NZ710925B2

NZ710925B2 - Chimeric antigen receptor and methods of use thereof

Info

Publication number: NZ710925B2
Application number: NZ710925A
Authority: NZ
Inventors: Wendell A Lim; James Onuffer; Chia Yung Wu
Original assignee: The Regents Of The University Of California
Priority date: 2013-02-15
Filing date: 2014-02-14
Publication date: 2021-06-29

Abstract

The present disclosure provides a heterodimeric, conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present disclosure provides cells genetically modified to produce the CAR. A CAR of the present disclosure can be used in various methods, which are also provided. The heterodimeric, conditionally active CAR comprises a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a first co-stimulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of a specific binding pair and the first co-stimulatory domain; and b) a second polypeptide comprising: i) a second member of the dimerization pair; and ii) an intracellular signaling domain; optionally further comprising a transmembrane domain and/or a second co-stimulatory domain, wherein the first and second members of the dimerization pair form a homodimer or a heterodimer in the presence of a dimerizing agent. arious methods, which are also provided. The heterodimeric, conditionally active CAR comprises a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a first co-stimulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of a specific binding pair and the first co-stimulatory domain; and b) a second polypeptide comprising: i) a second member of the dimerization pair; and ii) an intracellular signaling domain; optionally further comprising a transmembrane domain and/or a second co-stimulatory domain, wherein the first and second members of the dimerization pair form a homodimer or a heterodimer in the presence of a dimerizing agent.

Description

CHIMERIC ANTIGEN RECEPTOR AND METHODS OF USE THEREOF CROSS-REFERENCE This application claims the benefit of US. Provisional Patent Application No. 61/765,585, filed February 15, 2013, which application is incorporated herein by nce in its entirety.

STATEMENT REGARDING LLY SPONSORED RESEARCH This invention was made with government support under Grant Nos. EYOl6546 and GMlOl782 awarded by the National Institutes of Health. The ment has n rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE A Sequence Listing is provided herewith as a text file, “UCSF—464WO SeqList_ST25.txt” created on February 13, 2014 and having a size of 153 KB. The contents of the text file are incorporated by reference herein in their entirety.

INTRODUCTION In cell—based ve immunotherapy, immune cells isolated from a patient can be modified to express synthetic proteins that enable the cells to perform new therapeutic ons after they are subsequently transferred back into the patient. An example of such a synthetic protein is a chimeric antigen receptor (CAR). An example of a currently used CAR is a fusion of an extracellular recognition domain (e. g., an antigen—binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon n engagement, the intracellular signaling portion of the CAR can initiate an activation—related response in an immune cell, such are release of cytolytic molecules to induce tumor cell death, etc. r, such CARs are not capable of being pharmacologically controlled.

There is a need in the art for a conditionally activatable CAR that can be controlled pharmacologically.

The present sure provides a heterodimeric, conditionally active chimeric antigen or (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR.

The present disclosure provides cells genetically ed to produce the CAR. A CAR of the present disclosure can be used in various methods, which are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B provide nucleotide and amino acid sequences of the domains of construct #122.

Figures 2A and 2B provide nucleotide and amino acid sequences of the domains of construct #123.

Figures 3A and 3B provide nucleotide and amino acid sequences of the domains of construct #125.

Figure 4 es tide and amino acid sequences of the domains of construct #126.

Figures 5A and 5B provide nucleotide and amino acid ces of the s of construct #168.

Figures 6A—C provide nucleotide and amino acid sequences of the domains of construct #169.

Figures 7A and 7B e nucleotide and amino acid sequences of the domains of construct #170.

Figures 8A and 8B provide nucleotide and amino acid sequences of the domains of construct #197.

Figures 9A—C provide nucleotide and amino acid sequences of the domains of construct #206. s 10A and 10B provide nucleotide and amino acid ces of the domains of construct #207.

Figures 11A—C provide nucleotide and amino acid sequences of the domains of construct #199.

Figure 12 depicts IL—2 production triggered by five On—switch CAR variants.

Figure 13 depicts IL—2 production by control Jurkat lines.

Figure 14 depicts a comparison between CAR constructs “122 + 206” and “197 + 206”.

Figure 15 depicts cytotoxicity data with the On—switch CAR “197+206.” Figure 16 depicts T cell activation data using CAR constructs “122 + 199”; “197 + 199”; and “122 + 168.” Figure 17 is a schematic representation of an exemplary tch CAR.

Figures 18A and 18B depict various exemplary On—switch CAR.

Figures l9A—G depict IL—2 production triggered by 3 different On—switch CAR variants recognizing human mesothelin.

Figures 20A—C depict IL—2 production triggered by an On—switch CAR variant with a gibberellic acid responsive dimerization pair.

Figures 2lA—D depict exemplary On—switch CARs and conventional CARs with various co— stimulatory domains.

Figures 22A and 22B provide nucleotide and amino acid sequences of the domains of construct #270.

Figures 23A and 23B provide nucleotide and amino acid sequences of the domains of construct #300.

Figures 24A and 24B provide nucleotide and amino acid sequences of the domains of construct #336.

Figures 25A and 25B provide tide and amino acid sequences of the domains of construct #337.

Figures 26A and 26B e nucleotide and amino acid sequences of the domains of construct #357.

Figures 27A and 27B e tide and amino acid sequences of the domains of construct #365.

Figures 28A and 28B provide nucleotide and amino acid sequences of the domains of construct #366. s 29A and 29B provide nucleotide and amino acid sequences of the domains of construct #367.

Figures 30A and 30B provide nucleotide and amino acid sequences of the domains of uct #398.

Figures 31A and 31B e tide and amino acid sequences of the domains of construct #399.

Figures 32A and 32B provide nucleotide and amino acid sequences of the domains of construct #400.

Figures 33A and 33B provide nucleotide and amino acid sequences of the domains of uct #358.

DEFINITIONS The terms ucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or 2014/016527 deoxyribonucleotides. Thus, this term includes, but is not limited to, single—, double—, or stranded DNA or RNA, genomic DNA, cDNA, DNA—RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, ally or biochemically modified, non—natural, or derivatized nucleotide bases.

The terms "antibodies" and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to n, including, but not limited to, Fab, FV, scFV, and Fd fragments, chimeric antibodies, humanized dies, —chain antibodies, and fusion ns comprising an antigen—binding n of an antibody and a tibody protein.

"Antibody fragments" comprise a portion of an intact antibody, for e, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and FV fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057—1062 (1995)); single—chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain ion of antibodies produces two identical antigen—binding fragments, called "Fab" fragments, each with a single antigen—binding site, and a residual "Fc" fragment, a designation reﬂecting the ability to crystallize readily.

Pepsin treatment yields an 2 fragment that has two antigen combining sites and is still capable of cross—linking antigen.

"Single—chain FV" or "st" antibody fragments se the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some ments, the FV polypeptide further comprises a polypeptide linker between the VH and VL s, which s the sFV to form the desired structure for antigen binding.

For a reView of sFV, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (I994).

As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). ty can be at least l—fold greater, at least 2—fold greater, at least 3—fold greater, at least 4—fold greater, at least 5—fold greater, at least 6—fold greater, at least 7—fold greater, at least 8—fold greater, at least 9—fold greater, at least lO—fold greater, at least 20—fold r, at least 30—fold greater, at least 40—fold greater, at least 50—fold greater, at least 60—fold greater, at least 70—fold greater, at least 80—fold greater, at least 90—fold greater, at least lOO—fold greater, or at least lOOO—fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences. Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and/or antigen—binding fragments.

The term “binding” refers to a direct association n two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen—bond interactions, including interactions such as salt bridges and water bridges. Non—specific binding would refer to binding with an affinity of less than about 10'7 M, e. g., binding with an affinity of '6 M, 10'5 M, 10'4 M, etc.

As used herein, the term “hinge region” refers to a ﬂexible polypeptide connector region (also referred to herein as “hinge” or “spacer”) providing structural ﬂexibility and g to ﬂanking polypeptide regions and can consist of natural or synthetic polypeptides. A "hinge region" d from an immunoglobulin (e.g., IgGl) is generally d as stretching from Glu216 to Proz30 of human IgGl (Burton (1985) Molec. Immunol., 22: 161— 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter—heavy chain ide (S—S) bonds in the same positions. The hinge region may be of natural occurrence or non—natural occurrence, including but not limited to an d hinge region as described in US. Pat. No. ,677,425. The hinge region can include complete hinge region derived from an antibody of a different class or subclass from that of the CH1 . The term “hinge region” can also include regions derived from CD8 and other receptors that e a r function in providing ﬂexibility and spacing to ﬂanking regions.

An "isolated" polypeptide is one that has been identified and separated and/or recovered from a component of its natural nment. Contaminant components of its l environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the ptide will be purified (l) to greater than 90%, greater than 95%, or greater than 98%, by weight of antibody as determined by the Lowry method, for example, more than 99% by , (2) to a degree sufficient to obtain at least 15 residues of N—terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium l sulfate— polyacrylamide gel electrophoresis (SDS—PAGE) under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one ent of the polypeptide’s natural environment will not be present. In some instances, ed polypeptide will be prepared by at least one purification step.

As used herein, the term “immune cells” generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.

“Immune cells” includes, e. g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid—derived cells (neutrophil, eosinophil, basophil, te, macrophage, dendritic cells).

“T cell” includes all types of immune cells expressing CD3 including T—helper cells (CD4+ cells), cytotoxic T—cells (CD8+ cells), T—regulatory cells (Treg) and gamma—delta T cells.

A “cytotoxic cell” includes CD8+ T cells, natural—killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.

As used herein, the term “stem cell” generally includes pluripotent or multipotent stem cells. “Stem cells” includes, e. g., nic stem cells (ES); mesenchymal stem cells (MSC); induced—pluripotent stem cells (iPS); and committed progenitor cells (hematopoeitic stem cells (HSC); bone marrow d cells, etc.).

As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially ting a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or e effect attributable to the e. ment," as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and es: (a) preventing the disease from occurring in a subject which may be posed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the e, i.e., causing regression of the e.

The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, s (e. g., rats, mice), lagomorphs (e. g., rabbits), non—human primates, humans, canines, felines, ungulates (e. g., equines, bovines, ovines, porcines, caprines), etc.

A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary ing on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated. 2014/016527 Before the present ion is further described, it is to be understood that this invention is not limited to particular ments described, as such may, of course, vary. It is also to be tood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is tood that each intervening value, to the tenth of the unit of the lower limit unless the t clearly dictates otherwise, between the upper and lower limit of that range and any other stated or ening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and ific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and als are now described. All publications ned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the ar forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a chimeric antigen receptor” includes a plurality of such chimeric antigen receptor and reference to “the dimerizer—binding pair” includes reference to one or more dimerizer—binding pairs and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element.

As such, this statement is intended to serve as antecedent basis for use of such exclusive ology as “solely,77 4‘only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided tely or in any suitable sub—combination. All combinations of the embodiments pertaining to the invention are ically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub— combinations of the s embodiments and elements thereof are also ically embraced by the present invention and are disclosed herein just as if each and every such sub—combination was individually and explicitly disclosed .

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present ation. Nothing herein is to be ued as an admission that the present invention is not entitled to antedate such publication by virtue of prior ion.

Further, the dates of ation provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION The present disclosure provides a dimeric, conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide ce encoding the CAR.

The present disclosure provides cells genetically modified to produce the CAR. A CAR of the present disclosure can be used in various methods, which are also provided.

HETERODIMERIC, CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTOR.

The present disclosure provides a heterodimeric, ionally active chimeric antigen receptor, which, for simplicity, is referred to herein as “CAR.” In some embodiments, a CAR of the present disclosure comprises: a) a first polypeptide comprising: i) a member of a specific binding pair (e. g., an antigen—binding domain); ii) a first modulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the member of a specific g pair (e. g., an antigen—binding domain) and the first modulatory domain; and b) a second polypeptide comprising: i) a transmembrane domain; ii) a second modulatory domain; iii) a second member of the dimerization pair; and iv) an intracellular signaling domain. The modulatory domain can be a mulatory domain.

In some embodiments, a CAR of the present disclosure comprises: a) a first polypeptide comprising: i) a member of a ic binding pair (e. g., an antigen—binding domain); ii) a first co—stimulatory domain; iii) a first member of a dimerization pair (e.g., a dimerizer— binding pair); and iv) a transmembrane domain interposed between the member of a specific binding pair (e. g., an antigen—binding domain) and the first co—stimulatory domain; and b) a second polypeptide comprising: i) a embrane domain; ii) a second co—stimulatory domain; iii) a second member of the dimerization pair (e. g., the dimerizer—binding pair); and iV) an intracellular signaling domain.

In some embodiments, a CAR of the present disclosure comprises: a) a first ptide comprising: i) a member of a specific binding pair (e. g., an antigen—binding ); ii) a modulatory domain; iii) a first member of a dimerization pair (e.g., a dimerizer—binding pair); iV) a transmembrane domain interposed between the member of a specific binding pair (e. g., an n—binding domain) and the modulatory domain; and b) a second polypeptide comprising: i) a second member of the zation pair (e. g., the dimerizer— binding pair); and ii) an intracellular signaling domain. The modulatory domain can be a co— atory domain.

In some embodiments, a CAR of the present disclosure comprises: a) a first polypeptide comprising: i) a member of a specific binding pair (e. g., an antigen—binding domain); ii) a co—stimulatory domain; iii) a first member of a dimerization pair (e.g., a dimerizer—binding pair); iV) a transmembrane domain interposed between the member of a specific binding pair (e. g., an antigen—binding domain) and the co—stimulatory domain; and b) a second polypeptide comprising: i) a second member of the zation pair (e. g., the dimerizer— binding pair); and ii) an intracellular signaling domain.

An example of a subject CAR is ented schematically in Figure 17. A CAR of the present disclosure can be t in the plasma membrane of a eukaryotic cell, e. g., a mammalian cell, where suitable mammalian cells include, but are not limited to, a cytotoxic cell, a T lymphocyte, a stem cell, a progeny of a stem cell, a progenitor cell, a y of a itor cell, and an NK cell. When present in the plasma membrane of a eukaryotic cell, a CAR of the present disclosure is active in the presence of: l) a zing agent binds to the first and second members of the dimerizer—binding pair in the CAR, or otherwise induces dimerization of the first and second members of the dimer; and 2) a factor that binds the member of a specific binding pair (e. g., an antigen—binding domain), e. g., an antigen that binds the antigen—binding domain of the CAR. The factor that binds the member of the specific binding pair is a second member of the ic binding pair. The second member of the specific binding pair can be a soluble (e. g., not bound to a cell) factor; a factor present on the surface of a cell such as a target cell; a factor presented on a solid surface; a factor present in a lipid bilayer; and the like. Where the member of a specific binding pair is an antibody, and the second member of the specific binding pair is an antigen, the antigen can be a soluble (e. g., not bound to a cell) antigen; an n present WO 27261 on the surface of a cell such as a target cell; an antigen presented on a solid surface; an antigen present in a lipid bilayer; and the like.

