scaffold-based antigen-binding domain is an adnectin-based antigen-binding domain. The adnectin-based binding domain is generally derived from the tenth domain of fibronectin type III (10Fn3). This adnectin-based antigen-binding domain is generally a 94 amino acid binding domain that adopts a beta sandwich fold containing seven strands that are connected by six loops. Substitutions in three surface-exposed loops on one side of the adnectin domain may generate target antigen specific binding moieties.

[0089] In some embodiments, the alternative scaffold-based antigen-binding domain is a lipocalin-, affilin-, or anticalin-based antigen-binding domain. The anticalin scaffold generally displays a conserved [l-barrcl structure made up of eight anti-parallel [l-strands and generally consists of 160-180 amino acids. The ligand binding pocket of the anti callin-based binding scaffold may be composed of four loops, each containing up to 24 substitutions, that collectively confer target antigen recognition and binding specificity.Attorney Docket No. 48104-713601

[0090] In some embodiments, the alternative scaffold-based antigen-binding domain is an Avimer scaffold-based antigen-binding domain. Avimer scaffold-based binding domains are generally derived from the A-domain of cell surface receptors and are generally 35 amino acids in length. The structure of the Avimer-based binding domain is maintained by 12 conserved amino acids. Substitutions of up to all of the remaining 23 residues of the binding domain confer target antigen recognition and binding specificity. In some embodiments, the Avimer scaffoldbased binding domain comprises the sequence EFX3CX5NGX8CIPXi2Xi3WXi5CDGXi9DDCGDX25SDE, wherein X is any amino acid (SEQ ID NO: 21).

[0091] In some embodiments, the alternative scaffold-based antigen-binding domain is a fynomer scaffold-based antigen-binding domain. The fynomer binding domain is generally 60-75 amino acids in length and is composed of a pair of anti-parallel beta sheets joined by two flexible loops. Substitutions / insertions in the loops may confer target antigen recognition and binding specificity. In some embodiments, the fynomer-based antigen-binding domain comprises the sequence GVTLFVALYDYX12X13X14X15X16X17X18X19X20X21X22LSFHKGEKFQILSTHEYEX41X42X43X 44X45X46X47X48 WEARSLTTGETGXeilPSNYVAPVDSIQ, wherein X is any amino acid residue and X13-X21 and X42-X46, are optionally absent (SEQ ID NO: 22). In some embodiments, the fynomer-based antigen-binding domain comprises the sequence GVTLFVALYDYX12X13X14X15X16X17X18X19X20X21X22LSFHKGEKFQILSTHEYEDWWEAR SLTTGETGYIPSNYVAPVDSIQ, wherein X is any amino acid residue and X16-X21 and are optionally absent (SEQ ID NO: 23).

[0092] In some embodiments the alternative scaffold-based antigen-binding domain is a knottin scaffold-based antigen-binding domain. Knottin scaffold-based binding domains generally correspond to a 30-amino-acid protein fold composed of three anti-parallel ^-strands connected by loops of variable length and multiple disulfide bonds.

[0093] In some embodiments the alternative scaffold binding is a Kunitz domain-based antigenbinding domain. Kunitz domain-based binding domains are generally derived from the active motif of Kunitz-type protease inhibitors and are generally about 60 amino acids in length. The hydrophobic core of a Kunitz domain may be composed of a twisted two-stranded antiparallel fished and two a-helices stabilized by three pairs of disulfide bonds. Substitutions and insertions in the three loops may confer target antigen recognition and binding specificity. In some embodiments, the Kunitz domain-based antigen-binding domain comprises the sequenceAttorney Docket No. 48104-713601MHSFCAFKADXHGXBC X15Xi6Xi7Xi8Xi9RFFFNIFTRQCEEFX34YGGCX39X4oNQNRFESLEECKKMCTRDGA (SEQ ID NO: 24) sequence that is at least 85% identical to at positions other than X; Xu is one of: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; Xi3is one of: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; X15is one of: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; Xi6is one of: A, G, E, D, H, T; Xi7is one of: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; Xi8is one of: A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; Xi9is one of: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; X34is one of: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; X39 is one of: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; and X40is one of: G, and A.

[0094] In some embodiments the alternative scaffold binding is a WW domain-based antigenbinding domain. WW domain-based binding scaffolds are generally 30-35 amino acids in length. In some embodiments, the WW domain-based antigen binding scaffold comprises the sequence KLPPGWX7KX9WSX12X13XUGRVX18YX20NX22ITX25AX27QWERP (SEQ ID NO: 25), wherein X7, X9, X12, X13, Xi4, Xis, X20, X22, X25, and X27 represent any amino acid, and Xi4is optionally absent.

[0095] In some embodiments, the WW domain-based antigen binding scaffold comprises the sequence KLPPGWX7KX9WSX12X13GRVX17 YXi9NX2iITX24AX26QWERP (SEQ ID NO: 26), wherein X7, X9, X12, X13, X17, X19, X21, X24, and X26 represent any amino acid.Table 2. Exemplary Alternative Scaffold-Based Antigen-Binding Domains Antigen- SequenceBindingDomainsD-domain MGSWX5X6FKX9XioLAXi3lKXi6Xi7LEALGGSEAELAX3oFEX33X34IAX37FEX4oX4iLQ Fl X44YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 5)D-domain MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX32X33EIX36AFX39X4OELX43AYKGKG F2 NPEVEALX57X58EAX6IAIX64X65ELX68AYRHN (SEQ ID NO: 6)D-domain MGSWX5EFX8X9RLX12AIX15X16RLX19ALGGSEAELAAFEKEIAAFESELQAYKGKGNP F3 EVEX55LRX58X59AAX62IRX65X66LQAYRHN (SEQ ID NO: 7)D-domain MGSWX5X6FKX9XioLAXi3lKXi6Xi7LEALGGSEAELAAFX32X33EIX36AFX39X4OELX43A Cl YKGKGNPEVEX55LRX58X59AAX62IRX65X66LQAYRHN (SEQ ID NO: 8)D-domain MGSWX5EFX8X9RLXi2AIXi5Xi6RLXi9ALGGSEAELAX3oFEX33X34IAX37FEX4oX4iLQ C2 X44YKGKGNPEVEALX57X58EAX6IAIX64X65ELX68AYRHN (SEQ ID NO: 9)Attorney Docket No. 48104-713601D-domain MGSWX5X6FKX9X10LAX13IKX16X17LEALZ1EAELAX28FEX31X32IAX35FEX38X39LQ X42Y FILpX Z2NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 10)D-domain MGSWAEFKQRLAAIKTRLEALZ1EAELAAFX30X31EIX34AFX37X38ELX41AYZ2NPEV F2LpX EALX52X53EAX56AIX59X60ELX63AYRHN (SEQ ID NO: 11)D-domain MGSWX5EFX8X9RLX12AIX15X16RLX19ALZ1EAELAAFEKEIAAFESELQAY Z2NPEVE F3LpX X50LRX53X54AAX57IRX60X61LQAYRHN (SEQ ID NO: 12)D-domain MGSWX5X6FKX9X10LAX13IKX16X17LEALZ1EAELAAFX30X31EIX34AFX37X38ELX41 AYZ2 CILpX NPEVEX50LRX53X54AAX57IRX60X61LQAYRHN (SEQ ID NO: 13)D-domain MGSWX5EFX8X9RLX12AIX15X16RLX19ALZ1EAELAX28FEX31X32IAX35FEX38X39L QX42Y C2LpX Z2NPEVEALX52X53EAX56AIX59X60ELX63AYRHN (SEQ ID NO: 14)D-domain MGSWX5EFX8X9RLX12X13IX15X16RLX19ALGGSEAELAAFEKEIX36AFX39X40ELX43AYKG DD-WTF KGNPEVEALRX58EAX61X62IRX65ELX68X69YRX72X73 (SEQ ID NO: 15)Z-Domain VDNKFNKEX9X10X11AX13X14EIX17X18LPNLNX24X25QX27X28AFIX32SLX35DDPSQSANLLA AFFa EAKKLNDAQAPK (SEQ ID NO: 16)Z-DomainNKEX4X5X6AX8X9EIX12X13LPNLNX19X20QX22X23AFIX27SLX30DDP (SEQ ID NO: 17) AFFbDARPin DX2X3GX5TPLHLAAX13X14GHLEIVEVLLKZ26GADVNAX33 (SEQ ID NO: 18) wherein X is any amino acid but C, R or P and Z is H, N, or Y.Avimerl EFX3CX5NGX8CIPX12X13WX15CDGX19DDCGDX25SDE (SEQ ID NO: 21)GVTLFVALYDYX12X13X14X15X16X17X18X19X20X21X22LSFHKGEKFQILSTHEYEX41X42X43 Fynomerl X44X45X46X47X48WEARSLTTGETGX61IPSNYVAPVDSIQ wherein X= any amino acid residue and X13-X21 and X42-X46, are optionally absent (SEQ ID NO: 22)GVTLFVALYDYX12X13X14X15X16X17X18X19X20X21X22LSFHKGEKFQILSTHEYEDWWE Fynomer2 ARSLTTGETGYIPSNYVAPVDSIQ, wherein X16-X21 are optionally absent (SEQ ID NO:23)MHSFCAFKADX11GX13CX15X16X17X18X19RFFFNIFTRQCEEFX34 YGGCX39X40NQNR KunitzlFES LEECKK MCTRDGA (SEQ ID NO: 24)WW1 KLPPGWX7KX9WSX12X13X14GRVX18 YX20NX22ITX25AX27QWERP (SEQ ID NO: 25) WW2 KLPPGWX7KX9WSX12X13GRVX17 YX19NX21ITX24AX26QWERP (SEQ ID NO: 26) X = all amino acid residuesZi or Z2 = amino acid sequence corresponding to loopl (Z1) or Ioop2 (Z2) as described herein, comprising between about 2 to about 30 natural or non-natural amino acidsSignal Peptides

[0096] In some embodiments, the CAR comprises a signal peptide, which may be linked to the extracellular antigen-binding domain. In some embodiments, the signal peptide is cleavable, andAttorney Docket No. 48104-713601cleavage of the signal peptide may result in a CAR without a signal peptide. Suitable signal peptides may include but are not limited to chymotrypsinogen B.Transmembrane Domain

[0097] In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain is the region of a cell surface-expressed CAR that crosses the plasma membrane. In some embodiments, the transmembrane domain of the CAR is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments provided herein.

[0098] In some embodiments, the transmembrane domain of the CAR is fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain of the CAR is fused to the extracellular domain of the CAR via a linker. In some embodiments, the transmembrane domain of the CAR is fused to the extracellular domain of the CAR without a linker. In some embodiments, the transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some embodiments, the transmembrane domain in the CAR is the CD8 transmembrane domain. In some instances, the transmembrane domain of the CAR comprises the CD8 hinge domain. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to promote or inhibit association with other surface membrane proteins.

