BR112015019640B1 - HETERODIMER CONDITIONALLY ACTIVE CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTOR (CAR), METHOD OF ACTIVATING A T LYMPHOCYTE IN VITRO OR EX VIVO, METHOD OF PRODUCING A GENETICALLY MODIFIED MAMMALIAN CELL AND METHOD OF MODULATING THE ACTIVITY OF A T LYMPHOCYTE HOST CELL - Google Patents

Abstract

HETERODIMER CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTOR (CAR); MAMMALIAN CELL; NUCLEIC ACID; RECOMBINANT EXPRESSION VECTOR; METHOD OF ACTIVATING A T LYMPHOCYTE; METHOD OF PRODUCING A CELL; METHOD OF TREATMENT OF A CANCER IN AN INDIVIDUAL; AND METHOD FOR MODULATING THE ACTIVITY OF A HOST CELL. The present invention provides a heterodimeric conditionally active chimeric antigen receptor (CAR) and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present invention provides cells genetically modified to produce CAR. A CAR of the present disclosure can be used in various methods, which are also provided.

Description

Cross reference

[001] This application claims the benefit of US Provisional Patent Application No. 61/765,585, filed on February 15, 2013, which is incorporated in its entirety into this document by reference.

Statement on Federally Sponsored Research

[002] This invention was made with government support under Grants No. EY016546 and GM101782 awarded by the National Institutes of Health. The government has some rights to the invention.

Incorporation by reference of Sequence Listing provided as Text File

[003] A Sequence Listing is provided together with this document as a text file, “UCSF-464WO SeqList_ST25.txt” created on February 13, 2014, 153 KB. The contents of the text file are incorporated in their entirety into this document by reference.

Introduction

[004] In cell-based adoptive immunotherapy, leukocytes isolated from a patient can be modified to manifest synthetic proteins that allow the cells to perform new therapeutic functions after they have been subsequently transferred back to the patient. An example of such synthetic proteins is a chimeric antigen receptor (RQA). An example of an RQA currently in use 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 antigen binding, the intracellular signaling portion of RQA can initiate an activation response in a leukocyte, such as the release of cytolytic molecules to induce tumor cell death, etc. However, these RQAs are not capable of being pharmacologically controlled. There is a need in the art for conditionally activatable RQA that can be controlled pharmacologically.

Summary

[005] This invention provides a conditionally active and heterodimeric chimeric antigen receptor (RQA), and a nucleic acid composed of a nucleotide sequence that encodes the RQA. This invention provides genetically modified cells that produce RQA. An RQA of this invention can be used in several methods, which are also provided.

Brief description of the drawings

[006] Figures 1A and 1B provide amino acid and nucleotide sequences of the domains of construction #122.

[007] Figures 2A and 2B provide amino acid and nucleotide sequences of the domains of construction #123.

[008] Figures 3A and 3B provide amino acid and nucleotide sequences of the domains of construction #125.

[009] Figure 4 provides amino acid and nucleotide sequences of the domains of construction #126.

[010] Figures 5A and 5B provide amino acid and nucleotide sequences of the domains of construction #168.

[011] Figures 6A-C provide amino acid and nucleotide sequences of the domains of construct #169.

[012] Figures 7A and 7B provide amino acid and nucleotide sequences of the domains of construction #170.

[013] Figures 8A and 8B provide amino acid and nucleotide sequences of the domains of construction #197.

[014] Figures 9A-C provide amino acid and nucleotide sequences of the domains of construct #206.

[015] Figures 10A and 10B provide amino acid and nucleotide sequences of the domains of construction #207.

[016] Figures 11A-C provide amino acid and nucleotide sequences of the domains of construct #199.

[017] Figure 12 represents IL-2 production triggered by five activating RQA variants.

[018] Figure 13 represents IL-2 production by control Jurkat lines.

[019] Figure 14 represents a comparison between RQA constructions “122 + 206” and “197 + 206”.

[020] Figure 15 represents cytotoxicity data with the activation RQA “197+206.”

[021] Figure 16 represents T cell activation data using RQA constructs “122 + 199”; “197 + 199”; and “122 + 168.”

[022] Figure 17 is a schematic representation of an exemplary activation RQA.

[023] Figures 18A and 18B represent several examples of activation RQAs.

[024] Figures 19A-G represent IL-2 production triggered by 3 different activating RQA variants that recognize human mesothelin.

[025] Figures 20A-C represent IL-2 production triggered by an activating RQA variant with a gibberellin-responsive dimerization pair.

[026] Figures 21A-D represent exemplary activation RQAs and conventional RQAs with various costimulatory domains.

[027] Figures 22A and 22B provide amino acid and nucleotide sequences of the domains of construction #270.

[028] Figures 23A and 23B provide amino acid and nucleotide sequences of the domains of construction #300.

[029] Figures 24A and 24B provide amino acid and nucleotide sequences of the domains of construction #336.

[030] Figures 25A and 25B provide amino acid and nucleotide sequences of the domains of construct #337.

[031] Figures 26A and 26B provide amino acid and nucleotide sequences of the domains of construction #357.

[032] Figures 27A and 27B provide amino acid and nucleotide sequences of the domains of construction #365.

[033] Figures 28A and 28B provide amino acid and nucleotide sequences of the domains of construction #366.

[034] Figures 29A and 29B provide amino acid and nucleotide sequences of the domains of construction #367.

[035] Figures 30A and 30B provide amino acid and nucleotide sequences of the domains of construction #398.

[036] Figures 31A and 31B provide amino acid and nucleotide sequences of the domains of construction #399.

[037] Figures 32A and 32B provide amino acid and nucleotide sequences of the domains of construction #400.

[038] Figures 33A and 33B provide amino acid and nucleotide sequences of the domains of construction #358. Definitions

[039] The terms “polynucleotide” and “nucleic acid”, used interchangeably here, refer to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Therefore, this term includes, but is not limited to, single-stranded, bi-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer composed of purine and pyrimidine bases or other natural, chemically or biochemically modified nucleotide bases, unnatural or derived.

[040] The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to the antigen, including, among others, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies , single-chain antibodies, and fusion proteins composed of an antigen-binding portion of an antibody and a non-antibody protein.

[041] “Antibody fragments” consist of a portion of an intact antibody, for example, the antigen-bound region or a 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 digestion of antibodies produces two identical antigen-binding fragments, termed "Fab" fragments, each with a unique antigen-binding site, and a residual "Fc" fragment, a designation that reflects the ability to crystallize immediately. Pepsin treatment produces an F(ab')2 fragment that has two antigen-combining sites and is capable of cross-linking with antigen.

[042] "Single-chain Fv" or "sFv" antibody fragments consist of the antibody VH and VL domains, where these domains are present in a simple polypeptide chain. In some forms, the Fv polypeptide also includes a polypeptide linker between the VH and VL domains, which allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[043] As used in this document, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, and is expressed as a dissociation constant (Kd). The affinity can be at least 1 times greater, at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater , at least 80 times greater, at least 90 times greater, at least 100 times greater, at least 1000 times greater, or more, than the affinity of an antibody for non-amino acid sequences. The affinity of an antibody to a target protein may 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 fentomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably herein in relation to antibodies and/or antigen-binding fragments.

[044] The term "bond" refers to a direct association between 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 bindings refer to bindings with an affinity of less than 10-7 M, approximately, for example, bindings with an affinity of 10-6 M, 10-5 M, 10-4 M, etc.

[045] As used herein, the term "hinge region" refers to a flexible polypeptide connector region (also called a "hinge" or "spacer"), which provides structural flexibility and spacing to polypeptide flanking regions and can consist of natural or synthetic polypeptides. A "hinge region" derived from an immunoglobulin (e.g., IgG1) is generally defined as extending from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec Immunol., 22: 161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues forming interheavy chain disulfide (S-S) bonds at the same positions. The hinge region may be naturally occurring, or non-naturally occurring, including, but not limited to, an altered hinge region as described in U.S. Pat. No. 5,677,425. The hinge region may include the complete hinge region derived from an antibody of a class or subclass other than that of the CH1 domain. The term "hinge region" may also include regions derived from CD8 and other receptors that provide a similar function of flexibility and spacing to flanking regions.

[046] An "isolated" polypeptide is a polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminating components of its natural 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 forms, the polypeptide will be purified (1) to greater than 90%, greater than 95%, or greater than 98% by weight of antibody as determined by the Lowry method, e.g., greater than 99% by weight. (2) to a degree sufficient to obtain at least 15 residues of the internal or N-terminal amino acid sequence by use of a rotating cup sequencer, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis -PAGE) under reducing or non-reducing conditions, using Coomassie blue or silver impregnation. The isolated polypeptide includes the polypeptide in situ within recombinant cells, since at least one component of the polypeptide's natural environment will not be present. In some cases, the isolated polypeptide will be prepared by at least one purification step.

[047] As used herein, the term "leukocytes" generally includes white blood cells derived from hematopoietic stem cells (HSC) produced in the bone marrow. "Leukocytes" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendrites).

[048] "T cell" includes all types of leukocytes that express CD3, including helper T lymphocytes (CD4+ cells), cytotoxic T lymphocytes (CD8+ cells), regulatory T lymphocytes (Treg) and gamma-delta T lymphocytes.

[049] A "cytotoxic cell" includes CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxicity responses.

[050] As used herein, the term "stem cell" generally includes pluripotent or multipotent stem cells. “Stem cells” include, for example, embryonic stem (ES) cells; mesenchymal stem cells (MSC); induced pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); bone marrow-derived cells, etc.).

[051] As used herein, the term “treatment” and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of a disease or its symptom and/or may be therapeutic in terms of partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, includes any treatment of disease in a mammal, e.g., a human, and includes: (a) preventing the disease from occurring in an organism that may be predisposed to the disease, but in which the disease has not yet been diagnosed; (b) inhibit the disease, that is, stop its development; and (c) alleviate the disease, that is, cause the disease to regress.

[052] The terms "individual", "organism", "host" and "patient", used interchangeably in this document, refer to a mammal, including, but not limited to, murinae (such as rats, mice), lagomorphs (such as rabbits) , non-human primates, humans, canines, felines, ungulates (such as horses, cattle, sheep, pigs, goats), etc.

[053] A "therapeutically effective dose" or "effective dose" refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other organism for the treatment of a disease, is sufficient to carry out this treatment for the disease. The "therapeutically effective dose" will vary depending on the agent(s), the disease and its severity, and age, weight, etc. of the organism to be treated.

[054] Before we describe this invention in detail, it should be understood that it is not limited to the embodiments described in particular, as such embodiments can, obviously, vary. It should also be understood that the terminology used in this document is intended to describe only particular embodiments, and is not intended to be exhaustive, as the scope of this invention will be limited by the appended claims.

[055] When a range of values is provided, it is understood that each intervening value, to the tenth of a unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other value declared or intervening in this specified range, is encompassed by the invention. The upper and lower limits of these minor ranges may be independently included in the minor ranges and are also covered by the invention, subject to any limits specifically excluded in the indicated range. When the stated range includes one or both of these limits, ranges excluding either or both of those included limits are also included in the invention.

[056] Unless otherwise defined, all technical and scientific terms used in this document have the meanings generally understood by a person with normal knowledge in the area to which this invention belongs. Although many methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention, the preferred methods and materials are now described. All publications referenced herein are incorporated herein by reference to disclose and describe the methods and/or materials with respect to which the publications are cited.

[057] It should be noted that, as used in this documents and the attached requests, the singular articles “a”, “an”, “the” and “a” include their plural forms, unless the context clearly indicates otherwise . Thus, for example, reference to "a chimeric antigen receptor" includes a plurality of such chimeric antigen receptors, and reference to "the dimerizer binding pair" includes reference to one or more dimerizer binding pairs and known equivalents. by experts in this area, and so on. It is also noted that orders may be drafted to exclude any optional elements. Thus, this statement is intended to serve as a background background to the use of exclusionary terminology, such as “only,” “only,” and others, in connection with the recitation of the elements of the order, or use of a “negative” limitation.

[058] It should be noted that certain features of the invention, which are, for clarification, described in the context of separate embodiments, may also be provided in combination in a single embodiment. On the other hand, various features of the invention, which are, for objectivity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments of the invention are specifically encompassed by this invention and are disclosed herein as if each combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements of the invention are specifically encompassed by this invention and are disclosed herein as if each combination were individually and explicitly disclosed.

[059] The publications discussed in this document are provided only for the invention prior to the filing date of this application. Nothing in this document should be construed as an admission that this invention cannot predate such publication by reason of prior invention. Additionally, the publication dates provided may differ from the actual publication dates, which may need to be confirmed independently.

Detailed Description

[060] This invention provides a conditionally active and heterodimeric chimeric antigen receptor (RQA), and a nucleic acid composed of a nucleotide sequence that encodes the RQA. This invention provides genetically modified cells that produce RQA. An RQA of this invention can be used in several methods, which are also provided. Conditionally active and heterodimeric chimeric antigen receptor.

[061] This invention provides a conditionally active and heterodimeric chimeric antigen receptor, which, for simplicity, is referred to herein as “RQA”.

[062] In some embodiments, an RQA of this invention consists of: a) a first polypeptide consisting of: i) a member of a specific binding pair (for example, 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 binding pair (e.g., an antigen-binding domain) and the first modulatory domain; and b) a second polypeptide consisting of: i) a transmembrane domain; ii) a second modulating domain; iii) a second member of the dimerization pair; and iv) an intracellular signaling domain. The modulatory domain may be a costimulatory domain.

[063] In some embodiments, an RQA of this invention consists of: a) a first polypeptide consisting of: i) a member of a specific binding pair (for example, an antigen-binding domain); ii) a first costimulatory domain; iii) a first member of a dimerization 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 consisting of: i) a transmembrane domain; ii) a second costimulatory domain; iii) a second member of the dimerization pair (such as the dimerizer-binding pair); and iv) an intracellular signaling domain.

