Dual control for therapeutic cell activation and elimination

Abstract

To provide dual control for therapeutic cell activation and elimination.SOLUTION: The technology relates in part to methods for controlling the activity or elimination of therapeutic cells using molecular switches that employ distinct heterodimerizer ligands in conjunction with other multimeric ligands. The technology may be used, for example, to activate or eliminate cells used to promote engraftment, to treat diseases or conditions, or to control or modulate the activity of therapeutic cells that express chimeric antigen receptors or recombinant T cell receptors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications Priority is claimed to U.S. Provisional Patent Application No. 62 / 267,277, filed December 14, 2015, entitled "Dual Control for Therapeutic Cell Activation or Elimination," which is incorporated by reference in its entirety.

[0002] Field This technology relates in part to methods for regulating the activity or elimination of therapeutic cells using molecular switches that employ distinct heterodimerizer ligands in conjunction with other multimeric ligands. This technology can be used, for example, to activate or eliminate cells used to promote engraftment, to treat a disease or condition, or to regulate or modulate the activity of therapeutic cells expressing chimeric antigen receptors or recombinant T cell receptors. [Background technology]

[0003] background Cell therapy, in which modified or unmodified cells (e.g., T cells) are administered to patients, is increasingly being used. In some instances, cells are genetically engineered to express a heterologous gene, and these modified cells are then administered to patients. The heterologous gene can be used to express a chimeric antigen receptor (CAR). Chimeric antigen receptors are artificial receptors designed to convey antigen specificity to T cells without the need for MHC antigen presentation. They contain antigen-specific, transmembrane, and intracellular components selected to activate T cells and provide specific immunity. CAR-expressing T cells can be used in a variety of therapies, including cancer therapy. These treatments are used, for example, to target tumors for elimination and to treat cancer and blood disorders, but these therapies can have negative side effects.

[0004] In some cases of therapeutic cell-induced adverse events, rapid and nearly complete elimination of therapeutic cells is necessary. Excessive on-target effects (e.g., directed against large tumor masses) can result in cytokine storms associated with tumor lysis syndrome (TLS), cytokine release syndrome (CRS), or macrophage activation syndrome (MAS). As a result, the development of stable, reliable "suicide genes" that can eliminate transferred T cells or stem cells in the event that they cause a serious adverse event (SAE) or that can no longer be used after treatment is crucial. However, in some cases, the need for treatment may remain, and methods may exist to reduce negative effects while maintaining sufficient therapeutic levels.

[0005] In some cases, it is necessary to increase the activity of therapeutic cells.For example, costimulatory polypeptides can be used to enhance the activation of T cells and the activation of CAR-expressing T cells against target antigens.This increases the efficacy of adoptive immunotherapy.

[0006] Thus, there is a need for controlled activation or elimination of therapeutic cells to rapidly enhance activation or rapidly eliminate possible negative effects of donor cells used in cell therapy while retaining some or all of the beneficial effects of the therapy. Summary of the Invention [Means for solving the problem]

[0007] Abstract Chemical induction of dimerization (CID) with small molecules is a powerful technique used to generate switches in protein function and alter cellular physiology. A highly specific and efficient dimerizer is rimizuside (AP1903), which has two identical protein-binding surfaces arranged tail-to-tail, each of which binds to a mutant or variant of FKBP12: FKBP12(F36V) (FKBP12v36, F V36 or F v) have high affinity and specificity for rimizuside. Binding of one or more FV domains to one or more cell signaling molecules, which normally relies on homodimerization, can convert the protein to rimizuside regulation. Homodimerization with rimizuside is used in the context of inducible caspase safety switches and inducible activation switches for cell therapy, where costimulatory polypeptides including MyD88 and CD40 polypeptides are used to stimulate immune activity. Because both of these switches rely on the same ligand inducer, it is difficult to use these switches to regulate both functions in the same cell. In some embodiments, a molecular switch is provided that is regulated by a distinct dimerizer ligand based on a heterodimerizing small molecule, rapamycin, or a rapamycin analog ("rapalog"). Rapamycin binds to FKBP12 and its variants, and can induce heterodimerization of a signaling domain fused to FKBP12 by binding to both FKBP12 and a polypeptide containing the FKBP-rapamycin binding (FRB) domain of mTOR. In some embodiments of the present application, a molecular switch is provided that greatly increases the use of rapamycin, rapalogs, and rimizuside as drugs for therapeutic applications. In certain embodiments, the allele specificity of rimizuside is controlled by F v -fusions are used to allow selective dimerization. In other embodiments, a rapamycin- or rapalog-inducible pro-apoptotic polypeptide (such as caspase-9 or a rapamycin- or rapalog-inducible costimulatory polypeptide, such as MyD88 / CD40 (MC)) is used in combination with a rimizuside-inducible pro-apoptotic polypeptide (such as caspase-9) or a rimizuside-inducible chimeric stimulatory polypeptide (such as iMC) to generate a dual switch. These dual switches can be used to selectively regulate both cell proliferation and apoptosis by administration of either of two distinct ligand inducers.

[0008] In other embodiments, molecular switches are provided that allow the choice of activating a pro-apoptotic polypeptide (e.g., caspase-9) with either rimizuside or rapamycin or a rapalog, where the chimeric pro-apoptotic polypeptide includes both a rimizuside-inducible switch and a rapamycin-inducible or rapalog-inducible switch. Inclusion of both molecular switches in the same chimeric pro-apoptotic polypeptide provides flexibility in clinical practice. Clinicians can then choose to administer the appropriate drug based on its specific pharmacological properties or other considerations (e.g., availability). These chimeric pro-apoptotic polypeptides can, for example, include both the FKBP12-rapamycin binding domain (FRB) or an FRB variant of mTOR and an FKBP12 variant polypeptide (e.g., FKBP12v36). An FRB variant polypeptide refers to an FRB polypeptide that binds to a rapamycin analog (rapalog), such as the rapalogs provided herein. FRB variant polypeptides contain one or more amino acid substitutions and bind to a rapalog and may or may not bind to rapamycin.

[0009] In one embodiment of the dual switch technology, a (Fwt.FRBΔC9 / MC.FvFv) homodimerizer (e.g., AP1903 (rimizuside)) induces activation of the modified cells, and a heterodimerizer (e.g., rapamycin or a rapalog) activates the safety switch, causing apoptosis of the modified cells. In this embodiment, a chimeric pro-apoptotic polypeptide (e.g., caspase-9) containing both FKBP12 and FRB, or an FRB variant region (iFwtFRBC9), is expressed in cells along with MyD88 and CD40 polypeptides and an inducible chimeric MyD88 / CD40 costimulatory polypeptide (MC.FvFv) containing at least two copies of FKBP12v36. Upon contacting the cells with a dimerizer that binds to the Fv region, the MC.FvFv dimerizes or multimerizes, activating the cells. The cells can be, for example, T cells expressing a chimeric antigen receptor (CARζ) directed against a target antigen. As a safety switch, the cells can be contacted with a heterodimerizer (e.g., rapamycin or a rapalog that binds to the FRB region on the iFwtFRBC9 polypeptide and the FKBP12 region on the iFwtFRBC9 polypeptide), causing direct dimerization of caspase-9 polypeptides and inducing apoptosis (Figure 43(2), Figure 57). In another mechanism, the heterodimerizer binds to the FRB region on the iFwtFRBC9 polypeptide and the Fv region on the MC.FvFv polypeptide, causing anchorage-induced dimerization due to the scaffolding of two FKBP12v36 polypeptides on each MC.FvFv polypeptide (Figure 43(1)), inducing apoptosis. An FKBP12 variant polypeptide refers to an FKBP12 polypeptide that contains one or more amino acid substitutions and binds to a ligand (e.g., rimizuside) with at least 100-fold, 500-fold, or 1000-fold greater affinity than the ligand binds to the FKBP12 polypeptide region.

[0010] In another embodiment of the dual switch technology, a (FRBFwtMC / FvC9) heterodimerizer (e.g., rapamycin or a rapalog) induces activation of the modified cells, and a homodimerizer (e.g., AP1903) activates the safety switch, causing apoptosis of the modified cells. In this embodiment, for example, a chimeric pro-apoptotic polypeptide (e.g., caspase-9) containing an Fv region (iFvC9) is expressed in cells together with MyD88 and CD40 polypeptides, and an inducible chimeric MyD88 / CD40 costimulatory polypeptide (iFRBFwtMC) (MC.FvFv) containing both FKBP12 and an FRB or FRB variant region. When the cells are contacted with rapamycin or a rapalog that heterodimerizes the FKBP12 and FRB regions, iFRBFwtMC dimerizes or multimerizes, activating the cells. The cells can be, for example, T cells expressing a chimeric antigen receptor (CARζ) directed against a target antigen. As a safety switch, the cells can be contacted with a homodimerizer (such as AP1903) that binds to the iFvC9 polypeptide, causing direct dimerization of the caspase-9 polypeptide and inducing apoptosis (Figure 57 (right side)).

[0011] In yet another embodiment of the dual switch compositions and methods of the present application, dual switch apoptotic polypeptides, modified cells expressing the dual switch apoptotic polypeptides, and nucleic acids encoding the dual switch apoptotic polypeptides are provided. These dual switch chimeric pro-apoptotic polypeptides allow for the selection of ligand inducers. For example, in one embodiment, FRB.FKBP V ΔC9 polypeptide, or FKBP v Modified cells expressing the .FRBΔC9 polypeptide are provided; apoptosis can be induced by contacting the modified cells with either a heterodimer (e.g., rapamycin or a rapalog) or a homodimer (rimiduside).

[0012] Thus, in some embodiments, a dual-switch chimeric pro-apoptotic polypeptide (e.g., FRB.FKBP) V Modified cells are provided that contain a polynucleotide encoding an FRB polypeptide region (e.g., FKBPv.FRBΔC9 polypeptide, or FKBPv.FRBΔC9 polypeptide), wherein the FRB polypeptide region is an FRB variant polypeptide region (e.g., FKBPv.FRBΔC9 polypeptide). L Where FRB is indicated (e.g., in the nomenclature herein), other FRB derivatives may be used (e.g., FRB L It is understood that the Fed L provided as an example of a composition or method of the present application, a polypeptide comprising RB or FRB, together with an appropriate ligand (e.g., rapamycin or a rapalog), L It is understood that FKBP12 variants or derivatives other than FKBP12v36 may be used. It is also understood that FKBP12 variants other than FKBP12v36 may be used in place of FKBP12v36 where appropriate. The modified cells may further comprise a polynucleotide encoding a heterologous protein (e.g., a chimeric antigen receptor or a recombinant T cell receptor, etc.). The modified cells may further comprise a polynucleotide encoding a costimulatory polypeptide (e.g., a polypeptide comprising a MyD88 polypeptide region, or a truncated MyD88 polypeptide region lacking the TIR domain, or a polypeptide comprising a MyD88 polypeptide region, or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the extracellular domain, etc.). Also provided, in some embodiments, is a nucleic acid comprising a polynucleotide encoding a dual switch chimeric pro-apoptotic polypeptide (e.g., an FRB.FKBPV.ΔC9 polypeptide or an FKBPv.FRBΔC9 polypeptide), wherein the FRB polypeptide region is an FRB variant polypeptide region (e.g., an FRB LThe nucleic acid may further comprise a polynucleotide encoding a heterologous protein (e.g., a chimeric antigen receptor or a recombinant T cell receptor, etc.). The nucleic acid may further comprise a polynucleotide encoding a costimulatory polypeptide (e.g., a polypeptide comprising a MyD88 polypeptide region, or a truncated MyD88 polypeptide region lacking the TIR domain, or a polypeptide comprising a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the extracellular domain, etc.).

[0013] In some embodiments of the present application, chimeric polypeptides are provided, wherein a first chimeric polypeptide comprises a first multimerization region that binds to a first ligand; the first multimerization region comprises a first ligand binding unit and a second ligand binding unit; the first ligand is a multimeric ligand comprising a first portion and a second portion; the first ligand binding unit binds to the first portion of the first ligand and does not significantly bind to the second portion of the first ligand; and the second ligand binding unit binds to the second portion of the first ligand and does not significantly bind to the first portion of the first ligand. In some embodiments, a second chimeric polypeptide is also provided, wherein the second chimeric polypeptide comprises a second multimerization domain that binds to a second ligand; the second multimerization domain comprises a third ligand binding unit; the second ligand is a multimeric ligand comprising a third portion; and the third ligand binding unit binds to the third portion of the second ligand and does not significantly bind to the second portion of the first ligand. Examples of first ligand binding units include, but are not limited to, FKBP12 multimerization domains or variants (e.g., FKBP12v36), and examples of second ligand binding units include, but are not limited to, FKBP12 multimerization domains or variants (e.g., FKBP12v36). In certain embodiments, the first ligand-binding unit is FKBP12 and the third ligand-binding unit is FKBP12v36. In certain embodiments, the first ligand is rapamycin or a rapalog, and the second ligand is rimizuside (AP1903).

[0014] The multimerization domain (e.g., FKBP12 / FRB, FRB / FKBP12, and FKBP12v36) may be located amino-terminal to the pro-apoptotic or costimulatory polypeptide, or in other instances, carboxyl-terminal to the pro-apoptotic or costimulatory polypeptide. Additional polypeptides (e.g., linker polypeptides, stem polypeptides, spacer polypeptides, or in some instances, marker polypeptides) may be located in the chimeric polypeptide between the multimerization domain and the pro-apoptotic or costimulatory polypeptide.

[0015] Thus, in some embodiments, provided is a modified cell comprising: a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: (i) a pro-apoptotic polypeptide region; (ii) an FKBP12-rapamycin binding (FRB) domain polypeptide, or an FRB variant polypeptide region; and (iii) an FKBP12 or FKBP12 variant polypeptide region (FKBP12v); and a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises one or more (e.g., one, two, or three) FKBP12 variant polypeptide regions and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; or ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments, the modified cell further comprises a third polynucleotide encoding a chimeric antigen receptor or a recombinant T cell receptor.

[0016] Also provided, in some embodiments, is a nucleic acid comprising a promoter operably linked to a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: (i) a pro-apoptotic polypeptide region; (ii) an FKBP12-rapamycin binding (FRB) domain polypeptide or an FRB variant polypeptide region; and (iii) an FKBP12 or FKBP12 variant polypeptide region (FKBP12v); and a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises one or more (e.g., one, two, or three) FKBP12 variant polypeptide regions and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; or ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments, the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments, the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes a chimeric antigen receptor or a recombinant T cell receptor. In some embodiments, the pro-apoptotic polypeptide is a caspase-9 polypeptide, wherein the caspase-9 polypeptide lacks a CARD domain. In some embodiments, the cell is a T cell, a tumor-infiltrating lymphocyte, an NK-T cell, or an NK cell.Also provided, in some embodiments, are kits or compositions comprising nucleic acids comprising: a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: (i) a pro-apoptotic polypeptide region; (ii) an FKBP12-rapamycin binding (FRB) domain polypeptide region or a variant thereof; and (iii) an FKBP12 polypeptide or an FKBP12 variant polypeptide region (FKBP12v); and a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises one or more (e.g., one, two, or three) FKBP12 variant polypeptide regions and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; or ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.

[0017] In some embodiments, a method is provided for expressing a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises a pro-apoptotic polypeptide region; an FRB polypeptide or FRB variant polypeptide region and an FKBP12 polypeptide region of this embodiment; the method comprises contacting a nucleic acid of this embodiment with a cell under conditions such that the nucleic acid is incorporated into the cell, whereby the cell expresses the chimeric pro-apoptotic polypeptide from the incorporated nucleic acid.

[0018] In some embodiments, methods are provided for stimulating an immune response in a subject, comprising transplanting the modified cells of this embodiment into the subject, and after (a), administering an effective amount of a ligand that binds to an FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide to stimulate a cell-mediated immune response. In some embodiments, methods are provided for administering a ligand to a subject that has undergone cell therapy using modified cells, comprising administering to the human subject a ligand that binds to an FKBP variant region of the chimeric costimulatory polypeptide, wherein the modified cells comprise the modified cells of this embodiment (of the present embodiment). Also provided is a method for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, the method comprising: a) transplanting into the subject an effective amount of modified cells; wherein the modified cells comprise the modified cells of this embodiment, wherein the modified cells comprise a chimeric antigen receptor or a recombinant T cell receptor that includes an antigen recognition portion that binds to the target antigen; and b) after a), administering an effective amount of a ligand that binds to an FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide to reduce the number or concentration of the target antigen or target cells in the subject. Also provided is a method for reducing tumor size in a subject, the method comprising: a) administering to the subject the modified cells of this embodiment, wherein the cells comprise a chimeric antigen receptor or a recombinant T cell receptor that includes an antigen recognition portion that binds to an antigen on the tumor; and b) after a), administering an effective amount of a ligand that binds to an FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide, thereby reducing tumor size in the subject.Also provided is a method for regulating the survival of transplanted modified cells in a subject, the method comprising transplanting the modified cells of this embodiment into the subject; and administering to the subject rapamycin or a rapalog that binds to the FRB polypeptide or FRB variant polypeptide region of the chimeric apoptosis-promoting polypeptide in an amount effective to kill at least 30% of the modified cells that express the chimeric apoptosis-promoting polypeptide.

[0019] In other embodiments, modified cells are provided, the modified cells comprising: a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises i) a pro-apoptotic polypeptide region; and ii) an FKBP12 variant polypeptide region; and a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises an FKBP12-rapamycin binding (FRB) domain polypeptide or an FRB variant polypeptide region; an FKBP12 polypeptide or an FKBP12 variant polypeptide region; and a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, or a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments, the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments, the cell further comprises a third polynucleotide, wherein the third polynucleotide encodes a chimeric antigen receptor or a recombinant T cell receptor.

[0020] In some embodiments, a nucleic acid is provided, the nucleic acid comprising a promoter operably linked to a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, the chimeric pro-apoptotic polypeptide comprising: i) a pro-apoptotic polypeptide region; and i) an FKBP12 variant polypeptide region; and a second polynucleotide encoding a chimeric costimulatory polypeptide, the chimeric costimulatory polypeptide comprising: i) an FKBP12-rapamycin-binding (FRB) domain polypeptide or an FRB variant polypeptide region; ii) an FKBP12 polypeptide region; and ii) a second polynucleotide encoding a chimeric costimulatory polypeptide, the chimeric costimulatory polypeptide comprising: i) an FKBP12-rapamycin-binding (FRB) domain polypeptide or an FRB variant polypeptide region; ii) an FKBP12 polypeptide region; and ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, or a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments, the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments, the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes a chimeric antigen receptor or a recombinant T cell receptor. In some embodiments, the pro-apoptotic polypeptide is a caspase-9 polypeptide, wherein the caspase-9 polypeptide lacks a CARD domain. In some embodiments, the cell is a T cell, a tumor-infiltrating lymphocyte, an NK-T cell, or an NK cell. Kits or compositions containing nucleic acids comprising the polynucleotides of this embodiment are also provided.Also provided are methods for expressing chimeric pro-apoptotic polypeptides and chimeric costimulatory polypeptides, wherein a) the chimeric pro-apoptotic polypeptide comprises i) a pro-apoptotic polypeptide region; and ii) an FKBP12 variant polypeptide region; and b) the chimeric costimulatory polypeptide comprises an FRB or FRB variant polypeptide region; an FKBP12 polypeptide region; and a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, or a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. The method includes contacting a cell with a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a chimeric apoptosis-promoting polypeptide, wherein the chimeric apoptosis-promoting polypeptide comprises: a) a proapoptotic polypeptide region; b) an FKBP12-rapamycin binding domain (FRB) polypeptide or an FRB variant polypeptide region; and c) an FKBP12 variant polypeptide region, under conditions such that the nucleic acid is incorporated into the cell, whereby the cell expresses the chimeric apoptosis-promoting polypeptide and the chimeric costimulatory polypeptide from the incorporated nucleic acid.

[0021] In some embodiments, a method for stimulating an immune response in a subject is provided, the method comprising: a) transplanting the modified cells of this embodiment into the subject; and b) after (a), administering an effective amount of rapamycin or a rapalog that binds to the FRB polypeptide or FRB variant polypeptide region of the chimera stimulating polypeptide to stimulate a cell-mediated immune response. In some embodiments, a method is provided for administering a ligand to a subject that has undergone cell therapy using modified cells, the method comprising administering rapamycin or a rapalog to the subject, wherein the modified cells comprise the modified cells of this embodiment. In some embodiments, methods are provided for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, the method comprising: a) transplanting into the subject an effective amount of modified cells; wherein the modified cells comprise the modified cells of this embodiment, wherein the modified cells comprise a chimeric antigen receptor or a recombinant T cell receptor comprising an antigen recognition moiety that binds to the target antigen; and b) after a), administering an effective amount of rapamycin or a rapalog that binds to an FRB polypeptide or FRB variant region of the chimeric stimulating polypeptide to reduce the number or concentration of the target antigen or target cells in the subject. In some embodiments, methods are provided for reducing tumor size in a subject, the methods comprising: a) administering to the subject modified cells of the present embodiments, wherein the cells comprise a chimeric antigen receptor or a recombinant T cell receptor comprising an antigen recognition moiety that binds to an antigen on the tumor; and b) after a), administering an effective amount of rapamycin or a rapalog that binds to an FRB or FRB variant polypeptide region of the chimeric stimulating polypeptide to reduce tumor size in the subject.In some embodiments, a method is provided for regulating the survival of transplanted modified cells in a subject, the method comprising: a) transplanting the modified cells of this embodiment into the subject; and, after (a), administering to the subject a ligand that binds to the FKBP12 variant polypeptide region of the chimeric apoptosis-promoting polypeptide in an amount effective to kill at least 90% of the modified cells that express the chimeric apoptosis-promoting polypeptide.

[0022] In some embodiments of the present application, the chimeric costimulatory polypeptide comprises two FKBP12 variant polypeptide regions and a truncated MyD88 polypeptide region lacking the TIR domain. In some embodiments, the chimeric costimulatory polypeptide further comprises a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some embodiments of the present application, the chimeric costimulatory polypeptide comprises two FKBP12 variant polypeptide regions.

[0023] Also provided herein is a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: a) a pro-apoptotic polypeptide region; b) an FKBP12-rapamycin binding domain (FRB) polypeptide or an FRB variant polypeptide region; and c) an FKBP12 variant polypeptide region. In some embodiments, the FKBP12 variant comprises an amino acid substitution at amino acid residue 36. In some embodiments, the FKBP12 variant polypeptide region is an FKBP12v36 polypeptide region. In some embodiments, the FRB variant polypeptide region is selected from the group consisting of KLW(T2098L)(FRBL), KTF(W2101F), and KLF(T2098L,W2101F). In some embodiments, a chimeric pro-apoptotic polypeptide encoded by the nucleic acid of this embodiment is provided. In some embodiments, a modified cell transfected or transduced with the nucleic acid of this embodiment is provided. In some embodiments, the modified cells comprise a polynucleotide encoding a chimeric antigen receptor or a recombinant TCR.In some embodiments, a method for regulating survival of transplanted modified cells in a subject is provided, the method comprising: a) transplanting the modified cells of this embodiment into the subject, wherein the modified cells comprise a nucleic acid including a promoter operably linked to a polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: a) a pro-apoptotic polypeptide region; b) an FKBP12-rapamycin-binding domain (FRB) polypeptide or an FKBP12 variant polypeptide region; and c) an FKBP12 variant polypeptide region of this embodiment; and b) after (a), administering to the subject: i) a first ligand that binds to the FRB or FKBP12 variant polypeptide region of the chimeric pro-apoptotic polypeptide; or ii) a second ligand that binds to the FKBP12 variant polypeptide region of the chimeric pro-apoptotic polypeptide, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 30% of the modified cells that express the chimeric pro-apoptotic polypeptide.

[0024] Autologous T cells expressing chimeric antigen receptors (CARs) directed against tumor-associated antigens (TAAs) have had transformative effects in early clinical trials for the treatment of certain types of leukemia ("liquid tumors") and lymphomas, with objective response (OR) rates approaching 90%. Despite their significant clinical promise and predictable accompanying enthusiasm, this success is tempered by the observed high level of on-target, off-tumor adverse events, typical of cytokine release syndrome (CRS). To maintain the benefits of these groundbreaking treatments while minimizing risk, tunable safety switches have been developed to control the activity levels of CAR-expressing T cells. Inducible costimulatory chimeric polypeptides allow for sustained, regulated control of the chimeric antigen receptor (CAR) coexpressed in cells. The ligand inducer activates the CAR-expressing cells by multimerizing the inducible chimeric signaling molecule, which in turn induces the NF-κB signaling pathway and other intracellular signaling pathways, leading to the activation of target cells, such as T cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, or natural killer T (NK-T) cells. In the absence of the ligand inducer, T cells are quiescent or have a basal level of activity.

[0025] In the second level of control, a "dimer" switch can allow for continued cell therapy while reducing or eliminating serious side effects by eliminating therapeutic cells from the subject, if necessary. This dimer switch is dependent on a second ligand inducer. In some instances, if there is a need to rapidly eliminate therapeutic cells, an appropriate dose of this second ligand inducer is administered to eliminate more than 90% or more than 95% of the therapeutic cells from the patient. This second level of control can be "tunable." That is, the level of therapeutic cell elimination can be controlled to result in partial elimination of therapeutic cells. This second level of control can include, for example, a chimeric apoptosis-promoting polypeptide.

[0026] In some instances, the chimeric apoptotic polypeptide contains a binding site for rapamycin or a rapamycin analog (rapalog); an inducible chimeric polypeptide that activates the therapeutic cell upon induction by a ligand inducer is also present on the therapeutic cell; in some instances, the inducible chimeric polypeptide provides costimulatory activity to the therapeutic cell. The CAR may be present on a separate polypeptide expressed in the cell. In other instances, the CAR may be present as part of the same polypeptide as the inducible chimeric polypeptide. Using this controllable first level, the need for continued or stimulating treatment can be balanced with the need to eliminate or reduce the level of negative side effects.

[0027] In some embodiments, a rapamycin analog, a rapalog, is administered to a patient, which then binds to both the caspase polypeptide and the chimeric antigen receptor, thereby recruiting the caspase polypeptide to the location of the CAR and causing the caspase polypeptide to aggregate. Upon aggregation, the caspase polypeptide induces apoptosis. The amount of rapamycin or a rapamycin analog administered to a patient can vary; if lower cell levels of apoptosis are desired to reduce side effects and continue CAR therapy, lower levels of rapamycin or a rapalog can be administered to the patient.

[0028] In the second therapeutic cell elimination level, selective apoptosis can be induced in cells expressing a chimeric caspase-9 polypeptide fused to a dimeric ligand-binding polypeptide (such as the AP1903-binding polypeptide FKBP12v36) by administering rimizuside (AP1903). In some instances, the caspase-9 polypeptide, as part of the inducible chimeric polypeptide, contains amino acid substitutions that result in a lower basal apoptotic activity level than the wild-type caspase-9 polypeptide.

[0029] In some embodiments, the nucleic acid encoding the chimeric polypeptide of the present application further comprises a polynucleotide encoding a chimeric antigen receptor, a T cell receptor, or a T cell receptor-based chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises (i) a transmembrane domain, (ii) a T cell activation molecule, and (iii) an antigen recognition portion. Also provided are modified cells transfected or transduced with the nucleic acids discussed herein.

[0030] In some aspects of the present application, the cells are transduced or transfected with a viral vector, which may be, for example, but is not limited to, a retroviral vector (such as, but not limited to, a murine leukemia virus vector); an SFG vector; and an adenoviral vector or a lentiviral vector.

[0031] In some embodiments, the cells are isolated. In some embodiments, the cells are in a human subject. In some embodiments, the cells are transplanted in a human subject.

[0032] In some embodiments, personalized treatment is provided, wherein the stage or level of the disease or condition is determined prior to administering the multimeric ligand, prior to administering additional doses of the multimeric ligand, or in determining the method and dosage involved in administering the multimeric ligand. These methods may be used in any of the methods for any of the diseases or conditions of the present application. While these methods for evaluating the patient prior to administering the ligand are discussed in the context of graft-versus-host disease, it is understood that these methods may be applied to the treatment of other conditions and diseases as well. Thus, for example, in some embodiments of the present application, the method comprises administering therapeutic cells to a patient and further comprises identifying the presence or absence of a condition in the patient that requires the removal of transfected or transduced therapeutic cells from the patient; and administering a multimeric ligand that binds to the multimerization domain, maintaining a subsequent dosage of the multimeric ligand to the patient, or adjusting a subsequent dosage of the multimeric ligand based on the presence or absence of the identified condition in the patient. For example, in other embodiments of the present application, the method further comprises determining whether to administer an additional dose or additional doses of the multimeric ligand to the patient based on the appearance of symptoms of graft-versus-host disease in the patient. In some embodiments, the method further comprises identifying the presence or stage of graft-versus-host disease in the patient, and administering a multimeric ligand that binds to the multimerization domain, maintaining a subsequent dose of the multimeric ligand to the patient, or adjusting a subsequent dose of the multimeric ligand based on the presence or stage of graft-versus-host disease identified in the patient.In some embodiments, the method further comprises identifying the presence or stage of graft-versus-host disease in the patient, and determining whether a multimeric ligand that binds the multimerization domain should be administered to the patient, or whether a subsequent dose of the multimeric ligand administered to the patient should be adjusted, based on the presence or stage of graft-versus-host disease identified in the patient. In some embodiments, the method further comprises receiving information including the presence or stage of graft-versus-host disease in the patient; and administering a multimeric ligand that binds the multimerization domain, maintaining a subsequent dose of the multimeric ligand to the patient, or adjusting a subsequent dose of the multimeric ligand, based on the presence or stage of graft-versus-host disease identified in the patient. In some embodiments, the method further comprises identifying the presence or stage of graft-versus-host disease in the patient and communicating the presence or stage of graft-versus-host disease to a decision maker who administers a multimeric ligand that binds the multimerization domain, maintains a subsequent dose of the multimeric ligand administered to the patient, or adjusts a subsequent dose of the multimeric ligand based on the presence or stage of graft-versus-host disease identified in the subject. In some embodiments, the method further comprises identifying the presence or stage of graft-versus-host disease in the patient and communicating instructions to administer a multimeric ligand that binds the multimeric binding domain, maintains a subsequent dose of the multimeric ligand administered to the patient, or adjusts a subsequent dose of the multimeric ligand based on the presence or stage of graft-versus-host disease identified in the subject.

[0033] Also provided is a method for administering donor T cells to a human patient, said method comprising administering to the human patient the transduced or transfected T cells of the present application, wherein said cells are non-allodepleted human donor T cells.

[0034] In some embodiments, the therapeutic cells are administered to a subject with a non-malignant disorder, or wherein the subject has, for example, a primary immunodeficiency disorder (e.g., including, but not limited to, severe combined immunodeficiency (SCID), combined immunodeficiency (CID), congenital T-cell deficiency / deficiency, common variable immunodeficiency (CVID), chronic granulomatous disease, IPEX (immunodeficiency, polyendocrinopathy, enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich syndrome, CD40 ligand deficiency, leukocyte adhesion deficiency, DOCK8 deficiency, IL-10 deficiency / IL-10 receptor deficiency, GATA2 deficiency, X-linked lymphoproliferative disorder (XLP), cartilage hair hypoplasia, etc.), hemophagocytic lymphohistiocytosis (HLH) or other hemophagocytic disorders, inherited marrow failure disorders, etc. Have been diagnosed with a non-malignant disorder such as a cerebrovascular disorder (e.g., including but not limited to, Schwachman-Diamond syndrome, Diamond-Blackfan anemia, dyskeratosis congenita, Fanconi anemia, congenital neutropenia, etc.), a hemoglobinopathy (e.g., including but not limited to, sickle cell disease, thalassemia, etc.), a metabolic disorder (e.g., including but not limited to, mucopolysaccharidoses, sphingolipidoses, etc.), or an osteoclast disorder (e.g., including but not limited to, osteopetrosis).

