WO2017106185A2 - Dual controls for therapeutic cell activation or elimination - Google Patents

Dual controls for therapeutic cell activation or elimination Download PDF

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Publication number
WO2017106185A2
WO2017106185A2 PCT/US2016/066371 US2016066371W WO2017106185A2 WO 2017106185 A2 WO2017106185 A2 WO 2017106185A2 US 2016066371 W US2016066371 W US 2016066371W WO 2017106185 A2 WO2017106185 A2 WO 2017106185A2
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Prior art keywords
polypeptide
cell
ligand
nucleic acid
modified
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PCT/US2016/066371
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English (en)
French (fr)
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WO2017106185A3 (en
WO2017106185A8 (en
Inventor
Joseph Henri Bayle
MyLinh Thi DUONG
Matthew Robert COLLINSON-PAULZ
Aaron Edward FOSTER
David Michael SPENCER
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Bellicum Pharmaceuticals Inc
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Bellicum Pharmaceuticals Inc
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Priority to JP2018530845A priority Critical patent/JP2019506143A/ja
Application filed by Bellicum Pharmaceuticals Inc filed Critical Bellicum Pharmaceuticals Inc
Priority to EP16826509.8A priority patent/EP3390434A2/en
Priority to AU2016370470A priority patent/AU2016370470B2/en
Priority to KR1020187020145A priority patent/KR20180087423A/ko
Priority to CN202310839876.4A priority patent/CN117402829A/zh
Priority to SG11201804694SA priority patent/SG11201804694SA/en
Priority to CN201680081930.1A priority patent/CN108779163A/zh
Priority to CA3007473A priority patent/CA3007473A1/en
Publication of WO2017106185A2 publication Critical patent/WO2017106185A2/en
Publication of WO2017106185A3 publication Critical patent/WO2017106185A3/en
Anticipated expiration legal-status Critical
Publication of WO2017106185A8 publication Critical patent/WO2017106185A8/en
Priority to JP2021200645A priority patent/JP2022031906A/ja
Priority to JP2024105062A priority patent/JP2024120105A/ja
Priority to JP2024105063A priority patent/JP2024120106A/ja
Ceased legal-status Critical Current

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Definitions

  • 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 condition, or to control or modulate the activity of therapeutic cells that express chimeric antigen receptors or recombinant T cell receptors.
  • CARs chimeric antigen receptors
  • T cells are genetically engineered to express a heterologous gene, these modified cells are then administered to patients.
  • Heterologous genes may be used to express chimeric antigen receptors (CARs), which are artificial receptors designed to convey antigen specificity to T cells without the requirement for MHC antigen presentation. They include an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the T cell and provide specific immunity.
  • CAR-expressing T cells may be used in various therapies, including cancer therapies. These treatments are used, for example, to target tumors for elimination, and to treat cancer and blood disorders, but these therapies may have negative side effects.
  • TLS tumor lysis syndrome
  • CRS cytokine release syndrome
  • MAS macrophage activation syndrome
  • costimulating polypeptides may be used to enhance the activation of T cells, and of CAR- expressing T cells against target antigens, which would increase the potency of adoptive immunotherapy.
  • rimiducid which has two identical, protein-binding surfaces arranged tail-to-tail, each with high affinity and specificity for a mutant or vaiant of FKBP12: FKBP12(F36V) (FKBP12v36, Fv36 or F v ), Attachment of one or more Fv domains onto one or more cell signaling molecules that normally rely on homodimerization can convert that protein to rimiducid control.
  • Homodimerization with rimiducid is used in the context of an inducible caspase safety switch, and an inducible activation switch for cellular 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 control both functions using these switches within the same cell.
  • a molecular switch is provided that is controlled by a distinct dimerizer ligand, based on the heterodimerizing small molecule, rapamycin, or rapamycin analogs
  • Rapamycin binds to FKBP12, and its variants, and can induce heterodimerization of signaling domains that are fused to FKBP12 by binding to both FKBP12 and to polypeptides that contain the FKBP-rapamycin-binding (FRB) domain of mTOR.
  • FRB FKBP-rapamycin-binding
  • mTOR FKBP-rapamycin-binding
  • molecular switches that greatly augment the use of rapamycin, rapalogs and rimiducid as agents for therapeutic applications.
  • the allele specificity of rimiducid is used to allow selective dimerization of Fv-fusions.
  • a rapamycin or rapalog-inducible pro-apoptotic polypeptide such as, for example, Caspase-9 or a rapamycin or rapalog-inducible costimulatory polypeptide, such as, for example, MyD88/CD40 (MC) is used in combination with a rimiducid-inducible pro-apoptotic polypeptide, such as, for example, Caspase-9, or a rimiducid-inducible chimeric stimulating polypeptide, such as, for example, iMC to produce dual-switches.
  • dual-switches can be used to control both cell proliferation and apoptosis selectively by administration of either of two distinct ligand inducers.
  • a molecular switch that provides the option to activate a pro- apoptotic polypeptide, such as, for example, Caspase-9, with either rimiducid, or rapamycin or a rapalog, wherein the chimeric pro-apoptotic polypeptide comprises both a rimiducid-induced switch and a rapamycin-, or rapalog-, induced switch.
  • a pro- apoptotic polypeptide such as, for example, Caspase-9
  • the chimeric pro-apoptotic polypeptide comprises both a rimiducid-induced switch and a rapamycin-, or rapalog-, induced switch.
  • chimeric pro-apoptotic polypeptides may comprise, for example, both a FKBP12-Rapamycin-binding domain of mTOR (FRB), or an FRB variant, and an FKBP12 variant polypeptide, such as, for example, FKBP12v36.
  • FRB variant polypeptide is meant an FRB polypeptide that binds to a rapamycin analog (rapalog), for example, a rapalog provided in the present application.
  • FRB variant polypeptides comprise one or more amino acid substitutions, bind to a rapalog, and may bind, or may not bind to rapamycin.
  • a homodimerizer such as AP1903 (rimiducid) induces activation of a modified cell
  • a heterodimerizer such as rapamycin or a rapalog, activates a safety switch, causing apoptosis of the modified cell.
  • a chimeric pro-apoptotic polypeptide such as, for example, Caspase-9, comprising both an FKBP12 and an FRB, or FRB variant region (iFwtFRBC9) is expressed in a cell along with an inducible chimeric MyD88/CD40 costimulating polypeptide, that comprises MyD88 and CD40 polypeptides and at least two copies of FKBP12v36 (MC.FvFv).
  • MC.FvFv inducible chimeric MyD88/CD40 costimulating polypeptide
  • the MC.FvFv dimerizes or multimerizes, and activates the cell.
  • the cell may, for example, be a T cell that expresses a chimeric antigen receptor directed against a target antigen (CAR ⁇ ).
  • CAR ⁇ target antigen
  • the cell may be contacted with a heterodimerizer, such as, for example, rapamycin, or a rapalog, that binds to the FRB region on the iFwtFRBC9 polypeptide, as well as the FKBP12 region on the iFwtFRBC9 polypeptide, causing direct dimerization of the Caspase-9 polypeptide, and inducing apoptosis.
  • a heterodimerizer such as, for example, rapamycin, or a rapalog
  • the heterodimerizer binds to the FRB region on the iFwtFRBC9 polypeptide, and the Fv region on the MC.FvFv polypeptide, causing scaffold-induced dimerization, due to the scaffold of two FKBP12v36 polypeptides on each MC.FvFv polypeptide (Fig. 43 (1)), and inducing apoptosis.
  • FKBP12 variant polypeptide is meant an FKBP12 polypeptide that comprises one or more amino acid substitutions and that binds to a ligand such as, for example, rimiducid, with at least 100 times, 500 times, or 1000 times more affinity than the ligand binds to the FKBP12 polypeptide region.
  • a heterodimerizer such as rapamycin or a rapalog, induces activation of a modified cell
  • a homodimerizer such as AP1903 activates a safety switch, causing apoptosis of the modified cell.
  • a chimeric pro-apoptotic polypeptide such as, for example, Caspase-9, comprising an Fv region (iFvC9) is expressed in a cell along with an inducible chimeric MyD88/CD40 costimulating polypeptide, that comprises MyD88 and CD40 polypeptides and both an FKBP12 and an FRB or FRB variant region (iFRBFwtMC) (MC.FvFv).
  • iFRBFwtMC FRB or FRB variant region
  • the cell may, for example, be a T cell that expresses a chimeric antigen receptor directed against a target antigen (CAR ⁇ ).
  • CAR ⁇ target antigen
  • the cell may be contacted with a homodimerizer, such as, for example, AP1903, that binds to the iFvC9 polypeptide, causing direct dimerization of the Caspase-9 polypeptide, and inducing apoptosis. (Fig. 57 (right)).
  • dual switch compositions and methods of the present application dual switch apoptotic polypeptides, modified cells that express the dual switch apoptotic
  • modified cells that expresses a FRB.FKBPv. ⁇ C9 polypeptide, or a FKBP V .FRB ⁇ C9 polypeptide; apoptosis may be induced by contacting the modified cell with either a heterodimer, such as rapamycin or a rapalog, or the homodimer, rimiducid.
  • modified cells are provided that comprise polynucleotides that encode dual switch chimeric pro-apoptotic polypeptides, for example, FRB.FKBPv. ⁇ C9
  • FRB polypeptide or a FKBPv.FRB ⁇ C9 polypeptides, wherein the FRB polypeptide region may be an FRB variant polyeptide region, such as, for example, FRBL .
  • FRB is denoted, such as, for example, the table of nomenclature herein, other FRB derivatives may be used, such as, for example, FRBL
  • polypeptides comprising FRBL is provided as an example of a composition or method of the present application, it is understood that RB or FRB variants or derivatives other than FRBL may be used, with the appropriate ligand, such as rapamycin or a rapalog.
  • FKBP12 variants other than FKBP12v36 may be substituted for FKBP12v36, as appropriate
  • the modified cells may further comprise
  • polynucleotides that encode a heterologous protein such as, for example, a chimeric antigen receptor or a recombinant T cell receptor.
  • the modified cells may further comprise polynucleotides that encode a costimulatory polypeptide, such as, for example, a polypeptide that comprises a MyD88 polypeptide region, or a truncated MyD88 polypeptide region lacking the TIR domain, or, for example, a polypeptide that comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the extracellular domain.
  • nucleic acids that comprise polynucleotides that encode dual switch chimeric pro-apoptotic polypeptides for example,
  • FRB.FKBPV. ⁇ C9 polypeptide or a FKBPv.FRB ⁇ C9 polypeptides, wherein the FRB polypeptide region may be an FRB variant polyeptide region, such as, for example, FRBL .
  • the nucleic acids may further comprise polynucleotides that encode a heterologous protein such as, for example, a chimeric antigen receptor or a recombinant T cell receptor.
  • the nucleic acids may further comprise polynucleotides that encode a costimulatory polypeptide, such as, for example, a polypeptide that comprises a MyD88 polypeptide region, or a truncated MyD88 polypeptide region lacking the TIR domain, or, for example, a polypeptide that comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the extracellular domain.
  • a costimulatory polypeptide such as, for example, a polypeptide that comprises a MyD88 polypeptide region, or a truncated MyD88 polypeptide region lacking the TIR domain, or, for example, a polypeptide that comprises a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the extracellular domain.
  • chimeric polypeptides are provided, wherein a first chimeric polypeptide comprises a first multimerizing region that binds to a first ligand; the first multimerizing 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 bind significantly 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 bind significantly to the first portion of the first ligand.
  • a second chimeric polypeptide comprises a second multimerizing region that binds to a second ligand; the second multimerizing region 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 bind significantly to the second portion of the first ligand.
  • first ligand binding units include, but are not limited to, FKBP12 multimerizing regions, or variants, such as FKBP12v36
  • examples of second ligand binding units are, for example, FRB or FRB variant multimerizing regions.
  • Examples of a third ligand binding unit include, for example, but are not limited to, FKBP12 multimerizing regions, or variants, such as FKBP12v36.
  • the first ligand binding unit is FKBP12
  • the third ligand binding unit is FKBP12v36.
  • the first ligand is rapamycin, or a rapalog
  • the second ligand is rimiducid (AP1903).
  • the multimerizing regions such as FKBP12/FRB, FRB/FKBP12, and FKBP12v36, may be located amino terminal to the pro-apoptotic polypeptide or costimulatory polypeptide, or, in other examples, may be located carboxyl terminal to the pro-apoptotic polypeptide or costimulatory polypeptide.
  • Additional polypeptides such as, for example, linker polypeptides, stem polypeptides, spacer polypeptides, or in some examples, marker polypeptides, may be located between 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; (ii) a FKBP12-Rapamycin-Binding (FRB) domain polypeptide, or FRB variant polypeptide region; and (iii) a FKBP12 or FKBP12 variant polypeptide region (FKBP12v); and a second polynucleotide encoding a chimeric costimulating polypeptide, wherein the chimeric costimulating polypeptide comprises one or more, for example, 1 , 2, or 3 FKBP12 variant polypeptide regions and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; o rii) a MyD88 polypeptide region or a t
  • the modified cell further comprises a third polynucleotide encoding a chimeric antigen receptor or a recombinant T cell receptor.
  • 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) a FKBP12-Rapamycin- Binding (FRB) domain polypeptide, or FRB variant polypeptide region; and(iii) a FKBP12 or FKBP12 variant polypeptide region (FKBP12v); and a second polynucleotide encoding a chimeric costimulating polypeptide, wherein the chimeric costimulating polypeptide comprises one or more, for example, 1 , 2, or 3 FKBP12 variant polypeptide regions
  • the chimeric costimulating polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.
  • the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes a chimeric antigen receptor or a recombinant T cell receptor.
  • the pro-apoptotic polypeptide is a Caspase-9 polypeptide, wherein the Caspase-9 polypeptide lacks the CARD domain.
  • the cell is a T cell, tumor infiltrating lymphocyte, NK-T cell, or NK cell.
  • kits or compositions comprising nucleic acid comprising a first polynucleotide encoding a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises (i) a pro-apoptotic polypeptide region; (ii) a FKBP12-Rapamycin-Binding (FRB) domain polypeptide region, or variant thereof; and(iii) a FKBP12 polypeptide or FKBP12 variant polypeptide region (FKBP12v); and a second polynucleotide encoding a chimeric costimulating polypeptide, wherein the chimeric costimulating polypeptide comprises one or more, for example, 1 , 2, or 3 FKBP12 variant polypeptide regions and i) a MyD88 polypeptide region or a truncated MyD88 polypeptide region lacking the TIR domain; or
  • methods for expressing a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises a pro-apoptotic polypeptide region; a FRB polypeptide or FRB variant polypeptide region; and a FKBP12 polypeptide region of the present embodiments, comprising contacting a nucleic acid of the present embodiments with a cell under conditions in which the nucleic acid is incorporated into the cell, whereby the cell expresses the chimeric pro-apoptotic polypeptide from the incorporated nucleic acid.
  • methods are provided for stimulating an immune response in a subject, comprising: transplanting modified cells of the present embodiments into the subject, and after (a), administering an effective amount of a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulating polypeptide to stimulate a cell mediated immune response.
  • methods are provided for administering a ligand to a subject who has undergone cell therapy using modified cells, comprising administering a ligand that binds to the FKBP variant region of the chimeric costimulating polypeptide to the human subject, wherein the modified cells comprise modified cells of the present embodiments the present embodiments.
  • Also provided are methods for treating a subject having a disease or condition associated with an elevated expression of a target antigen expressed by a target cell comprising a) transplanting an effective amount of modified cells into the subject; wherein the modified cells comprise a modified cell of the present embodiments, wherein the modified cell comprises 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 a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulating polypeptide to reduce the number or concentration of target antigen or target cells in the subject.
  • Also provided are methods for reducing the size of a tumor in a subject comprising a) administering a modified cell of the present embodiments to the subject, wherein the cell comprises 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 a ligand that binds to the FKBP12 variant polypeptide region of the chimeric costimulating polypeptide to reduce the size of the tumor in the subject.
  • Also provided are methods for controlling survival of transplanted modified cells in a subject comprising transplanting modified cells of the present embodiments 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 pro-apoptotic polypeptide in an amount effective to kill at least 30% of the modified cells that express the chimeric pro-apoptotic polypeptide.
  • 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) a FKBP12 variant polypeptide region; and a second polynucleotide encoding a chimeric costimulating polypeptide, wherein the chimeric costimulating polypeptide comprises a FKBP12-Rapamycin Binding (FRB) domain polypeptide or FRB variant polypeptide region; a FKBP12 polypeptide or 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 chimeric costimulating polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.
  • the cell further comprises a third polynucleotide, wherein the third
  • nucleic acids encodes a chimeric antigen receptor or a recombinant T cell receptor.
  • nucleic acids are provided, wherein the nucleic acids comprise 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; and i) a FKBP12 variant polypeptide region; and a second polynucleotide encoding a chimeric
  • the chimeric costimulating polypeptide comprises i) a FKBP12- Rapamycin Binding (FRB) domain polypeptide or FRB variant polypeptide region; ii)a 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.
  • FRB FKBP12- Rapamycin Binding
  • the chimeric costimulating polypeptide comprises a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.
  • the promoter is operably linked to a third polynucleotide, wherein the third polynucleotide encodes chimeric antigen receptor or a recombinant T cell receptor.
  • the pro-apoptotic polypeptide is a Caspase-9 polypeptide, wherein the Caspase-9 polypeptide lacks the CARD domain.
  • the cell is a T cell, tumor infiltrating lymphocyte, NK-T cell, or NK cell.
  • kits or compositions comprising nucleic acids comprising polynucleotides of the present embodiments. Also provided are methods for expressing a chimeric pro-apoptotic polypeptide and a chimeric costimulating polypeptide, wherein a) the chimeric pro-apoptotic polypeptide comprises i) a pro- apoptotic polypeptide region; and ii) a FKBP12 variant polypeptide region; and b) the chimeric costimulating polypeptide comprises a FRB or FRB variant polypeptide region; a 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 comprising contacting a nucleic acid is a nucleic acid comprising
  • methods are provided of stimulating an immune response in a subject, comprising: a) transplanting modified cells of the present embodiments into the subject, and b)after (a), administering an effective amount of a 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.
  • methods are provided of administering a ligand to a subject who has undergone cell therapy using modified cells, comprising administering rapamycin or a rapalog to the subject, wherein the modified cells comprise modified cells of the present embodiments .
  • methods for treating a subject having a disease or condition associated with an elevated expression of a target antigen expressed by a target cell, comprising a) transplanting an effective amount of modified cells into the subject; wherein the modified cells comprise a modified cell of the present embodiments , wherein the modified cell comprises 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 the FRB polypeptide or FRB variant region of the chimeric stimulating polypeptide to reduce the number or concentration of target antigen or target cells in the subject.
  • methods for reducing the size of a tumor in a subject, comprising a) administering a modified cell of the present embodiments to the subject, wherein the cell comprises 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 the FRB or FRB variant polypeptide region of the chimeric stimulating polypeptide to reduce the size of the tumor in the subject.
  • methods are provided for controlling survival of transplanted modified cells in a subject, comprising a) transplanting modified cells of the present
  • embodiments into the subject and 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 at least 90% of the modified cells that express the chimeric pro-apoptotic polypeptide.
  • the chimeric costimulating polypeptide comprises two FKBP12 variant polypeptide regions, and a truncated MyD88 polypeptide region lacking the TIR domain. In some embodiments, the chimeric costimulating polypeptide further comprises a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. In some
  • the chimeric costimulating polypeptide comprises 2 FKBP12 variant polypeptide regions.
  • a nucleic acid comprising a promoter operably linked to a polynucleotide coding for a chimeric pro-apoptotic polypeptide, wherein the chimeric pro- apoptotic polypeptide comprises a) a pro-apoptotic polypeptide region; b) a FKBP12-Rapamycin binding domain (FRB) polypeptide or FRB variant polypeptide region; and c) a FKBP12 variant polypeptide region.
  • the FKBP12 variant comprises an amino acid substitution at amino acid residue 36.
  • the FKBP12 variant polypeptide region is a FKBP12v36 polypeptide region.
  • the FRB variant polypeptide region is selected from the group consisting of KLW (T2098L) (FRBL), KTF (W2101 F), and KLF (T2098L, W2101 F).
  • a chimeric pro-apoptotic polypeptide encoded by a nucleic acid of the present embodiments is provided.
  • modified cells are provided that are transfected or transduced with a nucleic acid of the present embodiments.
  • the modified cells comprise a polynucleotide that encodes a chimeric antigen receptor or a recombinant TCR.
  • methods are provided of controlling survival of transplanted modified cells in a subject, comprising: a) transplanting modified cells of the present embodiments, wherein the modified cells comprise a nucleic acid comprising a promoter operably linked to a polynucleotide coding for a chimeric pro-apoptotic polypeptide, wherein the chimeric pro-apoptotic polypeptide comprises a) a pro-apoptotic polypeptide region; b) a FKBP12-Rapamycin binding domain (FRB) polypeptide or FRB variant polypeptide region; and c) a FKBP12 variant polypeptide region, of the present embodiments 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
  • CARs chimeric antigen receptors
  • TAAs tumor-associated antigens
  • costimulatory chimeric polypeptide allows for a sustained, modulated control of a chimeric antigen receptor (CAR) that is co-expressed in the cell.
  • CAR chimeric antigen receptor
  • the ligand inducer activates the CAR-expressing cell by multimerizing the inducible chimeric signaling molecules, which, in turn, induces NF- ⁇ and other intracellular signaling pathways, leading to the activation of the target cells, for example, a T cell, a tumor-infiltrating lymphocyte (TIL), a natural killer (NK) cell, or a natural killer T (NK-T) cell.
  • TIL tumor-infiltrating lymphocyte
  • NK natural killer
  • NK-T natural killer T
  • a "dimmer" switch may allow for continued cell therapy, while reducing or eliminating significant side effects by eliminating the therapeutic cells from the subject, as needed.
  • This dimmer switch is dependent on a second ligand inducer.
  • an appropriate dose of the second ligand inducer is administered in order to eliminate over 90% or 95% of the therapeutic cells from the patient.
  • This second level of control may be "tunable," that is, the level of removal of the therapeutic cells may be controlled so that it results in partial removal of the therapeutic cells.
  • This second level of control may include, for example, a chimeric pro-apoptotic polypeptide.
  • the chimeric apoptotic polypeptide comprises a binding site for rapamycin, or a rapamycin analog (rapalog); also present in the therapeutic cell is an inducible chimeric polypeptide that, upon induction by a ligand inducer, activates the therapeutic cell; in some examples, 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 examples, 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 therapy, or the need to stimulate therapy, may be balanced with the need to eliminate or reduce the level of negative side effects.
  • a rapamycin analog is administered to the patient, which then binds to both the caspase polypeptide and the chimeric antigen receptor, thus recruiting the caspase polypeptide to the location of the CAR, and aggregating the caspase polypeptide. Upon aggregation, the caspase polypeptide induces apoptosis.
  • the amount of rapamycin or rapamycin analog administered to the patient may vary; if the removal of a lower level of cells by apoptosis is desired in order to reduce side effects and continue CAR therapy, a lower level of rapamycin or rapalog may be administered to the patient.
  • selective apoptosis may be induced in cells that express a chimeric Caspase-9 polypeptide fused to a dimeric ligand binding polypeptide, such as, for example, the AP1903-binding polypeptide FKBP12v36, by administering rimiducid (AP1903).
  • the Caspase-9 polypeptide includes amino acid substitutions that result in a lower level of basal apoptotic activity as part of the inducible chimeric polypeptide, than the wild type Caspase-9 polypeptide.
  • the nucleic acid encoding the chimeric polypeptides of the present application further comprise a polynucleotide encoding a chimeric antigen receptor, a T cell receptor, or a T cell receptor-based chimeric antigen receptor.
  • the chimeric antigen receptor comprises (i) a transmembrane region, (ii) a T cell activation molecule, and (iii) an antigen recognition moiety. Also provided are modified cells transfected or transduced with a nucleic acid discussed herein
  • the cells are transduced or transfected with a viral vector.