[064] In some embodiments, an RQA of this invention consists of: a) a first polypeptide consisting of: i) a member of a specific binding pair (for example, an antigen-binding domain); ii) a modulating domain; iii) a first member of a dimerization pair (such as 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 modulatory domain; and b) a second polypeptide consisting of: i) a second member of the dimerization pair (such as the dimerizer-binding pair); and ii) an intracellular signaling domain. The modulatory domain may be a costimulatory domain.

[065] In some embodiments, an RQA of this invention consists of: a) a first polypeptide consisting of: i) a member of a specific binding pair (for example, an antigen-binding domain); ii) a costimulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the member of a specific binding pair (e.g., an antigen-binding domain) and the costimulatory domain; and b) a second polypeptide consisting of: i) a second member of the dimerization pair (such as the dimerizer-binding pair); and ii) an intracellular signaling domain.

[066] An example of an RQA of an organism is represented schematically in Figure 17. An RQA of this invention may be present in the plasma membrane of a eukaryotic cell, for example, a mammalian cell, suitable mammalian cells being, among others, a cytotoxic cell, a T lymphocyte, a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of a progenitor cell, and an NK cell. When present in a plasma membrane, an RQA of this invention is activated in the presence of: 1) a dimerizing agent binds to the first and second elements of the dimerizer-binding pair in the RQA, or otherwise induces dimerization of the first and second members of the dimer; and 2) a factor that binds to the element of a specific binding pair (e.g., an antigen-binding domain), e.g., an antigen that binds to the antigen-binding domain of the RQA. The factor that binds the specific binding pair member is a second specific binding pair member. The second member of the specific binding pair may be a soluble factor (e.g., not bound to a cell); 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; It's similar. If one member of a specific binding pair is an antibody, and the second member of the specific binding pair is an antigen, the antigen may be soluble (e.g., not bound to a cell); an antigen present 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; It's similar.

[067] In some cases, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by a second member of a specific binding pair that binds to the specific binding pair member of the RQA (e.g. , an antigen that binds to the antigen-binding domain of RQA) and a dimerizing agent, increases the expression of at least one nucleic acid in the cell. For example, in some cases, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds to the antigen-binding domain of the RQA and a dimerizing agent, increases the expression of at least a 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared with the level of nucleic acid transcription in the absence of antigen and/or the dimerizing agent.

[068] As an example, the second polypeptide of an RQA of this invention may include an intracellular signaling polypeptide containing an immune receptor activating tyrosine motif (ITAM); In such cases, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds to the antigen-binding domain of the RQA and a dimerizing agent, increases nuclear factor-dependent transcription of T cells. activated (NFAT). NFAT-dependent transcription includes transcription induced by any member of the NFAT family, including, for example, NFATc1, NFATC2, NFATc3, NFATc4, NFAT5; AP-1; Sp1; NKKB; and the like.

[069] An RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds to the antigen-binding domain of the RQA and a dimerizing agent, may, in some cases, result in an increase the production of one or more cytokines by the cell. For example, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds to the antigen-binding domain of the RQA and a dimerizing agent, can increase the production of a cytokine by the cell in 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 at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the dose of cytokine produced by the cell in the absence of the antigen and/or the dimerizing agent. Cytokines whose production can be increased include, among others, an interferon, for example, IL-2, interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), IL-15, IL-12, IL-4, IL-5, IL-10; a chemokine; a growth factor; and the like.

[070] In some cases, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds to the antigen-binding domain of the RQA and a dimerizing agent, may result in an increase in transcription of a nucleic acid in the cell and increased production of a cytokine by the cell.

[071] In some cases, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by a dimerizing agent, results in cytotoxic activity by the cell to a target cell that expresses on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of RQA binds. For example, when the eukaryotic cell is a cytotoxic cell (e.g., an NK cell or a cytotoxic T lymphocyte), an RQA of this invention, when present in the plasma membrane of the cell, and, when activated by a dimerizing agent, increases the activity cytotoxicity of the cell to a target cell that expresses on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of RQA binds. For example, when the eukaryotic cell is an NK cell or a T lymphocyte, an RQA of this invention, when present on the plasma membrane of the cell, and when activated by a dimerizing agent, increases the 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 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the cytotoxic activity of the cell in the absence of the dimerizing agent.

[072] In some cases, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds to the antigen-binding domain of the RQA and a dimerizing agent, may result in other related events to RQA activation, such as proliferation and expansion (due to increased cell division or anti-apoptotic responses).

[073] In some cases, an RQA of this invention, when present in the plasma membrane of a eukaryotic cell, and when activated by an antigen that binds to the RQA binding domain and a dimerizing agent, may result in other activation-related events of RQA, such as modulation of intracellular signaling, cell differentiation or cell death.

[074] An RQA of this invention can be present in a eukaryotic cell membrane, in which the first and second polypeptides of the RQA are not covalently linked to each other. An RQA of this invention 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 RQA of this invention may be present in a eukaryotic cell membrane as a heterodimer that is covalently or non-covalently linked to a second RQA of this invention. In some cases, the first and second RQA are covalently linked through a disulfide bond formed between the cysteines present in a hinge region present in the first polypeptide of the first RQA and the first polypeptide of the second RQA.

[075] In some cases, an RQA of this invention may be present in a eukaryotic cell membrane, in which the first polypeptides of the RQA consist of an antibody fragment and the second polypeptide of the RQA consists of a signal transduction domain derived from a cytokine receptor, such that, upon dimerization, the RQA can represent a heterodimeric RQA signalobody, for example, a signalobody composed of at least two independent polypeptides. A "signalobody", as it is known in Aria, is a simple chimeric macromolecule composed of an antibody fragment and a signal transduction domain derived from a cytokine receptor. In certain cases, a heterodimeric signalobody RQA of this invention, when present in the cell membrane of a eukaryotic cell, dimerized by a dimerizer, and activated by an antigen, for example, an oligomerized antigen, can induce oligomerization of the heterodimeric signalobody RQA. Such ligand-induced oligomerization of a heterodimeric signalobody RQA can activate, e.g., enhance, or perpetuate, e.g., maintain, signal transduction, e.g., ligand-induced oligomerization of a heterodimeric signalobody RQA can transmit a signal to trigger a cellular response. In some cases, a plurality of heterodimeric signalobody RQAs can be used in a combinatorial manner to induce a desired cellular response.

Member of a specific binding pair

[076] An RQA of this invention 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 similar. Thus, a member of a specific binding pair suitable for use in an RQA of this invention includes an antigen; an antibody; a binder; and a ligand-receptor receptor.

Antigen-binding domain

[077] A suitable antigen-binding domain for use in an RQA of this invention can be any antigen-binding polypeptide, a wide variety of which are known in the art. In some cases, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains (variable domains of camelid antibodies cAb VHH) and humanized versions, IgNAR VH (variable domains of shark antibodies) and humanized versions, sdAb VH (variable domains of single-domain antibodies) and variable domains of "camelized" antibodies are suitable for use. In some cases, T cell receptor (TCR)-based recognition domains such as single-chain TCR (scTv, VDVD-containing two-domain single-chain TCR) are also suitable for use.

[078] An antigen-binding domain suitable for use in an RQA of this invention can have a variety of antigen-binding specificities. In some cases, the antigen-binding domain is specific for an epitope present on an antigen that is expressed by (synthesized by) a cancer cell, i.e., an antigen associated with the cancer cell. The cancer cell-associated antigen may be an antigen associated with, for example, a breast cancer cell, a B-cell lymphoma, a Hodgkin's lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a B-non-Hodgkin's lymphoma cell (B-NHL), 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. An antigen associated with a cancer cell can also be expressed by a non-cancerous cell.

[079] Non-exhaustive examples of antigens to which an antigen-binding domain of an RQA of an organism can bind include, for example, CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), surface adhesion molecule CD44, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular mass melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like.

Ligand

[080] In some cases, a member of a specific binding pair suitable for use in an RQA of an organism is a ligand for a receptor. Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF); and the like); an integrin-binding peptide (e.g., a peptide consisting of the sequence Arg-Gly-Asp); and the like.

[081] If the member of a specific binding pair in an RQA of an organism is a ligand, the RQA can be activated in the presence of a dimerizing agent and a second member of the specific binding pair, the second member of the binding pair being specific binding of a receptor to the ligand. For example, when the ligand is VEGF, the second member of the specific binding pair may be a VEGF receptor, including a soluble VEGF receptor. As another example, when the ligand is heregulin, the second member of the specific binding pair may be Her2.

Receivers

[082] As mentioned above, in some cases, the member of a specific binding pair that is included in an RQA of an organism is a receptor, for example, a receptor for a ligand, a coreceptor, etc. The receptor may be a ligand-binding fragment of a receptor. Suitable receptors include, among others, a growth factor receptor (e.g., a VEGF receptor); a killer cell lectin-like receptor subfamily K polypeptide member 1 (NKG2D) (receptor for MICA, MICB, and ULB6); a cytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor, etc.); Her2; CD27; a natural cytotoxicity receptor (NCR) polypeptide (e.g., NKP30 (NCR3/CD337) (receptor for HLA-B-associated transcription 3 (BAT3) and B7-H6); etc.); etc.

Hinge region

[083] In some cases, the first polypeptide of an RQA of an organism consists of a hinge region (also called a "spacer" herein), 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 hinge region derived from CD-8).

[084] The hinge region may have a length of about 4 amino acids to about 50 amino acids, for example, 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 30 aa to about 40 aa, or from about 40 aa to about 50 aa.

[085] Suitable spacers can be readily selected and can be of any of a number of suitable lengths, such as from one 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.

[086] Exemplary spacers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:37) and (GGGS)n (SEQ ID NO :38), where n is an integer greater than or equal to one), glycine-alanine polymers, alanine-serine polymers, and other flexible ligands known in the art. Glycine-serine and glycine polymers can be used; Both Gly and Ser are relatively unstructured and therefore can serve as a neutral link between the components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and are much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary spacers may be comprised of 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 NO:42) , GGGSG (SEQ ID NO:43), GSSSG (SEQ ID NO:44), and the like.

[087] In some cases, the hinge region in the first polypeptide of an RQA of an organism includes at least one cysteine. For example, in some cases, the hinge region may include the sequence Cis-Pro-Pro-Cis. If present, a cysteine in the hinge region of a first RQA may be available to form a disulfide bond with a hinge region in a second RQA.

[088] Amino acid sequences in the immunoglobulin hinge region are known in the art; see, for example, Tan et al. (1990) Proc. Natl. Academic. Sci USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14: 1779. As non-exhaustive examples, an immunoglobulin hinge region may include one of the following amino acid sequences: DKTHT (SEQ ID NO:45); CPPC (SEQ ID NO:46); CPEPKSCDTPPPCPR (SEQ ID NO:47) (see, for example, Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO:48); KSCDKTHTCP (SEQ ID NO:49); KCCVDCP (SEQ ID NO:50); KYGPPCP (SEQ ID NO:51); EPKSCDKTHTCPPCP (SEQ ID NO:52) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:53) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:54) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:55) (human IgG4 hinge); and the like.

[089] The hinge region can be composed of an amino acid sequence from an IgG1, IgG2, IgG3, or IgG4 hinge region. The hinge region may include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally occurring) hinge region. For example, human IgG1 hinge His229 can be replaced by Tir, so that the hinge region is composed of the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 52); see, for example, Yan et al. (2012) J. Biol. Chem. 287:5891.

[090] The hinge region may be composed of an amino acid sequence derived from human CD8; for example, the hinge region may include the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:56), or a variation thereof.

Transmembrane domain

[091] The first and second polypeptides of an RQA of this invention 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. When 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 polypeptide includes, in order, from the amino terminus (N-terminus) to the carboxyl terminus (C -terminal): an antigen-binding domain; a hinge region; a transmembrane domain; a first costimulatory domain; and a first member of a dimerizer bond pair.

[092] The transmembrane domain of the second polypeptide is at or near the N terminus of the polypeptide, so that the second polypeptide includes, in order, from the N terminus to the C terminus: a transmembrane domain; a second costimulatory domain; a second member of the dimerizer binding pair; and an intracellular signaling domain.

[093] Any transmembrane domain (TM) that provides for the insertion of a polypeptide into the cell membrane of a eukaryotic cell (e.g., mammalian) is suitable for use. As a non-exhaustive example, the sequence TM IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30) can be used. Additional non-exhaustive examples of suitable sequences include: a) CD8 beta derivative: LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO:57); b) CD4 derivative: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO:58); c) CD3 zeta derivative: LCYLLDGILFIYGVILTALFLRV (SEQ ID NO:59); d) CD28 derivative: WVLVVVGGVLACYSLLVTVAFIFIFVV (SEQ ID NO:60); e) CD134 (OX40) derivative: VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO:61); and f) CD7 derivative: ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO:62).

Ligands

[094] In some cases, a first polypeptide of an RQA of an organism includes a linker between any two adjacent domains. For example, a linker may be disposed between the transmembrane domain and the first co-stimulatory domain of the first polypeptide. As another example, a linker may be disposed between the first co-stimulatory domain and the first member of the dimerizer binding pair of the first polypeptide. As another example, a linker can be disposed between the transmembrane domain and the second co-stimulatory domain of the second polypeptide. As another example, 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 between the second member of the dimerizer binding pair and the intracellular signaling domain of the second polypeptide.