[0035] The therapeutic cells can be, for example, any cells administered to a patient for a desired therapeutic outcome. The cells can be, for example, T cells, natural killer cells, B cells, macrophages, peripheral blood cells, hematopoietic progenitor cells, bone marrow cells, or tumor cells. The modified caspase-9 polypeptide can also be used to directly kill tumor cells. In one application, a vector containing a polynucleotide encoding the inducible modified caspase-9 polypeptide is injected into a tumor, and 10 to 24 hours later (to allow protein expression), the ligand inducer (e.g., AP1903) is administered to induce apoptosis and release of tumor antigens into the tumor microenvironment. To further improve the tumor microenvironment and make it more immunogenic, treatment can be combined with one or more adjuvants (e.g., IL-12, TLR, IDO inhibitors, etc.). In some embodiments, the cells can be delivered to treat solid tumors (e.g., delivery of the cells to the tumor bed). In some embodiments, the polynucleotide encoding the chimeric caspase-9 polypeptide can be administered as part of a vaccine or by direct delivery to the tumor bed, resulting in expression of the chimeric caspase-9 polypeptide in the tumor cells, followed by apoptosis of the tumor cells after administration of the ligand inducer. Thus, in some embodiments, nucleic acid vaccines (e.g., DNA vaccines) are also provided, wherein the vaccine comprises a nucleic acid comprising a polynucleotide encoding an inducible or modified inducible caspase-9 polypeptide of the present application. The vaccine can be administered to a subject, thereby transforming or transducing target cells in vivo. The ligand inducer can then be administered according to the methods of the present application.

[0036] In some embodiments, the modified caspase-9 polypeptide is a truncated modified caspase-9 polypeptide. In some embodiments, the modified caspase-9 polypeptide lacks a caspase recruitment domain. In some embodiments, the caspase-9 polypeptide comprises the amino acid sequence of SEQ ID NO:9 or a fragment thereof, or is encoded by the nucleotide sequence of SEQ ID NO:8 or a fragment thereof.

[0037] In some embodiments, the method further comprises administering a multimeric ligand that binds to the multimeric ligand binding region. In some embodiments, the multimeric ligand binding region is selected from the group consisting of FKBP, a cyclophilin receptor, a steroid receptor, a tetracycline receptor, a heavy chain antibody subunit, a light chain antibody subunit, a single-chain antibody consisting of a tandem heavy chain variable region and a heavy chain variable region separated by a flexible linker domain, and mutated sequences thereof. In some embodiments, the multimeric ligand binding region is an FKBP12 region. In some embodiments, the multimeric ligand is an FK506 dimer or a dimeric FK506-like analog ligand. In some embodiments, the multimeric ligand is AP1903. In some embodiments, the number of therapeutic cells is reduced by about 60% to 99%, about 70% to 95%, 80% to 90%, or about 90% or more after administration of the multimeric ligand. In some embodiments, after administration of the multimeric ligand, donor T cells survive, are capable of expansion, and are reactive to viruses and fungi in the patient. In some embodiments, after administration of the multimeric ligand, donor T cells survive, are capable of expansion, and are reactive to tumor cells in the patient.

[0038] In some embodiments, the suicide gene used in the second level of control is a caspase polypeptide, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, or caspase 14. In certain embodiments, the caspase polypeptide is a caspase-9 polypeptide. In certain embodiments, the caspase-9 polypeptide comprises the amino acid sequence of a catalytically active (not catalytically dead) caspase variant polypeptide provided in Table 5 or Table 6 herein. In other embodiments, the caspase-9 polypeptide consists of the amino acid sequence of a catalytically active (not catalytically dead) caspase variant polypeptide provided in Table 5 or Table 6 herein. In other embodiments, a caspase polypeptide with lower basal activity in the absence of the ligand inducer may be used. For example, when included as part of an inducible chimeric caspase polypeptide, certain modified caspase-9 polypeptides may have lower basal activity compared to the wild-type caspase-9 in the chimeric construct. For example, the modified caspase-9 polypeptide may comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9 and may contain at least one amino acid substitution.

[0039] Certain embodiments are further described in the following description, examples, claims and drawings.