[0099] In some embodiments, the transmembrane domain is derived either from a natural or from a synthetic source. In some embodiments, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use for the purposes herein may be derived from (z.e., comprise at least the transmembrane region(s) of) a member selected from the group: the alpha, beta or zeta chain of the T cell receptor; CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD99 and LAT. Alternatively the transmembrane domain may be synthetic, in which case the CAR transmembrane domain may comprise predominantly hydrophobic residues such as leucine and valine. In further embodiments, the transmembrane domain comprises the triplet of phenylalanine, tryptophan and valine at each end of a synthetic transmembrane domain. In some embodiments, the CAR transmembrane domain comprises a 41BB or CD28 transmembrane domain.

[0100] In some embodiments, the CAR comprises an extracellular spacer domain (ESD). In some embodiments, the extracellular spacer domain is a hydrophilic region which is between the antigenspecific targeting region and the transmembrane domain. In some embodiments, the CAR does notAttorney Docket No. 48104-713601comprise an extracellular spacer domain. In some embodiments, the extracellular spacer domains include but are not limited to Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences or combinations thereof. Additional examples of extracellular spacer domains include but are not limited to CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly3 or CHI and CH3 domains of IgGs (such as human IgG4). In some embodiments, the extracellular spacer domain is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgGl, (vi) a hinge region of IgGl or (vi) a hinge and CH2 region of IgGl. Other extracellular spacer domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments, provided herein.

[0101] In some embodiments, a short polypeptide linker, from about 1 to 100 amino acids in length, is used to link together any of the domains of a CAR. In some embodiments, the linker is composed of flexible residues like glycine and serine (or any other amino acid) so that the adjacent protein domains are free to move relative to one another. The amino acid sequence composition of the linker may be selected to minimize potential immunogenicity of the CAR. In some embodiments, longer linkers are used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. In some embodiments, preferably between 2 and 10 amino acids in length forms the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In further embodiments, the linker is between 10 and 15 amino acids in length, or between 15 and 20, or between 20 and 30, or between 30 and 60, or between 60 and 100 amino acids in length (or any range in between those listed). In further embodiments, the linker is a glycine-serine doublet sequence. Further embodiments employ a fragment of the hinge region derived from the human T cell surface glycoprotein CD8 alpha-chain (for example ranging from amino acid positions 138-182 CD8 alpha chain; Swiss-Prot accession number P01732). Further embodiments employ a fragment of the CD8 hinge region that has been further modified, through amino acid substitution, to improve expression function or immunogenicity. Further embodiments employ a fragment of the extracellular region derived from the human CD28 Further embodiments employ a fragment of the CD28 extracellular region that has been further modified, through amino acid substitution, to improve expression function or immunogenicity.

[0102] In some embodiments with a pair of CARs, the first CAR in the pair comprises a transmembrane domain that does not dimerize with the transmembrane domain of the second CARAttorney Docket No. 48104-713601in the pair. In some embodiments, the transmembrane domain of the first CAR and the transmembrane domain of the second CAR promote dimerization when spatially colocalized.Intracellular Domain

[0103] In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the intracellular domain comprises a kinase domain. In other embodiments, the intracellular domain comprises a substrate for a kinase domain. In some embodiments, the intracellular domain comprises a binding site for PLCyl. In some embodiments, the binding site for PLCyl comprises a substrate for a kinase domain.Intracellular Domain with Kinase Domain

[0104] In some embodiments, the intracellular domain of the CAR comprises a kinase domain. In some embodiments, the kinase domain comprises a kinase. In some embodiments, the kinase domain comprises a biologically active domain of a kinase. In some embodiments, the kinase domain comprises a wild-type kinase. Suitable kinases include but are not limited to KIT, DDRI, FGFR1, TIE2, FLT1, ZAP-70, LCK, FYN, SYK. ErbB2, DDR2, PDGFRa, FLT1, ITK, or RLK In some embodiments, the kinase domain comprises an amino acid sequence of any one of SEQ ID NOs: 27 or 33-45 In some embodiments, the kinase domain comprises an amino acid sequence of any one of SEQ ID NOs: 33 or 46-49. In some embodiments, the kinase domain comprises a biologically active domain of a kinase selected from a group comprising KIT, FGFR1, TIE2, or SYK. In some embodiments, the kinase domain comprises a biologically active domain of KIT. In some embodiments, the domain of KIT is KITshort(i.e., a domain of KIT that is modified to delete the PLCyl binding site). In some embodiments, the KITshortcomprises SEQ ID NO: 27. In some embodiments, the kinase domain comprises a biologically active domain of FGFR1. In some embodiments, the kinase domain comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the kinase domain comprises an amino acid sequence of SEQ ID NO: 48. In some embodiments, the kinase domain comprises a biologically active domain of TIE2. In some embodiments, the kinase domain comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the kinase domain comprises an amino acid sequence of SEQ ID NO: 49. In some embodiments, the kinase domain comprises a biologically active domain of SYK.Attorney Docket No. 48104-713601Table 3. Exemplary Activated Kinase DomainsKinase SequenceDomainKTTshortPYDHKWEFPRNRLSFGKTLGAGAFGKWEATAYGLIKSDAAMTVAVKMLKPSAHLTE REALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSK TSPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDF GLARVIKNDSNYWKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPY PGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQIS ES (SEQ ID NO: 27) EEIRPKEVYLDRKLLTLEDKELGSGNFGTVKKGYYQMKKVVKTVAVKILKNEANDPAL KDELLAEANVMQQLDNPYIVRMIGICEAESWMLVMEMAELGPLNKYLQQNRHVKDKN IIELVHQVSMGMKYLEESNFVHRDLAARNVLLVTQHYAKISDFGLSKALRADENYYKA QTHGKWPVKWYAPECINYYKFSSKSDVWSFGVLMWEAFSYGQKPYRGMKGSEVTAM LEKGERMGCPAGCPREMYDLMNLCWTYDVENRPGFAAVELRLRNYYYDWN (SEQ SYK ID NO: 33)PNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANK EILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLN WCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADG GKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGER LPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFWIQNED (SEQ ID ErbB2 NO: 34)GKDVAVEEFPRKLLTFKEKLGEGQFGEVHLCEVEGMEKFKDKDFALDVSANQPVLVAV KMLRADANKNARNDFLKEIKIMSRLKDPNIIHLLAVCITDDPLCMITEYMENGDLNQFLS RHEPPNSSSSDVRTVSYTNLKFMATQIASGMKYLSSLNFVHRDLATRNCLVGKNYTIKI ADFGMSRNLYSGDFYRIQGRAVLPIRWMSWESILLGKFTTASDVWAFGVTLWETFTFC QEQPYSQLSDEQVIENTGEFFRDQGRQTYLPQPAICPDSVYKLMLSCWRRDTKNRPSFQ DDR 2 EIHLLLLQQGDE (SEQ ID NO: 35)DSRWEFPRDGLVLGRVLGSGAFGKWEGTAYGLSRSQPVMKVAVKMLKPTARSSEKQ ALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIIIEYCFYGDLVNYLHKNRDSFLSHHPEK PKKELDIFGLNPADESTRSYVILSFENNGDYMDMKQADTTQYVPMLERKEVSKYSDIQR SLYDRPASYKKKSMLDSEVKNLLSDDNSEGLTLLDLLSFTYQVARGMEFLASKNCVHR DLAARDVLLAQGKIVKICDFGLARDIMHDSNYVSKGSTFLPVKWMAPESIFDNLYTTLS DVWSYGILLWEIFSLGGTPYPGMMVDSTFYNKIKSGYRMAKPDHATSEVYEIMVKCWN PDGFRa SEPEKRPSFYHLSEIVENLLPGQY (SEQ ID NO: 36)PYDHKWEFPRNRLSFGKTLGAGAFGKWEATAYGLIKSDAAMTVAVKMLKPSAHLTE REALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSK KIT QEDHAEAALDKNLLHSKESSCSDSTNEDMDMKPGVSYWPTKADKRRSVRIGSYIERD(LONG) VTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFAttorney Docket No. 48104-713601GLARVIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPY PGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQIS ES (SEQ ID NO: 37) GDGPPRVDFPRSRLRFKEKLGEGQFGEVHLCEVDSPQDLVSLDFPLNVRKGHPLLVAVK ILRPDATKNARNDFLKEVKIMSRLKDPNIIRLLGVCVQDDPLCMITDYMENGDLNQFLS AHQLEDKAAEGAPGDGQAAQGPTISFPMLLHVAAQIASGMRYLATLNFVHRDLATRNC LVGENFTIKIADFGMSRNLYAGDYYRVQGRAVLPIRWMAWECILMGKFTTASDVWAF GVTLWEVLMLCRAQPFGQLTDEQVIENAGEFFRDQGRQVYLSRPPACPQGLYELMLRC DDR1 WSRESEQRPPFSQLHRFLAEDALNTV (SEQ ID NO: 38)SEYELPEDPRWELPRDRLVLGKPLGEGCFGQWLAEAIGLDKDKPNRVTKVAVKMLKS DATEKDLSDLISEMEMMKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLQARR PPGLEDCDNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDN VMKIADFGLARDIHHIDDDKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEI FTLGGSPYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQL FGFR1 VEDLDRIVALTSNQE (SEQ ID NO: 39)PTIYPVLDWNDIKFQDVIGEGNFGQVLKARIKKDGLWMDAAIKRMKEYASKDDHRDFA GELEVLCKLGHHPNIINLLGACEHRGYLYLAIEYAPHGNLLDFLRKSRVLETDPAFAIAN STASTLSSQQLLHFAADVARGMDYLSQKQFIHRDLAARNILVGENYVAKIADFGLSRGQ EVDVKKTMGRLPVRWMAIESLNYSVYTTNSDVWSYGVLLWEIVSLGGTPYCGMTCAE LYEKLPQGYRLEKPLNCDDEVYDLMRQCWREKPYERPSFAQILVSLNRMLEERKTYVN TIE2 (SEQ ID NO: 40)DASKWEFARERLKLGKSLGRGAFGKWQASAFGIKKSPTCRTVAVKMLKEGATASEYK ALMTELKILTHIGHHLNWNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLN KDAALHMEPKKEKMEPGLEQGKKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFY KEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNILLSENNWKICDFGLARDIYKNP DDVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFS SRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQ (SEQ ID FLT1 NO: 41)IDPSELTFVQEIGSGQFGLVHLGYWLNKDKVAIKTIREGAMSEEDFIEEAEVMMKLSHPK LVQLYGVCLEQAPICLVFEFMEHGCLSDYLRTQRGLFAAETLLGMCLDVCEGMAYLEE ACVIHRDLAARNCLVGENQVIKVSDFGMTRFVLDDQDTSSTGTKFPVKWASPEVFSFSR YSSKSDVWSFGVLMWEVFSEGKIPYENRSNSEWEDISTGFRLYKPRLASTHVYQIMNH ITK CWKERPEDRPAFSRLLRQLAEIAESGL (SEQ ID NO: 42)EKWEIDPSELAFIKEIGSGQFGVVHLGEWRSHIQVAIKAINEGSMSEEDFIEEAKVMMKL SHSKLVQLYGVCIQRKPLYIVTEFMENGCLLNYLRENKGKLRKEMLLSVCQDICEGMEY LERNGYIHRDLAARNCLVSSTCIVKISDFGMTRYVLDDEDVSSFGAKFPIKWSPPEVFLF NKYSSKSDVWSFGVLMWEVFTEGKMPFENKSNLQWEAISEGFRLYRPHLAPMSIYEV RLK MYSCWHEKPEGRPTFAELLRAVTEIAETW (SEQ ID NO: 43)Attorney Docket No. 48104-713601DKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMM REAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELL HQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYDTARSAG KWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGK RMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEG (SEQ ID ZAP70 NO: 44)EDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEAN LMKQLQHQRLVRLYAWTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAE GMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEDTAREGAKFPIKWTAPE AINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQ LCK LMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQFQPQP (SEQ ID NO: 45)Table 4. Exemplary Wild-Type Kinase DomainsKinase SequenceDomainSYK EEIRPKEVYLDRKLLTLEDKELGSGNFGTVKKGYYQMKKVVKTVAVKILKNEANDPAL KDELLAEANVMQQLDNPYIVRMIGICEAESWMLVMEMAELGPLNKYLQQNRHVKDKN IIELVHQVSMGMKYLEESNFVHRDLAARNVLLVTQHYAKISDFGLSKALRADENYYKA QTHGKWPVKWYAPECINYYKFSSKSDVWSFGVLMWEAFSYGQKPYRGMKGSEVTAM LEKGERMGCPAGCPREMYDLMNLCWTYDVENRPGFAAVELRLRNYYYDWN (SEQ ID NO: 33)KIT PYDHKWEFPRNRLSFGKTLGAGAFGKWEATAYGLIKSDAAMTVAVKMLKPSAHLTE(Long) REALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSK QEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYWPTKADKRRSVRIGSYIERD VTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDF GLARDIKNDSNYWKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPY PGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQIS ES (SEQ ID NO: 46)KIT PYDHKWEFPRNRLSFGKTLGAGAFGKWEATAYGLIKSDAAMTVAVKMLKPSAHLTE(Short) REALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSK TSPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDF GLARDIKNDSNYWKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPY PGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQIS ES (SEQ ID NO: 47)FGFR1 SEYELPEDPRWELPRDRLVLGKPLGEGCFGQWLAEAIGLDKDKPNRVTKVAVKMLKS DATEKDLSDLISEMEMMKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLQARR PPGLEYCYNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDN VMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIAttorney Docket No. 48104-713601FTLGGSPYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQL VEDLDRIVALTSNQE (SEQ ID NO: 48)TIE2 PTIYPVLDWNDIKFQDVIGEGNFGQVLKARIKKDGLRMDAAIKRMKEYASKDDHRDFA GELEVLCKLGHHPNIINLLGACEHRGYLYLAIEYAPHGNLLDFLRKSRVLETDPAFAIAN STASTLSSQQLLHFAADVARGMDYLSQKQFIHRDLAARNILVGENYVAKIADFGLSRGQ EVYVKKTMGRLPVRWMAIESLNYSVYTTNSDVWSYGVLLWEIVSLGGTPYCGMTCAE LYEKLPQGYRLEKPLNCDDEVYDLMRQCWREKPYERPSFAQILVSLNRMLEERKTYVN(SEQ ID NO: 49)