[095] The binding peptide can have a wide variety of amino acid sequences. Proteins can be linked by a spacer peptide, generally flexible in nature, although other chemical bonds are not excluded. A linker can be a peptide about 6 to 40 amino acids long, or about 6 to 25 amino acids long. These linkers can be produced by using synthetic linker-encoding oligonucleotides to bind proteins. Peptide linkers with a degree of flexibility can be used. Linker peptides can have virtually any amino acid sequence, keeping in mind that suitable linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, is common in creating a flexible peptide. The creation of these sequences is routine for experts in the field.

[096] Suitable linkers can be readily selected and can be of any suitable length, such as from one 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.

[097] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:37) and (GGGS)n (SEQ ID NO:38), where n is an integer greater than or equal to one), glycine-alanine polymers, alanine-serine polymers, and other flexible ligands known in the art. Glycine and serine-glycine polymers are of interest since both amino acids are relatively unstructured and therefore can serve as a neutral link between components. Glycine polymers are of particular interest, since glycine accesses significantly more phi-psi space than even alanine, and are much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Flexible linkers include, but are not limited to, GGSG (SEQ ID NO:39), GGSGG (SEQ ID NO:40), GSGSG (SEQ ID NO:41), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID NO:43 ), GSSSG (SEQ ID NO:44), and the like. One skilled in the art will recognize that the design of a peptide conjugated to any elements described above may include linkers that are completely or partially flexible, such that they may include a flexible linker, as well as one or more moieties that impart less flexible structure.

Modulating domains

[098] Modulating domains suitable for use in an RQA of this invention include costimulatory domains.

[099] In some cases, the modulating domain in the first polypeptide of an RQA of an organism has substantially the same amino acid sequence as the modulating domain in the second polypeptide of the RQA. For example, in some cases, the modulatory domain in the first polypeptide of an RQA includes 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 modulating domain on the second polypeptide of RQA. The modulating domain of the first polypeptide of an RQA of an organism may be substantially the same length as the modulating domain of the second polypeptide of an RQA of an organism; for example, the first and second modulator domains may differ in length from each other by less than 10 amino acids, or less than 5 amino acids. In some cases, the first and second modulator domains are the same length.

[0100] A modulator domain suitable for inclusion in the first and second polypeptides of an RQA of an organism may have a length of about 30 amino acids to about 70 amino acids (aa), for example, a modulator domain may have a length of about from 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 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In other cases, the modulatory domain may have a length of about 70 aa to 100 aa, about 100 aa to 200 aa, or greater than 200 aa.

[0101] Costimulatory domains suitable for use in the RQA of this invention are generally receptor-derived polypeptides. In some embodiments, co-stimulatory domains homodimerize. A costimulatory domain of an organism may be an intracellular portion of a transmembrane protein (that is, the costimulatory domain may be derived from a transmembrane protein). Non-exhaustive examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0102] In some cases, the costimulatory domain in the first polypeptide of an RQA of an organism has substantially the same amino acid sequence as the costimulatory domain in the second polypeptide of the RQA. For example, in some cases, the costimulatory domain in the first polypeptide of an RQA includes 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 costimulatory domain on the second polypeptide of RQA. The costimulatory domain of the first polypeptide of an RQA of an organism may be substantially the same length as the costimulatory domain of the second polypeptide of an RQA of an organism; for example, the first and second co-stimulatory domains may differ in length from each other by less than 10 amino acids, or less than 5 amino acids. In some cases, the first and second costimulatory domains are the same length.

[0103] A costimulatory domain suitable for inclusion in the first and second polypeptides of an RQA of an organism may have a length of about 30 amino acids to about 70 amino acids (aa), for example, a costimulatory domain may have a length of about from 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 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In other cases, the costimulatory domain may have a length of about 70 aa to 100 aa, about 100 aa to 200 aa, or greater than 200 aa.

[0104] In some cases, the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; 4-1BB; CDw137; ILA; etc.). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:24). In some such embodiments, the co-stimulatory domain of both the first and second polypeptides 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. 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.

[0105] In some cases, the costimulatory domain is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:63). In some such embodiments, the co-stimulatory domain of both the first and second polypeptides 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. 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.

[0106] In some cases, the costimulatory domain is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278 and CVID1). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO:64). In some such embodiments, the co-stimulatory domain of both the first and second polypeptides 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. 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.

[0107] In some cases, the costimulatory domain is derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:65). In some such embodiments, the co-stimulatory domain of both the first and second polypeptides 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. 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.

[0108] In some cases, the costimulatory domain is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIY DNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICV RS (SEQ ID NO:66).

[0109] In some cases, the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:67). In some such embodiments, the co-stimulatory domain of both the first and second polypeptides 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. 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.

[0110] In some cases, the costimulatory domain is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatory domain may include 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 at least about 98%, or 100% of the amino acid sequence identity of with a contiguous length of 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: EE EGKEDPLPTAASGK (SEQ ID NO:68).

[0111] In some cases, the costimulatory domain is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGD LWV (SEQ ID NO:69). In some such embodiments, the co-stimulatory domain of both the first and second polypeptides 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. 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.

[0112] In some cases, the costimulatory domain is derived from an intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable costimulatory domain may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO:70). In some such embodiments, the co-stimulatory domain of both the first and second polypeptides 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. 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

[0113] Dimer pairs suitable for use in an organism RQA include dimerizer binding pairs. Suitable dimerizer binding pairs for use in the RQA of this invention are, in some embodiments, polypeptides that bind to a different site on the same molecule (herein referred to as a “dimerizer”). dimmer switches connect to a different location on the dimmer and are thus brought closer to each other. In some embodiments, the connection to the dimerizer is reversible. In some embodiments, the connection to the dimerizer is irreversible. In some embodiments, the connection to the dimerizer is non-covalent. In some embodiments, the bond to the dimerizer is covalent.

[0114] Other dimer pairs suitable for use include dimerizer-binding pairs that dimerize 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 (covalently or non-covalently) to a second member of the dimer pair.

[0115] Other dimer pairs suitable for use include dimer pairs in which exposure to light (e.g., blue light) induces dimerization of the dimer pair.

[0116] Regardless of the mechanism, the pair of dimers 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. Therefore, for simplicity, the discussion below regarding “dimer bond pairs” includes dimer pairs that dimerize independently of the mechanism.

[0117] Non-exhaustive examples of suitable dimers (e.g., dimerizer binding pairs) include, among others: a) FK506 binding protein (FKBP) and FKBP; b) FKBP and calcineurin catalytic subunit A (CnA); c) FKBP and cyclophilin; d) FKBP and FKBP rapamycin-associated protein (FRB); e) rotate B (GyrB) and GyrB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB; h) PYL and ABI; i) Cry2 and CIB1; and j) GAI and GID1.

[0118] A first or second member of a dimer (e.g., a dimerizer binding pair) of an RQA of an organism may be about 50 amino acids long and about 300 amino acids or more; for example, a first or second member of a dimer (e.g., a dimerizer-binding pair) of an RQA of an organism may have a length of 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 to 250 aa, from about 250 aa to about 300 aa, or more than 300 aa.

[0119] In some cases, a member of a dimer (e.g., a dimerizer binding pair) of an RQA of an organism is derived from FKBP. For example, a member of a suitable dimerizer binding pair may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWE EGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:12).

[0120] In some cases, a member of a dimerizer-binding pair of an RQA of an organism is derived from a calcineurin catalytic A subunit (also known as catalytic subunit PPP3CA; CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B ;CAM-PRP; calcineurin A alpha; calcineurin A alpha isoform; catalytic subunit alpha isoform; For example, a member of a suitable dimerizer binding pair may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence (PP2Ac domain): GGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLED FGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTGF PSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFM (SEQ ID NO:71).

[0121] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from cytophilin (also known as cytophilin A, PPIA, CIPA, CIPH, PPIase A, etc.). For example, a member of a suitable dimerizer binding pair may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: NTVEAMERFGSRNGKTSKKTΠADCCTQLE (SEQ ID NO:72).

[0122] 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-rapamycin-associated protein 1 ; 12-rapamycin-associated protein 2; FK506-binding protein 1; For example, a member of a suitable dimerizer binding pair may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence (also known as “Frb”: Rapamycin Binding Domain Fkbp): 14).

[0123] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from GyrB (also as subunit B of gyrase DNA). For example, a member of a suitable dimerizer binding pair may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity with a contiguous length of 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, from about 700 aa at about 800 aa, from the following Escherichia coli GyrB amino acid sequence (or the B sequence of the DNA gyrase subunit): LELVIQREGKIHRQIYEHGVPQAPLAVTGETEKTGTMVRFWPSL ETFTNVTEFEYEILAKRLRELSFLNSGVSIRLRDKRDGKEDHFHYEGGIKAFVEYLNKNKT PIHPNIFYFSTEKDGIGVEVALQWNDGFQENIYCFTNNIPQRDGGTHLAGFRAAMTRTLNA YMDKEGYSKKAKVSATGDDAREGLIAVVSVKVPDPKFSSQ GK QEQYIKDDEAMDQYQISIALDGATLHTNASAPALAGEALEKLVSEYNATQKMINRMERR YPKAMLKELIYQPTLTEADLSDEQTVTRWVNALVSELNDKEQHGSQWKFDVHTNAEQNLFE PIVRVRTHGVDTDYPLDHEFITGGEYRRICTLGEKLRGLLEEDAFIERGERRQPVASFEQA LDWLVKESRRGLSIQRYKGLGEMN PEQLWETTMDPESRRMLRVTVKDAIAADQLFTTLMGD AVEPRRAFIEENALKAANIDI (SEQ ID NO:73). For example, a member of a dimerizer binding pair includes an amino acid sequence of 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 for amino acids 1-220 of the GirB amino acid sequence listed above from Escherichia coli.

[0124] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from DHFR (also known as dihydrofolate reductase, DHFRP1, and DYR). For example, a member of a suitable dimerizer binding pair may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: FFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVY EKND (SEQ ID NO:74).

[0125] 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 example, a member of a suitable dimerizer binding pair may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: MASRGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIR GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:75).

[0126] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from a PIL protein (also known as abscisic acid receptor and RCAR). For example, a member of an organism's dimerizer binding pair may be derived from proteins such as those from Arabidopsis thaliana: PYR1, RCAR1(PYL9), PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8 (RCAR3 ), PYL10, PYL11, PYL12, PYL13. For example, a member of a suitable dimerizer binding pair may include an amino acid sequence of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences:

[0127] PIL10: MNGDETKKVESEYIKKHHRHELVESQCSSTLVKHIKAPLHLVWSIVRRFDEPQKYKPFISR CVVQGKKLEVGSVREVDLKSGLPATKSTEVLEILDDNEHILGIRIVGGDHRLKNYSSTISL HSETIDGKTGTLAIESFVVDVPEGNTKEETCFFVEALIQCNLNSLADVTERLQAESMEKKI (SEQ ID NO:76).

[0128] PYL11: METSQKYHTCGSTLVQTIDAPLSLVWSILRRFDNPQAYKQFVKTCNLSSGDGGEGSVREVT VVSGLPAEFSRERLDELDDESHVMMISIIGGDHRLVNYRSKTMAFVAADTEEKTVVVESYV VDVPEGNSEEETTSFADTIVGFNLKSLAKLSERVAHLKL (SEQ ID NO:77)

[0129] PIL12: MKTSQEQHVCGSTVVQTINAPLPLVWSILRRFDNPKTFKHFVKTCKLRSGD GGEGSVREVTVVSDLPASFSLERLDELDDESHVMVISIIGGDHRLVNYQSKTTVFVAAEEE KTVVVESYVVDVPEGNTEEETTLFADTIVGCNLRSLAKLSEKMMELT (SEQ ID NO:78).

[0130] PIL13: MESSKQKRCRSSVVETIEAPLPLVWSILRSFDKPQAYQRFVKSCTMRSGGG GGKGGEGKGSVRDVTLVSGFPADFSTERLEELDDESHVMVVSIIGGNHRLVNYKSKTKVVA SPEDMAKKTVVVESYVVDVPEGTSEEDTIFFVDNIIRYNLTSLAKLTKKMMK (SEQ ID NO:79).

[0131] PIL1: MANSESSSSPVNEEENSQRISTLHHQTMPSDLTQDEFTQLSQSIAEFHTYQ LGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVIS GLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESY VVDVPEGNSEEDTRLFADTVIRL NLQKLASITEAMNRNNNNNNSSQVR (SEQ ID NO:80).

[0132] PIL2:MSSSPAVKGLTDEEQKTLEPVIKTYHQFEPDPTTCTSLITQRIHAPASVVW PLIRRFDNPERYKHFVKRCRLISGDGDVGSVREVTVISGLPASTSTERLEFVDDDHRVLSF RVVGGEHRLKNYKSVTSVNEFLNQDSGKVYTVVLESYTVDIPEGNTEEDTKMFVDTVVKLN LQKLGVAATSAPMHD OF (SEQ ID NO:81).

[0133] PIL3:MNLAPIHDPSSSSTTTTSSSTPYGLTKDEFSTLDSIIRTHHTFPRSPNTCT SLIAHRVDAPAHAIWRFVRDFANPNKYKHFIKSCTIRVNGNGIKEIKVGTIREVSVVSGLP ASTSVEILEVLDEEKRILSFRVLGGEHRLNNYRSVTSVNEFVVLEKDKKKRVYSVVLESYI VDIPQGNTEEDTRMF VDTVVKSNLQNLAVISTASPT (SEQ ID NO:82).

[0134] PIL4:MLAVHRPSSAVSDGDSVQIPMMIASFQKRFPSLSRDSTAARFHTHEVGPNQ CCSAVIQEISAPISTVWSVVRRFDNPQAYKHFLKSCSVIGGDGDNVGSLRQVHVVSGLPAA SSTERLDILDDERHVISFSVVGGDHRLSNYRSVTTLHPSPISGTVVVESYVVDVPPGNTKE ETCDFVDVIVRCNLQSLAK IAENTAAESKKKMSL (SEQ ID NO:83).