[0040] The drawings are illustrative of embodiments of the present technology, but are not limiting. For clarity and ease of illustration, the drawings have not been made to scale, and in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments. The present invention provides, for example, the following items. (Item 1) a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: (i) the pro-apoptotic polypeptide region; (ii) an FKBP12-rapamycin binding (FRB) domain polypeptide, or an FRB variant polypeptide region; and (iii) FKBP12 or FKBP12 variant polypeptide region (FKBP12v); a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises two FKBP12 variant polypeptide regions and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; or ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: A modified cell comprising: (Item 2) 2. The modified cell of item 1, wherein the chimeric costimulatory polypeptide comprises two FKBP12 variant polypeptide regions and a truncated MyD88 polypeptide region lacking a TIR domain. (Item 3) 2. The modified cell of item 1, wherein the chimeric costimulatory polypeptide comprises two FKBP12 variant polypeptide regions, a truncated MyD88 polypeptide region lacking a TIR domain, and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain. (Item 4) 4. The modified cell according to any one of items 1 to 3, wherein the chimeric pro-apoptotic polypeptide comprises (i) a pro-apoptotic polypeptide region, (ii) an FRB or FRB variant polypeptide region, and (iii) an FKBP12 polypeptide region. (Item 5) 6. The modified cell of any one of items 1 to 5, wherein the cell further comprises a third polynucleotide encoding a heterologous protein. (Item 6) 7. The modified cell of item 6, wherein the heterologous protein is a chimeric antigen receptor. (Item 7) 8. The modified cell of item 7, wherein the heterologous protein is a recombinant T cell receptor. (Item 8) a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: (i) the pro-apoptotic polypeptide region; (ii) an FKBP12-rapamycin binding (FRB) domain polypeptide, or an FRB variant polypeptide region; and (iii) FKBP12 or FKBP12 variant polypeptide region (FKBP12v); a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises two FKBP12 variant polypeptide regions, and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; or ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: A nucleic acid comprising a promoter operably linked to a (Item 9) 9. The nucleic acid of item 8, wherein the chimeric pro-apoptotic polypeptide comprises a pro-apoptotic polypeptide region, an FRB or FRB variant polypeptide region, and an FKBP12 polypeptide region. (Item 10) 10. The nucleic acid of any one of items 8 to 9, wherein the chimeric costimulatory polypeptide comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking a TIR domain. (Item 11) 10. The nucleic acid of any one of items 8 to 9, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain. (Item 12) 12. The nucleic acid of any one of items 8 to 11, wherein the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes a heterologous protein. (Item 13) 13. The nucleic acid of item 12, wherein the heterologous protein is a chimeric antigen receptor. (Item 14) 13. The nucleic acid of item 12, wherein the heterologous protein is a recombinant TCR. (Item 15) 15. The nucleic acid according to any one of Items 8 to 14, wherein the nucleic acid further comprises a polynucleotide encoding a linker polypeptide between the first polynucleotide and the second polynucleotide, wherein the linker polypeptide separates the translation products of the first polynucleotide and the second polynucleotide during or after translation. (Item 16) 16. The nucleic acid of item 15, wherein the nucleic acid further comprises a polynucleotide encoding a linker polypeptide between the third polynucleotide and the first polynucleotide or the second polynucleotide, wherein the linker polypeptide separates the translation product of the third polynucleotide from the translation product of the first polynucleotide or the second polynucleotide during or after translation. (Item 17) 17. The nucleic acid of any one of items 15 or 16, wherein the linker polypeptide is a 2A polypeptide. (Item 18) 18. A modified cell transduced or transfected with the nucleic acid of any one of items 8 to 17. (Item 19) 19. The modified cell or nucleic acid of any one of items 1 to 18, wherein the FRB polypeptide or FRB variant polypeptide region and the FKBP12 polypeptide or FKBP12 variant polypeptide region are amino-terminal to the pro-apoptotic polypeptide of the chimeric pro-apoptotic polypeptide. (Item 20) 20. The modified cell or nucleic acid of claim 19, wherein the FRB polypeptide or FRB variant polypeptide region is amino-terminal to the FKBP12 polypeptide or FKBP12 variant polypeptide region. (Item 21) 20. The modified cell or nucleic acid of claim 19, wherein the FKBP12 polypeptide or FKBP12 variant polypeptide region is amino-terminal to the FRB or FRB variant polypeptide region. (Item 22) 22. The modified cell or nucleic acid of any one of items 1 to 21, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 100-fold greater than the ligand binds to the FKBP12 polypeptide domain. (Item 23) 22. The modified cell or nucleic acid of any one of items 1 to 21, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 500 times greater than the ligand binds to the FKBP12 polypeptide domain. (Item 24) 22. The modified cell or nucleic acid of any one of items 1 to 21, wherein the FKBP12 variant polypeptide region binds to the ligand with an affinity that is at least 1000 times greater than the ligand binds to the wild-type FKBP12 polypeptide region. (Item 25) 25. The modified cell or nucleic acid of any one of items 1 to 24, wherein the FKBP12 variant polypeptide comprises an amino acid substitution at amino acid residue 36. (Item 26) 26. The modified cell or nucleic acid of item 25, wherein the amino acid substitution at position 36 is selected from the group consisting of valine, leucine, isoleucine, and alanine. (Item 27) 22. The modified cell or nucleic acid of any one of items 1 to 21, wherein the FKBP12 variant polypeptide region is an FKBP12v36 polypeptide region. (Item 28) 25. The modified cell or nucleic acid according to any one of items 22 to 24, wherein the ligand is rimizuside. (Item 29) 225. The modified cell or nucleic acid of any one of items 224, wherein the ligand is AP20187 or AP1510. (Item 30) 30. The modified cell or nucleic acid of any one of paragraphs 1 to 29, wherein the FRB variant polypeptide binds to a C7 rapalog. (Item 31) 31. The modified cell or nucleic acid of any one of items 1 to 30, wherein the FRB variant polypeptide comprises an amino acid substitution at position T2098 or W2101. (Item 32) The FRB variant polypeptide region is KLW(T2098L) (FRB L 32. The modified cell or nucleic acid of any one of items 1 to 31, wherein the modified cell or nucleic acid is selected from the group consisting of KTF(W2101F), KTF(W2101F), and KLF(T2098L, W2101F). (Item 33) The FRB variant polypeptide region comprises FRB L 33. The modified cell or nucleic acid according to any one of items 1 to 32, wherein (Item 34) 34. The modified cell of any one of paragraphs 1 to 33, wherein the FRB variant polypeptide region binds to a rapalog selected from the group consisting of So,p-dimethoxyphenyl (DMOP)-rapamycin, R-isopropoxyrapamycin, C7-isobutyloxyrapamycin, and S-butanesulfonamide rapamycin. (Item 35) 35. The modified cell or nucleic acid of any one of items 1 to 34, wherein the cell or the nucleic acid comprises a polynucleotide encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises (i) a transmembrane domain, (ii) a T cell activation molecule, and (iii) an antigen recognition portion. (Item 36) 34. The modified cell or nucleic acid of Item 33, wherein the T cell activation molecule is selected from the group consisting of an ITAM-containing molecule that confers signal 1, a Syk polypeptide, a ZAP70 polypeptide, a CD3ζ polypeptide, and an Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. (Item 37) 34. The modified cell or nucleic acid of claim 33, wherein the T cell activation molecule is selected from the group consisting of an ITAM-containing molecule that confers signal 1, a CD3ζ polypeptide, and an Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. (Item 38) 372. The modified cell or nucleic acid of any one of items 35 to 371, wherein the antigen recognition portion is a single-chain variable fragment. (Item 39) 39. The modified cell or nucleic acid according to any one of items 35 to 38, wherein the transmembrane domain is a CD8 transmembrane domain. (Item 40) 40. The modified cell or nucleic acid of any one of Items 35 to 39, wherein the antigen recognition moiety binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, MuclMuc1, Mucl, ROR1, mesothelin, GD2, CD123, Mucl6, CD33, CD38, and CD44v6. (Item 41) 31. The modified cell or nucleic acid of any one of Items 35 to 30, wherein the antigen recognition moiety binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, MuclMuc1, Mucl, ROR1, mesothelin, GD2, CD123, Mucl6, CD33, CD38, and CD44v6. (Item 42) 35. The modified cell of any one of items 1 to 34, wherein the cell comprises a polynucleotide encoding a recombinant T cell receptor, wherein the recombinant T cell receptor binds to an antigenic polypeptide selected from the group consisting of PRAME, Bob-1, and NY-ESO-1. (Item 43) 43. The modified cell or nucleic acid of any one of paragraphs 1 to 42, wherein the pro-apoptotic polypeptide is selected from the group consisting of caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, or caspase 14, FADD(DED), APAF1(CARD), CRADD / RAIDD CARD), ASC(CARD), Bax, Bak, Bcl-xL, Bcl-2, RIPK3, and RIPK1-RHIM. (Item 44) 44. The modified cell or nucleic acid of any one of items 1 to 43, wherein the pro-apoptotic polypeptide is a caspase polypeptide. (Item 45) 45. The modified cell or nucleic acid of item 44, wherein the pro-apoptotic polypeptide is a caspase-9 polypeptide. (Item 46) 46. ​​The nucleic acid or cell of item 45, wherein the caspase-9 polypeptide lacks a CARD domain. (Item 47) 47. The modified cell or nucleic acid of any one of items 45 or 46, wherein the caspase polypeptide comprises the amino acid sequence of SEQ ID NO: 300. (Item 48) 48. The modified cell or nucleic acid of any one of items 44 to 47, wherein the caspase polypeptide is a modified caspase-9 polypeptide comprising an amino acid substitution selected from the group consisting of catalytically active caspase variants in Table 5 or Table 6. (Item 49) 49. The modified cell or nucleic acid of item 48, wherein the caspase polypeptide is a modified caspase-9 polypeptide comprising an amino acid sequence selected from the group consisting of D330A, D330E, and N405Q. (Item 50) 50. The modified cell or nucleic acid of any one of items 1 to 49, wherein the truncated MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 214 or 305, or a functional fragment thereof. (Item 51) 50. The modified cell or nucleic acid of any one of items 1 to 49, wherein the MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 282 or a functional fragment thereof. (Item 52) 52. The modified cell or nucleic acid of any one of items 1 to 51, wherein the cytoplasmic CD40 polypeptide has the amino acid sequence of SEQ ID NO: 216 or a functional fragment thereof. (Item 53) a) the chimeric pro-apoptotic polypeptide is a caspase-9 polypeptide lacking a CARD domain, FRB L a polypeptide domain and an FKBP12 polypeptide domain; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and two FKBP12v36 polypeptide regions; Item 1. The modified cell of item 1. (Item 54) a) the chimeric pro-apoptotic polypeptide is a caspase-9 polypeptide lacking a CARD domain, FRB L a polypeptide domain and an FKBP12 polypeptide domain; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain, a CD40 cytoplasmic polypeptide region lacking an extracellular domain, and two FKBP12v36 polypeptide regions; Item 1. The modified cell of item 1. (Item 55) a) the chimeric pro-apoptotic polypeptide is a caspase-9 polypeptide lacking a CARD domain, FRB L a polypeptide domain and an FKBP12 polypeptide domain; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and two FKBP12v36 polypeptide regions; Item 19. The nucleic acid according to item 19. (Item 56) a) the chimeric pro-apoptotic polypeptide is a caspase-9 polypeptide lacking a CARD domain, FRB L a polypeptide domain and an FKBP12 polypeptide domain; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain, a CD40 cytoplasmic polypeptide region lacking an extracellular domain, and two FKBP12v36 polypeptide regions; Item 19. The nucleic acid according to item 19. (Item 57) 37. The modified cell of any one of paragraphs 1 to 8, 18, or 19 to 36, wherein the cell is a T cell, a tumor-infiltrating lymphocyte, an NK-T cell, or an NK cell. (Item 58) 37. The modified cell of any one of paragraphs 1 to 8, 18, or 19 to 36, wherein the cell is a T cell, an NK-T cell, or an NK cell. (Item 59) 37. The modified cell of any one of items 1 to 8, 18, or 19 to 36, wherein the cell is a T cell. (Item 60) 37. The modified cell of any one of items 1 to 8, 18, or 19 to 36, wherein the cell is a primary T cell. (Item 61) 37. The modified cell of any one of items 1 to 8, 18, or 19 to 36, wherein the cell is a cytotoxic T cell. (Item 62) 37. The modified cell of any one of items 1-8, 18, or 19-36, wherein the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a non-lymphocytic hematopoietic cell, a non-hematopoietic cell, a macrophage, a keratinocyte, a fibroblast, a melanoma cell, a tumor-infiltrating lymphocyte, a natural killer cell, a natural killer T cell, or a T cell. (Item 63) 37. The modified cell of any one of items 1 to 8, 18, or 19 to 36, wherein the T cell is a helper T cell. (Item 64) 37. The modified cells of any one of paragraphs 1-8, 18, or 19-36, wherein the cells are obtained or prepared from bone marrow. (Item 65) 37. The modified cells of any one of paragraphs 1-8, 18, or 19-36, wherein the cells are obtained or prepared from umbilical cord blood. (Item 66) 37. The modified cells of any one of paragraphs 1 to 8, 18, or 19 to 36, wherein the cells are obtained or prepared from peripheral blood. (Item 67) 37. The modified cell of any one of paragraphs 1 to 8, 18, or 19 to 36, wherein the cell is obtained or prepared from peripheral blood mononuclear cells. (Item 68) 68. The modified cell of any one of items 1 to 8, 18, 19 to 36 or 57 to 67, wherein the cell is a human cell. (Item 69) 69. The modified cell of any one of paragraphs 1-8, 18, 19-36 or 57-68, wherein the modified cell is transduced or transfected in vivo. (Item 70) 70. The modified cell of any one of paragraphs 1-8, 18, 19-36, or 57-69, wherein the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistic (e.g., gene gun with Au particles), lipid transfection, polymer transfection, nanoparticles, or polyplexes. (Item 71) a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: (i) the pro-apoptotic polypeptide region; (ii) an FKBP12-rapamycin binding (FRB) domain polypeptide region, or a variant thereof; and (iii) an FKBP12 polypeptide or an FKBP12 variant polypeptide region (FKBP12v); a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises two FKBP12 variant polypeptide regions and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; or ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: A kit or composition comprising a nucleic acid comprising: (Item 72) 72. The kit or composition of item 71, wherein the chimeric pro-apoptotic polypeptide comprises a pro-apoptotic polypeptide region, an FRB or FRB variant polypeptide region, and an FKBP12 polypeptide region. (Item 73) 73. The kit or composition of any one of items 71 to 72, wherein the chimeric costimulatory polypeptide comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain. (Item 74) 73. The kit or composition of any one of items 71 to 72, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain. (Item 75) 72. The kit or composition according to Item 71, wherein the nucleic acid is a nucleic acid according to any one of Items 8 to 17, 19 to 212, or 55 to 56. (Item 76) 76. The kit or composition of any one of items 71 to 75, further comprising a third polynucleotide, wherein the third polynucleotide encodes a heterologous protein. (Item 77) 73. The kit or composition of item 72, wherein the heterologous protein is a chimeric antigen receptor. (Item 78) 73. The kit or composition of item 72, wherein the heterologous protein is a recombinant TCR. (Item 79) 76. The kit or composition of any one of items 71 to 75, comprising a virus, wherein the virus comprises the first polynucleotide and the second polynucleotide. (Item 80) 79. The kit or composition of any one of items 72 to 78, comprising a virus, wherein the virus comprises the first polynucleotide, the second polynucleotide, and the third polynucleotide. (Item 81) 79. The kit or composition of any one of items 72 to 78, comprising a virus, wherein the virus comprises the first polynucleotide and the third polynucleotide. (Item 82) 79. The kit or composition of any one of items 72 to 78, comprising a virus, wherein the virus comprises the second polynucleotide and the third polynucleotide. (Item 83) 1. A method for expressing a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: a) a pro-apoptotic polypeptide region; an FRB polypeptide or FRB variant polypeptide region; and b) the FKBP12 polypeptide region; The method comprises: contacting a cell with the nucleic acid according to any one of items 8 to 17, 19 to 52, or 55 to 56 under conditions in which the nucleic acid is incorporated into the cell; whereby said cell expresses said chimeric pro-apoptotic polypeptide from said integrated nucleic acid. (Item 84) The cell further expresses a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises: a) two FKBP12 variant polypeptide regions; and b) a truncated MyD88 polypeptide region lacking the MyD88 polypeptide region or the TIR domain, or a truncated MyD88 polypeptide region lacking the MyD88 polypeptide region or the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; Item 84. The method according to Item 83, comprising: (Item 85) 85. The method of any one of paragraphs 83 or 84, wherein the nucleic acid is contacted with the cell ex vivo. (Item 86) 85. The method of any one of items 83 or 84, wherein the nucleic acid is contacted with the cell in vivo. (Item 87) 1. A method for stimulating an immune response in a subject, the method comprising: a) transplanting the modified cells of any one of items 1 to 8, 18, 19 to 36, or 57 to 70 into the subject; and b) after (a), administering an effective amount of a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide to stimulate a cell-mediated immune response; A method that encompasses (Item 88) 10. A method for administering a ligand to a subject who has undergone cell therapy using modified cells, the method comprising administering to the human subject a ligand that binds to the FKBP variant region of the chimeric costimulatory polypeptide, wherein the modified cells comprise the modified cells of any one of paragraphs 1-8, 18, 19-36, or 57-70. (Item 89) 1. A method for regulating the activity of transplanted modified cells in a subject, the method comprising: a) implanting the modified cells described in any one of items 1 to 8, 18, 19 to 36, or 57 to 70; and b) after (a), administering an effective amount of a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide to stimulate the activity of the transplanted modified cells; A method that encompasses (Item 90) 1. A method for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, the method comprising: a) administering to the subject an effective amount of modified cells; wherein the modified cells comprise the modified cells of any one of items 1-8, 18, 19-36, or 57-70, wherein the modified cells comprise a chimeric antigen receptor comprising an antigen recognition portion that binds to the target antigen; and b) following a), administering an effective amount of a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide to reduce the number or concentration of target antigens or target cells in the subject; A method that encompasses (Item 91) 91. The method of claim 90, wherein the target antigen is a tumor antigen. (Item 92) 1. A method for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, the method comprising: a) administering to the subject an effective amount of modified cells, wherein the modified cells comprise the modified cells of any one of items 1-8, 18, 19-36, or 57-70, wherein the modified cells comprise a recombinant T cell receptor that recognizes and binds to the target antigen; and b) following a), administering an effective amount of a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide to reduce the number or concentration of target antigens or target cells in the subject; A method that encompasses (Item 93) 1. A method for reducing tumor size in a subject, the method comprising: a) administering to the subject the modified cells of any one of items 1-8, 18, 19-36, or 57-70, wherein the cells comprise a chimeric antigen receptor comprising an antigen recognition portion that binds to an antigen on the tumor; and b) following a), administering an effective amount of a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulatory polypeptide to reduce the size of the tumor in the subject; A method that encompasses (Item 94) 94. The method of any one of items 90 to 93, comprising measuring the number or concentration of target cells in a first sample obtained from the subject before administration of a second ligand, measuring the number or concentration of target cells in a second sample obtained from the subject after administration of the ligand, and determining an increase or decrease in the number or concentration of target cells in the second sample compared to the number or concentration of target cells in the first sample. (Item 95) 95. The method of claim 94, wherein the concentration of target cells in the second sample is reduced compared to the concentration of target cells in the first sample. (Item 96) 95. The method of claim 94, wherein the concentration of target cells in the second sample is increased compared to the concentration of target cells in the first sample. (Item 97) 97. The method of any one of items 87 to 96, wherein the subject has undergone stem cell transplantation prior to or concurrently with administration of the modified cells. (Item 98) At least 1 x 10 6 98. The method of any one of items 87 to 97, wherein the transduced or transfected modified cells are administered to the subject. (Item 99) At least 1 x 10 7 98. The method of any one of items 87 to 97, wherein the transduced or transfected modified cells are administered to the subject. (Item 100) At least 1 x 10 8 98. The method of any one of items 87 to 97, wherein the modified cells are administered to the subject. (Item 101) 101. The method according to any one of items 87 to 100, wherein the FKBP12 variant polypeptide region is FKBP12v36, and the ligand that binds to the FKBP12 variant polypeptide region is AP1903. (Item 102) 1. A method for modulating survival of transplanted modified cells in a subject, the method comprising: a) transplanting the modified cells of any one of items 1-8, 18, 19-36, or 57-70 into the subject; and b) after a), administering to the subject rapamycin or a rapalog that binds to the FRB polypeptide or FRB variant polypeptide region of the chimeric pro-apoptotic polypeptide in an amount effective to kill at least 30% of the modified cells that express the chimeric pro-apoptotic polypeptide; A method that encompasses (Item 103) 103. The method of any one of items 87-102, further comprising, after b), administering to the subject rapamycin or a rapalog that binds to the FRB variant polypeptide region of the chimeric pro-apoptotic polypeptide in an amount effective to kill at least 30% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 104) 104. The method of claim 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 40% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 105) 104. The method of any one of paragraphs 102 or 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 50% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 106) 104. The method of any one of paragraphs 102 or 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 60% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 107) 104. The method of any one of paragraphs 102 or 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 70% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 108) 104. The method of any one of paragraphs 102 or 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 80% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 109) 104. The method of any one of paragraphs 102 or 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 90% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 110) 104. The method of any one of paragraphs 102 or 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 95% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 111) 104. The method of any one of paragraphs 102 or 103, wherein the rapamycin or rapalog is administered in an amount effective to kill at least 99% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 112) The chimeric pro-apoptotic polypeptide is FRB L 104. The method according to any one of items 102 to 103, comprising a region. (Item 113) 102. The method of any one of items 87 to 101, wherein more than one dose of the ligand is administered to the subject. (Item 114) 114. The method of any one of paragraphs 102 to 113, wherein more than one dose of the rapamycin or rapalog is administered to the subject. (Item 115) identifying the presence or absence of a condition in the subject that requires removal of the modified cells from the subject; and administering rapamycin or a rapalog to the subject, maintaining a subsequent dosage of rapamycin or the rapalog to the subject, or adjusting a subsequent dosage of the rapamycin or the rapalog to the subject based on the presence or absence of the condition identified in the subject; The method according to any one of items 87 to 113, further comprising: (Item 116) receiving information comprising the presence or absence of a condition in the subject that requires the modified cells to be removed from the subject; and administering said rapamycin or rapalog to said subject, maintaining a subsequent dosage of rapamycin or said rapalog to said subject, or adjusting a subsequent dosage of rapamycin or said rapalog to said subject based on the presence or absence of said condition identified in said subject; The method according to any one of items 87 to 113, further comprising: (Item 117) identifying the presence or absence of a condition in the subject that requires removal of the modified cells from the subject; and communicating the presence, absence, or stage of the condition identified in the subject to a decision maker who administers rapamycin or said rapalog to the subject, maintains the subsequent dosage of the rapamycin or rapalog administered to the subject, or adjusts the subsequent dosage of the rapamycin or rapalog administered to the subject based on the presence, absence, or stage of the condition identified in the subject; The method according to any one of items 87 to 113, further comprising: (Item 118) identifying the presence or absence of a condition in the subject that requires removal of the modified cells from the subject; and communicating instructions to administer the rapamycin or the rapalog to the subject, maintain a subsequent dosage of the rapamycin or the rapalog administered to the subject, or adjust a subsequent dosage of the rapamycin or the rapalog administered to the subject based on the presence, absence, or stage of the condition identified in the subject; The method according to any one of items 87 to 113, further comprising: (Item 119) 19. The method of any one of items 87 to 118, wherein the subject has cancer. (Item 120) 119. The method of any one of items 87 to 119, wherein the modified cells are delivered to a tumor bed. (Item 121) 121. The method of any one of paragraphs 119 or 120, wherein the cancer is present in the blood or bone marrow of the subject. (Item 122) 119. The method of any one of items 87 to 118, wherein the subject has a blood or bone marrow disease. (Item 123) 119. The method of any one of items 87-118, wherein the subject has been diagnosed with sickle cell anemia or metachromatic leukodystrophy. (Item 124) 119. The method of any one of paragraphs 87 to 118, wherein the patient has been diagnosed with a condition selected from the group consisting of a primary immunodeficiency condition, hemophagocytic lymphohistiocytosis (HLH) or other hemophagocytic condition, an inherited bone marrow failure condition, a hemoglobinopathy, a metabolic condition, and an osteoclast condition. (Item 125) 119. The method of any one of paragraphs 87 to 118, wherein the patient has been diagnosed with a disease or condition selected from the group consisting of severe combined immunodeficiency (SCID), combined immunodeficiency (CID), congenital T-cell deficiency / deficiency, common variable immunodeficiency (CVID), chronic granulomatous disease, IPEX (immunodeficiency, polyendocrinopathy, enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich syndrome, CD40 ligand deficiency, leukocyte adhesion deficiency, DOCA8 deficiency, IL-10 deficiency / IL-10 receptor deficiency, GATA2 deficiency, X-linked lymphoproliferative disorder (XLP), cartilage hair hypoplasia, Schwachman-Diamond syndrome, Diamond-Blackfan anemia, dyskeratosis congenita, Fanconi anemia, congenital neutropenia, sickle cell disease, thalassemia, mucopolysaccharidoses, sphingolipidoses, and osteopetrosis. (Item 126) a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: i) a pro-apoptotic polypeptide region; and ii) FKBP12 variant polypeptide region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises: i) an FKBP12-rapamycin binding (FRB) domain polypeptide or an FRB variant polypeptide region; ii) an FKBP12 polypeptide or an FKBP12 variant polypeptide region; and iii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, or a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: A modified cell comprising: (Item 127) 127. The modified cell of claim 126, wherein the chimeric costimulatory polypeptide comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain. (Item 128) 127. The modified cell of paragraph 126, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain. (Item 129) 129. The modified cell of any one of items 126 to 128, wherein the cell further comprises a third polynucleotide, wherein the third polynucleotide encodes a heterologous protein. (Item 130) 130. The modified cell of claim 129, wherein the heterologous protein is a chimeric antigen receptor. (Item 131) 130. The modified cell of claim 129, wherein the heterologous protein is a recombinant TCR. (Item 132) a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: i) a pro-apoptotic polypeptide region; and ii) FKBP12 variant polypeptide region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises: i) an FKBP12-rapamycin binding (FRB) domain polypeptide or an FRB variant polypeptide region; ii) an FKBP12 polypeptide region; and iii) a truncated MyD88 polypeptide domain lacking the MyD88 polypeptide domain or the TIR domain, or a truncated MyD88 polypeptide domain lacking the MyD88 polypeptide domain or the TIR domain and a CD40 cytoplasmic polypeptide domain lacking the CD40 extracellular domain; a second polynucleotide comprising A nucleic acid comprising a promoter operably linked to a (Item 133) 133. The nucleic acid of item 132, wherein the chimeric costimulatory polypeptide comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain. (Item 134) 133. The nucleic acid of item 132, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain. (Item 135) 135. The nucleic acid of any one of Items 132 to 134, wherein the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes a heterologous protein. (Item 136) 136. The nucleic acid of item 135, wherein the heterologous protein is a chimeric antigen receptor. (Item 137) 136. The nucleic acid of item 135, wherein the heterologous protein is a recombinant TCR. (Item 138) 138. The nucleic acid according to any one of Items 132 to 137, wherein the nucleic acid further comprises a polynucleotide encoding a linker polypeptide between the first polynucleotide and the second polynucleotide, wherein the linker polypeptide separates the translation products of the first polynucleotide and the second polynucleotide during or after translation. (Item 139) 139. The nucleic acid of item 138, wherein the nucleic acid further comprises a polynucleotide encoding a linker polypeptide between the third polynucleotide and the first polynucleotide or the second polynucleotide, wherein the linker polypeptide separates the translation product of the third polynucleotide from the translation product of the first polynucleotide or the second polynucleotide during or after translation. (Item 140) 139. The nucleic acid of any one of items 138 or 139, wherein the linker polypeptide is a 2A polypeptide. (Item 141) 141. A modified cell transduced or transfected with a nucleic acid according to any one of items 132 to 140. (Item 142) 142. The modified cell or nucleic acid of any one of paragraphs 126 to 141, wherein the FRB polypeptide or FRB variant polypeptide region and the FKBP12 polypeptide region are amino-terminal to the MyD88 polypeptide or truncated MyD88 polypeptide of the chimeric costimulatory polypeptide. (Item 143) 143. The modified cell or nucleic acid of claim 142, wherein the FRB polypeptide or FRB variant polypeptide region is amino-terminal to the FKBP12 polypeptide region. (Item 144) 143. The modified cell or nucleic acid of claim 142, wherein the FKBP12 polypeptide region is amino-terminal to the FRB or FRB variant polypeptide region. (Item 145) 145. The modified cell or nucleic acid of any one of items 126 to 144, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 100-fold greater than the ligand binds to the FKBP12 polypeptide domain. (Item 146) 145. The modified cell or nucleic acid of any one of items 126 to 144, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 500 times greater than the ligand binds to the FKBP12 polypeptide domain. (Item 147) 145. The modified cell or nucleic acid of any one of items 126 to 144, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 1000 times greater than the ligand binds to the FKBP12 polypeptide domain. (Item 148) 148. The modified cell or nucleic acid of any one of items 126 to 147, wherein the FKBP12 variant polypeptide comprises an amino acid substitution at amino acid residue 36. (Item 149) 149. The modified cell or nucleic acid of item 148, wherein the amino acid substitution at position 36 is selected from the group consisting of valine, leucine, isoleucine, and alanine. (Item 150) 145. The modified cell or nucleic acid of any one of items 126 to 144, wherein the FKBP12 variant polypeptide region is an FKBP12v36 polypeptide region. (Item 151) 148. The modified cell of any one of items 145 to 147, wherein the ligand is rimizuside. (Item 152) 148. The modified cell of any one of items 145 to 147, wherein the ligand is AP20187. (Item 153) 153. The modified cell of any one of paragraphs 126 to 152, wherein the FRB variant polypeptide binds to a C7 rapalog. (Item 154) 154. The modified cell of any one of items 126 to 153, wherein the FRB variant polypeptide comprises an amino acid substitution at position T2098 or W2101. (Item 155) 155. The modified cell of any one of items 126 to 154, wherein the FRB variant polypeptide region is selected from the group consisting of KLW(T2098L)(FRBL), KTF(W2101F), and KLF(T2098L,W2101F). (Item 156) The FRB variant polypeptide region comprises FRB L 156. The modified cell of any one of items 126 to 155, (Item 157) 157. The modified cell of any one of paragraphs 126 to 156, wherein the FRB variant polypeptide region binds to a rapalog selected from the group consisting of So,p-dimethoxyphenyl (DMOP)-rapamycin, R-isopropoxyrapamycin, C7-isobutyloxyrapamycin, and S-butanesulfonamide rapamycin. (Item 158) 158. The modified cell or nucleic acid of any one of Items 126 to 157, wherein the cell or the nucleic acid comprises a polynucleotide encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises (i) a transmembrane region, (ii) a T cell activation molecule, and (iii) an antigen recognition portion. (Item 159) 159. The modified cell or nucleic acid of any one of paragraphs 158, wherein the T cell activation molecule is selected from the group consisting of an ITAM-containing molecule that confers signal 1, a Syk polypeptide, a ZAP70 polypeptide, a CD3ζ polypeptide, and an Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. (Item 160) 160. The modified cell or nucleic acid of any one of items 158 or 159, wherein the antigen recognition portion is a single-chain variable fragment. (Item 161) 161. The modified cell or nucleic acid according to any one of items 158 to 160, wherein the transmembrane domain is a CD8 transmembrane domain. (Item 162) 162. The modified cell or nucleic acid of any one of paragraphs 158 to 161, wherein the antigen recognition moiety binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, MuclMuc1, Mucl, ROR1, mesothelin, GD2, CD123, Mucl6, CD33, CD38, and CD44v6. (Item 163) 163. The modified cell or nucleic acid of any one of paragraphs 158 to 162, wherein the antigen recognition moiety binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, MuclMuc1, Mucl, ROR1, mesothelin, GD2, CD123, Mucl6, CD33, CD38, and CD44v6. (Item 164) 158. The modified cell or nucleic acid of any one of items 126 to 157, wherein the cell comprises a polynucleotide encoding a recombinant T cell receptor, wherein the recombinant T cell receptor binds to an antigenic polypeptide selected from the group consisting of PRAME, Bob-1, and NY-ESO-1. (Item 165) 165. The modified cell or nucleic acid of any one of paragraphs 126-164, wherein the pro-apoptotic polypeptide is selected from the group consisting of caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, or caspase 14, FADD(DED), APAF1(CARD), CRADD / RAIDD CARD), ASC(CARD), Bax, Bak, Bcl-xL, Bcl-2, RIPK3, and RIPK1-RHIM. (Item 166) 166. The modified cell or nucleic acid of any one of items 126 to 165, wherein the pro-apoptotic polypeptide is a caspase polypeptide. (Item 167) 167. The modified cell or nucleic acid of item 166, wherein the pro-apoptotic polypeptide is a caspase-9 polypeptide. (Item 168) 168. The nucleic acid or cell of item 167, wherein the caspase-9 polypeptide lacks a CARD domain. (Item 169) 169. The modified cell or nucleic acid of any one of items 167 or 168, wherein the caspase polypeptide comprises the amino acid sequence of SEQ ID NO: 300. (Item 170) 169. The modified cell or nucleic acid of any one of items 166 to 168, wherein the caspase polypeptide is a modified caspase-9 polypeptide comprising an amino acid substitution selected from the group consisting of catalytically active caspase variants in Table 5 or Table 6. (Item 171) 171. The modified cell or nucleic acid of item 170, wherein the caspase polypeptide is a modified caspase-9 polypeptide comprising an amino acid sequence selected from the group consisting of D330A, D330E, and N405Q. (Item 172) 172. The modified cell or nucleic acid of any one of items 126 to 171, wherein the truncated MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 214 or 305, or a functional fragment thereof. (Item 173) 172. The modified cell or nucleic acid of any one of items 126 to 171, wherein the MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 282 or a functional fragment thereof. (Item 174) 174. The modified cell or nucleic acid of any one of items 126 to 173, wherein the cytoplasmic CD40 polypeptide has the amino acid sequence of SEQ ID NO: 216 or a functional fragment thereof. (Item 175) a) the chimeric pro-apoptotic polypeptide comprises a caspase-9 polypeptide lacking a CARD domain and an FKBP12v36 polypeptide region; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and an FRB L a polypeptide domain and an FKBP12 polypeptide domain, 127. The modified cell of item 126. (Item 176) a) the chimeric pro-apoptotic polypeptide comprises a caspase-9 polypeptide lacking a CARD domain and an FKBP12v36 polypeptide region; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain, a CD40 cytoplasmic polypeptide region lacking an extracellular domain, an FRB L a polypeptide domain and an FKBP12 polypeptide domain, 127. The modified cell of item 126. (Item 177) a) the chimeric pro-apoptotic polypeptide comprises a caspase-9 polypeptide lacking a CARD domain and an FKBP12v36 polypeptide region; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and an FRB L a polypeptide domain and an FKBP12 polypeptide domain, Item 142. The nucleic acid of item 142. (Item 178) a) the chimeric pro-apoptotic polypeptide comprises a caspase-9 polypeptide lacking a CARD domain and an FKBP12v36 polypeptide region; and b) the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain, a CD40 cytoplasmic polypeptide region lacking an extracellular domain, an FRB L a polypeptide domain and an FKBP12 polypeptide domain, Item 142. The nucleic acid according to item 142. (Item 179) Item 180: The modified cell of any one of Items 126 to 131, 141, or 142 to 176, wherein the cell is a T cell, a tumor-infiltrating lymphocyte, a NK-T cell, or a NK cell. 177. The modified cell of any one of paragraphs 126-131 or 141-176, wherein the cell is a T cell, an NK-T cell, or an NK cell. (Item 181) 177. The modified cell of any one of items 126 to 131 or 141 to 176, wherein the cell is a T cell. (Item 182) 177. The modified cell of any one of paragraphs 126 to 131 or 141 to 176, wherein the cell is a primary T cell. (Item 183) 177. The modified cell of any one of items 126 to 131 or 141 to 176, wherein the cell is a cytotoxic T cell. (Item 184) 177. The modified cell of any one of paragraphs 126-131 or 141-176, wherein the cell is selected from the group consisting of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphocytic hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, melanoma cells, tumor-infiltrating lymphocytes, natural killer cells, natural killer T cells, or T cells. (Item 185) 177. The modified cell of any one of items 126 to 131 or 141 to 176, wherein the T cell is a helper T cell. (Item 186) The cells are obtained from or prepared from bone marrow. The modified cells of any one of items 126-131 or 141-176. (Item 187) 177. The modified cells of any one of paragraphs 126-131 or 141-176, wherein the cells are obtained or prepared from umbilical cord blood. (Item 188) 177. The modified cells of any one of paragraphs 126-131 or 141-176, wherein the cells are obtained or prepared from peripheral blood. (Item 189) 177. The modified cell of any one of paragraphs 126-131 or 141-176, wherein the cell is obtained from or prepared from peripheral blood mononuclear cells. (Item 190) 177. The modified cell of any one of items 126 to 131 or 141 to 176, wherein the cell is a human cell. (Item 191) 191. The modified cell of any one of paragraphs 126-131, 141-176 or 179-190, wherein the modified cell is transduced or transfected in vivo. (Item 192) 190. The modified cell of any one of paragraphs 126-131, 141-174, or 179-190, wherein the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistic (e.g., gene gun with Au particles), lipid transfection, polymer transfection, nanoparticles, or polyplexes. (Item 193) a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: i) a pro-apoptotic polypeptide region; and ii) an FKBP12 variant polypeptide region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide comprises: i) an FRB polypeptide or FRB variant polypeptide region; ii) an FKBP12 polypeptide region; and iii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, or a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising A kit or composition comprising a nucleic acid comprising: (Item 194) 194. The kit or composition of claim 193, wherein the chimeric costimulatory polypeptide comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain. (Item 195) 195. The kit or composition of any one of items 193 to 194, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain. (Item 196) 196. The kit or composition of any one of items 193 to 195, further comprising a third polynucleotide, wherein the third polynucleotide encodes a heterologous protein. (Item 197) 197. The kit or composition of item 196, wherein the heterologous protein is a chimeric antigen receptor. (Item 198) Item 199. The kit or composition of Item 196, wherein the heterologous protein is a recombinant TCR. Item 194. The kit or composition according to Item 193, wherein the nucleic acid is a nucleic acid according to any one of Items 132 to 139, 142 to 174, or 177 to 178. (Item 200) 200. The kit or composition of any one of items 193 to 199, further comprising a third polynucleotide, wherein the third polynucleotide encodes a heterologous protein. (Item 201) 201. The kit or composition of claim 200, wherein the heterologous protein is a chimeric antigen receptor. (Item 202) Item 203. The kit or composition of Item 200, wherein the heterologous protein is a recombinant TCR. 200. The kit or composition of any one of items 194 to 199, comprising a virus, wherein the virus comprises the first polynucleotide and the second polynucleotide. (Item 204) 203. The kit or composition of any one of items 199 to 202, comprising a virus, wherein the virus comprises the first polynucleotide, the second polynucleotide, and the third polynucleotide. (Item 205) 203. The kit or composition of any one of items 200 to 202, comprising a virus, wherein the virus comprises the first polynucleotide and the third polynucleotide. (Item 206) 203. The kit or composition of any one of items 200 to 202, comprising a virus, wherein the virus comprises the second polynucleotide and the third polynucleotide. (Item 207) 203. The kit or composition of any one of items 200 to 202, comprising a virus, wherein the virus comprises the first polynucleotide, the second polynucleotide, and the third polynucleotide. (Item 208) 1. A method for expressing a chimeric pro-apoptotic polypeptide and a chimeric costimulatory polypeptide, comprising: a) the chimeric pro-apoptotic polypeptide comprises: i) a pro-apoptotic polypeptide region; and ii) an FKBP12 variant polypeptide region; and b) the chimeric costimulatory polypeptide comprises: i) an FRB or FRB variant polypeptide region; ii) an FKBP12 polypeptide region; and iii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, or a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; the method comprising contacting a cell with the nucleic acid of any one of items 132-139, 142-174, or 177-178 under conditions such that the nucleic acid is incorporated into the cell, whereby the cell expresses the chimeric pro-apoptotic polypeptide and the chimeric costimulatory polypeptide from the incorporated nucleic acid. (Item 209) 209. The method of claim 208, wherein the nucleic acid is contacted with the cell ex vivo. (Item 210) 209. The method of claim 208, wherein the nucleic acid is contacted with the cell in vivo. (Item 211) 1. A method for stimulating an immune response in a subject, the method comprising: a) transplanting the modified cells according to any one of items 126 to 131, 141 to 176, or 179 to 192 into the subject; and b) after (a), administering an effective amount of rapamycin or a rapalog that binds to the FRB polypeptide or FRB variant polypeptide region of the chimeric stimulating polypeptide to stimulate a cell-mediated immune response; A method that encompasses (Item 212) 192. A method for administering a ligand to a subject who has undergone cell therapy using modified cells, the method comprising administering rapamycin or a rapalog to the subject, wherein the modified cells comprise the modified cells of any one of paragraphs 126-131, 141-176, or 179-192. (Item 213) 1. A method for modulating the activity of transplanted, modified cells in a subject, the method comprising: a) implanting the modified cells according to any one of items 126 to 131, 141 to 176, or 179 to 192; and b) after (a), administering an effective amount of rapamycin or a rapalog that binds to the FRB or FRB variant polypeptide region of the chimera stimulating polypeptide to stimulate the activity of the transplanted, modified cells; A method that encompasses (Item 214) 1. A method for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, the method comprising: a) transplanting into the subject an effective amount of modified cells; wherein the modified cells comprise the modified cells of any one of items 126-131, 141-176, or 179-192, wherein the modified cells comprise a chimeric antigen receptor comprising an antigen recognition portion that binds to the target antigen; and b) following a), administering an effective amount of rapamycin or a rapalog that binds to the FRB polypeptide or FRB variant region of the chimera stimulating polypeptide to reduce the number or concentration of target antigens or target cells in the subject; A method that encompasses (Item 215) 215. The method of claim 214, wherein the target antigen is a tumor antigen. (Item 216) 1. A method for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, the method comprising: a) administering to the subject an effective amount of modified cells, wherein the modified cells comprise the modified cells of any one of items 126-131, 141-176, or 179-192, wherein the modified cells comprise a recombinant T cell receptor that recognizes and binds to the target antigen; and b) following a), administering an effective amount of rapamycin or a rapalog that binds to the FRB or FRB variant polypeptide region of the chimeric stimulating polypeptide to reduce the number or concentration of target antigens or target cells in the subject; A method that encompasses (Item 217) 1. A method for reducing tumor size in a subject, the method comprising: a) administering to the subject the modified cells of any one of items 126-131, 141-176, or 179-192, wherein the cells comprise a chimeric antigen receptor comprising an antigen recognition portion that binds to an antigen on the tumor; and b) following a), administering an effective amount of rapamycin or a rapalog that binds to the FRB or FRB variant polypeptide region of the chimera stimulating polypeptide to reduce the size of the tumor in the subject; A method that encompasses (Item 218) 218. The method of any one of items 214 to 217, comprising measuring the number or concentration of target cells in a first sample obtained from the subject before administration of a second ligand, measuring the number or concentration of target cells in a second sample obtained from the subject after administration of the ligand, and determining an increase or decrease in the number or concentration of target cells in the second sample compared to the number or concentration of target cells in the first sample. (Item 219) 219. The method of claim 218, wherein the concentration of target cells in the second sample is reduced compared to the concentration of target cells in the first sample. (Item 220) 219. The method of claim 218, wherein the concentration of target cells in the second sample is increased compared to the concentration of target cells in the first sample. (Item 221) 221. The method of any one of items 211 to 220, wherein the subject has undergone stem cell transplantation prior to or concurrently with administration of the modified cells. (Item 222) At least 1 x 10 6 222. The method of any one of items 211 to 221, wherein the transduced or transfected modified cells are administered to the subject. (Item 223) At least 1 x 10 7 222. The method of any one of items 211 to 221, wherein the transduced or transfected modified cells are administered to the subject. (Item 224) At least 1 x 10 8 222. The method of any one of items 211 to 221, wherein the modified cells are administered to the subject. (Item 225) 225. The method according to any one of items 211 to 224, wherein the FKBP12 variant polypeptide region is FKBP12v36, and the ligand that binds to the FKBP12 variant polypeptide region is AP1903. (Item 226) 1. A method for modulating survival of transplanted modified cells in a subject, the method comprising: a) administering to the subject the modified cells of any one of items 126 to 131, 141 to 176, or 179 to 192; and b) after (a), administering to the subject a ligand that binds to the FKBP12 variant polypeptide region of the chimeric pro-apoptotic polypeptide in an amount effective to kill less than 95% of the modified cells that express the chimeric pro-apoptotic polypeptide; A method that encompasses (Item 227) 226. The method of any one of items 211 to 225, further comprising, after (b), administering to the subject a ligand that binds to the FKBP12 variant polypeptide region of the chimeric apoptosis-promoting polypeptide in an amount effective to kill less than 95% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 228) 228. The method of any one of paragraphs 226 or 227, wherein the ligand that binds to the FKBP12 variant polypeptide region is administered in an amount effective to kill less than 40% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 229) 228. The method of any one of paragraphs 226 or 227, wherein the ligand that binds to the FKBP12 variant polypeptide region is administered in an amount effective to kill less than 50% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 230) 231. The method of claim 226 or 227, wherein the ligand that binds to the FKBP12 variant polypeptide region is administered in an amount effective to kill fewer than 60% of the modified cells that express the chimeric pro-apoptotic polypeptide. 232. The method of claim 226 or 227, wherein the ligand that binds to the FKBP12 variant polypeptide region is administered in an amount effective to kill fewer than 70% of the modified cells that express the chimeric pro-apoptotic polypeptide. 233. The method of claim 226 or 227, wherein the ligand that binds to the FKBP12 variant polypeptide region is administered in an amount effective to kill less than 90% of the modified cells that express the chimeric pro-apoptotic polypeptide. 228. The method of any one of paragraphs 226 or 227, wherein the ligand that binds to the FKBP12 variant polypeptide region is administered in an amount effective to kill at least 90% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 234) 228. The method of any one of paragraphs 226 or 227, wherein the ligand that binds to the FKBP12 variant polypeptide region is administered in an amount effective to kill at least 95% of the modified cells that express the chimeric pro-apoptotic polypeptide. (Item 235) The chimeric costimulatory polypeptide may comprise a FRB L 228. The method according to any one of items 226 to 227, comprising a region. (Item 236) 226. The method of any one of items 221 to 225, wherein more than one dose of the ligand is administered to the subject. (Item 237) 237. The method of any one of items 226 to 236, wherein more than one dose of the ligand that binds to the FKBP12 variant polypeptide region is administered to the subject. (Item 238) identifying the presence or absence of a condition in the subject that requires removal of the modified cells from the subject; and administering to the subject a ligand that binds to said FKBP12 variant polypeptide region, maintaining a subsequent dosage of said ligand to the subject, or adjusting a subsequent dosage of said ligand to the subject based on the presence or absence of said condition identified in said subject; The method according to any one of Items 211 to 236, further comprising: (Item 239) receiving information comprising the presence or absence of a condition in the subject that requires the modified cells to be removed from the subject; and administering to the subject a ligand that binds to said FKBP12 variant polypeptide region, maintaining a subsequent dosage of said ligand to the subject, or adjusting a subsequent dosage of said ligand to the subject based on the presence or absence of said condition identified in said subject; The method according to any one of Items 211 to 236, further comprising: (Item 240) identifying the presence or absence of a condition in the subject that requires removal of the modified cells from the subject; and communicating the presence, absence, or stage of the condition identified in the subject to a decision maker who administers to the subject a ligand that binds to said FKBP12 variant polypeptide region, maintains a subsequent dosage of the ligand administered to the subject, or adjusts a subsequent dosage of the ligand administered to the subject based on the presence, absence, or stage of the condition identified in the subject; The method according to any one of Items 211 to 236, further comprising: (Item 241) identifying the presence or absence of a condition in the subject that requires removal of the modified cells from the subject; and communicating instructions to administer to said subject a ligand that binds said FKBP12 variant polypeptide region, maintain a subsequent dosage of said ligand administered to said subject, or adjust a subsequent dosage of said ligand administered to said subject based on the presence, absence, or stage of said condition identified in said subject; The method according to any one of Items 211 to 236, further comprising: (Item 242) 242. The method of any one of items 211 to 241, wherein the subject has cancer. (Item 243) 242. The method of any one of items 211 to 241, wherein the modified cells are delivered to a tumor bed. (Item 244) 244. The method of any one of paragraphs 242 or 243, wherein the cancer is present in the blood or bone marrow of the subject. (Item 245) 242. The method of any one of items 211 to 241, wherein the subject has a blood or bone marrow disorder. (Item 246) 242. The method of any one of items 211 to 241, wherein the subject has been diagnosed with sickle cell anemia or metachromatic leukodystrophy. (Item 247) 242. The method of any one of paragraphs 211 to 241, wherein the patient has been diagnosed with a condition selected from the group consisting of a primary immunodeficiency condition, hemophagocytic lymphohistiocytosis (HLH) or other hemophagocytic condition, an inherited bone marrow failure condition, a hemoglobinopathy, a metabolic condition, and an osteoclast condition. (Item 248) 242. The method of any one of paragraphs 211 to 241, wherein the patient has been diagnosed with a disease or condition selected from the group consisting of severe combined immunodeficiency (SCID), combined immunodeficiency (CID), congenital T-cell deficiency / deficiency, common variable immunodeficiency (CVID), chronic granulomatous disease, IPEX (immunodeficiency, polyendocrinopathy, enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich syndrome, CD40 ligand deficiency, leukocyte adhesion deficiency, DOCA8 deficiency, IL-10 deficiency / IL-10 receptor deficiency, GATA2 deficiency, X-linked lymphoproliferative disorder (XLP), cartilage hair hypoplasia, Schwachman-Diamond syndrome, Diamond-Blackfan anemia, dyskeratosis congenita, Fanconi anemia, congenital neutropenia, sickle cell disease, thalassemia, mucopolysaccharidoses, sphingolipidoses, and osteopetrosis. (Item 249) 1. A nucleic acid comprising a promoter operably linked to a polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: a) the pro-apoptotic polypeptide region; b) an FKBP12-rapamycin binding domain (FRB) polypeptide or an FRB variant polypeptide region; and c) an FKBP12 variant polypeptide region; A nucleic acid comprising: (Item 250) 240. The nucleic acid of item 249, wherein the order of regions (a), (b), and (c) is, from the amino terminus to the carboxyl terminus of the chimeric pro-apoptotic polypeptide, (c), (b), (a). (Item 251) 240. The nucleic acid of item 249, wherein the order of regions (a), (b), and (c) is, from the amino terminus to the carboxyl terminus of the chimeric pro-apoptotic polypeptide, (b), (c), (a). (Item 252) 252. The nucleic acid of any one of items 250 or 251, wherein (b) and (c) are amino-terminal to the pro-apoptotic polypeptide. (Item 253) 252. The nucleic acid of any one of items 250 or 251, wherein (b) and (c) are carboxyl-terminal to the pro-apoptotic polypeptide. (Item 254) 255. The nucleic acid of any one of Items 259 to 253, wherein the chimeric apoptosis-promoting polypeptide further comprises a linker polypeptide between regions (a), (b), and (c). 255. The nucleic acid of any one of items 249 to 254, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 100 times greater than the ligand binds to the wild-type FKBP12 polypeptide domain. (Item 256) 255. The nucleic acid of any one of items 249 to 254, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 500 times greater than the ligand binds to the wild-type FKBP12 polypeptide domain. (Item 257) 255. The nucleic acid of any one of items 249 to 254, wherein the FKBP12 variant polypeptide domain binds to the ligand with an affinity that is at least 1000 times greater than the ligand binds to the wild-type FKBP12 polypeptide domain. (Item 258) 258. The nucleic acid according to any one of items 249 to 257, wherein the FKBP12 variant comprises an amino acid substitution at amino acid residue 36. (Item 259) 259. The nucleic acid of item 258, wherein the amino acid substitution at position 36 is selected from the group consisting of valine, leucine, isoleucine and alanine. (Item 260) 259. The nucleic acid of any one of Items 249 to 259, wherein the FKBP12 variant polypeptide region is an FKBP12v36 polypeptide region. (Item 261) 262. The nucleic acid according to any one of claims 255 to 260, wherein the ligand is rimizuside. 261. The nucleic acid according to any one of Items 255 to 260, wherein the ligand is AP20187 or N1510. (Item 263) 263. The nucleic acid of any one of items 249 to 262, wherein the FRB variant polypeptide binds to a C7 rapalog. (Item 264) 264. The nucleic acid of any one of items 249 to 263, wherein the FRB variant polypeptide comprises an amino acid substitution at position T2098 or W2101. (Item 265) 265. The nucleic acid of any one of items 249 to 264, wherein the FRB variant polypeptide region is selected from the group consisting of KLW(T2098L)(FRBL), KTF(W2101F), and KLF(T2098L, W2101F). (Item 266) The FRB variant polypeptide region comprises FRB L 266. The nucleic acid according to any one of Items 249 to 265, (Item 267) 267. The nucleic acid of any one of paragraphs 249 to 266, wherein the FRB variant polypeptide region binds to a rapalog selected from the group consisting of So,p-dimethoxyphenyl (DMOP)-rapamycin, R-isopropoxyrapamycin, C7-isobutyloxyrapamycin, and S-butanesulfonamide rapamycin. (Item 268) 268. The nucleic acid of any one of items 249 to 267, wherein the promoter is operably linked to a second polynucleotide, wherein the second polynucleotide encodes a heterologous protein. (Item 269) 269. The nucleic acid of item 268, wherein the heterologous protein is a chimeric antigen receptor. (Item 270) 269. The nucleic acid of item 268, wherein the heterologous protein is a recombinant TCR. (Item 271) 269. The nucleic acid according to any one of Items 249 to 268, wherein the nucleic acid further comprises a polynucleotide encoding a linker polypeptide between the polynucleotide encoding the chimeric apoptosis-promoting polypeptide and the second polynucleotide, wherein the linker polypeptide separates the translation products of the first polynucleotide and the second polynucleotide during or after translation. (Item 272) 272. The nucleic acid of claim 271, wherein the linker polypeptide is a 2A polypeptide. (Item 273) 273. The nucleic acid of any one of Items 269 or 271 to 272, wherein the chimeric antigen receptor comprises (i) a transmembrane domain, (ii) a T cell activation molecule, and (iii) an antigen recognition portion. (Item 274) 274. The nucleic acid of claim 273, wherein the T cell activation molecule is selected from the group consisting of an ITAM-containing molecule that confers signal 1, a Syk polypeptide, a ZAP70 polypeptide, a CD3ζ polypeptide, and an Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. (Item 275) 274. The nucleic acid of claim 273, wherein the T cell activation molecule is selected from the group consisting of an ITAM-containing molecule that confers signal 1, a CD3ζ polypeptide, and an Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. (Item 276) 276. The nucleic acid according to any one of Items 273 to 275, wherein the antigen recognition portion is a single-chain variable fragment. (Item 277) 277. The nucleic acid according to any one of Items 273 to 276, wherein the transmembrane domain is a CD8 transmembrane domain. (Item 278) 278. The nucleic acid of any one of Items 273 to 277, wherein the antigen recognition portion binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, MuclMuc1, Mucl, ROR1, mesothelin, GD2, CD123, Mucl6, CD33, CD38, and CD44v6. (Item 279) 278. The nucleic acid of any one of Items 273 to 277, wherein the antigen recognition portion binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, MuclMuc1, Mucl, ROR1, mesothelin, GD2, CD123, Mucl6, CD33, CD38, and CD44v6. (Item 280) 273. The nucleic acid of any one of Items 270 to 272, wherein the recombinant T cell receptor binds to an antigenic polypeptide selected from the group consisting of PRAME, Bob-1, and NY-ESO-1. (Item 281) 281. The nucleic acid of any one of items 249 to 280, further comprising a polynucleotide encoding a chimeric costimulatory polypeptide comprising a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain. (Item 282) 282. The nucleic acid of claim 281, wherein the chimeric costimulatory polypeptide further comprises a CD40 cytoplasmic polypeptide lacking the CD40 extracellular domain. (Item 283) 283. The nucleic acid of any one of Items 281 to 282, wherein the chimeric costimulatory polypeptide further comprises a membrane targeting region. (Item 284) 284. The nucleic acid of item 283, wherein the membrane targeting region comprises a myristoylation region. (Item 285) 285. The nucleic acid according to any one of Items 282 to 284, wherein the truncated MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 214 or 305, or a functional fragment thereof. (Item 286) 285. The nucleic acid according to any one of Items 282 to 284, wherein the MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 282 or a functional fragment thereof. (Item 287) 287. The nucleic acid of any one of Items 282 to 286, wherein the cytoplasmic CD40 polypeptide has the amino acid sequence of SEQ ID NO: 216 or a functional fragment thereof. (Item 288) 288. The nucleic acid of any one of Items 249 to 287, wherein the pro-apoptotic polypeptide is selected from the group consisting of caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, or caspase 14, FADD(DED), APAF1(CARD), CRADD / RAIDD (CARD), ASC(CARD), Bax, Bak, Bcl-xL, Bcl-2, RIPK3, and RIPK1-RHIM. (Item 289) 289. The nucleic acid according to any one of items 249 to 288, wherein the apoptosis-promoting polypeptide is a caspase polypeptide. (Item 290) 289. The nucleic acid of item 289, wherein the pro-apoptotic polypeptide is a caspase-9 polypeptide. (Item 291) 291. The nucleic acid of a cell according to claim 290, wherein the caspase-9 polypeptide lacks a CARD domain. (Item 292) 291. The nucleic acid of any one of items 289 or 290, wherein the caspase polypeptide comprises the amino acid sequence of SEQ ID NO: 300. (Item 293) 291. The nucleic acid of any one of items 289 to 290, wherein the caspase polypeptide is a modified caspase-9 polypeptide comprising an amino acid substitution selected from the group consisting of catalytically active caspase variants in Table 5 or Table 6. (Item 294) 295. The nucleic acid of item 294, wherein the caspase polypeptide is a modified caspase-9 polypeptide comprising an amino acid sequence selected from the group consisting of D330A, D330E, and N405Q. (Item 295) The chimeric pro-apoptotic polypeptide comprises a caspase-9 polypeptide lacking a CARD domain, an FKBP12v36 polypeptide region; and an FRB L 295. The nucleic acid of any one of items 249 to 294, comprising a polypeptide region. (Item 296) 296. A chimeric apoptosis-promoting polypeptide encoded by a nucleic acid according to any one of items 249 to 295. (Item 297) 296. A modified cell transfected or transduced with a nucleic acid according to any one of items 249 to 295. (Item 298) 298. The modified cell of Item 297, wherein the modified cell comprises a polynucleotide encoding a chimeric antigen receptor. (Item 299) 298. The modified cell of claim 297, wherein the modified cell comprises a polynucleotide encoding a recombinant TCR. (Item 300) 299. The modified cell of item 298, wherein the chimeric antigen receptor comprises (i) a transmembrane domain, (ii) a T cell activation molecule, and (iii) an antigen recognition portion. (Item 301) 301. The modified cell of paragraph 300, wherein the T cell activation molecule is selected from the group consisting of an ITAM-containing molecule that confers signal 1, a Syk polypeptide, a ZAP70 polypeptide, a CD3ζ polypeptide, and an Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. (Item 302) 301. The modified cell of paragraph 300, wherein the T cell activation molecule is selected from the group consisting of an ITAM-containing molecule that confers signal 1, a CD3ζ polypeptide, and an Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. (Item 303) 303. The modified cell of any one of items 300 to 302, wherein the antigen recognition moiety is a single-chain variable fragment. (Item 304) 304. The modified cell of any one of items 300 to 303, wherein the transmembrane domain is a CD8 transmembrane domain. (Item 305) 305. The modified cell of any one of paragraphs 300 to 304, wherein the antigen recognition moiety binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, Muc1Muc1, Muc1, ROR1, mesothelin, GD2, CD123, Muc16, CD33, CD38, and CD44v6. (Item 306) 305. The modified cell of any one of paragraphs 300 to 304, wherein the antigen recognition moiety binds to an antigen selected from the group consisting of an antigen on a tumor cell, an antigen on a cell involved in a hyperproliferative disease, a viral antigen, a bacterial antigen, CD19, PSCA, Her2 / Neu, PSMA, Muc1Muc1, Muc1, ROR1, mesothelin, GD2, CD123, Muc16, CD33, CD38, and CD44v6. (Item 307) 300. The modified cell of claim 299, wherein the recombinant T cell receptor binds to an antigenic polypeptide selected from the group consisting of PRAME, Bob-1, and NY-ESO-1. (Item 308) 298. The modified cell of Item 297, wherein the modified cell comprises a polynucleotide encoding a MyD88 polypeptide or a truncated MyD88 polypeptide region lacking the TIR domain. (Item 309) 309. The modified cell of claim 308, wherein the modified cell comprises a polynucleotide encoding a chimeric costimulatory polypeptide, wherein the chimeric costimulatory polypeptide encodes a MyD88 polypeptide or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide lacking the CD40 extracellular domain. (Item 310) 300. The modified cell of any one of items 308 to 309, wherein the truncated MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 214 or 305, or a functional fragment thereof. (Item 311) 311. The modified cell of any one of items 308 to 310, wherein the MyD88 polypeptide has the amino acid sequence of SEQ ID NO: 282 or a functional fragment thereof. (Item 312) 312. The modified cell of any one of items 309 to 311, wherein the cytoplasmic CD40 polypeptide has the amino acid sequence of SEQ ID NO: 216 or a functional fragment thereof. (Item 313) 313. The modified cell of any one of items 297 to 312, wherein the cell is a T cell, a tumor-infiltrating lymphocyte, an NK-T cell, or an NK cell. (Item 314) 313. The modified cell of any one of items 297 to 312, wherein the cell is a T cell, an NK-T cell, or an NK cell. (Item 315) 313. The modified cell of any one of items 297 to 312, wherein the cell is a T cell. (Item 316) 313. The modified cell of any one of items 297 to 312, wherein the cell is a primary T cell. (Item 317) 313. The modified cell of any one of items 297 to 312, wherein the cell is a cytotoxic T cell. (Item 318) 313. The modified cell of any one of items 297 to 312, wherein the cell is selected from the group consisting of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphocytic hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, melanoma cells, tumor-infiltrating lymphocytes, natural killer cells, natural killer T cells, or T cells. (Item 319) 313. The modified cell of any one of items 297 to 312, wherein the T cell is a helper T cell. (Item 320) 313. The modified cells of any one of items 297 to 312, wherein the cells are obtained or prepared from bone marrow. (Item 321) 313. The modified cells of any one of paragraphs 297 to 312, wherein the cells are obtained or prepared from umbilical cord blood. (Item 322) 313. The modified cells of any one of items 297 to 312, wherein the cells are obtained or prepared from peripheral blood. (Item 323) 313. The modified cell of any one of items 297 to 312, wherein the cell is obtained or prepared from peripheral blood mononuclear cells. (Item 324) 324. The modified cell of any one of items 297 to 323, wherein the cell is a human cell. (Item 325) 325. The modified cell of any one of items 297 to 324, wherein the modified cell is transduced or transfected in vivo. (Item 326) 326. The modified cell of any one of paragraphs 297-325, wherein the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistic (e.g., gene gun with Au particles), lipid transfection, polymer transfection, nanoparticles, or polyplexes. (Item 327) 1. A kit or composition comprising a nucleic acid comprising a polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: a) the pro-apoptotic polypeptide region; b) an FKBP12-rapamycin binding domain (FRB) polypeptide or an FRB variant polypeptide region; and c) an FKBP12 variant polypeptide region; A kit or composition comprising: (Item 328) 328. The kit or composition of item 327, wherein the FKBP12 variant comprises an amino acid substitution at amino acid residue 36. (Item 329) 329. The kit or composition of item 328, wherein the amino acid substitution at position 36 is selected from the group consisting of valine, leucine, isoleucine, and alanine. (Item 330) 320. The kit or composition according to any one of items 327 to 329, wherein the FKBP12 variant polypeptide region is an FKBP12v36 polypeptide region. (Item 331) 331. The kit or composition of any one of items 327 to 330, wherein the FKBP12 variant polypeptide region binds to rimizuside. (Item 332) 332. The kit or composition of any one of items 327 to 331, wherein the FKBP12 variant polypeptide region binds to AP20187 or AP1510. (Item 333) 333. The kit or composition of any one of items 327 to 332, wherein the FRB variant polypeptide binds to a C7 rapalog. (Item 334) 334. The kit or composition of any one of items 327 to 333, wherein the FRB variant polypeptide comprises an amino acid substitution at position T2098 or W2101. (Item 335) 335. The kit or composition of any one of items 327 to 334, wherein the FRB variant polypeptide region is selected from the group consisting of KLW(T2098L)(FRBL), KTF(W2101F), and KLF(T2098L, W2101F). (Item 336) The FRB variant polypeptide region comprises FRB L The kit or composition according to any one of items 327 to 335, (Item 337) 337. The kit or composition of any one of paragraphs 327 to 336, wherein the FRB variant polypeptide region binds to a rapalog selected from the group consisting of So,p-dimethoxyphenyl (DMOP)-rapamycin, R-isopropoxyrapamycin, C7-isobutyloxyrapamycin, and S-butanesulfonamidorapamycin. (Item 338) The kit or composition according to any one of Items 327 to 337, wherein the nucleic acid is a nucleic acid according to any one of Items 249 to N41. (Item 339) 1. A method for expressing a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises: a) the pro-apoptotic polypeptide region; b) an FRB or FRB variant polypeptide region; and c) an FKBP12 variant polypeptide region; 296. The method comprises contacting a cell with the nucleic acid of any one of items 249 to 295 under conditions in which the nucleic acid is incorporated into the cell, whereby the cell expresses the chimeric apoptosis-promoting polypeptide and the chimeric costimulatory polypeptide from the incorporated nucleic acid. (Item 340) 339. The method of claim 339, wherein the nucleic acid is contacted with the cell ex vivo. (Item 341) 339. The method of claim 339, wherein the nucleic acid is contacted with the cell in vivo. (Item 342) 1. A method for modulating survival of transplanted modified cells in a subject, the method comprising: a) transplanting the modified cells according to any one of items 297 to 326 into the subject; and b) after (a), administering to the subject: i) a first ligand that binds to the FRB or FRB variant polypeptide region of the chimeric pro-apoptotic polypeptide; or ii) a second ligand that binds to the FKBP12 variant polypeptide region of the chimeric pro-apoptotic polypeptide; administering wherein the first ligand or the second ligand is administered in an amount effective to kill at least 30% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 343) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 40% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 344) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 50% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 345) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 60% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 346) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 70% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 347) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 80% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 348) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 90% of the modified cells expressing the chimeric apoptosis-promoting polypeptide. (Item 349) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 95% of the modified cells expressing the chimeric apoptosis-promoting polypeptide. (Item 350) 343. The method of claim 342, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 99% of the modified cells expressing the chimeric apoptosis-promoting polypeptide. (Item 351) 1. A method for administering a first ligand or a second ligand to a subject who has undergone cell therapy using modified cells expressing a chimeric apoptosis-promoting polypeptide, wherein the modified cells comprise the nucleic acid of any one of paragraphs 249 to N45, and wherein the first ligand or the second ligand is administered in an amount effective to kill at least 30% of the modified cells expressing the chimeric apoptosis-promoting polypeptide. (Item 352) 352. The method of claim 351, wherein the first ligand binds to the FRB or FRB variant polypeptide region of the chimeric pro-apoptotic polypeptide and the second ligand binds to the FKBP12 variant polypeptide region of the chimeric pro-apoptotic polypeptide. (Item 353) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 40% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 354) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 50% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 355) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 60% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 356) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 70% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 357) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 80% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 358) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 90% of the modified cells that express the chimeric apoptosis-promoting polypeptide. (Item 359) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 95% of the modified cells expressing the chimeric apoptosis-promoting polypeptide. (Item 360) 353. The method of any one of paragraphs 351-352, wherein the first ligand or the second ligand is administered in an amount effective to kill at least 99% of the modified cells expressing the chimeric apoptosis-promoting polypeptide. (Item 361) 361. The method of any one of items 342 to 360, wherein more than one dose of the ligand is administered to the subject. (Item 362) 362. The method of any one of items 342 to 361, wherein the first ligand is rapamycin or a rapalog. (Item 363) 363. The method of claim 362, wherein the first ligand is a rapalog selected from the group consisting of So,p-dimethoxyphenyl (DMOP)-rapamycin, R-isopropoxyrapamycin, C7-isobutyloxyrapamycin, and S-butanesulfonamidorapamycin. (Item 364) 361. The method of any one of items 342 to 360, wherein the second ligand is rimizuside, AP20187, or AP1510. (Item 365) Item 365. The method of item 364, wherein the second ligand is rimizuside. (Item 366) 366. The method of any one of items 342 to 365, wherein more than one dose of the first ligand or the second ligand is administered. (Item 367) 367. The method of any one of items 342 to 366, wherein both the first ligand and the second ligand are administered. (Item 368) Identifying the presence or absence of a condition in the subject that requires removal of the modified cells from the subject; and administering the first ligand or the second ligand to the subject, or maintaining a subsequent dosage of the first ligand or the second ligand to the subject, or adjusting a subsequent dosage of the first ligand or the second ligand to the subject based on the presence or absence of the condition identified in the subject; The method according to any one of Items 342 to 367, further comprising: (Item 369) Identifying the presence or absence of a condition in the subject that requires removal of the transfected or transduced therapeutic cells from the subject; and determining whether the first ligand or the second ligand should be administered to the subject or whether a dosage of the first ligand or the second ligand subsequently administered to the subject is modulated based on the presence or absence of the condition identified in the subject; The method according to any one of Items 342 to 367, further comprising: (Item 370) receiving information comprising the presence or absence of a condition in the subject that requires removal of the transfected or transduced modified cells from the subject; and administering the first ligand or the second ligand to the subject, maintaining a subsequent dosage of the first ligand or the second ligand to the subject, or adjusting a subsequent dosage of the first ligand or the second ligand to the subject based on the presence or absence of the condition identified in the subject; The method according to any one of Items 342 to 369, further comprising: (Item 371) Identifying the presence or absence of a condition in the subject that requires removal of the transfected or transduced modified cells from the subject; and communicating the presence, absence, or stage of the condition identified in the subject to a decision maker who administers the first ligand or the second ligand to the subject, maintains a subsequent dosage of the first ligand or the second ligand administered to the subject, or adjusts a subsequent dosage of the first ligand or the second ligand administered to the subject based on the presence, absence, or stage of the condition identified in the subject; The method according to any one of Items 342 to 369, further comprising: (Item 372) Identifying the presence or absence of a condition in the subject that requires removal of the transfected or transduced modified cells from the subject; and communicating instructions to administer the first ligand or the second ligand to the subject, maintain a subsequent dosage of the first ligand or the second ligand administered to the subject, or adjust a subsequent dosage of the first ligand or the second ligand administered to the subject based on the presence, absence, or stage of the condition identified in the subject; The method according to any one of Items 342 to 39, further comprising: (Item 373) 369. The method of any one of items 342-369, wherein alloreactive modified cells are present in the subject and the number of alloreactive modified cells is reduced by at least 90% after administration of the first ligand or the second ligand. (Item 374) At least 1 x 10 6 369. The method of any one of paragraphs 342 to 369, wherein a number of transduced or transfected modified cells are administered to the subject. (Item 375) At least 1 x 10 7 374. The method of any one of items 342 to 373, wherein a number of transduced or transfected modified cells are administered to the subject. (Item 376) At least 1 x 10 8 374. The method of any one of items 342 to 373, wherein a number of transduced or transfected modified cells are administered to the subject. (Item 377) Identifying the presence, absence, or stage of graft-versus-host disease in said subject; and administering the first ligand or the second ligand to the subject, maintaining a subsequent dose of the first ligand or the second ligand to the subject, or adjusting a subsequent dose of the first ligand or the second ligand to the subject based on the presence, absence, or stage of the graft-versus-host disease identified in the subject; The method according to any one of Items 342 to 373, further comprising: (Item 378) 10. A method for administering a ligand to a subject who has undergone cell therapy using modified cells, the method comprising administering the ligand to the subject, wherein the modified cells comprise the modified cells of any one of paragraphs 297 to 326, and wherein the ligand binds to an FKBP12 variant polypeptide region. (Item 379) 10. A method for administering rapamycin or a rapalog to a subject who has undergone cell therapy using modified cells, the method comprising administering rapamycin or a rapalog to the subject, wherein the modified cells comprise the modified cells of any one of paragraphs 297-326, wherein the rapamycin or rapalog binds to an FRB polypeptide or an FRB variant polypeptide region. (Item 380) 379. The method of claim 378, wherein the ligand is selected from the group consisting of rapamycin, AP20187, and AP1510. (Item 381) 179. The method of any one of items 342 to 179, wherein at least 30% of cells expressing the chimeric apoptosis-promoting polypeptide are killed within 24 hours of administration of the first ligand or the second ligand. (Item 382) 382. The method of claim 381, wherein at least 40% of cells expressing the chimeric pro-apoptotic polypeptide are killed within 24 hours of administering the first ligand or the second ligand. (Item 383) 382. The method of claim 381, wherein at least 50% of cells expressing the chimeric pro-apoptotic polypeptide are killed within 24 hours of administration of the first ligand or the second ligand. (Item 384) 382. The method of claim 381, wherein at least 60% of cells expressing the chimeric pro-apoptotic polypeptide are killed within 24 hours of administration of the first ligand or the second ligand. (Item 385) 382. The method of claim 381, wherein at least 70% of cells expressing the chimeric pro-apoptotic polypeptide are killed within 24 hours of administration of the first ligand or the second ligand. (Item 386) 382. The method of claim 381, wherein at least 80% of cells expressing the chimeric apoptosis-promoting polypeptide are killed within 24 hours of administration of the first ligand or the second ligand. (Item 387) 382. The method of claim 381, wherein at least 90% of cells expressing the chimeric pro-apoptotic polypeptide are killed within 24 hours of administration of the first ligand or the second ligand. (Item 388) 382. The method of claim 381, wherein at least 95% of cells expressing the chimeric pro-apoptotic polypeptide are killed within 24 hours of administration of the first ligand or the second ligand. (Item 389) 389. The method of any one of items 381-388, wherein at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of cells expressing the chimeric apoptosis-promoting polypeptide are killed within 90 minutes of administration of the first ligand or the second ligand. (Item 390) a) the first ligand is administered to the subject, followed by the second ligand; or b) the second ligand is administered to the subject, followed by the first ligand; The method according to any one of items 342 to 389. (Item 391) 391. The method of any one of items 342 to 390, wherein the subject is a human. (Item 392) 392. The method of any one of items 342 to 391, wherein the subject is selected from the group consisting of non-human primates, mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, birds and fish. [Brief explanation of the drawings]