  • the viral vector may be, for example, but not limited to, a retroviral vector, such as, for example, but not limited to, a murine leukemia virus vector; an SFG vector; and adenoviral vector, or a lentiviral vector.
  • the cell is isolated. In some embodiments, the cell is in a human subject. In some embodiments, the cell is transplanted in a human subject.
  • personalized treatment wherein the stage or level of the disease or condition is determined before administration of the multimeric ligand, before the administration of an additional dose of the multimeric ligand, or in determining method and dosage involved in the administration of the multimeric ligand.
  • These methods may be used in any of the methods of any of the diseases or conditions of the present application. Where these methods of assessing the patient before administering the ligand are discussed in the context of graft versus host disease, it is understood that these methods may be similarly applied to the treatment of other conditions and diseases.
  • the method comprises administering therapeutic cells to a patient, and further comprises identifying a 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 multimerizing region, maintaining a subsequent dosage of the multimeric ligand, or adjusting a subsequent dosage of the multimeric ligand to the patient based on the presence or absence of the condition identified in the patient.
  • the method further comprises determining whether to administer an additional dose or additional doses of the multimeric ligand to the patient based upon the appearance of graft versus host disease symptoms in the patient.
  • the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and administering a multimeric ligand that binds to the multimerizing region, maintaining a subsequent dosage of the multimeric ligand, or adjusting a subsequent dosage of the multimeric ligand to the patient based on the presence, absence or stage of the graft versus host disease identified in the patient.
  • the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and determining whether a multimeric ligand that binds to the multimerizing region should be administered to the patient, or the dosage of the multimeric ligand subsequently administered to the patient is adjusted based on the presence, absence or stage of the graft versus host disease identified in the patient.
  • the method further comprises receiving information comprising the presence, absence or stage of graft versus host disease in the patient; and administering a multimeric ligand that binds to the multimerizing region, maintaining a subsequent dosage of the multimeric ligand, or adjusting a subsequent dosage of the multimeric ligand to the patient based on the presence, absence or stage of the graft versus host disease identified in the patient.
  • the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and transmitting the presence, absence or stage of the graft versus host disease to a decision maker who administers a multimeric ligand that binds to the multimerizing region, maintains a subsequent dosage of the multimeric ligand, or adjusts a subsequent dosage of the multimeric ligand administered to the patient based on the presence, absence or stage of the graft versus host disease identified in the subject.
  • the method further comprises identifying the presence, absence or stage of graft versus host disease in the patient, and transmitting an indication to administer a multimeric ligand that binds to the multimeric binding region, maintain a subsequent dosage of the multimeric ligand or adjust a subsequent dosage of the multimeric ligand administered to the patient based on the presence, absence or stage of the graft versus host disease identified in the subject.
  • a method for administering donor T cells to a human patient comprising administering a transduced or transfected T cell of the present application to a human patient, wherein the cells are non-allodepleted human donor T cells.
  • the therapeutic cells are administered to a subject having a non-malignant disorder, or where the subject has been diagnosed with a non-malignant disorder, such as, for example, a primary immune deficiency disorder (for example, but not limited to, Severe Combined Immune Deficiency (SCID), Combined Immune Deficiency (CID), Congenital T-cell
  • a primary immune deficiency disorder for example, but not limited to, Severe Combined Immune Deficiency (SCID), Combined Immune Deficiency (CID), Congenital T-cell
  • Defect/Deficiency Common Variable Immune Deficiency (CVID), Chronic Granulomatous Disease, IPEX (Immune deficiency, polyendocrinopathy, enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich Syndrome, CD40 Ligand Deficiency, Leukocyte Adhesion Deficiency, DOCK 8 Deficiency, IL-10 Deficiency/I L- 10 Receptor Deficiency, GATA 2 deficiency, X-linked lymphoproliferative disease (XLP), Cartilage Hair Hypoplasia, and the like), Hemophagocytosis Lymphohistiocytosis (HLH) or other hemophagocytic disorders, Inherited Marrow Failure Disorders (such as, for example, but not limited to, Shwachman Diamond Syndrome, Diamond Blackfan Anemia, Dyskeratosis
  • CVID Common Variable Immune Deficiency
  • IPEX Immune Deficiency
  • Congenita Fanconi Anemia, Congenital Neutropenia, and the like
  • Hemoglobinopathies such as, for example, but not limited to, Sickle Cell Disease, Thalassemia, and the like
  • Metabolic Disorders such as, for example, but not limited to, Mucopolysaccharidosis, Sphingolipidoses, and the like
  • an Osteoclast disorder such as, for example, but not limited to Osteopetrosis
  • the therapeutic cells may be, for example, any cell administered to a patient for a desired therapeutic result.
  • the cells may be, for example, T cells, natural killer cells, B cells,
  • the modified Caspase-9 polypeptide can also be used to directly kill tumor cells.
  • vectors comprising polynucleotides coding for the inducible modified Caspase-9 polypeptide would be injected into a tumor and after 10-24 hours (to permit protein expression), the ligand inducer, such as, for example, AP1903, would be administered to trigger apoptosis, causing the release of tumor antigens to the microenvironment.
  • the ligand inducer such as, for example, AP1903
  • the treatment may be combined with one or more adjuvants (e.g., IL-12, TLRs, IDO inhibitors, etc.).
  • the cells may be delivered to treat a solid tumor, such as, for example, delivery of the cells to a tumor bed.
  • a polynucleotide encoding the chimeric Caspase-9 polypeptide may be
  • nucleic acid vaccines such as DNA vaccines, wherein the vaccine comprises a nucleic acid comprising a polynucleotide that encodes an inducible, or modified inducible Caspase-9
  • the vaccine may be administered to a subject, thereby transforming or transducing target cells in vivo.
  • the ligand inducer is then administered following the methods of the present application.
  • the modified Caspase-9 polypeptide is a truncated modified Caspase-9 polypeptide. In some embodiments, the modified Caspase-9 polypeptide lacks the 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. In some embodiments, the methods further comprise administering a multimeric ligand that binds to the multimeric ligand binding region.
  • the multimeric ligand binding region is selected from the group consisting of FKBP, cyclophilin receptor, steroid receptor, tetracycline receptor, heavy chain antibody subunit, light chain antibody subunit, single chain antibodies comprised of heavy and light chain variable regions in tandem separated by a flexible linker domain, and mutated sequences thereof.
  • the multimeric ligand binding region is an FKBP12 region.
  • the multimeric ligand is an FK506 dimer or a dimeric FK506-like analog ligand.
  • the multimeric ligand is AP1903.
  • the number of therapeutic cells is reduced by from about 60% to 99%, about 70% to 95%, from 80% to 90% or about 90% or more after administration of the multimeric ligand.
  • donor T cells survive in the patient that are able to expand and are reactive to viruses and fungi.
  • donor T cells survive in the patient that are able to expand and are reactive to tumor cells in the patient.
  • the suicide gene used in the second level of control is a caspase polypeptide, for example, Caspase 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14.
  • the caspase polypeptide is a Caspase-9 polypeptide.
  • the Caspase-9 polypeptide comprises an amino acid sequence of a catalytically active (not catalytically dead) caspase variant polypeptide provided in Table 5 or 6 herein.
  • the Caspase-9 polypeptide consists of an amino acid sequence of a catalytically active (not catalytically dead) caspase variant polypeptide provided in Table 5 or 6 herein.
  • a caspase polypeptide may be used that has a lower basal activity in the absence of the ligand inducer. For example, when included as part of a chimeric inducible caspase
  • modified Caspase-9 polypeptides may have lower basal activity compared to wild type Caspase-9 in the chimeric construct.
  • the modified Caspase-9 polypeptide may comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and may comprise at least one amino acid substitution.
  • FIG. 1A illustrates various iCasp9 expression vectors as discussed herein.
  • FIG. 1 B illustrates a representative western blot of full length and truncated Caspase-9 protein produced by the expression vectors shown in FIG. 1A.
  • Fig. 2 is a schematic of the interaction of the suicide gene product and the CID to cause apoptosis.
  • Fig. 3 is a schematic depicting a two-tiered regulation of apoptosis.
  • the left section depicts rapalog-mediated recruitment of an inducible caspase polypeptide to FRBI-modified CAR.
  • the right section depicts a rimiducid (AP1903)-mediated inducible caspase polypeptide.
  • Fig. 4 is a plasmid map of a vector encoding FRBi_-modified CD19-MC-CAR and inducible
  • Fig. 5 is a plasmid map of a vector encoding FRB L -modified Her2-MC-CAR and an inducible Caspase-9 polypeptide.
  • Figs. 6A and 6B provide the results of an assay of two-tiered activation of apoptosis.
  • Fig. 6A shows recruitment of an inducible Caspase-9 polypeptide (iC9) with rapamycin, leading to more gradual apoptosis titration.
  • Fig. 6B shows complete apoptosis using rimiducid (AP1903).
  • Fig. 7 is a plasmid map of the pBP0545 vector,
  • Figs. 8A-8C illustrate that FRB or FKBP12-based scaffolds can multimerize signaling domains.
  • Fig. 8A Homodimerization of a signaling domain (red stick), like Caspase-9, can be achieved via a heterodimer that binds to the FRB-fused signaling domain on one side and FKBP12-fused domain on the other.
  • Fig. 8B Dimerization or multimerization of a signaling domain via 2 (left) or more (right) tandem copies of FRB (chevron).
  • the scaffold can contain subcellular targeting sequences to localize proteins to the plasma membrane (as depicted), the nucleus or organelles.
  • Fig. 8C Similar to Fig. 8B, but domain polarity is reversed.
  • Figs. 9A-9C provide schematics of iMC-mediated scaffolding of FRB L 2.
  • Fig. 9A In the presence of a heterodimer drug, such as a rapamycin, the FRBi_2-linked Caspase-9 binds with and clusters the FKBP-modified MyD88/CD40 (MC) signaling molecule. This clustering effect results in dimerization of FRBi_2. Caspase-9 and subsequent induction of cellular death via the apoptotic pathway.
  • Fig. 9B Similar to panel 9A, however the FKBP and FRB domains have been switched in relation to associated Caspase-9 and MC domains. The clustering effect still occurs in the presence of heterodimer drug.
  • Fig. 9C Similar to panel 9A; however there is only one FKBP domain attached to MC. Therefore, in the presence of heterodimer, Caspase-9 is no longer capable of being clustered and therefore apoptosis is not induced.
  • Fig. 10A-10E provide schematics of a rapalog-induced, FRB scaffold-based inducible Caspase-9 polypeptide.
  • Fig. 10A Rimiducid homodimerizes FKBPv-linked Caspase-9, resulting in dimerization and activation of Caspase-9 with subsequent induction of cellular death via the apoptotic pathway.
  • Fig. 10B Rapalogs heterodimerize FKBPv-linked Caspase-9 with FRB-linked Caspase-9, resulting in dimerization of Caspase-9 and cell death.
  • 10E are schematics illustrating that in the presence of a heterodimer drug, such as a rapalog, 2 or more FRB L domains act as a scaffold to recruit binding of FKBPv-linked Caspase-9, leading to dimerization or oligomerization of Caspase-9 and cell death.
  • a heterodimer drug such as a rapalog
  • Fig. 1 1 A is a schematic and Fig. 11 B is a line graph depicting activation of apoptosis by dimerization of a chimeric FRB-Caspase-9 polypeptide and a chimeric FKBP-Caspase-9 polypeptide (FRBi_-ACaspase-9 and FKBPv-ACaspase-9) with rapamycin.
  • Fig. 11 A Schematic representation of dimerization of FRB and FKBP12 with rapamycin to bring together fused Caspase-9 signaling domains and activation of apoptosis.
  • Fig. 1 1 B Schematic representation of dimerization of FRB and FKBP12 with rapamycin to bring together fused Caspase-9 signaling domains and activation of apoptosis.
  • Reporter assays were performed in HEK-293T cells transfected with the constitutive SRa-SEAP reporter (pBP046, 1 ⁇ g), a fusion of FRBL (L2098) and human ACaspase-9 (pBP0463, 2 ⁇ g) and a fusion of FKBP12 with ACaspase-9 (pBP0044, 2 ⁇ g).
  • Fig. 12A is a schematic and Figs. 12B and 12C are line graphs depicting assembly of FKBP- Caspase-9 on a FRB-based scaffold.
  • Fig. 12A Schematic of iterated FRB domains to provide scaffolds for rapamycin (or rapalog)-mediated multimerization of an FKBP12-Caspase-9 fusion protein.
  • Fig. 12A Schematic of iterated FRB domains to provide scaffolds for rapamycin (or rapalog)-mediated multimerization of an FKBP12-Caspase-9 fusion protein.
  • FIG. 12C Reporter assays were performed as in (B), but FRB-scaffolds were expressed from constructs encoding iterated FRBL domains with an amino-terminal myristoylation- targeting sequence and two (pBP0465) or four copies (pBP0721) of the FRBL domain.
  • Fig. 13A is a schematic and Fig. 13B is a line graph depicting assembly of FRB-ACaspase-9 on an FKBP scaffold.
  • Fig. 13A Schematic of iterated FKBP12 domains to produce scaffolds for assembly of rapamycin (or rapalog)-mediated multimerization of FRB-ACaspase-9 fusion protein, leading to apoptosis.
  • Fig. 13B Reporter assays were performed as in (B), but FRB-scaffolds were expressed from constructs encoding iterated FRBL domains with an amino-terminal myristoylation- targeting sequence and two (pBP0465) or four copies (
  • Reporter assays were performed as in Fig.s 12B and C with cultures of HEK-293T cells transfected with the constitutive SRa-SEAP reporter (pBP046, 1 ⁇ g), a fusion of FRBL (L2098) and CARD domain-deleted human ACaspase-9 (pBP0463, 2 ⁇ g) and FKBP expression constructs containing four tandem copies of FKBP12 (pBP722, 2 ⁇ g) or a control vector with one copy of FKBP (pS-SF1 E).
  • constitutive SRa-SEAP reporter pBP046, 1 ⁇ g
  • FRBL L2098
  • CARD domain-deleted human ACaspase-9 pBP0463, 2 ⁇ g
  • FKBP expression constructs containing four tandem copies of FKBP12 (pBP722, 2 ⁇ g) or a control vector with one copy of FKBP (pS-SF1 E).
  • Figs. 14A-14B provide line graphs showing that heterodimerization of FRB L scaffold with iCaspase9 induces cell death.
  • Primary T cells from three different donors (307, 582, 584) were transduced with pBP0220-pSFG-iC9.T2A- ⁇ CD19, pBP0756— pSFG-iC9.T2A- ⁇ CD19.P2A-FRB L , pBP0755— pSFG-iC9.T2A- ⁇ CD19.P2A-FRB L 2, or pBP0757— pSFG-iC9.T2A- ⁇ CD19.P2A-FRB L 3, containing iC9, CD19 marker, and 0-3 tandem copies of FRBL, respectively.
  • T Cells were plated with varying 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 initially gated on live lymphocytes by FSC vs SSC. Lymphocytes were then plotted as a CD19 histogram and subgated for high, medium and low expression within the CD19 + gate. Line graphs represent the relative percentage of the total cell population that express high levels of CD19, normalized to the no "0" drug control. All data points were done in duplicates.
  • Fig. 14A donor 307, 24 hr;
  • Fig. 14B donor 582, 24 hr;
  • Fig. 14C donor 584 24 hr;
  • Fig. 14D donor 582 48 hr;
  • Fig. 14E donor 584 48 hr.
  • Figs. 15A-15C provide line graphs and a schematic showing that rapamycin induces iC9 killing in the presence of tandem FRBL domains.
  • HEK-293 cells were transfected with 1 ⁇ g of SRa-SEAP constitutive reporter plasmid along with either negative (Neg) control, eGFP (pBP0047), iC9
  • Fig. 15B rapamycin
  • Fig. 15C schematic.
  • Fig.s 16A and 16B are line graphs showing that tandem FKBP scaffold mediates FRBi_2.Caspase activation in the presence of rapalogs.
  • Fig. 16A HEK-293 cells were transfected with 1 ⁇ g each of SRa-SEAP reporter plasmid, ⁇ myr.iMC.2A-anti-CD19.CAR.CD3 ⁇ (pBP0608), and
  • _2. Caspase-9 (pBP0467). After 24 hours, transfected cells were harvested and treated with varying concentrations of either rimiducid, rapamycin, or rapalog, C7-isopropoxy (IsoP)-rapamycin.
  • Fig. 16B Similar to the experiment described in (Fig. 16A), except that cells were transfected with a membrane-localized (myristoylated) iMC.2A-CD19.CAR.CD3 ⁇ (pBP0609), instead of non- myristoylated Amyr.iMC.2A-CD19.CAR.CD3C (pBP0608).
  • pBP0609 membrane-localized (myristoylated) iMC.2A-CD19.CAR.CD3 ⁇
  • Amyr.iMC.2A-CD19.CAR.CD3C pBP0608
  • Figs. 17A-17E provides line graphs and the results of FACs analysis showing that the iMC "switch", FKBP2.MyD88.CD40, creates a scaffold for FRBi_2.Caspase9 in the presence of rapamycin, inducing cell death.
  • Fig. 17A Primary T cells (2 donors) were transduced with ⁇ -RV, SFG-
  • AMyr.iMC.2A-CD19 from pBP0606
  • SFG-FRB L 2.Caspase9.2A-Q.8stm.zeta from pBP0668.
  • Cells were plated with 5-fold dilutions of rapamycin. After 24 hours, cells were harvested and analyzed by flow cytometry for expression of iMC (anti-CD19-APC), Caspase-9 (anti-CD34-PE), and T cell identity (anti-CD3-PerCPCy5.5). Cells were initially gated for lymphocyte morphology by FSC vs SSC, followed by CD3 expression (-99% of the lymphocytes).
  • CD3 + lymphocytes were plotted for CD19 (Amyr.iMC.2A-CD19) vs CD34 (FRB L 2.Caspase9.2A-Q.8stm.zeta) expression.
  • percentages of CD34 + CD19 + cells were divided by percent CD19 + CD34 " cells within each sample as an internal control. Those values were then normalized to drug free wells for each transduction which were set at 100%. Similar analysis was applied to the Hi-, Med-, and Lo-expressing cells within the CD34 + CD19 + gate.
  • Fig. 17B Representative example of how cells were gated for Hi, Med, and Lo expression.
  • Fig. 17C Representative scatter plots of final CD34 vs CD19 gates.
  • Fig. 17D and Fig. 17E T cells from a single donor were transduced with AMyr.iMC.2A-CD19 (pBP0606) or FRB L 2.Caspase9.2A-Q.8stm.zeta (pBP0668). Cells were plated in IL-2-containing media along with varying amounts of rapamycin for 24 or 48 hrs. Cells were then harvested and analyzed, as above.
  • Fig. 19 Plasmid map of pBP0463-pSH1-Fpk-Fpk'.LS.Fpk".Fpk"'.LS.HA
  • Fig. 24 Plasmid map of pBP0220-pSFG-iC9.T2A- ⁇ CD19 Fig. 25 Plasmid map of pBP0756-pSFG-iC9.T2A-dCD19.P2A-FRBI Fig. 26 Plasmid map of pBP0755-pSFG-iC9.T2A-dCD19.P2A-FRBI2 Fig. 27 Plasmid map of pBP0757-pSFG-iC9.T2A-dCD19.P2A-FRBI3 Fig. 28 Plasmid map of pBP0655-pSFG-AMyr.FRBI.MC.2A- ⁇ CD19 Fig.
  • Fig. 31 Plasmid map of pBP0467-pSH1-FRBI'.FRBI.LS.ACaspase9 Fig. 32 Plasmid map of pBP0606-pSFG-k-AMyr.iMC.2A- ⁇ CD19 Fig. 33 Plasmid map of pBP0607-pSFG-k-iMC.2A- ⁇ CD19
  • Fig. 34 Plasmid map of pBP0668 ⁇ pSFG-FRBIx2.Caspase9.2A-Q.8stm.CD3zeta
  • Fig. 35 Plasmid map of pBP0608-pSFG-AMyr.iMC.2A- ⁇ CD19.Q.8stm.CD3zeta
  • Fig. 37A provides a schematic of rimiducid binding to two copies of a chimeric Caspase-9 polypeptide, each having a FKBP12 multimerizing region.
  • Fig. 37B provides a schematic of rapamycin binding to two chimeric Caspase-9 polypeptides, one of which has a FKBP12 multimerizing region and the other which has a FRB multimerizing region.
  • Fig. 37C provides a graph of assay results using these chimeric polypeptides.
  • Fig. 38A provides a schematic of rapamycin or rapalog binding to two chimeric Caspase-9 polypeptides, one of which has a FKBP12v36 multimerizing region and the other which has a FRB variant (FRB L ) multimerizing region.
  • Fig. 38B provides a graph of assay results using this chimeric polypeptide.
  • Fig. 39A provides a schematic of rimiducid binding to two chimeric Caspase-9 polypeptides, each of which has a FKBP12v36 multimerizing region, and rapamycin binding to only one chimeric Caspase-9 polypeptide having a FKBP12v36 multimerizing region.
  • Fig. 39B provides a graph of assay results comparing the effects of rimiducid and rapamycin.
  • FIG. 40A provides a schematic of rimiducid binding to two chimeric Caspase-9 polypeptides, each of which has a FKBP12v36 multimerizing region, and rapamycin binding to only one chimeric Caspase-9 polypeptide having a FKBP12v36 multimerizing region in the presence of a FRB multimerization polypeptide.
  • Fig. 40B provides a graph of assay results using these polypeptides, comparing the effects of rimiducid and rapamycin.
  • Fig. 41 provides a plasmid map of pBP0463.pFRBI.LS.dCasp9.T2A.
  • Fig. 42 provides a plasmid map of pBP044-pSH1.iCasp9WT.
  • FIGs. 43A-43C Schematics of FwtFRBC9/MC.FvFv containing iFwtFRBC9 or iFRBFwtC9
  • tandem FKBP.FRB or FRB.FKBP domains are fused to Acaspase-9. Rapamycin or rapalogs can induce: 1) scaffold-induced dimerization of FKBP. FRB. ⁇ C9 (or FRB. FKBP. ⁇ C9) via the two FKBP domains fused to MC; 2) direct dimerization of FKBP. FRB. ⁇ C9 (or FRB. FKBP. ⁇ C9) to induce multimerization of the engineered caspase-9 fusion proteins.
  • Figs. 44A-44C Expression profile of iMC + CAR ⁇ -T, i9 + CAR ⁇ + MC, and FwtFRBC9/MC.FvFv T cells.
  • PBMCs from four different donors were activated and transduced with iMC + CAR ⁇ -T (608), i9 + CAR ⁇ + MC (844), and FwtFRBC9/MC.FvFv (1300)-containing vectors.
  • Fig. 48 For a vector schematic see Fig. 48.
  • iRC9 migrates the same as the endogenous caspase-9 and the added strength of the band denotes the level of the iRC9.
  • B CAR expression were analyzed 4, 7, 12, 21 , and 29 days post-transduction with anti-CD34-PE and anti-CD3-PerCPcy5 antibodies.
  • C T cell viability from cells growing in culture was assessed 3, 5, 12, 21 , and 29 days post- transduction using a Cellometer and AOPI viability dye.
  • Figs. 45A-45C Rapamycin induces robust apoptosis activation in FwtFRBC9/MC.FvFv T cells.
  • PBMCs from four different donors were activated and transduced with iMC + CAR ⁇ -T (608), i9 + CAR ⁇ + MC (844), and FwtFRBC9/MC.FvFv (1300)-containing vectors.
  • Five days post- transduction, T cells were seeded onto 96-well plates ⁇ rimiducid, ⁇ rapamycin, and in the presence of 2 ⁇ caspase 3/7 green reagent.
  • Figs. 46a-46C Q-LEHD-OPh efficiently inhibits caspase activation induced by iC9 and iRC9.