[0135] PIL5:MRSPVQLQHGSDATNGFHTLQPHDQTDGPIKRVCLTRGMHVPEHVAMHHTH DVGPDQCCSSVVQMIHAPPESVWALVRRFDNPKVYKNFIRQCRIVQGDGLHVGDLREVMVV SGLPAVSSTERLEILDEERHVISFSVVGGDHRLKNYRSVTTLHASDDEGTVVVESYIVDVP PGNTEEETLSFV DTIVRCNLQSLARSTNRQ (SEQ ID NO:84).

[0136] PIL6:MPTSIQFQRSSTAAEAANATVRNYPHHHQKQVQKVSLTRGMADVPEHVELS HTHVVGPSQCFSVVVQDVEAPVSTVWSILSRFEHPQAYKHFVKSCHVVIGDGREVGSVREV RVVSGLPAAFSLERLEIMDDDRHVISFSVVGGDHRLMNYKSVTTVHESEEDSDGKKRTRVV ESY VVDVPAGNDKEETCSFADTIVRCNLQSLAKLAENTSKFS (SEQ ID NO:85).

[0137] PIL7: MEMIGGDDTDTEMYGALVTAQSLRLRHLHHCRENQCTSVLVKYIQAPVHLV WSLVRRFDQPQKYKPFISRCTVNGDPEIGCLREVNVKSGLPATTSTERLEQLDDEEHILGI NIIGGDHRLKNYSSILTVHPEMIDGRSGTMVMESFVVDVPQGNTKDDTCYFVESLIKCNLK SLACVSERLAAQDITNSIAT FCNASNGYREKNHTETNL (SEQ ID NO:86).

[0138] PIL8: MEANGIENLTNPNQEREFIRRHHKHELVDNQCSSTLVKHINAPVHIVWSLV RRFDQPQKYKPFISRCVKGNMEIGTVREVDVKSGLPATRSTERLELLDDNEHILSIRIVG GDHRLKNYSSIISLHPETIEGRIGTLVIESFVVDVPEGNTKDETCYFVEALIKCNLKSLAD ISERLAVQDTTESRV (SEQ ID NO:87).

[0139] PIL9: MMDGVEGGTAMYGGLETVQYVRTHHQHLCRENQCTSALVKHIKAPLHLVWS LVRRFDQPQKYKPFVSRCTVIGDPEIGSLREVNVKSGLPATTSTERLELLDDEEHILGIKI IGGDHRLKNYSSILTVHPEIIEGRAGTMVIESFVVDVPQGNTKDETCYFVEALIRCNLKSL ADVSERLASQDITQ (SE QID NO:88).

[0140] PIR: QID NO:89).

[0141] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from an ABI (also known as abscisic acid insensitive) protein. For example, a member of an organism's dimerizer-binding pair may be derived from proteins such as those from Arabidopsis thaliana: ABI1 (Also known as ABSCISIC ACID INSENSITIVE 1, Protein Phosphatase 2C 56, AtPP2C56, P2C56, and PP2C ABI1) and/or ABI2 (also known as P2C77, Protein phosphatase 2C 77, AtPP2C77, ABSCISIC ACID INSENSITIVE 2, Protein phosphatase 2C ABI2, and PP2C ABI2). For example, a suitable dimerizer binding pair member may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about of 95%, at least about 98%, or 100% amino acid sequence identity with a contiguous length of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, of 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 from 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 one of the following amino acid sequences:

[0142] ABI: LAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTS VVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARV FGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRI LLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNIS VVVVDLKPRRK LKSKPLN (SEQ ID NO:90).

[0143] ABI2: MDEVSPAVAVPFRPFTDPHAGLRGYCNGESRVTLPESSCSGDGAMKDSSFEINTRQDSLTS SSSAMAGVDISAGDEINGSDEFDPRSMNQSEKKVLSRTESRSLFEFKCVPLYGVTSICGRR PEMEDSVSTIPRFLQVSSSSLLDGRVTNGFNPHLSAHFFGVYDGHGGSQVANYCRERMHLA LTEEIVKEKPEFCDGDTW QEKWKKALFNSFMRVDSEIETVAHAPETVGSTSVVAVVFPTHI FVANCGDSRAVLCRGKTPLALSVDHKPDRDDEAARIEAAGGKVIRWNGARVFGVLAMSRSI GDRYLKPSVIPDPEVTSVRRVKEDDCLILASDGLWDVMTNEEVCDLARKRILLWHKKNAMA GEALLPAEKRGEGKDPAAMSAAEYLSKMALQKGSKDNISVVVVDLK GIRKFKSKSLN (SEQ ID NO:91).

[0144] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from a Cri2 protein (also known as cryptochrome 2). For example, a member of a dimer from an organism (e.g., a dimerizer binding pair) can be derived from Cri2 proteins from any organism (e.g., a plant), such as, among others, those from Arabidopsis thaliana. For example, a suitable dimerizer binding pair member may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about of 95%, at least about 98%, or 100% amino acid sequence identity with a contiguous length of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, of 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 from 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 one of the following amino acid sequences:

[0145] Cri2 (Arabidopsis thaliana) MPITAAAEA IWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNST SLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICNFFP FTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFA VKFLLLPWKWGMKYFWDT LLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEG EYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRT REAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEE ERDMKKSRGFDERELFSTAESSSSVFFVSQSCSLASEGKNLEGIQDSSD QITTSLGKNG CK (SEQ ID NO:92).

[0146] In some cases, a member of a dimer (e.g., a dimerizer-binding pair) is derived from an Arabidopsis thaliana CIB1 protein (also known as bHLH63 transcription factor). For example, a suitable dimer member (e.g., a dimerizer binding pair) may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about of 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with a contiguous length of about 100 amino acids to about 110 amino acids (aa), of 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 approximately 200 aa from the following amino acid sequence: KISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEF LSMKLAIVNPRPDFDMDDIFAKEVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYL HVNPMQQVNTSSDPLSCFNNGEAPSMWDSHVQNLYGNLGV (SEQ ID NO:93).

[0147] In some cases, a member of a dimer (e.g., a dimerizer-binding pair) is derived from an Arabidopsis thaliana GAI protein (also known as Abscisic Acid Insensitive, and DELLA GAI protein). For example, a suitable dimerizer binding pair member may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about of 95%, at least about 98%, or 100% amino acid sequence identity with a contiguous length of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, of 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 from 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 as follows amino acid sequence:MKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLE QLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPPSSNAEYDLKAIPGDAILN QFAIDSASSSNQGGGGDTYTTNKRLKCSNGVVETTTATAESTRHVVLVDSQENGVRLVHAL LACAEAVQKENLTVAEALVKQIGFLAVSQIG AMRKVATYFAEALARRIYRLSPSQSPIDHS LSDTLQMHFYETCPYLKFAHFTANQAILEAFQGKKRVHVIDFSMSQGLQWPALMQALALRP GGPPVFRLTGIGPPAPDNFDYLHEVGCKLAHLAEAIHVEFEYRGFVANTLADLDASMLELR PSEIESVAVNSVFELHKLLGRPGAIDKVLGVVNQIKPEIFTVVEQESNHNSPIFLDR FTES LHYYSTLFDSLEGVPSGQDKVMSEVYLGKQICNVVACDGPDRVERHETLSQWRNRFGSAGF AAAHIGSNAFKQASMLLALFNGGEGYRVEESDGCLMLGWHTRPLIATSAWKLSTN (SEQ ID NO:94).

[0148] In some cases, a member of a dimer (e.g., a dimerizer-binding pair) is derived from an Arabidopsis thaliana GID1 protein (also known as the GID1 Gibberellin receptor). For example, a suitable dimer member may include 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 with a contiguous length of 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 sequences of amino acids:

[0149] GID1A:MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDR KVTANANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFF HGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRS HIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLD GKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGDLLIRDWQ LAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAEC (SEQ ID NO:95).

[0150] GID1B: MAGGNEVNLNECKRIVPLNTWVLISNFKLAYKVLRRPDGSFNRDLAEFLDR KVPANSFPLDGVFSFDHVDSTTNLLTRIYQPASLLHQTRHGTLELTKPLSTTEIVPVLIFF HGGSFTHSSANSAIYDTFCRRLVTICGVVVVSVDYRRSPEHRYPCAYDDGWNALNWVKSRV WLQSGKDSNVYVY LAGDSSGGNIAHNVAVRATNEGVKVLGNILLHPMFGGQERTQSEKTLD GKYFVTIQDRDWYWRAYLPEGEDRDHPACNPFGPRGQSLKGVNFPKSLVVVAGLDLVQDWQ LAYVDGLKKTGLEVNLLYLKQATIGFYFLPNNDHFHCLMEELNKFVHSIEDSQSKSSPVLL TP (SEQ ID NO:96)

[0151] GID1C:MAGSEEVNLIESKTVVPLNTWVLISNFKLAYNLLRRPDGTFNRHLAEFLDRKVPANANPVN GVFSFDVIIDRQTNLLSRVYRPADAGTSPSITDLQNPVDGEIVPVIVFFHGGSFAHSSANS AIYDTLCRRLVGLCGAVVVSVNYRRAPENRYPCAYDDGWAVLKWVNSSSWLRSKKDSKVRI FLAGDSSGGNIVH NVAVRAVESRIDVLGNILLNPMFGGTERTESEKRLDGKYFVTVRDRDW YWRAFLPEGEDREHPACSPFGPRSKSLEGLSFPKSLVVVAGLLDLIQDWQLKYAEGLKKAGQ EVKLLYLEQATIGFYLLPNNNHFHTVMDEIAAFVNAECQ (SEQ ID NO:97).

Dimmers

[0152] Dimerizers ("dimerizing agents") that can provide for the dimerization of a first member of a dimerizer-binding pair and a second member of a dimerizer-binding pair include, for example, (where the dimerizer is between parentheses after the dimerizer binding pair: a) FKBP and FKBP (rapamycin); b) FKBP and CnA (rapamycin); c) FKBP and cyclophilin (rapamycin): a) FKBP and FRG (rapamycin); (coumermycin); f) DHFR and DHFR (metrotexate); g) DmrB and DmrB (AP20187); .

[0153] As noted above, rapamycin can serve as a dimerizer. Alternatively, a rapamycin derivative or analogue can be used. See, for example, WO96/41865; WO 99/36553; WO 01/14387; and Ye et al (1999) Science 283: 88-91. For example, analogues, homologues, derivatives and other compounds structurally related to rapamycin ("rapalogs") include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or substitution of the methoxy at C7, C42 and/or C29; elimination, derivatization or substitution of hydroxy at C13, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative exchange in the cyclohexyl ring or replacement of the cyclohexyl ring with a substitute cyclopentyl ring. Additional information is provided in, for example, U.S. Pat. Us. 5,525,610; 5,310,903 5,362,718; and 5,527,907. Selective epimerization of the C-28 hydroxyl group has been described; see, for example, WO 01/14387. Additional synthetic dimerizing agents suitable for use as an alternative to rapamycin include those described in US Patent Publication No. 2012/0130076.

[0154] Rapamycin has the following structure:

[0155] Suitable Rapalogs include, for example:

[0156] Also suitable as a rapalog is the compound with the following formula:

[0157] where n is 1 or 2; R28 and R43 are independently H, or a substituted or unsubstituted aliphatic or acyl radical; one of R7a and R7b is H and the other AAAA AB AB is a halo, R , OR , SR , -OC(O)R , -OC(O)NR R , -NRR , - BABABABA B' 7a 7b NR C( OR)R , NR C(O)OR , -NR SO2R , or NR SO2NR R ; or R and R, together, are H in the tetraene group:

[0158] where RA is H or a substituted or unsubstituted aliphatic, heteroaliphatic group, aryl, or heteroaryl and where RB and RB' are independently H, OH, or a substituted or unsubstituted aliphatic, heteroaliphatic group, aryl, or heteroaryl.

[0159] As noted above, coumarmycin can serve as a dimerizing agent. Alternatively, a coumarmycin analogue can be used. See, for example, Farrar et al. (1996) Nature 383:178-181; and U.S. Pat. No. 6,916,846.

[0160] As noted above, in some cases, the dimerizing agent is methotrexate, for example, a homo-bifunctional non-cytotoxic methotrexate dimer. See, for example, US Patent No. 8,236,925.

Intracellular signaling domain

[0161] Suitable intracellular signaling domains for use in an RQA of this invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; changes in transcription of a target gene ; change in the activity of a protein; change in cell behavior, e.g., cell proliferation; cell survival; activated by antigen and dimerization agent). In some embodiments, the intracellular signaling domain includes at least one ITAM group (e.g., one, two, three, four, five, six, etc.), as described below. In some embodiments, the intracellular signaling domain includes DAP10/CD28-type signaling chains. In some embodiments, the intracellular signaling domain is not covalently linked to the membrane-bound RQA, but is diffuse in the cytoplasm.

ITAM

[0162] Suitable intracellular signaling domains for use in an RQA of this invention include immunoreceptor tyrosine-based activating group (ITAM) containing intracellular signaling polypeptides. An ITAM group is YX1X2L/I, where X1 and X2 are independently any amino acid (SEQ ID NO:130). In some cases, the intracellular signaling domain of an RQA of an organism includes 1, 2, 3, 4, or 5 ITAM groups. In some cases, an ITAM group is repeated twice in an intracellular signaling domain, and the first and second occurrences of the ITAM group are separated from each other 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 an RQA of an organism includes 3 ITAM groups.