[0105] In some embodiments, the kinase domain is capable of phosphorylating a tyrosine on an intracellular domain of an adjacent CAR. In some embodiments, the kinase domain is capable of being regulated by a regulatory domain on an adjacent CAR. In some embodiments, the kinase domain is capable of phosphorylating a tyrosine on PLCyl.

[0106] In some embodiments, the kinase domain is capable of dimerizing with a second kinase domain on an adjacent CAR. In some embodiments, the first kinase domain and the second kinase domain are different. In some embodiments, the first kinase domain and the second kinase domain are inactive when not dimerized. In some embodiments, the first kinase domain and the second kinase domain are active when dimerized.Intracellular Domain with Substrate for Kinase Domain and / or PLCyl Binding Site

[0107] In some embodiments, the intracellular domain of the CAR comprises a substrate for a kinase domain. In some embodiments, the intracellular domain comprises a binding site for PLCyl. In some embodiment, the intracellular domain comprises a domain that recruits PLCyl upon phosphorylation. In some embodiments, the binding site for PLCyl comprises a substrate for a kinase domain.

[0108] In some embodiments, the substrate for the kinase domain comprises a tyrosine. In some embodiments, the substrate is capable of being phosphorylated by a kinase domain on an adjacent CAR. In some embodiments, the substrate is capable of binding to PLCyl when the substrate is phosphorylated. Suitable intracellular domains for phosphorylation by a kinase include intracellular domains (e.g., PLCyl binding site) of LAT, VEGFR, PDGFRa, FGFR1, or KIT. In some embodiments, the intracellular domain comprises an amino acid sequence of any one of SEQ ID NOs: 28 or 50-53. In some embodiments, the intracellular domain comprises an intracellular domain of LAT. In some embodiments, the intracellular domain of LAT is a minimal LAT. In some embodiments, the minimal LAT comprises SEQ ID NO: 28. In some embodiments, the intracellular domain comprises an intracellular domain of VEGFR. In some embodiments, theAttorney Docket No. 48104-713601intracellular domain comprises an intracellular domain of PDGFRa. In some embodiments, the intracellular domain comprises an amino acid sequence of SEQ ID NO: 53. In some embodiments, the intracellular domain comprises an intracellular domain of FGFR1. In some embodiments, the intracellular domain comprises an intracellular domain of KIT. In some embodiments, the intracellular domain does not comprise an intracellular domain of LAT. In some embodiments, the intracellular domain does not comprise an intracellular domain of SLP-76. In some embodiments, the intracellular domain comprises an intracellular domain of any one of VEGFR, PDGFRa, FGFR1, or KIT positioned adjacent to an intracellular domain of minimal LAT. In some embodiments, the intracellular domain comprises an intercellular domain of minimal LAT with the PLCyl binding site of LAT is substituted for the PLCyl binding site of VEGFR, PDGFRa, FGFR1, or KIT.Table 5. Exemplary Intracellular Domains with Substrate Intracellular Sequence (PLCyl binding site in bold)Domain withSubstrateMinimal LAT HCHRLPGSYDSTSSDSLYPRGIQFKRPHTVARSPQPLGGSHRTPSSRRDSDGANSV ASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPG YLWLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDD (SEQ ID NO: 28) Minimal LATwith PLCyl HCHRLPGSYDSTSSDSLYPRGIQFKRPHTVARSPQPLGGSHRTPSSRRDSDGANSV binding site ASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDMKPGVS from KIT YWPTKASTPATSTAAPSAPALSTPGIRDSAFSMESIDD (SEQ ID NO: 50) Minimal LATwith PLCyl HCHRLPGSYDSTSSDSLYPRGIQFKRPHTVARSPQPLGGSHRTPSSRRDSDGANSV binding site ASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDSSDDVR from VEGFR YVNAFKFSTPATSTAAPSAPALSTPGIRDSAFSMESIDD (SEQ ID NO: 51) Minimal LATwith PLCyl HCHRLPGSYDSTSSDSLYPRGIQFKRPHTVARSPQPLGGSHRTPSSRRDSDGANSV binding site ASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDLTSNQE from FGFR1 YLDLSMPSTPATSTAAPSAPALSTPGIRDSAFSMESIDD (SEQ ID NO: 52) Minimal LATwith PLCyl HCHRLPGSYDSTSSDSLYPRGIQFKRPHTVARSPQPLGGSHRTPSSRRDSDGANSV binding site ASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDLSADSG from PDGFRa YIIPLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDD (SEQ ID NO: 53)Attorney Docket No. 48104-713601

[0109] In some embodiments, the intracellular domain comprises a kinase regulatory domain. In some embodiments, the kinase regulatory domain is capable of activating a kinase domain on an adjacent CAR. In some embodiments, the kinase regulatory domain is capable of repressing a kinase domain on an adjacent CAR.

[0110] In some embodiments, the intracellular domain comprises both (i) a substrate for a kinase domain and (ii) a kinase domain. In some embodiments, the kinase domain is capable of dimerizing with a second kinase domain on an adjacent CAR. In some embodiments, the first kinase domain and the second kinase domain are different. In some embodiments, the first kinase domain and the second kinase domain are inactive when not dimerized. In some embodiments, the first kinase domain and the second kinase domain are active when dimerized.Other Components of Intracellular Domain

[0111] In some embodiments, the intracellular domain comprises an intracellular signaling domain. In some embodiments, the intracellular domain transduces the effector function signal and directs the cell to perform its specialized function. In some embodiments, the intracellular domain (z.e., intracellular signaling domain) of a CAR is responsible for activation of at least one of the normal effector functions of an immune cell engineered to express a CAR. In some embodiments, the effector function is a specialized function of a cell. In some embodiments, the effector function of a T cell includes cytolytic activity and helper activity including the secretion of cytokines. In some embodiments, the intracellular signaling domain is the portion of a CAR protein which transduces the effector function signal and directs the cell to perform a specialized function. In some embodiments, an entire intracellular signaling domain corresponding to a naturally occurring receptor is employed. In some embodiments, the entire intracellular signaling domain corresponding to a naturally occurring receptor is not employed. In some embodiments, a truncated portion of the intracellular signaling domain is used. In some embodiments, the truncated portion is used in place of the intact chain as long as it transduces the effector function signal. In some embodiments, the intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. In some embodiments, an intracellular signaling domain in the CAR includes the cytoplasmic sequences of the T cell receptor (TCR) and also the sequence of co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, or any derivative or variant of these sequences that has functional capability. In some embodiments, the intracellular signaling domain comprises a chain of the T cell receptor complex or any of its homologs (e.g., | chain, FcsRly and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc I). In some embodiments, the intracellular signaling domainAttorney Docket No. 48104-713601comprises human CD3 zeta chain. In some embodiments, the intracellular signaling domain comprises CD3 polypeptides (A, B and c). In some embodiments, the intracellular signaling domain comprises syk family tyrosine kinases (Syk, ZAP 70, etc.). In some embodiments, the intracellular signaling domain comprises src family tyrosine kinases (Lek, Fyn, Lyn, efc.). In some embodiments, the intracellular signaling domain comprises other molecules involved in T cell transduction, such as CD2, CD5 and CD28.

[0112] It is known that signals generated through the TCR alone are often insufficient for full activation of the T cell and that a secondary or co-stimulatory signal may also be required. In some embodiments, T cell activation is mediated by cytoplasmic signaling sequences. In some embodiments, T cell activation is mediated by cytoplasmic signaling sequences that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences). In some embodiments, T cell activation is mediated by cytoplasmic signaling sequences that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0113] In some embodiments, primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in a stimulatory way. In some embodiments, primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in an inhibitory way. In some embodiments, primary cytoplasmic signaling sequences that act in a stimulatory manner may comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs).

[0114] In some embodiments, the ITAM comprising primary cytoplasmic signaling sequences comprise one or more ITAMs derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, or CD66d. In some embodiments, the ITAM comprising primary cytoplasmic signaling sequences comprise a cytoplasmic signaling sequence derived from CD3 zeta.