  • PBMCs were activated and transduced with i9 + CAF. ⁇ + MC (844) and FwtFRBC9/MC.FvFv (1300) vectors. Seven days post-transduction, T cells were seeded on 96-well plates (A) with increasing rimiducid/rapamycin concentration, (B) with increasing Q-LEHD-OPh concentration, and (C) with 20 nM rimiducid/rapamycin and increasing Q-LEHD-OPh concentration. Additionally, 2 ⁇ caspase 3/7 green reagent was added to monitor caspase cleavage by IncuCyte.
  • Figs. 47A-47D FRBL and caspase-9 N405Q mutants reduce iRC9 activity.
  • PBMCs were activated and transduced with plasmids 1300, 1308, 1316 and 1317.
  • T cells were seeded onto 96-well plates with 0 (A), 0.8 (B), 4 (C), and 20 nM (D) rapamycin. 2 ⁇ caspase 3/7 green reagent was included to monitor caspase activation over time in the IncuCyte.
  • Figs. 48A-48D iRC9 is a potent effector of rapamycin-induced apoptosis.
  • FwtFRBC9/MC.FvFv constructs (B-D) Activated T cells were transduced with retrovirus encoding iMC + CAR ⁇ -T, i9 + CAR ⁇ + MC, iFRBC9 and MC.FvFv, or FwtFRBC9/MC.FvFv and treated with no drug, 20 nM rapamycin or 20 nM rimiducid and cultured in the presence of 2.5 ⁇ caspase 3/7 green reagent. The 96-well microplate was placed inside the IncuCyte to monitor activated caspase activity (green fluorescence) for 48 hours.
  • Figs. 49A-49D iRC9 quickly and efficiently eliminates CAR-T cells in vivo.
  • a and B NSG mice were injected i.v. with 10 7 iMC + CAR ⁇ -T, i9 + CAR ⁇ + MC, iFRBC9 and MC.FvFv or
  • FwtFRBC9/MC.FvFv T cells co-transduced with GFP-Ffluc per mouse. Bioluminescence of CAR T cells was assessed 18 hours (-18h) prior to drug treatment, immediately before drug treatment (Oh) and 4.5h, 18h, 27h, and 45h post-drug treatment.
  • i9 + CAR ⁇ + MC T cell injection 5 mg/kg rimiducid was injected i.p. per mouse.
  • mice receiving iMC + CAR ⁇ -T, (iFRBC9 and MC.FvFv) and FwtFRBC9 MC.FvFv T cells 10 mg/kg rapamycin was injected i.p. per mouse.
  • mice were euthanized and (C) blood and (D) spleen were collected for flow cytometry analysis with antibodies to hCD3, hCD34, and mCD45.
  • Figs. 50A-50D The on- and off-switches in FwtFRBC9/MC.FvFv are efficiently controlled by rimiducid and rapamycin, respectively.
  • PBMCs from donor 920 were activated and co-transduced with GFP-Ffluc and iMC + CAF ⁇ -T (189), i9 + CAF ⁇ + MC (873), or FwtFRBC9/MC.FvFv (1308)- encoding vectors.
  • T cells were seeded onto 96-well plates at 1 :2 and 1 :5 E:T ratios with HPAC-RFP cells in the presence of 0, 2, or 10 nM rimiducid and placed in the IncuCyte to monitor the kinetics of T cell-GFP and HPAC-RFP growth.
  • a & B Two days post- seeding, culture supernatants were analyzed for IL-2, IL-6, and IFN- ⁇ production by ELISA.
  • Figs. 51A-51 E iRC9 activates apoptosis via direct self-dimerization independent of scaffold- induced dimerization in FwtFRBC9/MC.FvFv.
  • PBMCs from donor 920 were activated and transduced with various vectors de in (A).
  • B Protein expression of the CAR T cells was analyzed by Western blot using antibodies to hMyD88, hCaspase-9 and ⁇ -actin.
  • C-D Five days post- transduction, T cells were seeded on 96-well plates with increasing rapamycin concentrations. Additionally, 2 ⁇ caspase 3/7 green reagent was added to monitor caspase cleavage by
  • IncuCyte Line graphs depict caspase activation over 24 hours post-rapamycin treatment of MC variants (C) and FRB.FKBP. ⁇ C9 versus FKBP.FRB. ⁇ C9 iRC9(D).
  • E Seven days post- transduction, T cells were seeded onto 96-well plates with increasing rimiducid concentrations and IL-2 and IL-6 secretion were quantified by ELISA 48 hours post-rimiducid treatment.
  • Figs. 52A-52B Relatively high (> 100 nM) rimiducid concentration is required to activate iRC9. 293 cells were seeded at 300,000 cells/well in a 6-well plate and allowed to grow for 2 days. After 48h, cells were transfected with 1 ⁇ g of experimental plasmids. Cells were harvested 48h after transfection and diluted 2.5X their original volume. (A) For the lncucyte/casp3/7 assay, 50 ⁇ of cells were plated per well including either rimiducid or rapamycin drug and caspase 3/7 green reagent (2.5 ⁇ final concentration).
  • FIGs. 53A-53B Schematic of MC-Rap, a CAR-costimulation strategy inducible with rapamycin or rapalogs.
  • tandem FKBP. FRB (or FRB. FKBP) domains are fused to MyD88-CD40 (MC) (right).
  • Rapamycin or rapalogs can induce direct dimerization of FKBP in MC-FKBP-FRB (or MC-FRB-FKBP) with FRB in a second molecule of MC- FKBP-FRB to induce multimerization of the engineered MC fusion proteins.
  • FRB can be present as the wild-type or as a mutant such as FRBL inducible with rapalogs that have reduced affinity for mTOR. This strategy is contrasted with homodimerization directed by rimiducid and FKBPvse in the iMC + CAR ⁇ platform (left).
  • Figs. 54A-54B Induction of MC costimulatory activity with a rapalog and a MC-Rap-CAR.
  • Human PBMCs were activated and transduced with iMC + CAR ⁇ constructs (BP0774 and BP1433), MC- rap-CAR (BP1440) or an noninducible MC only construct (BP1151).
  • Cells were allowed to rest for 6 days then aliquots were stimulated with rimiducid or the rapalog C7-dimethoxy-7- isobutyloxyrapamycin.
  • Supernatant media was harvested 24 hours later and the amount of secreted IL-6 determined by ELISA as an indicator of MC activity.
  • MC activity in iMC + CAR ⁇ -T cells is stimulated strongly with rimiducid and not with the rapalog.
  • MC activity in MC-rap-T cells is not stimulated with rimiducid because FKBP12 in pBP1440 is the wild-type rather than the rimiducid sensitive allele V36.
  • MC-Rap activity is instead strongly responsive to
  • Figs. 55A-55B Protein expression of MC from iMC + CAR ⁇ .
  • Human PBMCs were activated and transduced with iMC + CAR ⁇ constructs (BP0774, BP1433 and BP1439), MC-rap-CAR (BP1440) or an noninducible MC only constructs (BP1151 oriented at the 5' end of the retrovirus and 1414 oriented 3' relative to the CAR).
  • Cells were expanded for 2 weeks then extracts were prepared for SDS-PAGE. Western blots were probed with antibodies to MyD88.
  • the MC-FKBP-FRB fusion protein was expressed at a similar level to the MC-FKBP V fusions from iMC + CAR ⁇ constructs.
  • Figs. 56A -56B esponsiveness of MC-rap to dosage of rapamycin and rapamycin analog.
  • 293T cells were transfected with 1 ⁇ g of reporter construct NF- ⁇ SeAP and 4 ⁇ g of the iMC + CAR ⁇ construct pBP0774 or the MC-rap-CAR construct pBP1440 using the GeneJuice protocol
  • NF- ⁇ reporter activity was stimulated with a subnanomolar EC50 with both the rapalog and rapamycin while up to 50 nM rimiducid could not direct MC-rap dimerization.
  • FIGs. 57A-57B Schematic of MC-Rap, a CAR-costimulation strategy inducible with rapamycin or rapalogs.
  • FwtFRBC9/MC.FvFv tandem FKBP.FRB (or FRB.FKBP) domains are fused to Caspase 9 and tandem Fv moieties are fused to MC.
  • Caspase 9 can be activated by
  • FRBFwtMC/FvC9 uses rapamycin or rapalogs can to induce MC-rap while iC9 induced by rimiducid for a cell suicide switch.
  • FRBFwtMC/FvC9 can effectively control tumor growth but is abrogated by activation of iC9 with rimiducid.
  • PBMCs from donor 676 were activated and transduced with a CD19 directed i9 + CAR ⁇ + MC (BP0844), FRBFwtMC/FvC9 (BP1460) or FwtFRBC9/MC.FvFv (BP1300).
  • T cells were seeded onto 24-well plates at 1 :5 E:T ratios with Raji-GFP cells in the presence of 2 nM rimiducid, 2 nM isobutyloxyrapamycin or 2 nM rapamycin.
  • Fig. 59 Schematic of plasmid pBP1300-pSFG-FKBP.FRB. ⁇ C9.T2A-aCD19.Q.CD8stm. ⁇ .P2A-iMC
  • Fig. 60 Schematic of plasmid pBP1308-pSFG-FKBP.FRB. ⁇ C9.T2A-aPSCA.Q.CD8stm. ⁇ .P2A-iMC
  • Fig. 61 Schematic of plasmid pBP1310-pSFG.FRB.FKBP. ⁇ C9.T2A- ⁇ CD19
  • Fig. 62 Schematic of plasmid pBP131 1-pSFG.FKBP.FRB. ⁇ C9.T2A- ⁇ CD19
  • Fig. 63 Schematic of plasmid pBP1316-pSFG-FKBP.FRB L . ⁇ C9.T2A-aPSCA.Q.CD8stm. ⁇ .P2A-iMC
  • Fig. 64 Schematic of plasmid pBP1317-pSFG-FKBP.FRB. ⁇ C9 Q .T2A-aPSCA.Q.CD8stm. ⁇ .P2A- iMC
  • Fig. 65 Schematic of plasmid pBP1319-pSFG-FKBP.FRB. ⁇ C9.T2A-aPSCA.Q.CD8stm. ⁇ .P2A- MC.FKBPv
  • Fig. 66 Schematic of plasmid pBP1320 ⁇ pSFG-FKBP.FRB. ⁇ C9.T2A-aPSCA.Q.CD8stm. ⁇ .P2A-MC
  • Fig. 67 Schematic of plasmid pBP1321--pSFG-FKBP.FRB. ⁇ C9.T2A-aPSCA.Q.CD8stm. ⁇ .P2A- MC.FKBPv.FKBP
  • Fig. 68A provides a graph of drug-dependent CAR-T cell killing of tumor cells.
  • Fig. 68B provides schematics of of inducible MyD88-CD40 polyeptides.
  • Fig. 69A provides a schematic representation of retroviral vectors that express inducible MyD88- CD40 polypeptides.
  • Fig. 69B provides a bar graph of results of a reporter assay of costimulatory signaling.
  • Fig. 69C provides a bar graph of CAR-T cell cytokine secretion.
  • Fig. 69D provides a graph of a CAR-T cell killing assay.
  • Fig. 70A provides a schematic representation of retroviral vectors that express inducible MyD88- CD40 polypeptides.
  • Fig. 70B provides a graph of a reporter assay of costimulatory signaling.
  • Fig. 70C provides a graph of a PSCA-CAR-T cell killing assay.
  • Fig. 70D provides a graph of a PSCA CAR-T cell killing assay.
  • Fig. 70E provides a graph of a HER2-CAR-T cell killing assay.
  • Fig. 70F provides a graph of a HER2-CAR-T cell killing assay.
  • Fig. 70G provides a graph of a HER2-CAR-T cell killing assay.
  • Fig. 70A provides a schematic representation of retroviral vectors that express inducible MyD88- CD40 polypeptides.
  • Fig. 70B provides a graph of a reporter assay of costimulatory signaling.
  • Fig. 70C provides
  • 71A provides a graph of apoptosis activity directed by inducible Caspase-9in the presence of rimiducid.
  • Fig. 71 B provides a graph of apoptosis activity directed by inducible Caspase-9 in the presence of C7-isobutyloxyrapamycin.
  • Fig. 72A provides a schematic of polypeptides expressed on a single vector, including a CAR polypeptide, a iRC9 polypeptide, and an iMC polypeptide.
  • Fig. 72B provides schematics of the polypeptides expressed on two separate vectors.
  • Fig. 73A provides a schematic of inducible Caspase 9 retroviral constructs.
  • Fig. 73B provides data showing fluorescent conversion of cells that express Caspase 9 in the presence of rapamycin.
  • Fig. 73C provides a graph of relative apoptosis activity of Fig. 73B.
  • Fig. 73D provides a Western blot of Caspase-9 transgene expression in T cells.
  • Fig. 74A provides a graph of IL-6 secretion in the presence of rimiducid.
  • Fig. 74B provides a graph of IL-2 secretion in the presence of rimiducid.
  • Fig. 74C provides a graph of IFN- ⁇ secretion in the presence of rimiducid.
  • Fig. 74D provides a graph of CAR-T cell killing in thepresence of rimiducid.
  • Fig. 74E provides a Western blot of expression of iMC and iRC9.
  • Fig. 75A provides cell sorting results from non-transduced T cells, or T cells transduced with retroviruses that encode iRC9, iMC, and CAR, as indicated.
  • Fig. 75B provides a graph of the results of Fig. 75A.
  • Fig. 75C provides cell sorting results of an apoptosis assay.
  • Fig.75D provides a graphical representation of an apopotosis assay.
  • Fig. 76A provides micrographs of tumor bearing animals determined by bioluminescence imaging.
  • Fig. 76B provides graphs of average tumor growth.
  • Fig. 76C provides graphs of human T cells in spleens at termination.
  • Fig. 76D provides graphs of vector copy number.
  • Fig. 77A provides micrographs of tumor-bearing animals determined by bioluminescence imaging.
  • Fig. 77B provides graphs of average radiance.
  • Fig. 77C provides a graph of a Kaplan-Meier analysis from Fig. 77A.
  • Fig. 77D provides a representative FACS analysis at termination.
  • Fig.78A provides micrographs of tumor-bearing animals determined by bioluminescence imaging.
  • Fig. 78B provides graphical representations of the average calculated radiance from Fig. 78A.
  • Fig.78C provides a graph of human T cell counts in mouse spleens.
  • Fig. 79A provides micrographs of tumor-bearing animals determined by bioluminescence imaging.
  • Fig. 79B provides a graphical representation of the average calculated radiance from Fig. 79A.
  • Fig. 79C provides a graph of the number of human T cells in mouse spleens at termination.
  • Fig. 79D provides graphs of vector copy number from DNA derived from mouse spleens.
  • Fig. 80 provides a plasmid map of pBP1151-pSFG-MC-T2A-aCD19.Q.CD8stm. ⁇
  • Fig. 81 provides a plasmid map of pBP1152--pSFG-MC-T2A-aCD19.Q.CD8stm. ⁇
  • Fig. 82 provides a plasmid map of pBP1414--pSFG-aCD19.Q.CD8stm.- ⁇ P2A-MC
  • Fig. 83 provides a plasmid map of pBP1414--pSFG-aCD19.Q.CD8stm.- ⁇ P2A-MC
  • Fig. 84 provides a plasmid map of pBP1433--pSFG— Fv-Fv-MC-T2A-aCD19.Q.CD8stm. ⁇
  • Fig. 85 provides a plasmid map of pBP1439-pSFG-MC.FKBP v -T2A-aCD19.Q.CD8stm. ⁇
  • Fig. 86 provides a plasmid map of pBP1440--pSFG-FKBPv. ⁇ C9.T2A- aCD19.Q.CD8stm. ⁇ .T2A.P2A-MC.FKBP wt .FRB L
  • Fig. 87 provides a plasmid map of pBP1460--pSFG-FKBPv. ⁇ C9.T2A- aCD19.Q.CD8stm. ⁇ .T2A.P2A-MC.FKBP wt .FRB L
  • Fig. 88 provides a plasmid map of pBP1293--pSFG-iMC.T2A-ahCD33(My9.6). ⁇
  • Fig. 89 provides a plasmid map of pBP1296-pSFG-iMC.T2A-ahCD123(32716). ⁇
  • Fig. 90 provides a plasmid map of pBP1327-pSFG-FRB.FKBP v . ⁇ C9.2A- ⁇ CD19
  • Fig. 91 provides a plasmid map of pBP1328-pSFG-FKBP v .FRB. ⁇ C9.2A- ⁇ CD19
  • Fig. 92 provides a plasmid map of pBP1351-pSFG-SP163.FKBP.FRB. ⁇ C9.T2A- ahPSCA.Q.CD8stm. ⁇ 2A-iMC
  • Fig. 93 provides a plasmid map of pBP1373-pSFG-sp-FKBP.FRB. ⁇ C9.T2A- ahPSCAscFv.Q.CD8stm. ⁇
  • Fig. 94 provides a plasmid map of pBP1385-pSFG-FRB.FKBP. ⁇ C9.T2A- ⁇ CD19
  • Fig. 97 provides a plasmid map of pBP1474--pSFG-FKBPv. ⁇ C9.T2A-aHER2.Q.CD8stm.
  • Fig. 98 provides a plasmid map of pBP1475--pSFG-FKBPv. ⁇ C9.T2A-aPSCA.Q.CD8stm.
  • Fig. 99 provides a plasmid map of pBP1488--pSFG-FRB L .FKBP wt .MC-T2A-aPSCA.Q.CD8stm. ⁇ Fig.
  • 100 provides a plasmid map of pBP1491-pSFG- - FKBPv. ⁇ C9.P2A.MC.FKBP wt .FRB L .T2A- aHER2.Q.CD8stm. ⁇
  • Fig. 101 provides a plasmid map of pBP1493-pSFG- MC.FKBP wt .FRB L -P2A.FKBPv. ⁇ C9.T2A- aHER2.Q.CD8stm. ⁇
  • Fig. 102 provides a plasmid map of pBP1494-pSFG- MC.FKBP wt .FRB L -P2A.FKBPv. ⁇ C9.T2A- PSCA.Q.CD8stm. ⁇
  • Fig. 103 provides a plasmid map of pBP1757-pSFG- FRB L .FKBP wt .MC-P2A.FKBPv. ⁇ C9.T2A- aPSCA.Q.CD8stm. ⁇
  • Fig. 104 provides a plasmid map of pBP1759--pSFG--FRB L .FKBP wt .MC-P2A.FKBPv. ⁇ C9.T2A- aHER2.Q.CD8stm. ⁇
  • Fig. 105 provides a plasmid map of pBP1796-pSFG--FKBP wt .FRB L -MC. P2A.FKBPv. ⁇ C9.T2A- aPSCA.Q.CD8stm. ⁇
  • Fig. 106A provides a schematic of various inducible chimeric Caspase-9 constructs.
  • Fig. 106 provides graphs of caspase activation assays.
  • Fig.106C is a photo of a Western blot showing protein expression.
  • Fig. 107A provides graphs of caspase activity.
  • Fig. 107B provides graphs of SEAP activity.
  • Fig. 108A provides graphs of SEAP activity.
  • Fig. 108B provides graphs of caspase activity.
  • Fig. 108C provides a Western blot showing protein expression.
  • Fig. 109A provides a FACS analysis of transduction efficiency.
  • Fig. 109B provides graphs of bioiluminesence.
  • Fig. 109C provides photos of bioiluminesence in mice.
  • Fig. 109D provides graphs of FACs analysis of mice spleen cells.
  • Fig. 1 1 OA provides a FACs analysis of transduction efficiency.
  • Fig. 11 OB provides graphs of bioiluminescence.
  • Fig.HOC provides photos of bioiluminescence in mice.
  • Fig. 110D provides a graph of FACs analysis of mice spleen cells.
  • Fig.1 11 provides a schematic of a vector encoding a CD123-CAR ⁇ and an iMC polypeptide.
  • Fig. 1 12A provides a graph of IL-6 production;
  • Fig. 1 12 B provides a graph of IL-2 production;
  • Fig. 112C provides a graph of total green fluorescence intensity of THP1-GP.FIuc, and
  • Fig. 112D provides a graph of number of HPAC-RFP cells.
  • Fig. 1 13A provides a graph of IL-2 production
  • Fig. 1 13B provides a graph of THP1-FP.FIuc cells
  • Fig. 1 13C provides a graph of T cells-RFP
  • Fig.D provides a graph of THP1-GFP. Flue green fluorescence
  • Fig. E provides a graph of T cell-RFP red fluorescence.
  • Fig. 1 14A provides a FACs anlysis
  • Fig. 114B provides a schematic of tumor growth via IVIS monitoring
  • Fig. 114C provides photos of bioiluminescence in mice
  • Fig. 1 14D provides a graph of CAR-T cell presence as measured by flow cytometry
  • Fig. 114E provides a graph of vector copy number.
  • Fig. 1 15A provides photos of bioiluminescence in mice;
  • Fig. 115B provides a graph of vector copy number.
  • Fig. 1 16 provides a schematic of inducible MC expressed with a recombinant TCR.
  • Fig. 1 17A provides a schematic of a PRAM E TCR polypeptide ;
  • Fig. 117B provides a schematic of an iMC polypeptide;
  • Fig. 117C provides a schematic of a PRAME-TCR polypeptide co-expressed with an iMC polypeptide;
  • Fig. 1 17D provides a graph of IL-2 production, items listed along the X- axis are in the same order as the legend.
  • Fig. 1 18A provides a schematic of trans-well assay set-up;
  • Fig. 118B provides a graph of HLA-A, B, C levels.
  • Fig. 1 19 A provides a graph of specific lysis.
  • Fig. 1 19B provides a graph of IL-2 production.
  • Fig. 120A provides a graph of specific lysis
  • Fig. 120 B provides a graph of IL-2 production.
  • Fig. 121 A provides a schematic of an immune-deficient NSG xenographt model
  • Fig. 121 B provides graphs of average radiance in non-transduced and transduced cells
  • Fig. 121C provides a graph of the number of ⁇ 1 + CD8 + cells/spleen
  • Fig. 121 D provides a graph of the number of ⁇ 1 + CD8 + cells/spleen.
  • Transduction of signals is governed by enzymatic processes, such as amino acid side chain phosphorylation, acetylation, or proteolytic cleavage that lack intrinsic specificity. Furthermore, many proteins or factors are present at cellular concentrations or at subcellular locations that preclude spontaneous generation of a sufficient substrate/product relationship to activate or propagate signaling. An important component of activated signaling is the recruitment of these components to signaling "nodes” or spatial signaling centers that efficiently transmit (or attenuate) the pathway via appropriate upstream signals.
  • CID chemically induced dimerization
  • a single protein would be modified to contain one or more structurally identical ligand binding domains, which would then be the basis of homodimerization or oligomerization, respectively, in the presence of a cognate homodimeric ligand (Spencer DM et al (93) Science 262, 1019-24).
  • Spcer DM et al (93) Science 262, 1019-24 A slightly more complicated version of this concept would involve placing one or more distinct ligand binding domains on two different proteins to enable
  • a method to induce multimerization of proteins in this case, two or more heterodimer ligand binding regions (or “domains") in tandem are used as a "molecular scaffold" to dimerize or oligomerize a second, signaling domain-containing protein that is fused to one or more copies of the second binding site for the heterodimeric ligand.
  • the molecular scaffold can be expressed as an isolated multimer of ligand binding domains (Fig. 8), either localized within the cell or unlocalized (Fig. 8B, 8C), or it can be attached to another protein that provides a structural, signaling, cell marking, or more complex combinatorial function (Fig. 9).
  • scaffold is meant a polypeptide that comprises at least two, for example, two or more, heterodimer ligand binding regions; in certain examples the ligand binding regions are in tandem, that is, each ligand binding region is located directly proximal to the next ligand binding region. In other examples, each ligand binding region may be located close to the next ligand binding region, for example, separated by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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, but retain the scaffold function of dimerization of an inducible caspase molecule in the presence of a dimerizer.