[0163] A suitable intracellular signaling domain may be an ITAM group-containing portion derived from a polypeptide that contains an ITAM group. For example, an intracellular signaling domain may be an ITAM group-containing domain of any ITAM group-containing protein. Therefore, a suitable intracellular signaling domain need not contain the complete sequence of the entire protein from which it is derived. Examples of suitable ITAM group-containing polypeptides include, among others: DAP12; FCER1G (epsilon I Fc receptor gamma chain); CD3D (CD3 delta); CD3e (CD3 epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A (antigen-receptor complex-associated protein alpha chain).

[0164] In some cases, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; tyrosine kinase binding protein TYRO protein; KARAP; PLOSL; DNAX activation protein 12; KAR-associated protein; binding protein TYRO protein tyrosine kinase-binding protein; TYRO protein tyrosine kinase-associated protein; For example, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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 sequences (4 isoforms): MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAV YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:98); MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDL VLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYY K (SEQ ID NO:99); MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVY FLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:100); or MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVY FLGRLVPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:101), where ITAM groups are in bold and underlined.

[0165] Likewise, an intracellular signaling domain polypeptide can include an ITAM group-containing portion of the complete DAP12 amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: ESPYQELQGQRSDVYSDLNTQ (SEQ ID NO:102), where the ITAM groups are in bold and underlined.

[0166] In some cases, the intracellular signaling domain is derived from FCER1G (also known as FCRG; epsilon receptor gamma chain Fc; epsilon receptor gamma chain Fc; gamma RI epsilon fc; fcRgamma; fceRI gamma; gamma subunit of high-affinity immunoglobulin receptor; high-affinity immunoglobulin E receptor, gamma chain, etc.). For example, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVR KAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO:103), where ITAM groups are in bold and underlined.

[0167] Likewise, an intracellular signaling domain polypeptide can include an ITAM group-containing portion of the complete FCER1G amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: DGVYTGLSTRNQETYETLKHE (SEQ ID NO: 104), where the ITAM groups are in bold and underlined.

[0168] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, polypeptide delta (TiT3 complex); OKT3 delta chain; T-cell receptor delta chain; T-cell surface glycoprotein CD3; For example, a suitable intracellular signaling domain polypeptide may include 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 of having a contiguous length of about 100 amino acids to about 110 amino acids (aa), of about 110 aa to about 115 aa, of 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 any of the following amino acid sequences (2 isoforms): MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTL LSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDV lATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYS HLGGNWARNK (SEQ ID NO:105) or MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHLGGN WARNK (SEQ ID NO:106), where the ITAM groups are in bold and underlined.

[0169] Likewise, a suitable intracellular signaling domain polypeptide may include an ITAM group-containing portion of the complete CD3 amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: DQVYQPLRDRDDAQYSHLGGN (SEQ ID NO:107), where the ITAM groups are in bold and underlined.

[0170] In some cases, the intracellular signaling domain is derived from a T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen epsilon T3/Leu-4 chain, CD3 epsilon chain of T cell surface glycoprotein, AI504783, CD3, CD3epsilon, T3e, etc.). For example, a suitable intracellular signaling domain polypeptide may include 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 of having a contiguous length of about 100 amino acids to about 110 amino acids (aa), of about 110 aa to about 115 aa, of 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: NKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:108), where ITAM groups are in bold and underlined.

[0171] Likewise, an intracellular signaling domain polypeptide may include an ITAM group-containing portion of the complete CD3 amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: NPDYEPIRKGQRDLYSGLNQR (SEQ ID NO:109), where the ITAM groups are in bold and underlined.

[0172] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMA, T3G, gamma polypeptide (complex TiT3), etc.). For example, a suitable intracellular signaling domain polypeptide may include 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 of having a contiguous length of about 100 amino acids to about 110 amino acids (aa), of about 110 aa to about 115 aa, of 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 following amino acid sequence: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAK NITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNA ATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKD REDDQYSH LQGNQLRRN (SEQ ID NO:110), where ITAM groups are in bold and underlined.

[0173] Likewise, a suitable intracellular signaling domain polypeptide may include an ITAM group-containing portion of the complete CD3 amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: DQLYQPLKDREDDQYSHLQGN (SEQ ID NO:111), where the ITAM groups are in bold and underlined.

[0174] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3GZ T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z , TCRZ, etc.). For example, a suitable intracellular signaling domain polypeptide may include 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 of having a contiguous length of about 100 amino acids to about 110 amino acids (aa), of about 110 aa to about 115 aa, of 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 any of the following amino acid sequences (2 isoforms): either Q ID NO:113), where ITAM groups are in bold and underlined.

[0175] Likewise, an intracellular signaling domain polypeptide may include an ITAM group-containing portion of the complete CD3 amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:18); NQLYNELNLGRREEYDVLDKR (SEQ ID NO:114); EGLYNELQKDKMAEAYSEIGMK (SEQ ID NO:115); or DGLYQGLSTATKDTYDALHMQ (SEQ ID NO:116), where ITAM groups are in bold and underlined.

[0176] In some cases, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; For example, a suitable intracellular signaling domain polypeptide may include 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 with a contiguous length of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about from 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 of 220 aa, of any of the following amino acid sequences (2 isoforms):

[0177] MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPAS LMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGG IYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLL LFRKRWQNEKLGLDAGDEYEDENLYEGLNLD DCSMYEDISRGLQGTYQDVGSLNIGDVQLE KP (SEQ ID NO:117); or

(SE Q ID NO:118), where ITAM groups are in bold and underlined.

[0179] Likewise, an intracellular signaling domain polypeptide can include an ITAM group-containing portion of the complete DA79A amino acid sequence. Therefore, a suitable intracellular signaling domain polypeptide may include an amino acid sequence of 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: ENLYEGLNLDDCSMYEDISRG (SEQ ID NO:119), where the ITAM groups are in bold and underlined.DAP10/CD28

[0180] Intracellular signaling domains suitable for use in an RQA of this invention include a DAP10/CD28-type signaling chain.

[0181] An example of a DAP10 signaling chain is the amino acid sequence: RPRRSPAQDGKVYINMPGRG (SEQ ID NO:120). In some embodiments, a suitable intracellular signaling domain includes an amino acid sequence of 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 complete amino acid sequence RPRRSPAQDGKVYINMPGRG (SEQ ID NO:120).

[0182] An example of a CD28 signaling chain is the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRS (SEQ ID NO:121). In some embodiments, a suitable intracellular signaling domain includes an amino acid sequence of 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 complete amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRS (SEQ ID NO:121).ZAP70

[0183] Suitable intracellular signaling domains for use in an RQA of this invention include a ZAP70 polypeptide, for example, a polypeptide including 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 for a contiguous stretch of about 300 amino acids and about 400 amino acids, from about 400 amino acids to about 500 amino acids , or from about 500 amino acids to about 619 amino acids of the following amino acid sequence: LTREEA ERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQ LVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPE PARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGV YRMRKKQIDVAIKVLKQ GTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMA GGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKIS DFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKP YKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDC WIYKWEDRPDFLTVEQRMRACYYSL ASKVEGPPGSTQKAEAACA (SEQ ID NO:36).

Additional sequences

[0184] The first and/or second polypeptide of an RQA of an organism may further include one or more additional polypeptide domains, where such domains include, among others, a signal sequence; an epitope identification; an affinity domain; and a polypeptide that produces a detectable signal.

Signal sequences

[0185] Signal sequences that are suitable for use in an RQA of an organism, for example, in the first polypeptide of an RQA of an organism, include any eukaryotic signal sequence, including a naturally occurring signal sequence, a synthetic sign (e.g. man-made), etc.

Epitope identification

[0186] Suitable epitope identifications include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO:122); FLAG (e.g., DYKDDDDK (SEQ ID NO:123); c-myc (e.g., EQKLISEEDL ; SEQ ID NO:4), and the like.

Affinity Domain

[0187] Affinity domains include peptide sequences that can interact with a binding partner, for example, as one immobilized on a solid support, useful for identification or purification. DNA sequences encoding multiple consecutive individual amino acids, such as histidine, when fused to the expressed protein, can be used for single-step purification of the recombinant protein by high-affinity binding to a resin column, such as nickel sepharose. . Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 124), HisX6 (HHHHHH) (SEQ ID NO: 125), C-myc (EQKLISEEDL) (SEQ ID NO: 4), Flag (DYKDDDDK) (SEQ ID NO:123), StrepTag (WSHPQFEK) (SEQ ID NO:126), hemagglutinin, 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 identification, WEAAAREACCRECCARA (SEQ ID NO: 129 ), metal-binding domains, e.g., zinc-binding domains or ligands, or calcium-binding domains, such as those from calcium-binding proteins, e.g., calmodulin large subunit, troponin C, calcineurin B, light chain myosin, recoverin, S-modulin, visinin, VILIP, neurocalcin, hypocalcin, frequenin, caltractin, calpain, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, inteins, biotin, streptavidin, MyoD, Id, zipper sequences leucine, and maltose binding protein.

Detectable signal-producing polypeptides

[0188] Suitable detectable signal-producing proteins include, for example, fluorescent proteins; enzymes that catalyze a reaction that generates a detectable signal as a product; and the like.

[0189] Suitable fluorescent proteins include, among others, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), GFP enhanced (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP ) , mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J- Red, dimer2, t-dimer2(12), mRFP1, pocyloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and killer protein, phycobiliprotein and phycobiliprotein conjugates including B-phycoerythrin, R-Phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mMelada, mBanana, mLorange, dTomate, tdTomate, mTangerine, mMstrawberry, mCherry, mUva1, mRaspberry, mUva2, mPlum (Shaner et al (2005) Nat. Methods: 2:905-909), and the like. Any of the variety of fluorescent and colored proteins from Anthozoan species, as described in, for example, Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

[0190] Suitable enzymes include, among others, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like.

Sequence recombination

[0191] In some cases, polypeptide sequences of an RQA, for example, RQA domains, can be rearranged or deleted in a cell by the use of site-specific recombination technology. In certain embodiments, the cellular activation-related response to a particular RQA can be altered by site-specific recombination, e.g., the first intracellular signaling domain of an RQA inducing a first activation-related response can be exchanged for a second domain. of intracellular signaling inducing a second response related to activation. In certain embodiments, a response to a particular dimerizer an RQA can be altered by site-specific recombination, for example, a first dimerizer binding pair causing dimerization of an RQA in the presence of a first dimerizer can be exchanged for a second lifation pair to the dimerizer causing the dimerization of an RQA 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 an RQA for any other domain or sequence as shown herein. As will also be clear to a specialist, site-specific recombination can be used in a cell to delete any domain or sequence from an RQA. This exchange and excision of domain sequences is known in the field, see, for example, domain exchange in signal bodies as described in Tone et al. (2013) Biotechnology and Bioengineering, 3219-3226, information about which is included herein by reference. Mechanisms and requirements for carrying out site-specific recombination in vivo are also well known in the art, see, for example, Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), information about which is incorporated herein by reference.

NUCLEIC ACIDS

[0192] This invention provides a nucleic acid that includes a nucleotide sequence that encodes the first and/or second polypeptide of a heterodimeric, conditionally active RQA of this invention. A nucleic acid including a nucleotide sequence encoding the first and/or second polypeptide of a heterodimeric, conditionally active RQA of this invention will in some embodiments be DNA, including, for example, a recombinant expression vector. A nucleic acid including a nucleotide sequence encoding the first and/or second polypeptide of a heterodimeric, conditionally active RQA of this invention will in some embodiments be RNA, for example, RNA synthesized in vitro.

[0193] In some cases, a nucleic acid of this invention includes a nucleotide sequence that encodes the first polypeptide (and not the second polypeptide) of a heterodimeric, conditionally active RQA of this invention. In some cases, a nucleic acid of this invention includes a nucleotide sequence that encodes only the second polypeptide (and not the first polypeptide) of a heterodimeric, conditionally active RQA of this invention. In some cases, a nucleic acid of this invention includes a nucleotide sequence that encodes both the first polypeptide and the second polypeptide of a heterodimeric, conditionally active RQA of this invention.

[0194] In some cases, a nucleic acid from an organism makes it possible to produce an RQA of this invention, for example, in a mammalian cell. In other cases, a nucleic acid from an organism enables amplification of an RQA decoding nucleic acid.

[0195] A nucleotide sequence encoding the first and/or second polypeptide of an RQA of this invention can be operatively linked to a transcriptional control element, for example, a promoter, and enhancer, etc.

[0196] Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, among others, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, among others, light or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; SV40 early and late promoters; promoter present in long terminal repeats of a retrovirus; mouse metallothionein-I promoter; and several tissue-specific promoters known in the art.

[0197] Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters can be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters, but also including additional control proteins, include, among others, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters that respond to alcohol transactivating proteins alcohol (AlcR), etc.), portetracycline-regulated promoters, (e.g., promoter systems, including TetActivators TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoters, retinoid promoter systems, thyroid promoter systems, ecdysone 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 inducible promoters (e.g., HSP-70, HSP-90, the soy heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like .

[0198] In some cases, the locus or transgene construct containing the appropriate promoter is irreversibly linked through the induction of an inducible system. Systems suitable for inducing an irreversible switch are well known in the art, for example, induction of an irreversible switch can make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., PNAS (2000) 28:e99, the invention of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known in the area can be used to obtain an irreversibly convertible promoter. Methods, mechanisms and requirements for carrying out site-specific recombination, described elsewhere than this document, find use in the generation of irreversibly convertible promoters and are well known in the art, see, for example, Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), information about which is incorporated herein by reference.

[0199] In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used, e.g., Salmon et al. (1993) Proc. Natl. Academic. Sci. USA 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell specific expression can be achieved through the use of an Ncrl (p46) promoter, e.g., Eckelhart et al. (2011) Blood 117:1565.

[0200] In some embodiments, for example, for expression in a yeast cell, a suitable promoter is a constitutive promoter, such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter, such as a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia) . Selection of the appropriate vector and promoter is well within the standard level of knowledge in the field.