[0115] In some embodiments, the CAR comprises a co-stimulatory domain (CSD). In some embodiments, the CAR is the portion of a CAR which enhances the proliferation, survival and / or development of memory cells. The CAR may comprise one or more co-stimulatory domains. Each co-stimulatory domain may comprise the costimulatory domain of any one or more of, for example, a member of the TNFR superfamily, selected from CD28, CD137 (4-1BB), CD134 (0X40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1(CD1 la / CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, and CD40 or a combination thereof. Other co-stimulatory domains (e.g., from other proteins) will be apparentAttorney Docket No. 48104-713601to those of skill in the art and may be used in connection with alternate embodiments encompassed by the disclosure.

[0116] In some embodiments, the CAR comprises an intracellular signaling domain selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 4 IBB domain; a human CD28 domain; LAT; SLP-76; and any combination thereof. In some embodiments, the CAR intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

[0117] In some embodiments, the intracellular signaling domain is selected from the group consisting of a human CD3 zeta domain, 41BB domain, a CD28 domain, LAT, SLP-76 and / or any combination thereof. Depending on the embodiment, the costimulatory signaling region may comprise, for example, the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA 1), CD2, CD7, LIGHT, NKG2C, B7H3, a ligand that specifically binds with CD83, and any combination thereof.

[0118] In some embodiments, the intracellular domain of a CAR comprises the CD3-zeta signaling domain or variant thereof by itself. In some embodiments, the intracellular domain of a CAR comprises the CD3-zeta signaling domain or variant thereof in combination with any other desired intracellular domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR may comprise a CD3 zeta chain portion and a costimulatory signaling region. In some embodiments, the costimulatory signaling region is a portion of the CAR comprising the intracellular domain of a costimulatory molecule. In some embodiments, a costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. In some embodiments, the costimulatory molecule may be any one of CD27, CD28, 41BB (CD 137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3, TIM1, or LAG3 or any combination thereof.Linkers

[0119] In some embodiments, the CAR comprises a linker. Generally, the linkers are chemical linkages located between two or more otherwise independent functional domains of the CAR.Attorney Docket No. 48104-713601

[0120] Suitable linkers include but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine- and serine-rich linkers or linkers composed of largely polar polypeptide fragments.

[0121] In one embodiment, the linker is made up of a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In one embodiment, one or more linkers in the CAR is made up of a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In one embodiment, one or more linkers in the CAR is made up of one or more amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In another embodiment, one or more linkers in the CAR is made up of a majority of amino acids that are sterically unhindered. In another embodiment, a linker in which the majority of amino acids are glycine, serine, and / or alanine. In some embodiments, one or more linkers in a CAR linker comprises polyglycines (such as (Gly)s (SEQ ID NO: 29), and (Gly)s (SEQ ID NO: 30), poly(Gly-Ala), and polyalanines. In some embodiments, the peptide linker contains the sequence of Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 31). In some embodiments, one or more linkers in the CAR comprises the sequence of Gly-Gly-Gly-Gly-Asp-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 32).

[0122] In one embodiment, the CAR comprises an antigen-binding domain directly attached (i.e., without a linker) to another component of the CAR, respectively. In one embodiment, the CAR contains at least 2, at least 3, at least 4, or at least 5 antigen-binding domains directly attached to another domain of the CAR, respectively.

[0123] In another embodiment, an antigen-binding domain can be operably linked to another component of the CAR through a linker. CARs can contain a single linker, multiple linkers, or no linkers. In one embodiment, the CAR comprises an antigen-binding domain operably linked to another component of the CAR, respectively, through a linker peptide. In one embodiment, the CAR contains at least 2, at least 3, at least 4, or at least 5 antigen-binding domains operably linked to another domain of the CAR, respectively, through the same or different linkers.

[0124] Linkers can be of any size or composition so long as they are able to operably link a functional domain of the CAR in a manner that enables the functional domain to function (e.g., the ability of an antigenic determinant binding domain to bind a target of interest). In some embodiments, linker(s) are about 1 to about 100 amino acids, about 1 to 50 amino acids, about 1 to 20 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2Attorney Docket No. 48104-713601to 5 amino acids. It should be clear that the length, the degree of flexibility and / or other properties of the linker(s) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for a target of interest, or for one or more other target proteins of interest. When two or more linkers are used in the CAR, these linkers may be the same or different. In the context and disclosure provided herein, a person skilled in the art will be able to routinely determine the optimal linker composition and length for the purpose of operably linking the functional domains of a CAR.

[0125] The linker can also be a non-peptide linker such as an alkyl linker, or a PEG linker. For example, alkyl linkers such as -NH-(CH2)s-C(O)-, wherein s=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl e.g., C1-C6) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc. An exemplary non- peptide linker is a PEG linker. In certain embodiments, the PEG linker has a molecular weight of about 100 to 5000 kDa, or about 100 to 500 kDa.

[0126] Suitable linkers for coupling CAR functional domains by chemical cross-linking include, but are not limited to, homo-bifunctional chemical cross-linking compounds such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and dimethyl pimelimidate (DMP) or N-hydroxysuccinimide (NHS) esters such as dithiobis(succinimidylpropionate)(DSP) and dithiobis (sulfosuccini- midylpropionate)(DTSSP). Examples of suitable linkers for coupling CAR functional domains include but are not limited to cross-linkers with one amine-reactive end and a sulfhydryl-reactive moiety at the other end, or with an NHS ester at one end and an SH-reactive group (e.g., a maleimide or pyridyl).

[0127] In additional embodiments, one or more of the linkers in the CAR is cleavable.Examples of cleavable linkers include, include but are not limited to a peptide sequence recognized by proteases (in vitro or in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other corporeal compartments.

[0128] In some embodiments, the linker is a cleavable linker that facilitates the release of a functional domain or cytotoxic agent in a cell or at the cell surface. For example, an acid-labile linker (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) linker, photolabile linker, dimethyl linker or disulfide-containing linker (see, e.g., Chari, Can. Res. 52: 127131 (1992); U. S. Pat. No. 5,208,020; and U. S. Appl. Pub. No. 20090110753; the contents of each of which is herein incorporated by reference in its entirety) can be used.Attorney Docket No. 48104-713601

[0129] In additional embodiments, one or more of the linkers in the CAR is cleavable.Examples of cleavable linkers include, include but are not limited to a peptide sequence recognized by proteases (in vitro or in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other corporeal compartments.

[0130] In some embodiments, a short oligo- or polypeptide linker, from about 1 to 100 amino acids in length, is used to link together any of the domains of a CAR. Linkers can be composed of flexible residues like glycine and serine (or any other amino acid) so that the adjacent protein domains are free to move relative to one another. The amino acids sequence composition of the linker may be selected to minimize potential immunogenicity of the CAR. Longer linkers can be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another.

[0131] In some embodiments, preferably between 2 and 10 amino acids in length forms the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In further embodiments, the linker is between 10 and 15 amino acids in length, or between 15 and 20, or between 20 and 30, or between 30 and 60, or between 60 and 100 amino acids in length (or any range in between those listed). In further embodiments, the linker is a glycineserine doublet sequence. In some embodiments, the ESD corresponds to the human T cell surface glycoprotein CD8 alpha-chain ESD region (e.g., amino acid residues 138 to 182 CD8 alpha chain; Swiss-Prot Acc. No. P01732). In some embodiments, the ESD corresponds to the CD8 ESD region that has been further modified, through amino acid substitution, to improve expression function or immunogenicity. In further embodiments, the ESD corresponds to the CD28 ESD or sequences containing modifications of the CD28 ESD that confer improved expression function or immunogenicity.

[0132] Linker optimization can be evaluated using techniques described herein and / or otherwise known in the art. In some embodiments, linkers do not disrupt the ability of a CAR to bind a target antigen and / or another CAR functional domain to function appropriately.

[0133] Polypeptide linkers may be positioned between adjacent elements of the CAR. For example, linkers may be positioned between DD and the transmembrane domain or between the transmembrane domain and the intracellular domain or between adjacent intracellular domains. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR may be linked to each other in a random or specified order. Optionally, a short linker, preferably betweenAttorney Docket No. 48104-7136012 and 10 amino acids in length may form the linkage. In some embodiments, the linker is a glycineserine doublet.Recombinant Expression of AND-Gated CARs

[0134] In some embodiments, recombinant vectors that encode CARs are used to express the CARs. CARs may be intentionally cell associated and used in the context of the cell in which they are expressed. In some embodiments, the CARs are operably linked to a cell membrane anchor or transmembrane domain have the potential to remain cell associated. In some embodiments, a strategy of adoptive cell transfer of T cells is used. In some embodiments, the cell may be genetically modified to stably express CAR(s) on its surface, conferring novel target specificity that is MHC independent.

[0135] In some embodiments, multiple CARs are expressed in a cell simultaneously. In some embodiments, a pair of AND-gated CARs as disclosed herein is expressed in a cell. In some embodiments, vectors that encode multiple CARs (e.g., a pair of AND-gated CARs) on the same vector are used. In some embodiments, vectors that encode only one CAR are used in combination (e.g., a first vector encoding a first CAR of a pair of AND-gated CARs and a second vector encoding a second CAR of a pair of AND-gated CARs ).

[0136] A variety of viral-derived vectors may be used in applications in which viruses are used for transfection and integration into a mammalian cell genome. Viruses, which may be useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Lentiviral vectors are particularly suitable to achieving long-term gene transfer (e.g., adoptive T cell immune therapy) since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors may have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they may transduce non-proliferating cells, such as hepatocytes. They may also have the added advantage of low immunogenicity. In some embodiments, a vector comprises elements selected from a group consisting of an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WOOl / 96584 and W001 / 29058; and U. S. Pat. No. 6,326,193). In some embodiments, a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence may be a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. In some embodiments, a suitable promoter is EFla. Alternatively, other constitutive promoter sequences may also be used, including, but notAttorney Docket No. 48104-713601limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Inducible promoters include, but are not limited to a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0137] In order to assess the expression of a CAR or portions thereof, the expression vector to be introduced into a cell may also comprise either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0138] Prior to expansion and genetic modification of the T cells, a source of T cells may be obtained from a subject. T cells may be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In various embodiments, any number of T cell lines available in the art, may be used.

[0139] Nucleic acids comprising a polynucleotide sequence encoding CAR(s) are also provided in this disclosure. Such polynucleotides optionally further comprise, one or more expression control elements. For example, the polynucleotide may comprise one or more promoters or transcriptional enhancers, ribosomal binding sites, transcription termination signals, and polyadenylation signals, as expression control elements. The polynucleotide may be inserted within any suitable vector, which may be contained within any suitable host cell for expression.