  • a scaffold may comprise, 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 may also be linked to another polypeptide, such as, for example, a marker polypeptide, a costimulating molecule, a chimeric antigen receptor, a T cell receptor, or the like.
  • the first polypeptide consists essentially of at least two, three, four, five, six, seven, eight, nine, ten, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 units of the first multimerizing region.
  • first polypeptide consists essentially of the scaffold region.
  • the first polypeptide consists essentially of a membrane association region or a membrane targeting region.
  • linker polypeptides at either terminus of the scaffold, or between the units, and can optionally include small polypeptides such as, for example stem polypeptides as shown in Figs. 10B, 10C, 10D, and 10E.
  • a tandem multimer of the ⁇ 89 aa FK506-rapamycin binding (FRB) domain derived from the protein kinase mTOR (Chen J et al (95) PNAS, 92, 4947-51) is used to recruit multiple FKBPv36-fused Caspase-9 (iC9/iCaspase-9) in the presence of rapamycin or a rapamycin-based analogue ("rapalog")
  • Library 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) (Figs.
  • tandem FRB domains are fused to a chimeric antigen receptor (CAR) and this provides rapalog-driven iC9 activation to cells expressing both fusion proteins (Fig. 15, inset).
  • CAR chimeric antigen receptor
  • Fig. 15, inset the polarity of the two proteins are reversed so that two or more copies of
  • FKBP12 are used to recruit and multimerize FRB-modified signaling molecules in the presence of rapamycin (Fig. 8C, 9A).
  • a chimeric polypeptide may comprise a single ligand binding region, or a scaffold comprising more than one ligand binding region may be, 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.
  • MyD88, or MyD88 polypeptide is meant the polypeptide product of the myeloid differentiation primary response gene 88, for example, but not limited to the human version, cited as ncbi Gene ID 4615.
  • truncated is meant that the protein is not full length and may lack, for example, a domain.
  • a truncated MyD88 is not full length and may, for example, be missing the TIR domain.
  • An example of a truncated MyD88 polypeptide amino acid sequence is presented as SEQ ID NO: 305.
  • a nucleic acid sequence coding for "truncated MyD88” is meant the nucleic acid sequence coding for the truncated MyD88 peptide, the term may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by the linkers.
  • a method or construct refers to a truncated MyD88 polypeptide
  • the method may also be used, or the construct designed to refer to another MyD88 polypeptide, such as a full length MyD88 polypeptide.
  • the method may also be used, or the construct designed to refer to a truncated MyD88 polypeptide.
  • the CD40 portion of the peptide may be located either upstream or downstream from the MyD88 or truncated MyD88 polypeptide portion.
  • unstable FRB variants e.g., FRBL2098
  • FRBL2098 FRBL2098
  • Fig. 9, 10 the unstable fusion molecule is stabilized leading to aggregation as before, but with lower background signaling.
  • ligands to direct signaling proteins may be generally applied to activate or attenuate many signaling pathways. Examples are provided herein that demonstrate a utility of the approach by controlling apoptosis or programmed cell death with the "initiating caspase", Caspase-9 as the primary target. Control of apoptosis by dimerization of proapoptotic proteins with widely available rapamycin or more proprietary rapalogs, should permit an experimenter or clinician to tightly and rapidly control the viability of a cell-based implant that displays unwanted effects. Examples of these effects include, but are not limited to, Graft versus Host (GvH) immune responses against off-target tissue or excessive, uncontrolled growth or metastasis of an implant.
  • GvH Graft versus Host
  • Apoptosis is tightly regulated and naturally uses scaffolds, such as Apaf-1 , CRADD/RAIDD, or FADD/Mort1 , to oligomerize and activate the caspases that can ultimately kill the cell.
  • Apaf-1 can assemble the apoptotic protease Caspase-9 into a latent complex that then forms an active oligomeric apoptosome upon recruitment of cytochrome C to the scaffold.
  • the key event is oligomerization of the scaffold units causing dimerization and activation of the caspase.
  • Similar adapters such as CRADD, can oligomerize Caspase-2, leading to 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 permit the spontaneous dimerization and activation of caspase units present as FRB or FKBP fusions upon recruitment with rapamycin.
  • caspase activation occurs only when rapamycin or rapalogs are present to recruit the FRB or FKBP-fused caspase to the scaffold.
  • the FRB or FKBP polypeptides must be present as a multimeric unit not as monomers to drive FKBP- or FRB-caspase dimerization (except when FRB-Caspase-9 is dimerized with FKBP-Caspase-9).
  • the FRB or FKBP-based scaffold can be expressed in a targeted cell as a fusion with other proteins and retains its capacity to serve as a scaffold to assemble and activate proapoptotic molecules.
  • the FRB or FKBP scaffold may be localized within the cytosol as a soluble entity or present in specific subcellular locales, such as the plasma membrane through targeting signals.
  • the components used to activate apoptosis and the downstream components that degrade the cell are shared by all cells and across species. With regard to Caspase-9 activation, these methods can be broadly utilized in cell lines, in normal primary cells, such as, for example, but not limited to, T cells, or in cell implants.
  • FKBP-fused Caspases can be dimerized by homodimerizer molecules, such as AP1510, AP20187 or AP1903 (Fig. 6 (right panel), 10A (schematic) (A similar proapototic switch can be directed via heterodimerization of a binary switch using rapamycin or rapalogs by coexpression of a FRB-Caspase-9 fusion protein along with FKBP-Caspase-9, leading to homodimerization of the caspase domains within the chimeric proteins (Fig. 8A (schematic), 10B (schematic), (1 1).
  • allogeneic refers to HLA or MHC loci that are antigenically distinct. Thus, cells or tissue transferred from the same species can be antigenically distinct. Syngeneic mice can differ at one or more loci (congenics) and allogeneic mice can have the same
  • antigen as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • an "antigen recognition moiety” may be any polypeptide or fragment thereof, such as, for example, an antibody fragment variable domain, either naturally-derived, or synthetic, which binds to an antigen.
  • antigen recognition moieties include, but are not limited to, polypeptides derived from antibodies, such as, for example, single-chain variable fragments (scFv), Fab, Fab', F(ab')2, and Fv fragments; polypeptides derived from T Cell receptors, such as, for example, TCR variable domains; and any ligand or receptor fragment that binds to the extracellular cognate protein.
  • cancer as used herein is defined as a hyperproliferation of cells whose unique trait— loss of normal controls— results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis.
  • examples include but are not limited to, melanoma, non-small cell lung, small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck, breast, pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon, sarcoma or bladder.
  • Donor refers to a mammal, for example, a human, that is not the patient recipient.
  • the donor may, for example, have HLA identity with the recipient, or may have partial or greater HLA disparity with the recipient.
  • Haploidentical refers to cells sharing a haplotype or cells having substantially the same alleles at a set of closely linked genes on one chromosome. A haploidentical donor does not have complete HLA identity with the recipient, there is a partial HLA disparity.
  • Blood disease refers to conditions that affect the production of blood and its components, including but not limited to, blood cells, hemoglobin, blood proteins, the mechanism of coagulation, production of blood, production of blood proteins, the like and combinations thereof.
  • blood diseases include anemias, leukemias, lymphomas, hematological neoplasms, albuminemias, haemophilias and the like.
  • Bone marrow disease refers to conditions leading to a decrease in the production of blood cells and blood platelets.
  • normal bone marrow architecture can be displaced by infections (e.g., tuberculosis) or malignancies, which in turn can lead to the decrease in production of blood cells and blood platelets.
  • infections e.g., tuberculosis
  • malignancies e.g., malignancies
  • Non-limiting examples of bone marrow diseases include leukemias, bacterial infections (e.g., tuberculosis), radiation sickness or poisoning, apnocytopenia, anemia, multiple myeloma and the like.
  • T cells and Activated T cells include that this means CD3 + cells: T cells (also referred to as T lymphocytes) belong to a group of white blood cells referred to as lymphocytes. Lymphocytes generally are involved in cell-mediated immunity.
  • the "T” in “T cells” refers to cells derived from or whose maturation is influenced by the thymus. T cells can be distinguished from other
  • lymphocytes types such as B cells and Natural Killer (NK) cells by the presence of cell surface proteins known as T cell receptors.
  • activated T cells refers to T cells that have been stimulated to produce an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant presented in the context of a Class II major histo- compatibility (MHC) marker.
  • MHC major histo- compatibility
  • T-cells are activated by the presence of an antigenic determinant, cytokines and/or lymphokines and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, the like and combinations thereof).
  • CD3 and CD4 proteins are cell surface receptors or co-receptors that may be directly and/or indirectly involved in signal transduction in T cells.
  • peripheral blood refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow.
  • red blood cells e.g., red blood cells, white blood cells and platelets
  • platelets e.g., red blood cells, white blood cells and platelets
  • Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver or bone marrow.
  • Cord blood often contains stem cells including hematopoietic cells.
  • cytoplasmic CD40 or "CD40 lacking the CD40 extracellular domain” is meant a CD40 polypeptide that lacks the CD40 extracellular domain. In some examples, the terms also refer to a CD40 polypeptide that lacks both the CD40 extracellular domain and a portion of, or all of, the CD40 transmembrane domain.
  • obtained or prepared as, for example, in the case of cells, is meant that the cells or cell culture are isolated, purified, or partially purified from the source, where the source may be, for example, umbilical cord blood, bone marrow, or peripheral blood. The terms may also apply to the case where the original source, or a cell culture, has been cultured and the cells have replicated, and where the progeny cells are now derived from the original source.
  • kill or “killing” as in a percent of cells killed, is meant the death of a cell through apoptosis, as measured using any method known for measuring apoptosis, and, for example, using the assays discussed herein, such as, for example the SEAP assays or T cell assays discussed herein.
  • the term may also refer to cell ablation.
  • Allodepletion refers to the selective depletion of alloreactive T cells.
  • alloreactive T cells refers to T cells activated to produce an immune response in reaction to exposure to foreign cells, such as, for example, in a transplanted allograft.
  • the selective depletion generally involves targeting various cell surface expressed markers or proteins, (e.g., sometimes cluster of differentiation proteins (CD proteins), CD19, or the like), for removal using immunomagnets, immunotoxins, flow sorting, induction of apoptosis, photodepletion techniques, the like or combinations thereof.
  • the cells may be transduced or transfected with the chimeric protein-encoding vector before or after allodepletion. Also, the cells may be transduced or transfected with the chimeric protein- encoding vector without an allodepletion step, and the non-allodepleted cells may be administered to the patient. Because of the added "safety switch" it is, for example, possible to administer the non-allo-depleted (or only partially allo-depleted) T cells because an adverse event such as, for example, graft versus host disease, may be alleviated upon the administration of the multimeric ligand.
  • Graft versus host disease refers to a transplant versus host disease
  • aGVHD Acute GVHD
  • Chronic GVHD often begins 100 days or later post transplant or transfusion and can attack the same organs as acute GvHD, but also can affect connective tissue and exocrine glands.
  • Acute GvHD of the skin can result in a diffuse maculopapular rash, sometimes in a lacy pattern.
  • Donor T cell refers to T cells that often are administered to a recipient to confer anti-viral and/or anti-tumor immunity following allogeneic stem cell
  • Donor T cells often are utilized to inhibit marrow graft rejection and increase the success of alloengraftment, however the same donor T cells can cause an alloaggressive response against host antigens, which in turn can result in graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • Certain activated donor T cells can cause a higher or lower GvHD response than other activated T cells. Donor T cells may also be reactive against recipient tumor cells, causing a beneficial graft vs. tumor effect.
  • Mesenchymal stromal cell The terms "mesenchymal stromal cell” or "bone marrow derived mesenchymal stromal cell” as used herein, refer to multipotent stem cells that can differentiate ex vivo, in vitro and in vivo into adipocytes, osteoblasts and chondroblasts, and may be further defined as a fraction of mononuclear bone marrow cells that adhere to plastic culture dishes in standard culture conditions, are negative for hematopoietic lineage markers and are positive for CD73, CD90 and CD105.
  • Embryonic stem cell refers to pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo of between 50 to 150 cells. Embryonic stem cells are characterized by their ability to renew themselves indefinitely and by their ability to differentiate into derivatives of all three primary germ layers, ectoderm, endoderm and mesoderm. Pluripotent is distinguished from mutipotent in that pluripotent cells can generate all cell types, while multipotent cells (e.g., adult stem cells) can only produce a limited number of cell types.
  • inducible pluripotent stem cell refers to adult, or differentiated cells, that are “reprogrammed” or induced by genetic (e.g., expression of genes that in turn activates pluripotency), biological (e.g., treatment viruses or retroviruses) and/or chemical (e.g., small molecules, peptides and the like) manipulation to generate cells that are capable of differentiating into many if not all cell types, like embryonic stem cells.
  • Inducible pluripotent stem cells are distinguished from embryonic stem cells in that they achieve an intermediate or terminally differentiated state (e.g., skin cells, bone cells, fibroblasts, and the like) and then are induced to dedifferentiate, thereby regaining some or all of the ability to generate multipotent or pluripotent cells.
  • an intermediate or terminally differentiated state e.g., skin cells, bone cells, fibroblasts, and the like
  • CD34 + cell refers to a cell expressing the CD34 protein on its cell surface.
  • CD34 refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entrance into lymph nodes, and is a member of the "cluster of differentiation" gene family. CD34 also may mediate the attachment of stem cells to bone marrow, extracellular matrix or directly to stromal cells.
  • a cell surface glycoprotein e.g., sialomucin protein
  • CD34 + cells often are 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 lymphatics (except pleural lymphatics), mast cells, a sub-population of dendritic cells (which are factor Xllla negative) in the interstitium and around the adnexa of dermis of skin, as well as cells in certain soft tissue tumors (e.g., alveolar soft part sarcoma, pre-B acute lymphoblastic leukemia (Pre-B-ALL), acute myelogenous leukemia (AML), AML-M7, dermatofibrosarcoma protuberans, gastrointestinal stromal tumors, giant cell fibroblastoma, granulocytic sarcoma, Kaposi's sarcoma, liposarcoma, malignant fibrous histiocyto
  • Gene expression vector The terms "gene expression vector”, “nucleic acid expression vector”, or “expression vector” as used herein, which can be used interchangeably throughout the document, generally refers 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 in a host cell and be utilized to introduce a gene or genes into a host cell.
  • ARS autonomously replicating sequence
  • ARS autonomously replicating sequence
  • YAC yeast artificial chromosome
  • the genes introduced on the expression vector can be endogenous genes (e.g., a gene normally found in the host cell or organism) or heterologous genes (e.g., genes not normally found in the genome or on extra-chromosomal nucleic acids of the host cell or organism).
  • the genes introduced into a cell by an expression vector can be native genes or genes that have been modified or engineered.
  • the gene expression vector also can be engineered to contain 5' and 3' untranslated regulatory sequences that sometimes can function as enhancer sequences, promoter regions and/or terminator sequences that can facilitate or enhance efficient transcription of the gene or genes carried on the expression vector.
  • a gene expression vector sometimes also is 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 maintenance of the vector in the host or recipient cell.
  • Developmentally regulated promoter refers to a promoter that acts as the initial binding site for RNA polymerase to transcribe a gene which is expressed under certain conditions that are controlled, initiated by or influenced by a developmental program or pathway. Developmentally regulated promoters often have additional control regions at or near the promoter region for binding activators or repressors of transcription that can influence transcription of a gene that is part of a development program or pathway.
  • developmentally differentiated cells refers to cells that have undergone a process, often involving expression of specific developmentally regulated genes, by which the cell evolves from a less specialized form to a more specialized form in order to perform a specific function.
  • Non-limiting examples of developmentally differentiated cells are liver cells, lung cells, skin cells, nerve cells, blood cells, and the like.
  • Changes in developmental differentiation generally involve changes in gene expression (e.g., changes in patterns of gene expression), genetic re-organization (e.g., remodeling or chromatin to hide or expose genes that will be silenced or expressed, respectively), and occasionally involve changes in DNA sequences (e.g., immune diversity differentiation).
  • Cellular differentiation during development can be understood as the result of a gene regulatory network.
  • a regulatory gene and its cis-regulatory modules are nodes in a gene regulatory network that receive input (e.g., protein expressed upstream in a development pathway or program) and create output elsewhere in the network (e.g., the expressed gene product acts on other genes downstream in the developmental pathway or program).
  • cell may be used interchangeably. All of these terms also include their progeny, which are any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • rapalog is meant as an analog of the natural antibiotic rapamycin.
  • Certain rapalogs in the present embodiments have properties such as stability in serum, a poor affinity to wildtype FRB (and hence the parent protein, mTOR, leading to reduction or elimination of immunosuppressive properties), and a relatively high affinity to a mutant FRB domain.
  • the rapalogs have useful scaling and production properties.
  • rapalogs include, but are not limited to, S-o,p-dimethoxyphenyl (DMOP)- rapamycin: EC 50 (wt FRB (K2095 T2098 W2101) ⁇ 1000 nM), EC 50 (FRB-KLW - 5 nM) Luengo Jl (95) Chem & Biol 2:471-81 ; Luengo Jl (94) J.
  • DMOP S-o,p-dimethoxyphenyl
  • FRB refers to the FKBP12-Rapamycin-Binding (FRB) domain (residues 2015—2114 encoded within mTOR), and analogs thereof.
  • FRB analogs or variants are provided.
  • the properties of an FRB analog or variant variant are stability (some variants are more labile than others) and ability to bind to various rapalogs.
  • the FRB analog or variant binds to a C7 rapalog, such as, for example, those provided in the present application, and those referred to in publications that are incorporated by reference herein.
  • the FRB analog or variant comprises an amino acid substitution at position T2098.
  • FRB variant polypeptide regions of the present embodiments include, but are not limited to, KLW (with L2098); KTF (with F2101); and KLF (L2098, F2101).
  • FRB variant KLW corresponds to the FRBL polypeptide, for example, consisting of the amino acid of SEQ I D NO: 303, and has a substitution of an L residue at position 2098.
  • the KLW variant of SEQ I D NO: 303 with the wild type FRB polypeptide, for example, the polypeptide consisting of the amino acid sequence of SEQ I D NO: 304, one can determine the sequence of the other FRB variants listed herein.
  • Each ligand can include two or more portions (e.g., defined portions, distinct portions), and sometimes includes two, three, four, five, six, seven, eight, nine, ten, or more portions.
  • the first ligand and second ligand each, independently, can consist of two portions (i.e., dimer), consist of three portions (i.e., trimer) or consist of four portions (i.e., tetramer).
  • the first ligand sometimes includes a first portion and a second portion and the second ligand sometimes includes a third portion and a fourth portion.
  • the first portion and the second portion often are different (i.e., heterogeneous (e.g., heterodimer)), the first portion and the third portion sometimes are different and sometimes are the same, and the third portion and the fourth portion often are the same (i.e., homogeneous (e.g., homodimer)).
  • Portions that are different sometimes have a different function (e.g., bind to the first multimerizing region, bind to the second multimerizing region, do not significantly bind to the first multimerizing region, do not significantly bind to the second multimerizing region (e.g., the first portion binds to the first multimerizing region but does not significantly bind to the second multimerizing region) and sometimes have a different chemical structure.
  • the first portion sometimes binds to the first multimerizing region and sometimes does not bind significantly to the second multimerizing region.
  • Each portion sometimes is referred to as a "side.” Sides of a ligand may sometimes be adjacent to each other, and may sometimes be located at opposing locations on a ligand.
  • ligand binding region By being “capable of binding”, as in the example of a multimeric or heterodimeric ligand binding to a multimerizing region or ligand binding region is meant that the ligand binds to the ligand binding region, for example, a portion, or portions, of the ligand bind to the multimerizing region, and that this binding may be detected by an assay method including, but not limited to, a biological assay, a chemical assay, or physical means of detection such as, for example, x-ray crystallography.
  • a ligand is considered to "not significantly bind” is meant that there may be minor detection of binding of a ligand to the ligand binding region, but that this amount of binding, or the stability of binding is not significantly detectable, and, when occurring in the cells of the present embodiment, does not activate the modified cell or cause apoptosis.
  • the ligand does not "significantly bind,” upon administration of the ligand, the amount of cells undergoing apoptosis is less than 10, 5, 4, 3, 2, or 1 %.
  • region'Or “domain” is meant a polypeptide, or fragment thereof, that maintains the function of the polypeptide as it relates to the chimeric polypeptides of the present application. That is, for example, an FKBP12 binding domain, FKBP12 domain, FKBP12 region, FKBP12 multimerizing region, and the like, refer to an FKBP12 polypeptide that binds to the CID ligand, such as, for example, rimiducid, or rapamycin, to cause, or allow for, dimerization or multimerization of the chimeric polypeptide.
  • CID ligand such as, for example, rimiducid, or rapamycin
  • region or domain of a pro-apoptotic polypeptide for example, the Caspase-9 polypeptides or truncated Caspase-9 polypeptides of the present applications, is meant that upon dimerization or multimerization of the Caspase-9 region as part of the chimeric polypeptide, or chimeric pro-apoptotic polypeitde, the dimerized or multimerized chimeric polypeptide can participate in the caspase cascade, allowing for, or causing, apoptosis.
  • iCaspase-9 molecule, polypeptide, or protein is defined as an inducible Caspase-9.
  • the term “iCaspase-9” embraces iCaspase-9 nucleic acids, iCaspase-9 polypeptides and/or iCaspase-9 expression vectors. The term also encompasses either the natural iCaspase-9 nucleotide or amino acid sequence, or a truncated sequence that is lacking the CARD domain.
  • iCaspase 1 molecule As used herein, the term “iCaspase 1 molecule”, “iCaspase 3 molecule”, or “iCaspase 8 molecule” is defined as an inducible Caspase 1 , 3, or 8, respectively.
  • the term iCaspase 1 , iCaspase 3, or iCaspase 8 embraces iCaspase 1 , 3, or 8 nucleic acids, iCaspase 1 , 3, or 8 polypeptides and/or iCaspase 1 , 3, or 8 expression vectors, respectively.
  • the term also encompasses either the natural CaspaseiCaspase-1 , -3, or -8 nucleotide or amino acid sequence, respectively, or a truncated sequence that is lacking the CARD domain.
  • wild type Caspase-9 in the context of the experimental details provided herein, is meant the Caspase-9 molecule lacking the CARD domain.
  • Modified Caspase-9 polypeptides comprise at least one amino acid substitution that affects basal activity or IC 50 , in a chimeric polypeptide comprising the modified Caspase-9 polypeptide. Methods for testing basal activity and IC 50 are discussed herein. Non-modified Caspase-9 polypeptides do not comprise this type of amino acid substitution. Both modified and non-modified Caspase-9 polypeptides may be truncated, for example, to remove the CARD domain.
  • “Function-conservative variants” are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, 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 as compared to the properties of the genetically encoded amino acids.
  • amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 70%, at least 80%, at least 90%, and at least 95%, as determined according to an alignment scheme.
  • sequence similarity means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation.
  • Sequence identity herein means the extent to which two nucleotide or amino acid sequences are invariant.
  • Sequence alignment means the process of lining up two or more sequences to achieve maximal levels 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 such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using any of these programs, the settings may be selected that result in the highest sequence similarity.
  • the amino acid residue numbers referred to herein reflect the amino acid position in the non- truncated and non-modified Caspase-9 polypeptide, for example, that of SEQ ID NO: 9.
  • SEQ ID NO: 9 provides an amino acid sequence for the truncated Caspase-9 polypeptide, which does not include the CARD domain. Thus SEQ ID NO: 9 commences at amino acid residue number 135, and ends at amino acid residue number 416, with reference to the full length Caspase-9 amino acid sequence.
  • Those of ordinary skill in the art may align the sequence with other sequences of Caspase-9 polypeptides to, if desired, correlate the amino acid residue number, for example, using the sequence alignment methods discussed herein.
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • expression construct or "transgene” is defined as any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed can be inserted into the vector.
  • the transcript is translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product.
  • expression only includes transcription of the nucleic acid encoding genes of interest.
  • therapeutic construct may also be used to refer to the expression construct or transgene.