[0201] Suitable promoters for use in prokaryotic host cells include, among others, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, for example, a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as a ssaG promoter or a related promoter (see, for example, US Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol, 1991: 173(1): 86 -93; Alpuche-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, for example, Dunstan et al (1999) Infect Immun 67:5133-5141; McKelvie et al (2004) Vaccine 22:3243-3255; a sigma70 promoter, for example, a consensus sigma70 promoter (see, for example, GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, for example, a dps promoter, an spv promoter, and the like; a promoter derived from the SPI-2 pathogenicity island (see, for example, WO96/17951); an actA promoter (see, for example, Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, for example, Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter 9vide, 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, for example, Melton et al. (1984) Nucl. Acids Res. 12:7035); and the like. Strong promoters suitable for use in prokaryotes such as Escherichia coli include, among others, Trc, Tac, T5, T7, and PLambda. Non-exhaustive examples of operators for use in bacterial host cells include a lactose promoter operator (LacI repressor protein changes conformation when in contact with lactose, thus preventing the LacI repressor protein from binding to the operator), a tryptophan promoter operator ( when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind the operator), and a tac promoter operator (see, for example, , deBoer et al (1983) Natl. Sci. U.S.A. 80: 21-25).

[0202] A nucleotide sequence encoding an RQA of an organism can be presented in an expression vector and/or a cloning vector. When an RQA of an organism includes two separate polypeptides, the nucleotide sequences encoding the two polypeptides can be cloned into the same or separate vectors. An expression vector may include a selectable marker, an origin of replication, and other characteristics that provide for replication and/or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

[0203] A large number of suitable vectors and promoters are known to those skilled in the art; Many are commercially available for the generation of recombinant constructs in organisms. The following vectors are provided as an example. Bacterial: PBS, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryote: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0204] Expression vectors generally have convenient restriction sites located close to the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, among others, viral vectors (e.g., vaccine virus-based viral vectors; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al. ., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; /19191; WO 94/28938; WO 95/11984 and WO 95/00655); 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; 1988) 166:154-165; and Flotte et al, PNAS (1993) 90:10613-10617); 94:10319 23, 1997; Takahashi et al, J. Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma, and mammary tumor virus); and the like.

[0205] As noted above, in some embodiments, a nucleic acid including a nucleotide sequence encoding the first and/or second polypeptide of a heterodimeric, conditionally active RQA of this invention will in some embodiments be RNA, e.g., RNA synthesized in vitro. Methods for the in vitro synthesis of RNA are known in the art; Any known method can be used to synthesize RNA including a nucleotide sequence encoding the first and/or second polypeptide of a heterodimeric, conditionally active RQA of this invention. Methods for introducing RNA into a host cell are known in the art. See, for example, Zhao et al. (2010) Cancer Res. 15:9053. Introduction of the RNA including a nucleotide sequence encoding the first and/or second polypeptide of a heterodimeric, conditionally active RQA of this invention into a host cell can be done in vitro or ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo, with RNA comprising a nucleotide sequence encoding the first and/or second polypeptide of a conditionally active heterodimeric RQA of this invention.

Cells

[0206] This invention provides a mammalian cell that is genetically modified to produce a conditionally active heterodimeric RQA of this invention.

[0207] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent cell lines (e.g., mouse, rat), and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) no. CCL-2), CHO cells (e.g., ATCC nos. CRL9618, CCL61, CRL9096), 293 cells ( e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC no. CRL1721), COS cells, COS-7 cells (ATCC no. CRL1651), RAT1 cells, mouse L cells (ATCC no. CCLI.3), human embryonic kidney (HEK) cells (ATCC no. CRL1573), HLHepG2, Hut-78, Jurkat, HL-60 cells, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0208] In some cases, the cell is not an immortalized cell line, but rather a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is a leukocyte obtained from an individual. For example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem or progenitor cell obtained from an individual.

Methods for activating a leukocyte

[0209] This invention provides methods for activating a leukocyte in vitro, in vivo or ex vivo. The methods generally involve contacting a leukocyte (in vitro, in vivo, or ex vivo) with a dimerizing agent and an antigen, with the leukocyte being genetically modified to produce a conditionally active heterodimeric RQA of this invention. In the presence of the dimerizing agent and the antigen, the conditionally active heterodimeric RQA dimerizes and activates the leukocyte, thus producing an active leukocyte. Leukocytes include, for example, a cytotoxic T lymphocyte, an NK cell, a CD4+ T cell, a T regulatory cell (Treg), etc.

[0210] Contact to the genetically modified leukocyte (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 the production of a cytokine by the leukocyte 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times compared to the amount of cytokine produced by leukocytes in the absence of the second member of a specific binding pair and/or the dimerizing agent. Cytokines whose production can be increased include, among others, IL-2 and IFN-γ,

[0211] Contacting a genetically modified leukocyte (e.g., a T lymphocyte, an NK cell) with a dimerizing agent and an antigen can increase the production of a cytokine by the leukocyte by at least about 10%, at least about of 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the amount of cytokine produced by leukocytes in the absence of the antigen and/or the dimming agent. Cytokines whose production can be increased include, among others, IL-2 and IFN-Y.

[0212] Contacting the genetically modified leukocyte (e.g., 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 the cytotoxic activity of the cell cytotoxic 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the cytotoxic activity of the cytotoxic cell in the absence of the dimerizing agent.

[0213] Contacting the genetically modified leukocyte (e.g., T lymphocyte) with a dimerizing agent and an antigen can increase the cytotoxic activity of the cytotoxic 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 times, at least about of 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the cytotoxic activity of the cytotoxic cell in the absence of the dimerizing agent.

[0214] In other embodiments, for example, depending on the host leukocyte, contacting a genetically modified leukocyte with a dimerizing agent and an antigen can increase or decrease cell proliferation, cell survival, cell death, and the like.

Methods for generating conditionally activatable cells

[0215] This invention 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 transcribed RNA), including nucleotide sequences encoding a conditionally active heterodimeric RQA of this invention. The genetically modified cell is conditionally activatable in the presence of: a) an antigen to which the first polypeptide of RQA binds; and b) a dimerizer (a dimming agent). Genetic modification can be carried out in vivo, in vitro or ex vivo. The cell can be a leukocyte (e.g. a T lymphocyte or NK cell), a stem cell, a progenitor cell, etc.

[0216] In some cases, genetic modification is performed ex vivo. For example, a T lymphocyte, stem cell, or NK cell is obtained from an individual; and the cell obtained from the individual is genetically modified to express an RQA of this invention. The genetically modified cell is conditionally activatable in the presence of: a) an antigen to which the first polypeptide of RQA 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 (for example, the individual from whom the cell was obtained); and the genetically modified cell is activated in vivo, for example, by administering a dimerizer to the individual. For example, where the antigen is present on the surface of a cell in the individual, there is no need to administer an antigen. The genetically modified cell comes into contact with the antigen present on the surface of a cell in the individual; and, by administering a dimerizer to the individual, the genetically modified cell is activated. For example, when the genetically modified cell is a T lymphocyte, the genetically modified cell may exhibit cytotoxicity towards a cell that presents an antigen on its surface to which RQA binds.

Treatment methods

[0217] This invention provides various methods of treatment using an RQA in an organism.

Cytotoxicity methods

[0218] An RQA of this invention, when present in a T lymphocyte or an NK cell, can mediate cytotoxicin to a target cell. An RQA of this invention binds to an antigen present on a target cell, thereby mediating the killing of a target cell by a T lymphocyte or an NK cell genetically modified to produce the RQA. The antigen-binding domain of RQA binds to an antigen present on the surface of a target cell.

[0219] Target cells include, among others, cancer cells. Thus, this invention provides methods for killing, or inhibiting the growth of, a target cancer cell, with a method that involves contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell, or an NK cell) that is genetically modified. to produce an RQA of an organism, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of a target cancer cell, and mediates the death of the target cell.

[0220] This invention provides a method of treating cancer in an individual that consists of: i) genetically modifying T lymphocytes obtained from the individual with an expression vector with nucleotide sequences encoding the conditionally active heterodimeric RQA of this invention, in the which antigen-binding domain of the conditionally active heterodimeric RQA is specific for an epitope on a cancer cell of the subject, and in which genetic modification is carried out ex vivo; ii) introduce genetically modified T lymphocytes into the individual; and iii) administering to the individual an effective amount of a dimerizing agent, wherein the dimerizing agent induces dimerization of the conditionally active heterodimeric RQA, wherein such dimerization provides activation of genetically modified T lymphocytes and death of the cancer cell, thereby treating the cancer.

[0221] Carcinomas that can be treated with therapy by a method disclosed in this invention include, among others, 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 and non-small cell lung carcinoma, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, 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.

[0222] Sarcomas that can be treated with therapy by a method of this invention include, among others, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0223] Other solid tumors that can be treated with therapy by a method of this invention include, among others, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0224] Leukemias that can be treated with therapy by a method of this invention include, among others, a), chronic myeloproliferative syndromes (neoplastic diseases 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 cell CLL -B, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated using a method of this invention include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin lymphoma; non-Hodgkin's lymphoma, and the like.

[0225] Other cancers that can be treated in accordance with the methods of this invention include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchyma (intestine), hepatocellular carcinoma (liver), hepatoblastoma ( liver), clear cell carcinoma (kidney), and mediastinal neurofibroma.

Immunomodulatory methods

[0226] A method of this invention can also be used to treat inflammatory conditions and autoimmune disease. An RQA of this invention is expressed in a helper T cell or a regulatory T lymphocyte for use in an immunomodulation method. Immunomodulatory methods include, for example, enhancing an immune response in a mammal to a pathogen; improve an immune response in an organism that is immunocompromised; reduce an inflammatory response; reducing an immunological response in a mammal to a self antigen, for example, to treat an autoimmune disease; and reducing an immunological response in a mammal to a transplanted organ or tissue, to reduce tissue or organ rejection.

[0227] When the method involves reducing an immune response to an antigen, the antigen used to activate the RQA is a self-antigen. When the method involves reducing an immune response to a transplanted organ or tissue, the antigen used to activate RQA is an antigen specific to the transplanted organ.

Formulas, dosages, and routes of administration

[0228] As discussed above, a treatment method of this invention involves administering an effective amount of a dimerizing agent to an individual in need of treatment, and may also involve administering an antigen.

[0229] An "effective amount" of a dimerizing 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 a T lymphocyte expressing an RQA of 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the activity cytotoxicity of T lymphocytes in the absence of the dimerizing agent.

[0230] An "effective amount" of a dimerizing 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 expressing an RQA of 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the activity cytotoxicity of NK cells in the absence of the dimerizing agent.

[0231] An "effective amount" of a dimming 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 the mass of the tumor in the individual, 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%, or more than 75%, compared to the number of cancer cells and/or tumor mass in the absence of the dimerizing agent.

[0232] 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 in reducing one or more tumor growth rate, number of cancer cells, and tumor mass, by at least about 5%, 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 of 70%, at least about 80%, at least about 90%, or more, compared to the tumor growth rate, number of cancer cells, or tumor mass in the absence of treatment with the dimerizer.

Formulas

[0233] In the methods of this invention, a dimerizer can be administered to the host through any convenient means capable of eliciting the desired therapeutic effect or diagnostic effect. Thus, the dimerizer can be incorporated into a variety of formulas for therapeutic administration. More particularly a dimerizer may be formulated into pharmaceutical compositions by combination with suitable and pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions. , suppositories, injections, inhalants and aerosols.

[0234] In pharmaceutical dosage forms, a dimerizer can be administered in the form of its pharmaceutically acceptable salts, or can also be used alone or in suitable association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way exhaustive.

[0235] Suitable excipient carriers are, for example, water, saline solution, dextrose, glycerol, ethanol, or the like, and combinations thereof. Furthermore, if desired, the carrier may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formula to be administered will, in any case, contain an amount of dimerizer adequate to achieve the desired state in the organism being treated. .

[0236] Pharmaceutically acceptable excipients, such as vehicles, adjuvants or diluents, are readily available to the public. Furthermore, 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.

[0237] 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 conventional 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 diluents, buffering agents, wetting agents, preservatives and flavoring agents.

[0238] A dimerizer can be formulated into preparations for injection by dissolving, suspending or emulsifying in an aqueous or non-aqueous solvent, such as vegetable oils or the like, glycerides of synthetic aliphatic acids, esters of higher aliphatic acids or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0239] Pharmaceutical compositions including 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 non-toxic to receptors at the dosages and concentrations used, 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-chloro-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids, such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular mass polypeptides (less than about 10 residues); proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine and neuraminic acid; and/or nonionic sulfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0240] 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 must be reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (normally equivalent to the volume removed during lyophilization); however, solutions that include antibacterial agents can be used to produce pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0241] The term "unit dosage form", as used herein, refers to physically discrete units suitable as unitary dosages for human and animal organisms, each containing a predetermined amount of a dimerizer calculated in an amount sufficient to produce the desired effect. in association with a pharmaceutically acceptable diluent or carrier. The specifications for a given dimerizer may depend on the particular dimerizer used and the effect to be achieved, and the pharmacodynamics associated with each dimerizer in the host.

[0242] In some embodiments, a dimerizer is formulated in a controlled release formula. Sustained release preparations can be made using methods well known in the art. Suitable examples of sustained release preparations include semipermeable matrices of hydrophobic solid polymers containing the dimerizer in which the matrices are in the form of molded articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, L-glutamic acid and ethyl-L-glutamate copolymers, non-degradable satin vinyl foam, hydrogels, polylates, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)- acid 3-hydroxybutyric. Possible loss of biological activity can be prevented by the use of appropriate additives, by controlling the moisture content of and developing specific polymer matrix compositions.