[0140] Additionally provided in this disclosure is a host cell comprising nucleic acids encoding CAR(s) described herein. Compositions comprising a nucleic acid sequence encoding the CAR(s) are also provided in this disclosure. In some embodiments, the nucleic acid encoding the CAR(s) is co-expressed with at least one other protein coding sequence. In some embodiments, the nucleic acid encoding the CAR(s) and the at least one other protein-coding sequence encode a single protein or a chimeric protein as a single polypeptide chain.Attorney Docket No. 48104-713601

[0141] In some embodiments, prior to expansion and genetic modification or other modification, a source of cells (e.g., T cells or natural killer cells) can routinely be obtained from a subject using techniques known in the art. T cells may be obtained from sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0142] The expression of nucleic acids encoding CAR(s) may be achieved by operably linking a nucleic acid encoding the CAR(s) to a promoter in an expression vector. In some embodiments, the expression vector comprises one or more of transcription and translation terminators, initiation sequences, or promoters useful for regulation of the expression of the desired nucleic acid sequence. Methods known in the art may be used to routinely construct expression vectors comprising the nucleic acid sequence encoding a CAR(s) along with appropriate transcriptional / translational control signals. These methods include, but are not limited to in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. In some embodiments, a nucleic acid sequence encoding CAR(s) is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and / or translated into CAR(s) in a host.

[0143] In some embodiments, a vector comprising a CAR-encoding nucleic acid is introduced into a host cell for expression of CAR(s). In some embodiments, the vector may remain episomal or become chromosomally integrated, as long as the insert encoding therapeutic agent may be transcribed. Vectors may be constructed by standard recombinant DNA technology. Vectors may be plasmids, phages, cosmids, phagemids, viruses, or any other types, which are used for replication and expression in prokaryotic or eukaryotic cells. It will be appreciated by one of skill in the art that a wide variety of components known in the art (such as expression control elements) may be included in such vectors, including a wide variety of transcription signals, such as promoters and other sequences that regulate the binding of RNA polymerase onto the promoter. Any promoter known or demonstrated to be effective in the cells in which the vector will be expressed may be used to initiate expression of the CAR. Suitable promoters may be inducible (e.g., regulated) or constitutive. Non-limiting examples of suitable promoters include the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, EFla, the HSV-1 (herpes simplex virus-1) thymidine kinase promoter, the regulatory sequences of the metallothionein gene, etc., as well as the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region which is active in pancreatic beta cells, mouse mammary tumor virus control region which is active in testicular,Attorney Docket No. 48104-713601breast, lymphoid and mast cells, albumin gene control region which is active in liver, alphafetoprotein gene control region which is active in liver, alpha 1 -antitrypsin gene control region which is active in the liver, beta-globin gene control region which is active in erythroid cells, myelin basic protein gene control region which is active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region which is active in skeletal muscle, and gonadotropin releasing hormone gene control region which is active in the hypothalamus. In some embodiments, the promoter is an immunoglobulin gene control region which is active in lymphoid cells.

[0144] In some embodiments, one or several nucleic acids encoding CAR(s) is expressed under the control of a constitutive promoter or, alternately, a regulated expression system. Suitable regulated expression systems include, but are not limited to, a tetracycline-regulated expression system, an ecdysone inducible expression system, a lac-switch expression system, a glucocorticoid-inducible expression system, a temperature-inducible promoter system, and a metallothionein metal-inducible expression system. If several different nucleic acids encoding CAR(s) are contained within the host cell system, some of the nucleic acids may be expressed under the control of a constitutive promoter, while others may be expressed under the control of a regulated promoter. Expression levels may be determined by methods known in the art, including Western blot analysis and Northern blot analysis.

[0145] A variety of host-expression vector systems may be utilized to express a nucleic acid encoding CAR(s). Vectors comprising the nucleic acids encoding the CAR(s) or portions or fragments thereof, may include plasmid vectors, a single and double-stranded phage vectors, as well as single and double-stranded RNA or DNA viral vectors. Phage and viral vectors may also be introduced into host cells in the form of packaged or encapsulated virus using known techniques for infection and transduction. Moreover, viral vectors may be replication competent or alternatively, replication defective. Alternatively, cell-free translation systems may also be used to produce the protein using RNAs derived from the DNA expression constructs (see, e.g., W086 / 05807 and W089 / 01036; and U. S. Pat. No. 5,122,464; the contents of each of which is herein incorporated by reference in its entirety).

[0146] Generally, any type of cells or cultured cell line may be used to express CAR(s) provided herein. In some embodiments, the background cell line used to generate an engineered host cells is a phage, a bacterial cell, a yeast cell or a mammalian cell. A variety of host-expression vector systems may be used to express the coding sequence CAR(s).

[0147] The cells may be primary isolates from organisms (including human), cultures, or cell lines of transformed or transgenic nature. In some embodiments, the host cell is a human cell. InAttorney Docket No. 48104-713601some embodiments, the host cell is human T cell. In some embodiments, the host cell is derived from a human subject. In particular embodiments, the mammalian cell systems are used to produce the CAR(s). Mammalian cell systems may utilize recombinant expression constructs comprising promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0148] In some embodiments, physical methods for introducing a nucleic acid into a host cell (e.g., a mammalian host cell) are used. In some embodiments, the methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0149] In some embodiments, biological methods for introducing a polynucleotide of interest into a host cell e.g., DNA and RNA vectors) are used. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U. S. Pat, Nos. 5,350,674 and 5,585,362, the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, clustered regularly interspaced short palindromic repeats (CRISPR) is used.

[0150] In some embodiments, methods for introducing a DNA and RNA polynucleotides of interest into a host cell include electroporation of cells, in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or polynucleotides to be introduced into the cell. DNA or RNA constructs comprising nucleic acids encoding CAR(s) may be introduced into cells using electroporation.

[0151] In some embodiments, electroporation of cells results in the expression of CAR(s) on the surface of T cells, NK cells, NKT cells. Such expression may be transient or stable over the life of the cell. Electroporation may be accomplished with methods known in the art including MaxCyte GT® and STX® Transfection Systems (MaxCyte, Gaithersburg, MD, USA).

[0152] In some embodiments, chemical means for introducing a polynucleotide into a host cell may be used. In some embodiments, the chemical means includes colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidalAttorney Docket No. 48104-713601system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g, an artificial membrane vesicle). In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. In some embodiments, lipid formulations are used for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo'). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution comprising a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a collapsed structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids may be naturally occurring or synthetic lipids. For example, lipids may include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which comprise long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0153] Lipids suitable for use may be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine (" DMPC”), dicetyl phosphate (" DCP"), cholesterol, dimyristoyl phosphatidylglycerol (" DMPG") and other lipids may be used. Stock solutions of lipids in chloroform or chloroform / methanol may be stored at about -20°C. Chloroform may be used as the only solvent since it is more readily evaporated than methanol. In some embodiments, liposomes are used. In some embodiments, liposomes are single or multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They may form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components may undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-510 (1991)). In some embodiments, compositions that have different structures in solution than the normal vesicular structure are used. For example, the lipids may assume a micellar structure or merely exist as non-uniform aggregates of lipid molecules. In some embodiments, lipofectamine-nucleic acid complexes are used.Attorney Docket No. 48104-713601

[0154] Regardless of the method used to introduce exogenous nucleic acids into a host cell, or the presence of the recombinant nucleic acid sequence in the host cell may routinely be confirmed through a variety of assays known in the art. Such assays include, for example, molecular biological assays known in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the provided embodiments.

[0155] In some embodiments, reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In some embodiments, a reporter gene is a gene that is not present in or expressed by the recipient organism, tissue, or cell and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene may be assayed at a suitable time after the DNA has been introduced into the recipient cells. A non-limiting list of suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Lett. 479: 79-82 (2000)). Suitable expression systems are known in the art and may be prepared using known techniques or obtained commercially. In some embodiments, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may routinely be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0156] A number of selection systems may be used in mammalian host-vector expression systems, including, but not limited to, the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817 (1980)) genes, which may be employed in tk', hgprt" or aprf cells, respectively. Additionally, antimetabolite resistance may be used as the basis of selection for e.g., dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase), and the glutamine synthase system.Therapeutic Use of Cells Engineered to Express AND-Gated CARs

[0157] In one aspect, this disclosure provides therapeutic methods of using cells engineered to express CAR(s) disclosed herein. In some embodiments, the cells express a pair of AND-gated CARs. In some embodiments, the therapeutic method utilizes the target-binding specificity of the target binding domain(s).Attorney Docket No. 48104-713601

[0158] In some embodiments, the CAR(s) described herein are useful for treating a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is a disease or disorder of the immune system, such as inflammation or an autoimmune disease.

[0159] In some embodiments, the disclosure provides a method of treating a subject suffering from a disease or disorder, comprising administering to the subject a therapeutically effective amount of a cell engineered to express CAR(s) (e.g., a pair of AND-gated CARs as described herein). In some embodiments, the cell expresses a pair of AND-gated CARs that bind to a first antigen and a second antigen which are both expressed by a disease-associated target cell. In some embodiments, the first antigen and the second antigen are different. In some embodiments, the disease-associated target cells are killed by the administered cells. In some embodiments, cells that do not express both the first antigen and the second antigen are not killed by the administered cells. In some embodiments, the disease-associated target cells are selectively killed by the administered cells relative to cells that do not express both the first antigen and the second antigen.

[0160] In some embodiments, the disclosure provides a method of depleting cells in a subject, comprising administering to the subject a therapeutically effective amount of a cell engineered to express CAR(s) (e.g., a pair of / AND-gated CARs as described herein). In some embodiments, the cell expresses a pair of AND-gated CARs that bind to a first antigen and a second antigen which are both expressed by a target cell. In some embodiments, the first antigen and the second antigen are different. In some embodiments, the target cells expressing both the first antigen and the second antigen are killed by the administered cells. In some embodiments, cells that do not express both the first antigen and the second antigen are not killed by the administered cells.

[0161] In some embodiments, the administered cell engineered to express CAR(s) is an immune effector cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the administered ceil engineered to express CAR(s) is not a T cell or an NK cell. In further embodiments, a combination of different cell types (e.g., NK cells and T cells) engineered to express CAR(s) is administered to the subject.

[0162] In some embodiments, the target binding domain of the administered cell engineered to express CAR(s) specifically binds antigens expressed by a disease-associated target cell of the subject, and induces the cell engineered to express CAR(s) to generate a cytotoxic signal that results in cytotoxic effects on the disease-associated target cell, thereby treating the disease.

[0163] In some embodiments, wherein cells expressing CAR(s) (e.g., a pair of AND-gated CARs as described herein) are administered to the subject having a disease, the binding of the CAR(s) toAttorney Docket No. 48104-713601their target antigens (e.g., when both CARs in the pair of AND-gated CARs interact with their target antigens) stimulates the cells expressing CAR(s) to initiate intracellular signaling. In further embodiments, the binding of the CAR(s) to their target antigens (e.g., when both CARs in the pair of AND-gated CARs interact with their target antigens) stimulates the cells expressing CAR(s) to initiate intracellular signaling and produce cytokines. In further embodiments, the binding of the CAR(s) to their target antigens (e.g., when both CARs in the pair of AND-gated CARs interact with their target antigens) stimulates the cells expressing CAR(s) to initiate intracellular signaling, produce cytokines, and degranulate, leading to cytotoxic effect on the target cell. In some embodiments, the cells expressing CAR(s) proliferate in response to binding to their target antigens (e.g., when both CARs in the pair of AND-gated CARs interact with their target antigens). Advantageously, in some embodiments, the activity of the cells expressing CAR(s) does not result in the cells expressing CAR(s) exhibiting a phenotype associated with T cell exhaustion.