In some instances, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell, and when activated by a second member of a specific binding pair that binds the member of the specific—binding pair of the CAR (e. g., an n that binds the antigen—binding domain of the CAR) and a dimerizing agent, increases expression of at least one nucleic acid in the cell. For example, in some cases, a CAR of the present disclosure, when present in the plasma ne of a eukaryotic cell, and when activated by an n that binds the antigen—binding domain of the CAR and a dimerizing agent, increases expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2—fold, at least about 2.5—fold, at least about 5—fold, at least about lO—fold, or more than lO—fold, compared with the level of transcription of the nucleic acid in the absence of the antigen and/or the dimerizing agent.

As an example, the second polypeptide of a CAR of the present disclosure can include an immunoreceptor tyrosine—based activation motif (ITAM)—containing intracellular signaling polypeptide; in such cases, a CAR of the present disclosure, when present in the plasma ne of a eukaryotic cell, and when activated by an antigen that binds the antigen— binding domain of the CAR and a dimerizing agent, increases nuclear factor of activated T cells (NFAT)—dependent transcription. NFAT—dependent transcription es transcription induced by any member of the NFAT family, ing, e. g., , NFATc2, NFATc3, NFATc4, NFAT5; AP—l; Spl; NKKB; and the like.

A CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds the n—binding domain of the CAR and a dimerizing agent, can, in some instances, result in increased production of one or more cytokines by the cell. For example, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds the antigen—binding domain of the CAR and a dimerizing agent, can increase production of a ne by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about , at least about 2.5—fold, at least about 5—fold, at least about lO—fold, or more than d, compared with the amount of cytokine produced by the cell in the absence of the antigen and/or the dimerizing agent. Cytokines whose tion can be increased include, but are not limited to, an interferon, e. g., IL—2, interferon gamma (IFN—y), tumor necrosis factor—alpha (TNF—(x), IL—l5, IL—12, IL—4, IL—5, IL—lO; a chemokine; a growth factor; and the like.

In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds the antigen—binding domain of the CAR and a dimerizing agent, can result in both an increase in transcription of a nucleic acid in the cell and an increase in production of a cytokine by the cell.

In some instances, a CAR of the present disclosure, when present in the plasma ne of a eukaryotic cell, and when activated by a dimerizing agent, s in cytotoxic activity by the cell toward a target cell that expresses on its cell e an antigen to which the antigen—binding domain of the first polypeptide of the CAR binds. For example, where the eukaryotic cell is a cytotoxic cell (e. g., an NK cell or a cytotoxic T lymphocyte), a CAR of the present sure, when present in the plasma membrane of the cell, and when activated by a dimerizing agent, increases cytotoxic activity of the cell toward a target cell that expresses on its cell surface an antigen to which the n—binding domain of the first polypeptide of the CAR binds. For example, where the eukaryotic cell is an NK cell or a T lymphocyte, a CAR of the present disclosure, when present in the plasma membrane of the cell, and when activated by a dimerizing agent, increases cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about %, at least about 40%, at least about 50%, at least about 75%, at least about 2—fold, at least about 2.5—fold, at least about 5—fold, at least about lO—fold, or more than lO—fold, compared to the cytotoxic activity of the cell in the e of the dimerizing agent.

In some cases, a CAR of the t disclosure, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds the antigen—binding domain of the CAR and a dimerizing agent, can result in other CAR activation d events such as proliferation and expansion (either due to increased cellular division or anti—apoptotic ses).

In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds the antigen—binding domain of the CAR and a dimerizing agent, can result in other CAR tion related events such as intracellular signaling tion, cellular differentiation, or cell death.

A CAR of the present sure can be present in a otic cell membrane, where the first and second polypeptides of the CAR are not covalently linked to one another. A CAR of the present disclosure can be present in a eukaryotic cell membrane as a single heterodimer that is not covalently linked to any other polypeptide in the membrane.

Alternatively, a first CAR of the t disclosure can be present in a eukaryotic cell ne as a heterodimer that is covalently or valently linked to a second CAR of the present disclosure. In some cases, the first and the second CAR are covalently linked via a disulfide bond formed between cysteines present in a hinge region present in both the first polypeptide of the first CAR and the first polypeptide of the second CAR.

In some cases, a CAR of the present disclosure can be present in a eukaryotic cell membrane, where the first polypeptides of the CAR comprise an antibody fragment and the second polypeptides of the CAR comprise a signal transducing domain derived from a cytokine receptor, such that, upon zation, the CAR may represent a heterodimeric— signalobody CAR, e. g., a signalobody composed of at least two independent polypeptides.

A “signalobody”, as it is known in the art, is a single chimeric macromolecule composed of an antibody fragment and a signal uction domain derived from a cytokine receptor. In n instances, a heterodimeric—signalobody CAR of the present disclosure, when present in the cell membrane of a otic cell, dimerized by a dimerizer, and ted by an antigen, e. g., an oligomerized antigen, may induce the oligomerization of the dimeric— signalobody CAR. Such ligand—induced oligomerization of a heterodimeric—signalobody CAR may activate, e. g., increase, or perpetuate, e. g., maintain, signal transduction, e.g., ligand—induced oligomerization of a heterodimeric—signalobody CAR may transmit a signal eliciting a cellular response. In some instances, a ity of dimeric—signalobody CARs may be utilized combinatorially to elicit a desired cellular response.

Member of a specific binding pair A CAR of the present disclosure includes a member of a specific binding pair. Specific binding pairs include, but are not limited to, antigen—antibody binding pairs; ligand—receptor binding pairs; and the like. Thus, a member of a specific binding pair suitable for use in a CAR of the present disclosure includes an n; an antibody; a ligand; and a ligand— binding receptor.

Antigen-binding domain An antigen—binding domain suitable for use in a CAR of the present disclosure can be any antigen—binding polypeptide, a wide variety of which are known in the art. In some instances, the antigen—binding domain is a single chain Fv . Other antibody based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable s are suitable for use. In some instances, T—cell receptor (TCR) based recognition domains such as single chain TCR (sch, single chain two—domain TCR containing VOLVB) are also suitable for use.

An antigen—binding domain suitable for use in a CAR of the present disclosure can have a variety of n—binding specificities. In some cases, the n—binding domain is specific for an epitope present in an antigen that is expressed by (synthesized by) a cancer cell, i.e., a cancer cell associated n. The cancer cell ated antigen can be an antigen associated with, e. g., a breast cancer cell, a B cell ma, a Hodgkin lymphoma cell, an n cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e. g., a small cell lung cancer cell), a non—Hodgkin B—cell lymphoma (B—NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancer cell associated antigen may also be expressed by a non—cancerous cell.

Non—limiting examples of antigens to which an antigen—binding domain of a subject CAR can bind include, e.g., CD19, CD20, CD38, CD30, Her2/neu, ERBBZ, CA125, MUC—l, prostate—specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, ar endothelial growth factor receptor—2 (VEGFRZ), high molecular weight—melanoma ated antigen (HMW—MAA), MAGE—Al, IL—l3R—a2, GD2, and the like.

Ligand In some cases, a member of a specific binding pair suitable for use in a subject CAR is a ligand for a receptor. Ligands include, but are not limited to, cytokines (e. g., IL—l3, etc.); growth factors (e. g., heregulin; vascular endothelial growth factor (VEGF); and the like); an integrin—binding peptide (e. g., a peptide comprising the sequence Arg—Gly—Asp); and the like.

Where the member of a specific g pair in a subject CAR is a ligand, the CAR can be activated in the presence of both a dimerizer agent and a second member of the specific binding pair, where the second member of the specific binding pair is a receptor for the ligand. For e, where the ligand is VEGF, the second member of the specific binding pair can be a VEGF receptor, including a e VEGF receptor. As another example, where the ligand is heregulin, the second member of the specific binding pair can be Her2.

Receptors As noted above, in some cases, the member of a specific g pair that is included in a t CAR is a or, e. g., a receptor for a ligand, a co—receptor, etc. The receptor can be a —binding fragment of a receptor. Suitable receptors include, but are not limited to, a growth factor receptor (e.g., a VEGF receptor); a killer cell lectin—like receptor subfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); a ne receptor (e. g., an IL—13 receptor; an IL—2 receptor; etc.); Her2; CD27; a natural cytotoxicity receptor (NCR) (e. g., NKP30 (NCR3/CD337) ptide (receptor for HLA—B—associated transcript 3 (BAT3) and B7—H6); etc.); etc.

Hinge region In some cases, the first polypeptide of a subject CAR comprises a hinge region (also referred to herein as a “spacer”), where the hinge region is interposed between the antigen— binding domain and the transmembrane domain. In some cases, the hinge region is an immunoglobulin heavy chain hinge region. In some cases, the hinge region is a hinge region polypeptide derived from a receptor (e. g., a CD8—derived hinge region).

The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about aa to about 40 aa, or from about 40 aa to about 50 aa.

Suitable spacers can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e. g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary spacers include glycine polymers (G)n, glycine—serine polymers (including, for example, (GS)n, (GSGGS)Il (SEQ ID NO:37) and (GGGS)Il (SEQ ID , where n is an integer of at least one), glycine—alanine polymers, alanine—serine polymers, and other ﬂexible linkers known in the art. Glycine and glycine—serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine es significantly more phi—psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. ational Chem. 11173—142 ). ary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:39), GGSGG (SEQ ID NO:40), GSGSG (SEQ ID NO:41), GSGGG (SEQ ID , GGGSG (SEQ ID NO:43), GSSSG (SEQ ID NO:44), and the like.

In some cases, the hinge region in the first polypeptide of a subject CAR includes at least one ne. For example, in some cases, the hinge region can include the sequence Cys— o—Cys. If present, a cysteine in the hinge region of a first CAR can be available to form a disulfide bond with a hinge region in a second CAR.

Irnmunoglobulin hinge region amino acid sequences are known in the art; see, e. g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14: 1779. As non—limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO:45); CPPC (SEQ ID NO:46); CDTPPPCPR (SEQ ID NO:47) (see, e. g., Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO:48); KSCDKTHTCP (SEQ ID NO:49); P (SEQ ID NO:50); KYGPPCP (SEQ ID NO:51); EPKSCDKTHTCPPCP (SEQ ID NO:52) (human IgGl hinge); ERKCCVECPPCP (SEQ ID NO:53) (human IgG2 hinge); GDTTHTCPRCP (SEQ ID NO:54) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:55) (human IgG4 hinge); and the like.

The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. The hinge region can e one or more amino acid substitutions and/or insertions and/or deletions compared to a wild—type (naturally—occurring) hinge region. For example, H18229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO:52); see, e. g., Yan et al. (2012) J. Biol. Chem. 287:5891.

The hinge region can comprise an amino acid sequence derived from human CD8; e. g., the hinge region can comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:56), or a variant thereof.

Transmembrane domain The first and the second polypeptides of a CAR of the present disclosure include transmembrane domains for insertion into a eukaryotic cell membrane. The transmembrane domain of the first polypeptide is interposed between the antigen—binding domain and the co—stimulatory domain. Where the first polypeptide includes a hinge region, the transmembrane domain is interposed between the hinge region and the co—stimulatory domain, such that the first ptide comprises, in order from the amino us (N— terrninus) to the carboxyl terminus (C—terminus): an antigen—binding domain; a hinge region; a transmembrane domain; a first co—stimulatory domain; and a first member of a dimerizer—binding pair.

The transmembrane domain of the second polypeptide is at or near the N—terminus of the polypeptide, such that the second polypeptide comprises, in order from N—terminus to C— terrninus: a transmembrane domain; a second co—stimulatory domain; a second member of the dimerizer—binding pair; and an intracellular signaling domain.

Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e. g., mammalian) cell is suitable for use. As one non—limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30) can be used. Additional non—limiting examples of suitable TM sequences include: a) CD8 beta derived : LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO:57); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO:58); c) CD3 zeta derived: GILFIYGVILTALFLRV (SEQ ID ; d) CD28 derived: WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:60); e) CD134 (0X40) derived: VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID ; and f) CD7 derived: ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO:62).

Linkers In some cases, a first polypeptide of a subject CAR includes a linker between any two adjacent domains. For example, a linker can be disposed between the transmembrane domain and the first co—stimulatory domain of the first polypeptide. As another example, a linker can be disposed between the first co—stimulatory domain and the first member of a dimerizer—binding pair of the first polypeptide. As another example, a linker can be disposed n the embrane domain and the second co—stimulatory domain of the second polypeptide. As r e, a linker can be disposed between the second co— stimulatory domain and the second member of the dimerizer—binding pair of the second polypeptide. As another example, a linker can be disposed n the second member of the dimerizer—binding pair and the intracellular signaling domain of the second polypeptide.

The linker peptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer e, generally of a ﬂexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in , or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker—encoding oligonucleotides to couple the ns.

Peptide linkers with a degree of ﬂexibility can be used. The linking peptides may have lly any amino acid sequence, bearing in mind that suitable linkers will have a WO 27261 sequence that results in a generally ﬂexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a ﬂexible peptide. The creation of such ces is routine to those of skill in the art.

Suitable linkers can be y selected and can be of any of a le of different s, such as from 1 amino acid (e. g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary ﬂexible linkers include glycine polymers (G)n, glycine—serine polymers ding, for example, (GS)n, GSGGSIl (SEQ ID NO:37) and GGGSIl (SEQ ID NO:38), where n is an integer of at least one), glycine—alanine polymers, e—serine polymers, and other ﬂexible linkers known in the art. Glycine and e—serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components. Glycine polymers are of particular st since glycine accesses significantly more phi—psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. lll73—l42 (1992)). Exemplary ﬂexible linkers include, but are not limited GGSG (SEQ ID NO:39), GGSGG (SEQ ID NO:40), GSGSG (SEQ ID NO:4l), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID NO:43), GSSSG (SEQ ID NO:44), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially ﬂexible, such that the linker can include a ﬂexible linker as well as one or more portions that confer less ﬂexible structure.

Modulatory s Modulatory domains suitable for use in a CAR of the present disclosure include co— stimulatory domains.

In some cases, the modulatory domain on the first polypeptide of a subject CAR has substantially the same amino acid sequence as the tory domain on the second polypeptide of the CAR. For example, in some cases, the tory domain on the first polypeptide of a CAR ses an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, identical to the amino acid sequence of the modulatory domain on the second polypeptide of the CAR. The modulatory domain of the first polypeptide of a subject CAR can have substantially the same length as the modulatory domain of the second polypeptide of a subject CAR; e. g., the first and second modulatory domains can differ in length from one another by fewer than 10 amino acids, or fewer than 5 amino acids. In some cases, the first and second modulatory domains have the same length.

A modulatory domain suitable for inclusion in the first and the second polypeptide of a subject CAR can have a length of from about 30 amino acids to about 70 amino acids (aa), e.g., a modulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In other cases, modulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

Co—stimulatory domains suitable for use in a CAR of the t disclosure are generally ptides derived from receptors. In some embodiments, co—stimulatory domains homodimerize. A t co—stimulatory domain can be an intracellular portion of a transmembrane protein (i.e., the mulatory domain can be derived from a transmembrane protein). Non—limiting examples of suitable co—stimulatory polypeptides e, but are not limited to, 4—lBB (CD137), CD28, ICOS, OX—40, BTLA, CD27, CD30, GITR, and HVEM.