[0041] [Figure 1A] FIG. 1A illustrates various iCasp9 expression vectors as discussed herein. [Figure 1B] FIG. 1B depicts a representative Western blot of full-length and truncated caspase-9 proteins produced by the expression vector shown in FIG. 1A.

[0042] [Figure 2] FIG. 2 is a schematic representation of the interaction of suicide gene products and CID to trigger apoptosis.

[0043] [Figure 3]Figure 3 is a schematic diagram showing the two-stage regulation of apoptosis. The left part shows rapalog-mediated recruitment of inducible caspase polypeptides to FRBI-modified CAR. The right part shows rimizuside (AP1903)-mediated inducible caspase polypeptides.

[0044] [Figure 4] Figure 4 is a plasmid map of the vector encoding FRBL-modified CD19-MC-CAR and inducible caspase-9: pSFG-iCasp9-2A-CD19-Q-CD28stm-MCz-FRBL2.

[0045] [Figure 5] Figure 5 is a plasmid map of the vector encoding FRBL-modified Her2-MC-CAR and the inducible caspase-9 polypeptide: pSFG-iCasp9-2A-aHer2-Q_CD28stm-mMCz-FRBL2.

[0046] [Figure 6] Figures 6A and 6B provide the results of an assay for the two-stage activation of apoptosis. Figure 6A shows recruitment of inducible caspase-9 polypeptide (iC9) with rapamycin, resulting in a more gradual apoptosis titration. Figure 6B shows complete apoptosis using rimizuside (AP1903).

[0047] [Figure 7] Figure 7 is a plasmid map of the pBP0545 vector, pBP0545.pSFG.iCasp9.2A.Her2scFv.Q.CD8stm.MC-Zeta.

[0048] [Figure 8]Figures 8A-8C illustrate that FRB- or FKBP12-based scaffolds can multimerize signaling domains. Figure 8A. Homodimerization of signaling domains (red bars) can be achieved through heterodimers, such as caspase-9, that bind to an FRB-fused signaling domain on one side and an FKBP12-fused domain on the other. Figure 8B. Dimerization or multimerization of signaling domains via two (left) or more than two (right) tandem copies (chevrons) of FRB. The scaffolds can contain subcellular targeting sequences to localize proteins to the plasma membrane (as shown), nucleus, or organelles. Figure 8C. Same as Figure 8B, but with reversed domain polarity.

[0049] [Figure 9] Figures 9A-9C provide a schematic of iMC-mediated scaffolding of FRBL2.caspase-9. Figure 9A. In the presence of a heterodimeric drug (e.g., rapamycin), FRBL2-linked caspase-9 binds and clusters with the FKBP-modified MyD88 / CD40 (MC) signaling molecule. This clustering effect results in dimerization of FRBL2.caspase-9 and subsequent induction of cell death via the apoptotic pathway. Figure 9B. Similar to panel 9A, except the FKBP and FRB domains are swapped relative to the associated caspase-9 and MC domains. The clustering effect still occurs in the presence of the heterodimeric drug. Figure 9C. Similar to panel 9A, except there is only one FKBP domain bound to the MC. Thus, in the presence of the heterodimer, caspase-9 can no longer cluster, and therefore apoptosis is not induced.

[0050] [Figure 10]Figures 10A-10E provide schematic diagrams of rapalog-induced FRB scaffold-based inducible caspase-9 polypeptides. Figure 10A: Rimizuside homodimerizes FKBPv-linked caspase-9, resulting in caspase-9 dimerization and activation with subsequent induction of cell death via the apoptotic pathway. Figure 10B: Rapalogs heterodimerize FKBPv-linked caspase-9 with FRB-linked caspase-9, resulting in caspase-9 dimerization and cell death. Figures 10C, 10D, and 10E are schematic diagrams illustrating that in the presence of a heterodimeric drug (e.g., a rapalog), two or more FRBL domains act as scaffolds to recruit the binding of FKBPv-linked caspase-9, leading to caspase-9 dimerization and oligomerization and cell death.

[0051] [Figure 11] Figure 11A is a schematic diagram showing activation of apoptosis by dimerization of chimeric FRB-caspase-9 and chimeric FKBP-caspase-9 polypeptides (FRBL-Δcaspase-9 and FKBPv-Δcaspase-9) with rapamycin, and Figure 11 is a line graph showing this activation. Figure 11A. Representative schematic diagram of dimerization of FRB and FKBP12 with rapamycin and activation of apoptosis to fuse the caspase-9 signaling domains together. Figure 11B. Reporter assays were performed in HEK-293T cells transfected with a constitutive SRα-SEAP reporter (pBP046, 1 μg), a fusion of FRBL (L2098) and human Δcaspase-9 (pBP0463, 2 μg), and a fusion of FKBP12 and Δcaspase-9 (pBP0044, 2 μg).

[0052] [Figure 12]Figure 12A is a schematic diagram showing the assembly of FKBP-caspase-9 on an FRB-based scaffold, and Figures 12B and 12C are line graphs. Figure 12A: Schematic of the repeated FRB domain to provide a scaffold for rapamycin (or rapalog)-mediated multimerization of FKBP12-caspase-9 fusion proteins. Figure 12B: Cultures of HEK-293 cells were transfected (via Genejuice (Novagen)) with a constitutive SRα-SEAP reporter plasmid (pBP0046, 1 μg), a fusion of human FKBP12 with human caspase-9 (pBP0044, 2 μg), and an FRB-encoding expression construct containing four copies of FRBL (pBP0725, 2 μg), or a control vector encoding zero or one copy of FRBL. Twenty-four hours after transfection, cells were distributed into 96-well plates, and rapamycin or a derivative rapalog with specificity for mutant FRBL, C7-isopropoxyrapamycin (Liberles et al., 1997), was administered to triplicate wells. Placental SEAP reporter activity was determined 24 hours after drug administration. Figure 12C: Reporter assays were performed as in (B), except that the FRB scaffold was expressed from a construct encoding a repeat FRBL domain with an amino-terminal myristoylation targeting sequence and two (pBP0465) or four (pBP0721) copies of the FRBL domain.

[0053] [Figure 13]Figure 13A is a schematic diagram showing the assembly of FRB-Δcaspase-9 on an FKBP scaffold, and Figure 13B is a line graph showing that assembly. Figure 13A. Schematic of repeated FKBP12 domains generating a scaffold for assembly of FRB-Δcaspase-9 fusion proteins, leading to apoptosis. Figure 13B. Reporter assays were performed as in Figures 12B and 12C in cultures of HEK-293T cells transfected with a constitutive SRα-SEAP reporter (pBP046, 1 μg), a fusion of FRBL (L2098) and CARD domain-deleted human Δcaspase-9 (pBP0463, 2 μg), and an FKBP expression construct containing four tandem copies of FKBP12 (pBP722, 2 μg) or a control vector with one copy of FKBP (pS-SF1E).

[0054] [Figure 14]Figures 14A-14B provide line graphs showing that heterodimerization of the FRBL scaffold with iCaspase 9 induces cell death. Primary T cells derived from three different donors (307, 582, and 584) were transduced with pBP0220-pSFG-iC9.T2A-ΔCD19, pBP0756-pSFG-iC9.T2A-ΔCD19.P2A-FRBL, pBP0755-pSFG-iC9.T2A-ΔCD19.P2A-FRBL2, or pBP0757-pSFG-iC9.T2A-ΔCD19.P2A-FRBL3, which contain iC9 and CD19 markers and zero to three tandem copies of FRBL, respectively. T cells were plated with various concentrations of rapamycin, and after 24 and 48 hours, cell aliquots were harvested, stained with APC-CD19 antibody, and analyzed by flow cytometry. Cells were first gated for live lymphocytes by FSC vs. SSC. Lymphocytes were then plotted as CD19 histograms and subgated for high, moderate, and low expression within the CD19+ gate. Line graphs represent the relative percentage of the total cell population expressing high levels of CD19 normalized to the "0" drug-free control. All data points were performed in duplicate. Figure 14A: Donor 307, 24 hours; Figure 14B: Donor 582, 24 hours; Figure 14C: Donor 584, 24 hours; Figure 14D: Donor 582, 48 hours; Figure 14E: Donor 584, 48 hours.

[0055] [Figure 15]Figures 15A-15C provide line graphs and a schematic diagram showing that rapamycin induces iC9 killing in the presence of tandem FRBL domains. HEK-293 cells were transfected with 1 μg of the SRα-SEAP constitutive reporter plasmid along with either a negative (Neg) control, eGFP (pBP0047), iC9 (iC9 / pBP0044) alone, or iC9 in addition to iMC.FRBL (pBP0655) + anti-HER2.CAR.Fpk2 (pBP0488) or iMC.FRBL2 (pBP0498) + anti-HER2.CAR.Fpk2. Cells were then plated with half-log dilutions of rimizuside or rapamycin and assayed for SEAP as previously described. A decrease in SEAP activity correlates with cell elimination. The schematic represents one possible rapamycin-mediated complex of signaling domains, which leads to caspase-9 clustering and apoptosis. Figure 15A: rimizuside; Figure 15B: rapamycin; Figure 15C: schematic.

[0056] [Figure 16] Figures 16A and 16B are line graphs showing that tandem FKBP scaffolds mediate FRBL2.caspase activation in the presence of a rapalog. Figure 16A. HEK-293 cells were transfected with 1 μg each of the SRα-SEAP reporter plasmid, Δmyr.iMC.2A-anti-CD19.CAR.CD3ζ (pBP0608), and FRBL2.caspase-9 (pBP0467). After 24 hours, transfected cells were harvested and treated with various concentrations of either rimizuside, rapamycin, or the rapalog C7-isopropoxy (IsoP)-rapamycin. After ON incubation, cell supernatants were assayed for SEAP activity as previously described. Figure 16B. Similar to the experiment described in (Figure 16A), except that cells were transfected with membrane-localized (myristoylated) iMC.2A-CD19.CAR.CD3ζ(pBP0609) instead of non-myristoylated Δmyr.iMC.2A-CD19.CAR.CD3ζ(pBP0608).

[0057] [Figure 17]Figures 17A-17E provide line graphs and FACs analysis results showing that the iMC "switch," FKBP2.MyD88.CD40, creates a scaffold for FRBL2.Caspase-9 in the presence of rapamycin and induces cell death. Figure 17A. Primary T cells (two donors) were transduced with γ-RV, SFG-ΔMyr.iMC.2A-CD19 (derived from pBP0606), and SFG-FRBL2.Caspase-9.2AQ.8stm.ζ (derived from pBP0668). Cells were plated with 5-fold dilutions of rapamycin. 24 hours later, cells were harvested and analyzed by flow cytometry for iMC (anti-CD19-APC), caspase-9 (anti-CD34-PE) expression, and T cell identity (anti-CD3-PerCPCy5.5). Cells were first gated for lymphocyte morphology by FSC vs. SSC, followed by CD3 expression (approximately 99% of the lymphocytes). CD3+ lymphocytes were plotted for CD19 (Δmyr.iMC.2A-CD19) vs. CD34 (FRBL2.caspase9.2AQ.8stm.ζ) expression. To normalize the gated population, the percentage of CD34+CD19+ cells was divided by the percentage of CD19+CD34- cells in each sample as an internal control. These values ​​were then normalized to drug-free wells for each transduction, which were set to 100%. Similar analysis was applied to high-, medium-, and low-expressing cells within the CD34+CD19+ gate. (Figure 17B.) Representative examples of how cells were gated for high, medium, and low expression. (Figure 17C.) Representative scatter plots of the final CD34 vs. CD19 gate. As rapamycin increased, the % CD34+CD19+ cells decreased, indicating cell elimination. (Figures 17D and 17E). T cells from a single donor were transduced with ΔMyr.iMC.2A-CD19 (pBP0606) or FRBL2.caspase9.2AQ.8stm.ζ (pBP0668). Cells were plated in IL-2-containing medium with various amounts of rapamycin for 24 or 48 hours.Cells were then harvested and analyzed as described above.

[0058] [Figure 18] Figure 18. Plasmid map of pBP0044:pSH1-iCaspase9wt.

[0059] [Figure 19] Figure 19. Plasmid map of pBP0463--pSH1-Fpk-Fpk'.LS.Fpk''.Fpk'''.LS.HA.

[0060] [Figure 20] Figure 20. Plasmid map of pBP0725--pSH1-FRBl.FRBl'.LS.FRBl''.FRBl'''.

[0061] [Figure 21] Figure 21. Plasmid map of pBP0465--pSH1-M-FRBl.FRBl'.LS.HA.

[0062] [Figure 22] Figure 22. Plasmid map of pBP0721--pSH1-M-FRBl.FRBl'.LS.FRBl''.FRBl'''HA.

[0063] [Figure 23] Figure 23. Plasmid map of pBP0722--pSH1-Fpk-Fpk'.LS.Fpk''.Fpk'''.LS.HA.

[0064] [Figure 24] Figure 24. Plasmid map of pBP0220--pSFG-iC9.T2A-ΔCD19.

[0065] [Figure 25] Figure 25. Plasmid map of pBP0756--pSFG-iC9.T2A-dCD19.P2A-FRBl.

[0066] [Figure 26] Figure 26. Plasmid map of pBP0755--pSFG-iC9.T2A-dCD19.P2A-FRBl2.

[0067] [Figure 27] Figure 27. Plasmid map of pBP0757--pSFG-iC9.T2A-dCD19.P2A-FRBl3.

[0068] [Figure 28] Figure 28. Plasmid map of pBP0655--pSFG-ΔMyr.FRBl.MC.2A-ΔCD19.

[0069] [Figure 29] Figure 29. Plasmid map of pBP0498--pSFG-ΔMyr.iMC.FRBl2.P2A-ΔCD19.

[0070] [Figure 30] Figure 30. Plasmid map of pBP0488--pSFG-aHER2.Q.8stm.CD3zeta.Fpk2.

[0071] [Figure 31] Figure 31. Plasmid map of pBP0467--pSH1-FRBl'.FRBl.LS.ΔCaspase9.

[0072] [Figure 32] Figure 32. Plasmid map of pBP0606--pSFG-k-ΔMyr.iMC.2A-ΔCD19.

[0073] [Figure 33] Figure 33. Plasmid map of pBP0607--pSFG-k-iMC.2A-ΔCD19.

[0074] [Figure 34] Figure 34. Plasmid map of pBP0668--pSFG-FRBlx2.Caspase9.2AQ.8stm.CD3ζ.

[0075] [Figure 35] Figure 35. Plasmid map of pBP0608--pSFG-ΔMyr.iMC.2A-ΔCD19.Q.8stm.CD3ζ.

[0076] [Figure 36] Figure 36. Plasmid map of pBP0609:pSFG-iMC.2A-ΔCD19.Q.8stm.CD3ζ.

[0077] [Figure 37] Figure 37A provides a schematic diagram of rimizuside binding to two copies of a chimeric caspase-9 polypeptide (each having an FKBP12 multimerization domain). Figure 37B provides a schematic diagram of rapamycin binding to two chimeric caspase-9 polypeptides (one of which has an FKBP12 multimerization domain and the other of which has an FRB multimerization domain). Figure 37C provides a graph of the results of an assay using these chimeric polypeptides.

[0078] [Figure 38] Figure 38A provides a schematic diagram of rapamycin or a rapalog binding to two chimeric caspase-9 polypeptides, one of which has an FKBP12v36 multimerization domain and the other of which has an FRB variant (FRBL) multimerization domain, and Figure 38B provides a graph of the results of an assay using this chimeric polypeptide.

[0079] [Figure 39] Figure 39A is a schematic diagram of rimizuside binding to two chimeric caspase-9 polypeptides (each of which has an FKBP12v36 multimerization domain) and rapamycin binding to only one chimeric caspase-9 polypeptide having an FKBP12v36 multimerization domain. Figure 39B provides a graph of assay results comparing the effects of rimizuside and rapamycin.

[0080] [Figure 40] Figure 40A provides a schematic diagram of rimizuside binding to two chimeric caspase-9 polypeptides (each of which has an FKBP12v36 multimerization domain) and rapamycin binding to only one chimeric caspase-9 polypeptide having an FKBP12v36 multimerization domain in the presence of an FRB multimerization polypeptide. Figure 40B provides a graph of the results of an assay using these polypeptides, comparing the effects of rimizuside and rapamycin.

[0081] [Figure 41] Figure 41 provides a plasmid map of pBP0463.pFRBl.LS.dCasp9.T2A.

[0082] [Figure 42] Figure 42 provides a plasmid map of pBP044-pSH1.iCasp9WT.

[0083] [Figure 43] Figures 43A-43C are schematic diagrams of FwtFRBC9 / MC.FvFv containing iFwtFRBC9 or iFRBFwtC9 (collectively, iRC9). In this version of the rapamycin-inducible chimeric pro-apoptotic polypeptide, tandem FKBP.FRB (or FRB.FKBP) domains are fused to Δcaspase-9. Rapamycin or a rapalog can induce 1) the anchorage-induced dimerization of FKBP.FRB.ΔC9 (or FRB.FKBP.ΔC9) via the two FKBP domains fused to MC; or 2) the direct dimerization of FKBP.FRB.ΔC9 (or FRB.FKBP.ΔC9) to induce multimerization of engineered caspase-9 fusion proteins.

[0084] [Figure 44]Figures 44A-44C. Expression profiles of iMC+CARζ-T, i9+CARζ+MC, and FwtFRBC9 / MC.FvFv T cells. PBMCs from four different donors were activated and transduced with vectors containing iMC+CARζ-T (608), i9+CARζ+MC (844), and FwtFRBC9 / MC.FvFv (1300). For a schematic representation of the vectors, see Figure 48. (A) Five days after transduction, T cell lysates were subjected to Western blot analysis with antibodies against MyD88, caspase-9, and β-actin (which serves to demonstrate equal protein loading in all lanes). Note that iRC9 migrates similarly to endogenous caspase-9, and the added intensity of the band indicates the level of iRC9. (B) CAR expression was analyzed with anti-CD34-PE and anti-CD3-PerCPcy5 antibodies at 4, 7, 12, 21, and 29 days after transduction. (C) T cell viability from cells growing in culture was assessed using a Cellometer and AOPI viability dye at 3, 5, 12, 21, and 29 days after transduction.

[0085] [Figure 45]Figures 45A-45C. Rapamycin induces robust apoptotic activation in FwtFRBC9 / MC.FvFv T cells. PBMCs from four different donors were activated and transduced with vectors containing iMC+CARζ-T (608), i9+CARζ+MC (844), and FwtFRBC9 / MC.FvFv (1300). Five days after transduction, T cells were seeded onto 96-well plates ± rimizuside ± rapamycin in the presence of 2 μM caspase 3 / 7 green reagent. (A) Plates were placed inside an IncuCyte to monitor green fluorescence over time, reflecting cleaved caspase 3 / 7 reagent. (B) After 48 hours, cells were stained with anti-CD34-PE (FL2), PI (FL4), and Annexin V-PacBlue (FL9), and cleaved caspase 3 / 7 was detected in the FL1 channel on a Galios cytometer. (C) Culture supernatants were collected 48 hours after plating, and IL-2 and IL-6 cytokine production was analyzed by ELISA.