Dosages

[0243] An appropriate dosage can be determined by a treating physician or other qualified professional based on various clinical factors. As is well known in the medical field, dosages for any patient depend on many factors, including the patient's size, body surface area, age, the particular dimmer being administered, the patient's sex, time and route of administration. , general health, and other medications being administered concomitantly. A dimerizer may be administered in amounts of between 1 ng/kg of body weight and 20 mg/kg of body weight per dose, e.g. between 0.1 mg/kg of body weight to 10 mg/kg of body weight, e.g. , between 0.5 mg/kg of body weight to 5 mg/kg of body weight; however, doses below or above this exemplary range are anticipated, especially considering the aforementioned factors. If the regimen is continuous infusion, it can also be between 1 μg and 10 mg per kilogram of body weight per minute.

[0244] Experts will easily appreciate that dosage levels may vary depending on the specific dimerizer, the severity of symptoms and the organism's susceptibility to side effects. Preferred dosages for a given compound are readily determinable by those skilled in the art by a variety of means.

Routes of administration

[0245] A dimerizer is administered to an individual using any suitable and available mode and route for drug administration, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.

[0246] Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intra-arterial, rectal, nasal, oral and other enteral and parenteral routes of administration. Routes of administration can be combined, if desired, or adjusted depending on the dimerizer and/or the desired effect. A dimer may be administered in a single dose or in multiple doses. In some embodiments, a dimerizer is administered orally. In some embodiments, a dimerizer is administered by inhalation. In some embodiments, a dimerizer is administered intranasally. In some embodiments, a dimerizer is administered locally. In some embodiments, a dimerizer is administered intrathymorally. In some embodiments, a dimerizer is administered peritumorally. In some embodiments, a dimerizer is administered intracranially. In some embodiments, a dimerizer is administered intravenously.

[0247] The agent can be administered to a host using any conventional methods and routes available and suitable for the administration of conventional medicines, including systemic or localized routes. In general, the routes of administration contemplated by this invention include, but are not limited to, enteral, parenteral, or by inhalation.

[0248] Parenteral routes of administration other than administration by inhalation include, among others, topical, transdermal, subcutaneous, intramuscular, intra-orbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous, i.e., any route of administration other than the alimentary canal. Parenteral administration can be carried out for systemic or local administration of a dimerizer. When systemic administration is desired, it typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

[0249] A dimerizer can also be delivered to the body by enteral administration. Enteral routes of administration include, but are not limited to, oral and rectal (e.g., using a suppository).

[0250] By treatment is meant at least an improvement in the symptoms associated with the pathological condition that afflicts the host, with the term improvement being used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. example, symptom, associated with the pathological condition to be treated, such as cancer. As such, treatment also includes situations in which the pathological condition, or at least symptoms associated with it, are completely inhibited, e.g. prevented from happening, or stopped, e.g. eradicated, so that the host does not suffer from the disease. pathological condition, or, at least, the symptoms that characterize the pathological condition.

[0251] In some embodiments, a dimerizer is administered by injection and/or administration, for example, at a site in a cerebral artery or directly into brain tissue. A dimerizer can also be administered directly to a target site, for example, by direct injection, by implantation of a drug delivery device such as an osmotic pump or slow release particles, by biolistic delivery to the target site, etc.

Combination therapy

[0252] In some embodiments, a dimerizer is administered as an adjuvant therapy to standard cancer therapy. Standard cancer therapies include surgery (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapeutic treatment, antibody treatment, biological response modification treatment, and certain combinations of the above.

[0253] Radiation therapy includes, among others, x-rays or gamma rays administered by an externally applied source as a beam, or by implantation of small radioactive sources.

[0254] Antibodies suitable for use in the treatment of cancer include, among others, naked antibodies, for example, trastuzumab (Herceptin), bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), ipilimumab (Yervoy™) , rituximab (Rituxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), Oregovomab (OvaRex™), Lambrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™) etc., and conjugated antibodies, for example, gemtuzumab ozogamicin (Mylortarg™), Brentuximab vedotin (Adcetris™), 90Y-ibritumomab labeled tiuxetan (Zevalin™), 131I-labeled tositumoma (Bexxar™), etc. Antibodies suitable for use in the treatment of cancer include, but are not limited to, antibodies raised against tumor-associated antigens. Such antigens include, among others, 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-1, EGFR, ERBB2, ERBB3,MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, etc .

[0255] Biological response modifiers suitable for use in connection with the methods of this invention include, among others, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) antagonists of tumor-associated antigens, such as antibodies that specifically bind to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) interferon-a; (7) interferon -Y; (8) colony-stimulating factors; (9) angiogenesis inhibitors; and (10) tumor necrosis factor antagonists.

[0256] Chemotherapeutic agents are non-peptide (that is, non-protein) compounds that reduce the proliferation of cancer cells, and include cytotoxic and cytostatic agents. Non-exhaustive examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant alkaloids (vinca), and steroid hormones.

[0257] Agents that act to reduce cell 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, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysine), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, triethylenemelamine, triethylenethiophosphoramine, dacarbazine, and temozolomide.

[0258] Antimetabolite agents include folic acid analogues, pyrimidine analogues, purine analogues and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR ), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-didesazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid ( DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0259] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins), include, among others, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brake; alkaloids, for example, vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, for example, anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxyzone 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, for example, cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.

[0260] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, Letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0261] Agents that affect microtubules and that have antiproliferative activity are also suitable for use and include, among others, allocolchicine (NSC 406042), Halicondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g. NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterine, sulfate vinblastine, vincristine sulfate, natural and synthetic epothilones, including, among others, eoptilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

[0262] Hormone modulators and steroids (including synthetic analogues) that are suitable for use include, among others, adrenocorticosteroids, for example, prednisone, dexamethasone, etc.; estrogens and progestins, for example, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, for example, aminoglutethimide; 17α- ethinyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and . Estrogens stimulate proliferation and differentiation, therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0263] Other chemotherapeutic agents include metal complexes, for example, cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, for example N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, for example, mycophenolic acid, thalidomide, deoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); etc.

[0264] "Taxanes" include paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOLTM, TAXOTERETM (a formulation of docetaxel), 10-desacetyl analogues of paclitaxel, and 3'N-desbenzoyl-analogs paclitaxel 3'N-t-butoxycarbonyl) can be readily prepared using techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; variety of commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 of Taxus brevifolia, or T-1912 of Taxus yannanensis).

[0265] Paclitaxel should be understood to refer to not only the commonly chemically available form of paclitaxel, but analogues and derivatives (e.g., TaxotereTM docetaxel, as mentioned above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel -dextran, or paclitaxel-xylose).

[0266] Also included within the term "taxane" are a variety of known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, among others, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, 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. 5,415,869. It also includes paclitaxel prodrugs, including, among others, those described in WO 98/58927; WO 98/13059; and US Patent No. 5,824,701.

Suitable organisms for treatment

[0267] A variety of organisms are suitable for treatment with this method of combating cancer. Suitable organisms include any individual, for example, a human or non-human animal with cancer, who has been diagnosed with cancer, who is at risk of developing cancer, who has had cancer and is at risk of cancer recurrence, who has been treated with an agent that is not a cancer dimer and has not responded to treatment, or who has been treated with an agent that is not a cancer dimer but has relapsed after the initial response to treatment.

[0268] Organisms suitable for treatment with an organism immunomodulatory method include individuals who have an autoimmune disease; individuals receiving transplanted organs or tissues; and the like; immunocompromised individuals; and individuals who are infected with a pathogen.

Examples

[0269] The following examples are presented in order to provide those skilled in the art with a complete presentation and description of how to prepare and use this invention, and are not intended to limit the scope of what the inventors consider to be their invention nor are they intended to to declare that the experiments below are all or the only ones carried out. Efforts have been made to ensure accuracy regarding the numbers used (e.g. quantities, temperature, etc.), but some experimental errors and deviations must be considered. Unless otherwise noted, parts are parts by weight, molecular mass is the average molecular mass, temperature is in degrees Celsius, and pressure is close to atmospheric. Conventional abbreviations may be used, for example, 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(mind); i.p., intraperitoneal(mind); s.c., subcutaneous (mind); intravenous i.v. (mind); and the like.

Example 1: Generation of RQA Materials and methods

[0270] The anti-human CD19 scFv was selected as the antigen recognition domain in RQAs throughout the design optimization process. Figures 18A and 18B summarize the molecular structure of each RQA consisting of two numerically identified polypeptides. All membrane-anchored polypeptides are disulfide-linked homodimers. Membrane-anchored polypeptides are described as monomers for graphical simplicity.

Generation of RQA constructs

[0271] The sequence encoding the anti-human CD19 scFv was cloned from a construct. Human 4-1BB co-stimulation and CD3 zeta ITAM signaling chains were cloned from cDNAs provided by Open Biosystems FKBP- and sequences encoding FRB were cloned from plasmids provided by Addgene.

[0272] Standard molecular cloning techniques (polymerase chain reaction (RCP), restriction digestion, ligation, etc.) were applied to generate lentivirus expression plasmids.

Target and effector cell culture conditions

[0273] Human primary CD8+ T cells were isolated from anonymous donor blood after apheresis (Trima waste from Blood Centers of the Pacific, San Francisco, CA) by negative selection using a RosetteSep Human CD8+ T Cell Enrichment Cocktail (STEMCELL Technologies #15063), as approved by the University Institutional Review Board. Cells were cultured in human T cell medium, which consisted of X-VIVO15 (Lonza #04-418Q), 5% human AB serum (Valley Biomedical Inc., #HP1022), 10mM N-acetyl L-Cysteine (Sigma -Aldrich #A9165) and 100 IU/mL of recombinant human IL-2 (NCI/BRB Preclinical Repositories). A Jurkat cell line expressing Green Fluorescent Protein (PFV) under NFAT activation was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. U. Penn K562 target cells were cultured in IMDM supplemented with 10% FBS.

Effector and target cell engineering with lentivirus

[0274] The pantropic VSV-G pseudotyped lentivirus was produced from Lenti-X 293T cells (Clontech Laboratories #632180) co-transfected with a pHR'SIN:CSW transgene expression vector, viral packaging plasmids pCMVdR8.91 and pMD2.G using Lipofectamine LTX (Life Technologies #15338). Supernatant infection medium was collected 48 hours after transfection and used directly for transduction.

[0275] Twenty-four hours before viral transduction, primary human T cells were activated using Dynabeads CD3/CD28 human T Activators (Life Technologies #111-31D) at a 1:3 cell:bead ratio. Jurkat and K562 cells were split 1~2 days in advance to ensure that the cultures would be in the recording 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 ~10^6/mL in human T cell medium for about two weeks until the cells returned to a resting state. The expression levels of RQA encoded in the lentiviral constructs were quantified by detecting fluorophore-conjugated antibodies or fluorescent reporter proteins using a flow cytometer.

Quantification of IL-2 production and NFAT activity

[0276] Jurkat CD4+ T cells expressing RQAs were mixed with U. Penn cognate or non-cognate K562 target cells at a 1:2 effector:target ratio. Rapalog A/C Heterodimerizer (Clontech Laboratories #635055) was serially diluted in medium and added to reaction mixtures. After 20~24 hours of incubation, the medium supernatants were collected and analyzed with BD OptEIA Human IL-2 ELISA Set (BD Biosciences #555190). Flow cytometry was performed to quantify NFAT-dependent GFP reporter expression in Jurkat cells as a specific indicator for RQA activity.

Flow cytometry-based redirected cytotoxicity assay

[0277] Cognate and non-cognate K562 target cells were engineered to express distinct fluorescent proteins so that both cell types in a mixture could be quantified simultaneously by flow cytometry. Target cell types were mixed at a 1:1 ratio and co-incubated with primary human CD8+ effector T cells at a 5:2 effector:target ratio. 100 IU/mL human IL-2 and different amounts of rapalog (Clontech Laboratories #635055) were added to the reaction mixtures. After 24 hours of incubation, the samples were centrifuged at 400g for 5 minutes. Pelletized 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 surviving cognate target cells to non-cognate target cells were calculated for each sample to enumerate redirected cytotoxic activities of effector cells.

Results

[0278] IL-2 production induced by the various RQA constructs was evaluated. The data is presented in Figure 12.

[0279] Figure 12. IL-2 production triggered by five activating RQA variants. Effector = human Jurkat CD4+ T cells engineered with RQAs. Target= K562 cell lines with or without the cognate CD19 antigen. The amounts of IL-2 secreted by effector cells were quantified by enzyme-linked immunosorbent assay (ELISA).

[0280] Figure 13. Production of IL-2 by control Jurkat lines in the same experiment described in Figure 12. The "125" construct encodes a conventional control currently used in clinical trials.

[0281] Figure 14. Comparison between "122 + 206" and "197 + 206" in a separate experiment under conditions identical to those described in Figure 12.

[0282] Figure 15 demonstrates pharmacologically titratable cytotoxicity conferred by the "197+206" trigger RQA. In the presence of the rapalog small molecule, the RQA efficiently mediates redirected cytotoxicity to cognate target cells. At high doses of rapalog, this trigger RQA can output a signal as strong as that of the conventional "125" RQA. Effectors = human primary CD8+ T cells engineered with RQAs or a control vector. Target = fluorescent derivatives of K562 cell lines expressing the cognate human CD19 antigen or the non-cognate human mesothelin antigen.

[0283] Figure 16 shows the data for the RQA constructed with the cytoplasmic tyrosine kinase Zap70 from the T cell receptor passage as the intracellular signaling domain.