In some cases, the co—stimulatory domain on the first polypeptide of a t CAR has substantially the same amino acid sequence as the co—stimulatory domain on the second polypeptide of the CAR. For e, in some cases, the co—stimulatory domain on the first polypeptide of a CAR comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, identical to the amino acid ce of the co—stimulatory domain on the second polypeptide of the CAR. The co— stimulatory domain of the first polypeptide of a subject CAR can have substantially the same length as the co—stimulatory domain of the second polypeptide of a subject CAR; e.g., the first and second co—stimulatory domains can differ in length from one another by fewer than 10 amino acids, or fewer than 5 amino acids. In some cases, the first and second co— stimulatory domains have the same length.

A co—stimulatory domain suitable for inclusion in the first and the second polypeptide of a subject CAR can have a length of from about 30 amino acids to about 70 amino acids (aa), e. g., a co—stimulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In other cases, the co— WO 27261 stimulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

In some cases, the co—stimulatory domain is derived from an intracellular portion of the transmembrane protein 4—lBB (also known as TNFRSF9; CDl37; 4—lBB; Cle37; ILA; etc.). For example, a suitable co—stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:24). In some of these embodiments, the co—stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some cases, the co—stimulatory domain is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable co— stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid ce identity to the following amino acid sequence: RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:63).

In some of these embodiments, the co—stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some cases, the co—stimulatory domain is derived from an intracellular portion of the embrane protein ICOS (also known as AILIM, CD278, and CVIDl). For example, a suitable co—stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence ty to the following amino acid TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO:64). In some of these embodiments, the co—stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 In some cases, the co—stimulatory domain is derived from an intracellular portion of the transmembrane protein OX—40 (also known as TNFRSF4, RP5—902P8.3, ACT35, CDl34, 0X40, TXGPlL). For example, a suitable co—stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid ce identity to the following amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:65). In some of these ments, the co—stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 In some cases, the co—stimulatory domain is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLAl and . For example, a suitable co—stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid ce: CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPD LCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVR S (SEQ ID NO:66).

In some cases, the co—stimulatory domain is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, Tl4, TNFRSF7, and Tp55). For example, a suitable co—stimulatory domain can comprise an amino acid ce having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:67). In some of these embodiments, the co—stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some cases, the co—stimulatory domain is derived from an intracellular portion of the transmembrane protein CD30 (also known as 8, DlSl66E, and Ki—l). For example, a suitable co—stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, or from about 160 aa to about 185 aa of the following amino acid sequence: RIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMS QPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKA DTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSV EEEGKEDPLPTAASGK (SEQ ID NO:68).

In some cases, the co—stimulatory domain is derived from an ellular portion of the transmembrane protein GITR (also known as TNFRSFl8, RP5—902P8.2, AITR, CD357, and GITR—D). For example, a suitable co—stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO:69). In some of these embodiments, the co—stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some cases, the co—stimulatory domain derived from an intracellular portion of the transmembrane protein HVEM (also known as TNFRSFl4, RP3—395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable co—stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid ce identity to the ing amino acid ce: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRS PNH (SEQ ID NO:70). In some of these embodiments, the co—stimulatory domain of both the first and the second polypeptide has a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

Dimer pairs Dimer pairs suitable for use in a subject CAR include dimerizer—binding pairs.

Dimerizer—binding pairs suitable for use in a CAR of the present disclosure are in some embodiments polypeptides that bind to a different site of the same molecule (referred to herein as a “dimerizer”). In the presence of a dimerizer, both members of the dimerizer— binding pair bind to a different site of the dimerizer and are thus t into ity with one another. In some ments, binding to the dimerizer is reversible. In some embodiments, g to the zer is irreversible. In some embodiments, binding to the dimerizer is non—covalent. In some embodiments, binding to the dimerizer is nt.

Other dimer pairs suitable for use e dimerizer—binding pairs that ze upon binding of a first member of a dimer pair to a dimerizing agent, where the dimerizing agent induces a conformational change in the first member of the dimer pair, and where the conformational change allows the first member of the dimer pair to bind ently or non— covalently) to a second member of the dimer pair.

Other dimer pairs suitable for use include dimer pairs in which exposure to light (e. g., blue light) induces dimerization of the dimer pair.

Regardless of the mechanism, the dimer pair will dimerize upon exposure to an agent that induces dimerization, where the agent is in some cases a small molecule, or, in other cases, light. Thus, for simplicity, the discussion below referring to “dimerizer—binding pairs” includes dimer pairs that dimerize regardless of the mechanism.

Non—limiting examples of suitable dimers (e. g., dimerizer—binding pairs) include, but are not limited to: a) FK506 binding protein (FKBP) and FKBP; b) FKBP and calcineurin tic subunit A (CnA); c) FKBP and cyclophilin; d) FKBP and FKBP—rapamycin associated protein (FRB); e) gyrase B (GyrB) and GyrB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB; 2014/016527 h) PYL and ABI; i) Cry2 and CIBl; and j) GAI and GIDl.

A first or a second member of a dimer (e.g., a dimerizer—binding pair) of a subject CAR can have a length of from about 50 amino acids to about 300 amino acids or more; e. g., a first or a second member of a dimer (e.g., a dimerizer—binding pair) of a subject CAR can have a length of from about 50 aa to about 100 aa, from about 100 aa to about 150 aa, from about 150 aa to about 200 aa, from about 200 aa toa bout 250 aa, from about 250 aa to about 300 aa, or more than 300 aa.

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) of a subject CAR is derived from FKBP. For example, a suitable zer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 12).

In some cases, a member of a dimerizer—binding pair of a subject CAR is derived from calcineurin catalytic subunit A (also known as PPP3CA; CALN; CALNA; CALNAl; CCNl; CNAl; PPP2B; CAM—PRP catalytic subunit; calcineurin A alpha; calmodulin— dependent calcineurin A subunit alpha isoform; protein phosphatase 2B, catalytic subunit, alpha isoform; etc.). For e, a suitable dimerizer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence (PP2Ac domain): LRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRY VDRGYFSIECVLYLWALKILYPKTLFLLRGNHECRHLTEYFTFKQECKIKY SERVYDACMDAFDCLPLAALMNQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGP SDPLEDFGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHE AQDAGYRMYRKSQTTGFPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSP HPYWLPNFM (SEQ ID NO:7l).

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from cyclophilin (also known cyclophilin A, PPIA, CYPA, CYPH, PPIase A, etc.). For example, a suitable dimerizer—binding pair member can se an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRII PGFMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFI CTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE (SEQ ID NO:72).

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from MTOR (also known as FKBP—rapamycin associated protein; FK506 binding protein 12— cin associated protein 1; FK506 binding protein 12—rapamycin associated protein 2; FK506—binding protein 12—rapamycin x—associated n 1; FRAP; FRAPl; FRAPZ; RAFTl; and RAPTl). For example, a suitable dimerizer—binding pair member can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence (also known as “Frb”: Fkbp— Rapamycin g Domain): MILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 14).

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from GyrB (also known as DNA gyrase subunit B). For example, a suitable dimerizer—binding pair member can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous h of from about 100 amino acids to about 200 amino acids (aa), from about 200 aa to about 300 aa, from about 300 aa to about 400 aa, from about 400 aa to about 500 aa, from about 500 aa to about 600 aa, from about 600 aa to about 700 aa, or from about 700 aa to about 800 aa, of the following GyrB amino acid sequence from Escherichia coli (or to the DNA gyrase subunit B sequence from any organism): MSNSYDSSSIKVLKGLDAVRKRPGMYIGDTDDGTGLHHMVFEVVDNAIDEALAGH CKEIIVTIHADNSVSVQDDGRGIPTGIHPEEGVSAAEVIMTVLHAGGKFDDNSYKVS GGLHGVGVSVVNALSQKLELVIQREGKIHRQIYEHGVPQAPLAVTGETEKTGTMV ETFTNVTEFEYEILAKRLRELSFLNSGVSIRLRDKRDGKEDHFHYEGGIKAF VEYLNKNKTPIHPNIFYFSTEKDGIGVEVALQWNDGFQENIYCFTNNIPQRDGGTHL AGFRAAMTRTLNAYMDKEGYSKKAKVSATGDDAREGLIAVVSVKVPDPKFSSQT KDKLVSSEVKSAVEQQMNELLAEYLLENPTDAKIVVGKIIDAARAREAARRAREM TRRKGALDLAGLPGKLADCQERDPALSELYLVEGDSAGGSAKQGRNRKNQAILPL KGKILNVEKARFDKMLSSQEVATLITALGCGIGRDEYNPDKLRYHSIIIMTDADVDG LLTFFYRQMPEIVERGHVYIAQPPLYKVKKGKQEQYIKDDEAMDQYQISIA LDGATLHTNASAPALAGEALEKLVSEYNATQKMINRMERRYPKAMLKELIYQPTL TEADLSDEQTVTRWVNALVSELNDKEQHGSQWKFDVHTNAEQNLFEPIVRVRTHG VDTDYPLDHEFITGGEYRRICTLGEKLRGLLEEDAFIERGERRQPVASFEQALDWLV KESRRGLSIQRYKGLGEMNPEQLWETTMDPESRRMLRVTVKDAIAADQLFTTLMG DAVEPRRAFIEENALKAANIDI (SEQ ID NO:73). In some cases, a member of a dimerizer—binding pair ses an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to amino acids 1—220 of the above—listed GyrB amino acid sequence from Escherichia coli.

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from DHFR (also known as dihydrofolate reductase, DHFRPl, and DYR). For example, a suitable dimerizer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMTTTSSVEGKQNLVIMGK KTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDM VWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEYPGVLS DVQEEKGIKYKFEVYEKND (SEQ ID NO:74).

] In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from the DmrB binding domain (i.e., DmrB homodimerization domain). For e, a le dimerizer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: QVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLG KQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID .

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from a PYL protein (also known as abscisic acid receptor and as RCAR). For example a member of a subject dimerizer—binding pair can be derived from proteins such as those of Arabidopsis thaliana: PYRl, RCAR1(PYL9), PYLl, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8 (RCAR3), PYL10, PYLl 1, PYL12, PYL13. For example, a suitable dimerizer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid ces: PYL10: MNGDETKKVESEYIKKHHRHELVESQCSSTLVKHIKAPLHLVWSIVRRFDEPQKYK PFISRCVVQGKKLEVGSVREVDLKSGLPATKSTEVLEILDDNEHILGIRIVGGDHRLK NYSSTISLHSETIDGKTGTLAIESFVVDVPEGNTKEETCFFVEALIQCNLNSLADVTE RLQAESMEKKI (SEQ ID NO:76).

PYLl 1: METSQKYHTCGSTLVQTIDAPLSLVWSILRRFDNPQAYKQFVKTCNLSSGDGGEGS VREVTVVSGLPAEFSRERLDELDDESHVMMISIIGGDHRLVNYRSKTMAFVAADTE ESYVVDVPEGNSEEETTSFADTIVGFNLKSLAKLSERVAHLKL (SEQ ID NO:77) PYL12: MKTSQEQHVCGSTVVQTINAPLPLVWSILRRFDNPKTFKHFVKTCKLRSGDGGEGS VREVTVVSDLPASFSLERLDELDDESHVMVISIIGGDHRLVNYQSKTTVFVAAEEEK TVVVESYVVDVPEGNTEEETTLFADTIVGCNLRSLAKLSEKMMELT (SEQ ID NO:78).

PYL13: MESSKQKRCRSSVVETIEAPLPLVWSILRSFDKPQAYQRFVKSCTMRSGGGGGKGG EGKGSVRDVTLVSGFPADFSTERLEELDDESHVMVVSIIGGNHRLVNYKSKTKVVA KKTVVVESYVVDVPEGTSEEDTIFFVDNIIRYNLTSLAKLTKKMMK (SEQ ID NO:79).

PYL1: MANSESSSSPVNEEENSQRISTLHHQTMPSDLTQDEFTQLSQSIAEFHTYQLGNGRC SSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGL PANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLE SYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMNRNNNNNNSSQVR (SEQ ID PYL2: MSSSPAVKGLTDEEQKTLEPVIKTYHQFEPDPTTCTSLITQRIHAPASVVWPLIRRFD NPERYKHFVKRCRLISGDGDVGSVREVTVISGLPASTSTERLEFVDDDHRVLSFRVV GGEHRLKNYKSVTSVNEFLNQDSGKVYTVVLESYTVDIPEGNTEEDTKMFVDTVV KLNLQKLGVAATSAPMHDDE (SEQ ID N081).

PYL3: MNLAPIHDPSSSSTTTTSSSTPYGLTKDEFSTLDSIIRTHHTFPRSPNTCTSLIAHRVDA PAHAIWRFVRDFANPNKYKHFIKSCTIRVNGNGIKEIKVGTIREVSVVSGLPASTSVE ILEVLDEEKRILSFRVLGGEHRLNNYRSVTSVNEFVVLEKDKKKRVYSVVLESYIVD IPQGNTEEDTRMFVDTVVKSNLQNLAVISTASPT (SEQ ID N082).

PYL4: MLAVHRPSSAVSDGDSVQIPMMIASFQKRFPSLSRDSTAARFHTHEVGPNQCCSAVI QEISAPISTVWSVVRRFDNPQAYKHFLKSCSVIGGDGDNVGSLRQVHVVSGLPAAS STERLDILDDERHVISFSVVGGDHRLSNYRSVTTLHPSPISGTVVVESYVVDVPPGNT FVDVIVRCNLQSLAKIAENTAAESKKKMSL (SEQ ID NO:83).

PYLS: MRSPVQLQHGSDATNGFHTLQPHDQTDGPIKRVCLTRGMHVPEHVAMHHTHDVG PDQCCSSVVQMIHAPPESVWALVRRFDNPKVYKNFIRQCRIVQGDGLHVGDLREV MVVSGLPAVSSTERLEILDEERHVISFSVVGGDHRLKNYRSVTTLHASDDEGTVVV ESYIVDVPPGNTEEETLSFVDTIVRCNLQSLARSTNRQ (SEQ ID N084).

PYL6: MPTSIQFQRSSTAAEAANATVRNYPHHHQKQVQKVSLTRGMADVPEHVELSHTHV VGPSQCFSVVVQDVEAPVSTVWSILSRFEHPQAYKHFVKSCHVVIGDGREVGSVRE VRVVSGLPAAFSLERLEIMDDDRHVISFSVVGGDHRLMNYKSVTTVHESEEDSDGK KRTRVVESYVVDVPAGNDKEETCSFADTIVRCNLQSLAKLAENTSKFS (SEQ ID N085).

PYL7: MEMIGGDDTDTEMYGALVTAQSLRLRHLHHCRENQCTSVLVKYIQAPVHLVWSL VRRFDQPQKYKPFISRCTVNGDPEIGCLREVNVKSGLPATTSTERLEQLDDEEHILGI NIIGGDHRLKNYSSILTVHPEMIDGRSGTMVMESFVVDVPQGNTKDDTCYFVESLIK ACVSERLAAQDITNSIATFCNASNGYREKNHTETNL (SEQ ID NO:86).