[0086] [Figure 46] Figures 46a-46C show that Q-LEHD-OPh efficiently inhibits caspase activation induced by iC9 and iRC9. PBMCs were activated and transduced with i9+CARζ+MC(844) and FwtFRBC9 / MC.FvFv(1300) vectors. Seven days after transduction, T cells were seeded into 96-well plates containing (A) increasing concentrations of rimizuside / rapamycin, (B) increasing concentrations of Q-LEHD-OPh, and (C) 20 nM rimizuside / rapamycin and increasing concentrations of Q-LEHD-OPh. Additionally, 2 μM Caspase 3 / 7 Green reagent was added to monitor caspase cleavage using an IncuCyte™ system.

[0087] [Figure 47]Figures 47A-47D. FRBL and the caspase-9 N405Q mutant reduce iRC9 activity. PBMCs were activated and transduced with plasmids 1300, 1308, 1316, and 1317. Five days after transduction, T cells were seeded into 96-well plates with 0 nM (A), 0.8 nM (B), 4 nM (C), and 20 nM (D) rapamycin. Caspase activation was monitored over time in an IncuCyte with the inclusion of 2 μM caspase 3 / 7 green reagent.

[0088] [Figure 48] Figures 48A-48D. iRC9 is a potent effector of rapamycin-induced apoptosis. (A) Schematic diagram of iMC+CARζ-T, i9+CARζ+MC, iFRBC9 and MC.FvFv, and FwtFRBC9 / MC.FvFv constructs. (B-D) Activated T cells were transduced with retroviruses encoding iMC+CARζ-T, i9+CARζ+MC, iFRBC9 and MC.FvFv, or FwtFRBC9 / MC.FvFv, and treated with no drug, 20 nM rapamycin, or 20 nM rimizuside and cultured in the presence of 2.5 μM caspase 3 / 7 green reagent. The 96-well microplate was placed in an IncuCyte™ system to monitor activated caspase activity (green fluorescence) for 48 hours.

[0089] [Figure 49-1]Figures 49A-49D. iRC9 rapidly and efficiently eliminates CAR-T cells in vitro. (A and B) NSG mice were injected i.v. with 107 iMC+CARζ-T, i9+CARζ+MC, iFRBC9, and MC.FvFv or FwtFRBC9 / MC.FvFv T cells co-transduced with GFP-Ffluc per mouse. CAR T cell bioluminescence was assessed 18 hours before drug treatment (-18 hours), immediately before drug treatment (0 hours), and at 4.5, 18, 27, and 45 hours after drug treatment. Mice receiving i9+CARζ+MC T cell injections were injected i.p. with 5 mg / kg rimizuside per mouse. For mice receiving iMC+CARζ-T (iFRBC9 and MC.FvFv) and FwtFRBC9 MC.FvFv T cells, 10 mg / kg rapamycin was injected i.p. per mouse. Forty-five hours after drug treatment, mice were euthanized, and (C) blood and (D) spleens were collected for flow cytometry analysis using antibodies against hCD3, hCD34, and mCD45. [Figure 49-2] Same as above.

[0090] [Figure 50-1]Figures 50A-50D show that the on- and off-switches in FwtFRBC9 / MC.FvFv are efficiently regulated by rimizuside and rapamycin, respectively. PBMCs from donor 920 were activated and co-transduced with vectors encoding GFP-Ffluc and iMC+CARζ-T(189), i9+CARζ+MC(873), or FwtFRBC9 / MC.FvFv(1308). Seven days after transduction, T cells were seeded in 96-well plates with HPAC-RFP cells at E:T ratios of 1:2 and 1:5 in the presence of 0 nM, 2 nM, or 10 nM rimizuside and placed in an IncuCyte to monitor the kinetics of T cell-GFP and HPAC-RFP growth. (A and B) Two days after seeding, culture supernatants were analyzed by ELISA for the production of IL-2, IL-6, and IFN-γ. On day 7, 10 nM rimizuside was added to i9+CARζ+MC cultures, and 10 nM rapamycin was added to GFP, iMC+CARζ-T, and FwtFRBC9 / MC.FvFv cultures, followed by monitoring by IncuCyte until day 8. The numbers of HPAC-RFP and T cell-GFP at an E:T 1:2 ratio were analyzed using IncuCyte's Basic Analyzer software on day 7 (Ci) and on day 8 with 0 nM suicide drug (Cii) and 10 nM suicide drug (Ciii). A similar analysis was also performed at a 1:5 E:T ratio (D). (Note: The y-axes for Ci and Di are logarithmic.) [Figure 50-2] Same as above.

[0091] [Figure 51-1]Figures 51A-51E. iRC9 activates apoptosis through direct self-dimerization in FwtFRBC9 / MC.FvFv, independent of anchorage-induced dimerization. PBMCs from donor 920 were activated and transduced with various vectors as described in (A). (B) Protein expression of CAR T cells was analyzed by Western blot using antibodies against hMyD88, hCaspase-9, and β-actin. (C-D) Five days after transduction, T cells were seeded into 96-well plates with increasing concentrations of rapamycin. Additionally, 2 μM Caspase 3 / 7 Green reagent was added to monitor caspase cleavage by IncuCyte. Line graphs show caspase activity over 24 hours after rapamycin treatment for MC variants (C) and FRB.FKBP.ΔC9 vs. FKBP.FRB.ΔC9 iRC9 (D). (E) Seven days after transduction, T cells were seeded into 96-well plates with increasing concentrations of rimizuside, and IL-2 and IL-6 secretion was quantified by ELISA 48 hours after rimizuside treatment. [Figure 51-2] Same as above.

[0092] [Figure 52] Figures 52A-52B. Relatively high (>100 nM) concentrations of rimizuside are required to activate iRC9. 293 cells were seeded at 300,000 cells / well in 6-well plates and grown for 2 days. After 48 hours, cells were transfected with 1 μg of experimental plasmid. Cells were harvested 48 hours post-transfection and diluted 2.5-fold from their original volume. (A) For the Incucyte / casp3 / 7 assay, 50 μl of cells were plated per well containing either rimizuside or rapamycin drug and caspase 3 / 7 green reagent (2.5 μM final concentration). (B) For the SEAP assay, 100 μl of cells were plated in 96-well plates with (half-log) rimiduside (or rapamycin) drug dilutions, and after drug exposure, the plates were heat-inactivated approximately 18 hours before the addition of substrate (4-MUP).

[0093] [Figure 53] Figures 53A-53B. Schematic of the MC-Rap, rapamycin, or rapalog-inducible CAR costimulation strategy. In this version of the inducible costimulation switch, tandem FKBP.FRB (or FRB.FKBP) domains are fused to MyD88-CD40 (MC) (right side). Rapamycin or a rapalog can induce direct dimerization of the FKBP in MC-FKBP-FRB (or MC-FRB-FKBP) with the FRB in the second molecule of MC-FKBP-FRB to induce multimerization of the engineered MC fusion protein. Note that FRB can be present as wild-type or as a rapalog-inducible mutant (e.g., FRBL) with reduced affinity for mTOR. This strategy contrasts with the homodimerization directed by FKBPV36 in the rimizuside and iMC+CARζ platforms (left side).

[0094] [Figure 54] Figures 54A-54B Induction of MC costimulatory activity with rapalogs and MC-Rap-CAR. Human PBMCs were activated and transduced with iMC+CARζ constructs (BP0774 and BP1433), MC-rap-CAR (BP1440), or a non-inducible MC-only construct (BP1151). Cells were rested for 6 days, and then aliquots were stimulated with rimizuside or the rapalog C7-dimethoxy-7-isobutyloxyrapamycin. Supernatant medium was collected 24 hours later, and the amount of secreted IL-6 was determined by ELISA as an indicator of MC activity. MC activity in iMC+CARζ-T cells is strongly stimulated by rimizuside, but not by rapalogs. MC activity in MC-rap-T cells is not stimulated by rimizuside. Because FKBP12 in pBP1440 is wild-type, rather than the rimiduside-sensitive allele V36, MC-Rap activity instead responds strongly to isobutyloxyrapamycin, to an extent similar to that of iMC+CARζ-T on rimiduside.

[0095] [Figure 55] Figures 55A-55B Protein expression of MCs from iMC+CARζ. Human PBMCs were activated and transduced with the iMC+CARζ construct (BP0774, BP1433, and BP1439), MC-rap-CAR (BP1440), or a non-inducible MC-only construct (BP1151 oriented at the 5' end of the retrovirus and 1414 oriented 3' to the CAR). Cells were expanded for 2 weeks, and then extracts were prepared for SDS-PAGE. Western blots were probed with an antibody against MyD88. The MC-FKBP-FRB fusion protein was expressed from the iMC+CARζ construct at levels similar to the MC-FKBPV fusion.

[0096] [Figure 56] Figures 56A-56B. Responsiveness of MC-rap to doses of rapamycin and rapamycin analogs. 293T cells were transfected with 1 μg of the reporter construct NF-κB SeAP and 4 μg of the iMC+CARζ construct pBP0774 or the MC-rap-CAR construct pBP1440 using the GeneJuice protocol (Novagen). 24 h after transfection, cells were split into 96-well plates and incubated with increasing concentrations of rimizuside, rapamycin, or isobutyloxyrapamycin. After a further 24 h of incubation, SeAP activity was determined from cell supernatants. NF-κB reporter activity was stimulated with both rapalogs and rapamycin at subnanomolar EC50s, while up to 50 nM rimizuside failed to induce MC-rap dimerization.

[0097] [Figure 57]Figures 57A-57B: Schematic of MC-Rap, rapamycin, or rapalog-inducible CAR costimulation strategies. In FwtFRBC9 / MC.FvFv (left), tandem FKBP.FRB (or FRB.FKBP) domains are fused to caspase-9, and the tandem Fv portion is fused to MC. Caspase-9 can be activated by homodimerization via rapamycin-directed FRB and wild-type FKBP ligation or by scaffolding with iMC. Rimizuside dimerizes the FKBPV36 portion to activate MC. FRBFwtMC / FvC9 (right) can induce MC-Rap using rapamycin or a rapalog, while iC9 can be induced by rimizuside as a cell suicide switch.

[0098] [Figure 58] Figures 58A-58C show that FRBFwtMC / FvC9 can effectively regulate tumor growth, but activation of iC9 with rimizuside abrogates it. PBMCs from donor 676 were activated and transduced with CD19-directed i9+CARζ+MC (BP0844), FRBFwtMC / FvC9 (BP1460), or FwtFRBC9 / MC.FvFv (BP1300). Seven days after transduction, T cells were seeded in 24-well plates with Raji-GFP cells at a 1:5 E:T ratio in the presence of 2 nM rimizuside, 2 nM isobutyloxyrapamycin, or 2 nM rapamycin. After 7 days of incubation, viable cells were analyzed for the percentage of GFP-labeled tumor cells (left) and total T cells (CD3+, right) and transduced CAR-T cells (CD34, not shown). Rimizuside caused cell death of CAR-T cells in i9+CARζ+MC or FRBFwtMC / FvC9 and tumor cells dominated the cultures, while rapamycin or isobutyloxyrapamycin caused cell death in FwtFRBC9 / MC.FvFv.

[0099] [Figure 59]Figure 59. Schematic diagram of plasmid pBP1300--pSFG-FKBP.FRB.ΔC9.T2A-αCD19.Q.CD8stm.ζ.P2A-iMC.

[0100] [Figure 60] Figure 60. Schematic diagram of plasmid pBP1308--pSFG-FKBP.FRB.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.P2A-iMC.

[0101] [Figure 61] Figure 61. Schematic diagram of plasmid pBP1310--pSFG.FRB.FKBP.ΔC9.T2A-ΔCD19.

[0102] [Figure 62] Figure 62. Schematic diagram of plasmid pBP1311--pSFG.FKBP.FRB.ΔC9.T2A-ΔCD19.

[0103] [Figure 63] Figure 63. Schematic diagram of plasmid pBP1316--pSFG-FKBP.FRBL.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.P2A-iMC.

[0104] [Figure 64] Figure 64. Schematic diagram of plasmid pBP1317--pSFG-FKBP.FRB.ΔC9Q.T2A-αPSCA.Q.CD8stm.ζ.P2A-iMC.

[0105] [Figure 65] Figure 65. Schematic diagram of plasmid pBP1319--pSFG-FKBP.FRB.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.P2A-MC.FKBPV.

[0106] [Figure 66] Figure 66. Schematic diagram of plasmid pBP1320--pSFG-FKBP.FRB.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.P2A-MC.

[0107] [Figure 67] Figure 67. Schematic diagram of plasmid pBP1321--pSFG-FKBP.FRB.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.P2A-MC.FKBPV.FKBP.

[0108] [Figure 68] Figure 68A provides a graph of drug-dependent CAR-T cell killing of tumor cells. Figure 68B provides a schematic of the inducible MyD88-CD40 polypeptide.

[0109] [Figure 69-1] Figure 69A provides a schematic diagram of a retroviral vector expressing an inducible MyD88-CD40 polypeptide. Figure 69B provides a bar graph of the results of a reporter assay of costimulatory signaling. Figure 69C provides a bar graph of CAR-T cell cytokine secretion. Figure 69D provides a graph of a CAR-T cell killing assay. [Figure 69-2] Same as above.

[0110] [Figure 70-1] Figure 70A provides a schematic of a retroviral vector expressing an inducible MyD88-CD40 polypeptide. Figure 70B provides a graph of a reporter assay of costimulatory signaling. Figure 70C provides a graph of a PSCA-CAR-T cell killing assay. Figure 70D provides a graph of a PSCA CAR-T cell killing assay. Figure 70E provides a graph of a HER2-CAR-T cell killing assay. Figure 70F provides a graph of a HER2-CAR-T cell killing assay. Figure 70G provides a graph of a HER2-CAR-T cell killing assay. [Figure 70-2] Same as above. [Figure 70-3] Same as above.

[0111] [Figure 71]Figure 71A provides a graph of apoptotic activity directed by inducible caspase-9 in the presence of rimizuside, and Figure 71B provides a graph of apoptotic activity directed by inducible caspase-9 in the presence of C7-isobutyloxyrapamycin.

[0112] [Figure 72] Figure 72A provides a schematic diagram of polypeptides expressed on a single vector, including a CAR polypeptide, an iRC9 polypeptide, and an iMC polypeptide. Figure 72B provides a schematic diagram of polypeptides expressed on two separate vectors.

[0113] [Figure 73] Figure 73A provides a schematic diagram of an inducible caspase-9 retroviral construct. Figure 73B provides data showing the fluorescence conversion of cells expressing caspase-9 in the presence of rapamycin. Figure 73C provides a graph of the relative apoptotic activity of Figure 73B. Figure 73D provides a Western blot of caspase-9 transgene expression in T cells.

[0114] [Figure 74] Figure 74A provides a graph of IL-6 secretion in the presence of limizuside. Figure 74B provides a graph of IL-2 secretion in the presence of limizuside. Figure 74C provides a graph of IFN-γ secretion in the presence of limizuside. Figure 74D provides a graph of CAR-T cell killing in the presence of limizuside. Figure 74E provides a Western blot of iMC and iRC9 expression.

[0115] [Figure 75A] Figure 75A provides cell sorting results for untransduced T cells or T cells transduced with retroviruses encoding iRC9, iMC, and CAR, as indicated. Figure 75B provides a graph of the results in Figure 75A. Figure 75C provides cell sorting results of an apoptosis assay. Figure 75D provides a graphical representation of the apoptosis assay. [Figure 75B] Same as above. [Figure 75C] Same as above. [Figure 75D] Same as above.

[0116] [Figure 76A] Figure 76A provides micrographs of tumor-bearing animals as determined by bioluminescence imaging. Figure 76B provides a graph of mean tumor growth. Figure 76C provides a graph of human T cells in the spleen at termination. Figure 76D provides a graph of vector copy number. [Figure 76B] Same as above. [Figure 76C] Same as above. [Figure 76D] Same as above.

[0117] [Figure 77A] Figure 77A provides photomicrographs of tumor-bearing animals as determined by bioluminescence imaging. Figure 77B provides a graph of mean radiance. Figure 77C provides a graph of Kaplan-Meier analysis from Figure 77A. Figure 77D provides a representative FACS analysis at termination. [Figure 77B] Same as above.

[0118] [Figure 78] Figure 78A provides photomicrographs of tumor-bearing animals as determined by bioluminescence imaging. Figure 78B provides a graphical representation of the calculated mean radiance from Figure 78A. Figure 78C provides a graph of human T cell counts in mouse spleens.

[0119] [Figure 79A]Figure 79A provides photomicrographs of tumor-bearing animals as determined by bioluminescence imaging. Figure 79B provides a graphical representation of the calculated mean radiance from Figure 79A. Figure 79C provides a graph of the number of human T cells in the spleens of mice at termination. Figure 79D provides a graph of the vector copy number of DNA derived from the spleens of mice. [Figure 79B] Same as above. [Figure 79C] Same as above. [Figure 79D] Same as above.

[0120] [Figure 80] Figure 80 provides a plasmid map of pBP1151--pSFG--MC-T2A-αCD19.Q.CD8stm.ζ.

[0121] [Figure 81] Figure 81 provides a plasmid map of pBP1152--pSFG--MC-T2A-αCD19.Q.CD8stm.ζ.

[0122] [Figure 82] Figure 82 provides a plasmid map of pBP1414--pSFG-αCD19.Q.CD8stm.ζ-P2A-MC.

[0123] [Figure 83] Figure 83 provides a plasmid map of pBP1414--pSFG-αCD19.Q.CD8stm.ζ-P2A-MC.

[0124] [Figure 84] Figure 84 provides a plasmid map of pBP1433--pSFG-Fv-Fv-MC-T2A-αCD19.Q.CD8stm.ζ.

[0125] [Figure 85] Figure 85 provides a plasmid map of pBP1439--pSFG--MC.FKBPv-T2A-αCD19.Q.CD8stm.ζ.

[0126] [Figure 86] Figure 86 provides a plasmid map of pBP1440--pSFG-FKBPv.ΔC9.T2A-αCD19.Q.CD8stm.ζ.T2A.P2A-MC.FKBPwt.FRBL.

[0127] [Figure 87] Figure 87 provides a plasmid map of pBP1460--pSFG-FKBPv.ΔC9.T2A-αCD19.Q.CD8stm.ζ.T2A.P2A-MC.FKBPwt.FRBL.

[0128] [Figure 88] Figure 88 provides a plasmid map of pBP1293--pSFG-iMC.T2A-αhCD33(My9.6).ζ.

[0129] [Figure 89] Figure 89 provides a plasmid map of pBP1296--pSFG-iMC.T2A-αhCD123(32716).ζ.

[0130] [Figure 90] Figure 90 provides a plasmid map of pBP1327--pSFG-FRB.FKBPV.ΔC9.2A-ΔCD19.

[0131] [Figure 91] Figure 91 provides a plasmid map of pBP1328--pSFG-FKBPV.FRB.ΔC9.2A-ΔCD19.

[0132] [Figure 92] Figure 92 provides a plasmid map of pBP1351--pSFG-SP163.FKBP.FRB.ΔC9.T2A-αhPSCA.Q.CD8stm.ζ.2A-iMC.

[0133] [Figure 93]Figure 93 provides a plasmid map of pBP1373--pSFG-sp-FKBP.FRB.ΔC9.T2A-αhPSCAscFv.Q.CD8stm.ζ.

[0134] [Figure 94] Figure 94 provides a plasmid map of pBP1385--pSFG-FRB.FKBP.ΔC9.T2A-ΔCD19.

[0135] [Figure 95] Figure 95 provides a plasmid map of pBP1455--pSFG-MC.FKBPwt.FRBL.T2A-αPSCA.Q.CD8stm.ζ.

[0136] [Figure 96] Figure 96 provides a plasmid map of pBP1466--pSFG-FKBPv.ΔC9.T2A-PSCA.Q.CD8stm.ζ.P2A-MC.FKBPwt.FRBL.

[0137] [Figure 97] Figure 97 provides a plasmid map of pBP1474--pSFG-FKBPv.ΔC9.T2A-αHER2.Q.CD8stm.ζ.

[0138] [Figure 98] Figure 98 provides a plasmid map of pBP1475--pSFG-FKBPv.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.

[0139] [Figure 99] Figure 99 provides the plasmid map of pBP1488--pSFG-FRBL.FKBPwt.MC-T2A-αPSCA.Q.CD8stm.ζ.

[0140] [Figure 100]Figure 100 provides a plasmid map of pBP1491--pSFG--FKBPv.ΔC9.P2A.MC.FKBPwt.FRBL.T2A-αHER2.Q.CD8stm.ζ.

[0141] [Figure 101] Figure 101 provides a plasmid map of pBP1493--pSFG-MC.FKBPwt.FRBL-P2A.FKBPv.ΔC9.T2A-αHER2.Q.CD8stm.ζ.

[0142] [Figure 102] Figure 102 provides a plasmid map of pBP1494--pSFG-MC.FKBPwt.FRBL-P2A.FKBPv.ΔC9.T2A-PSCA.Q.CD8stm.ζ.

[0143] [Figure 103] Figure 103 provides a plasmid map of pBP1757--pSFG-FRBL.FKBPwt.MC-P2A.FKBPv.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.

[0144] [Figure 104] Figure 104 provides a plasmid map of pBP1759--pSFG--FRBL.FKBPwt.MC-P2A.FKBPv.ΔC9.T2A-αHER2.Q.CD8stm.ζ.

[0145] [Figure 105] Figure 105 provides a plasmid map of pBP1796--pSFG--FKBPwt.FRBL-MC.P2A.FKBPv.ΔC9.T2A-αPSCA.Q.CD8stm.ζ.

[0146] [Figure 106] Figure 106A provides a schematic diagram of various inducible chimeric caspase-9 constructs. Figure 106 provides a graph of a caspase activation assay. Figure 106C is a photograph of a Western blot showing protein expression.

[0147] [Figure 107] Figure 107A provides a graph of caspase activity. Figure 107B provides a graph of SEAP activity.

[0148] [Figure 108] Figure 108A provides a graph of SEAP activity, Figure 108B provides a graph of caspase activity, and Figure 108C provides a Western blot showing protein expression.

[0149] [Figure 109-1] Figure 109A provides a FACS analysis of transduction efficiency. Figure 109B provides a graph of bioluminescence. Figure 109C provides a photograph of bioluminescence in mice. Figure 109D provides a graph of FACS analysis of mouse splenocytes. [Figure 109-2] Same as above. [Figure 109-3] Same as above.

[0150] [Figure 110-1] Figure 110A provides a FACs analysis of transduction efficiency. Figure 110B provides a graph of bioluminescence. Figure 110C provides a photograph of bioluminescence in mice. Figure 110D provides a graph of FACs analysis of mouse splenocytes. [Figure 110-2] Same as above. [Figure 110-3] Same as above.

[0151] [Figure 111] Figure 111 provides a schematic diagram of vectors encoding CD123-CAR-ζ and iMC polypeptides.

[0152] [Figure 112-1]Figure 112A provides a graph of IL-6 production; Figure 112B provides a graph of IL-2 production; Figure 112C provides a graph of total green fluorescence intensity of THP1-GP.Fluc, and Figure 112D provides a graph of HPAC-RFP cell number. [Figure 112-2] Same as above.

[0153] [Figure 113-1] Figure 113A provides a graph of IL-2 production; Figure 113B provides a graph of THP1-FP.Fluc cells; Figure 113C provides a graph of T cell-RFP; Figure D provides a graph of THP1-GFP.Fluc green fluorescence; Figure E provides a graph of T cell-RFP red fluorescence. [Figure 113-2] Same as above.

[0154] [Figure 114-1] Figure 114A provides FACs analysis; Figure 114B provides a schematic of tumor growth via IVIS monitoring; Figure 114C provides a photograph of bioluminescence in mice; Figure 114D provides a graph of the presence of CAR-T cells as measured by flow cytometry; Figure 114E provides a graph of vector copy number. [Figure 114-2] Same as above. [Figure 114-3] Same as above.

[0155] [Figure 115] Figure 115A provides a photograph of bioluminescence in mice; Figure 115B provides a graph of vector copy number.

[0156] [Figure 116] Figure 116 provides a schematic diagram of inducible MCs expressed with recombinant TCRs.

[0157] [Figure 117-1]Figure 117A provides a schematic diagram of a PRAME TCR polypeptide; Figure 117B provides a schematic diagram of an iMC polypeptide; Figure 117C provides a schematic diagram of a PRAME-TCR polypeptide co-expressed with an iMC polypeptide; Figure 117D provides a graph of IL-2 production, with items listed along the X-axis in the same order as in the legend. [Figure 117-2] Same as above.

[0158] [Figure 118] Figure 118A provides a schematic of the transwell assay setup; Figure 118B provides a graph of HLA-A, B, C levels.

[0159] [Figure 119] Figure 119A provides a graph of specific lysis. Figure 119B provides a graph of IL-2 production.

[0160] [Figure 120] Figure 120A provides a graph of specific lysis; Figure 120B provides a graph of IL-2 production.

[0161] [Figure 121-1] Figure 121A provides a schematic of the immunodeficient NSG xenograft model; Figure 121B provides a graph of the mean radiance in non-transduced and transduced cells; Figure 121C provides a graph of the number of Vβ1+CD8+ cells / spleen; Figure 121D provides a graph of the number of Vβ1+CD8+ cells / spleen. [Figure 121-2] Same as above. [Figure 121-3] Same as above. [Figure 121-4] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0162] (Detailed explanation) Regulated protein-protein interactions have evolved to control most, if not all, signaling pathways as a mechanism for transferring information from the external environment to the interior of the cell. Signaling depends on enzymatic processes (e.g., amino acid side chain phosphorylation, acetylation, or proteolytic cleavage that lack inherent specificity). Furthermore, many proteins or factors exist at cellular concentrations or subcellular locations that preclude the spontaneous generation of sufficient substrate / product relationships to activate or propagate signaling. A key component of activated signaling is the recruitment of these components to signaling "nodes" or spatial signaling centers that efficiently propagate (or attenuate) the pathway through appropriate upstream signals.

[0163] As a tool for artificially isolating and manipulating individual protein-protein interactions and, therefore, individual signaling proteins, the chemically induced dimerization (CID) technique has been developed to impose homotypic or heterotypic interactions on target proteins to recapitulate natural biological regulation. In its simplest form, a single protein is engineered to contain one or more structurally identical ligand-binding domains. This then becomes the basis for homodimerization or oligomerization, respectively, in the presence of the cognate homodimeric ligand (Spencer DM et al. (93) Science 262, 1019-24). A slightly more complex version of this concept involves placing one or more distinct ligand-binding domains on two different proteins and allowing heterodimerization of these signaling molecules using a small molecule, a heterodimeric ligand, that simultaneously binds both distinct domains (Ho SN et al. (96) Nature 382, ​​822-6). This drug-mediated dimerization generates very high local concentrations of the ligand-binding domain-tagged components sufficient to allow their inducible or spontaneous assembly and control.

[0164] In some embodiments, provided herein are methods for inducing protein multimerization. In this case, two or more heterodimeric ligand-binding regions (or "domains") in tandem are used as a "molecular scaffold" to dimerize or oligomerize a second signaling domain-containing protein fused to one or more copies of a second binding site for the heterodimeric ligand. This molecular scaffold can be expressed as an isolated multimer of ligand-binding domains (FIG. 8), either localized or not (FIGS. 8B, 8C), or can be attached to another protein that provides structural, signaling, cell-marking, or more complex combinatorial functions (FIG. 9). By "scaffold" is meant a polypeptide comprising at least two, e.g., two or more, heterodimeric ligand-binding regions; in certain instances, the ligand-binding regions are in tandem, i.e., each ligand-binding region is located immediately proximal to the next. In other examples, each ligand-binding region can be located proximally relative to the next ligand-binding region, e.g., separated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more amino acids, yet retain scaffolding function for dimerization of inducible caspase molecules in the presence of a dimerizing agent. The scaffold can include, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more ligand binding regions, and can also be linked to another polypeptide (such as, for example, a marker polypeptide, a costimulatory molecule, a chimeric antigen receptor, a T cell receptor, etc.).

[0165] In some embodiments, the first polypeptide consists essentially of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 units of the first multimerization region. In some embodiments, the first polypeptide consists essentially of the scaffold region. In some embodiments, the first polypeptide consists essentially of a membrane-association region or a membrane-targeting region. By "consists essentially of," we mean that the scaffold unit or scaffold can be present alone or can optionally include linker polypeptides, and optionally small polypeptides (such as stem polypeptides as shown in Figures 10B, 10C, 10D, and 10E), either at the ends of the scaffold or between the units.

[0166] In one example, a tandem multimer of approximately 89aa FK506-rapamycin-binding (FRB) domains from the protein kinase mTOR (Chen J et al. (95) PNAS, 92, 4947-51) was used to recruit multiple FKBPv36-fused caspase-9s (iC9 / iCaspase-9) in the presence of rapamycin or rapamycin-based analogs ("rapalogs") (Liberles SD (97) PNAS, 94, 7825-30; Rivera VM (96) Nat Med, 2, 1028-1032; Stankunas K (03) Mol Cell, 12, 1615-24; Bayle JH (06) Chem & Biol, 13, 99-107) (Figures 1-3). This recruitment leads to spontaneous caspase dimerization and activation.

[0167] In a second example, the tandem FRB domains are fused to a chimeric antigen receptor (CAR), which provides rapalog-driven iC9 activation to cells expressing both fusion proteins (Figure 15, inset).

[0168] In the third example, the polarity of the two proteins is reversed, such that two or more copies of FKBP12 are used to recruit and dimerize FRB-modified signaling molecules in the presence of rapamycin (Figures 8C, 9A).

[0169] In some examples, a chimeric polypeptide may comprise a single ligand-binding domain, or a scaffold may be present that comprises more than one ligand-binding domain, where the chimeric polypeptide comprises a polypeptide such as, for example, a MyD88 polypeptide, a truncated MyD88 polypeptide, a cytoplasmic CD40 polypeptide, a chimeric MyD88 / cytoplasmic CD40 polypeptide, or a chimeric truncated MyD88 / cytoplasmic CD40 polypeptide.

[0170] MyD88 or MyD88 polypeptide refers to the polypeptide product (e.g., but not limited to, the human version) of myeloid differentiation primary response gene 88, cited as ncbi Gene ID 4615. "Truncated" means that the protein is not full-length and may, for example, lack a domain. For example, truncated MyD88 may not be full-length and may, for example, lack the TIR domain. An example amino acid sequence of a truncated MyD88 polypeptide is set forth as SEQ ID NO: 305. A nucleic acid sequence encoding "truncated MyD88" refers to a nucleic acid sequence that encodes a truncated MyD88 peptide; this term can also refer to a nucleic acid sequence that includes portions that encode any amino acids added as a cloning artifact (including any amino acids encoded by a linker). Where a method or construct refers to a truncated MyD88 polypeptide, it is understood that the method can also be used, or the construct can be designed, to refer to another MyD88 polypeptide (e.g., a full-length MyD88 polypeptide). Where a method or construct refers to a full-length MyD88 polypeptide, the method can also be used or the construct can be designed to refer to a truncated MyD88 polypeptide.

[0171] In the methods herein, the CD40 portion of the peptide can be located either upstream or downstream from the MyD88 or truncated MyD88 polypeptide portion.

[0172] In a fourth example, an unstable FRB variant (e.g., FRBL2098) is used to destabilize the signaling molecule before rapalog administration (Stankunas K (03) Mol Cell 12, 1615-24; Stankunas K (07) ChemBioChem 8, 1162-69) (Figures 9, 10). After rapalog exposure, the unstable fusion molecule is stabilized, resulting in aggregation as before, but with lower background signaling.

[0173] The use of ligands to target signaling proteins can be generally applied to activate or attenuate many signaling pathways. An example demonstrating the utility of this approach is provided herein by controlling apoptosis, or programmed cell death, with the "initiator caspase," caspase-9, as its primary target. Controlling apoptosis through dimerization of pro-apoptotic proteins with the widely available rapamycin or more proprietary rapalogs should enable experimenters or clinicians to tightly and rapidly control the viability of cell-based implants that exhibit undesirable effects. Examples of these effects include, but are not limited to, off-target tissues or graft-versus-host (GvH) immune responses to excessive, uncontrolled growth or metastasis of the implant. Rapid induction of apoptosis tightly attenuates the function of undesirable cells and allows natural clearance of dead cells by phagocytes (e.g., macrophages) without excessive inflammation.