[0284] Figure 16 shows data from Jurkat cells designed with several variants of the activation RQAs. The designed Jurkat cells were co-incubated with K562 target cells with or without the cognate antigen (CD19) and the indicated concentrations of rapalog. As a component of RQA, the kinase Zap70 (first and second structures from the left numbered “199”) was as effective as ITAM (third structure from the left numbered “168”) in activating NFAT function. Addition of the 4-1BB signaling domain increased surface expression of the antigen recognition portion of the receptor and led to stronger signaling by “197+199”. A non-signaling RQA (far to the right) was included as a negative control.

Example 2: Mesothelin with RQA as target Materials and methods

[0285] A number of chimeric antigen receptor constructs have been made and tested. The constructs shown here encode three different anti-human scFv mesothelins as the antigen recognition domains. Figures 19A, 19B and 19C summarize the molecular structure of each anti-human mesothelin RQA, with each RQA consisting of two polypeptides. The intercellular portion of each anti-human mesothelin RQA consists of two 4-1BB costimulatory domains, an FKBP and FRB dimerizer-binding pair, and an ITAM intracellular signaling domain. The three different antigen recognition domains shown in this document are HN1 scFv, SS1 scFv, and m912 anti-mysothelin scFv. All membrane-anchored polypeptides are disulfide-linked homodimers.

Generation of RQA constructs

[0286] The sequences encoding anti-mesothelin were cloned from constructs synthesized by gene clustering by PCR. The human 4-1BB costimulation and CD3 zeta ITAM signaling chains were cloned from cDNAs provided by Open Biosystems. HN1 scFv-, SS1 scFv-, and m912 scFv coding sequences were synthesized by PCR and, in some cases, optimized by condon. Sequences encoding FKBP- and FRB were cloned from Addgene plasmids.

[0287] Standard molecular cloning techniques (polymerase chain reaction (RCP), restriction digestion, ligation, etc.) were applied to generate lentivirus expression plasmids.

Target and effector cell culture conditions

[0288] A Jurkat cell line expressing GFP under NFAT activation was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. K562 target cells were cultured in IMDM supplemented with 10% FBS.

Effector and target cell engineering with lentivirus

[0289] The pantropic VSV-G pseudotyped lentivirus was produced from Lenti-X 293T cells (Clontech Laboratories #632180) co-transfected with a pHR'SIN:CSW transgene expression vector, viral packaging plasmids pCMVdR8.91 and pMD2.G using Lipofectamine LTX (Life Technologies #15338). Supernatant infection medium was collected 48 hours after transfection and used directly for transduction.

[0290] Jurkat and K562 cells were split 1~2 days in advance to ensure that the cultures would be in the recording phase at the time of transduction. Transduced Jurkat and K562 cells were cultured for at least 7 days before experiments were conducted. The expression levels of RQA encoded in the lentiviral constructs were quantified by detecting fluorophore-conjugated antibodies or fluorescent reporter proteins using a flow cytometer.

Quantification of IL-2 production

[0291] Jurkat CD4+ T cells expressing RQAs were mixed with cognate or non-cognate K562 target cells at a 1:2 effector:target ratio. Rapalog A/C Heterodimerizer (Clontech Laboratories #635055) was serially diluted in medium and added to reaction mixtures. After 20~24 hours of incubation, the medium supernatants were collected and analyzed with BD OptEIA Human IL-2 ELISA Set (BD Biosciences #555190).

Results

[0292] IL-2 production induced by anti-mesothelin RQA constructs was evaluated. The data is presented in Figure 19D-F.

[0293] Figure 19. IL-2 production driven by HN1 scFv (Fig. 19D), SS1 scFv (Fig. 19E), and m912 scFv (Fig. 19F) Drive RQA variants. IL-2 production by conventional RQA (Fig. 19G, construct #358) was measured and included for comparison to trigger RQAs (Fig. 19D). Effector = human Jurkat CD4+ T cells engineered with RQAs. Target= K562 cell lines with or without the cognate mesothelin antigen. The amounts of IL-2 secreted by effector cells were quantified by enzyme-linked immunosorbent assay (ELISA).

Example 3: Gibberellic acid as a dimmer for driving RQAs Materials and methods

[0294] Figure 20A summarizes the molecular structure of the organism's gibberellic acid dimerizing RQA. The antigen-binding portion includes the CD19 scFv. anti-human. The intercellular portion consists of two 4-1BB costimulatory domains, a GID1 and GAI dimerizer-binding pair, and an ITAM intracellular signaling domain. All membrane-anchored polypeptides are disulfide-linked homodimers.

Generation of RQA constructs

[0295] The sequences encoding the gibberellic acid dimerizer RQA were cloned from constructs. The anti-CD19 scFv was cloned from a plasmid. The human 4-1BB costimulation and CD3 zeta ITAM signaling chains were cloned from cDNAs provided by Open Biosystems. GID1- and GAI coding sequences were cloned from Addgene plasmids. Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentivirus expression plasmids.

Target and effector cell culture conditions

[0296] A Jurkat cell line expressing GFP under NFAT activation was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. K562 target cells were cultured in IMDM supplemented with 10% FBS.

Effector and target cell engineering with lentivirus

[0297] The pantropic VSV-G pseudotyped lentivirus was produced from Lenti-X 293T cells (Clontech Laboratories #632180) co-transfected with a pHR'SIN:CSW transgene expression vector, viral packaging plasmids pCMVdR8.91 and pMD2.G using Lipofectamine LTX (Life Technologies #15338). Supernatant infection medium was collected 48 hours after transfection and used directly for transduction.

[0298] Jurkat and K562 cells were split 1~2 days in advance to ensure that the cultures would be in the recording phase at the time of transduction. Transduced Jurkat and K562 cells were cultured for at least 7 days before experiments were conducted. The expression levels of RQA encoded in the lentiviral constructs were quantified by detecting fluorophore-conjugated antibodies or fluorescent reporter proteins using a flow cytometer.

Quantification of IL-2 production

[0299] Jurkat CD4+ T cells expressing RQAs were mixed with cognate or non-cognate K562 target cells at a 1:2 effector:target ratio. Gibberellic acid 3-acetoxymethyl ester (3-AM gibberellic acid) predissolved 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, the medium supernatants were collected and analyzed with BD OptEIA Human IL-2 ELISA Set (BD Biosciences #555190).

Results

[0300] IL-2 production induced by the gibberellic acid dimerizer RQA construct was evaluated. The data is presented in Figure 20.

[0301] Figure 20. IL-2 production triggered by the gibberellic acid dimerizer RQA variant (Fig. 20B). IL-2 production by conventional RQA (Fig. 20C, construct “125”) was measured and included for comparison to trigger RQA. Effector = human Jurkat CD4+ T cells engineered with RQAs. Target= K562 cell lines with or without the cognate CD19 antigen. The amounts of IL-2 secreted by effector cells were quantified by enzyme-linked immunosorbent assay (ELISA).

Example 4: Activation RQAs with multiple costimulation domains Materials and methods

[0302] A series of chimeric antigen receptor constructs were made essentially as described for Example 1, except several other costimulatory domains were exchanged for the 4-1BB costimulatory domains. Figures 21A and 21B summarize the molecular structure of the RQAs described in this document.

Generation of RQA constructs

[0303] The sequences encoding the anti-human CD19 scFv were cloned from a plasmid. The ITAM zeta CD3 signaling chain and the coding sequences for the human costimulatory domains CD28 and OX-40 were cloned from cDNAs provided by Open Biosystems. Sequences encoding FKBP- and FRB- were cloned from Addgene plasmids.

[0304] Standard molecular cloning techniques (polymerase chain reaction (RCP), restriction digestion, ligation, etc.) were applied to generate lentivirus expression plasmids.

RQA Construct Tests

[0305] Effector and target cells are cultured and transfected according to Example 1 using the RQA CD28 and OX-40 activation costimulatory domain containing described constructs (Fig. 21A-B, constructs "365 + 367" and "399+ 400", respectively) and the corresponding conventional RQA controls (Fig. 21C-D, constructs "366" and "398", respectively). IL-2 production, NFAT activity assays, and flow cytometry-based assays can also be performed with the CD28 costimulatory domain-containing OX-40 costimulatory domain and construct-containing construct as described for Example 1. Alternatively, subunits of RQA CD28 and OX-40 activating costimulatory domain containing constructs can be paired with subunits of constructs from Example 1 (e.g., "197+367", "365+206", "197+400", "399+206, " etc.).

Example 5: In vivo assessment of activation RQA

[0306] An activation RQA can be evaluated by its ability to mediate the in vivo killing of a target tumor cell. In vivo tumor cell killing caused by injection of T cells expressing the activating RQA is evaluated. Tumor cell lines that have been confirmed in vitro to express the cognate antigen and can be killed by CD8+ T cells expressing the corresponding RQA are used. Tumor cells engineered to express firefly or Renilla luciferase to enable bio-luminescence imaging to quantify tumor burden in vivo can be used. Tumor cells are injected into immunocompromised mice (e.g., 6~10 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 appropriate number of tumor cells for implantation can be based on the ideal conditions for the tumor cell line used. Tumor burden can be monitored twice weekly by bioluminescence imaging and by caliper measurement when applicable. Once tumor burden is detectable, 0.5~2.5 x 10^7 (1:1 CD4+:CD8+) total T cells expressing the activating RQA are injected intravenously into mice to initiate treatment. A dimerizing small molecule drug (e.g., rapalog) is administered intraperitoneally in a vehicle formulation. Activating RQA-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.

[0307] Although this invention has been described with reference to the specific embodiments of this document, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adapt a particular situation, material, composition of matter, process, step or process steps to the purpose, spirit and scope of this invention. All such modifications must be within the scope of the attached requests.

Claims (13)

1. HETERODIMER CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTOR (CAR), characterized by comprising: 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 transmembrane domain; ii) a second co-stimulatory domain; iii) a second member of the dimerization pair; and iv) an intracellular signaling 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 between the first member of a specific binding pair and the costimulatory domain; and b) a second polypeptide comprising: i) a second member of the dimerization pair; and ii) an intracellular signaling domain, in which the first and second members of the dimerization pair form a homodimer or a heterodimer in the presence of a dimerization agent. 2. CONDITIONALLY ACTIVE HETERODIMER CAR, according to claim 1, characterized in that the order of the domains from the amino terminal to the carboxy terminal is: the first member of the specific binding domain, the transmembrane domain, the first co-stimulatory domain and the first member of the dimming pair. 3. HETERODIMER CONDITIONALLY ACTIVE CAR according to any one of claims 1 or 2, characterized in that the first polypeptide comprises a hinge region, such as an IgG immunoglobulin hinge region or a CD8-derived hinge interposed between the first member of the pair. specific binding domain and the transmembrane domain. 4. HETERODIMER CONDITIONALLY ACTIVE CAR, according to any one of claims 1 to 3, characterized in that the first member of the specific binding pair is an antibody or antibody fragment, a ligand or a receptor. 5. CONDITIONALLY ACTIVE HETERODIMER CAR, according to any one of claims 1 to 4, characterized in that the first and second co-stimulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR and HVEM. 6. CONDITIONALLY ACTIVE HETERODIMER CAR according to any one of claims 1 to 5, characterized in that the intracellular signaling domain comprises a tyrosine-based immunoreceptor activation motif (ITAM) and is selected from CD3-zeta and ZAP70. 7. CONDITIONALLY ACTIVE HETERODIMER CAR, according to any one of claims 1 to 6, characterized in that the first and second members of the dimerization pair are selected from: a) FKBP and FKBP-rapamycin associated protein (FRB); b) GAI and GID1; c) FKBP and catalytic subunit calcineurin A (CnA); d) FKBP and cyclophilin; e) FK506 binding protein (FKBP) and FKBP; f) gyrase B (GyrB) and GyrB; g) dihydrofolate reductase (DHFR) and DHFR; h) DmrB and DmrB; i) PYL and ABI; and j) Cry2 and CIP. 8. CONDITIONALLY ACTIVE HETERODIMER CAR, according to claim 1, characterized in that the first member of the specific binding pair is a single-chain Fv and: i) the first and second co-stimulatory domains are derived from 4-1BB; ii) the first and second members of the dimerization pair are FKBP and FRB; and ii) the signaling domain comprises an ITAM. 9. CONDITIONALLY ACTIVE HETERODIMER CAR according to any one of claims 1 to 8, characterized in that the first member of the specific binding pair binds to an epitope present on a cell such as a cancer cell, on a solid surface, or a lipid bilayer. 10. METHOD OF ACTIVATING A T LYMPHOCYTE IN VITRO OR EX VIVO, characterized by comprising the contact of T lymphocytes with a dimerization agent and a second member of a specific binding pair, in which the T lymphocyte is genetically modified to produce a Heterodimeric conditionally active CAR as defined in any one of claims 1 to 9, and wherein, in the presence of the dimerizing agent and the second member of a specific binding pair, the heterodimeric conditionally active CAR dimerizes and activates the T lymphocyte, producing , thus, an activated T lymphocyte. 11. METHOD OF PRODUCING A GENETICALLY MODIFIED MAMMALIAN CELL, characterized by comprising the genetic modification of a mammalian cell in vitro or ex vivo with an expression vector comprising nucleotide sequences encoding the heterodimeric conditionally active CAR, as defined in 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, as defined with any one of claims 1 to 9. 12. METHOD OF MODULATING THE ACTIVITY OF A T LYMPHOCYTE HOST CELL, characterized by comprising the contact of the host cell in vitro or ex vivo with a dimerization agent and a second member of a specific binding pair, in which the T lymphocyte is genetically modified to produce a heterodimeric conditionally active CAR as defined in any one of claims 1 to 9, and wherein, in the presence of the dimerizing agent and the second member of a specific binding pair, the heterodimeric conditionally active CAR dimerizes and modulates at least one host cell activity. 13. METHOD according to claim 12, characterized in that the activity is proliferation, cell survival, apoptosis, gene expression or immune activation, or in which the second member of a specific binding pair is an antigen.