  • the expression construct or transgene may be used, for example, as a therapy to treat
  • the expression construct or transgene is a therapeutic construct or a prophylactic construct.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are discussed infra.
  • ex vivo refers to "outside” the body.
  • ex vivo and in vitro can be used interchangeably herein.
  • functionally equivalent as it relates to Caspase-9, or truncated
  • Caspase-9 refers to a Caspase-9 nucleic acid fragment, variant, or analog, refers to a nucleic acid that codes for a Caspase-9 polypeptide, or a Caspase-9 polypeptide, that stimulates an apoptotic response.
  • “Functionally equivalent” refers, for example, to a Caspase-9 polypeptide that is lacking the CARD domain, but is capable of inducing an apoptotic cell response.
  • the term “functionally equivalent” when applied to other nucleic acids or polypeptides, such as, for example, CD19, the 5'LTR, the multimeric ligand binding region, or CD3, it refers to fragments, variants, and the like that have the same or similar activity as the reference polypeptides of the methods 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 mutants.
  • hyperproliferative disease is defined as a disease that results from a hyperproliferation of cells.
  • 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.
  • immunogen refers to a substance that is capable of provoking an immune response.
  • immunogens include, e.g., antigens, autoantigens that play a role in induction of autoimmune diseases, and tumor-associated antigens expressed on cancer cells.
  • immunocompromised as used herein is defined as a subject that has reduced or weakened immune system.
  • the immunocompromised condition may be due to a defect or dysfunction of the immune system or to other factors that heighten susceptibility to infection and/or disease. Although such a categorization allows a conceptual basis for evaluation,
  • immunocompromised individuals often do not fit completely into one group or the other. More than one defect in the body's defense mechanisms may be affected. For example, individuals with a specific T-lymphocyte defect caused by HIV may also have neutropenia caused by drugs used for antiviral therapy or be immunocompromised because of a breach of the integrity of the skin and mucous membranes.
  • An immunocompromised state can result from indwelling central lines or other types of impairment due to intravenous drug abuse; or be caused by secondary malignancy, malnutrition, or having been infected with other infectious agents such as tuberculosis or sexually transmitted diseases, e.g., syphilis or hepatitis.
  • 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 an animal or a human.
  • 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 therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • polynucleotide is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable.
  • Nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.”
  • the monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means. Furthermore,
  • polynucleotides include mutations of the polynucleotides, include but are not limited to, mutation of the nucleotides, or nucleosides by methods well known in the art.
  • a nucleic acid may comprise one or more polynucleotides.
  • polypeptide is defined as a chain of amino acid residues, usually having a defined sequence.
  • polypeptide is interchangeable with the terms “peptides” and “proteins”.
  • 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.
  • transfection and “transduction” are interchangeable and refer to the process by which an exogenous DNA sequence is introduced into a eukaryotic host cell. Transfection (or transduction) can be achieved by any one of a number of means including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, superfection and the like.
  • sergeneic refers to cells, tissues or animals that have genotypes that are identical or closely related enough to allow tissue transplant, or are immunologically compatible.
  • identical twins or animals of the same inbred strain can be used interchangeably.
  • the terms "patient” or “subject” are interchangeable, and, as used herein include, but are not limited to, an organism or animal; a mammal, including, e.g., a human, non-human primate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; a non-mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate.
  • a mammal including, e.g., a human, non-human primate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep,
  • T cell activation molecule is meant a polypeptide that, when incorporated into a T cell expressing a chimeric antigen receptor, enhances activation of the T cell.
  • T cell activation molecule examples include, but are not limited to, ITAM-containing, Signal 1 conferring molecules such as, for example, CD3 ⁇ polypeptide, and Fc receptor gamma, such as, for example, Fc epsilon receptor gamma (FceRlv) subunit (Haynes, N.M., et al. J. Immunol. 166: 182-7 (2001)) J. Immunology).
  • under transcriptional control or "operatively linked” is defined as the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • the terms “treatment”, “treat”, “treated”, or “treating” refer to prophylaxis and/or therapy.
  • the term “vaccine” refers to a formulation that contains a composition presented herein which is in a form that is capable of being administered to an animal.
  • the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition is suspended or dissolved.
  • the composition can be used conveniently to prevent, ameliorate, or otherwise treat a condition.
  • the vaccine Upon introduction into a subject, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies, cytokines and/or other cellular responses.
  • the nucleic acid is contained within a viral vector.
  • the viral vector is a retroviral vector.
  • the viral vector is an adenoviral vector or a lentiviral vector. It is understood that in some embodiments, the antigen-presenting cell is contacted with the viral vector ex vivo, and in some embodiments, the antigen-presenting cell is contacted with the viral vector in vivo.
  • Hematopoietic stem cells include hematopoietic progenitor cells, immature, multipotent cells that can differentiate into mature blood cell types. These stem cells and progenitor cells may 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 inducible pluripotent stem cells.
  • Bone marrow derived mesenchymal stromal cells have been defined as a fraction of mononuclear bone marrow cells that adhere to plastic culture dishes in standard culture conditions, are negative for hematopoietic lineage markers and positive for CD73, CD90 and CD105, and able to differentiate in vitro into adipocytes, osteoblasts, and chondroblasts. While one physiologic role is presumed to be the support of hematopoiesis, several reports have also established that MSCs are able to incorporate and possibly proliferate in areas of active growth, such as cicatricial and neoplastic tissues, and to home to their native microenvironment and replace the function of diseased cells.
  • MSCs Their differentiation potential and homing ability make MSCs attractive vehicles for cellular therapy, either in their native form for regenerative applications, or through their genetic modification for delivery of active biological agents to specific microenvironments such as diseased bone marrow or metastatic deposits.
  • MSCs possess potent intrinsic properties
  • MSCs have been infused in hundreds of patients with minimal reported side effects.
  • follow-up is limited, long term side effects are unknown, and little is known of the consequences that will be associated with future efforts to induce their in vivo differentiation, for example to cartilage or bone, or to genetically modify them to enhance their functionality.
  • Several animal models have raised safety concerns. For instance, spontaneous osteosarcoma formation in culture has been observed in murine derived MSCs.
  • ectopic ossification and calcification foci have been discussed in mouse and rat models of myocardial infarction after local injection of MSC, and their proarrhythmic potential has also been apparent in co-culture experiments with neonatal rat ventricular myocytes.
  • T cells transduced with a nucleic acid encoding a chimeric antigen receptor have been administered to patients to treat cancer (Zhong, X.-S., (2010)
  • Chimeric antigen receptors are artificial receptors designed to convey antigen specificity to T cells without the requirement for MHC antigen presentation. They include an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the T cell and provide specific immunity. Chimeric antigen receptor-expressing T cells may be used in various therapies, including cancer therapies. Costimulating polypeptides may be used to enhance the activation of CAR-expressing T cells against target antigens, and therefore increase the potency of adoptive immunotherapy.
  • T cells expressing a chimeric antigen receptor based on the humanized monoclonal antibody Trastuzumab has been used to treat cancer patients. Adverse events are possible, however, and in at least one reported case, the therapy had fatal consequences to the patient (Morgan, R.A., et al., (2010) Molecular Therapy 18:843-851). Transducing the cells with a chimeric Caspase-9-based safety switch as presented herein, would provide a safety switch that could stop the adverse event from progressing. Therefore, in some embodiments are provided nucleic acids, cells, and methods wherein the modified T cell also expresses an inducible
  • an inducible ligand may be administered to the patient, thereby inducing apoptosis of the modified T cells.
  • T cells engineered to express chimeric antigen receptors have steadily improved as CAR molecules have incorporated additional signaling domains to increase their potency.
  • Second generation CAR T cells that incorporate the intracellular costimulating domains from either CD28 or 4-1 BB (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 U S A 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.
  • TNF tumor necrosis factor
  • chimeric antigen receptor or “CAR” is meant, for example, a chimeric polypeptide which comprises a polypeptide sequence that recognizes a target antigen (an antigen-recognition domain) linked to a transmembrane polypeptide and intracellular domain polypeptide selected to activate the T cell and provide specific immunity.
  • the antigen-recognition domain may be a single- chain variable fragment (scFv), or may, for example, be derived from other molecules such as, for example, a T cell receptor or Pattern Recognition Receptor.
  • the intracellular domain comprises at least one polypeptide which causes activation of the T cell, such as, for example, but not limited to, CD3 zeta, and, for example, co-stimulatory molecules, for example, but not limited to, CD28, OX40 and 4-1 BB.
  • chimeric antigen receptor may also refer to chimeric receptors that are not derived from antibodies, but are chimeric T cell receptors.
  • chimeric T cell receptors may comprise a polypeptide sequence that recognizes a target antigen, where the recognition sequence may be, for example, but not limited to, the recognition sequence derived from a T cell receptor or an scFv.
  • the intracellular domain polypeptides are those that act to activate the T cell. Chimeric T cell receptors are discussed in, for example, Gross, G., and Eshar, Z., FASEB Journal 6:3370-3378 (1992), and Zhang, Y., et al., PLOS Pathogens 6: 1- 13 (2010).
  • VH and VL variable heavy chains for a tumor-specific monoclonal antibody are fused in-frame with the CD3 zeta chain ( ⁇ ) from the T cell receptor complex.
  • T he VH and VL are generally connected together using a flexible glycine-serine linker, and then attached to the transmembrane domain by a spacer (CH2CH3) to extend the scFv away from the cell surface so that it can interact with tumor antigens.
  • CH2CH3 spacer
  • T cells now express the CAR on their surface, and upon contact and ligation with a tumor antigen, signal through the CD3 zeta chain inducing cytotoxicity and cellular activation.
  • CD28 enhanced expansion in vivo, in part due to increased IL-2 production following activation.
  • the inclusion of CD28 is called a 2nd generation CAR.
  • the most commonly used costimulating molecules include CD28 and 4-1 BB, which, following tumor recognition, can initiate a signaling cascade resulting in NF- ⁇ activation, which promotes both T cell proliferation and cell survival.
  • co-stimulating polypeptides 4-1 BB or OX40 in CAR design has further improved T cell survival and efficacy.
  • 4-1 BB in particular appears to greatly enhance T cell proliferation and survival.
  • This 3rd generation design (with 3 signaling domains) has been used in PSMA CARs (Zhong XS, et al., Mol Ther. 2010 Feb; 18(2):413-20) and in CD19 CARs, most notably for the treatment of CLL (Milone, M.C., et al., (2009) Mol. Ther. 17:1453-1464; Kalos, M., et al., Sci.
  • the intracellular domain comprises at least one polypeptide which causes activation of the T cell, such as, for example, but not limited to, CD3 zeta, and, for example, co-stimulatory molecules, for example, but not limited to, CD28, OX40 and 4-1 BB.
  • T cell receptors are molecules composed of two different polypeptides that are on the surface of T cells. They recognize antigens bound to major histocompatibility complex molecules; upon recognition with the antigen, the T cell is activated.
  • recognition is meant, for example, that the T cell receptor, or fragment or fragments thereof, such as TCRa polypeptide and TCR& together, is capable of contacting the antigen and identifying it as a target.
  • TCRs may comprise a and ⁇ polypeptides, or chains.
  • the a and ⁇ polypeptides include two extracellular domains, the variable and the constant domains.
  • the variable domain of the a and ⁇ polypeptides has three
  • CDRs complementarity determining regions
  • the a polypeptide includes the V and J regions, generated by VJ recombination, and the ⁇ polypeptide includes the V, D, and J regions, generated by VDJ recombination.
  • the intersection of the VJ regions 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, the 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).
  • Chimeric T cell receptors may bind to, for example, antigenic polypeptides such as Bob-1 , PRAME, and NY-ESO-1.
  • antigenic polypeptides such as Bob-1 , PRAME, and NY-ESO-1.
  • T cells are modified so that they express a non-functional TGF- beta receptor, rendering them resistant to TGF-beta.
  • it also could result in a T cell lymphoma, or other adverse effect, as the modified T cells now lack part of the normal cellular control; these therapeutic T cells could themselves become malignant.
  • Natural Killer cells are modified to express the membrane-targeting polypeptide.
  • the heterologous membrane bound polypeptide is a NKG2D receptor.
  • NKG2D receptors can bind to stress proteins (e.g. MICA/B) on tumor cells and can thereby activate NK cells.
  • the extracellular binding domain can also be fused to signaling domains (Barber, A., et al., Cancer Res 2007;67: 5003-8; Barber A, et al., Exp
  • VEGF-R could be used as a docking site for FRB domains to enhance tumor- dependent clustering in the presence of hypoxia-triggered VEGF, found at high levels within many tumors.
  • Cells used in cellular therapy that express a heterologous gene, such as a modified receptor, or a chimeric receptor, may be transduced with nucleic acid that encodes a chimeric Caspase-9-based safety switch before, after, or at the same time, as the cells are transduced with the heterologous gene.
  • a heterologous gene such as a modified receptor, or a chimeric receptor
  • graft rejection may be overcome by a combination of appropriate conditioning and large doses of stem cells, while graft versus host disease (GvHD) may be prevented by extensive T cell-depletion of the donor graft.
  • GvHD graft versus host disease
  • the immediate outcomes of such procedures have been gratifying, with engraftment rate > 90% and a severe GvHD rate of ⁇ 10% for both adults and children even in the absence of post transplant immunosuppression.
  • the profound immunosuppression of the grafting procedure coupled with the extensive T cell-depletion and HLA mismatching between donor and recipient lead to an extremely high rate of post-transplant infectious complications, and contributed to high incidence of disease relapse.
  • Donor T cell infusion is an effective strategy for conferring anti-viral and anti-tumor immunity following allogeneic stem cell transplantation.
  • Simple addback of T cells to the patients after haploidentical transplantation cannot work; 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 accelerate immune reconstitution by administrating donor T cells that have first been depleted of alloreactive ceils.
  • One method of achieving this is stimulating donor T cells with recipient EBV-transformed B lymphoblastoid cell lines (LCLs).
  • Alloreactive T cells upregulate CD25 expression, and are eliminated by a CD25 Mab immunotoxin conjugate, RFT5-SMPT-dgA.
  • This compound consists of a murine lgG1 anti-CD25 (IL-2 receptor alpha chain) conjugated via a hetero-bifunctional crosslinker [N-succinimidyloxycarbonyl-alpha- methyl-d- (2-pyridylthio) toluene] to chemically deglycosylated ricin A chain (dgA).
  • Treatment with CD25 immunotoxin after LCL stimulation depletes >90% of alloreactive cells.
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • Graft versus Host Disease is a condition that sometimes occurs after the transplantation of donor immunocompetent cells, for example, 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 the beneficial effects, such as, for example, the reconstitution of an immune system and the graft anti-tumor effect. But, the number of T cells that can be transplanted can be limited by the concern that the transplant will result in severe graft versus host disease.
  • Graft versus Host Disease may be staged as indicated in the following tables:
  • Acute GvHD grading may be performed by the consensus conference criteria (Przepiorka D et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995; 15:825- 828).
  • Inducible Caspase-9 as a "Safety Switch" for Cell Therapy and for Genetically Engineered Cell Transplantation
  • reducing the effect of graft versus host disease is meant, for example, a decrease in the GvHD symptoms so that the patient may be assigned a lower level stage, or, for example, a reduction of a symptom of graft versus host disease by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • a reduction in the effect of graft versus host disease may also be measured by detection of a reduction in activated T cells involved in the GvHD reaction, such as, for example, a reduction of cells that express the marker protein, for example CD19, and express CD3 (CD3 + CD19 + cells, for example) by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • a reduction in activated T cells involved in the GvHD reaction such as, for example, a reduction of cells that express the marker protein, for example CD19, and express CD3 (CD3 + CD19 + cells, for example) by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • variants may include, for example, an FKBP region that has an amino acid substitution at position 36 selected from the group consisting of valine, leucine, isoleuceine and alanine
  • AP1903 is a synthetic molecule that has proven safe in healthy volunteers (luliucci JD, et al., J Clin Pharmacol. 2001 , 41 :870-879). Administration of this small molecule results in cross-linking and activation of the proapoptotic target molecules.
  • the application of this inducible system in human T lymphocytes has been explored using Fas or the death effector domain (DED) of the Fas-associated death domain-containing protein (FADD) as proapoptotic molecules.
  • DED death effector domain
  • FADD Fas-associated death domain-containing protein
  • This suicide gene strategy may be used in any appropriate cell used for cell therapy including, for example, hematopoietic stem cells, and other progenitor cells, including, for example, mesenchymal stromal cells, embryonic stem cells, and inducible pluripotent stem cells.
  • AP20187 and AP1950 a synthetic version of AP1903, may also be used as the ligand inducer.
  • this safety switch catalyzed by Caspase-9, may be used where there is a condition in the cell therapy patient that requires the removal of the transfected or transduced therapeutic cells.
  • Conditions where the cells may need 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.
  • tissue specific promoters For example, where a progenitor cell differentiates into bone and fat cells, and the fat cells are not desired, the vector used to transfect or transduce the progenitor cell may have a fat cell specific promoter that is operably linked to the Caspase-9 nucleotide sequence. In this way, should the cells differentiate into fat cells, upon administration of the multimer ligand, apoptosis of the inappropriately differentiated fat cells should result.
  • the methods may be used, for example, for any disorder that can be alleviated by cell therapy, including cancer, cancer in the blood or bone marrow, other blood or bone marrow borne diseases such as sickle cell anemia and metachromic leukodystrophy, and any disorder that can be alleviated by a stem cell transplantation, for example blood or bone marrow disorders such as sickle cell anemia or metachromal leukodystrophy.
  • cancer cancer in the blood or bone marrow
  • other blood or bone marrow borne diseases such as sickle cell anemia and metachromic leukodystrophy
  • any disorder that can be alleviated by a stem cell transplantation for example blood or bone marrow disorders such as sickle cell anemia or metachromal leukodystrophy.
  • the efficacy of adoptive immunotherapy may be enhanced by rendering the therapeutic T cells resistant to immune evasion strategies employed by tumor cells.
  • immune evasion strategies employed by tumor cells In vitro studies have shown that this can be achieved by transduction with a dominant-negative receptor or an immunomodulatory cytokine (Bollard CM, et al., Blood. 2002, 99:3179-3187: Wagner HJ, et al., Cancer Gene Ther. 2004, 1 1 :81-91).
  • transfer of antigen-specific T-cell receptors allows for the application of T-cell therapy to a broader range of tumors (Pule M, et al., Cytotherapy. 2003, 5:211-226;
  • the gene is derived from a prokaryotic or eukaryotic source such as a bacterium, a virus, yeast, a parasite, a plant, or even an animal.
  • the heterologous DNA also is derived from more than one source, i.e., a multigene construct or a fusion protein.
  • the heterologous DNA also may include a regulatory sequence, which is derived from one source and the gene from a different source. Or, the heterologous DNA may include regulatory sequences that are used to change the normal expression of a cellular endogenous gene.
  • Other Caspase molecules are used to change the normal expression of a cellular endogenous gene.
  • Caspase polypeptides other than Caspase-9 that may be encoded by the chimeric polypeptides of the current technology include, for example, Caspase-1 , Caspase-3, and Caspase-8. Discussions of these Caspase polypeptides may be found in, for example, MacCorkle, R.A., et al., Proc. Natl. Acad. Sci. U.S.A. (1998) 95:3655-3660; and Fan, L, et al. (1999) Human Gene Therapy 10:2273- 2285).
  • Expression constructs encode a multimeric ligand binding region and a Caspase-9 polypeptide, or, in certain embodiments a multimeric ligand binding region and a Caspase-9 polypeptide linked to a marker polypeptide, all operatively linked.
  • the term "operably linked” is meant to indicate that the promoter sequence is functionally linked to a second sequence, wherein, for example, the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence.
  • the Caspase-9 polypeptide may be full length or truncated.
  • the marker polypeptide is linked to the Caspase-9 polypeptide.
  • the marker polypeptide may be linked to the Caspase-9 polypeptide via a polypeptide sequence, such as, for example, a cleavable 2A-like sequence.
  • the marker polypeptide may be, for example, CD19, or may be, for example, a heterologous protein, selected to not affect the activity of the chimeric caspase polypeptide.
  • the polynucleotide may encode the Caspase-9 polypeptide and a heterologous protein, which may be, for example a marker polypeptide and may be, for example, a chimeric antigen receptor.
  • the heterologous polypeptide for example, the chimeric antigen receptor, may be linked to the Caspase-9 polypeptide via a polypeptide sequence, such as, for example, a cleavable 2A-like sequence.
  • a nucleic acid comprising a polynucleotide coding for a chimeric antigen receptor is included in the same vector, such as, for example, a viral or plasmid vector, as a polynucleotide coding for a second polypeptide.
  • This second polypeptide may be, for example, a caspase polypeptide, as discussed herein, or a marker polypeptide.
  • the construct may be designed with one promoter operably linked to a nucleic acid comprising a polynucleotide coding for the two polypeptides, linked by a cleavable 2A polypeptide.
  • the first and second polypeptides are separated during translation, resulting in a chimeric antigen receptor polypeptide, and the second polypeptide.
  • the two polypeptide may be, for example, a caspase polypeptide, as discussed herein, or a marker polypeptide.
  • the construct may be designed with one promoter operably linked to a nucle
  • polypeptides may be expressed separately from the same vector, where each nucleic acid comprising a polynucleotide coding for one of the polypeptides is operably linked to a separate promoter.
  • one promoter may be operably linked to the two nucleic acids, directing the production of two separate RNA transcripts, and thus two polypeptides. Therefore, the expression constructs discussed herein may comprise at least one, or at least two promoters.
  • 2A-like sequences are derived from, for example, many different viruses, including, for example, from Thosea asigna. These sequences are sometimes also known as “peptide skipping sequences.”
  • peptide skipping sequences When this type of sequence is placed within a cistron, between two peptides that are intended to be separated, the ribosome appears to skip a peptide bond, in the case of Thosea asigna sequence, 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.
  • the peptide that is encoded 5' of the 2A sequence may end up with additional amino acids at the carboxy terminus, including the Gly residue and any upstream in the 2A sequence.
  • the peptide that is encoded 3' of the 2A sequence may end up with additional amino acids at the amino terminus, including the Pro residue and any 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 may be used in the polypeptides of the present application.
  • 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, the2A 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, in other embodiments, the 2A linker comprises a GSGPR amino acid sequence at the amino terminus of the polypeptide.
  • a "2A" sequence the term may refer to the2A sequence as listed herein, or may also refer to a 2A sequence as listed herein further comprising a GSG or GSGPR sequence at the amino terminus of the linker.
  • the expression construct may be inserted into a vector, for example a viral vector or plasmid.
  • a vector for example a viral vector or plasmid.
  • the steps of the methods provided may be performed using any suitable method; these methods include, without limitation, methods of transducing, transforming, or otherwise providing nucleic acid to the antigen-presenting cell, presented herein.
  • 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 DNA linkers, or has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28or a functionally equivalent fragment thereof.
  • the CD19 polypeptide is encoded by the nucleotide sequence of SEQ ID NO 14, or a functionally equivalent fragment thereof, with or without DNA linkers, or has the amino acid sequence of SEQ ID NO: 15, or a functionally equivalent fragment thereof.
  • a functionally equivalent fragment of the Caspase-9 polypeptide has substantially the same ability to induce apoptosis as the polypeptide of SEQ ID NO: 9, with at least 50%, 60%, 70%, 80%, 90%, or 95% of the activity of the polypeptide of SEQ ID NO: 9.
  • a functionally equivalent fragment of the CD19 polypeptide has substantially the same ability as the polypeptide of SEQ ID No: 15, to act as a marker to be used to identify and select transduced or transfected cells, with 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.
  • ligand binding domain or multimerizing region may be used in the expression construct.
  • the expression construct contains a membrane-targeting sequence.
  • Appropriate expression constructs may include a co-stimulatory polypeptide element on either side of the above FKBP ligand binding elements.
  • the polynucleotide coding for the inducible caspase polypeptide is included in the same vector, such as, for example, a viral or plasmid vector, as a polynucleotide coding for a chimeric antigen receptor.