[0174] Apoptosis is tightly regulated and naturally uses scaffolds (e.g., Apaf-1, CRADD / RAIDD, or FADD / Mort1) to oligomerize and activate caspases, which can ultimately kill the cell. Apaf-1 can assemble the apoptotic protease caspase-9 into a latent complex, which then forms an active oligomeric apoptosome upon recruitment of cytochrome C to the scaffold. The key event is oligomerization of the scaffold units, which leads to dimerization and activation of the caspases. Similar adaptors (e.g., CRADD) can oligomerize caspase-2, resulting in apoptosis. The compositions and methods provided herein use, for example, multimeric versions of the ligand-binding domains FRB or FKBP to serve as scaffolds that enable spontaneous dimerization and activation of caspase units present as FRB or FKBP fusions upon recruitment with rapamycin.

[0175] Using certain methods provided in the Examples herein, caspase activation occurs only when rapamycin or a rapalog is present to recruit FRB- or FKBP-fused caspases to the scaffold. In these methods, the FRB or FKBP polypeptide must exist as a multimeric unit, not as a monomer, to drive FKBP-caspase or FRB-caspase dimerization (except when FRB-caspase-9 dimerizes with FKBP-caspase-9). The FRB- or FKBP-based scaffold can be expressed in targeted cells as a fusion with other proteins and retain its ability to act as a scaffold to assemble and activate pro-apoptotic molecules. The FRB or FKBP scaffold can be localized in the cytosol as a soluble entity or can reside in a specific subcellular location (e.g., the plasma membrane) through a targeting signal. The components used to activate apoptosis and the downstream components that degrade the cell are shared by all cells and across species. With respect to caspase-9 activation, these methods may be widely utilized in cell lines, in normal primary cells (such as, but not limited to, T cells), or in cell transplants.

[0176] In certain instances where FKBP-caspases are directly dimerized with rapamycin to direct apoptosis, the FKBP-fused caspases are fused to homodimerizer molecules (e.g., AP1510, AP20187, or AP1 903) (Fig. 6 (right panel), 10A (schematic diagram) (a similar proapoptotic switch can be found in the chimeric protein). It has been shown that binary switching can be directed via heterodimerization using rapamycin or a rapalog by coexpression of an FRB-caspase-9 fusion protein along with FKBP-caspase-9, which leads to primary homodimerization (Figures 8A (schematic), 10B (schematic), (11)).

[0177] As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Still further, the terms "having," "including," "containing," and "comprising" are interchangeable, and those of skill in the art recognize these terms as open-ended terms.

[0178] The following table outlines the nature of some of the switch nomenclature and acronyms discussed in this and following examples. [Table 11]

[0179] As used herein, the term "allogeneic" refers to antigenically distinct HLA or MHC loci.

[0180] Thus, cells or tissues transplanted from the same species can be antigenically different. Syngeneic mice can differ at one or more genetic loci (congenic), and allogeneic mice can have the same background.

[0181] The term "antigen," as used herein, is defined as a molecule that provokes an immune response, which may involve either antibody production or activation of specific immunologically competent cells, or both.

[0182] An "antigen recognition moiety" can be any polypeptide or fragment thereof that binds to an antigen, such as a naturally occurring or synthetic antibody fragment variable domain. Examples of antigen recognition moieties include, but are not limited to, antibody-derived polypeptides, such as single-chain variable fragments (scFv), Fab, Fab', F(ab')2, and Fv fragments; T-cell receptor-derived polypeptides, such as TCR variable domains; and any ligand or receptor fragment that binds to an extracellular cognate protein.

[0183] The term "cancer" used herein is defined as the hyperproliferation of cells, whose unique characteristic is the loss of normal control, resulting in uncontrolled growth, lack of differentiation, local tissue invasion and metastasis.Examples include but are not limited to melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, hepatocellular carcinoma, leukemia, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, digestive cancer, lymphoma, brain cancer, colon cancer, sarcoma or bladder cancer.

[0184] Donor: The term "donor" refers to a mammal (e.g., a human) that is not a patient-recipient. The donor may, for example, be HLA-identical to the recipient, or may have a partial or greater HLA mismatch with the recipient.

[0185] Haploidentical: The term "haploidentical," when used in reference to a cell, cell type, and / or cell lineage herein, refers to cells that share a haplotype or have substantially the same alleles in a set of closely linked genes on a single chromosome. A haploidentical donor does not have complete HLA identity with the recipient; there is a partial HLA mismatch.

[0186] Hematological Disorders: As used herein, the terms "hematological disorder," "blood disease," and / or "disorder of the blood" refer to conditions that affect the production of blood and its components (including, but not limited to, blood cells, hemoglobin, blood proteins), clotting mechanisms, blood production, blood protein production, and the like, and combinations thereof. Non-limiting examples of hematological disorders include anemia, leukemia, lymphoma, hematological neoplasms, albuminemias, hemophilia, and the like.

[0187] Bone marrow disease: As used herein, the term "bone marrow disease" refers to a condition that results in a decrease in the production of blood cells and platelets. In some bone marrow diseases, the normal bone marrow structure may be replaced by infection (e.g., tuberculosis) or malignant disease, which may then lead to a decrease in the production of blood cells and platelets. Non-limiting examples of bone marrow diseases include leukemia, bacterial infection (e.g., tuberculosis), radiation sickness or radiation poisoning, pancytopenia, anemia, multiple myeloma, etc.

[0188] T cells and activated T cells (including those that refer to CD3+ cells): T cells (also called T lymphocytes) belong to a group of white blood cells called lymphocytes. Lymphocytes are generally involved in cell-mediated immunity. The "T" in "T cell" refers to cells that originate from the thymus or whose maturation is influenced by the thymus. T cells can be distinguished from other lymphocyte types (e.g., B cells and natural killer (NK) cells) by the presence of a cell surface protein known as the T cell receptor. As used herein, the term "activated T cell" refers to a T cell that has been stimulated to produce an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant displayed on a class II major histocompatibility (MHC) marker. T cells are activated by the presence of antigenic determinants, cytokines and / or lymphokines, and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, etc., and combinations thereof). Cells that express a cluster of differential proteins are often said to be "positive" for expression of that protein on the T cell surface (e.g., cells that are positive for expression of CD3 or CD4 are said to be "positive" for CD3 expression). + or CD4 + The CD3 and CD4 proteins are cell surface receptors or co-receptors that can be directly and / or indirectly involved in signal transduction in T cells.

[0189] Peripheral blood: As used herein, the term "peripheral blood" refers to the cellular components of blood (e.g., red blood cells, white blood cells, and platelets) obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow.

[0190] Umbilical Cord Blood: Umbilical cord blood is distinct from peripheral blood and from blood sequestered within the lymphatic system, spleen, liver, or bone marrow. The terms "umbilical blood," "umbilical blood," or "cord blood," which may be used interchangeably, refer to the blood remaining in the placenta and the umbilical cord after birth. Umbilical cord blood often contains stem cells, including hematopoietic cells.

[0191] "Cytoplasmic CD40" or "CD40 lacking the CD40 extracellular domain" refers to a CD40 polypeptide that lacks the CD40 extracellular domain. In some instances, this term also refers to a CD40 polypeptide that lacks both the CD40 extracellular domain and part or all of the CD40 transmembrane domain.

[0192] For example, "obtained or prepared," as in the case of cells, means that the cells or cell culture are isolated, purified, or partially purified from their source, where the source may be, for example, umbilical cord blood, bone marrow, or peripheral blood. These terms also apply when the original source or cell culture is cultured, the cells replicate, and progeny cells are derived from that original source.

[0193] "Kill" or "killing," as in the case of a percentage of cells being killed, refers to the death of cells by apoptosis, as measured using any method known for measuring apoptosis, and for example, using an assay discussed herein (such as, for example, the SEAP assay or T cell assay discussed herein). The term can also refer to cell elimination.

[0194] Allodepletion: As used herein, the term "allo-depletion" refers to the selective depletion of alloreactive T cells. As used herein, the term "allo-reactive T cells" refers to T cells activated to generate an immune response in response to exposure to foreign cells (such as in a transplanted allograft). Selective depletion generally involves targeting various cell surface-expressed markers or proteins (e.g., sometimes cluster of differentiation proteins (CD proteins), CD19, etc.) for removal using immunomagnetic forces, immunotoxins, flow sorting, induction of apoptosis, photodepletion techniques, etc., or a combination thereof. In this method, the cells can be transduced or transfected with a chimeric protein-encoding vector before or after allodepletion. Alternatively, the cells can be transduced or transfected with a chimeric protein-encoding vector without the allodepletion step, and non-allo-depleted cells can be administered to the patient. Due to the added "safety switch," it is possible to administer, for example, non-allo-depleted (or only partially allo-depleted) T cells, since adverse events, such as graft-versus-host disease, can be mitigated upon administration of the multimeric ligand.

[0195] Graft-versus-host disease: The term "graft-versus-host disease" or "GvHD" refers to a complication often associated with allogeneic bone marrow transplantation and occasionally with transfusion of non-irradiated blood to immunocompromised patients. Graft-versus-host disease can sometimes occur when functional immune cells in the transplanted bone marrow recognize the recipient as "foreign" and mount an immune response. GvHD can be divided into acute and chronic forms. Acute GVHD (aGVHD) is often observed within the first 100 days after transplantation or transfusion and can affect the liver, skin, mucous membranes, immune system (e.g., hematopoietic system, bone marrow, thymus, etc.), lungs, and gastrointestinal tract. Chronic GVHD (cGVHD) often begins 100 days or more after transplantation or transfusion and can attack the same organs as acute GvHD, but can also affect connective tissue and exocrine glands. Acute GvHD of the skin can produce a diffuse maculopapular rash, sometimes with a lacy pattern.

[0196] Donor T cells: As used herein, the term "donor T cells" refers to T cells that are often administered to recipients after allogeneic stem cell transplantation to provide antiviral and / or antitumor immunity. Donor T cells are often utilized to inhibit bone marrow graft rejection and increase the success of allogeneic engraftment; however, the same donor T cells can elicit an alloaggressive response to host antigens, which can subsequently result in graft-versus-host disease (GvHD). Certain activated donor T cells may elicit a higher or lower GvHD response than other activated T cells. Donor T cells may also be reactive against recipient tumor cells, resulting in a beneficial graft-versus-tumor effect.

[0197] Mesenchymal stromal cells: As used herein, the term "mesenchymal stromal cells" or "bone marrow-derived mesenchymal stromal cells" refers to multipotent stem cells that can differentiate ex vivo, in vitro, and in vivo into adipocytes, osteoblasts, and chondroblasts, and can be further defined as a subset of mononuclear bone marrow cells that adhere to plastic culture dishes under standard culture conditions, are negative for hematopoietic markers, and are positive for CD73, CD90, and CD105.

[0198] Embryonic stem cells: As used herein, the term "embryonic stem cells" refers to pluripotent stem cells derived from the inner cell mass of the blastocyst, an early embryo of 50-150 cells. Embryonic stem cells are characterized by the ability to renew themselves indefinitely and to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. Pluripotent is distinguished from mutipotent in that pluripotent cells can give rise to all cell types, while multipotent cells (e.g., adult stem cells) can only give rise to a limited number of cell types.

[0199] Induced pluripotent stem cells: As used herein, the term "induced pluripotent stem cells" or "induced pluripotent stem cells" refers to adult or differentiated cells that have been "reprogrammed" or induced by genetic manipulation (e.g., expression of genes that subsequently activate pluripotency), biological manipulation (e.g., treatment with viruses or retroviruses), and / or chemical manipulation (e.g., small molecules, peptides, etc.) to generate cells that can differentiate into many, but not all, cell types, such as embryonic stem cells. Induced pluripotent stem cells are distinguished from embryonic stem cells in that they achieve an intermediately or terminally differentiated state (e.g., skin cells, bone cells, fibroblasts, etc.) and then are induced to dedifferentiate, thereby regaining some or all of their ability to generate multipotent or pluripotent cells.

[0200] CD34 + Cells: As used herein, the term "CD34 + "CD34" refers to a cell that expresses the CD34 protein on its cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as an intercellular adhesion factor, is involved in T cell entry into lymph nodes, and is a member of the "cluster of differentiation" gene family. CD34 may also mediate stem cell attachment to bone marrow, extracellular matrix, or directly to stromal cells. CD34 + The cells are frequently found in the umbilical cord and bone marrow as hematopoietic cells, a subset of mesenchymal stem cells, endothelial progenitor cells, endothelial cells of blood vessels but not lymphatic vessels (except those of the pleura), mast cells, a subpopulation of dendritic cells (factor XIIIa negative) in the stroma of the dermis of the skin and around the adnexa, and cells in certain soft tissue tumors (e.g., alveolar soft part sarcoma, pre-B acute lymphoblastic leukemia (Pre-B-ALL), acute myeloid leukemia (AML), AML-M7, dermatofibrosarcoma protuberans, gastrointestinal stromal tumor, giant cell fibroblastoma, granulocytic sarcoma, Kaposi's sarcoma, liposarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, meningeal hemangiopericytoma, meningioma, neurofibroma, schwannoma, and papillary thyroid carcinoma).

[0201] Gene Expression Vector: As used herein, the terms "gene expression vector," "nucleic acid expression vector," or "expression vector," which may be used interchangeably throughout this document, broadly refer to a nucleic acid molecule (e.g., a plasmid, phage, autonomously replicating sequence (ARS), artificial chromosome, yeast artificial chromosome (e.g., YAC)) that can be replicated within a host cell and utilized to introduce one or more genes into the host cell. The gene introduced on the expression vector can be an endogenous gene (e.g., a gene normally found in the host cell or host organism) or a heterologous gene (e.g., a gene not normally found in the genome or on an extrachromosomal nucleic acid of the host cell or host organism). The gene introduced into a cell by the expression vector can be a native gene or a modified or engineered gene. Gene expression vectors can also be engineered to contain 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences, which can facilitate or enhance efficient transcription of the gene or genes carried on the expression vector. Gene expression vectors are sometimes engineered for replication and / or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors sometimes include a selectable marker for maintaining the vector in the host or recipient cell.

[0202] Developmentally-regulated promoter: As used herein, the term "developmentally-regulated promoter" refers to a promoter that serves as the initial binding site for RNA polymerase, which transcribes genes that are expressed under certain conditions that are controlled, initiated, or influenced by a developmental program or pathway. Developmentally-regulated promoters often have additional regulatory regions in or near the promoter region for binding transcriptional activators or repressors that can affect the transcription of genes that are part of a developmental program or pathway. Developmentally-regulated promoters are sometimes involved in the transcription of genes whose gene products affect the development and differentiation of cells.

[0203] Developmentally differentiated cells: As used herein, the term "developmentally differentiated cells" refers to cells that have undergone a process of developing from a less specialized form to a more specialized form to perform a specific function, often accompanied by the expression of specific genes that are developmentally controlled. Non-limiting examples of developmentally differentiated cells include liver cells, lung cells, skin cells, nerve cells, blood cells, etc. Developmental differentiation changes usually involve changes in gene expression (e.g., changes in the pattern of gene expression), gene rearrangements (e.g., remodeling or chromatin that conceals or exposes silenced or expressed genes, respectively), and sometimes changes in DNA sequence (e.g., differentiation of immune diversity). Cell differentiation during development can be understood as the result of a gene regulatory network. Regulatory genes and their cis-regulatory modules are nodes in a gene regulatory network that receive inputs (e.g., proteins expressed upstream in a developmental pathway or program) and produce outputs elsewhere in the network (e.g., expressed gene products act on other genes downstream in that developmental pathway or program).

[0204] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny of any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.

[0205] As used herein, the term "rapalog" refers to an analog of the natural antibiotic rapamycin. Certain rapalogs in this embodiment have properties such as serum stability, poor affinity for wild-type FRB (and thus the parent protein mTOR, resulting in reduced or eliminated immunosuppressive properties), and relatively high affinity for mutant FRB domains. For commercial purposes, in certain embodiments, the rapalogs have useful memory and production properties. Examples of rapalogs include So,p-dimethoxyphenyl (DMOP)-rapamycin:EC 50 (wt FRB(K2095 T2098 W2101) approx. 1000nM), EC 50 (FRB-KLW approx. 5nM) Luengo JI (95) Chem & Biol 2:471-81; Luengo JI (94) J. Org Chem 59:6512-6513; U.S. Patent No. 6,187,757; R-isopropoxyrapamycin: EC 50 (wt FRB (K2095 T2098 W2101) approximately 300 nM), EC50 (FRB-PLF approximately 8.5 nM); Liberles S (97) PNAS 94: 7825-30; and S-Butanesulfonamidorap (AP23050): EC 50 (wt FRB(K2095 T2098 W2101) approx. 2.7nM), EC 50 (FRB-KTF approximately >200 nM) Bayle (06) Chem & Bio. 13: 99-107, but are not limited thereto.

[0206] The term "FRB" refers to the FKBP12-rapamycin binding (FRB) domain (residues 2015-2114 encoded in mTOR) and analogs thereof. In certain embodiments, FRB variants are provided. Characteristics of FRB analogs or variants are their stability (some variants are less stable than others) and their ability to bind to various rapalogs. In certain embodiments, the FRB analog or variant binds to a C7 rapalog (such as those provided in this application and those referenced in the publications incorporated herein by reference). In certain embodiments, the FRB analog or variant contains an amino acid substitution at position T2098. Based on the crystal structure of a protein bound to rapamycin, there are three key rapamycin-interacting residues that have been most analyzed (K2095, T2098, and W2101). All three mutations result in an unstable protein that can be stabilized in the presence of rapamycin or some rapalogs. This feature can be used to further increase the signal to noise ratio in some applications. Examples of variants are discussed in Bayle et al. (06) Chem & Bio 13: 99-107; Stankunas et al. (07) Chembiochem 8:1162-1169; and Liberles S (97) PNAS 94:7825-30). Examples of FRB variant polypeptide regions in this embodiment include, but are not limited to, KLW (having L2098); KTF (having F2101); and KLF (L2098, F2101). The FRB variant KLW is a FRB variant. L It corresponds to a polypeptide (e.g., consisting of the amino acids of SEQ ID NO: 303) with a substitution of an L residue at position 2098. By comparing the KLW variant of SEQ ID NO: 303 with a wild-type FRB polypeptide (e.g., a polypeptide consisting of the amino acid sequence of SEQ ID NO: 304), the sequences of other FRB variants listed herein can be determined.

[0207] Each ligand can include two or more moieties (e.g., defined moieties, distinct moieties), and sometimes includes two, three, four, five, six, seven, eight, nine, ten, or more moieties. The first and second ligands can each independently consist of two moieties (i.e., a dimer), three moieties (i.e., a trimer), or four moieties (i.e., a tetramer). The first ligand sometimes includes a first moiety and a second moiety, and the second ligand sometimes includes a third moiety and a fourth moiety. The first and second moieties are often different (i.e., heterogeneous, e.g., heterodimers), the first and third moieties are sometimes different and sometimes the same, and the third and fourth moieties are often the same (i.e., homogeneous, e.g., homodimers). The different moieties sometimes have different functions (e.g., bind to the first multimerization domain, bind to the second multimerization domain, do not significantly bind to the first multimerization domain, do not significantly bind to the second multimerization domain (e.g., the first moiety binds to the first multimerization domain but does not significantly bind to the second multimerization domain)) and sometimes have different chemical structures. The different moieties sometimes have different chemical structures but can bind to the same multimerization domain (e.g., the second moiety and the third moiety can bind to the second multimerization domain but can have different structures). The first moiety sometimes binds to the first multimerization domain and sometimes does not significantly bind to the second multimerization domain. Each moiety is sometimes referred to as a "monomer" (e.g., the first, second, third, and fourth monomers that track the first, second, third, and fourth moieties, respectively). Each moiety is sometimes referred to as a "flank." The flanks of a ligand can sometimes be adjacent to each other and sometimes be located at opposite positions on the ligand.

[0208] By "capable of binding," as in the example of a multimeric or heterodimeric ligand that binds to a multimerization domain or a ligand-binding domain, it is meant that the ligand binds to the ligand-binding domain, e.g., one or more portions of the ligand bind to the multimerization domain, and that this binding can be detected by an assay method, including, but not limited to, a biological assay, a chemical assay, or a physical detection means (e.g., x-ray crystallography, etc.). Furthermore, when a ligand is considered to "not bind significantly," it means that although there may be slight detection of binding of the ligand to the ligand-binding domain, the amount or stability of this binding is not significantly detectable, and, when occurring in the cells of this embodiment, does not activate the modified cells or cause apoptosis. In certain examples, when the ligand does not "significantly bind" upon administration of the ligand, the amount of cells that undergo apoptosis is less than 10%, 5%, 4%, 3%, 2%, or 1%.

[0209] A "region" or "domain," when referring to a chimeric polypeptide of the present application, refers to a polypeptide or a fragment thereof that maintains the function of the polypeptide. For example, an FKBP12-binding domain, an FKBP12 domain, an FKBP12 region, an FKBP12 multimerization region, etc., refers to an FKBP12 polypeptide that binds to a CID ligand (e.g., rimizuside or rapamycin) to cause or enable dimerization or multimerization of the chimeric polypeptide. A "region" or "domain" of a pro-apoptotic polypeptide, such as a caspase-9 polypeptide or truncated caspase-9 polypeptide of the present application, means that upon dimerization or multimerization of the chimeric polypeptide, or the caspase-9 region as part of the chimeric pro-apoptotic polypeptide, the dimerized or multimerized chimeric polypeptide can participate in the caspase cascade and enable or cause apoptosis.

[0210] As used herein, the term "iCaspase-9" molecule, polypeptide, or protein is defined as inducible caspase-9. The term "iCaspase-9" encompasses iCaspase-9 nucleic acids, iCaspase-9 polypeptides, and / or iCaspase-9 expression vectors. The term also encompasses either native iCaspase-9 nucleotide or amino acid sequences, or truncated sequences lacking the CARD domain.

[0211] As used herein, the terms "iCaspase-1 molecule," "iCaspase-3 molecule," or "iCaspase-8 molecule" are defined as inducible caspase-1, 3, or 8, respectively. The terms iCaspase-1, iCaspase-3, or iCaspase-8 encompass iCaspase-1, 3, or 8 nucleic acids, iCaspase-1, 3, or 8 polypeptides, and / or iCaspase-1, 3, or 8 expression vectors, respectively. The terms also encompass either native caspase iCaspase-1, -3, or -8 nucleotide or amino acid sequences, respectively, or truncated sequences lacking the CARD domain. In the context of the experimental details provided herein, "wild-type" caspase-9 refers to a caspase-9 molecule lacking the CARD domain.

[0212] The modified caspase-9 polypeptide may be used to enhance basal activity or IC in chimeric polypeptides containing the modified caspase-9 polypeptide. 50 Contains at least one amino acid substitution affecting basal activity and IC 50 Methods for testing for this are discussed herein. Unmodified caspase-9 polypeptides do not contain this type of amino acid substitution. Both modified and unmodified caspase-9 polypeptides can be truncated, for example, to remove the CARD domain.

[0213] A "function-conservative variant" is a protein or enzyme in which a given amino acid residue has been altered without changing the overall conformation and function of the protein or enzyme, including, but not limited to, replacing an amino acid with an amino acid having similar properties, including polar or non-polar nature, size, shape, and charge. Conservative amino acid substitutions for many of the commonly known non-genetically encoded amino acids are well known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on their physical properties compared to those of the genetically encoded amino acids.

[0214] Amino acids other than those designated as conserved amino acids may differ in proteins or enzymes, and the percent similarity of protein or amino acid sequences between any two proteins with similar functions may vary, e.g., at least 70%, at least 80%, at least 90%, and at least 95%, as determined according to an alignment scheme. As referred to herein, "sequence similarity" refers to the degree to which nucleotide or protein sequences are related. The degree of similarity between two sequences may be based on the percent sequence identity and / or conservation. "Sequence identity" herein refers to the degree to which two nucleotide or amino acid sequences are invariant. "Sequence alignment" refers to the process of aligning two or more sequences to achieve the maximum level of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity / identity are known in the art (e.g., the Cluster method, in which similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA). When using any of these programs, settings can be selected as those that result in the highest sequence similarity.

[0215] The amino acid residue numbers referred to herein reflect the amino acid positions in a non-truncated and unmodified caspase-9 polypeptide (e.g., that of SEQ ID NO: 9). SEQ ID NO: 9 provides the amino acid sequence of a truncated caspase-9 polypeptide, which does not include the CARD domain. Thus, SEQ ID NO: 9 begins at amino acid residue 135 and ends at amino acid residue 416 with reference to the full-length caspase-9 amino acid sequence. One skilled in the art can align the sequence with other sequences of caspase-9 polypeptides and, if desired, correlate the amino acid residue numbers using, for example, the sequence alignment methods discussed herein.

[0216] As used herein, the term "cDNA" is intended to refer to the DNA prepared by using messenger RNA (mRNA) as a template.The advantage of using cDNA as opposed to genomic DNA or DNA polymerized from genomic RNA template, unprocessed RNA template or partially processed RNA template is that cDNA mainly contains the coding sequence of corresponding protein.There are cases where complete or partial genomic sequence is used, for example, when non-coding region is required for optimal expression, or when non-coding region such as intron is targeted in antisense strategy.

[0217] As used herein, the term "expression construct" or "transgene" is defined as any type of genetic construct containing a nucleic acid encoding a gene product, where part or all of the nucleic acid coding sequence can be transcribed, and the construct can be inserted into a vector. The transcript can be, but is not necessarily, translated into a protein. In certain embodiments, expression includes both transcription of the gene and translation of mRNA into a gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest. The term "therapeutic construct" can also be used to refer to an expression construct or transgene. An expression construct or transgene can be used, for example, as a therapy to treat a hyperproliferative disease or disorder (e.g., cancer), and thus the expression construct or transgene is a therapeutic or prophylactic construct.

[0218] As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product capable of being transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, for example, in the production of antisense molecules or ribozymes, these sequences are not translated. Expression vectors can contain various regulatory sequences, which refer to nucleic acid sequences necessary to transcribe and possibly translate an operably linked coding sequence in a particular host organism. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that perform other functions as well, which are discussed below.

[0219] As used herein, the term "ex vivo" refers to "outside" the body. The terms "ex vivo" and "in vitro" may be used interchangeably herein.

[0220] As used herein, the term "functionally equivalent" when referring to caspase-9 or truncated caspase-9 refers to, for example, a caspase-9 nucleic acid fragment, variant, or analog, and refers to a caspase-9 polypeptide or a nucleic acid encoding a caspase-9 polypeptide that stimulates an apoptotic response. "Functionally equivalent" refers, for example, to a caspase-9 polypeptide that lacks the CARD domain but can induce an apoptotic cellular response. When applied to other nucleic acids or polypeptides (e.g., CD19, 5'LTR, multimeric ligand-binding region, or CD3, etc.), it refers to fragments, variants, etc. that have the same or similar activity as the reference polypeptide in the methods herein.

[0221] As used herein, the term "gene" is defined as a functional protein, polypeptide, or peptide-encoding unit. As will be understood, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express or are adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants.

[0222] The term "hyperproliferative disease" is defined as a disease caused by excessive cell proliferation. Exemplary hyperproliferative diseases include, but are not limited to, cancer or autoimmune diseases. Other hyperproliferative diseases may include vascular occlusion, restenosis, atherosclerosis, or inflammatory bowel disease.

[0223] The term "immunogenic composition" or "immunogen" refers to a substance capable of eliciting an immune response. Examples of immunogens include, for example, antigens, autoantigens that play a role in the induction of autoimmune disease, and tumor-associated antigens expressed on cancer cells.

[0224] The term "immunocompromised" as used herein is defined as a subject with a weakened or weakened immune system. The immunocompromised state may be due to immune system deficiencies or dysfunction or other factors that increase susceptibility to infection and / or disease. While such classifications provide a conceptual basis for assessment, immunocompromised individuals often do not fit completely into one group or the other. More than one deficiency in the body's defense mechanisms may be affected. For example, an individual with a specific T-lymphocyte deficiency caused by HIV may also have neutropenia caused by drugs used for antiviral treatment, or may be immunocompromised because the integrity of the skin and mucous membranes has been breached. The immunocompromised state may be due to other types of dysfunction resulting from indwelling central lines or intravenous drug abuse; or may be caused by secondary malignancies, malnutrition, or infection with other infectious agents (e.g., tuberculosis) or sexually transmitted diseases such as syphilis or hepatitis.

[0225] As used herein, the term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans.

[0226] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells presented herein, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0227] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. A nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., conventional cloning techniques and PCR. TM These include, but are not limited to, cloning nucleic acid sequences from recombinant libraries or cell genomes using techniques such as gene expression, as well as synthetic means. Additionally, polynucleotides include mutations of polynucleotides, including, but not limited to, mutations of nucleotides or nucleosides by methods well known in the art. A nucleic acid can comprise one or more polynucleotides.

[0228] As used herein, the term "polypeptide" is defined as a chain of amino acid residues, usually having a defined sequence. As used herein, the term polypeptide is interchangeable with the terms "peptide" and "protein."

[0229] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a gene.

[0230] The terms "transfection" and "transduction" are used interchangeably and refer to the process by which exogenous DNA sequences are introduced into eukaryotic host cells. Transfection (or transduction) can be accomplished by any one of several means, including electroporation, microinjection, biolistic delivery, retroviral infection, lipofection, superfection, etc.

[0231] As used herein, the term "syngeneic" refers to cells, tissues, or animals having identical genotypes, or genotypes that are closely related enough to allow tissue transplantation, or immunologically compatible genotypes. For example, identical twins or animals of the same inbred strain. Syngeneic and isogenic may be used interchangeably.

[0232] The terms "patient" or "subject" are interchangeable and, as used herein, refer to organisms or animals; including, but not limited to, mammals (including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep or other non-human mammals); non-mammals (including, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates).

[0233] "T cell activation molecule" refers to a polypeptide that enhances T cell activation when incorporated into T cells expressing a chimeric antigen receptor. Examples include, but are not limited to, ITAM-containing molecules that confer signal 1 (e.g., CD3ζ polypeptide) and Fc receptor gamma (e.g., Fc epsilon receptor gamma (FcεR1γ) subunit) (Haynes, NM et al., J. Immunol. 166:182-7 (2001)). J. Immunology).

[0234] As used herein, the term "under transcriptional control" or "operably linked" is defined as a promoter being in the correct position and orientation relative to a nucleic acid that controls initiation of RNA polymerase and expression of a gene.

[0235] As used herein, the terms "treatment," "treat," "treated," or "treating" refer to prophylaxis and / or therapy.

[0236] As used herein, the term "vaccine" refers to a formulation containing the composition presented herein in a form that can be administered to an animal. Typically, a vaccine contains a conventional saline or buffered aqueous solution medium in which the composition is suspended or dissolved. In this form, the composition can be conveniently used to prevent, ameliorate, or otherwise treat a condition. When introduced into a subject, a vaccine can induce an immune response, including, but not limited to, the production of antibodies, cytokines, and / or other cellular responses.

[0237] In some embodiments, the nucleic acid is contained within a viral vector. In certain embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is an adenoviral vector or a lentiviral vector. It is understood that in some embodiments, the antigen-presenting cells are contacted with the viral vector ex vivo, and in some embodiments, the antigen-presenting cells are contacted with the viral vector in vivo.

[0238] Hematopoietic Stem Cells and Cell Therapy Hematopoietic stem cells include hematopoietic progenitor cells, immature multipotent cells that can differentiate into mature blood cell types. These stem and progenitor cells can be isolated from bone marrow and umbilical cord blood, and in some cases, from peripheral blood. Other stem and progenitor cells include, for example, mesenchymal stromal cells, embryonic stem cells, and induced pluripotent stem cells.