PYL8: MEANGIENLTNPNQEREFIRRHHKHELVDNQCSSTLVKHINAPVHIVWSLVRRFDQ FISRCVVKGNMEIGTVREVDVKSGLPATRSTERLELLDDNEHILSIRIVGGD HRLKNYSSIISLHPETIEGRIGTLVIESFVVDVPEGNTKDETCYFVEALIKCNLKSLAD ISERLAVQDTTESRV (SEQ ID N087).

PYL9: MMDGVEGGTAMYGGLETVQYVRTHHQHLCRENQCTSALVKHIKAPLHLVWSLV RRFDQPQKYKPFVSRCTVIGDPEIGSLREVNVKSGLPATTSTERLELLDDEEHILGIKI IGGDHRLKNYSSILTVHPEIIEGRAGTMVIESFVVDVPQGNTKDETCYFVEALIRCNL KSLADVSERLASQDITQ (SEQ ID NO:88).

PYRl: MPSELTPEERSELKNSIAEFHTYQLDPGSCSSLHAQRIHAPPELVWSIVRRFDKPQTY KHFIKSCSVEQNFEMRVGCTRDVIVISGLPANTSTERLDILDDERRVTGFSIIGGEHR LTNYKSVTTVHRFEKENRIWTVVLESYVVDMPEGNSEDDTRMFADTVVKLNLQKL ATVAEAMARNSGDGSGSQVT (SEQ ID NO:89).

] In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from an ABI protein (also known as Abscisic Acid—Insensitive). For example a member of a subject dimerizer—binding pair can be d from proteins such as those of Arabidopsis thaliana: ABIl (Also known as IC ACID—INSENSITIVE 1, Protein phosphatase 2C 56, AtPP2C56, P2C56, and PP2C ABIl) and/or AB12(also known as P2C77, Protein phosphatase 2C 77, AtPP2C77, ABSCISIC NSENSITIVE 2, Protein phosphatase 2C ABIZ, and PP2C ABIZ). For example, a suitable dimerizer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of any of the following amino acid sequences: ABIl: AIAGPFRPFSETQMDFTGIRLGKGYCNNQYSNQDSENGDLMVSLPETSSCS VSGSHGSESRKVLISRINSPNLNMKESAAADIVVVDISAGDEINGSDITSEKKMISRT ESRSLFEFKSVPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFF GVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLR VDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDRE DEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDD CLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPA AMSAAEYLSKLAIQRGSKDNISVVVVDLKPRRKLKSKPLN (SEQ ID NO:90).

ABIZ: MDEVSPAVAVPFRPFTDPHAGLRGYCNGESRVTLPESSCSGDGAMKDSSFEINTRQ DSLTSSSSAMAGVDISAGDEINGSDEFDPRSMNQSEKKVLSRTESRSLFEFKCVPLY GVTSICGRRPEMEDSVSTIPRFLQVSSSSLLDGRVTNGFNPHLSAHFFGVYDGHGGS QVANYCRERMHLALTEEIVKEKPEFCDGDTWQEKWKKALFNSFMRVDSEIETVAH APETVGSTSVVAVVFPTHIFVANCGDSRAVLCRGKTPLALSVDHKPDRDDEAARIE AAGGKVIRWNGARVFGVLAMSRSIGDRYLKPSVIPDPEVTSVRRVKEDDCLILASD GLWDVMTNEEVCDLARKRILLWHKKNAMAGEALLPAEKRGEGKDPAAMSAAEY LSKMALQKGSKDNISVVVVDLKGIRKFKSKSLN (SEQ ID NO:9l).

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from a Cry2 protein (also known as cryptochrome 2). For example a member of a subject dimer (e.g., a dimerizer—binding pair) can be derived from Cry2 proteins from any organism (e.g., a plant) such as, but not limited to, those of Arabidopsis thaliana. For example, a suitable dimerizer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid ce ty to a uous h of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of any of the following amino acid sequences: Cry2 (Arabidopsis thaliana) MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWW MKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRD LVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVML PPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFI EKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTG YPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADL ECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHH PWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPDEIVA DSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFD ERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK (SEQ ID NO:92).

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from the CIBl Arabidopsis na protein (also known as transcription factor bHLH63). For example, a le dimer (e.g., a dimerizer—binding pair) member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a uous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of the following amino acid sequence: GDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYL STAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMT MNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKT DYIHVRARRGQATDSHSIAERVRREKISERMKFLQDLVPGCDKITGKAGMLDEIINY VQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAKEVASTPMTVVPSPEMVLSGYSHEM VHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPSMWDSHVQNLYGNLG V (SEQ ID NO:93).

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is derived from the GAIArabidopsis thaliana protein (also known as Gibberellic Acid Insensitive, and DELLA protein GAI). For example, a suitable dimerizer—binding pair member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of the following amino acid sequence: MKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQ LEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPPSSNAEYDLKAIPG DAILNQFAIDSASSSNQGGGGDTYTTNKRLKCSNGVVETTTATAESTRHVVLVDSQ VHALLACAEAVQKENLTVAEALVKQIGFLAVSQIGAMRKVATYFAEALA RRIYRLSPSQSPIDHSLSDTLQMHFYETCPYLKFAHFTANQAILEAFQGKKRVHVIDF WO 27261 SMSQGLQWPALMQALALRPGGPPVFRLTGIGPPAPDNFDYLHEVGCKLAHLAEAIH VEFEYRGFVANTLADLDASMLELRPSEIESVAVNSVFELHKLLGRPGAIDKVLGVV NQIKPEIFTVVEQESNHNSPIFLDRFTESLHYYSTLFDSLEGVPSGQDKVMSEVYLGK ACDGPDRVERHETLSQWRNRFGSAGFAAAHIGSNAFKQASMLLALFNGG EGYRVEESDGCLMLGWHTRPLIATSAWKLSTN (SEQ ID NO:94).

In some cases, a member of a dimer (e.g., a dimerizer—binding pair) is d from a GIDl Arabidopsis thaliana protein (also known as Gibberellin receptor GIDl). For example, a suitable dimer member can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of any of the following amino acid sequences: GIDlA: MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTAN ANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHG GSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWV NSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNE RTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKS LVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVM DEISAFVNAEC (SEQ ID NO:95).

GIDlB: MAGGNEVNLNECKRIVPLNTWVLISNFKLAYKVLRRPDGSFNRDLAEFLDRKVPA NSFPLDGVFSFDHVDSTTNLLTRIYQPASLLHQTRHGTLELTKPLSTTEIVPVLIFFHG GSFTHSSANSAIYDTFCRRLVTICGVVVVSVDYRRSPEHRYPCAYDDGWNALNWV KSRVWLQSGKDSNVYVYLAGDSSGGNIAHNVAVRATNEGVKVLGNILLHPMFGG EKTLDGKYFVTIQDRDWYWRAYLPEGEDRDHPACNPFGPRGQSLKGVNF PKSLVVVAGLDLVQDWQLAYVDGLKKTGLEVNLLYLKQATIGFYFLPNNDHFHCL MEELNKFVHSIEDSQSKSSPVLLTP (SEQ ID NO:96) GIDlC: MAGSEEVNLIESKTVVPLNTWVLISNFKLAYNLLRRPDGTFNRHLAEFLDRKVPAN ANPVNGVFSFDVIIDRQTNLLSRVYRPADAGTSPSITDLQNPVDGEIVPVIVFFHGGS FAHSSANSAIYDTLCRRLVGLCGAVVVSVNYRRAPENRYPCAYDDGWAVLKWVN SSSWLRSKKDSKVRIFLAGDSSGGNIVHNVAVRAVESRIDVLGNILLNPMFGGTERT ESEKRLDGKYFVTVRDRDWYWRAFLPEGEDREHPACSPFGPRSKSLEGLSFPKSLV VVAGLDLIQDWQLKYAEGLKKAGQEVKLLYLEQATIGFYLLPNNNHFHTVMDEIA AFVNAECQ (SEQ ID NO:97).

Dimerizers Dimerizers (“dimerizing agents) that can provide for dimerization of a first member of a zer—binding pair and a second member of a dimerizer—binding pair include, e.g. (where the dimerizer is in parentheses following the dimerizer—binding pair: a) FKBP and FKBP (rapamycin); b) FKBP and CnA (rapamycin); c) FKBP and cyclophilin (rapamycin); d) FKBP and FRG (rapamycin); e) GyrB and GyrB (coumermycin); f) DHFR and DHFR (methotrexate); g) DmrB and DmrB (AP20187); h) PYL and ABI (abscisic acid); i) Cry2 and CIBl (blue light); and j) GAI and GIDl (gibberellin).

As noted above, rapamycin can serve as a dimerizer. Alternatively, a rapamycin derivative or analog can be used. See, e.g., WO96/41865; WO 99/36553; WO 01/14387; and Ye et al (1999) Science —91. For example, analogs, homologs, derivatives and other compounds related structurally to rapamycin logs") include, among , variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, ation or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or ement of the hydroxy at C13, C43 and/or C28; ion, elimination or derivatization of the ketone at C14, C24 and/or C30; ement of the 6—membered pipecolate ring with a 5—membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Additional information is presented in, e.g., US. Pat. Nos. 5,525,610; ,310,903 718; and 5,527,907. Selective epimerization of the C—28 hydroxyl group has been described; see, e.g., W0 01/14387. Additional tic dimerizing agents suitable for use as an alternative to rapamycin include those described in US. Patent Publication No. 2012/0130076.

Rapamycin has the structure: Rapamycin le rapalogs include, e.g., 28—epirapamycin Also suitable as a rapalog is a compound of the formula: where n is l or 2; R28 and R43 are ndently H, or a substituted or unsubstituted aliphatic or acyl moiety; one of R7a and R7b is H and the other is halo, RA, ORA, SRA, — 2014/016527 OC(O)RA, —OC(O)NRARB, —NRARB, —NRBC(OR)RA, )ORA, —NRBSOZRA, or NRARB’; or R7a and R7b, taken together, are H in the tetraene moiety: where RA is H or a substituted or tituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety and where RB and RB, are independently H, OH, or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety.

As noted above, coumermycin can serve as a dimerizing agent. Alternatively, a coumermycin analog can be used. See, e.g., Farrar et al. (1996) Nature 383: 178-181; and US. Pat. No. 846.

As noted above, in some cases, the dimerizing agent is methotrexate, e.g., a non— cytotoxic, ifunctional methotrexate dimer. See, e. g., US. Patent No. 8,236,925.

Intracellular signaling domain Intracellular signaling domains suitable for use in a CAR of the present disclosure include any d signaling domain that provides a distinct and able signal (e. g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e. g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to tion of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e. g., one, two, three, four, five, siX, etc.) ITAM motifs as described below. In some embodiments, the intracellular signaling domain es DAPlO/CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

ITAM Intracellular signaling domains suitable for use in a CAR of the present disclosure include immunoreceptor tyrosine—based activation motif (lTAM)—containing intracellular signaling polypeptides. An ITAM motif is YXngL/I, where X1 and X2 are independently any amino acid (SEQ ID NO: 130). In some cases, the intracellular signaling domain of a subject CAR ses 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e. g., (YX1X2L/I)(X3)n(YX1X2L/I), where n is an integer from 6 to 8, and each of the 6—8 X3 can be any amino acid (SEQ ID NO: 131). In some cases, the intracellular signaling domain of a subject CAR comprises 3 ITAM motifs.

A suitable intracellular signaling domain can be an ITAM motif—containing n that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif—containing domain from any ITAM motif—containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif—containing polypeptides include, but are not limited to: DAP12; FCERlG (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A (antigen receptor complex—associated protein alpha chain).

In some cases, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding n; KARAP; PLOSL; DNAX—activation protein 12; KAR—associated protein; TYRO protein tyrosine — binding n; killer ting receptor associated protein; killer—activating receptor— associated protein; etc.). For example, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to any of the ing amino acid sequences (4 isoforms): MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLI ALAVYFLGRLVPRGRGAAEAATRKQRITETESPMQGQRSDVYSiNTQRPYY K (SEQ ID NO:98); MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLI LGRLVPRGRGAAEATRKQRITETESPwQGQRSDVYSiNTQRPYYK (SEQ ID NO:99); MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLV PRGRGAAEAATRKQRITETESPwQGQRSDVYSiNTQRPYYK (SEQ ID NO:100); or MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLV AEATRKQRITETESPwQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 101), where the ITAM motifs are in bold and are underlined. se, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif—containing portion of the full length DAP12 amino acid sequence. Thus, a suitable intracellular ing domain ptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to the following amino acid ce: ESPMQGQRSDVYSiNTQ (SEQ ID NO: 102), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from FCERlG (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma—chain; fc—epsilon RI—gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). For example, a suitable ellular signaling domain polypeptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid ce identity to the following amino acid sequence: MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSY EKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 103), where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif—containing portion of the full length FCERlG amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise an amino acid ce having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid ce ty to the following amino acid sequence: DGVYTGLSTRNQETYETLKHE (SEQ ID NO: 104), where the ITAM motifs are in bold and are ined.

In some cases, the intracellular signaling domain is derived from T—cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3—DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 compleX); OKT3, delta chain; T—cell receptor T3 delta chain; T—cell surface glycoprotein CD3 delta chain; etc.). For example, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 170 aa, of either of the following amino acid sequences (2 isoforms): MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRL DLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIAT LLLALGVFCFAGHETGRLSGAADTQALLRNDQVMRDRDDAQﬂGGNWAR NK (SEQ ID NO:105) or MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRL LDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVMRDRDD AQﬂGGNWARNK (SEQ ID NO: 106) where the ITAM motifs are in bold and are underlined.

] Likewise, a suitable ellular signaling domain polypeptide can comprise an ITAM motif—containing portion of the full length CD3 delta amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to the following amino acid sequence: DQVMRDRDDAQﬂGGN (SEQ ID NO: 107), where the ITAM motifs are in bold and are underlined.

] In some cases, the intracellular signaling domain is derived from T—cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T—cell surface antigen T3/Leu—4 epsilon chain, T—cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, ilon, T3e, etc.). For example, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a uous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 205 aa, of the following amino acid sequence: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSE ILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYL YLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAG AGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 108), where the ITAM motifs are in bold and are underlined.

Likewise, a suitable ellular signaling domain ptide can comprise an ITAM motif—containing portion of the full length CD3 epsilon amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise an amino acid ce haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to the following amino acid ce: NPDYEPIRKGQRDLYSGLNQR (SEQ ID NO: 109), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T—cell surface glycoprotein CD3 gamma chain (also known as CD3G, T—cell receptor T3 gamma chain, MMA, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 180 aa, of the ing amino acid sequence: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWF KDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIEL NAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLMKDRE DDQﬂQGNQLRRN (SEQ ID NO:110), where the ITAM motifs are in bold and are underlined.