[0164] In some embodiments, the disclosed CARs are used for the purpose of redirecting transduced T cells to a target cell defined by the binding specificity of the CAR(s). In some embodiments, primary T cells are transduced with lentiviral vector(s) encoding CAR(s). The resultant population of transduced T cells may elicit a CAR-mediated T cell response. In some embodiments, both CARs in the pair of AND-gated CARs must interact with their target antigens to elicit a CAR-mediated T cell response. In some embodiments, T cells are genetically modified to express CAR(s) and the CAR T cell is infused to a recipient in need thereof. In further embodiments, the infused cell is able to kill tumor cells in the recipient. Particularly advantageous properties of CARs include one, several or all of the following benefits: (i) target-binding specificity, (ii) enhanced therapeutic efficacy, (iii) reduced off-target side effects, (iv) customizability for markers of a particular subject or subject population, (v) enhanced stability during production and processing, and (vi) ability to target one, two, or more specific targets to enhance target-directed therapy.

[0165] In some embodiments, the disclosed CARs or CAR therapeutics are used to treat a disease. In some embodiments, the CAR(s) are expressed in a T cell and provides a method for treating or preventing a disease, comprising the administration of host cells expressing CAR(s) to a subject in which target cells express target antigen(s) on its surface, and wherein the CAR(s) specifically bind the target antigen(s).

[0166] In one embodiment of the methods of killing a target cell in a patient or the methods of redirecting target cell killing in a patient, the patient has been diagnosed with cancer and the target cell is a cancer cell. In one embodiment, the cancer cell is a prostate cancer cell, a breastAttorney Docket No. 48104-713601cancer cell, a colorectal cancer cell, a lung cancer cell, an osteosarcoma cell, or a glioblastoma cell. Thus, in some embodiments, the methods provided herein treat cancer.

[0167] Cancers that can be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers can comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or can comprise solid tumors. Types of cancers to be treated include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0168] Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases). In some embodiments, the solid tumor is breast cancer. In some embodiments, the solid tumor is ovarian cancer.

[0169] In another embodiment, the methods described herein are useful for treating a patient having a hematological cancer. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. Further examples of hematological (orAttorney Docket No. 48104-713601hematogenous) cancers include acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM). In some embodiments, the hematological (or hematogenous) cancer is AML. In some embodiments, the hematological (or hematogenous) cancer is CLL. In some embodiments, the hematological (or hematogenous) cancer is MM.

[0170] In some embodiments, the cancer is a relapsed of refractory cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer has relapsed following chemotherapy. In some embodiments, the cancer has relapsed following treatment with a biological agent. In some embodiments, the biological agent is a therapeutic antibody or a CAR-T cell. In some embodiments, the cancer is a refractory cancer. In some embodiments, the cancer is refractory to chemotherapy. In some embodiments, the cancer is refractory to treatment with a biological agent. In some embodiments, the biological agent is a therapeutic antibody or a CAR-T cell.

[0171] In some embodiments, the cancer is a relapsed of refractory hematological cancer. In some embodiments, the cancer is a relapsed hematological cancer. In some embodiments, the cancer is a hematological cancer that has relapsed following chemotherapy. In some embodiments, the cancer is a hematological cancer that has relapsed following treatment with a biological agent. In some embodiments, the biological agent is a therapeutic antibody or a CAR-T cell. In some embodiments, the cancer is a hematological cancer that has relapsed following autologous bone marrow transplantation. In some embodiments, the cancer is a hematological cancer that has relapsed following allogeneic bone marrow transplantation. In some embodiments, the cancer is a hematological cancer that has relapsed following hematopoietic stem cell transplantation (HSCT). In some embodiments, the HSCT is autologous HSCT. In some embodiments, the cancer is a refractory hematological cancer. In some embodiments, the cancer is a hematological cancer that is refractory to chemotherapy. In some embodiments, the cancer is a hematological cancer that is refractory to treatment with a biological agent. In some embodiments, the biological agent is a therapeutic antibody or a CAR-T cell. In some embodiments, the hematological cancer is acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM). In some embodiments, the hematological cancer is AML. In some embodiments, the hematological cancer is CLL. In some embodiments, the hematological cancer is MM. In some embodiments, the target cell cell is a B cell. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a naive T cell. In some embodiments, the target cell is a memory T cell. In some embodiments, the target cell is a myeloma cell.Attorney Docket No. 48104-713601

[0172] In another embodiment, the methods described herein are useful for treating a patient having an autoimmune disorder. Examples of autoimmune diseases include but are not limited to, Addison’s disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others.

[0173] In some embodiments, the disclosure provides a method of determining the suitability of a subject for treatment with a cell therapy. In some embodiments, the method comprises (a) obtaining a cancerous sample from the subject; (b) testing the cancerous sample for the presence of a first antigen and a second antigen on a cell in the cancerous sample; and (c) determining that the subject is suitable for treatment with the cellular therapy if both the first antigen and the second antigen are present on the cell. In some embodiments, the cellular therapy is administered to the subject if the first antigen and the second antigen are detected.Therapeutic Compositions

[0174] Also provided are therapeutic compositions useful for practicing therapeutic methods described herein. In some embodiments, therapeutic compositions provided herein comprise a physiologically tolerable carrier together with a cell engineered to express CAR(s) disclosed herein, dispersed therein as an active ingredient. In some embodiments, therapeutic compositions provided herein comprise a physiologically tolerable carrier together with a cell engineered to express a first CAR comprising a kinase domain and a second CAR as described herein, dispersed therein as an active ingredient. In some embodiments, therapeutic composition is not immunogenic when administered to a human subject for therapeutic purposes.

[0175] The preparation of a pharmacological composition that comprises active ingredients dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous. However, solid forms suitable for solution, or suspensions, in liquid prior to use may also be prepared. The preparation may also be emulsified.

[0176] The cell engineered to express (CARs) may be mixed other active ingredients and / or excipients that are pharmaceutically acceptable and compatible with the active ingredient and inAttorney Docket No. 48104-713601amounts suitable for use in therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.

[0177] Therapeutic compositions may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0178] Physiologically tolerable carriers are known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers may contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol, and other solutes.

[0179] Liquid compositions may also contain liquid phases in addition to, and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.

[0180] The cells engineered to express CAR(s) provided herein may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as chemotherapeutics, antibodies, cytokines or cell populations. Compositions provided herein are preferably formulated for intravenous administration that may be administered one or more times.Articles of Manufacture

[0181] Articles of manufacture, including kits, are provided herein. The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials or syringes. The containers may be formed from a variety of materials such as glass or plastic. The container holds one or more nucleic acids encodingAttorney Docket No. 48104-713601CAR(s) disclosed herein and / or vectors or host cells of the present disclosure. The label or package insert may include directions for administration to a subject.EXAMPLES

[0182] The application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concepts.Example 1. Phosphorylation analysis of PLCyl and LAT in the presence of constitutively activated kinases

[0183] To assess whether constitutively active kinases on the intracellular domain of a transmembrane protein could phosphorylate both PLCyl and LAT, the phosphorylation state of PLCyl and LAT after exposure to various kinases was assessed by Western blotting.

[0184] First, HEK293 cells were transfected with DNA expressing either an activated kinase domain, an activated kinase domain and minimal LAT domain (SEQ ID NO: 28), or a minimal LAT domain. The constructs that had the activated kinase domain comprised (i) an extracellular domain with a FLAG epitope and (ii) an intracellular domain with the activated kinase domain. The kinases SYK, ErbB2 (HER2), DDR2, PDGFRa, KIT, DDR1, FGFR1, FLT1, ITK, TIE2, and RLK (SEQ ID NOs: 33-35, 37-41, and 43-45) were tested. The kinase domains were forced to be active by either phosphomimetic mutation, mutation of the activation tyrosine to aspartic acid, or literature reported activating mutations (i.e., mutations found in cancer that promote an active kinase). The constructs that had the minimal LAT domain comprised (i) an extracellular domain with a V5 epitope and (ii) an intracellular domain with the minimal LAT domain.

[0185] Following transfection, HEK293 cells expressed the constructs for approximately 48 hrs to allow for strong expression and ample time for the kinase to phosphorylate the minimal LAT substrate and PLCyl. The cells were then harvested, washed, and lysed. Equal total protein for each condition was used for Western blot analysis in which the expression of the construct was confirmed via V5 signal for the kinase or FLAG signal for the minimal LAT. The expression of endogenous PLCyl was assessed via an anti-PLCyl antibody. The phosphorylation status of the minimal LAT and PLCyl was assessed via phospho-specific antibodies.

[0186] Kinases expressed with the LAT minimal substrate that produced a high ratio of phosphorylated PLCyl divided by total PLCyl and a high ratio of phosphorylated minimal LATAttorney Docket No. 48104-713601divided by total minimal LAT were identified. For example, SYK, KIT, TIE2, FGFR1, and RLK demonstrated this property.Example 2. Phosphorylation analysis of PLCyl and LAT in the presence of wildtype kinases.

[0187] To assess if kinases could self-activate and then phosphorylate both PLCyl and minimal LAT, the phosphorylation state of the kinase, PLCyl, and minimal LAT after exposure to kinases was assessed by Western blotting.

[0188] First, HEK293 cells were transfected with DNA expressing (i) a wild-type or a catalytically inactive kinase domain alone, (ii) (a) a wild-type or a catalytically inactive kinase domain and (b) minimal LAT domain (SEQ ID NO: 28), or (iii) a minimal LAT domain alone. The constructs that had the wild-type or catalytically inactive kinase domain comprised (i) an extracellular domain with a FLAG epitope and (ii) an intracellular domain with the kinase domain. The kinases being tested were SYK, KITshort, FGFR1, and TIE2 (SEQ ID NOs: 33, 47-49, and 54-57). The constructs that had the minimal LAT domain comprised (i) an extracellular domain with a V5 epitope and (ii) an intracellular domain with the minimal LAT domain.Table 6. Exemplary Catalytically Inactive KinasesKinase SequenceDomainSYK EEIRPKEVYLDRKLLTLEDKELGSGNFGTVKKGYYQMKKVVKTVAVKILKNEANDPAL KDELLAEANVMQQLDNPYIVRMIGICEAESWMLVMEMAELGPLNKYLQQNRHVKDKN IIELVHQVSMGMKYLEESNFVHRNLAARNVLLVTQHYAKISDFGLSKALRADENYYKA QTHGKWPVKWYAPECINYYKFSSKSDVWSFGVLMWEAFSYGQKPYRGMKGSEVTAM LEKGERMGCPAGCPREMYDLMNLCWTYDVENRPGFAAVELRLRNYYYDVVN (SEQ ID NO: 54)KIT PYDHKWEFPRNRLSFGKTLGAGAFGKWEATAYGLIKSDAAMTVAVKMLKPSAHLTE(Short) REALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSK TSPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRNLAARNILLTHGRITKICDF GLARDIKNDSNYWKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPY PGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQIS ES (SEQ ID NO: 55)FGFR1 SEYELPEDPRWELPRDRLVLGKPLGEGCFGQVVLAEAIGLDKDKPNRVTKVAVKMLKS DATEKDLSDLISEMEMMKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLQARR PPGLEYCYNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRNLAARNVLVTEDN VMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEI FTLGGSPYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQL VEDLDRIVALTSNQE (SEQ ID NO: 56)Attorney Docket No. 48104-713601TIE2 PTIYPVLDWNDIKFQDVIGEGNFGQVLKARIKKDGLRMDAAIKRMKEYASKDDHRDFA GELEVLCKLGHHPNIINLLGACEHRGYLYLAIEYAPHGNLLDFLRKSRVLETDPAFAIAN STASTLSSQQLLHFAADVARGMDYLSQKQFIHRNLAARNILVGENYVAKIADFGLSRGQ EVYVKKTMGRLPVRWMAIESLNYSVYTTNSDVWSYGVLLWEIVSLGGTPYCGMTCAE LYEKLPQGYRLEKPLNCDDEVYDLMRQCWREKPYERPSFAQILVSLNRMLEERKTYVN(SEQ ID NO: 57)