[0239] Bone marrow-derived mesenchymal stromal cells (MSCs) have been defined as a fraction of mononuclear bone marrow cells that adhere to plastic culture dishes under standard culture conditions, are negative for hematopoietic lineage markers, positive for CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, osteoblasts, and chondroblasts. While one physiological role is presumed to be supporting hematopoiesis, several reports have also established that MSCs can integrate and possibly proliferate in areas of active growth (e.g., scar tissue and neoplastic tissue), home to their native microenvironment, and replace the function of pathological cells. Their differentiation potential and homing ability make MSCs attractive vehicles for cell therapy, either for regenerative applications in their native form or through their genetic modification for the delivery of active biological factors to specific microenvironments (e.g., pathological bone marrow or metastatic deposits). Furthermore, MSCs have potent intrinsic immunosuppressive activity, and to date their most frequent application has found itself in the experimental treatment of graft-versus-host disease and autoimmune disorders (Pittenger, MF et al. (1999). Science 284: 143-147; Dominici, M. et al. (2006). Cytotherapy 8: 315-317; Prockop, DJ (1997). Science 276: 71-74; Lee, RH et al. (2006). Proc Natl Acad Sci USA 103: 17438-17443; Studeny, M. et al. (2002). Cancer Res 62: 3603-3608; Studeny, M. et al. (2004). J Natl Cancer Inst 96: 1593-1603; Horwitz, EM et al. (1999). Nat Med 5: 309-313; Chamberlain, G. et al., (2007). Stem Cells 25: 2739-2749; Phinney, DG, and Prockop, DJ (2007). Stem Cells 25: 2896-2902; Horwitz, EM et al. (2002). Proc Natl Acad Sci USA 99: 8932-8937; Hall, B. et al. (2007). Int J Hematol 86: 8-16; Nauta, A. J., and Fibbe, W.E. (2007). Blood 110: 3499-3506; Le Blanc, K. et al. (2008). Lancet 371: 1579-1586; Tyndall, A., and Uccelli, A. (2009). Bone Marrow Transplant).

[0240] MSCs have been infused in hundreds of patients with minimal reported side effects. However, follow-up is limited, long-term side effects are unknown, and the consequences of genetically modifying MSCs to induce their in vivo differentiation (e.g., into cartilage or bone) or enhance their functionality are largely unknown. Some animal models have raised safety concerns. For example, spontaneous osteosarcoma formation in culture has been observed in mouse-derived MSCs. Furthermore, heterotopic ossification and calcification lesions have been reported after local injection of MSCs in mouse and rat models of myocardial infarction, and their proarrhythmic potential has also been evident in coculture experiments with neonatal rat ventricular myocytes. Furthermore, bilateral diffuse pulmonary bone formation has been observed after bone marrow transplantation in dogs, likely due to transplanted stromal components (Horwitz, E. M. et al. (2007). Biol Blood Marrow Transplant 13: 53-57; Tolar, J. et al. (2007). Stem Cells 25: 371-379; Yoon, Y.-S. et al. (2004). Circulation 109: 3154-3157; Breitbach, M. et al. (2007). Blood 110: 1362-1369; Chang, M. G. et al. (2006). Circulation 113: 1832-1841; Sale, G.E., and Storb, R. (1983). Exp Hematol 11: 961-966).

[0241] In another example of cell therapy, T cells transduced with nucleic acids encoding chimeric antigen receptors have been administered to patients to treat cancer (Zhong, X.-S., (2010) Molecular Therapy 18:413-420). Chimeric antigen receptors (CARs) are artificial receptors designed to convey antigen specificity to T cells without the need for MHC antigen presentation. They contain an antigen-specific component, a transmembrane component, and an intracellular component selected to activate T cells and provide specific immunity. Chimeric antigen receptor-expressing T cells can be used in a variety of therapies, including cancer therapy. Costimulatory polypeptides can be used to enhance the activation of CAR-expressing T cells against target antigens, thus increasing the efficacy of adoptive immunotherapy.

[0242] For example, T cells expressing chimeric antigen receptors based on the humanized monoclonal antibody trastuzumab (Herceptin) have been used to treat cancer patients. While adverse events are possible, in at least one reported case, this treatment resulted in a fatal outcome for the patient (Morgan, RA et al., (2010) Molecular Therapy 18:843-851). Transducing cells with a chimeric caspase-9-based safety switch as described herein provides a safety switch that can stop adverse events from progressing. Thus, in some embodiments, nucleic acids, cells, and methods are provided in which the modified T cells also express an inducible caspase-9 polypeptide. For example, if there is a need to reduce the number of chimeric antigen receptor-modified T cells, an inducible ligand can be administered to the patient, thereby inducing apoptosis of the modified T cells.

[0243] The antitumor efficacy of immunotherapy with T cells engineered to express chimeric antigen receptor (CAR) molecules has steadily improved when additional signaling domains have been incorporated into the CAR molecules to enhance their potency. Because tumor cells often lack essential costimulatory molecules required for full T cell activation, T cells transduced with first-generation CARs containing only the CD3ζ intracellular signaling molecule clearly exhibit poor persistence and expansion in vivo after adoptive transfer (Till BG, Jensen MC, Wang J, et al.: CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. Blood 119:3940-50, 2012; Pule MA, Savoldo B, Myers GD, et al.: Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma. Nat Med 14:1264-70, 2008; Kershaw MH, Westwood JA, Parker LL, et al.: A phase I study on adoptive immunotherapy using gene-modified T cells for (ovarian cancer. Clin Cancer Res 12:6106-15, 2006). Second-generation CAR T cells were designed to improve the proliferation and survival of these cells. Second-generation CAR T cells incorporating intracellular costimulatory domains derived from CD28 or 4-1BB (Carpenito C, Milone MC, Hassan R, et al.: Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA 106:3360-5, 2009; Song DG, Ye Q, Poussin M, et al.: CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo. Blood 119:696-706, 2012) have shown improved survival and in vivo proliferation after adoptive transfer, and more recent clinical trials using T cells engineered with anti-CD19 CARs containing these costimulatory molecules have demonstrated improved survival and in vivo proliferation. + Remarkable efficacy has been demonstrated for the treatment of leukemia (Kalos M, Levine BL, Porter DL, et al.: T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Sci Transl Med 3:95ra73,2011;Porter DL,Levine BL,Kalos M et al.:Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med 365:725-33,2011;Brentjens RJ,Davila ML,Riviere I et al.:CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci Transl Med 5:177ra38,2013).

[0244] Other researchers have explored additional signaling molecules derived from tumor necrosis factor (TNF) family proteins (e.g., OX40 and 4-1BB), termed "third-generation" CAR T cells (Finney HM, Akbar AN, Lawson AD: Activation of resting human primary T cells With chimeric receptors (costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain. J Immunol 172:104-13, 2004; Guedan S, Chen X, Madar A, et al.: ICOS-based chimeric antigen receptors program bipolar TH17 / TH1 cells. Blood, 2014), other molecules that induce distinct T cell signaling pathways, such as the CD3 zeta nuclear factor-activated T cell (NFAT) pathway, may provide the costimulation necessary for T cell survival and proliferation, potentially endowing CAR T cells with additional valuable functions not provided by conventional costimulatory molecules. Some second- and third-generation CAR T cells have been implicated in patient deaths due to cytokine storms and tumor lysis syndrome caused by highly activated T cells.

[0245] "Chimeric antigen receptor" or "CAR" refers to a chimeric polypeptide comprising a polypeptide sequence that recognizes a target antigen (antigen recognition domain) linked to a transmembrane polypeptide and an intracellular domain polypeptide selected, for example, to activate T cells and confer specific immunity. The antigen recognition domain can be a single-chain variable fragment (ScFv) or can be derived, for example, from other molecules (e.g., T cell receptors or pattern recognition receptors). The intracellular domain contains at least one polypeptide that triggers T cell activation (e.g., but not limited to, CD3 zeta, and costimulatory molecules such as CD28, OX40, and 4-1BB). The term "chimeric antigen receptor" can also refer to chimeric receptors that are derived from chimeric T cell receptors rather than antibodies. These chimeric T cell receptors can comprise a polypeptide sequence that recognizes a target antigen, where the recognition sequence can be, for example, but not limited to, a recognition sequence derived from a T cell receptor or scFv. The intracellular domain polypeptide is a polypeptide that acts to activate T cells. Chimeric T cell receptors are discussed, for example, in Gross, G., and Eshar, Z., FASEB Journal 6:3370-3378 (1992) and Zhang, Y. et al., PLOS Pathogens 6:1-13 (2010).

[0246] In one type of chimeric antigen receptor (CAR), the variable heavy (VH) and light (VL) chains of a tumor-specific monoclonal antibody are fused in-frame with the CD3 zeta chain (ζ) derived from the T cell complex. The VH and VL are typically connected to each other using a glycine-serine flexible linker, and then a spacer (CH2CH3) is attached to the transmembrane domain, extending the scFv away from the cell surface so that it can interact with tumor antigens. After transduction, T cells now express the CAR on their surface, which, upon contact and ligation with tumor antigens, signals via the CD3 zeta chain, inducing cytotoxicity and cell activation.

[0247] Researchers have noted that CD3 zeta-mediated T cell activation is sufficient to induce tumor-specific killing, but not sufficient to induce T cell proliferation and survival. Early clinical trials using T cells engineered with first-generation CARs expressing only the zeta chain showed that the genetically engineered T cells exhibited poor survival and proliferation in vivo.

[0248] Because costimulation via the B7 axis is necessary for complete T cell activation, researchers have added the costimulatory polypeptide CD28 signaling domain to CAR constructs. This region typically contains a transmembrane domain (instead of the CD3 zeta version) and a YMNM motif for binding to PI3K and Lck. In vivo comparisons between T cells expressing CARs bearing only zeta or both zeta and CD28 demonstrated that CD28 promotes proliferation in vivo, partly due to increased IL-2 production after activation. Those containing CD28 are called second-generation CARs. The most commonly used costimulatory molecules include CD28 and 4-1BB, which can initiate a signaling cascade that activates NF-κB after tumor recognition, promoting both T cell proliferation and cell survival.

[0249] The use of costimulatory polypeptides 4-1BB or OX40 in CAR design further improved T cell survival and efficacy. 4-1BB, in particular, appears to significantly enhance T cell proliferation and survival. This third-generation design (with three signaling domains) has been used in PSMA CARs (Zhong XS et al., Mol Ther. 2010 Feb;18(2):413-20) and CD19 CARs, most notably for the treatment of CLL (Milone, MC et al. (2009) Mol. Ther. 17:1453-1464; Kalos, M. et al., Sci. Transl. Med. (2011) 3:95ra73; Porter, D. et al. (2011) N. Engl. J. Med. 365:725-533). These cells expanded over 1000-fold in vivo and demonstrated remarkable function in three patients, resulting in sustained remission in all three patients.

[0250] It is understood that "derived from" means that the nucleotide sequence or amino acid sequence can be derived from the sequence of the molecule. The intracellular domain contains at least one polypeptide that causes T cell activation (such as, but not limited to, CD3 zeta) and, for example, a costimulatory molecule (such as, but not limited to, CD28, OX40, and 4-1BB).

[0251] T cell receptors are molecules composed of two different polypeptides present on the surface of T cells. They recognize antigens bound to major histocompatibility complex molecules; upon recognition, the T cell is activated. "Recognize" means, for example, that a T cell receptor or its fragment(s), such as a TCRα polypeptide and a TCRβ polypeptide, together contacts the antigen and can identify it as a target. A TCR can comprise α and β polypeptides, or chains. The α and β polypeptides comprise two extracellular domains, a variable domain, and a constant domain. The variable domains of the α and β polypeptides have three complementarity-determining regions (CDRs); CDR3 is considered to be the primary CDR responsible for epitope recognition. The α polypeptide comprises a V region and a J region generated by VJ recombination, while the β polypeptide comprises a V region, a D region, and a J region generated by VDJ recombination. The intersection of the VJ and VDJ regions corresponds to the CDR3 region. TCRs are often named using the International Immunogenetics (IMGT) TCR nomenclature (IMGT database, www.IMGT.org; Giudicelli, V. et al., IMGT / LIGM-DB, IMGT® Comprehensive Database of Immunoglobulin and T Cell Receptor Nucleotide Sequences, Nucl. Acids Res., 34, D781-D784 (2006). PMID: 16381979; T Cell Receptor Factsbook, LeFranc and LeFranc, Academic Press ISBN 0-12-441352-8).

[0252] Chimeric T cell receptors can bind, for example, antigenic polypeptides (e.g., Bob-1, PRAME, and NY-ESO-1) (U.S. Patent Application No. 14 / 930,572, filed November 2, 2015, entitled "T Cell Receptors Directed Against Bob1 and Uses Thereof," and U.S. Provisional Patent Application No. 62 / 130,884, filed March 10, 2015, entitled "T Cell Receptors Directed Against the Preferentially-Expressed Antigen of Melanoma and Uses Thereof"), each of which is incorporated herein by reference in its entirety.

[0253] In another example of cell therapy, T cells are modified to express non-functional TGF-β receptors, making them resistant to TGF-β. This allows the modified T cells to avoid cytotoxicity caused by TGF-β, allowing the cells to be used in cell therapy (Bollard, CJ et al., (2002) Blood 99:3179-3187; Bollard, CM et al., (2004) J. Exptl. Med. 200:1623-1633). However, it can also result in T cell lymphoma or other adverse effects because the modified T cells now lack some of the normal cellular controls; these therapeutic T cells may themselves become malignant. Transducing these modified T cells with a chimeric caspase-9-based safety switch as presented herein provides a safety switch that can avoid this outcome.

[0254] In another example, natural killer cells are modified to express a membrane-targeted polypeptide. Instead of a chimeric antigen receptor, in certain embodiments, the heterologous membrane-bound polypeptide is an NKG2D receptor. The NKG2D receptor can bind to stress proteins (e.g., MICA / B) on tumor cells, thereby activating NK cells. The extracellular binding domain can also be fused to a signaling domain (Barber, A. et al., Cancer Res 2007;67:5003-8; Barber A et al., Exp Hematol. 2008; 36:1318-28; Zhang (T. et al., Cancer Res. 2007; 67:11029-36.), which in turn can be linked to an FRB domain similar to FRB-linked CAR. Furthermore, other cell surface receptors (e.g., VEGF-R) can be used as docking sites for the FRB domain to enhance tumor-dependent clustering in the presence of hypoxia-induced VEGF, which is found at high levels in many tumors.

[0255] Cells used in cell therapy that express a heterologous gene (e.g., a modified or chimeric receptor) can be transduced with a nucleic acid encoding a chimeric caspase-9-based safety switch before, after, or simultaneously with transduction of the cells with the heterologous gene.

[0256] (Haploidentical stem cell transplantation) While stem cell transplantation has proven an effective means of treating a wide variety of diseases involving hematopoietic stem cells and their progeny, the shortage of histocompatible donors has proven a major obstacle to the widest application of this approach. The introduction of large pools of unrelated stem cell donors and / or umbilical cord blood banks has helped to alleviate the problem, but many patients remain unmatched by either source. Even when a matched donor can be found, the time elapsed between the initiation of the search and the collection of stem cells typically exceeds three months, a delay that can doom many of the most needy patients. Therefore, there is considerable importance in utilizing HLA-haploidentical familial donors. Such donors can be parents, siblings, or second-degree relatives. The problem of transplant rejection can be overcome by appropriate conditioning and the combination of large doses of stem cells, while graft-versus-host disease (GvHD) can be prevented by extensive T-cell depletion of the donor graft. The immediate results of such procedures are gratifying, with engraftment rates >90% and severe GvHD rates <10% for both adults and children, even in the absence of post-transplant immunosuppression. Unfortunately, the substantial immunosuppression of the transplant procedure, coupled with extensive T-cell depletion and HLA mismatch between donor and recipient, results in an extremely high rate of post-transplant infectious complications and contributes to a high incidence of disease recurrence.

[0257] Donor T cell infusion is an effective strategy for conferring antiviral and antitumor immunity after allogeneic stem cell transplantation. However, simply adding back T cells to patients after haploidentical transplantation is not functional; the frequency of alloreactive T cells is several orders of magnitude higher than the frequency of, for example, virus-specific T lymphocytes. Methods are being developed to promote immune reconstitution by administering donor T cells that have first been depleted of alloreactive cells. One way to achieve this is to stimulate donor T cells with the recipient's EBV-transformed B lymphoblastoid cell line (LCL). Alloreactive T cells upregulate CD25 expression and are eliminated by the CD25 MAb immunotoxin conjugate, RFT5-SMPT-dgA. This compound consists of murine IgG1 anti-CD25 (IL-2 receptor α chain) conjugated to chemically deglycosylated ricin A chain (dgA) via a heterobifunctional cross-linker [N-succinimidyloxycarbonyl-α-methyl-d-(2-pyridylthio)toluene].

[0258] Treatment with CD25 immunotoxin after LCL stimulation depletes >90% of alloreactive cells. In a phase I clinical study, CD25 immunotoxin was used to deplete alloreactive lymphocytes, and allodepleted, reconstituted donor T cells were infused at two dose levels into recipients of T cell-depleted haploidentical SCT. Eight patients were randomly assigned to receive 10 4 cells / kg / dose, and 8 patients were treated with 10 5 cells / kg / dose given. 10 5 Patients receiving cells / kg / dose were 10 4Compared with patients receiving 100 cells / kg / dose, patients showed significantly improved T cell recovery at 3, 4, and 5 months after SCT (P<0.05). The accelerated T cell recovery occurred as a result of the expansion of effector memory (CD45RA(-)CCR-7(-)) populations (P<0.05), suggesting that protective T cell responses are likely to be long-lived. T cell receptor signal joint excision circles (TRECs) were not detected in T cell reconstitution in patients at dose level 2, indicating that they likely originated from the infused allogeneically depleted cells. Spectratyping of T cells at 4 months revealed a polyclonal Vβ repertoire. Using tetramer and enzyme-linked immunospot (ELISpot) assays, cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-specific responses were observed as early as 2–4 months after transplant in four of six evaluable patients at dose level 2, whereas such responses were not observed until 6–12 months in patients at dose level 1. The incidence of significant acute graft-versus-host disease (2 of 16) and chronic graft-versus-host disease (GvHD; 2 of 15) was low. These data indicate that allogeneic depleted donor T cells can be safely used to improve T-cell recovery after haploidentical SCT. The amount of cells infused was subsequently increased to 10% without evidence of GvHD. 6 cells / kg.

[0259] Although this approach reconstituted antiviral immunity, relapse remained a major problem, and six patients died of disease after transplant due to high-risk leukemia relapse. Therefore, higher T cell doses are useful for reconstituting antitumor immunity and providing the desired antitumor effect, because the estimated frequency of tumor-reactive precursors is 1-2 logs lower than the frequency of virus-reactive precursors. However, in some patients, these doses of cells are sufficient to induce GvHD even after allogeneic depletion (Hurley et al., 2011). CK et al., Biol Blood Marrow Transplant 2003;9:610-615; Dey BR et al., Br.J Haematol. 2006;135:423-437; Aversa F et al., N Engl J Med 1998;339:1186-1193; Aversa F et al., JC lin.On col. 2005;23:3447-3454; Lang P, Mol.Dis. 2004;33:281-287; Kolb HJ et al., Blood 2004;103:767-776; Gottschalk S et al., Annu.Rev.Med 2005;56:29-44; Bleakley M et al., Nat.Rev.Cancer 2004;4:371-380; Andre-Schmutz I et al., Lancet 2002;360:130-137; Solomon SR et al., Blood 2005;106:1123-1129; Amrolia PJ et al., Blood 2006;108:1797-1808; Amrolia PJ et al., Blood 2003; Ghetie V et al., J Immunol Methods 1991;142:223-230; Molldrem JJ et al., Cancer Res 1999;59:2675-2681; Rezvani K et al., CIin.Cancer Res. 2005;1 1:8799-8807; Rezvani K et al., Blood 2003;102:2892-2900).

[0260] (Graft-versus-host disease (GvHD)) Graft-versus-host disease (GVHD) is a condition that sometimes occurs after transplantation of donor immunocompetent cells (e.g., T cells) into a recipient. The transplanted cells recognize the recipient's cells as foreign and attack and destroy them. This condition can be a dangerous effect of T cell transplantation, especially when associated with haploidentical stem cell transplantation. Sufficient T cells should be infused to provide beneficial effects (e.g., immune system reconstitution and antitumor effects of the graft). However, the number of T cells that can be transplanted may be limited by concerns that the transplant will result in severe GVHD.

[0261] Graft-versus-host disease can be staged as shown in the following table: [Table 12]

[0262] Acute GvHD grading can be performed according to the consensus conference criteria (Przepiorka D et al., 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825-828). [Table 13]

[0263] Inducible caspase-9 as a "safety switch" for cell therapy and genetically engineered cell transplantation

[0264] Reducing the effects of graft-versus-host disease means a reduction in the symptoms of GvHD, or, for example, a reduction in the symptoms of graft-versus-host disease by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, such that, for example, a patient can be assigned to a lower level stage. Reducing the effects of graft-versus-host disease also means a reduction in activated T cells involved in the GvHD response (e.g., cells expressing marker proteins (e.g., CD19) and cells expressing CD3 (e.g., CD3+ CD19 + The expression level may be measured by detection of at least a 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction in the number of cells.

[0265] Provided herein is an alternative suicide gene strategy based on a human pro-apoptotic molecule fused to an FKBP variant optimized for binding a chemical inducer of dimerization (CID). The variant can include, for example, an FKBP region with an amino acid substitution at position 36 selected from the group consisting of valine, leucine, isoleucine, and alanine (Clackson T et al., Proc Natl Acad Sci US A. 1998, 95:10437-10442). AP1903 is a synthetic molecule that has been shown to be safe in healthy volunteers (Iuliucci JD et al., J Clin Pharmacol. 2001, 41:870-879). Administration of this small molecule results in cross-linking and activation of pro-apoptotic target molecules. The application of this inducible system in human T lymphocytes was explored using Fas or the death effector domain (DED) of Fas-associated death domain-containing protein (FADD) as the pro-apoptotic molecule. Up to 90% of T cells transduced with these inducible death molecules underwent apoptosis after administration of CID (Thomis DC et al., Blood. 2001, 97:1249-1257; Spencer DM et al., Curr Biol. 1996, 6: 839-847; Fan L et al., Hum Gene Ther. 1999, 10: 2273-2285; Berger C et al., Blood. 2004, 103: 1261-1269; Junker K et al., Gene Ther. 2003, 10: 1189-197). This suicide gene strategy can be used in any suitable cell used for cell therapy, including hematopoietic stem cells and other precursor cells, such as mesenchymal stromal cells, embryonic stem cells, and induced pluripotent stem cells. AP20187 and AP1950 (synthetic versions of AP1903) can also be used as ligand inducers (Amara JF (97) PNAS 94: 10618-23; Clontech Laboratories-Takara Bio).

[0266] Thus, this safety switch (catalyzed by caspase-9) can be used when a condition in a cell therapy patient requires the removal of transfected or transduced therapeutic cells. Conditions that may require the cells to be removed include, for example, GvHD, inappropriate differentiation of the cells into more mature cells of the wrong tissue or cell type, and other toxicities. It is possible to use a tissue-specific promoter to activate the caspase-9 switch in the event of inappropriate differentiation. For example, if precursor cells differentiate into bone and adipocytes and adipocytes are undesirable, the vector used to transfect or transduce the precursor cells can have an adipocyte-specific promoter operably linked to a caspase-9 nucleotide sequence. Thus, if the cells differentiate into adipocytes upon administration of the multimeric ligand, apoptosis of the inappropriately differentiated adipocytes should occur.

[0267] The methods can be used, for example, for any disorder that can be alleviated by cell therapy, including cancer, cancer of the blood or bone marrow, other blood- or bone marrow-derived diseases (e.g., sickle cell anemia and metachromic leukodystrophy), and any disorder that can be alleviated by stem cell transplantation (e.g., blood or bone marrow disorders such as sickle cell anemia or metachromal leukodystrophy).

[0268] The efficacy of adoptive immunotherapy can be enhanced by rendering therapeutic T cells resistant to immune evasion strategies used by tumor cells. In vitro studies have shown that this can be achieved by transduction with dominant-negative receptors or immunomodulatory cytokines (Bollard CM et al., Blood. 2002, 99:3179-3187; Wagner HJ et al., Cancer Gene Ther. 2004, 11:81-91). Furthermore, the transfer of antigen-specific T cell receptors allows T cell therapy to be applied to a wider range of tumors (Pule M et al., Cytotherapy. 2003, 5:211-226; Schumacher TN, Nat Rev Immunol. 2002, 2:512-519). A suicide system for engineered human T cells was developed and tested to enable the subsequent use of the T cells in clinical studies. Modification of caspase-9 was shown to be stably expressed in human T lymphocytes without impairing their functional and phenotypic properties, even though T cells with upregulated anti-apoptotic molecules were susceptible to CID (Straathof, KC et al., 2005, Blood 105:4248-54).

[0269] In genetically modified cells used for gene therapy, the gene may be a heterologous polynucleotide sequence derived from a source other than the cell used to express the gene. The gene may be derived from a prokaryotic or eukaryotic source, such as bacteria, viruses, yeast, parasites, plants, or even animals. The heterologous DNA may also be derived from more than one source (i.e., a multi-gene construct or fusion protein). The heterologous DNA may also contain a regulatory sequence derived from one source and a gene derived from a different source. Alternatively, the heterologous DNA may contain a regulatory sequence used to alter the normal expression of a cell's endogenous gene.

[0270] (other caspase molecules) Caspase polypeptides other than caspase-9 that can be encoded by state-of-the-art chimeric polypeptides include, for example, caspase-1, caspase-3, and caspase-8. For a discussion of these caspase polypeptides, see, for example, MacCorkle, RA et al., Proc. Natl. Acad. Sci. USA (1998) 95:3655-3660; and Fan, L. et al. (1999) Human Gene Therapy 10:2273-2285).

[0271] Engineering Expression Constructs The expression construct encodes a multimeric ligand-binding region and a caspase-9 polypeptide, or in certain embodiments, a caspase-9 polypeptide linked to a multimeric ligand-binding region and a marker polypeptide (all operably linked). Generally, the term "operably linked" refers to a promoter sequence functionally linked to a second sequence (e.g., where the promoter sequence initiates and mediates transcription of DNA corresponding to the second sequence). The caspase-9 polypeptide may be full-length or truncated. In certain embodiments, the marker polypeptide is linked to the caspase-9 polypeptide. For example, the marker polypeptide may be linked to the caspase-9 polypeptide via a polypeptide sequence (e.g., a cleavable 2A-like sequence). The marker polypeptide may be, for example, CD19, or may be, for example, a heterologous protein, selected so as not to affect the activity of the chimeric caspase polypeptide.

[0272] In some embodiments, the polynucleotide may encode a caspase-9 polypeptide and a heterologous protein (which may be, for example, a marker polypeptide, such as a chimeric antigen receptor). The heterologous polypeptide, e.g., the chimeric antigen receptor, may be linked to the caspase-9 polypeptide via a polypeptide sequence, such as a cleavable 2A-like sequence.

[0273] In certain examples, a nucleic acid comprising a polynucleotide encoding a chimeric antigen receptor is contained within the same vector (e.g., a viral vector or a plasmid vector) as a polynucleotide encoding a second polypeptide. This second polypeptide can be, for example, a caspase polypeptide (as discussed herein) or a marker polypeptide. In these examples, the construct can be designed with a single promoter operably linked to a nucleic acid comprising a polynucleotide encoding the two polypeptides linked by a cleavable 2A polypeptide. In this example, the first and second polypeptides are separated during translation to produce the chimeric antigen receptor polypeptide and the second polypeptide. In other examples, the two polypeptides can be expressed separately from the same vector, where each nucleic acid comprising a polynucleotide encoding one of the polypeptides is operably linked to a separate promoter. In yet other examples, a single promoter can be operably linked to the two nucleic acids and direct the production of two separate RNA transcripts and thus two polypeptides. Thus, the expression constructs discussed herein can comprise at least one or at least two promoters.

[0274] 2A-like sequences, or "cleavable" 2A sequences, are derived, for example, from many different viruses, including Thosea asigna. These sequences are sometimes known as "peptide skipping sequences." When this type of sequence is placed intracistronically between two peptides intended to be cleaved, the ribosome appears to skip the peptide bond; in the case of Thosea asigna sequences, the bond between the Gly and Pro amino acids is omitted. This leaves two polypeptides, in this case the caspase-9 polypeptide and the marker polypeptide. When this sequence is used, the peptide encoded 5' of the 2A sequence can terminate with additional amino acids (including Gly residues) at the carboxy terminus and anywhere upstream in the 2A sequence. The peptide encoded 3' of the 2A sequence can terminate with additional amino acids (including Pro residues) at the amino terminus and anywhere downstream in the 2A sequence. "2A" or "2A-like" sequences are part of a large family of peptides that can cause peptide bond skipping. Various 2A sequences have been characterized (e.g., F2A, P2A, T2A) and are examples of 2A-like sequences that can be used in the polypeptides of the present application. In certain embodiments, the 2A linker comprises the amino acid sequence of SEQ ID NO: 306; in certain embodiments, the 2A linker consists of the amino acid sequence of SEQ ID NO: 306. In some embodiments, the 2A linker comprises the amino acid sequence of SEQ ID NO: 307; in some embodiments, the 2A linker consists of the amino acid sequence of SEQ ID NO: 307. In certain embodiments, the 2A linker further comprises a GSG amino acid sequence at the amino terminus of the polypeptide, and in other embodiments, the 2A linker comprises a GSGPR amino acid sequence at the amino terminus of the polypeptide. Thus, by "2A" sequence, the term may refer to a 2A sequence as enumerated herein, or to a 2A sequence as enumerated herein that further comprises a GSG or GSGPR sequence at the amino terminus of the linker.

[0275] The expression construct can be inserted into a vector (e.g., a viral vector or a plasmid vector). The provided methods can be performed using any suitable method, including, but not limited to, methods for transducing, transforming, or otherwise providing a nucleic acid into an antigen-presenting cell (as provided herein). In some embodiments, the truncated caspase-9 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:8, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or a functionally equivalent fragment thereof, with or without a DNA linker, or has the amino acid sequence of SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28, or a functionally equivalent fragment thereof. In some embodiments, the CD19 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:14, or a functionally equivalent fragment thereof, with or without a DNA linker, or has the amino acid sequence of SEQ ID NO:15, or a functionally equivalent fragment thereof. A functionally equivalent fragment of a caspase-9 polypeptide has substantially the same ability to induce apoptosis as the polypeptide of SEQ ID NO: 9, having at least 50%, 60%, 70%, 80%, 90%, or 95% of the ability of the polypeptide of SEQ ID NO: 9. A functionally equivalent fragment of a CD19 polypeptide has substantially the same ability as the polypeptide of SEQ ID NO: 15, having at least 50%, 60%, 70%, 80%, 90%, or 95% of the marker polypeptide being detected, when compared to the polypeptide of SEQ ID NO: 15 using standard detection techniques, to act as a marker to be used to identify and select transduced or transfected cells.

[0276] More specifically, more than one ligand-binding domain or multimerization region can be used in the expression construct. Still further, the expression construct can include a membrane targeting sequence. Suitable expression constructs can include costimulatory polypeptide elements on either side of the FKBP ligand-binding element.

[0277] In certain instances, the polynucleotide encoding the inducible caspase polypeptide is contained within the same vector (e.g., a viral vector or a plasmid vector) as the polynucleotide encoding the chimeric antigen receptor. In these instances, the construct can be designed with a single promoter operably linked to a nucleic acid comprising nucleotide sequences encoding two polypeptides linked by a cleavable 2A polypeptide. In this example, the first and second polypeptides are cleaved after expression to produce the chimeric antigen receptor polypeptide and the inducible caspase-9 polypeptide. In other instances, the two polypeptides can be expressed separately from the same vector, with each nucleic acid comprising a nucleotide sequence encoding one of the polypeptides operably linked to a separate promoter. In yet other instances, a single promoter can be operably linked to two nucleic acids and direct the production of two separate RNA transcripts, and thus two polypeptides. Thus, the expression constructs discussed herein can comprise at least one or at least two promoters.

[0278] In yet other examples, two polypeptides can be expressed in a cell using two separate vectors, and the cells can be co-transfected or co-transformed with the vectors, or the vectors can be introduced into the cells at different times.

[0279] (ligand binding domain) The ligand-binding ("dimerization") domain or multimerization region of the expression construct can be any convenient domain that can allow induction with natural or non-natural ligands, e.g., non-natural synthetic ligands. The multimerization region can be internal or external to the cell membrane, depending on the nature of the construct and the choice of ligand. A wide variety of ligand-binding proteins are known, including receptors that comprise ligand-binding proteins associated with the cytoplasmic regions shown above. As used herein, the term "ligand-binding domain" can be interchangeable with the term "receptor." Of particular interest are ligand-binding proteins for which the ligand (e.g., small organic ligand) is known or can be easily produced. These ligand-binding domains or receptors include FKBP and cyclophilin receptors, steroid receptors, tetracycline receptors, other receptors set forth above, etc., as well as "unnatural" receptors that can be obtained from antibodies, particularly heavy or light chain subunits, mutated sequences thereof, random amino acid sequences obtained by stochastic procedures, combinatorial synthesis, etc. In certain embodiments, the ligand-binding region is selected from the group consisting of an FKBP ligand-binding region, a cyclophilin receptor ligand-binding region, a steroid receptor ligand-binding region, a cyclophilin receptor ligand-binding region, and a tetracycline receptor ligand-binding region. Often, the ligand-binding region is selected from the group consisting of FKBP and cyclophilin receptor ligand-binding regions, steroid receptor ligand-binding regions, cyclophilin receptor ligand-binding regions, and tetracycline receptor ligand-binding regions. v F vls Contains sequences. Occasionally, F v’ F vlsThe sequence further comprises an additional Fv' sequence. Examples include those discussed in, for example, Kopytek, SJ et al., Chemistry & Biology 7:313-321 (2000) and Gestwicki, JE et al., Combinatorial Chem. & High Throughput Screening 10:667-675 (2007); Clackson T (2006) Chem Biol Drug Des 67:440-2; Clackson, T., in Chemical Biology: From Small Molecules to Systems Biology and Drug Design (Schreiber, S. et al., eds., Wiley, 2007).