] Likewise, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif—containing portion of the full length CD3 gamma amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence ty to the following amino acid sequence: DQLMKDREDDQﬂQGN (SEQ ID NO:111), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T—cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T—cell receptor T3 zeta chain, CD247, CD3—ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa, of either of the following amino acid sequences (2 isoforms): MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRS QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLm ﬂQKDKMAEAYSEIGMKGERRRGKGHDGLMSTATKDTYDALHMQALPPR (SEQ ID NO:112) or MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLX ﬂQKDKMAEAYSEIGMKGERRRGKGHDGLMSTATKDTYDALHMQALPPR( SEQ ID NO:113) where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif—containing portion of the full length CD3 zeta amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise an amino acid ce haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid ce ty to any of the following amino acid sequences: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAEGMKGERRRGKGHDGLMSTATKDTYDALHMQ ALPPR (SEQ ID NO: 18); NQLYNELNLGRREEYDVLDKR (SEQ ID NO:114); EGLYNELQKDKMAEAEGMK (SEQ ID NO:115); or DGLMSTATKDTYDALHMQ (SEQ ID NO:116), where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from CD79A (also known as B—cell antigen or x—associated protein alpha chain; CD79a n (immunoglobulin—associated alpha); MB—l membrane glycoprotein; ig—alpha; membrane— bound immunoglobulin—associated protein; surface IgM—associated protein; etc.). For example, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 150 aa, from about 150 aa to about 200 aa, or from about 200 aa to about 220 aa, of either of the following amino acid sequences (2 ms): MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDA HFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYV CRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLL LFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMMSRGLQGTYQDVGSLN IGDVQLEKP (SEQ ID NO:117); or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDA HFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNR IITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYE DISRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO:118) where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif—containing portion of the full length CD79A amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise an amino acid sequence haVing at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to the ing amino acid sequence: ENLYEGLNLDDCSMYEDISRG (SEQ ID ), where the ITAM motifs are in bold and are underlined.

DAP10/CD28 Intracellular signaling domains suitable for use in a CAR of the present disclosure include a DAP10/CD28 type ing chain.

An e of a DAP10 ing chain is the amino acid sequence is: RPRRSPAQDGKVYINMPGRG (SEQ ID NO: 120). In some embodiments, a suitable intracellular signaling domain ses an amino acid sequence haVing at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to the entire length of the amino acid sequence RPRRSPAQDGKVYINMPGRG (SEQ ID NO: 120).

] An example of a CD28 signaling chain is the amino acid sequence is FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRS (SEQ ID NO: 121). In some embodiments, a suitable intracellular signaling domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to the entire length of the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRS (SEQ ID NO: 121).

ZAP70 Intracellular signaling s suitable for use in a CAR of the present disclosure include a ZAP70 polypeptide, e. g., a polypeptide sing an amino acid sequence haVing at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 300 amino acids to about 400 amino acids, from about 400 amino acids to about 500 amino acids, or from about 500 amino acids to 619 amino acids, of the following amino acid sequence: MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRF VFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSL TREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIP EGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP QRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNL LGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQ LDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSM GMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKW PLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQG PECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGS TQKAEAACA (SEQ ID NO:36).

Additional sequences The first and/or the second polypeptide of a subject CAR can further include one or more onal polypeptide domains, where such domains include, but are not limited to, a signal sequence; an epitope tag; an affinity domain; and a polypeptide that produces a detectable signal.

Signal sequences Signal sequences that are suitable for use in a subject CAR, e. g., in the first polypeptide of a subject CAR, include any otic signal sequence, including a naturally—occurring signal sequence, a synthetic (e.g., de) signal sequence, etc.

Epitope tag Suitable epitope tags e, but are not limited to, hemagglutinin (HA; e. g., YPYDVPDYA (SEQ ID NO:l22); FLAG (e.g., DYKDDDDK (SEQ ID NO:l23); c-myc (e. g., EQKLISEEDL; SEQ ID NO:4), and the like.

Aﬂinity domain ] Affinity domains include peptide sequences that can interact with a binding partner, e. g., such as one lized on a solid support, useful for identification or purification.

DNA ces encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one—step purification of the recombinant protein by high affinity binding to a resin , such as nickel sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 124), HisX6 H) (SEQ ID NO:l25), C-myc (EQKLISEEDL) (SEQ ID NO:4), Flag (DYKDDDDK) (SEQ ID NO:l23), StrepTag (WSHPQFEK) (SEQ ID NO:l26), hemagluttinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO: 122), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 127), Phe—His—His—Thr (SEQ ID NO: 128), chitin binding domain, S—peptide, T7 peptide, SH2 domain, C—end RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 129), metal binding domains, e. g., zinc binding domains or calcium binding s such as those from calcium—binding proteins, e. g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S—modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large—subunit, S100 ns, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.

Detectable signal-producing ptides Suitable detectable signal—producing proteins include, e. g., ﬂuorescent proteins; enzymes that catalyze a reaction that generates a detectable signal as a product; and the like.

Suitable cent proteins include, but are not limited to, green ﬂuorescent protein (GFP) or ts thereof, blue cent variant of GFP (BFP), cyan cent variant of GFP (CFP), yellow ﬂuorescent variant of GFP (YFP), enhanced GFP (EGFP), ed CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T—Sapphire, CyPet, YPet, mKO, HcRed, t—HcRed, DsRed, DsRed2, DsRed—monomer, J—Red, dimer2, t—dimer2(l2), mRFPl, oporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B—Phycoerythrin, R—Phycoerythrin and 2014/016527 Allophycocyanin. Other examples of ﬂuorescent proteins e dew, mBanana, mOrange, dTomato, o, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Not. Methods 2:905—909), and the like.

Any of a variety of ﬂuorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969—973, is le for use.

Suitable enzymes include, but are not d to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta—galactosidase (GAL), glucose—6—phosphate dehydrogenase, beta—N—acetylglucosaminidase, B—glucuronidase, invertase, Xanthine Oxidase, fireﬂy luciferase, glucose oxidase (GO), and the like.

Recombination of sequences In certain instances, ces of the ptides of a CAR, e. g., CAR domains, may be rearranged or deleted in a cell through the use of site—specific recombination technology. In certain embodiments, the cellular activation—related response to a particular CAR can be changed by site—specific recombination, e. g., a first intracellular signaling domain of a CAR eliciting a first tion—related response may be exchanged for a second intracellular signaling domain eliciting a second activation—related response. In certain instances, the response to a particular dimerizer of a CAR can be changed by site—specific recombination, e. g., a first dimerizer—binding pair causing the dimerization of a CAR in the presence of a first dimerizer may be exchanged for a second dimerizer—binding pair g the dimerization of the CAR in the presence of a second dimerizer. As will be clear to one skilled in the art, site—specific recombination can be used in a cell to exchange any domain or sequence of a CAR with any other domain or sequence as disclosed herein. As will also be clear to one skilled in the art, site—specific ination can be used in a cell to delete any domain or sequence of a CAR. Such exchange and excision of sequences and domains is known in the art, see, e. g., domain switching in signalobodies as described in Tone et al. (2013) Biotechnology and Bioengineering, 3219—3226, the disclosure of which is disclosed herein by reference. Mechanisms and requirements for performing site— specific ination in vivo are also well known in the art, see, e. g., Grindley et al. (2006) Annual Review ofBiochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), the disclosures of which are incorporated herein by reference.

NUCLEIC ACIDS The present disclosure provides a nucleic acid that comprises a nucleotide sequence encoding the first and/or the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure. A nucleic acid comprising a nucleotide ce encoding the first and/or the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure will in some ments be DNA, ing, e. g., a recombinant expression . A nucleic acid comprising a nucleotide ce encoding the first and/or the second polypeptide of a dimeric, conditionally active CAR of the present disclosure will in some embodiments be RNA, e.g., in vitro synthesized RNA.

In some cases, a nucleic acid of the present sure comprises a nucleotide sequence encoding only the first polypeptide (and not the second polypeptide) of a heterodimeric, conditionally active CAR of the present disclosure. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding only the second polypeptide (and not the first polypeptide) of a heterodimeric, conditionally active CAR of the present disclosure. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding both the first polypeptide and the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure.

] In some cases, a subject nucleic acid provides for production of a CAR of the present disclosure, e. g., in a mammalian cell. In other cases, a subject nucleic acid provides for amplification of the CAR—encoding nucleic acid.

A nucleotide sequence encoding the first and/or the second polypeptide of a CAR of the present disclosure can be operably linked to a transcriptional control element, e. g., a promoter, and enhancer, etc.

Suitable promoter and enhancer elements are known in the art. For expression in a ial cell, suitable promoters include, but are not d to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early er; herpes simplex virus ine kinase promoter; early and late SV40 ers; promoter present in long terminal repeats from a irus; mouse othionein—I promoter; and various art—known tissue specific promoters.

Suitable reversible promoters, including reversible inducible ers are known in the art. Such reversible promoters may be isolated and derived from many organisms, e. g., eukaryotes and prokaryotes. Modification of ible promoters derived from a first organism for use in a second organism, e. g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible ers but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e. g., alcohol 2014/016527 dehydrogenase I (ach) gene promoter, promoters responsive to l transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e. g., promoter systems including TetActivators, TetON, , etc.), steroid regulated promoters (e. g., rat glucocorticoid receptor promoter systems, human en receptor er systems, retinoid er systems, d promoter systems, ne promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e. g., metallothionein promoter systems, etc.), pathogenesis—related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e. g., heat shock ble promoters (e. g., HSP—70, HSP—90, soybean heat shock promoter, etc.), light regulated promoters, synthetic ble promoters, and the like.

In some instances, the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an rsible switch are well known in the art, e. g., induction of an irreversible switch may make use of a Cre—lox—mediated recombination (see, e. g., Fuhrmann—Benzakein, et al., PNAS (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable er. Methods, mechanisms, and requirements for performing site—specific recombination, described elsewhere herein, find use in generating rsibly switched promoters and are well known in the art, see, e. g., Grindley et al. (2006) Annual Review of Biochemistry, 567—605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), the disclosures of which are incorporated herein by reference.

In some cases, the er is a CD8 pecific promoter, a CD4 cell—specific promoter, a neutrophil—specific promoter, or an cific promoter. For example, a CD4 gene promoter can be used; see, e. g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter can be used. NK pecific expression can be achieved by use of an Ncrl (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood ll7:1565.

In some embodiments, e. g., for expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADHl promoter, a PGKl promoter, an ENO promoter, a PYKl promoter and the like; or a regulatable promoter such as a GALl promoter, a GALlO promoter, an ADH2 promoter, a PHOS promoter, a CUPl promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYCl promoter, a HIS3 promoter, an ADHl promoter, a PGK promoter, a GAPDH promoter, an ADCl promoter, a TRPl promoter, a URA3 promoter, a LEU2 promoter, an ENO er, a TPl promoter, and AOXl (e. g., for use in Picliia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid er, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in viva regulated promoters, such as an ssaG promoter or a related promoter (see, e. g., US. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J.

Bacterial, 1991: 173(1): 86—93; e—Aranda et al., PNAS, 1992; 89(21): 10079—83), a nirB promoter (Harborne et al. (1992) Mol. Micro. 6:2805—2813), and the like (see, e. g., n et al. (1999) Infect. Immun. 67:5133—5141; McKelvie et al. (2004) Vaccine 3—3255; and Chatfield et al. (1992) Biotechnol. 10:888—892); a sigma70 promoter, e.g., a sus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a nary phase promoter, e. g., a dps promoter, an spv promoter, and the like; a er derived from the pathogenicity island SPI—2 (see, e.g., 7951); an actA promoter (see, e. g., Shetron—Rama et al. (2002) Infect. Immun. 70:1087—1096); an rpsM promoter (see, e.g., ia and Falkow (1996). Mal. Microbial. 22:367); a tet promoter (see, e. g., Hillen,W. and Wissmann,A. (1989) In Saenger,W. and Heinemann,U. (eds), Topics in Molecular and Structural Biology, Protein—Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143—162); an SP6 promoter (see, e. g., Melton et al. (1984) Nucl. Acids Res. 12:7035); and the like. Suitable strong promoters for use in yotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda. Non—limiting examples of operators for use in bacterial host cells include a e promoter operator (Lacl sor n changes mation when contacted with lactose, thereby preventing the Lacl repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA. 80:21—25).

A nucleotide sequence encoding a subject CAR can be present in an expression vector and/or a cloning vector. Where a subject CAR comprises two separate polypeptides, nucleotide sequences encoding the two polypeptides can be cloned in the same or separate vectors. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression s include, e. g., plasmids, viral vectors, and the like.

Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a t recombinant ucts. The ing vectors are provided by way of example. Bacterial: sz, phagescript, PsiX174, pBluescript SK, sz KS, pNH8a, , pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223—3, pKK233—3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXRl, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites d near the promoter sequence to provide for the insertion of nucleic acid sequences encoding logous proteins. A selectable marker operative in the expression host may be present.

Suitable expression vectors include, but are not limited to, viral vectors (e. g. viral vectors based on vaccinia virus; irus; adenovirus (see, e. g., Li et al., Invest Opthalmol Vis Sci :2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 0 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno- associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822—3828; Mendelson et al., Virol. (1988) 166:154—165; and Flotte et al., PNAS (1993) 90:10613—10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e. g., Murine Leukemia Virus, spleen is virus, and s derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

As noted above, in some embodiments, a nucleic acid comprising a nucleotide sequence encoding the first and/or the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure will in some embodiments be RNA, e. g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA comprising a nucleotide sequence ng the first and/or the second ptide of a heterodimeric, ionally active CAR of the present disclosure. s for introducing RNA into a host cell are known in the art. See, e. g., Zhao et al. (2010) Cancer Res. 15:9053. Introducing RNA comprising a nucleotide sequence encoding the first and/or the second polypeptide of a heterodimeric, conditionally active CAR of the present disclosure into a host cell can be carried out in vitra or ex viva or in viva. For example, a host cell (e. g., an NK cell, a xic T cyte, etc.) can be electroporated in vitra or ex viva with RNA comprising a nucleotide sequence encoding the first and/or the second polypeptide of a heterodimeric, ionally active CAR of the present disclosure.

CELLS The present disclosure provides a mammalian cell that is genetically modified to produce a heterodimeric, ionally active CAR of the present disclosure.

Suitable ian cells include primary cells and immortalized cell lines.

Suitable mammalian cell lines include human cell lines, non—human primate cell lines, rodent (e. g., mouse, rat) cell lines, and the like. le mammalian cell lines include, but are not limited to, HeLa cells (e. g., American Type e Collection (ATCC) No. CCL—2), CHO cells (e.g., ATCC Nos. CRL9618, CCL6l, CRL9096), 293 cells (e.g., ATCC No.

CRL—1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL—l658), Huh—7 cells, BHK cells (e.g., ATCC No. CCLlO), PC12 cells (ATCC No. CRLl721), COS cells, COS—7 cells (ATCC No. CRLl65 l), RATl cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRLlS73), HLHepG2 cells, , Jurkat, HL—60, NK cell lines (e. g., NKL, NK92, and YTS), and the like.

] In some instances, the cell is not an immortalized cell line, but is instead a cell (e. g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an dual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.

METHODS OF ACTIVATING AN IMMUNE CELL The present disclosure provides methods of activating an immune cell in vitra, in viva, or ex viva. The methods generally involve contacting an immune cell (in vitra, in viva, or ex viva) with a dimerizing agent and an antigen, where the immune cell is genetically modified to produce a heterodimeric, conditionally active CAR of the present disclosure. In the presence of the dimerizing agent and the antigen, the heterodimeric, conditionally active CAR dimerizes and activates the immune cell, thereby producing an activated immune cell.