  • the construct may be designed with one promoter operably linked to a nucleic acid comprising a nucleotide sequence coding for the two polypeptides, linked by a cleavable 2A polypeptide.
  • the first and second polypeptides are cleaved after expression, resulting in a chimeric antigen receptor polypeptide and an inducible Caspase-9 polypeptide.
  • the two polypeptides may be expressed separately from the same vector, where each nucleic acid comprising a nucleotide sequence coding for one of the polypeptides is operably linked to a separate promoter.
  • one promoter may be operably linked to the two nucleic acids, directing the production of two separate RNA transcripts, and thus two polypeptides. Therefore, the expression constructs discussed herein may comprise at least one, or at least two promoters.
  • two polypeptides may be expressed in a cell using two separate vectors. The cells may be co-transfected or co-transformed with the vectors, or the vectors may be introduced to the cells at different times.
  • the ligand binding (“dimerization") domain, or multimerizing region, of the expression construct can be any convenient domain that will allow for induction using a natural or unnatural ligand, for example, an unnatural synthetic ligand.
  • the multimerizing region can be internal or external to the cellular membrane, depending upon the nature of the construct and the choice of ligand.
  • ligand binding proteins including receptors, are known, including ligand binding proteins associated with the cytoplasmic regions indicated above.
  • the term "ligand binding domain” can be interchangeable with the term "receptor".
  • ligand binding proteins for which ligands for example, small organic ligands
  • ligand binding domains or receptors include the FKBPs and cyclophilin receptors, the steroid receptors, the tetracycline receptor, the other receptors indicated above, and the like, as well as "unnatural" receptors, which can be obtained from antibodies, particularly the heavy or light chain subunit, mutated sequences thereof, random amino acid sequences obtained by stochastic procedures, combinatorial syntheses, and the like.
  • the ligand binding region is selected from the group consisting of FKBP ligand binding region, cyclophilin receptor ligand binding region, steroid receptor ligand binding region, cyclophilin receptors ligand binding region, and tetracycline receptor ligand binding region.
  • the ligand binding region comprises a F V F V
  • S sequence further comprises an additional F v > sequence. Examples include, for example, those discussed in Kopytek, S.J., et al., Chemistry & Biology 7:313-321 (2000) and in Gestwicki, J.E., et al., Combinatorial Chem. & High Throughput Screening 10:667-675 (2007); Clackson T (2006) Chem Biol Drug Des 67:440-2;
  • the ligand binding domains or receptor domains will be at least about 50 amino acids, and fewer than about 350 amino acids, usually fewer than 200 amino acids, either as the natural domain or truncated active portion thereof.
  • the binding domain may, for example, be small ( ⁇ 25 kDa, to allow efficient transfection in viral vectors), monomeric, nonimmunogenic, have synthetically accessible, cell permeable, nontoxic ligands that can be configured for dimerization.
  • the receptor domain can be intracellular or extracellular depending upon the design of the expression construct and the availability of an appropriate ligand.
  • 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, unless there is a transport system for internalizing the ligand in a form in which it is available for binding.
  • the construct can encode a signal peptide and transmembrane domain 5' or 3' of the receptor domain sequence or may have a lipid attachment signal sequence 5' of the receptor domain sequence. Where the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain will be extracellular.
  • the portion of the expression construct encoding the receptor can be subjected to mutagenesis for a variety of reasons.
  • the mutagenized protein can provide for higher binding affinity, allow for discrimination by the ligand of the naturally occurring receptor and the mutagenized receptor, provide opportunities to design a receptor-ligand pair, or the like.
  • the change in the receptor can involve changes in amino acids known to be at the binding site, random mutagenesis using combinatorial techniques, where the codons for the amino acids associated with the binding site or other amino acids associated with conformational changes can be subject to mutagenesis by changing the codon(s) for the particular amino acid, either with known changes or randomly, expressing the resulting proteins in an appropriate prokaryotic host and then screening the resulting proteins for binding.
  • Antibodies and antibody subunits e.g., heavy or light chain, particularly fragments, more particularly all or part of the variable region, or fusions of heavy and light chain to create high- affinity binding, can be used as the binding domain.
  • Antibodies that are contemplated include ones that are an ectopically expressed human product, such as an extracellular domain that would not trigger an immune response and generally not expressed in the periphery (i.e., outside the CNS/brain area). Such examples, include, but are not limited to low affinity nerve growth factor receptor (LNGFR), and embryonic surface proteins (i.e., carcinoembryonic antigen).
  • LNGFR low affinity nerve growth factor receptor
  • embryonic surface proteins i.e., carcinoembryonic antigen
  • antibodies can be prepared against haptenic molecules, which are physiologically acceptable, and the individual antibody subunits screened for binding affinity.
  • the cDNA encoding the subunits can be isolated and modified by deletion of the constant region, portions of the variable region, mutagenesis of the variable region, or the like, to obtain a binding protein domain that has the appropriate affinity for the ligand.
  • almost any physiologically acceptable haptenic compound can be employed as the ligand or to provide an epitope for the ligand.
  • natural receptors can be employed, where the binding domain is known and there is a useful ligand for binding.
  • the transduced signal will normally result from ligand-mediated oligomerization of the chimeric protein molecules, i.e., as a result of oligomerization following ligand binding, although other binding events, for example allosteric activation, can be employed to initiate a signal.
  • the construct of the chimeric protein will vary as to the order of the various domains and the number of repeats of an individual domain.
  • the ligand for the ligand binding domains/receptor domains of the chimeric surface membrane proteins will usually be multimeric in the sense that it will have at least two binding sites, with each of the binding sites capable of binding to the ligand receptor domain.
  • multimeric ligand binding region is meant a ligand binding region that binds to a multimeric ligand.
  • multimeric ligands include dimeric ligands. A dimeric ligand will have two binding sites capable of binding to the ligand receptor domain.
  • the subject ligands will be a dimer or higher order oligomer, usually not greater than about tetrameric, of small synthetic organic molecules, the individual molecules typically being at least about 150 Da and less than about 5 kDa, usually less than about 3 kDa.
  • a variety of pairs of synthetic ligands and receptors can be employed.
  • dimeric FK506 can be used with an FKBP12 receptor
  • dimerized cyclosporin A can be used with the cyclophilin receptor
  • dimerized estrogen with an estrogen receptor
  • dimerized glucocorticoids with a glucocorticoid receptor
  • dimerized tetracycline with the tetracycline receptor
  • dimerized vitamin D with the vitamin D receptor
  • higher orders of the ligands e.g., trimeric can be used.
  • any of a large variety of compounds can be used.
  • a significant characteristic of these ligand units is that each binding site is able to bind the receptor with high affinity and they are able to be dimerized chemically. Also, methods are available to balance the hydrophobicity/hydrophilicity of the ligands so that they are able to dissolve in serum at functional levels, yet diffuse across plasma membranes for most applications.
  • the present methods utilize the technique of chemically induced dimerization (CID) to produce a conditionally controlled protein or polypeptide.
  • CID chemically induced dimerization
  • this technique is inducible, it also is reversible, due to the degradation of the labile dimerizing agent or administration of a monomeric competitive inhibitor.
  • the CID system uses synthetic bivalent ligands to rapidly crosslink signaling molecules that are fused to ligand binding domains. This system has been used to trigger the oligomerization and activation of cell surface (Spencer, D. M., et al., Science, 1993. 262: p. 1019-1024; Spencer D. M. et al., Curr Biol 1996, 6:839-847; Blau, C. A. et al., Proc Natl Acad.Sci. USA 1997, 94:3076-3081), or cytosolic proteins (Luo, Z. et al., Nature 1996,383: 181-185; MacCorkle, R. A.
  • the CID system is based upon the notion that surface receptor aggregation effectively activates downstream signaling cascades.
  • the CID system uses a dimeric analog of the lipid permeable immunosuppressant drug, FK506, which loses its normal bioactivity while gaining the ability to crosslink molecules genetically fused to the FK506-binding protein, FKBP12.
  • FK506 lipid permeable immunosuppressant drug
  • FKBP12 lipid permeable immunosuppressant drug
  • FKBP12 third-generation AP20187/AP1903 CIDs for their binding domain, FKBP12, permits specific activation of the recombinant receptor in vivo without the induction of non-specific side effects through endogenous FKBP12.
  • FKBP12 variants having amino acid substitutions and deletions, such as FKBP12v36, that bind to a dimerizer drug, may also be used.
  • FKBP12 variants include, but are not limited to, those having amino acid substitutions at position 36, selected from the group consisting of valine, leucine, isoleuceine, and alanine.
  • the synthetic ligands are resistant to protease degradation, making them more efficient at activating receptors in vivo than most delivered protein agents.
  • FKBP12 is meant the wild type FKBP12 polypeptide, or analogs or derivatives thereof that may comprise amino acid substitutions, that maintains FKBP12 binding activity to rapamycin; FKBP12 polypeptides or polypeptide regions bind to rimiducid with at least 100 times less affinity than
  • FKBP12v36 polypeptides binds to a ligand, such as rimiducid, with at least 100 times less affinity than an FKBP12 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 302.
  • FKBP12 variant polypeptide if meant an FKBP12 polypeptide that binds to a ligand, such as rimiducid with at least 100 times more affinity than a wild type FKBP12 polypeptide, such as, for example, the wild type FKBP12 polypeptide consisting of the amino acid sequence of SEQ ID NO: 301.
  • the ligands used are capable of binding to two or more of the ligand binding domains.
  • the chimeric proteins may be able to bind to more than one ligand when they contain more than one ligand binding domain.
  • the ligand is typically a non-protein or a chemical.
  • Exemplary ligands include, but are not limited to FK506 (e.g., FK1012).
  • Other ligand binding regions may be, for example, dimeric regions, or modified ligand binding regions with a wobble substitution, such as, for example, FKBP12(V36): The human 12 kDa FK506-binding protein with an F36 to V substitution, the complete mature coding sequence (amino acids 1-107), provides a binding site for synthetic dimerizer drug AP1903 (Jemal, A. et al., CA Cancer J. Clinic.
  • Two tandem copies of the protein may also be used in the construct so that higher-order oligomers are induced upon cross-linking by AP1903.
  • FKBP12 variants may also be used in the FKBP12/FRB multimerizing regions. Variants used in these fusions, in some embodiments, will bind to rapamycin, or rapalogs, but will bind to less affinity to rimiducid than, for example, FKBP12v36. Examples of FKBP12 variants include those from many species, including, for example, yeast. In one embodiment, the FKBP12 variant is FKBP12.6 (calstablin). Other heterodimers are contemplated in the present application. In one embodiment, a
  • the first unit of the first multimerizing region is a calcineurin-A polypeptide.
  • the first unit of the first multimerizing region is a calcineurin-A polypeptide region and the second unit of the first multimerizing region is a FKBP12 or FKBP12 variant multimerizing region.
  • the first unit of the first multimerizing region is a FKBP12 or FKBP12 variant multimerizing region and the second unit of the first multimerizing region is a calcineuring-A polypeptide region.
  • the first ligand comprises, for example, cyclosporine.
  • F36V-FKBP is a codon-wobbled version of F36V-FKBP. It encodes the identical polypeptide sequence as F36V-FKPB but has only 62% homology at the nucleotide level.
  • F36V-FKBP was designed to reduce recombination in retroviral vectors (Schellhammer,
  • F36V-FKBP was constructed by a PCR assembly procedure.
  • the transgene contains one copy of F36V-FKBP linked directly to one copy of F36V- FKBP.
  • the ligand is a small molecule.
  • the appropriate ligand for the selected ligand binding region may be selected. Often, the ligand is dimeric, sometimes, the ligand is a dimeric FK506 or a dimeric FK506-like analog.
  • 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[(1 R)-3-(3,4- dimethoxyphenyl)propylidene]]] ester, [2S-[1 (R*),2R*[S*[S*[1 (R*),2R*]]]]]]]-(9CI)
  • the ligand is AP20187.
  • the ligand is an AP20187 analog, such as, for example, AP1510.
  • certain analogs will be appropriate for the FKBP12, and certain analogs appropriate for the wobbled version of FKBP12.
  • one ligand binding region is included in the chimeric protein.
  • two or more ligand binding regions are included. Where, for example, the ligand binding region is FKBP12, where two of these regions are included, one may, for example, be the wobbled version.
  • dimerization systems contemplated include the coumermycin/DNA gyrase B system.
  • a membrane-targeting sequence or region provides for transport of the chimeric protein to the cell surface membrane, where the same or other sequences can encode binding of the chimeric protein to the cell surface membrane.
  • Molecules in association with cell membranes contain certain regions that facilitate the membrane association, and such regions can be incorporated into a chimeric protein molecule to generate membrane-targeted molecules.
  • some proteins contain sequences at the N-terminus or C-terminus that are acylated, and these acyl moieties facilitate membrane association.
  • Such sequences are recognized by acyltransferases and often conform to a particular sequence motif.
  • Certain acylation motifs are capable of being modified with a single acyl moiety (often followed by several positively charged residues (e.g.
  • human c-Src M-G-S-N-K-S-K-P-K-D-A-S-Q-R-R-R) to improve association with anionic lipid head groups
  • others are capable of being modified with multiple acyl moieties.
  • the N- terminal sequence of the protein tyrosine kinase Src can comprise a single myristoyl moiety.
  • Dual acylation regions are located within the N-terminal regions 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 often are located within the first eighteen amino acids of such proteins, and conform to the sequence motif Met-Gly-Cys-Xaa-Cys, where the Met is cleaved, the Gly is N- acylated and one of the Cys residues is S-acylated. The Gly often is myristoylated and a Cys can be palmitoylated.
  • Acylation regions conforming to the sequence motif Cys-Ala-Ala-Xaa (so called "CAAX boxes"), which can modified with C15 or C10 isoprenyl moieties, from the C-terminus of G- protein gamma subunits and other proteins (e.g., World Wde Web address
  • acylation motifs include, for example, those discussed in 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 1 10: 673-679 (1997), and can be incorporated in chimeric molecules to induce membrane localization.
  • a native sequence from a protein containing an acylation motif is incorporated into a chimeric protein.
  • an N-terminal portion of Lck, Fyn or Yes or a G-protein alpha subunit such as the first twenty-five N-terminal amino acids or fewer from such proteins (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), may be incorporated within the N-terminus of a chimeric protein.
  • 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 with optional mutations) can be linked to the C-terminus of a chimeric protein.
  • an acyl moiety has a log p value of +1 to +6, and sometimes has a log p value of +3 to +4.5.
  • Log p values are a measure of hydrophobicity and often are derived from octanol/water partitioning studies, in which molecules with higher hydrophobicity partition into octanol with higher frequency and are characterized as having a higher log p value.
  • Log p values are published for a number of lipophilic molecules and log p values can be calculated using known partitioning processes (e.g., Chemical Reviews, Vol. 71 , Issue 6, page 599, where entry 4493 shows lauric acid having a log p value of 4.2).
  • acyl moiety can be linked to a peptide composition discussed above and tested for antimicrobial activity using known methods and those discussed hereafter.
  • the acyl moiety sometimes is a 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, for example.
  • Any acyl-containing moiety sometimes is a fatty acid, and examples of fatty acid moieties are propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), heptyl (C7), octyl (C8), nonyl (C9), decyl (C10), undecyl (C1 1), lauryl (C12), myristyl (C14), palmityl (C16), stearyl (C18), arachidyl (C20), behenyl (C22) and lignoceryl moieties (C24), and each moiety can contain 0, 1 , 2, 3, 4, 5, 6, 7 or 8 unsaturations (i.e., double bonds).
  • An acyl moiety sometimes is a lipid molecule, such as a phosphatidyl lipid (e.g., phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl choline), sphingolipid (e.g., shingomyelin, sphingosine, ceramide, ganglioside, cerebroside), or modified versions thereof.
  • a phosphatidyl lipid e.g., phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl choline
  • sphingolipid e.g., shingomyelin, sphingosine, ceramide, ganglioside, cerebroside
  • one, two, three, four or five or more acyl moieties are linked to a membrane association region.
  • a chimeric protein herein also may include a single-pass or multiple pass transmembrane sequence (e.g., at the N-terminus or C-terminus of the chimeric protein).
  • Single pass transmembrane regions are found in certain CD molecules, tyrosine kinase receptors, serine/threonine kinase receptors, TGFbeta, BMP, activin and phosphatases.
  • Single pass transmembrane regions often include a signal peptide region and a transmembrane region of about 20 to about 25 amino acids, many of which are hydrophobic amino acids and can form an alpha helix.
  • a short track of positively charged amino acids often follows the transmembrane span to anchor the protein in the membrane.
  • Multiple pass proteins include ion pumps, ion channels, and transporters, and include two or more helices that span the membrane multiple times. All or substantially all of a multiple pass protein sometimes is incorporated in a chimeric protein.
  • Sequences for single pass and multiple pass transmembrane regions are known and can be selected for incorporation into a chimeric protein molecule.
  • membrane-targeting sequence can be employed that is functional in the host and may, or may not, be associated with one of the other domains of the chimeric protein.
  • such sequences include, but are not limited to myristoylation-targeting sequence, palmitoylation- targeting sequence, prenylation sequences (i.e., farnesylation, geranyl-geranylation, CAAX Box), protein-protein interaction motifs or transmembrane sequences (utilizing signal peptides) from receptors. Examples include those discussed in, for example, ten Klooster JP et al, Biology of the Cell (2007) 99, 1-12, Vincent, S., et al., Nature Biotechnology 21 :936-40, 1098 (2003).
  • PH domains can increase protein retention at various membranes.
  • an ⁇ 120 amino acid pleckstrin homology (PH) domain is found in over 200 human proteins that are typically involved in intracellular signaling.
  • PH domains can bind various phosphatidylinositol (PI) lipids within membranes (e.g. PI (3, 4,5)-P3, PI (3,4)-P2, PI (4,5)-P2) and thus play a key role in recruiting proteins to different membrane or cellular compartments.
  • PI phosphatidylinositol
  • PI phosphatidylinositol
  • PI phosphatidylinositol
  • PTEN phosphatidylinositol
  • interaction of membranes with PH domains are not as stable as by acyl lipids.
  • AP1903 for Injection 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, this formulation is a clear, slightly yellow solution. Upon refrigeration, this formulation undergoes a reversible phase transition, resulting in a milky solution. This phase transition is reversed upon re-warming to room temperature. The fill is 2.33 ml_ in a 3 ml_ glass vial (-10 mg AP1903 for Injection total per vial).
  • AP1903 is removed from the refrigerator the night before the patient is dosed and stored at a temperature of approximately 21 °C overnight, so that the solution is clear prior to dilution.
  • the solution is prepared within 30 minutes of the start of the infusion in glass or polyethylene bottles or non-DEHP bags and stored at approximately 21 °C prior to dosing.
  • All study medication is maintained at a temperature between 2 degrees C and 8 degrees C, protected from excessive light and heat, and stored in a locked area with restricted access.
  • patients may be, for example, administered a single fixed dose of AP1903 for Injection (0.4 mg/kg) via IV infusion over 2 hours, using a non-DEHP, non-ethylene oxide sterilized infusion set.
  • the dose of AP1903 is calculated individually for all patients, and is not to be recalculated unless body weight fluctuates by ⁇ 10%.
  • the calculated dose is diluted in 100 ml_ in 0.9% normal saline before infusion.
  • AP1903 plasma levels were directly proportional to dose, with mean C ma x values ranging from approximately 10 - 1275 ng/mL over the 0.01 - 1.0 mg/kg dose range.
  • blood concentrations demonstrated a rapid distribution phase, with plasma levels reduced to approximately 18, 7, and 1 % of maximal concentration at 0.5, 2 and 10 hours post- dose, respectively.
  • AP1903 for Injection was shown to be safe and well tolerated at all dose levels and demonstrated a favorable pharmacokinetic profile, luliucci JD, et al., J Clin Pharmacol. 41 : 870-9, 2001.
  • the fixed dose of AP1903 for injection used may be 0.4 mg/kg intravenously infused over 2 hours.
  • the amount of AP1903 needed in vitro for effective signaling of cells is 10 - 100 nM (1600 Da MW). This equates to 16 - 160 ⁇ g/L or -0.016 - 1.6 mg/kg (1.6 - 160 Mg/kg). Doses up to 1 mg/kg were well-tolerated in the Phase 1 study of AP1903 discussed above. Therefore, 0.4 mg/kg may be a safe and effective dose of AP1903 for this Phase I study in combination with the therapeutic cells.
  • the expression constructs contain nucleic acid constructs whose expression is identified in vitro or in vivo by including a marker in the expression construct.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants.
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as Herpes Simplex Virus-I thymidine kinase (tk) are employed.
  • Immunologic surface markers containing the extracellular, non-signaling domains or various proteins (e.g.
  • CD34, CD19, LNGFR also can be employed, permitting a straightforward method for magnetic or fluorescence antibody-mediated sorting.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.
  • Further examples of selectable markers include, for example, reporters such as GFP, EGFP, beta-gal or chloramphenicol acetyltransferase (CAT).
  • the marker protein, such as, for example, CD19 is used for selection of the cells for transfusion, such as, for example, in immunomagnetic selection.
  • a CD19 marker is distinguished from an anti-CD19 antibody, or, for example, an scFv, TCR, or other antigen recognition moiety that binds to CD19.
  • a polypeptide may be included in the expression vector to aid in sorting cells.
  • the CD34 minimal epitope may be incorporated into the vector.
  • the expression vectors used to express the chimeric antigen receptors or chimeric stimulating molecules provided herein further comprise a polynucleotide that encodes the 16 amino acid CD34 minimal epitope.
  • the CD34 minimal epitope is incorporated at the amino terminal position of the CD8 stalk.
  • a chimeric antigen receptor herein may include a single-pass or multiple pass transmembrane sequence (e.g., at the N-terminus or C-terminus of the chimeric protein).
  • transmembrane regions are found in certain CD molecules, tyrosine kinase receptors,
  • transmembrane regions often include a signal peptide region and a transmembrane region of about 20 to about 25 amino acids, many of which are hydrophobic amino acids and can form an alpha helix.
  • a short track of positively charged amino acids often follows the transmembrane span to anchor the protein in the membrane.
  • Multiple pass proteins include ion pumps, ion channels, and transporters, and include two or more helices that span the membrane multiple times. All or substantially all of a multiple pass protein sometimes is incorporated in a chimeric protein.
  • Sequences for single pass and multiple pass transmembrane regions are known and can be selected for incorporation into a chimeric protein molecule.
  • the transmembrane domain is fused to the extracellular domain of the CAR.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • a transmembrane domain that is not naturally associated with one of the domains in the CAR is used.
  • the transmembrane domain can be selected or modified by amino acid substitution (e.g., typically charged to a hydrophobic residue) to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • Transmembrane domains may, for example, be derived from the alpha, beta, or zeta chain of the T cell receptor, CD3-£, CD3 ⁇ , CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137, or CD154.
  • the transmembrane domain may be synthesized de novo, comprising mostly hydrophobic residues, such as, for example, leucine and valine.
  • a short polypeptide linker may form the linkage between the transmembrane domain and the intracellular domain of the chimeric antigen receptor.
  • the chimeric antigen receptors may further comprise a stalk, that is, an extracellular region of amino acids between the extracellular domain and the transmembrane domain.
  • the stalk may be a sequence of amino acids naturally associated with the selected transmembrane domain.
  • the chimeric antigen receptor comprises a CD8 transmembrane domain
  • the chimeric antigen receptor comprises a CD8 transmembrane domain, and additional amino acids on the extracellular portion of the
  • the chimeric antigen receptor comprises a CD8 transmembrane domain and a CD8 stalk.
  • the chimeric antigen receptor may further comprise a region of amino acids between the transmembrane domain and the cytoplasmic domain, which are naturally associated with the polypeptide from which the transmembrane domain is derived. Control Regions
  • Promoters The particular promoter employed to control the expression of a polynucleotide sequence of interest is not believed to be important, so long as it is capable of directing the expression of the polynucleotide in the targeted cell.