[0280] Generally, a ligand-binding or receptor domain, as a naturally occurring domain or a truncated active portion thereof, can be at least about 50 amino acids and less than about 350 amino acids, usually less than 200. The binding domain can be, for example, small (<25 kDa to allow efficient transfection with viral vectors), monomeric, non-immunogenic, and can have synthetically available, cell-permeable, non-toxic ligands that can be formed due to dimerization.

[0281] The receptor domain can be intracellular or extracellular, depending on the design of the expression construct and the availability of an appropriate ligand. For hydrophobic ligands, the binding domain can be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding domain will usually be external to the cell membrane if no transport system exists to internalize the ligand in a form available for binding. For intracellular receptors, the construct can encode a signal peptide and transmembrane domain 5' or 3' to the receptor domain sequence, or can have a lipid attachment signal sequence 5' to the receptor domain sequence. If the receptor domain is located between the signal peptide and the transmembrane domain, the receptor domain can be extracellular.

[0282] Portions of expression constructs encoding receptors can be subjected to mutagenesis for a variety of reasons. Mutagenized proteins can provide higher binding affinity, allow differentiation by ligands between naturally occurring and mutagenized receptors, provide opportunities to design receptor-ligand pairs, etc. Receptor alterations can include random mutagenesis using combinatorial methods in the binding site and known amino acid alterations, where codons for amino acids involved in the binding site or other amino acids involved in conformational changes can be subjected to mutagenesis by altering the codon(s) for a particular amino acid by known changes or randomly, expressing the resulting protein in a suitable prokaryotic host, and then screening the resulting protein for binding.

[0283] Antibodies and antibody subunits, such as heavy or light chains, particularly fragments, more particularly, all or part of the variable region, or fusions of heavy and light chains that result in high-affinity binding, can be used as binding domains.Contemplated antibodies include those that are ectopically expressed human products, such as extracellular domains that do not provoke immune responses and are not generally expressed in the periphery (i.e., outside the CNS / brain region).Such examples include, but are not limited to, low-affinity nerve growth factor receptor (LNGFR) and embryonic surface proteins (i.e., carcinoembryonic antigen).

[0284] Furthermore, antibodies can be prepared against physiologically acceptable hapten molecules, and individual antibody subunits screened for binding affinity. The cDNA encoding the subunits can be isolated and modified by deletion of portions of the constant region, variable region, mutagenesis of the variable region, etc., to obtain binding protein domains with appropriate affinity for the ligand. In this way, almost any physiologically acceptable hapten compound can be used as a ligand or to provide an epitope for the ligand. Instead of antibody units, natural receptors can be used whose binding domains are known and for which useful ligands exist for binding.

[0285] (oligomerization) The transduced signal will usually occur through ligand-mediated oligomerization of the chimeric protein molecule, i.e., as a result of oligomerization after ligand binding, although other binding events, such as allosteric activation, can be used to initiate the signal. The construction of the chimeric protein can vary with regard to the order of the various domains and the number of repeats of individual domains.

[0286] When receptors are multimerized, the ligand for the ligand-binding domain / receptor domain of the chimeric surface membrane protein can be multimeric in the sense that it usually has at least two binding sites, each of which can bind to the ligand-receptor domain. A "multimeric ligand-binding region" refers to a ligand-binding region that binds to a multimeric ligand. The term "multimeric ligand" includes dimeric ligands. A dimeric ligand can have two binding sites that can bind to the ligand-receptor domain. Desirably, the ligand of interest can be a small, synthetic organic molecule that is a dimer or higher oligomer, usually about tetramer or smaller, with individual molecules typically being at least about 150 Da and less than about 5 kDa, usually less than about 3 kDa. A variety of synthetic ligand-receptor pairs can be used. For example, in embodiments involving natural receptors, dimeric FK506 may be used with the FKBP12 receptor, dimerized cyclosporin A may be used with the cyclophilin receptor, dimerized estrogen may be used with the estrogen receptor, dimerized glucocorticoid may be used with the glucocorticoid receptor, dimerized tetracycline may be used with the tetracycline receptor, dimerized vitamin D may be used with the vitamin D receptor, etc. Alternatively, higher order ligands, e.g., trimers, may be used. For embodiments involving non-natural receptors, such as antibody subunits, modified antibody subunits, single-chain antibodies comprising tandem heavy and light chain variable regions separated by a flexible linker domain, or modified receptors and mutated sequences thereof, any of a wide variety of compounds may be used. A significant feature of these ligand units is that each binding site can bind to a receptor with high affinity, and that the ligand units can be chemically dimerized.Methods are also available to balance the hydrophobicity / hydrophilicity of ligands so that they can be dissolved in serum at functional levels and still diffuse across the plasma membrane for most applications.

[0287] In certain embodiments, the methods utilize a chemically induced dimerization (CID) approach to generate conditionally controlled proteins or polypeptides that is inducible and reversible due to decomposition of a labile dimerizer or administration of a competitive inhibitor of the monomer.

[0288] The CID system uses synthetic bivalent ligands to rapidly crosslink signaling molecules fused to ligand-binding domains. This system can bind to cell surface proteins (Spencer, DM et al., Science, 1993, 262:1019-1024; Spencer DM et al., Curr Biol 1996, 6:839-847; Blau, CA et al., Proc Natl Acad. Sci. USA 1997, 94:3076-3081) or cytosolic proteins (Luo, Z. et al., Nature 1996, 383:181-185; MacCorkle, RA et al., Proc Natl Acad Sci USA 1998, 95:3655-3660), recruitment of transcription factors to DNA elements to regulate transcription (Ho, SN et al., Nature 1996, 382:822-826; Rivera, VM et al., Nat. Med. 1996, 2:1028-1032), or recruitment of signaling molecules to the plasma membrane to stimulate signal transduction (Spencer DM et al., Proc. Natl. Acad. Sci. USA 1995, 92:9805-9809; Holsinger, LJ et al., Proc. Natl. Acad. Sci. USA 1995, 95:9810-9814).

[0289] The CID system is based on the concept that aggregation of surface receptors efficiently activates downstream signaling cascades. In its simplest embodiment, the CID system uses a dimeric analog of the lipid-permeable immunosuppressant FK506, which loses its normal biological activity but gains the ability to crosslink molecules genetically fused to the FK506-binding protein, FKBP12. By fusing one or more FKBPs to caspase-9, caspase-9 activity can be stimulated in a dimerization-drug-dependent but ligand- and ectodomain-independent manner. This provides a system with temporal control, reversibility using monomeric drug analogs, and high specificity. The high affinity of the third-generation AP20187 / AP1903 CIDs for their binding domain, FKBP12, allows for specific activation of recombinant receptors in vivo without inducing nonspecific side effects due to endogenous FKBP12. FKBP12 variants with amino acid substitutions and deletions that bind to dimerizing drugs (e.g., FKBP12 v FKBP12 variants, including but not limited to those with an amino acid substitution at position 36 selected from the group consisting of valine, leucine, isoleucine, and alanine, can also be used. Furthermore, synthetic ligands are resistant to protease degradation, making them more efficient at activating receptors in vivo than most delivered protein agents.

[0290] FKBP12 refers to a wild-type FKBP12 polypeptide, or an analog or derivative thereof that may contain amino acid substitutions and maintain FKBP12 binding activity to rapamycin; the FKBP12 polypeptide or polypeptide region binds to rimizuside with an affinity that is at least 100-fold lower than that of an FKBP12v36 polypeptide. In some examples, the FKBP12 polypeptide binds to a ligand (e.g., rimizuside) with an affinity that is at least 100-fold lower than that of an FKBP12 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 302.

[0291] By FKBP12 variant polypeptide is meant an FKBP12 polypeptide that binds to a ligand (e.g., rimizuside) with an affinity that is at least 100 times higher than a wild-type FKBP12 polypeptide (e.g., a wild-type FKBP12 polypeptide consisting of the amino acid sequence of SEQ ID NO: 301).

[0292] The ligand used can bind to two or more of the ligand-binding domains. When a chimeric protein contains two or more ligand-binding domains, it may be able to bind to two or more ligands. The ligand is typically a non-protein or chemical substance. Exemplary ligands include, but are not limited to, FK506 (e.g., FK1012).

[0293] Other ligand-binding regions can be, for example, dimerization regions or ligand-binding regions modified by wobble substitutions, such as FKBP12(V36): the human 12 kDa FK506-binding protein in which F36 is replaced by V; the fully mature coding sequence (amino acids 1-107) provides a binding site for the synthetic dimerization drug AP1903 (Jemal, A. et al., CA Cancer J. Clinic. 58, 71-96 (2008); Scher, HI and Kelly, WK, Journal of Clinical Oncology 11, 1566-72 (1993)). Two tandem copies of the protein can also be used in the construct so that higher-order oligomers are induced upon crosslinking by AP1903.

[0294] FKBP12 variants can also be used in the FKBP12 / FRB multimerization region. In some embodiments, the variants used in these fusions bind to rapamycin or rapalogs, but bind with lower affinity to rimizuside than, for example, FKBP12v36. Examples of FKBP12 variants include those from many species, including yeast. In one embodiment, the FKBP12 variant is FKBP12.6 (calstablin).

[0295] Other heterodimers are contemplated in the present application. In one embodiment, a calcineurin A polypeptide or domain can be used in place of the FRB multimerization domain. In some embodiments, the first unit of the first multimerization domain is a calcineurin-A polypeptide. In some embodiments, the first unit of the first multimerization domain is a calcineurin-A polypeptide domain, and the second unit of the first multimerization domain is an FKBP12 or FKBP12 variant multimerization domain. In some embodiments, the first unit of the first multimerization domain is an FKBP12 or FKBP12 variant multimerization domain, and the second unit of the first multimerization domain is a calcineurin-A polypeptide domain. In these embodiments, the first ligand comprises, for example, cyclosporine. F36V'-FKBP: F36V'-FKBP is a codon-wobble version of F36V-FKBP. It encodes the same polypeptide sequence as F36V-FKPB, but shares only 62% homology at the nucleotide level. F36V'-FKBP was designed to reduce recombination in retroviral vectors (Schellhammer, PF et al., J. Urol. 157, 1731-5 (1997)). F36V'-FKBP was constructed by PCR assembly. The transgene contains one copy of F36V'-FKBP directly linked to one copy of F36V-FKBP.

[0296] In some embodiments, the ligand is a small molecule. An appropriate ligand for the selected ligand-binding region can be selected. Often, the ligand is a dimer, and occasionally the ligand is a dimeric FK506 or a dimeric FK506-like analog. In certain embodiments, the ligand is AP1903 (CAS index name: 2-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylidene]] ester, [2S-[1(R * ), 2R * [S * [S * [1(R * ), 2R * ]]]]]-(9CI) CAS Registry Number: 195514-63-7; Molecular Formula: C78H98N4O20 Molecular Weight: 1411.65). In certain embodiments, the ligand is AP20187.

[0297] In certain embodiments, the ligand is an AP20187 analog, such as AP1510. In some embodiments, certain analogs may be suitable for FKBP12, and certain analogs may be suitable for a wobble version of FKBP12. In certain embodiments, one ligand-binding region is included in the chimeric protein. In other embodiments, two or more ligand-binding regions are included. For example, if the ligand-binding region is FKBP12, and two of these regions are included, one may be, for example, a wobble version.

[0298] Other contemplated dimerization systems include the coumermycin / DNA gyrase B system. Coumermycin-induced dimerization activates modified Raf proteins and stimulates the MAP kinase cascade. Farrar, MA, et al. (1996) See Nature 383, 178-181. In other embodiments, the abscisic acid (ABA) system developed by G.R. Crabtree and coworkers (Liang FS et al., Sci Signal. 2011 Mar 15;4(164):rs2) can be used, but, like DNA gyrase B, it relies on foreign proteins that are immunogenic.

[0299] (membrane targeting) The membrane targeting sequence or region provides transport of the chimeric protein to the cell surface membrane, where the same or another sequence can encode binding of the chimeric protein to the cell surface membrane. Molecules associated with cell membranes contain certain regions that facilitate membrane association, and such regions can be incorporated into chimeric protein molecules to result in membrane-targeted molecules. For example, some proteins contain acylation sequences at the N- or C-terminus, and these acyl moieties facilitate membrane association. Such sequences are recognized by acyltransferases and often correspond to specific sequence motifs. Certain acylation motifs can be modified with a single acyl moiety (often followed by several positively charged residues that improve association with anionic lipid head groups (e.g., human c-Src:MGSNKSKPKDASQRRR)), while others can be modified with multiple acyl moieties. For example, the N-terminal sequence of protein tyrosine kinase Src can contain a single myristoyl moiety. Dual acylation regions are located within the N-terminal region of certain protein kinases, such as a subset of Src family members (e.g., Yes, Fyn, Lck) and G protein alpha subunits. Such dual acylation regions are often located within the first 18 amino acids of such proteins and conform to the sequence motif Met-Gly-Cys-Xaa-Cys, where Met is cleaved, Gly is N-acylated, and one of the Cys residues is S-acylated. Gly is often myristoylated, and Cys can be palmitoylated. An acylation region conforming to the sequence motif Cys-Ala-Ala-Xaa (the so-called "CAAX box") can be modified with a C15 or C10 isoprenyl moiety from the C-terminus of G protein gamma subunits and other proteins (e.g., World Wide Web address ebi.ac.uk / interpro / DisplayIproEntry?ac=IPR001230) can also be used.These and other acylation motifs, including those discussed in, for example, Gauthier-Campbell et al., Molecular Biology of the Cell 15:2205-2217 (2004); Glabati et al., Biochem. J. 303:697-700 (1994); and Zlakine et al., J. Cell Science 110:673-679 (1997), can be incorporated into chimeric molecules to induce membrane localization. In certain embodiments, a native sequence from a protein containing an acylation motif is incorporated into the chimeric protein. For example, in some embodiments, the N-terminal portion of Lck, Fyn, or Yes or a G protein alpha subunit (e.g., the first 25 N-terminal amino acids or fewer amino acids from such a protein (e.g., about 5 to about 20 amino acids, about 10 to about 19 amino acids, or about 15 to about 19 amino acids of the native sequence, with optional mutations)) can be incorporated into the N-terminus of the chimeric protein. In certain embodiments, a C-terminal sequence of about 25 amino acids or less from a G protein gamma subunit containing a CAAX box motif sequence (e.g., about 5 to about 20 amino acids, about 10 to about 18 amino acids, or about 15 to about 18 amino acids of the native sequence, optionally with mutations) can be linked to the C-terminus of the chimeric protein.

[0300] In some embodiments, the acyl moiety has a log p value of +1 to +6, and occasionally a log p value of +3 to +4.5. Log p values ​​are a measure of hydrophobicity and are often derived from octanol / water partitioning studies, where molecules with higher hydrophobicity are characterized as partitioning more frequently into octanol and having higher log p values. Log p values ​​have been published for several lipophilic molecules, and log p values ​​can be calculated using known partitioning processes (e.g., Chemical Reviews, Vol. 71, Issue 6, page 599, entry 4493 shows lauric acid to have a log p value of 4.2). Any acyl moiety can be linked to the peptide compositions discussed above and tested for antibacterial activity using known methods and those discussed hereinafter. The acyl moiety is sometimes, for example, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C4-C12 cyclalkylalkyl, aryl, substituted aryl, or aryl(C1-C4)alkyl. The optional acyl-containing moiety is sometimes a fatty acid; examples of fatty acid moieties are propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), heptyl (C7), octyl (C8), nonyl (C9), decyl (C10), undecyl (C11), lauryl (C12), myristyl (C14), palmityl (C16), stearyl (C18), arachidyl (C20), behenyl (C22), and lignoceryl moieties (C24), each of which may contain 0, 1, 2, 3, 4, 5, 6, 7, or 8 unsaturations (i.e., double bonds). The acyl moiety is sometimes a lipid molecule (e.g., a phosphatidyl lipid (e.g., phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine), a sphingolipid (e.g., sphingomyelin, sphingosine, ceramide, ganglioside, cerebroside)), or a modified version thereof. In certain embodiments, one, two, three, four, five, or more acyl moieties are linked to the membrane-associated region.

[0301] The chimeric proteins herein may also contain single-pass or multiple-pass transmembrane sequences (e.g., at the N- or C-terminus of the chimeric protein). Single-pass transmembrane regions are found in certain CD molecules, tyrosine kinase receptors, serine / threonine kinase receptors, TGF-beta, BMP, activins, and phosphatases. Single-pass transmembrane regions often contain a signal peptide region and a transmembrane region of about 20 to about 25 amino acids (many of which are hydrophobic and can form an alpha helix). A short track of positively charged amino acids often follows the transmembrane span to anchor the protein to the membrane. Multipass proteins include ion pumps, ion channels, and transporters, and contain two or more helices that span the membrane multiple times. All or substantially all of the multipass proteins are sometimes incorporated into the chimeric protein. Sequences for single-spanning and multi-spanning membrane spanning regions are known and can be selected for incorporation into chimeric protein molecules.

[0302] Any membrane targeting sequence that is functional in the host and may or may not be associated with one of the other domains of the chimeric protein can be used. In some embodiments, such sequences include, but are not limited to, myristoylation targeting sequences, palmitoylation targeting sequences, prenylation sequences (i.e., farnesylation, geranyl-geranylation, CAAX box), protein-protein interaction motifs, or receptor-derived transmembrane sequences (using signal peptides). Examples include those discussed in, for example, Klooster JP et al., Biology of the Cell (2007) 99, 1-12, Vincent, S. et al., Nature Biotechnology 21: 936-40, 1098 (2003).

[0303] Additional protein domains exist that can enhance protein retention in various membranes. For example, the approximately 120 amino acid pleckstrin homology (PH) domain is found in over 200 human proteins, typically involved in intracellular signal transduction. PH domains can bind to various phosphatidylinositol (PI) lipids (e.g., PI(3,4,5)-P3, PI(3,4)-P2, PI(4,5)-P2) within membranes, potentially playing an important role in recruiting proteins to various membrane or intracellular compartments. Often, the phosphorylation state of PI lipids is controlled by, for example, PI-3 kinase or PTEN, so the interaction of PH domains with membranes is less stable than that of acyl lipids. AP1903 for Injection

[0304] AP1903 API is manufactured by Alphora Research Inc., and AP1903 Drug Product for Injection is made by Formatech Inc. It is formulated as a 5 mg / mL solution of AP1903 in a 25% solution of the non-ionic solubilizer Solutol HS 15 (250 mg / mL, BASF). At room temperature, the formulation is a clear, slightly yellow solution. When refrigerated, the formulation undergoes a reversible phase transition, becoming a milky white solution. This phase transition is reversible upon rewarming to room temperature. The fill contains: 2.33 mL in a 3 mL glass vial (approximately 10 mg total AP1903 for injection per vial).

[0305] The night before administration to patients, AP1903 is removed from the refrigerator and stored overnight at a temperature of approximately 21°C so that the solution becomes clear before dilution. The solution is prepared in glass or polyethylene bottles or non-DEHP bags within 30 minutes of the start of the infusion and stored at approximately 21°C until administration.

[0306] All study medications will be maintained at a temperature between 2°C and 8°C, protected from excessive light and heat, and stored in a locked area with limited access.

[0307] Upon determining the need to administer AP1903 to induce inducible caspase-9 polypeptide, patients may receive, for example, a single fixed dose of injectable AP1903 (0.4 mg / kg) via IV infusion over 2 hours using a non-DEHP, non-ethylene oxide sterile infusion set. The AP1903 dose is calculated individually for every patient and should not be recalculated unless body weight fluctuates by ≥10%. The calculated dose is diluted in 100 mL of 0.9% normal saline prior to infusion.

[0308] In a previous Phase 1 study of AP1903, 24 healthy vol...

Claims

1. a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: i) a Δcaspase-9 (ΔCasp9) polypeptide lacking the CARD domain; and ii) the FKBP12 or FKBP12v36 ligand-binding region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein said chimeric costimulatory polypeptide comprises: i) an FKBP12-rapamycin binding (FRB) domain or a variant polypeptide region thereof that binds to rapamycin or a rapalog; ii) the FKBP12 or FKBP12v36 ligand-binding region; and iii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: A modified cell comprising: wherein the cell is a modified cell selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

2. 2. The modified cell of claim 1, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain.

3. 2. The modified cell of claim 1, wherein the cell further comprises a third polynucleotide, wherein the third polynucleotide encodes a chimeric antigen receptor (CAR) or a recombinant T cell receptor.

4. The modified cell of any one of claims 1 to 3, wherein the cell is a natural killer (NK) cell, a T cell, a tumor-infiltrating lymphocyte, or an NK-T cell.

5. 1. A composition comprising a nucleic acid for transfecting or transducing the nucleic acid into a cell, comprising: wherein the nucleic acid is: a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: i) a Δcaspase-9 (ΔCasp9) polypeptide lacking the CARD domain; and ii) the FKBP12 or FKBP12v36 ligand-binding region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein said chimeric costimulatory polypeptide comprises: i) an FKBP12-rapamycin binding (FRB) domain or a variant polypeptide region thereof that binds to rapamycin or a rapalog; ii) the FKBP12 or FKBP12v36 ligand-binding region; and iii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising a promoter operably linked to wherein the cells are selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

6. The composition of claim 5, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain.

7. 6. The composition of claim 5, wherein the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes a CAR or a recombinant T cell receptor.

8. 1. A kit or composition comprising a viral vector comprising a nucleic acid for transfecting or transducing a cell, said nucleic acid comprising: a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: i) a Δcaspase-9 (ΔCasp9) polypeptide lacking the CARD domain; and ii) the FKBP12 or FKBP12v36 ligand-binding region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein said chimeric costimulatory polypeptide comprises: i) an FKBP12-rapamycin binding (FRB) domain or a variant polypeptide region thereof that binds to rapamycin or a rapalog; ii) the FKBP12 or FKBP12v36 ligand-binding region; and iii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising Including, wherein the cells are selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

9. 1. A composition for use in a method for expressing a chimeric pro-apoptotic polypeptide and a chimeric costimulatory polypeptide in a cell, said composition comprising a first polynucleotide encoding said chimeric pro-apoptotic polypeptide and a second polynucleotide encoding said chimeric costimulatory polypeptide, wherein: a) the chimeric pro-apoptotic polypeptide comprises: i) a Δcaspase-9 (ΔCasp9) polypeptide lacking the CARD domain; and ii) the FKBP12 or FKBP12v36 ligand-binding region; and b) the chimeric costimulatory polypeptide is i) an FKBP12-rapamycin binding (FRB) domain or a variant thereof that binds to rapamycin or a rapalog; ii) the FKBP12 or FKBP12v36 ligand-binding region; and iii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; Including, wherein the cells are selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

10. 10. A composition for use in a method for stimulating an immune response in a subject, said composition comprising the modified cells of any one of claims 1 to 4, wherein said method comprises: a) transplanting the modified cells into the subject; and b) after step a), administering an effective amount of rapamycin or a rapalog that binds to the FRB domain or variant thereof of the chimeric costimulatory polypeptide to stimulate a cell-mediated immune response; A composition comprising:

11. 10. A composition for use in a method of administering a ligand to a subject who has undergone cell therapy using modified cells, the composition comprising rapamycin or a rapalog, wherein the composition is administered to the subject, and the modified cells comprise the modified cells of any one of claims 1 to 4.

12. 10. A composition for use in a method for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, the composition comprising a modified cell comprising the modified cell of any one of claims 1 to 4, wherein the method comprises: a) transplanting the modified cells into the subject, wherein the modified cells comprise a CAR or a recombinant T cell receptor comprising an antigen recognition moiety that binds to the target antigen; and b) after step a), administering an effective amount of rapamycin or a rapalog that binds to the FRB domain or variant thereof of the chimeric costimulatory polypeptide to reduce the number or concentration of target antigens or target cells in the subject; A composition comprising:

13. 10. A composition for use in a method for reducing tumor size in a subject, said composition comprising the modified cells of any one of claims 1 to 4, wherein said method comprises: a) administering the composition to the subject, wherein the modified cells comprise a CAR or a recombinant T cell receptor that includes an antigen recognition moiety that binds to an antigen on the tumor; and b) after step a), administering an effective amount of rapamycin or a rapalog that binds to the FRB domain or variant thereof of the chimeric costimulatory polypeptide to reduce the size of the tumor in the subject; A composition comprising:

14. 1. A composition for use in a method for modulating survival of transplanted modified cells in a subject, said composition comprising the modified cells of any one of claims 1 to 4, said method comprising: a) transplanting the modified cells into the subject; and b) after step a), administering to the subject a ligand that binds to the FKBP12 or FKBP12v36 ligand-binding region of the chimeric pro-apoptotic polypeptide in an amount effective to kill at least 90% of the modified cells that express the chimeric pro-apoptotic polypeptide; A composition comprising:

15. a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: (i) a Δcaspase-9 (ΔCasp9) polypeptide lacking the CARD domain; (ii) an FKBP12-rapamycin binding (FRB) domain or a variant thereof that binds to rapamycin or a rapalog; and (iii) the FKBP12 or FKBP12v36 ligand-binding region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein said chimeric costimulatory polypeptide comprises: (i) an FKBP12 or FKBP12v36 ligand-binding region, and (ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: A modified cell comprising: wherein the cell is a modified cell selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

16. The modified cell of claim 15, wherein the chimeric costimulatory polypeptide comprises an FKBP12 or FKBP12v36 ligand-binding region, a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.

17. 16. The modified cell of claim 15, wherein the cell further comprises a third polynucleotide encoding a CAR or a recombinant T cell receptor.

18. 1. A composition for transfecting or transducing a cell, comprising a nucleic acid, said nucleic acid comprising: a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: (i) a Δcaspase-9 (ΔCasp9) polypeptide lacking the CARD domain; (ii) an FKBP12-rapamycin binding (FRB) domain or a variant thereof that binds to rapamycin or a rapalog; and (iii) the FKBP12 or FKBP12v36 ligand-binding region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein said chimeric costimulatory polypeptide comprises: (i) an FKBP12 or FKBP12v36 ligand-binding region, and (ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: a promoter operably linked to wherein the cells are selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

19. 20. The composition of claim 18, wherein the chimeric costimulatory polypeptide comprises a truncated MyD88 polypeptide region lacking a TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40 extracellular domain.

20. 19. The composition of claim 18, wherein the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes a CAR or a recombinant T cell receptor.

21. 1. A kit or composition comprising a viral vector containing a nucleic acid for transfecting or transducing a cell, said nucleic acid comprising: a) a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein said chimeric pro-apoptotic polypeptide comprises: (i) a Δcaspase-9 (ΔCasp9) polypeptide lacking the CARD domain; (ii) an FKBP12-rapamycin binding (FRB) domain or a variant thereof that binds to rapamycin or a rapalog; and (iii) the FKBP12 or FKBP12v36 ligand-binding region; a first polynucleotide comprising: b) a second polynucleotide encoding a chimeric costimulatory polypeptide, wherein said chimeric costimulatory polypeptide comprises: (i) an FKBP12 or FKBP12v36 ligand-binding region, and (ii) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain, and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; a second polynucleotide comprising: Including, wherein the cells are selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

22. 22. A composition for use in a method for expressing a chimeric pro-apoptotic polypeptide in a cell, said composition comprising a nucleic acid according to any one of claims 18 to 21, wherein said chimeric pro-apoptotic polypeptide is a) a delta-caspase-9 (ΔCasp9) polypeptide lacking the CARD domain; an FRB domain or a variant thereof; and b) the FKBP12 or FKBP12v36 ligand-binding region; the method comprising contacting the composition with a cell under conditions such that the nucleic acid is incorporated into the cell, whereby the cell expresses the chimeric apoptosis-promoting polypeptide from the incorporated nucleic acid; wherein the cells are selected from the group consisting of natural killer cells, natural killer T cells, T cells, tumor-infiltrating lymphocytes, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), non-lymphoid hematopoietic cells, non-hematopoietic cells, macrophages, keratinocytes, fibroblasts, and melanoma cells.

23. 18. A composition for use in a method for stimulating an immune response in a subject, said composition comprising the modified cells of any one of claims 15 to 17, wherein said method comprises: a) transplanting the modified cells into the subject; and b) after step a), administering an effective amount of a ligand that binds to the FKBP12 or FKBP12v36 ligand-binding region of the chimeric costimulatory polypeptide to stimulate a cell-mediated immune response; A composition comprising:

24. 18. A composition for use in a method of administering a ligand to a subject who has undergone cell therapy using modified cells, the composition comprising a ligand that binds to the FKBP12 or FKBP12v36 ligand-binding region of the chimeric costimulatory polypeptide, the composition being administered to the subject, and the modified cells comprising the modified cells of any one of claims 15 to 17.

25. 18. A composition for use in a method for treating a subject having a disease or condition associated with elevated expression of a target antigen expressed by a target cell, said composition comprising modified cells comprising modified cells according to any one of claims 15 to 17, wherein said method comprises: a) transplanting the modified cells into the subject, wherein the modified cells comprise a CAR or a recombinant T cell receptor comprising an antigen recognition moiety that binds to the target antigen; and b) after step a), administering an effective amount of a ligand that binds to the FKBP12 or FKBP12v36 ligand-binding region of the chimeric costimulatory polypeptide to reduce the number or concentration of target antigens or target cells in the subject; A composition comprising:

26. 18. A composition for use in a method for reducing the size of a tumor in a subject, said composition comprising the modified cells of any one of claims 15 to 17, wherein said method comprises: a) administering the composition to the subject, wherein the modified cells comprise a CAR or a recombinant T cell receptor that includes an antigen recognition moiety that binds to an antigen on the tumor; and b) after step a), administering an effective amount of a ligand that binds to the FKBP12 or FKBP12v36 ligand-binding region of the chimeric costimulatory polypeptide to reduce the size of the tumor in the subject; A composition comprising:

27. 18. A composition for use in a method for modulating survival of transplanted modified cells in a subject, said composition comprising the modified cells of any one of claims 15 to 17, said method comprising: a) transplanting the modified cells into the subject; and b) after step a), administering to the subject rapamycin or a rapalog that binds to the FRB domain or variant thereof of the chimeric pro-apoptotic polypeptide in an amount effective to kill at least 30% of the modified cells that express the chimeric pro-apoptotic polypeptide; A composition comprising:

28. The composition of any one of claims 5 to 7 and 18 to 20, wherein the FKBP12v36 ligand binding region comprises an amino acid substitution at amino acid residue 36.

29. The composition of any one of claims 5 to 7 and 18 to 20, wherein the FKBP12v36 ligand binding region comprises the sequence shown in SEQ ID NO:

3.

30. The FRB domain variant is KLW (SEQ ID NO: 303, amino acid T2098L substitution in the FRB domain from the wild-type mTOR protein) (FRB Ｌ 21. The composition of any one of claims 5 to 7 and 18 to 20, wherein the mTOR domain is selected from the group consisting of: KTF (an amino acid W2101F substitution in the FRB domain derived from the wild-type mTOR protein); and KLF (an amino acid T2098L substitution and an amino acid W2101F substitution in the FRB domain derived from the wild-type mTOR protein).

31. A modified cell transfected or transduced with the composition of any one of claims 5 to 7, 18 to 20 and 28 to 30.

32. 32. The modified cell of claim 31 , wherein the modified cell comprises a polynucleotide encoding a CAR or a recombinant TCR.

33. A modified cell according to any one of claims 1 to 4, 15 to 17, and 31 to 32, or a composition according to any one of claims 5 to 14, and 18 to 30, or a kit according to claim 8 or 21, wherein the cell is a natural killer cell, a natural killer T cell, a T cell, or a tumor-infiltrating lymphocyte. Modified cells, compositions, or kits.

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