Immune cells include, e. g., a cytotoxic T cyte, an NK cell, a CD4+ T cell, a T regulatory (Treg) cell, etc.

Contacting the genetically ed immune cell (e.g., a T lymphocyte, an NK cell) with a dimerizing agent and a second member of a specific binding pair (e. g., an antigen, a ligand, a receptor) can increase tion of a cytokine by the immune cell by at least abouthQb,atleastabout159b,atleastabout209%,atleastabout2595,atleastabout309%,at leastabout4095,atleastabout509b,atleastabout7595,atleastabout2—ﬁﬂd,atleastabout 2.5—fold, at least about 5—fold, at least about lO—fold, or more than lO—fold, compared with the amount of cytokine produced by the immune cell in the absence of the second member of a specific binding pair and/or the dimerizing agent. Cytokines whose production can be increased include, but are not limited to, IL—2 and IFN—y.

Contacting the genetically modified immune cell (e.g., a T lymphocyte, an NK cell) with a dimerizing agent and an n can increase production of a cytokine by the immune cellbyzﬁleastabout109b,atleastabout159%,atleastabout2095,atleastabout259%,at bout3095,atleastabout4095,atleastaboutSOQb,atleastabout759b,atleastabout2— fold, at least about 2.5—fold, at least about 5—fold, at least about lO—fold, or more than 10— fold, compared with the amount of ne produced by the immune cell in the absence of the antigen and/or the dimerizing agent. Cytokines whose production can be increased e, but are not limited to, IL—2 and IFN—y.

Contacting a genetically modified cytotoxic cell (e.g., cytotoxic T lymphocyte) with a dimerizing agent and a second member of a specific binding pair (e. g., an antigen, a ligand, a receptor) can increase cytotoxic actiVity of the cytotoxic cell by at least about 10%, atleastabout159b,atleastabout209b,atleastabout259b,atleastabout3095,atleastabout 40%, at least about 50%, at least about 75%, at least about 2—fold, at least about 2.5—fold, at least about 5—fold, at least about lO—fold, or more than lO—fold, compared to the cytotoxic actiVity of the cytotoxic cell in the absence of the zing agent.

Contacting a genetically modified cytotoxic cell (e. g., cytotoxic T lymphocyte) with a dimerizing agent and an antigen can increase xic actiVity of the cytotoxic cell by at leastabout109%,atleastabout159%,atleastabout2095,atleastabout2595,atleastabout tleastabout4095,atleastabout509b,atleastabout759b,atleastabout2aﬁﬂd,at least about 2.5—fold, at least about 5—fold, at least about lO—fold, or more than lO—fold, compared to the xic actiVity of the cytotoxic cell in the absence of the dimerizing agent.

WO 27261 In other embodiments, e. g., depending on the host immune cell, contacting a genetically modified host cell with a dimerizing agent and an antigen can increase or decrease cell proliferation, cell survival, cell death, and the like.

METHODS OF GENERATING A CONDITIONALLY ACTIVATABLE CELL The present disclosure provides a method of generating a conditionally activatable cell. The method generally involves genetically modifying a mammalian cell with an expression vector, or an RNA (e. g., in vitro ribed RNA), comprising nucleotide sequences encoding a heterodimeric, conditionally active CAR of the present disclosure.

The genetically modified cell is conditionally activatable in the presence of: a) an antigen to which the first ptide of the CAR binds; and b) a dimerizer (a zing agent). The c modification can be carried out in vivo, in vitro, or ex vivo. The cell can be an immune cell (e. g., a T lymphocyte or NK cell), a stem cell, a progenitor cell, etc.

In some cases, the genetic cation is carried out ex vivo. For example, a T lymphocyte, a stem cell, or an NK cell is obtained from an individual; and the cell obtained from the individual is genetically modified to express a CAR of the present disclosure. The cally modified cell is ionally activatable in the presence of: a) an antigen to which the first polypeptide of the CAR binds; and b) a dimerizer. In some cases, the genetically modified cell is activated ex vivo. In other cases, the genetically modified cell is introduced into an individual (e.g., the individual from whom the cell was obtained); and the genetically modified cell is activated in viva, e. g., by administering to the individual a dimerizer. For example, where the antigen is present on the e of a cell in the individual, there is no need to administer the antigen. The genetically modified cell comes into contact with the antigen present on the surface of a cell in the individual; and, upon administration to the individual of a dimerizer, the cally modified cell is activated. For example, where the genetically modified cell is a T lymphocyte, the genetically modified cell can exhibit cytotoxicity toward a cell that presents an antigen on its surface to which the CAR binds.

TREATMENT METHODS The t sure provides various treatment methods using a subject CAR.

Cytotoxicity methods A CAR of the present disclosure, when present in a T cyte or an NK cell, can mediate cytotoxicity toward a target cell. A CAR of the t disclosure binds to an antigen present on a target cell, y mediating killing of a target cell by a T lymphocyte or an NK cell genetically modified to produce the CAR. The antigen—binding domain of the CAR binds to an antigen present on the surface of a target cell.

] Target cells include, but are not limited to, cancer cells. Thus, the present disclosure provides methods of killing, or inhibiting the growth of, a target cancer cell, the method involving contacting a cytotoxic immune effector cell (e. g., a cytotoxic T cell, or an NK cell) that is cally modified to produce a subject CAR, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of a target cancer cell, and mediates g of the target cell.

The present disclosure provides a method of treating cancer in an dual having a cancer, the method comprising: i) genetically modifying T lymphocytes obtained from the individual with an expression vector comprising nucleotide sequences encoding the heterodimeric, conditionally active CAR of the present disclosure, where the antigen— g domain of the heterodimeric, conditionally active CAR is specific for an epitope on a cancer cell in the individual, and where the genetic cation is carried out ex vivo; ii) introducing the genetically modified T cytes into the individual; and iii) administering to the individual an effective amount of a dimerizing agent, n the dimerizing agent s dimerization of the heterodimeric, conditionally active CAR, wherein said zation provides for activation of the genetically modified T lymphocytes and killing of the cancer cell, thereby ng the cancer. omas that can be amenable to therapy by a method disclosed herein include, but are not d to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non—small cell oma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic oma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be le to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, osarcoma, chordoma, osteogenic sarcoma, arcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, osarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be amenable to therapy by a method disclosed herein e, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Leukemias that can be amenable to therapy by a method disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential; c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B— cell CLL, T—cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias cterized by accumulation of lymphoblasts). Lymphomas that can be treated using a subject method include, but are not limited to, B—cell lymphomas (e. g., Burkitt's ma); Hodgkin's lymphoma; non—Hodgkin's lymphoma, and the like.

] Other cancers that can be amenable to ent according to the methods disclosed herein include atypical meningioma (brain), islet cell oma eas), medullary carcinoma id), mesenchymoma (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and neurofibroma mediastinum.

Immunomodulatory methods ] A subject method can also be used to treat inﬂammatory conditions and autoimmune disease. A t CAR is sed in a er cell or a Tregs for use in an immunomodulatory method. Immunomodulatory methods include, e. g., enhancing an immune response in a mammalian subject toward a pathogen; enhancing an immune response in a subject who is immunocompromised; reducing an inﬂammatory response; reducing an immune response in a mammalian subject to an autoantigen, e. g., to treat an autoimmune e; and reducing an immune response in a mammalian subject to a lanted organ or tissue, to reduce organ or tissue rejection.

Where the method involves reducing an immune response to an autoantigen, the antigen used to activate the CAR is an autoantigen. Where the method involves reducing an immune se to a transplanted organ or tissue, the antigen used to activate the CAR is an antigen specific to the transplanted organ.

Formulations, dosages, and routes of administration As discussed above, a treatment method of the present disclosure involves stration to an individual in need thereof of an effective amount of a dimerizer agent, and may also involve administration of an antigen.

An “effective amount” of a dimerizer agent is in some cases an amount that, when administered in one or more doses to an individual in need thereof, increases the level of cytotoxic ty of a T lymphocyte expressing a subject CAR by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2—fold, at least about 2.5—fold, at least about 5—fold, at least about 10—fold, or more than 10—fold, compared to the cytotoxic ty of the T lymphocyte in the absence of the dimerizing agent.

An “effective amount” of a dimerizer agent is in some cases an amount that, when administered in one or more doses to an individual in need thereof, increases the level of cytotoxic activity of an NK cell sing a subject CAR by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2—fold, at least about ld, at least about 5—fold, at least about 10—fold, or more than 10—fold, compared to the cytotoxic activity of the NK cell in the e of the dimerizing agent.

An “effective amount” of a dimerizer agent is in some cases an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual and/or reduces tumor mass in the individual, by at least about %, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or more than 75%, compared to the number of cancer cells and/or tumor mass in the absence of the zing agent.

In some embodiments, an effective amount of a dimerizer is an amount that, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents, in one or more doses, is effective to reduce one or more of tumor growth rate, cancer cell number, and tumor mass, by at least about %, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the tumor growth rate, cancer cell number, or tumor mass in the e of treatment with the dimerizer.

Formulations ] In the subject methods, a dimerizer can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect or diagnostic .

Thus, the dimerizer can be incorporated into a variety of formulations for therapeutic administration. More particularly, a dimerizer can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi—solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, ions, inhalants and aerosols.

In pharmaceutical dosage forms, a dimerizer can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely ary and are in no way limiting.

Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering . Actual methods of preparing such dosage forms are known, or will be nt, to those skilled in the art. E, gg, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, lvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of a dimerizer te to achieve the desired state in the subject being treated.

The ceutically acceptable excipients, such as es, adjuvants, carriers or ts, are readily available to the public. er, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

For oral preparations, a dimerizer can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with tional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with ts, buffering agents, moistening agents, vatives and ﬂavoring agents.

A dimerizer can be formulated into preparations for injection by dissolving, suspending or fying them in an aqueous or nonaqueous t, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher tic acids or propylene ; and if d, with conventional additives such as lizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

Pharmaceutical compositions comprising a dimerizer are prepared by mixing the dimerizer having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m—cresol, p—chlor—m—cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, ine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, phan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other ydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, e, mannose, e, galactose, fructose, sorbose, raffinose, glucosamine, N— methylglucosamine, galactosamine, and neuraminic acid; and/or non—ionic surfactants such as Tween, Brij Pluronics, Triton—X, or polyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The rd procedure for reconstituting a lyophilized composition is to add back a volume of pure water ally equivalent to the volume removed during lyophilization); r solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 131 l—54.

The term “unit dosage form,” as used herein, refers to physically discrete units suitable as y dosages for human and animal subjects, each unit containing a predetermined quantity a dimerizer calculated in an amount ient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or e. The specifications for a given dimerizer may depend on the ular dimerizer employed and the effect to be achieved, and the pharmacodynamics associated with each dimerizer in the host.

In some embodiments, a dimerizer is formulated in a controlled release formulation.

Sustained—release preparations may be prepared using s well known in the art.

Suitable examples of sustained—release preparations include semipermeable es of solid hydrophobic polymers containing the dimerizer in which the es are in the form of shaped articles, e. g. films or microcapsules. es of sustained—release matrices include polyesters, copolymers of amic acid and ethyl—L—glutamate, non—degradable ethylene—vinyl e, hydrogels, polylactides, degradable lactic acid—glycolic acid copolymers and —(—)—3—hydroxybutyric acid. Possible loss of biological activity may be prevented by using appropriate additives, by controlling moisture content and by developing specific polymer matrix compositions.

Dosages A suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular dimerizer to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. A dimerizer may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between 0.5 mg/kg body weight to 5 mg/kg body ; r, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it can also be in the range of 1 ug to 10 mg per kilogram of body weight per minute.

Those of skill will readily appreciate that dose levels can vary as a function of the ic dimerizer, the ty of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

Routes of administration A dimerizer is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and zed routes of administration.

Conventional and pharmaceutically acceptable routes of administration include umoral, peritumoral, intramuscular, intratracheal, intracranial, aneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other l and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the dimerizer and/or the desired effect. A dimerizer can be administered in a single dose or in le doses. In some ments, a dimerizer is administered orally. In some ments, a dimerizer is administered via an inhalational route. In some embodiments, a dimerizer is administered intranasally. In some embodiments, a zer is administered locally. In some embodiments, a dimerizer is administered intratumorally. In some embodiments, a dimerizer is administered peritumorally. In some embodiments, a dimerizer is administered intracranially. In some embodiments, a dimerizer is administered intravenously.

The agent can be administered to a host using any available conventional methods and routes suitable for ry of conventional drugs, ing systemic or localized . In general, routes of administration contemplated by the ion include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous routes, i.e., any route of administration other than through the tary canal.

Parenteral administration can be carried to effect systemic or local delivery of a dimerizer.

Where systemic ry is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of ceutical preparations.

A dimerizer can also be delivered to the subject by l administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

] By treatment is meant at least an amelioration of the symptoms associated with the pathological condition afﬂicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, ated with the pathological condition being treated, such as cancer. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely ted, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the ms that characterize the pathological condition.

In some embodiments, a dimerizer is administered by injection and/or delivery, e. g., to a site in a brain artery or directly into brain tissue. A dimerizer can also be administered directly to a target site e. g., by direct injection, by implantation of a drug delivery device such as an c pump or slow release particle, by tic delivery to the target site, etc.

Combination therapy In some embodiments, a dimerizer is administered as an adjuvant therapy to a standard cancer therapy. Standard cancer therapies include surgery (e. g., surgical removal of ous tissue), radiation therapy, bone marrow transplantation, chemotherapeutic treatment, antibody treatment, biological response modifier ent, and certain combinations of the foregoing.

] Radiation therapy includes, but is not d to, x—rays or gamma rays that are delivered from either an externally applied source such as a beam, or by implantation of small radioactive sources.

Suitable antibodies for use in cancer ent e, but are not limited to, naked antibodies, e. g., zumab (Herceptin) bevacizumab (AvastinTM), mab (ErbituxTM), panitumumab (VectibixTM), Ipilimumab (YervoyTM), rituximab (Rituxan), alemtuzumab (LemtradaTM), Ofatumumab (ArzerraTM), Oregovomab (OvaRexTM), Lambrolizumab (MK—3475), pertuzumab (PerjetaTM), ranibizumab (LucentisTM) etc., and conjugated antibodies, e. g., gemtuzumab ozogamicin (MylortargTM), Brentuximab vedotin (AdcetrisTM), 90Y—labelled ibritumomab tiuxetan (ZevalinTM), 131I—labelled tositumoma rTM), etc. Suitable antibodies for use in cancer treatment include, but are not limited to, antibodies raised against tumor—associated ns. Such antigens include, but are not limited to, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, Mucins, TAG-72, CAIX, PSMA, Folate—binding protein, Gangliosides (e. g., GD2, GD3, GM2, etc.), Le y , VEGF, VEGFR, Integrin alpha—V—beta—3, Integrin alpha—5—beta—l, EGFR, ERBB2, ERBB3, MET, IGFlR, EPHA3, TRAILRl, TRAILRZ, RANKL, FAP, Tenascin, etc.

Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor—associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; ( 4) apoptosis receptor ts; (5) interleukin—2; (6) interferon—(1.; (7) interferon —y; (8) colony— stimulating factors; (9) inhibitors of angiogenesis; and (10) antagonists of tumor necrosis factor.