  • the polynucleotide sequence-coding region may, for example, be placed adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.
  • Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product.
  • a transgene or transgenes when a multicistronic vector is utilized, is toxic to the cells in which the vector is produced in, it is desirable to prohibit or reduce expression of one or more of the transgenes.
  • transgenes that are toxic to the producer cell line are pro-apoptotic and cytokine genes.
  • inducible promoter systems are available for production of viral vectors where the transgene products are toxic (add in more inducible promoters).
  • the ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility.
  • the system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained.
  • both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter, which drives expression of the gene of interest, is on another plasmid.
  • Engineering of this type of system into the gene transfer vector of interest would therefore be useful.
  • Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene.
  • expression of the transgene could be activated with ecdysone or muristeron A.
  • Tet-OffTM or Tet-OnTM system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-5551 , 1992; Gossen et al., Science, 268: 1766-1769, 1995).
  • This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline.
  • Tet-OnTM system gene expression is turned on in the presence of doxycycline
  • Tet-OffTM system gene expression is turned on in the absence of doxycycline.
  • tetracycline resistance operon of E. coli he tetracycline operator sequence to which the tetracycline repressor binds
  • tetracycline repressor protein The gene of interest is cloned into a plasmid behind a promoter that has tetracycline-responsive elements present in it.
  • a second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet-OffTM system, of the VP16 domain from the herpes simplex virus and the wild-type tertracycline repressor.
  • the tetracycline-controlled transactivator which is composed, in the Tet-OffTM system, of the VP16 domain from the herpes simplex virus and the wild-type tertracycline repressor.
  • the tetracycline repressor is not wild type and in the presence of doxycycline activates transcription.
  • the Tet-OffTM system may be used so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on.
  • a transgene in a gene therapy vector.
  • different viral promoters with varying strengths of activity are utilized depending on the level of expression desired.
  • the CMV immediate early promoter is often used to provide strong transcriptional activation.
  • the CMV promoter is reviewed in Donnelly, J.J. , et al., 1997. Annu. Rev. Immunol. 15:617-48. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLV or MMTV are often used.
  • viral promoters that are used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAV LTR, HSV-TK, and avian sarcoma virus.
  • promoters may be selected that are developmentally regulated and are active in particular differentiated cells.
  • a promoter may not be active in a pluripotent stem cell, but, for example, where the pluripotent stem cell differentiates into a more mature cell, the promoter may then be activated.
  • tissue specific promoters are used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues. These promoters may result in reduced expression compared to a stronger promoter such as the CMV promoter, but may also result in more limited expression, and immunogenicity (Bojak, A., et al., 2002. Vaccine.
  • tissue specific promoters such as the PSA associated promoter or prostate-specific glandular kallikrein, or the muscle creatine kinase gene may be used where appropriate.
  • tissue specific or differentiation specific promoters include, but are not limited to, the following: B29 (B cells); CD14 (monocytic cells); CD43 (leukocytes and platelets); CD45
  • CD68 hematopoietic cells
  • desmin muscle
  • elastase-1 pancreatic acinar cells
  • endoglin endothelial cells
  • fibronectin differentiated cells, healing tissues
  • Flt-1 endothelial cells
  • GFAP astrocytes
  • telomeres are hormone or cytokine regulatable.
  • Cytokine and inflammatory protein responsive promoters that can be used include K and T kininogen (Kageyama et al., (1987) J. Biol. Chem., 262,2345-2351), c-fos, TNF-alpha, C- reactive protein (Arcone, et al., (1988) Nucl.
  • haptoglobin (Oliviero et al., (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C/EBP alpha, IL-1 , IL-6 (Poli and Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86,8202-8206), Complement C3 (Wlson et al., (1990) Mol. Cell. Biol., 6181-6191), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, (1988) Mol Cell Biol, 8,42-51), alpha-1 antitrypsin, lipoprotein lipase (Zechner et al., Mol. Cell.
  • angiotensinogen (Ron, et al., (1991) Mol. Cell. Biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1 anti-chymotrypsin.
  • Other promoters include, for example, SV40, MMTV, Human
  • MV Immunodeficiency Virus
  • Moloney virus Moloney virus
  • ALV Epstein Barr virus
  • Rous Sarcoma virus human actin
  • myosin myosin
  • hemoglobin and creatine.
  • Promoters are selected such that they are functional in the desired cells or tissue.
  • this list of promoters should not be construed to be exhaustive or limiting; other promoters that are used in conjunction with the promoters and methods disclosed herein. Enhancers
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Early examples include the enhancers associated with immunoglobulin and T cell receptors that both flank the coding sequence and occur within several introns. Many viral promoters, such as CMV, SV40, and retroviral LTRs are closely associated with enhancer activity and are often treated like single elements. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole stimulates transcription at a distance and often independent of orientation; this need not be true of a promoter region or its component elements. On the other hand, a promoter has one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities.
  • LCRs locus-control regions
  • Any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) can be used to drive expression of the gene, although many will restrict expression to a particular tissue type or subset of tissues (reviewed in, for example, Kutzler, M.A., and Weiner, D.B., 2008. Nature Reviews Genetics 9:776-88). Examples include, but are not limited to, enhancers from the human actin, myosin, hemoglobin, muscle creatine kinase, sequences, and from viruses CMV, RSV, and EBV. Appropriate enhancers may be selected for particular applications. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • Polyadenylation Signals Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the present methods, and any such sequence is employed such as human or bovine growth hormone and SV40 polyadenylation signals and LTR polyadenylation signals.
  • One non-limiting example is the SV40 polyadenylation signal present in the pCEP3 plasmid (Invitrogen, Carlsbad, California).
  • a terminator contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • Termination or poly(A) signal sequences may be, for example, positioned about 1 1-30 nucleotides downstream from a conserved sequence (AAUAAA) at the 3' end of the mRNA (Montgomery, D.L., et al., 1993. DNA Cell Biol. 12:777-83; Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88).
  • a specific initiation signal also may be required for efficient translation of coding sequences.
  • Exogenous translational control signals including the ATG initiation codon, may need to be provided.
  • the initiation codon is placed in-frame with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • the exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • IRES internal ribosome entry sites
  • IRES elements are able to bypass the ribosome-scanning model of 5' methylated cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature, 334:320-325, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been discussed (Pelletier and
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating
  • each open reading frame is accessible to ribosomes for efficient translation.
  • Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and
  • Protein production may also be increased by optimizing the codons in the transgene. Species specific codon changes may be used to increase protein production. Also, codons may be optimized to produce an optimized RNA, which may result in more efficient translation. By optimizing the codons to be incorporated in the RNA, elements such as those that result in a secondary structure that causes instability, secondary mRNA structures that can, for example, inhibit ribosomal binding, or cryptic sequences that can inhibit nuclear export of mRNA can be removed (Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88; Yan, J. et al., 2007. Mol. Ther.
  • Leader sequences may be added to enhance the stability of mRNA and result in more efficient translation.
  • the leader sequence is usually involved in targeting the mRNA to the endoplasmic reticulum. Examples include the signal sequence for the HIV-1 envelope glycoprotein (Env), which delays its own cleavage, and the IgE gene leader sequence (Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88; Li, V., et al., 2000. Virology 272:417-28; Xu, Z.L, et al. 2001. Gene 272: 149-56; Malin, A.S., et al., 2000. Microbes Infect.
  • the IgE leader may be used to enhance insertion into the endoplasmic reticulum (Tepler, I, et al. (1989) J. Biol. Chem. 264:5912).
  • Expression of the transgenes may be optimized and/or controlled by the selection of appropriate methods for optimizing expression. These methods include, for example, optimizing promoters, delivery methods, and gene sequences, (for example, as presented in Laddy, D.J., et al., 2008. PLoS.ONE 3 e2517; Kutzler, M.A., and Weiner, D.B., 2008. Nature Rev. Gen. 9:776-88).
  • nucleic acid generally refers to a molecule (one, two or more strands) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • a nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil "U” or a C).
  • nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.” Nucleic acids may be, be at least, be at most, or be about 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91
  • Nucleic acids herein provided may have regions of identity or complementarity to another nucleic acid. It is contemplated that the region of complementarity or identity can be at least 5 contiguous residues, though it is specifically contemplated that the region is, is at least, is at most, or is about 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91
  • hybridization As used herein, “hybridization”, “hybridizes” or “capable of hybridizing” is understood to mean forming a double or triple stranded molecule or a molecule with partial double or triple stranded nature.
  • anneal as used herein is synonymous with “hybridize.”
  • hybridization encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”
  • stringent condition(s) or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are known, and are often used for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
  • Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.5 M NaCI at temperatures of about 42 degrees C to about 70 degrees C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and the presence or
  • low stringency or “low stringency conditions”
  • non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCI at a temperature range of about 20 degrees C. to about 50 degrees C.
  • the low or high stringency conditions may be further modified to suit a particular application.
  • any of the modifications discussed below may be applied to a nucleic acid.
  • modifications include alterations to the RNA or DNA backbone, sugar or base, and various combinations thereof. Any suitable number of backbone linkages, sugars and/or bases in a nucleic acid can be modified (e.g., independently about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to 100%).
  • An unmodified nucleoside is any one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1 ' carbon of beta-D-ribo-furanose.
  • a modified base is a nucleotide base other than adenine, guanine, cytosine and uracil at a T position.
  • modified bases include inosine, purine, pyridin-4-one, pyridin-2- one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e. g., 5-methylcytidine), 5-alkyluridines (e. g., ribothymidine), 5- halouridine (e.
  • modified bases include nitropyrrolyl (e.g., 3-nitropyrrolyl), nitroindolyl (e.g., 4-, 5-, 6-nitroindolyl), hypoxanthinyl, isoinosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4- methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrily
  • a nucleic acid may comprise modified nucleic acid molecules, with phosphate backbone modifications.
  • backbone modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxy methyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl modifications.
  • a ribose sugar moiety that naturally occurs in a nucleoside is replaced with a hexose sugar, polycyclic heteroalkyl ring, or cyclohexenyl group.
  • the hexose sugar is an allose, altrose, glucose, mannose, gulose, idose, galactose, talose, or a derivative thereof.
  • the hexose may be a D- hexose, glucose, or mannose.
  • the polycyclic heteroalkyl group may be a bicyclic ring containing one oxygen atom in the ring.
  • the polycyclic heteroalkyl group is a bicyclo[2.2.1]heptane, a bicyclo[3.2.1]octane, or a bicyclo[3.3.1]nonane.
  • Nitropyrrolyl and nitroindolyl nucleobases are members of a class of compounds known as universal bases. Universal bases are those compounds that can replace any of the four naturally occurring bases without substantially affecting the melting behavior or activity of the
  • oligonucleotide duplexes containing 3-nitropyrrolyl nucleobases may be stabilized solely by stacking interactions. The absence of significant hydrogen-bonding interactions with nitropyrrolyl nucleobases obviates the specificity for a specific complementary base. In addition, 4-, 5- and 6-nitroindolyl display very little specificity for the four natural bases. Procedures for the preparation of 1-(2'-0-methyl-.beta.-D-ribofuranosyl)-5- nitroindole are discussed in Gaubert, G.; Wengel, J.
  • Difluorotolyl is a non-natural nucleobase that functions as a universal base.
  • Difluorotolyl is an isostere of the natural nucleobase thymine. But unlike thymine, difluorotolyl shows no appreciable selectivity for any of the natural bases.
  • Other aromatic compounds that function as universal bases are 4-fluoro-6-methylbenzimidazole and 4-methylbenzimidazole.
  • the relatively hydrophobic isocarbostyrilyl derivatives 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, and 3- methyl-7-propynyl isocarbostyrilyl are universal bases which cause only slight destabilization of oligonucleotide duplexes compared to the oligonucleotide sequence containing only natural bases.
  • nucleobases include 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9- methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7- azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivates thereof.
  • cross-linking agents may be used to add further stability or irreversibility to the reaction.
  • cross-linking agents include, for example, 1 , 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N- maleimido-1 ,8-octane and agents such as methyl-3-[(p-azidophenyl) dithio]propioimidate.
  • a nucleotide analog may also include a "locked" nucleic acid.
  • Certain compositions can be used to essentially “anchor” or “lock” an endogenous nucleic acid into a particular structure.
  • Anchoring sequences serve to prevent disassociation of a nucleic acid complex, and thus not only can prevent copying but may also enable labeling, modification, and/or cloning of the endogeneous sequence.
  • the locked structure may regulate gene expression (i.e. inhibit or enhance transcription or replication), or can be used as a stable structure that can be used to label or otherwise modify the endogenous nucleic acid sequence, or can be used to isolate the endogenous sequence, i.e. for cloning.
  • Nucleic acid molecules need not be limited to those molecules containing only RNA or DNA, but further encompass chemically-modified nucleotides and non-nucleotides.
  • the percent of non- nucleotides or modified nucleotides may be from 1 % to 100% (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%).
  • a nucleic acid is provided for use as a control or standard in an assay, or therapeutic, for example.
  • a nucleic acid may be made by any technique known in the art, such as for example, chemical synthesis, enzymatic production or biological production.
  • Nucleic acids may be recovered or isolated from a biological sample. The nucleic acid may be recombinant or it may be natural or endogenous to the cell (produced from the cell's genome). It is contemplated that a biological sample may be treated in a way so as to enhance the recovery of small nucleic acid molecules. Generally, methods may involve lysing cells with a solution having guanidinium and a detergent.
  • Nucleic acid synthesis may also be performed according to standard methods.
  • Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques or via deoxynucleoside H-phosphonate intermediates.
  • a synthetic nucleic acid e.g., a synthetic oligonucleotide
  • Various different mechanisms of oligonucleotide synthesis have been disclosed elsewhere.
  • Nucleic acids may be isolated using known techniques. In particular embodiments, methods for isolating small nucleic acid molecules, and/or isolating RNA molecules can be employed.
  • Chromatography is a process used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography. If a nucleic acid from cells is to be used or evaluated, methods generally involve lysing the cells with a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA.
  • a chaotropic e.g., guanidinium isothiocyanate
  • detergent e.g., N-lauroyl sarcosine
  • Methods may involve the use of organic solvents and/or alcohol to isolate nucleic acids.
  • the amount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well.
  • a solid support may be any structure, and it includes beads, filters, and columns, which may include a mineral or polymer support with electronegative groups. A glass fiber filter or column is effective for such isolation procedures.
  • a nucleic acid isolation processes may sometimes include: a) lysing cells in the sample with a lysing solution comprising guanidinium, where a lysate with a concentration of at least about 1 M guanidinium is produced; b) extracting nucleic acid molecules from the lysate with an extraction solution comprising phenol; c) adding to the lysate an alcohol solution to form a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%; d) applying the lysate/alcohol mixture to a solid support; e) eluting the nucleic acid molecules from the solid support with an ionic solution; and, f) capturing the nucleic acid molecules.
  • compositions or kits that comprise nucleic acid comprising the polynucleotides of the present application.
  • compositions or kits may, for example, comprise both the first and second polynucleotides, encoding the first and second chimeric polypeptides.
  • the nucleic acid may comprise more than one nucleic acid species, that is, for example, the first nucleic acid species comprises the first polynucleotide, and the second nucleic acid species comprises the second polynucleotide. In other examples, the nucleic acid may comprise both the first and second polynucleotides.
  • kits may, in addition, comprise the first or second ligand, or both.
  • the kits may, in some embodiments, provide a nucleic acid composition, such as, for example, a virus, for example, a retrovirus, that comprises at least two polynucleotides, wherein the polynucleotides express, for example, an inducible pro-apoptotic polypeptide and a chimeric antigen receptor; an inducible pro-apoptotic polypeptide and a recombinant TCR; an inducible pro-apoptotic polypeptide and a chimeric costimulating polypeptide such as, for example an inducible chimeric MyD88 polypeptide, an inducible chimeric truncated MyD88 polypeptide, and optionally a CD40 polypeptide.
  • a nucleic acid composition such as, for example, a virus, for example, a retrovirus, that comprises at least two polynucleotides, wherein the polynucleo
  • the nucleic acid composition may comprise polynucleotides encoding an inducible pro-apoptotic polypeptide, an inducible chimeric MyD88 polypeptide or an inducible chimeric truncated MyD88 polypeptide, and optionally a CD40 polypeptide, and a chimeric antigen receptor or a recombinant T cell receptor.
  • kits comprise a nucleic acid composition such as, for example a virus, for example, a retrovirus, that comprises a polynucleotide that encodes 1) an iRC9 or iRmC9 polypeptide and an iM (MyD88FvFv) or iMC polypeptide; 2) an RC9 or iRmC9 polypeptide and a chimeric antigen receptor; 3) an iRC9 or iRmC9 polypeptide and a recombinant TCR; 4) an iC9 polypeptide and an iRMC or iRM (iRMyD88) polypeptide; 5) an iC9 polypeptide and an iRMC or iRM (iRMyD88) polypeptide and a chimeric antigen receptor; or 6) an iC9 polypeptide and an iRMC or iRM (iRMyD88) polypeptide and a recombinant T cell receptor.
  • a virus for example,
  • a transformed cell comprising an expression vector is generated by introducing into the cell the expression vector.
  • Suitable methods for polynucleotide delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current methods include virtually any method by which a polynucleotide (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism.
  • a polynucleotide e.g., DNA
  • a host cell can, and has been, used as a recipient for vectors.
  • Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded polynucleotide sequences.
  • Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials.
  • ATCC American Type Culture Collection
  • An appropriate host may be determined. Generally, this is based on the vector backbone and the desired result.
  • a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
  • Bacterial cells used as host cells for vector replication and/or expression include DH5alpha, JM 109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK Gold Cells (STRATAGENE®, La Jolla, CA).
  • bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
  • Eukaryotic cells that can be used as host cells include, but are not limited to yeast, insects and mammals.
  • mammalian eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12.
  • yeast strains include, but are not limited to, YPH499, YPH500 and YPH501.
  • Nucleic acid vaccines may include, for example, non-viral DNA vectors, "naked” DNA and RNA, and viral vectors. Methods of transforming cells with these vaccines, and for optimizing the expression of genes included in these vaccines are known and are also discussed herein. Examples of Methods of Nucleic Acid or Viral Vector Transfer
  • Any appropriate method may be used to transfect or transform the cells, or to administer the nucleotide sequences or compositions of the present methods.
  • Certain examples are presented herein, and further include methods such as delivery using cationic polymers, lipid like molecules, and certain commercial products such as, for example, IN-VIVO-JET PEL
  • an antigen presenting cell or a nucleic acid or viral vector may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, subcutaneous, intradermal, intramuscular, intravenous, intraprotatic, intratumor, intraperitoneal, etc.
  • injections i.e., a needle injection
  • Methods of injection include, for example, injection of a composition comprising a saline solution.
  • Further embodiments include the introduction of a polynucleotide by direct microinjection.
  • the amount of the expression vector used may vary upon the nature of the antigen as well as the organelle, cell, tissue or organism used.
  • Intradermal, intranodal, or intralymphatic injections are some of the more commonly used methods of DC administration. Intradermal injection is characterized by a low rate of absorption into the bloodstream but rapid uptake into the lymphatic system. The presence of large numbers of Langerhans dendritic cells in the dermis will transport intact as well as processed antigen to draining lymph nodes. Proper site preparation is necessary to perform this correctly (i.e., hair is clipped in order to observe proper needle placement). Intranodal injection allows for direct delivery of antigen to lymphoid tissues. Intralymphatic injection allows direct administration of DCs. Electroporation
  • a polynucleotide is introduced into an organelle, a cell, a tissue or an organism via electroporation.
  • Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
  • certain cell wall- degrading enzymes such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Patent No. 5,384,253, incorporated herein by reference).
  • Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81 , 7161-7165), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., (1986) Mol. Cell Biol., 6,716-718) in this manner.
  • a DNA vector encoding a polypeptide is injected intradermal. ⁇ in a patient. Then electrodes apply electrical pulses to the intradermal space causing the cells localized there, especially resident dermal dendritic cells, to take up the DNA vector and express the encoded polypeptide. These polypeptide-expressing cells activated by local inflammation can then migrate to lymph-nodes, presenting antigens, for example.
  • a nucleic acid is electroporetically administered when it is administered using electroporation, following, for example, but not limited to, injection of the nucleic acid or any other means of administration where the nucleic acid may be delivered to the cells by electroporation
  • a polynucleotide is introduced to the cells using calcium phosphate precipitation.
  • Human KB cells have been transfected with adenovirus 5 DNA (Graham and van der Eb, (1973) Virology, 52,456-467) using this technique.
  • mouse L(A9), mouse C127, CHO, CV-1 , BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., Mol. Cell Biol., 10:689-695, 1990).
  • a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol.
  • reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, T.V., Mol Cell Biol. 1985 May;5(5):1 188-90).
  • Additional embodiments include the introduction of a polynucleotide by direct sonic loading.
  • LTK- fibroblasts have been transfected with the thymidine kinase gene by sonication loading
  • a polynucleotide may be entrapped in a lipid complex such as, for example, a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. pp. 87-104).
  • a polynucleotide complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).
  • a polynucleotide may be delivered to a target cell via receptor-mediated delivery vehicles.
  • receptor-mediated delivery vehicles take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity.
  • Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a polynucleotide-binding agent. Others comprise a cell receptor-specific ligand to which the polynucleotide to be delivered has been operatively attached.
  • ligands have been used for receptor-mediated gene transfer (Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA, 91 :4086-4090, 1994; Myers, EPO 0273085), which establishes the operability of the technique.
  • a ligand is chosen to correspond to a receptor specifically expressed on the target cell population.
  • polynucleotide-targeting vehicle may comprise a specific binding ligand in combination with a liposome.
  • the polynucleotide(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane.
  • the liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell.
  • the polynucleotide delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which may, for example, comprise one or more lipids or glycoproteins that direct cell-specific binding.
  • lipids or glycoproteins that direct cell-specific binding.
  • lactosyl-ceramide, a galactose-terminal asialoganglioside have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., (1987) Methods Enzymol., 149,157-176). It is contemplated that the tissue-specific transforming constructs may be specifically delivered into a target cell in a similar manner.
  • Microprojectile bombardment techniques can be used to introduce a polynucleotide into at least one, organelle, cell, tissue or organism (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO 94/09699; each of which is incorporated herein by reference). This method depends on the ability to accelerate DNA-coated
  • microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., (1987) Nature, 327, 70-73).
  • microprojectile bombardment techniques known in the art, many of which are applicable to the present methods.
  • one or more particles may be coated with at least one polynucleotide and delivered into cells by a propelling force.
  • Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., (1990) Proc. Nat'l Acad. Sci. USA, 87, 9568-9572).
  • microprojectiles used have consisted of biologically inert substances such as tungsten or gold particles or beads.
  • Exemplary particles include those comprised of tungsten, platinum, and, in certain examples, gold, including, for example, nanoparticles. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.
  • Any viral vector suitable for administering nucleotide sequences, or compositions comprising nucleotide sequences, to a cell or to a subject, such that the cell or cells in the subject may express the genes encoded by the nucleotide sequences may be employed in the present methods.
  • a transgene is incorporated into a viral particle to mediate gene transfer to a cell.
  • the virus simply will be exposed to the appropriate host cell under physiologic conditions, permitting uptake of the virus.
  • the present methods are advantageously employed using a variety of viral vectors, as discussed below.
  • Adenovirus Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized DNA genome, ease of manipulation, high titer, wide target-cell range, and high infectivity.
  • the roughly 36 kb viral genome is bounded by 100-200 base pair (bp) inverted terminal repeats (ITR), in which are contained cis-acting elements necessary for viral DNA replication and packaging.
  • ITR inverted terminal repeats
  • E and late (L) regions of the genome that contain different transcription units are divided by the onset of viral DNA replication.
  • the E1 region encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan, M. J. (1990) Radiother Oncol., 19, 197-218).
  • the products of the late genes (L1 , L2, L3, L4 and L5), including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).