[0189] Following transfection, HEK293 cells expressed the constructs for approximately 48 hrs to allow for strong expression and ample time for the kinase to phosphorylate the minimal LAT substrate and PLCyl. The cells were then harvested, washed, and lysed. Equal total protein for each condition was used for Western blot analysis in which the expression of the construct was confirmed via V5 signal for the kinase or FLAG signal for the minimal LAT. The expression of endogenous PLCyl was accessed via an anti-PLCyl antibody. The phosphorylation status of the kinase, minimal LAT, and PLCyl was assessed via phospho-specific antibodies.

[0190] Kinases were identified that demonstrated three properties: (i) wild-type kinases selfactivated upon expression, (ii) wild-type kinases phosphorylated both the minimal LAT substrate and PLCyl, and (iii) the catalytically inactive kinase did not meet any of the previous two criteria. Catalytically inactive and wild-type kinases were assessed for self-activation by phospho-specific antibodies for the kinase activation loop (anti-KinasePY211). Catalytically inactive and wild-type kinases expressed with the LAT minimal substrate were assessed for phosphorylating the LAT minimal substrate and PLCyl by dividing the phosphorylated minimal LAT divided by total minimal LAT and the phosphorylated PLCyl by total PLCyl. For example, SYKWT, FGFR1WT, and TIE2WTdemonstrated all three properties.Example 3. T-Cells mediated cytotoxicity of AND-gated CARs in the Presence of CAR Ligands

[0191] To assess whether T cells engineered to express a pair of AND-gated CARs exhibit logicgated activation, a T-cell cytotoxicity assay was conducted.

[0192] First, multiple primary T cells were engineered to express either (1) pairs of AND-gated CARs, (2) only one of the CARs in the pair, or (3) no CARs. The first CAR in each pair comprised (i) an intracellular domain with a kinase domain and (ii) an extracellular domain with a HER2-binding scFv (SEQ ID NO: 1). The kinase domains tested included SYK, FGFR1, TIE2 (SEQ ID NOs: 33, 39, and 40). The second CAR in each pair comprised (i) an intracellular domain with a minimal LAT domain (SEQ ID NO: 28) and (ii) an extracellular domain with a CD19-binding scFvAttorney Docket No. 48104-713601(SEQ ID NO: 2). The minimal LAT domain has a tyrosine that can be phosphorylated, and when the tyrosine is phosphorylated, the minimal LAT domain can act as a binding site for PLCyl.

[0193] The engineered T-cell populations are cultured in an Incucyte instrument for 96 hours in the presence fluorescent-positive (GFP or NIR) NALM6 target cells at effector-to-target (E: T) ratios of 1:2 or 1:8.

[0194] The NALM6 cells used in the assay expressed (1) CD19 only, (2) HER2 only, or (3) both CD 19 and HER2. Throughout the incubation, a cytotoxicity assay was conducted.

[0195] In the assay, the “Total Green / NIR Object” measurement corresponded to the proportion of GFP / NIR-positive NALM6 cells relative to timepoint 0 (prior to CAR-T cell addition). At an E: T ratio of 1:2, in the presence of CD19+HER2+NALM6 cells, cytotoxic activity was observed for T cells expressing LAT+SYK, LAT+TIE2, LAT+FGFR1, and the FMC63 positive-control CAR, whereas no cytotoxicity was observed for LAT alone, FGFR1 alone, TIE2 alone, or negative-control T cells.

[0196] Against CDI9 HER2 NALM6 cells or CD 19 HER2 NALM6 cells, T cells expressing LAT+TIE2 did not demonstrate substantial cytotoxic activity, indicating that the AND-gating system did not result in cytotoxicity when only one antigen was present.

[0197] At an E: T ratio of 1:8, in the presence of CD19+HER2+NALM6 cells, cytotoxicity is observed for LAT+TIE2, LAT+FGFR1, LAT+SYK, and the FMC63 CAR. Against CD19+HER2 NALM6 cells, LAT+TIE2 and LAT+FGFR1, did not demonstrate target-cell killing.

[0198] These results indicate that combinations comprising LAT+TIE2 or LAT+FGFR1 can enhance the selectivity of the AND-gated CAR system toward target cells expressing both HER2 and CD 19.Example 4. Phosphorylation analysis of PLCyl and various substrates in the presence of wildtype FGFR1 and TIE2

[0199] To assess alternative recruitment sites for PLCyl within the LAT minimal domain, four other phospho-PLCyl binding sites were assessed: KIT, VEGFR, FGFR1, and PDGFRa. The sites were assessed for their ability to be phosphorylated by previously identified kinases, SYK, KITshort, FGFR1, and TIE2 and the ability to recruit PLCyl based on the phosphorylation level of PLCyl.

[0200] First, HEK293 cells were transfected with DNA expressing either a wild-type kinase domain, a wild-type kinase domain and minimal LAT domain, or a minimal LAT domain. The constructs that had the wild-type kinase domain comprised (i) an extracellular domain with a FLAG epitope and (ii) an intracellular domain with the kinase domain. The kinases tested wereAttorney Docket No. 48104-713601SYK, KITshort, FGFR1, and TIE2 (SEQ ID NOs: 33 and 47-49). The constructs that had the minimal LAT domain comprised (i) an extracellular domain with a V5 epitope and (ii) an intracellular domain with the minimal LAT domain with the PLCyl binding site from LAT, KIT, VEGFR, FGFR1, or PDGFRa (SEQ ID NOs: 28 or 50-53).

[0201] Following transfection, HEK293 cells expressed the constructs for approximately 48 hrs to allow for strong expression and ample time for the kinase to phosphorylate the PLCyl binding site and PLCyl. The cells were then harvested, washed, and lysed. Equal total protein for each condition was used for Western blot analysis in which the expression of the construct was confirmed via V5 signal for the kinase or FLAG signal for the minimal LAT. The expression of endogenous PLCyl was assessed via an anti-PLCyl antibody. The phosphorylation status of the kinase, PLCyl binding site, and PLCyl was assessed via phospho-specific antibodies.

[0202] Substrates were identified that demonstrated three properties: (i) the level of phosphorylation of PLCyl was equal to or better than the minimal LAT PLCyl binding site, (ii) the PLCyl binding site had an equal or lower level of phosphorylation in the absence of kinase when compared to the minimal LAT, and (iii) the kinase was able to phosphorylate the PLCyl binding site. Wild-type kinases were assessed for self-activation by phospho-specific antibodies for the kinase activation loop (anti-I<inasepYal). Wild-type kinases expressed with the various PLCyl binding sites were assessed for phosphorylation level by dividing the phosphorylated PLCyl binding site by total PLCyl binding site expression. The level of PLCyl phosphorylation was assessed by dividing phosphorylated PLCyl by total PLCyl. The KIT PLCyl binding site with SYK and TIE2 demonstrated all three properties. The VEGFR PLCyl binding site with FGFR1 and TIE2 demonstrated all three propertiesPLCyl PLCyl. The FGFR1 PLCyl binding site with SYK, FGFR1, and TIE2 demonstrated all three properties. The PDGFRa PLCyl binding site with SYK and TIE2 demonstrated three properties.Example 5. T-Cells mediated cytotoxicity of AND-gated CARs with various PLCyl substrates

[0203] To assess the role of PLCyl recruitment substrates, T-cells were engineered to express a pair of AND-gated CARs, and a T-cell cytotoxicity assay was conducted.

[0204] First, multiple primary T cells were engineered to express (1) pairs of AND-gated CARs, (2) only one of the CARs in the pair, or (3) no CARs. The first CAR in each pair comprised (i) an intracellular domain with a TIE2 kinase domain or a FGFR1 kinase domain (SEQ ID NOs: 39 and 40) and (ii) an extracellular domain with a HER2 -binding scFv (SEQ ID NO: 1). The second CARAttorney Docket No. 48104-713601in each pair comprised (i) an intracellular domain with a minimal LAT domain, KIT domain, VEGFR domain or PDGFRa domain (SEQ ID NOs: 28, 50-51, and 53) and (ii) an extracellular domain with a CD19-binding scFv (SEQ ID NO: 2). Each substrate had a tyrosine that can be phosphorylated, and when the tyrosine is phosphorylated, the substrate can act as a binding site for PLCyl.

[0205] The engineered T-cell populations were cultured in an Incucyte instrument for 48-96 hours in the presence of fluorescent-positive (GFP) target cells at effector-to-target (E: T) ratios of 1: 1 or 1:2. The NALM6 cells used in the assay expressed (1) CD19 only or (2) both CD19 and HER2. Throughout the incubation, a cytotoxicity assay was conducted.

[0206] In the assay, the “Total Green Object” measurement corresponds to the proportion of Fluorescent-positive NALM6 cells relative to timepoint 0 (prior to CAR-T cell addition). At an E: T ratio of 1:1, in the presence of CD19+HER2+NALM6 cells, cytotoxic activity was observed for T cells expressing LAT+TIE2, PDGFRa+TIE2, and the FMC63 positive-control CAR, whereas no cytotoxicity was observed for substrates alone or negative-control T cells. At an E: T ratio of 1:1, in the presence of CD19+HER2+NALM6 cells, cytotoxic activity was observed for T cells expressing LAT+FGFR1, KIT+ FGFR1, VEGFR1+ FGFR1, PDGFRa+ FGFR1, and the FMC63 positive-control CAR, whereas no cytotoxicity was observed for substrates alone, or the negativecontrol T cells.

[0207] At an E: T ratio of 1:2, in the presence of CD19+HER2- NALM6 cells, no cytotoxicity was observed for PDGFRa+TIE2 demonstrating no leakiness induced by either substrate.

[0208] These results indicate that combinations comprising PDGFRa+TIE2 enhance the selectivity of the AND-gated CAR system toward target cells expressing both HER2 and CD 19.