Chemotherapeutic agents are non—peptidic (i.e., non—proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic .

Non—limiting es of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, mor antibiotics, plant (vinca) ids, and steroid hormones.

Agents that act to reduce cellular proliferation are known in the art and Widely used.

Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, rethamine, cyclophosphamide (CytoxanTM), melphalan (L—sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl—CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and lomide.

Antimetabolite agents include folic acid s, pyrimidine analogs, purine analogs, and adenosine deaminase tors, including, but not limited to, cytarabine (CYTOSAR—U), cytosine arabinoside, ﬂuorouracil (5—FU), ﬂoxuridine (FudR), 6— anine, 6—mercaptopurine (6—MP), pentostatin, 5—ﬂuorouracil (5—FU), methotrexate, l0—propargyl—5,8—dideazafolate (PDDF, ), 5,8—dideazatetrahydrofolic acid (DDATHF), leucovorin, ﬂudarabine ate, pentostatine, and gemcitabine. le natural products and their derivatives, (e. g., vinca alkaloids, antitumor otics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not d to, Ara—C, paclitaxel ®), docetaxel (Taxotere®), deoxycoformycin, mitomycin—C, L— asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, eta; podophyllotoxins, e.g. etoposide, side, eta; antibiotics, e.g. anthracycline, daunorubicin hloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, eta; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK—506 (tacrolimus, prograf), rapamycin, etc; and the like.

Other anti—proliferative cytotoxic agents are navelbene, CPT—l l, anastrazole, ole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

Microtubule affecting agents that have oliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC ), maytansine (NSC l53858), rhizoxin (NSC 332598), axel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

WO 27261 Hormone modulators and steroids (including synthetic analogs) that are le for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, eta; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, rol acetate, iol, clomiphene, tamoxifen; etc; and adrenocortical suppressants, e.g. aminoglutethimide; l70t—ethinylestradiol; diethylstilbestrol, terone, ﬂuoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl— testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation, therefore compounds that bind to the estrogen receptor are used to block this ty. Corticosteroids may inhibit T cell proliferation.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin (cis—DDP), carboplatin, eta; ureas, e.g. hydroxyurea; and ines, e.g. ylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; r; etc.. Other roliferative agents of st include immunosuppressants, e.g. mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leﬂunomide, mizoribine, rane (SKF ); Iressa® (ZD 1839, hloro—4—ﬂuorophenylamino)—7— methoxy—6—(3—(4—morpholinyl)propoxy)quinazoline); etc.

"Taxanes" include paclitaxel, as well as any active taxane derivative or pro—drug.

"Paclitaxel" (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOLTM, TAXOTERETM (a formulation of docetaxel), lO—desacetyl analogs of paclitaxel and 3'N—desbenzoyl—3'N—t—butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/0788l, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; US. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; ,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T—l9l2 from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e. g., TaxotereTM docetaxel, as noted above) and axel conjugates (e. g., paclitaxel—PEG, paclitaxel—dextran, or paclitaxel—xylose).

Also included within the term “taxane” are a y of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives bed in W0 99/14209; taxane tives described in WO 99/09021, WO 51, and US. Patent No. 5,869,680; 6—thio derivatives described in WO 98/28288; sulfenamide derivatives described in US. Patent No. 5,821,263; and taxol derivative described in US. Patent No. ,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and US. Patent No. 5,824,701.

SUBJECTS SUITABLE FOR TREATMENT A variety of subjects are suitable for treatment with a subject method of treating cancer. Suitable subjects e any dual, e. g., a human or non—human animal who has cancer, who has been diagnosed with cancer, who is at risk for developing cancer, who has had cancer and is at risk for recurrence of the cancer, who has been treated with an agent other than a zer for the cancer and failed to respond to such treatment, or who has been treated with an agent other than a dimerizer for the cancer but relapsed after initial response to such treatment.

Subjects suitable for treatment with a subject immunomodulatory method include individuals who have an autoimmune er; individuals who are organ or tissue transplant recipients; and the like; duals who are immunocompromised; and individuals who are infected with a en.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. s have been made to ensure accuracy with respect to numbers used (e. g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight e molecular weight, temperature is in degrees Celsius, and re is at or near atmospheric. Standard abbreviations may be used, e. g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); i.v., intravenous(1y); and the like.

Example 1: Generation of CAR MATERIALS AND METHODS The anti—human CD19 scFv was selected as the antigen recognition domain in CARs throughout the design optimization process. Figures 18A and 18B summarize the molecular structure of each CAR consisting of two numerically identified polypeptides. All membrane—anchored polypeptides are di— sulfide bonded homo—dimers. The membrane— ed ptides are depicted as monomers for graphical simplicity.

Generation of CAR constructs Sequence encoding the anti—human CD19 scFv was cloned from a construct. The human 4—1BB co—stimulation and CD3 zeta lTAM ing chains were cloned from cDNAs supplied by Open Biosystems. FKBP— and FRB—encoding sequences were cloned from plasmids supplied by e.

Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction ion, ligation, etc.) were d to generate lentiviral expression plasmids.

Effector and target cell ing conditions Human primary CD8+ T cells were isolated from anonymous donor’s blood after apheresis (Trima residuals from Blood Centers of the Pacific, San Francisco, CA) by negative selection using RosetteSep Human CD8+ T Cell Enrichment Cocktail (STEMCELL Technologies #15063) as approved by University Institutional Review Board.

Cells were cultured in human T cell medium, consisting of l5 (Lonza #04—418Q), % human AB serum (Valley Biomedical Inc., #HP1022), 10mM N—acetyl L—Cysteine (Sigma—Aldrich #A9165) and 100 IU/mL recombinant human IL—2 (NCI/BRB Preclinical Repository). A Jurkat cell line sing the Green Fluorescent Protein (GFP) upon NFAT activation was maintained in 640 medium supplemented with 10% fetal bovine serum (FBS), llin and streptomycin. K562 target cells from U. Penn were ed in IMDM supplemented with 10% FBS.

Effector and target cell engineering with irus Pantropic VSV—G pseudotyped lentivirus was produced from Lenti—X 293T cells ech Laboratories #632180) co—transfected with a pHR’SINzCSW transgene expression vector, viral packaging plasmids pCMVdR8.91 and pMD2.G using Lipofectamine LTX (Life Technologies #15338). Infection medium supernatant was collected 48 hours after transfection and used directly for transduction.

Twenty four hours prior to viral transduction, primary human T cells were activated using the human T—Activator CD3/CD28 Dynabeads (Life Technologies #111—31D) at a 1:3 cell:bead ratio. Jurkat and K562 cells were split l~2 days in advance to ensure that cultures would be in log phase at the time of transduction. Transduced Jurkat and K562 cells were cultured for at least 7 days before experiments were conducted. Primary T cells were maintained at ~lOA6/mL in human T cell medium for about two weeks until cells returned to a resting state. Expression levels of CARs d in the iral constructs were quantified by detecting either ﬂuorophore—conjugated antibodies or ﬂuorescent reporter proteins using a ﬂow cytometer.

Quantitation of IL—2 production and NFAT activity Jurkat CD4+ T cells expressing CARs were mixed with cognate or non—cognate K562 target cells from U. Penn at a 1:2 effector:target ratio. The rapalog A/C Heterodimerizer (Clontech tories #635055) were serially diluted in medium and added to reaction mixtures. After 20~24 hours of incubation, medium supematants were collected and ed with ED OptEIA Human IL—2 ELISA Set (BD Biosciences #555 190). Flow cytometry was performed to quantify NFAT—dependent GFP reporter expression in Jurkat cells as a separate indicator for CAR activity.

Flow cytometry—based re—directed cytotoxicity assay The cognate and non—cognate K562 target cells were engineered to express distinct cent proteins so that both cell types in a mixture could be simultaneously quantified by ﬂow cytometry. The target cell types were mixed at a 1:1 ratio and ubated with human primary CD8+ effector T cells at a 5:2 or:target ratio. 100 IU/mL human IL—2 and varying amounts of the g (Clontech Laboratories #635055) were added to reaction mixtures. After 24 hours of incubation, samples were fuged at 400g for 5 minutes. Pelleted cells were resuspended in wash buffer (PBS + 0.5% BSA + 0.1% sodium azide) and fixed with an equal volume of BD Cytofix (BD cat #554655) prior to ﬂow cytometry. Ratios of the surviving cognate target cells to gnate target cells were ated for each sample to enumerate re—directed cytotoxic activities of the effector cells.

RESULTS IL—2 production elicited by the various CAR constructs was assessed. The data are presented in Figure 12.

] Figure 12. IL—2 production triggered by five On—switch CAR variants. Effector 2 human CD4+ Jurkat T cells engineered with CARs. Target 2 K562 cell lines with or without the cognate CD19 antigen. Amounts of secreted IL—2 by effector cells were quantified by enzyme—linked immunosorbent assay (ELISA).

WO 27261 Figure 13. IL—2 production by control Jurkat lines in the same experiment as that bed in Figure 12. uct “125” encodes a conventional l currently used in clinical trials.

Figure 14. ison between “122 + 206” and “197 + 206” in a separate experiment under conditions identical to those described in Figure 12.

Figure 15 demonstrates pharmacologically titratable cytoxicity conferred by the On— switch CAR “197+206” In the presence of the small molecule rapalog, the CAR effectively mediates re—directed xicity towards cognate target cells. At high dosages of rapalog, this On—switch CAR can signal as strongly as the “125” conventional CAR. Effector 2 human primary CD8+ T cells engineered with CARs or a control vector. Target 2 ﬂuorescent derivatives of K562 cell lines expressing either the cognate human CD19 antigen or the non—cognate human mesothelin antigen.

Figure 16 depicts data for CARs constructed with the asmic tyrosine kinase Zap70 from the T cell receptor pathway as the intracellular signaling domain.

Figure 16 shows data from Jurkat cells ered with several variants of On— switch CARs. The engineered Jurkat cells were co—incubated with K562 target cells with or without the cognate antigen (CD19) and the indicated concentrations of g. As a CAR ent, the Zap70 kinase (first and second structures from left ing “199”) was as effective as the ITAM (third structure from left featuring “168”) in activating NFAT function. Addition of the 4—1BB signaling domain increased surface expression of the antigen ition portion of the receptor and led to stronger signaling by “197+199”. A non—signaling CAR (far—right) was ed as a negative control. e 2: CARs targeting mesothelin MATERIALS AND METHODS A number of chimeric antigen receptor constructs were made and tested. The constructs shown here encode three different anti—human mesothelin scFv as the antigen recognition domains. Figures 19A, 19B, and 19C summarize the molecular structure of each anti—human mesothelin CAR, with each CAR comprising two polypeptides. The intercellular portion of each anti—human mesothelin CAR comprises two 4—1BB co— stimulatory domains, an FKBP and FRB dimerizer—binding pair, and an ITAM intracellular signaling domain. The three different antigen recognition domains shown here are anti— mesothelin HNl scFv, SSl scFv, and m912 scFv. All membrane—anchored polypeptides are di—sulfide bonded homo—dimers.

Generation of CAR constructs Sequences encoding the anti—mesothelin were cloned from ucts or synthesized via gene assembly by PCR. The human 4—1BB co—stimulation and CD3 zeta lTAM signaling chains were cloned from cDNAs supplied by Open Biosystems. HNl scFv—, SS1 scFv—, and m912 scFv—encoding sequences were synthesized by PCR and, in some cases, codon zed. FKBP— and FRB—encoding sequences were cloned from Addgene plasmids.

] Standard lar cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

Effector and target cell culturing conditions A Jurkat cell line expressing GFP upon NFAT activation was maintained in RPMI— 1640 medium supplemented with 10% FBS, penicillin and streptomycin. K562 target cells were cultured in IMDM supplemented with 10% fetal bovine serum (FBS).

Effector and target cell engineering with lentivirus Pantropic VSV—G pseudotyped lentivirus was ed from Lenti—X 293T cells (Clontech Laboratories #632180) co—transfected with a pHR’SINzCSW transgene expression vector, viral packaging ds pCMVdR8.91 and pMD2.G using Lipofectamine LTX (Life Technologies #15338). Infection medium supernatant was collected 48 hours after transfection and used ly for transduction.

Jurkat and K562 cells were split 1~2 days in advance to ensure that cultures would be in log phase at the time of transduction. Transduced Jurkat and K562 cells were cultured for at least 7 days before experiments were conducted. Expression levels of CARs encoded in the lentiviral constructs were quantified by detecting either ﬂuorophore—conjugated dies or ﬂuorescent reporter proteins using a ﬂow cytometer.

Quantitation of IL—2 tion Jurkat CD4+ T cells expressing CARs were mixed with cognate or non—cognate K562 target cells at a 1:2 effector:target ratio. The rapalog A/C dimerizer (Clontech Laboratories #635055) were serially diluted in medium and added to reaction mixtures.

After 20~24 hours of incubation, medium supernatants were collected and analyzed with ED OptEIA Human IL—2 ELISA Set (BD Biosciences #555190).

IL—2 production elicited by the anti—mesothelin CAR constructs was assessed. The data are presented in Figure 19D—F. 2014/016527 Figure 19. IL—2 production triggered by HNl scFv (Fig. 19D), SS1 scFv (Fig. 19E), and m912 scFv (Fig. 19F) On—switch CAR variants. IL—2 production by a conventional CAR (Fig. 19G, construct #358) was measured and included for comparison to tch CARs (Fig. 19D). Effector 2 human CD4+ Jurkat T cells engineered with CARs. Target 2 K562 cell lines with or without the cognate mesothelin antigen. Amounts of secreted IL—2 by effector cells were quantified by enzyme—linked immunosorbent assay (ELISA).

Example 3: Gibberellic acid as a dimerizer of On—switch CARs MATERIALS AND S ] Figure 20A summarizes the lar structure of the t ellic acid dimerizer CAR. The antigen binding portion comprises the anti—human CD19 scFv. The intracellular portion comprises two 4— 1BB co—stimulatory domains, a GIDl and GAI dimerizer—binding pair, and an ITAM intracellular signaling domain. All membrane— anchored polypeptides are di—sulfide bonded homo—dimers.

Generation of CAR constructs ces encoding the gibberellic acid dimerizer CAR were cloned from constructs. The anti—CD19 scFv was cloned from a plasmid. The human 4—1BB co— stimulation and CD3 zeta ITAM signaling chains were cloned from cDNAs supplied by Open Biosystems. GIDl— and GAI—encoding ces were cloned from Addgene plasmids. Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

Effector and target cell engineering with lentivirus Pantropic VSV—G pseudotyped lentivirus was produced from Lenti—X 293T cells (Clontech Laboratories #632180) co—transfected with a pHR’SINzCSW transgene expression , viral ing plasmids pCMVdR8.91 and pMD2.G using ctamine LTX (Life Technologies ). Infection medium supernatant was collected 48 hours after transfection and used directly for transduction.

Jurkat and K562 cells were split 1~2 days in advance to ensure that cultures would be in log phase at the time of uction. Transduced Jurkat and K562 cells were cultured for at least 7 days before experiments were conducted. Expression levels of CARs encoded in the lentiviral constructs were quantified by detecting either ﬂuorophore—conjugated antibodies or cent reporter proteins using a ﬂow cytometer. tation of IL—2 production Jurkat CD4+ T cells expressing CARs were mixed with cognate or non—cognate K562 target cells at a 1:2 effector:target ratio. The gibberellic acid—3 acetoxymethyl ester (gibberrelic acid—3 AM) pre—dissolved in ethanol (Toronto Research Chemicals #G377500) was diluted in growth medium and added to reaction mixtures. Gibberellic acid (gibberellic acid—3 AM) was used at 10 mM. After 20~24 hours of incubation, medium supematants were collected and analyzed with ED OptEIA Human IL—2 ELISA Set (BD ences #555 190).

RESULTS IL—2 production elicited by the gibberellic acid dimerizer CAR construct was assessed. The data are presented in Figure 20.

Figure 20. IL—2 production triggered by gibberellic acid dimerizer CAR variant (Fig. 20B). IL—2 production by a conventional CAR (Fig. 20C, construct “125”) was measured and ed for comparison to On—switch CAR. or 2 human CD4+ Jurkat T cells engineered with CARs. Target 2 K562 cell lines with or t the cognate CD19 antigen.

Amounts of secreted IL—2 by effector cells were quantified by enzyme—linked immunosorbent assay (ELISA). e 4: On—switch CARs with various co—stimulatog domains MATERIALS AND METHODS A number of chimeric antigen receptor constructs were made essentially as described for Example 1, except various other co—stimulatory domains were exchanged for the 4—1BB co—stimulatory domains. Figures 21A and 21B summarize the molecular structure of the CARs described here.

Generation of CAR constructs Sequences ng the anti—human CD19 scFv were cloned from a plasmid. The human CD3 zeta lTAM signaling chain and the human co—stimulatory s CD28 and OX—40 encoding sequences were cloned from cDNAs supplied by Open Biosystems.

FKBP— and FRB—encoding ces were cloned from plasmids from Addgene.

Standard molecular cloning techniques erase chain reaction (PCR), restriction digestion, ligation, etc.) were d to generate lentiviral expression plasmids.

Testing of CAR constructs Effector and target cells are cultured and transfected according to e 1 using the on—switch CAR CD28 and OX—40 co—stimulatory domain containing constructs described (Fig. 2lA—B, constructs ”365+367” and ”399+400”, respectively) and corresponding conventional CAR controls (Fig. 2lC—D, constructs “366” and “398”, respectively). IL—2 tion, NFAT activity assays, and ﬂow cytometry—based assays can also be performed with the CD28 co—stimulatory domain containing construct and OX—40 co—stimulatory domain ning construct as described for Example 1. Alternatively, subunits of on—switch CAR CD28 and OX—40 co—stimulatory domain containing constructs can be paired with subunits of constructs from Example 1 (e. g., 67”, “ 365+206,” “197+400”, “399+206,” etc.).

Example 5: In vivo assessment of On—switch CAR An On—switch CAR can be assessed for its y to mediate in viva killing of a target tumor cell. In vivo tumor cell g elicited by injection of T cells expressing the ON—switch CAR is assessed. Tumor cell lines that have been confirmed in vitro to s the cognate antigen and can be killed by CD8+ T cells expressing the corresponding CAR are used. Tumor cells engineered to express either the fireﬂy or Renilla luciferase to enable bio—luminescence imaging to quantify tumor burden in viva can be used. Tumor cells are injected into immunocompromised mice (e. g., 6~lO week old female NOD scid gamma (NSG) mice) either subcutaneously for subcutaneous tumor models or intravenously for systemic tumor models. The method of tumor implantation and the optimal number of tumor cells to implant can be based on conditions optimal for the tumor cell line used.

Tumor burden can be monitored twice a week by bio—luminescence imaging and by r measurement when applicable. As soon as tumor burden is detectable, 0.5~2.5 x lOA7 total T cells (lzl CD4+:CD8+) expressing the ON—switch CAR are intravenously ed into mice to begin ent. A dimerizing small molecule drug (e. g., rapalog) is administered intraperitoneally in a vehicle formulation. On—switch CAR—expressing T cells can be injected repeatedly during the experiment to enhance the anti—tumor effect. Interleukin—2 (IL—2) can be administered to enhance the anti—tumor effect.

While the present invention has been described with nce to the specific embodiments f, it should be tood by those skilled in the art that various s may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present ion. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is d is:

1. A heterodimeric, conditionally active chimeric antigen receptor (CAR) comprising: a) a first polypeptide comprising: i) a first member of a ic binding pair; ii) a first co-stimulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of a specific binding pair and the first co-stimulatory domain; and b) a second polypeptide comprising: i) a transmembrane domain; ii) a second co-stimulatory domain; iii) a second member of the dimerization pair; and iv) an intracellular ing domain; or comprising: a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a co-stimulatory domain; iii) a first member of a dimerization pair; iv) a transmembrane domain interposed n the first member of a specific binding pair and the co-stimulatory ; and b) a second polypeptide comprising: i) a second member of the dimerization pair; and ii) an intracellular signaling domain, wherein the first and second members of the dimerization pair form a homodimer or a heterodimer in the presence of a dimerizing agent.

2. The heterodimeric, conditionally active CAR of claim 1, wherein the order of the domains in the first polypeptide from the amino terminus to the carboxyl terminus is: the first member of the specific g domain, the transmembrane domain, the first co-stimulatory domain and the first member of the dimerization pair.

3. The heterodimeric, ionally active CAR of claim 1 or 2, wherein the first polypeptide comprises a hinge region such as an immunoglobulin lgG hinge region or a hinge derived from CD8 interposed between the first member of the specific binding pair and the transmembrane domain.

4. The heterodimeric, conditionally active CAR of any one of the preceding claims, wherein the first member of the ic binding pair is an antibody or antibody fragment, a ligand, or a receptor.

5. The dimeric, conditionally active CAR of any one of the preceding claims, wherein the first and second co-stimulatory domains are selected from 4-1BB (CD137), CD28, ble T-Cell Costimulator Precursor (ICOS), B- and T-Lymphocyte Attenuator (BTLA), OX-40, CD27, CD30, glucocorticoid-induced TNFR-Related (GITR) and Herpes virus entry mediator (HVEM).

6. The heterodimeric, conditionally active CAR of any one of the preceding claims, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM), and is selected from CD3-zeta and ZAP70.

7. The heterodimeric, conditionally active CAR of any one of the preceding claims, wherein the first and second members of the dimerization pair are ed from: a) FK506 binding protein (FKBP) and FKBP-rapamycin associated protein (FRB); b) Gibberellic Acid Insensitive (GAI) and Gibberelin-Insensitive Dwarf 1 (GID1). c) FKBP and eurin tic subunit A (CnA); d) FKBP and cyclophilin; e) FKBP and FKBP; f) gyrase B (GyrB) and GyrB; g) dihydrofolate reductase (DHFR) and DHFR; h) Dihydromethanopterin reduc tase B (DmrB) and DmrB; i) Pyrabactin Resistance 1-like (PYL) and abscisic acid insensitive (ABI); and j) chrome-2 (Cry2) and cryptochrome-interacting basic-helix-loop-helix 1 (CIB1).

8. The heterodimeric, conditionally active CAR of claim 1, wherein the first member of the specific g pair is a single-chain Fv and: i) the first and second co-stimulatory s are derived from 4-1BB; ii) the first and second members of the zation pair are FKBP and FRB; iii) the signaling domain comprises an ITAM.

9. The heterodimeric, conditionally active CAR of any one of the preceding claims, wherein the first member of the ic binding pair binds an epitope present on a cell such as a cancer cell, on a solid surface, or a lipid bilayer.

10. An in vitro or ex vivo mammalian cell genetically modified to produce the heterodimeric, conditionally active CAR of any one of the preceding claims, wherein the genetically modified mammalian cell is not a genetically modified human tent cell.

11. The genetically modified mammalian cell of claim 10, wherein the cell is a stem cell, a hematopoietic stem cell (HSC), a progenitor cell, a cell d from a stem cell or from a progenitor cell, a T lymphocyte, or a NK cell.

12. One or more nucleic acids comprising a nucleotide sequence(s) encoding the heterodimeric, conditionally active CAR of any one of claims 1 to 9.

13. A nucleic acid comprising nucleotide sequences ng the heterodimeric, ionally active CAR of any one of claims 1 to 9, wherein the nucleic acid is optionally linked to a T lymphocyte-specific promoter or an NK cell-specific promoter.

14. The nucleic acid of claim 12 or claim 13, wherein the nucleic acid is in vitro transcribed RNA or is present in a recombinant expression .

15. A method of making the cally modified mammalian cell of claim 10, the method comprising genetically modifying a mammalian cell in vitro or ex vivo with an expression vector comprising nucleotide sequences encoding the heterodimeric, conditionally active CAR of any one of claims 1 to 9, or genetically modifying a mammalian cell with an RNA comprising nucleotide sequences encoding the heterodimeric, conditionally active CAR of any one of claims 1 to 9.

16. A heterodimeric, conditionally active chimeric antigen receptor (CAR) comprising: a first polypeptide comprising a first member of a dimerization pair, a first transmembrane domain, and an intracellular ing domain; and a second polypeptide sing an antigen-binding domain that comprises a single chain antibody variable region that specifically binds to CD19 or an antigen expressed by an immune cell, a second member of the dimerization pair, and a second transmembrane domain; wherein the first ptide does not se an n-binding domain and the second polypeptide does not comprise an intracellular signaling domain capable of inducing immune activation, wherein a dimerizing agent dimerizes the first and second polypeptides to form the heterodimeric CAR when the first and second polypeptides are expressed by a cell with the dimerizing agent bound between the dimerization pair members of the first and second polypeptides, and wherein activation of the cell by the formed heterodimeric CAR binding CD19 or said antigen is increased as compared to tion of the cell in the absence of the dimerizing agent.

17. The heterodimeric, conditionally active CAR of claim 16, wherein the immune cell is a cell of a cyte lineage.

18. The heterodimeric, conditionally active CAR of claim 17, wherein the lymphocyte lineage is a B cell lineage.

19. A dimeric, conditionally active chimeric antigen receptor (CAR) sing: a first polypeptide comprising a first member of a dimerization pair, a first transmembrane domain, and an intracellular signaling domain; and a second polypeptide comprising an antigen-binding domain that comprises a single chain antibody variable region, a second member of the dimerization pair, and a second transmembrane domain; wherein the single chain antibody variable region specifically binds to an antigen selected from the group consisting of: CD19, CD20, CD38, CD30, Her2/neu, Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), Cancer Antigen 125 ), Mucin 1 (MUC-1), prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor or (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), Melanoma- Associated Antigen 1 A1), Interleukein 13 or Subunit Alpha 2 (IL-13R- a2), and Ganglioside GD2; and wherein the first polypeptide does not comprise an antigen-binding domain and the second polypeptide does not comprise an intracellular signaling domain capable of inducing immune activation, wherein a dimerizing agent zes the first and second polypeptides to form the heterodimeric CAR when the first and second polypeptides are expressed by a cell with the dimerizing agent bound between the dimerization pair members of the first and second polypeptides, and wherein activation of the cell by the formed heterodimeric CAR binding said antigen is increased as compared to activation of the cell in the absence of the zing agent.

20. The heterodimeric, conditionally active CAR of claim 19, wherein the antigen is CD19 or elin.

21. The heterodimeric, conditionally active CAR of claim 19 or claim 20, wherein the antigen is CD19.

22. The heterodimeric, conditionally active CAR of claim 19 or claim 20, wherein the antigen is mesothelin.

23. The heterodimeric, conditionally active CAR of any one of claims 16 to 22, wherein the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.

24. The heterodimeric, conditionally active CAR of any one of claims 16 to 22, wherein the intracellular signaling domain comprises an immunoreceptor tyrosinebased activation motif (ITAM).

25. The heterodimeric, conditionally active CAR of any one of claims 16 to 24, wherein the first polypeptide, the second polypeptide or both comprise an ellular costimulatory polypeptide.

26. The heterodimeric, ionally active CAR of claim 25, wherein the intracellular ulatory polypeptide is selected from the group consisting of: 4-1BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

27. The heterodimeric, conditionally active CAR of claim 25, wherein the first polypeptide comprises a first costimulatory polypeptide comprising an amino acid sequence and the second ptide comprises a second costimulatory polypeptide sing an amino acid sequence that has at least 95% amino acid identity to the amino acid ce of the first costimulatory polypeptide in the first polypeptide.

28. The heterodimeric, conditionally active CAR of any one of claims 16 to 27, wherein the dimerizing agent is selected from the group consisting of: rapamycin, coumermycin, methotrexate, AP20187, ic acid, gibberellin and analogs thereof.

29. The heterodimeric, conditionally active CAR of any one of claims 16 to 28, wherein the first and second members of the dimerization pair are selected from the group consisting of: a) FK506 binding n (FKBP) and apamycin associated protein (FRB); b) a Gibberellic Acid Insensitive (GAI) protein and a gibberellin receptor (GID1) protein; c) FKBP and calcineurin catalytic subunit A (CnA); d) an abscisic acid receptor (PYL) protein and an abscissic acid insensitive (ABI) protein; and e) FKBP and cyclophilin.

30. The heterodimeric, conditionally active CAR of any one of claims 16 to 28, wherein the first and second s of the dimerization pair are selected from the group consisting of: a) FK506 binding protein (FKBP) and FKBP; b) gyrase B (GyrB) and GyrB; c) dihydrofolate reductase (DHFR) and DHFR; and d) DmrB and DmrB.

31. One or more nucleic acids sing a nucleotide sequence(s) encoding the heterodimeric, conditionally active CAR of any one of claims 16 to 30.

32. A nucleic acid of claim 31, wherein the nucleic acid is optionally linked to a T lymphocyte-specific promoter or an NK cell-specific promoter.

33. The one or more nucleic acids of claim 31, wherein at least one of the one or more c acids is in vitro transcribed RNA or is present in a recombinant expression vector.

34. An isolated cell comprising the one or more nucleic acids of any one of claims 31 to 33, wherein the isolated cell is not a tent human cell.

35. The isolated cell of claim 34, n the isolated cell is an isolated immune cell.

36. The isolated cell of claim 35, wherein the isolated immune cell is a cyte or a NK cell.

37. The isolated cell of any one of claims 34 to 36, wherein the isolated cell is a human cell.

38. A pharmaceutical composition comprising the one or more nucleic acids of any one of claims 12 to 14, and 31 to 33; and/or the genetically modified mammalian cell of any one of claims 10, 11, and 34 to 37.

39. The pharmaceutical composition of claim 38, further comprising the dimerizing agent.

40. A kit comprising: (i) the heterodimeric, ionally active CAR of any of claims 1 to 9 and 16 to 30; the one or more nucleic acids of any one of claims 12 to 14, and 31 to 33; and/or the genetically modified ian cell of any one of claims 10, 11, and 34 to 37; or (ii) a dimerizing agent selected from the group consisting of: rapamycin, coumermycin, methotrexate, AP20187, abscisic acid, gibberellin and analogs thereof, and (i).

41. A method of making the isolated cell of any one of claims 34 to 37, the method comprising genetically modifying a mammalian cell in vitro or ex vivo with the one or more nucleic acids of claim 31 to 33.