[0209] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No. 48104-713601CLAIMS WHAT IS CLAIMED IS:

1. A cell comprising:a first chimeric antigen receptor comprising a first extracellular domain and a first intracellular domain, wherein the first extracellular domain comprises a first antigenbinding domain, and wherein the first intracellular domain comprises a first kinase domain; anda second chimeric antigen receptor comprising a second extracellular domain and a second intracellular domain, wherein the second extracellular domain comprises a second antigen-binding domain, wherein the second intracellular domain comprises a tyrosine, and wherein the tyrosine is a substrate for phosphorylation by the first kinase domain.

2. A cell comprising:a first chimeric antigen receptor comprising a first extracellular domain and a first intracellular domain, wherein the first extracellular domain comprises a first antigenbinding domain, and wherein the first intracellular domain comprises a first kinase domain;a second chimeric antigen receptor comprising a second extracellular domain and a second intracellular domain, wherein the second extracellular domain comprises a second antigen-binding domain; anda PLCyl, wherein the PLCyl is phosphorylated by the first kinase domain when the first chimeric antigen receptor is in contact with the second chimeric antigen receptor.

3. The cell of claim 1 or claim 2, wherein the first kinase domain comprises a biologically active domain of any one of KIT, DDR1, FGFR1, TIE2, FLT1, ZAP-70, LCK, FYN, SYK, ErbB2, DDR2, PDGFRa, FLT1, ITK, or RLK proteins.

4. The cell of claim 1 or claim 2, wherein the first kinase domain comprises a biologically active domain of any one of KIT, DDR1, FGFR1, TIE2, FLT1, ZAP-70, LCK, FYN, SYK, or RLK proteins.Attorney Docket No. 48104-7136015. The cell of any one of claims 1 to 3, wherein the first kinase domain comprises the amino acid sequence of any one of SEQ ID NOs: 27 or 33-45.

6. The cell of one of claims 1 to 3, wherein the first kinase domain comprises the amino acid sequence of any one of SEQ ID NOs: 33 or 46-49.

7. The cell of claim 1 or claim 2, wherein the first kinase domain comprises a biologically active domain of any one of KIT, FGFR1, TIE2, or SYK proteins.

8. The cell of claim 7, wherein the first kinase domain comprises a biologically active domain of TIE2.

9. The cell of claim 8, wherein the first kinase domain comprises the amino acid sequence of SEQ ID NO: 40.

10. The cell of claim 8, wherein the first kinase domain comprises the amino acid sequence of SEQ ID NO: 49.

11. The cell of claim 7, wherein the first kinase domain comprises a biologically active domain of FGFR1.

12. The cell of claim 11, wherein the first kinase domain comprises the amino acid sequence of SEQ ID NO: 39.

13. The cell of claim 11, wherein the first kinase domain comprises the amino acid sequence of SEQ ID NO: 48.

14. The cell of any one of claims 1 to 13, wherein the second intracellular domain comprises a domain that recruits PLCyl upon phosphorylation.

15. The cell of any one of claims 1 to 11, wherein the second intracellular domain comprises an intracellular domain of any one of LAT, VEGFR, PDGFRa, FGFR1, or KIT proteins.

16. The cell of claim 15, wherein the second intracellular domain comprises the amino acid sequence of any one of SEQ ID NOs: 28 or 50-53.

17. The cell of claim 15, wherein the second intracellular domain comprises an intracellular domain of LAT.

18. The cell of claim 15, wherein the second intracellular domain comprises the amino acid sequence of SEQ ID NO: 28.Attorney Docket No. 48104-71360119. The cell of claim 17 or claim 18, wherein the second intracellular domain comprises an intracellular domain of LAT and wherein the first kinase domain comprises a biologically active domain of TIE2.

20. The cell of claim 17 or claim 18, wherein the second intracellular domain comprises an intracellular domain of LAT and wherein the first kinase domain comprises a biologically active domain of FGFR1.

21. The cell of claim 15, wherein the second intracellular domain comprises an intracellular domain of any one of VEGFR, PDGFRa, FGFR1, or KIT proteins.

22. The cell of claim 21, wherein the second intracellular domain comprises an intracellular domain of PDGFRa.

23. The cell of claim 22, wherein the second intracellular domain comprises an amino acid sequence of SEQ ID NO: 53.

24. The cell of claim 22 or claim 23, wherein the second intracellular domain comprises an intracellular domain of PDGFRa and wherein the first kinase domain comprises a biologically active domain of TIE2.

25. The cell of claim 22 or claim 23, wherein the second intracellular domain comprises an intracellular domain of PDGFRa and wherein the first kinase domain comprises a biologically active domain of FGFR1.

26. The cell of any one of claims 1 to 25, wherein the second chimeric antigen receptor comprises a second kinase domain.

27. The cell of claim 26, wherein the second kinase domain comprises a kinase regulatory domain.

28. The cell of claim 27, wherein the kinase regulatory domain is an activator of the first kinase domain.

29. The cell of claim 27, wherein the kinase regulatory domain is a repressor of the first kinase domain.

30. The cell of claim 26, wherein the first kinase domain and the second kinase domain are activated when dimerized to each other.Attorney Docket No. 48104-71360131. The cell of any one of claims 1 or 3 to 30, wherein the tyrosine binds to a PLCyl when phosphorylated.

32. The cell of claim 31, wherein the PLCyl comprises a substrate for the first kinase domain.

33. The cell of claim 31, wherein the PLCyl comprises a substrate for the second kinase domain.

34. The cell of any one of claims 1 to 33, wherein the first chimeric antigen receptor comprises a first transmembrane domain, and wherein the second chimeric antigen receptor comprises a second transmembrane domain.

35. The cell of claim 34, wherein the first transmembrane domain and the second transmembrane domain do not promote dimerization to each other in the absence of binding of the cell to both a first antigen that binds to the first antigen-binding domain and a second antigen that binds to the second antigen-binding domain.

36. The cell of claim 34, wherein the first transmembrane domain and the second transmembrane domain promote dimerization when spatially colocalized.

37. The cell of any one of claims 34 to 35, wherein one or both of the first transmembrane domain and the second transmembrane domain comprise a CD4, CD8, 4 IBB, or CD28 transmembrane domain or variations thereof.

38. The cell of any one of claims 1 to 37, wherein one or both of the first intracellular domain and the second intracellular domain comprises an intracellular signaling domain.

39. The cell of claim 38, wherein the intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; LAT; SLP-76; and any combination thereof.

40. The cell of any one of claims 1 to 39, wherein the intracellular domain comprises a costimulatory molecule selected from the group consisting of CD27, CD28, 4 IBB, 0X40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

41. The cell of any one of claims 1 to 40, wherein the cell is an immune cell.Attorney Docket No. 48104-71360142. The cell of claim 41, wherein the immune cell is a T cell.

43. The cell of claim 41, wherein the immune cell is a natural killer (NK) cell.

44. The cell of any one of claims 1 to 43, wherein the first antigen-binding domain and the second antigen-binding domain bind to different antigens.

45. The cell of claim 44, wherein one of the first binding domain or the second binding domain binds to CD 19, and wherein one of the first binding domain or the second binding domain binds to HER2.

46. The cell of any one of claims 1 to 45, wherein one or both of the first antigen-binding domain and the second antigen-binding domain comprises an antibody, an antibody fragment, or an alternative scaffold-based antigen-binding domain.

47. The cell of claim 46, wherein the alternative scaffold-based antigen-binding domain comprises a D domain.

48. The cell of any one of claims 1 to 47, wherein the cell is selectively activated in an AND- gated manner.

49. A method of treating a subject having a cancer, the method comprising:administering to the subject an effective amount of the cell of any one of claims 1 to 48.

50. The method of claim 49, wherein cancer cells of the subject comprise a first extracellular antigen that binds to the first antigen-binding domain of the cell of any one of claims 1 to 48 and a second extracellular antigen that binds to the second antigen-binding domain of the cell of any one of claims 1 to 48, wherein the first extracellular antigen and the second extracellular antigen are different.

51. A method of treating a cancer patient having cancer cells, the method comprising:administering to the cancer patient an effective amount of the cell of any one of claims 1 to 48.

52. The method of claim 51, wherein the cancer cells comprise a first extracellular antigen that binds to the first antigen-binding domain of the cell of any one of claims 1 to 48 and a second extracellular antigen that binds to the second antigen-binding domain of the cell of any one of claims 1 to 48, wherein the first extracellular antigen and the second extracellular antigen are different.Attorney Docket No. 48104-71360153. The method of claim 52, wherein the cancer cells are killed by the administered cells, and wherein cells without one or more of the first extracellular antigen and the second extracellular antigen are not killed by the administered cells.

54. A method of treating a subject having an autoimmune disorder, the method comprising:administering to the subject an effective amount of the cell of any one of claims 1 to 48.

55. The method of claim 54, wherein immune cells of the subject comprise a first extracellular antigen that binds to the first antigen-binding domain of the cell of any one of claims 1 to 48 and a second extracellular antigen that binds to the second antigenbinding domain of the cell of any one of claims 1 to 48, wherein the first extracellular antigen and the second extracellular antigen are different.

56. A method of treating an autoimmune patient having immune cells, the method comprising:administering to the autoimmune patient an effective amount of the cell of any one of claims 1 to 48.

57. The method of claim 56, wherein the immune cells comprise a first extracellular antigen that binds to the first antigen-binding domain of the cell of any one of claims 1 to 48 and a second extracellular antigen that binds to the second antigen-binding domain of the cell of any one of claims 1 to 48, wherein the first extracellular antigen and the second extracellular antigen are different.

58. The method of claim 57, wherein the immune cells are killed by the administered cells, and wherein cells without one or more of the first extracellular antigen and the second extracellular antigen are not killed by the administered cells.

59. A method for depleting cells of a subject, the method comprising:administering to the subject an effective amount of the cell of any one of claims 1 to 48, wherein the cells of the subject comprise a first extracellular antigen that binds to the first antigen-binding domain of the cell of any one of claims 1 to 48 and a second extracellular antigen that binds to the second antigen-binding domain of the cell of any one of claims 1 to 48, wherein the first extracellular antigen and the second extracellular antigen are different.Attorney Docket No. 48104-71360160. The method of claim 59, wherein the cells of the subject comprising the first extracellular antigen and the second extracellular antigen are killed by the administered cells, and wherein cells without one or more of the first extracellular antigen and the second extracellular antigen are not killed by the administered cells.

61. A vector comprising a nucleic acid sequence encoding the first chimeric antigen receptor of any one of claims 1 to 48 and a nucleic acid sequence encoding the second chimeric antigen receptor of any one of claims 1 to 48.

62. A method for determining the suitability of a subject for treatment with a cell therapy, the method comprising: (a) obtaining a sample from the subject; (b) testing the sample for the presence of a first antigen and a second antigen on a cell in the sample; and (c) determining that the subject is suitable for treatment with the cellular therapy if both the first antigen and the second antigen are present on the cell; wherein the cellular therapy is administered to the subject if the first antigen and the second antigen are detected, wherein the cell therapy comprises administering the cell of any one of claims 1 to 48.

63. The method of claim 62, wherein the sample comprises a cancerous sample.

64. The method of claim 62, wherein the sample comprises immune cells.

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