  • MLP located at 16.8 map units
  • TL tripartite leader
  • adenovirus In order for adenovirus to be optimized for gene therapy, it is necessary to maximize the carrying capacity so that large segments of DNA can be included. It also is very desirable to reduce the toxicity and immunologic reaction associated with certain adenoviral products.
  • the two goals are, to an extent, coterminous in that elimination of adenoviral genes serves both ends. By practice of the present methods, it is possible to achieve both these goals while retaining the ability to manipulate the therapeutic constructs with relative ease.
  • ITR inverted terminal repeats
  • the packaging signal for viral encapsulation is localized between 194-385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al., J. (1987) Virol., 67, 2555-2558).
  • This signal mimics the protein recognition site in bacteriophage lambda DNA where a specific sequence close to the left end, but outside the cohesive end sequence, mediates the binding to proteins that are required for insertion of the DNA into the head structure.
  • E1 substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome could direct packaging in 293 cells (Levrero et al., Gene, 101 : 195-202, 1991).
  • adenoviral genome can be incorporated into the genome of mammalian cells and the genes encoded thereby expressed. These cell lines are capable of supporting the replication of an adenoviral vector that is deficient in the adenoviral function encoded by the cell line. There also have been reports of complementation of replication deficient adenoviral vectors by "helping" vectors, e.g., wild-type virus or conditionally defective mutants.
  • Replication-deficient adenoviral vectors can be complemented, in trans, by helper virus. This observation alone does not permit isolation of the replication-deficient vectors, however, since the presence of helper virus, needed to provide replicative functions, would contaminate any preparation. Thus, an additional element was needed that would add specificity to the replication and/or packaging of the replication-deficient vector. That element derives from the packaging function of adenovirus.
  • the mutations are point mutations or deletions.
  • helper viruses with low efficiency packaging When helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at reduced rates compared to wild-type virus, thereby permitting propagation of the helper.
  • helper viruses When these helper viruses are grown in cells along with virus that contains wild-type packaging signals, however, the wild-type packaging signals are recognized preferentially over the mutated versions. Given a limiting amount of packaging factor, the virus containing the wild-type signals is packaged selectively when compared to the helpers. If the preference is great enough, stocks approaching homogeneity may be achieved.
  • the receptor-binding fiber sequences can often be substituted between adenoviral isolates. For example the
  • Coxsackie-adenovirus receptor (CAR) ligand found in adenovirus 5 can be substituted for the CD46-binding fiber sequence from adenovirus 35, making a virus with greatly improved binding affinity for human hematopoietic cells.
  • the resulting "pseudotyped" virus, Ad5f35 has been the basis for several clinically developed viral isolates.
  • various biochemical methods exist to modify the fiber to allow re-targeting of the virus to target cells. Methods include use of bifunctional antibodies (with one end binding the CAR ligand and one end binding the target sequence), and metabolic biotinylation of the fiber to permit association with customized avidin- based chimeric ligands.
  • ligands e.g. anti-CD205 by
  • heterobifunctional linkers e.g. PEG-containing
  • Retrovirus The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, (1990) In: Virology, ed., New York: Raven Press, pp. 1437-1500). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes - gag, pol and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene functions as a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).
  • the present technology includes, for example, cells whereby the polynucleotide used to transduce the cell is integrated into the genome of the cell.
  • a nucleic acid encoding a promoter is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol and env genes but without the LTR and psi components is constructed (Mann et al., (1983) Cell, 33, 153-159).
  • a recombinant plasmid containing a human cDNA, together with the retroviral LTR and psi sequences is introduced into this cell line (by calcium phosphate precipitation for example), the psi sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas, J.F., and Rubenstein, J.L.R., (1988) In: Vectors: a Survey of Molecular Cloning Vectors and Their Uses, Rodriquez and Denhardt, Eds.). Nicolas and Rubenstein; Temin et al., (1986) In: Gene Transfer, Kucherlapati (ed.), and New York: Plenum Press, pp.
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression of many types of retroviruses require the division of host cells (Paskind et al., (1975) Virology, 67,242-248). An approach designed to allow specific targeting of retrovirus vectors recently was developed based on the chemical modification of a retrovirus by the chemical addition of galactose residues to the viral envelope. This modification could permit the specific infection of cells such as hepatocytes via asialoglycoprotein receptors, may be desired.
  • Adeno-associated Virus AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1 , VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.
  • the three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, p19 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced.
  • the splice site derived from map units 42-46, is the same for each transcript.
  • the four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.
  • AAV is not associated with any pathologic state in humans. Interestingly, for efficient replication, AAV requires "helping" functions from viruses such as herpes simplex virus I and II,
  • the terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201 , which contains a modified AAV genome (Samulski et al., J. Virol., 61 :3096-3101 (1987)), or by other methods, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV. It can be
  • the minimum sequence or part of the AAV ITRs which is required to allow function i.e., stable and site-specific integration. It can also be determined which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.
  • AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo (Carter and Flotte, (1995) Ann. N.Y. Acad. Sci., 770; 79-90; Chatteijee, et al., (1995) Ann. N.Y. Acad. Sci., 770,79-90; Ferrari et al., (1996) J. Virol., 70,3227-3234; Fisher et al., (1996) J. Virol. , 70,520-532; Flotte et al., Proc. Nat'l Acad. Sci.
  • AAV-mediated efficient gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1995; Flotte et al., Proc. Nat'l Acad. Sci. USA, 90, 10613-10617, (1993)).
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, (1988) In: Vectors: A survey of molecular cloning vectors and their uses, pp. 467-492; Baichwal and Sugden, (1986) In, Gene Transfer, pp. 117-148; Coupar et al., Gene, 68: 1-10, 1988) canary poxvirus, and herpes viruses are employed. These viruses offer several features for use in gene transfer into various mammalian cells. Once the construct has been delivered into the cell, the nucleic acid encoding the transgene are positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the transgene is stably integrated into the genome of the cell.
  • nucleic acid is stably maintained in the cell as a separate, episomal segment of DNA.
  • nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the present methods also encompass methods of treatment or prevention of a disease where administration of cells by, for example, infusion, may be beneficial.
  • Cells such as, for example, T cells, tumor infiltrating lymphocytes, natural killer cells, natural killer T cells, or progenitor cells, such as, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and embryonic stem cells may be used for cell therapy.
  • the cells may be from a donor, or may be cells obtained from the patient.
  • the cells may, for example, be used in regeneration, for example, to replace the function of diseased cells.
  • the cells may also be modified to express a heterologous gene so that biological agents may be delivered to specific microenvironments such as, for example, diseased bone marrow or metastatic deposits.
  • Mesenchymal stromal cells have also, for example, been used to provide immunosuppressive activity, and may be used in the treatment of graft versus host disease and autoimmune disorders.
  • the cells provided in the present application contain a safety switch that may be valuable in a situation where following cell therapy, the activity of the therapeutic cells needs to be increased, or decreased.
  • T cells that express a chimeric antigen receptor are provided to the patient, in some situations there may be an adverse event, such as off-target toxicity.
  • Ceasing the administration of the ligand would return the therapeutic T cells to a non-activated state, remaining at a low, non-toxic, level of expression.
  • the therapeutic cell may work to decrease the tumor cell, or tumor size, and may no longer be needed.
  • the ligand may cease, and the therapeutic cells would no longer be activated. If the tumor cells return, or the tumor size increases following the initial therapy, the ligand may be administered again, in order to activate the chimeric antigen receptor-expressing T cells, and retreat the patient.
  • terapéutica cell is meant a cell used for cell therapy, that is, a cell administered to a subject to treat or prevent a condition or disease. In such cases, where the cells have a negative effect, the present methods may be used to remove the therapeutic cells through selective apoptosis.
  • T cells are used to treat various diseases and conditions, and as a part of stem cell transplantation.
  • An adverse event that may occur after haploidentical T cell transplantation is graft versus host disease (GvHD).
  • GvHD graft versus host disease
  • the likelihood of GvHD occurring increases with the increased number of T cells that are transplanted. This limits the number of T cells that may be infused.
  • a greater number of T cells may be infused, increasing the number to greater than 10 6 , greater than 10 7 , greater than 10 8 , or greater than 10 9 cells.
  • the number of T cells/kg body weight that may be administered may be, for example, from about 1 x 10 4 T cells/kg body weight to about 9 x 10 7 T cells/kg body weight, for example about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 4 ; about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 5 ; about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 6 ; or about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 7 T cells/kg body weight.
  • therapeutic cells other than T cells may be used.
  • the number of therapeutic cells/kg body weight that may be administered may be, for example, from about 1 x 10 4 T cells/kg body weight to about 9 x 10 7 T cells/kg body weight, for example about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 4 ; about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 5 ; about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 6 ; or about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 x 10 7 therapeutic cells/kg body weight.
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of pharmaceutical composition calculated to produce the desired immunogenic effect in association with the required diluent.
  • the specifications for the unit dose of an inoculum are dictated by and are dependent upon the unique characteristics of the pharmaceutical composition and the particular immunologic effect to be achieved.
  • an effective amount of the pharmaceutical composition such as the multimeric ligand presented herein, would be the amount that achieves this selected result of selectively removing the cells that include the Caspase-9 vector, such that over 60%, 70%, 80%, 85%, 90%, 95%, or 97% of the Caspase-9 expressing cells are killed.
  • the term is also synonymous with "sufficient amount.”
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition. One can empirically determine the effective amount of a particular composition presented herein without necessitating undue experimentation.
  • contacted and “exposed,” when applied to a cell, tissue or organism are used herein to discuss the process by which the pharmaceutical composition and/or another agent, such as for example a chemotherapeutic or radiotherapeutic agent, are delivered to a target cell, tissue or organism or are placed in direct juxtaposition with the target cell, tissue or organism.
  • the pharmaceutical composition and/or additional agent(s) are delivered to one or more cells in a combined amount effective to kill the cell(s) or prevent them from dividing.
  • the administration of the pharmaceutical composition may precede, be co-current with and/or follow the other agent(s) by intervals ranging from minutes to weeks.
  • the pharmaceutical composition and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the pharmaceutical composition and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one or more agents may be administered within of from substantially simultaneously, about 1 minute, to about 24 hours to about 7 days to about 1 to about 8 weeks or more, and any range derivable therein, prior to and/or after administering the expression vector.
  • various combination regimens of the pharmaceutical composition presented herein and one or more agents may be employed. Optimized and Personalized Therapeutic Treatment
  • the induction of apoptosis after administration of the dimer may be optimized by determining the stage of graft versus host disease, or the number of undesired therapeutic cells that remain in the patient.
  • determining that a patient has GvHD, and the stage of the GvHD provides an indication to a clinician that it may be necessary to induce Caspase-9 associated apoptosis by administering the multimeric ligand.
  • determining that a patient has a reduced level of GvHD after treatment with the multimeric ligand may indicate to the clinician that no additional dose of the multimeric ligand is needed.
  • determining that the patient continues to exhibit GvHD symptoms, or suffers a relapse of GvHD may indicate to the clinician that it may be necessary to administer at least one additional dose of multimeric ligand.
  • the term "dosage" is meant to include both the amount of the dose and the frequency of administration, such as, for example, the timing of the next dose
  • the multimeric ligand may be administered to the patient.
  • the methods comprise determining the presence or absence of a negative symptom or condition, such as Graft vs Host Disease, or off target toxicity, and administering a dose of the multimeric ligand.
  • the methods may further comprise monitoring the symptom or condition and administering an additional dose of the multimeric ligand in the event the symptom or condition persists. This monitoring and treatment schedule may continue while the therapeutic cells that express chimeric antigen receptors or chimeric signaling molecules remain in the patient.
  • An indication of adjusting or maintaining a subsequent drug dose can be provided in any convenient manner.
  • An indication may be provided in tabular form (e.g., in a physical or electronic medium) in some embodiments.
  • the graft versus host disease observed symptoms may be provided in a table, and a clinician may compare the symptoms with a list or table of stages of the disease. The clinician then can identify from the table an indication for subsequent drug dose.
  • an indication can be presented (e.g., displayed) by a computer, after the symptoms or the GvHD stage is provided to the computer (e.g., entered into memory on the computer).
  • this information can be provided to a computer (e.g., entered into computer memory by a user or transmitted to a computer via a remote device in a computer network), and software in the computer can generate an indication for adjusting or maintaining a subsequent drug dose, and/or provide the subsequent drug dose amount.
  • a computer e.g., entered into computer memory by a user or transmitted to a computer via a remote device in a computer network
  • software in the computer can generate an indication for adjusting or maintaining a subsequent drug dose, and/or provide the subsequent drug dose amount.
  • a clinician may administer the subsequent dose or provide instructions to adjust the dose to another person or entity.
  • the term "clinician" as used herein refers to a decision maker, and a clinician is a medical professional in certain embodiments.
  • a decision maker can be a computer or a displayed computer program output in some embodiments, and a health service provider may act on the indication or subsequent drug dose displayed by the computer.
  • a decision maker may administer the subsequent dose directly (e.g., infuse the subsequent dose into the subject) or remotely (e.g., pump parameters may be changed remotely by a decision maker).
  • a dose, or multiple doses of the ligand may be administered before clinical manifestations of GvHD, or other symptoms, such as CRS symptoms, are apparent.
  • cell therapy is terminated before the appearance of negative symptoms.
  • the therapy may be terminated after the transplant has made progress toward
  • the ligand may be administered to eliminate the modified cells in order to eliminate on target/off-tumor cells, such as, for example, healthy B cells co-expressing the B cell-associated target antigen.
  • an effective amount of an activated cell is defined as that amount sufficient to detectably and repeatedly to achieve the stated desired result, for example, to ameliorate, reduce, minimize or limit the extent of the disease or its symptoms. Other more rigorous definitions may apply, including elimination, eradication or cure of disease. In some embodiments there may be a step of monitoring the biomarkers to evaluate the effectiveness of treatment and to control toxicity. Dual Control of Therapeutic Cells and Heterdimerizer Control of Apoptosis for Controlled Therapy
  • Nucleic acids and cells provided herein may be used to achieve dual control of therapeutic cells for controlled therapy.
  • the subject may be diagnosed with a condition, such as a tumor, where there is a need to deliver targeted chimeric antigen receptor therapy.
  • Methods discussed herein provide several examples of ways to control therapy in order to induce activity of the CAR- expressing therapeutic cells, and also to provide a safety switch should there be a need to discontinue therapy completely, or to reduce the number or percent of the therapeutic cells in the subject.
  • modified T cells are administered to a subject that express the following polypeptides: 1.
  • a chimeric polypeptide iMyD88/CD40, or " iMC" that comprises two or more FKBP12 ligand binding regions and a costimulatory polypeptide or polypeptides, such as, for example, MyD88 or truncated MyD88 and CD40; 2.
  • a chimeric proapoptotic polypeptide that comprises one or more FRB ligand binding regions and a Caspase-9 polypeptide;
  • a chimeric antigen receptor polypeptide comprising an antigen recognition moiety that binds to a target antigen.
  • the target antigen is a tumor antigen present on tumor cells in the subject.
  • the ligand AP1903 may be administered to the subject, which induces iMC activation of the CAR-T cell.
  • the therapy is monitored, for example, the tumor size or growth may be assessed during the course of therapy.
  • One or more doses of the ligand may be administered during the course of therapy.
  • the safety switch— chimeric Caspase-9 polypeptide may be activated by administering a rapalog, which binds to the FRB ligand binding region.
  • the amount and dosing schedule of the rapalog may be determined based on the level of CAR-T cell therapy that is needed.
  • the dose of the rapalog is an amount effective to remove at least 90%, 95%, 97%, 98%, or 99% of the administered modified cells.
  • the dose is an amount effective to remove up to 30%, 40%, 50%, 60%, 70%, 80%, 90, 95%, or 100% of the cells that express the chimeric caspase polypeptide, if there is a need to reduce the level of CAR-T cell therapy, but not completely stop the therapy.
  • This may be measured, for example, by obtaining a sample from the subject before inducing the safety switch, before administering the rapamycin or rapalog, and obtaining a sample following administration of the rapamycin or rapalog, at, for example 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 hours, or 1 , 2, 3, 4, 5 days following administration, and comparing the number or concentration of chimeric caspase- expressing cells between the two samples by, for example, any method available, including, for example, detecting the presence of a marker.
  • This method of determining percent removal of the cells may also be used where the inducing ligand is AP1903 or binds to the FKBP12 or FKBP12 variant multimerizing region.
  • the inducible MyD88/CD40 chimeric polypeptide also comprises the chimeric antigen receptor.
  • the chimeric polypeptide may comprise one or more ligand binding regions.
  • CID protein Dimerization
  • AP1903 small molecule homodimerizing ligand, rimiducid
  • This technology underlies the "safety switch" incorporated as a gene therapy adjunct in cell transplants (1 , 2).
  • the central tenet of the technology is that normal cellular regulatory pathways that rely on protein-protein interaction as part of a signaling pathway can be adapted to ligand- dependent, conditional control if a small molecule dimerizing drug is used to control the protein- protein oligomerization event (3-5).
  • Caspase-9 is an initiating caspase that acts as a "gate-keeper" of the apoptotic process (6).
  • pro-apoptotic molecules e.g., cytochrome c
  • pro-apoptotic molecules released from the mitochondria of apoptotic cells alter the conformation of Apaf-1 , a caspase-9-binding scaffold, leading to its oligomerization and formation of the "apoptosome".
  • This alteration facilitates caspase-9 dimerization and cleavage of its latent form into an active molecule that, in turn, cleaves the "downstream" apoptosis effector, caspase-3, leading to irreversible cell death.
  • Rimiducid binds directly with two FKBP12-V36 moieties and can direct the dimerization of fusion proteins that include FKBP12-V36 (1 , 2).
  • iC9 engagement with rimiducid circumvents the need for Apafl conversion to the active apoptosome.
  • the fusion of caspase-9 to protein moieties that engage a heterodimerizing ligand is assayed for its ability to direct its activation and cell death with similar efficacy to rimiducid-mediated iC9 activation.
  • MyD88 and CD40 were chosen as the basis of the iMC activation switch.
  • MyD88 plays a central signaling role in the detection of pathogens or cell injury by antigen-presenting cells (APCs), like dendritic cells (DCs).
  • APCs antigen-presenting cells
  • DCs dendritic cells
  • TLRs Toll-Like Receptors
  • iMC also MC.FvFv
  • Rapamycin is a natural product macrolide that binds with high affinity ( ⁇ 1 nM) to FKBP12 and together initiates the high-affinity, inhibitory interaction with the FKBP-Rapamycin-Binding (FRB) domain of mTOR (8).
  • FRB is small (89 amino acids) and can thereby be used as a protein "tag” or “handle” when appended to many proteins (9-1 1). Coexpression of a FRB-fused protein with a FKBP12-fused protein renders their approximation rapamycin-inducible (12-16). This and the examples that follow provide experiments and results designed to test whether expression of
  • Caspase-9 bound with FKBP and FRB in tandem can also direct apoptosis and serve as the basis for a cell safety switch regulated by the orally available ligand, rapamycin.
  • an inducible MyD88/CD40 rapamycin-sensitive costimulatory polypeptide was developed by fusing FKBP and FRB in tandem with the MyD88/CD40 polypeptide.
  • derivatives of rapamycin may also be used that do not inhibit mTOR at a low, therapeutic dose.
  • rapamycin or these rapamycin analogs may bind with selected, MC-FKBP-fused mutant FRB domains, using a heterdimerizer to homodimerize two MC-FKBP- FRB polypeptides.
  • a heterdimerizer to homodimerize two MC-FKBP- FRB polypeptides.
  • RAFT1 a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell. 1994;78(1):35-43.
  • Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem Biol. 2006; 13(1 ):99- 107.
  • FRB. FKBP. ⁇ C9 were assayed in response to either the heterodimer, rapamycin, or the
  • Rimiducid or AP1903 is a highly specific and efficient dimerizer composed of two identical protein-binding surfaces (based on FK506) arranged tail-to-tail, each with high affinity and specificity for an FKBP mutant, FKBP12v36 or FKBPv.
  • FKBP12v36 is a modified version of FKBP12, in which phenylalanine 36, is replaced with the smaller hydrophobic residue, valine, which accommodates the bulky modification on the FKBP12-binding site of AP1903 [1].
  • Rapamycin binds to FKBP12, but unlike rimiducid, rapamycin also binds to the FKBP12- Rapamycin-Binding (FRB) domain of mTOR and can induce heterodimerization of signaling domains that are fused to FKBP12 with fusions containing FRB.
  • FRB Rapamycin-Binding
  • Expression of Caspase-9 fused with FKBP and FRB in tandem in both orientations: FKBP. FRB. ⁇ C9 or FRB. FKBP. ⁇ C9
  • FKBP. ⁇ C9 FKBP.
  • rimiducid contains a bulky modification on the FKBP12-binding site, this dimerizer is not able to bind to wild type FKBP12.
  • the FRB.FKBPv. ⁇ C9 switch provides the option to activate caspase-9 with either rimiducid or rapamycin by mutating the FKBP domain to FKBPv. This flexibility in terms of choice of activating drug may be important in a clinical setting where the clinician can choose to administer the drug based on its specific pharmacological properties. Additionally, this switch provides a molecule to allow for direct comparison between the drug-activating kinetics of rimiducid and rapamycin where the effector is contained within a single molecule. 1. D. Spencer, et al., Science, vol. 262, pp. 1019-1024, 1993.
  • compositions expression constructs, expression vectors, fused proteins, transfected or transduced cells, in a form appropriate for the intended application.
  • this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • the multimeric ligand such as, for example, AP1903 (INN rimiducid, may be delivered, for example at doses of about 0.1 to 10 mg/kg subject weight, of about 0.1 to 5 mg/kg subject weight, of about 0.2 to 4 mg/kg subject weight, of about 0.3 to 3 mg/kg subject weight, of about 0.3 to 2 mg/kg subject weight, or about 0.3 to 1 mg/kg subject weight, for example, about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mg/kg subject weight.
  • the ligand is provided at 0.4mg/kg per dose, for example at a concentration of 5mg/ml_.
  • Vials or other containers may be provided containing the ligand at, for example, a volume per vial of about 0.25 ml to about 10 ml, for example, about 0.25, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 ml, for example, about 2 ml.
  • One may generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also may be employed when recombinant cells are introduced into a patient.
  • Aqueous compositions comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • a 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 known. Except insofar as any conventional media or agent is incompatible with the vectors or cells, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • the active compositions may include classic pharmaceutical preparations. Administration of these compositions will be via any common route so long as the target tissue is available via that route. This includes, for example, oral, nasal, buccal, rectal, vaginal or topical. Alternatively,
  • administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • Such compositions would normally be administered as pharmaceutically acceptable compositions, discussed herein.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form is sterile and is fluid to the extent that easy syringability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
  • compositions may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient also may be dispersed in dentifrices, including, for example: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include, for example, water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • a paste dentifrice may include, for example, water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include, for example, the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media can be employed.
  • one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570- 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards. Examples
  • HVTK herpes simplex virus l-derived thymidine kinase
  • HSV-TK-directed immune responses have resulted in elimination of HSV-TK-transduced cells, even in immunosuppressed human immunodeficiency virus and bone marrow transplant patients, compromising the persistence and hence efficacy of the infused T cells.
  • HSV-TK is also virus-derived, and therefore potentially immunogenic (Bonini C, et al., Science. 1997, 276: 1719-1724; Riddell SR, et al., Nat Med.
  • E coli-derived cytosine deaminase gene has also been used clinically (Freytag SO, et al., Cancer Res. 2002, 62:4968-4976), but as a xenoantigen it may be immunogenic and thus incompatible with T-cell- based therapies that require long-term persistence.
  • Transgenic human CD20 which can be activated by a monoclonal chimeric anti-CD20 antibody, has been proposed as a nonimmunogenic safety system (Introna M, et al., Hum Gene Ther. 2000, 11 : 61 1-620).
  • the following section provides examples of method of providing a safety switch in cells used for cellular therapy, using a Caspase-9 chimeric protein.
  • a safety switch that can be stably and efficiently expressed in human T cells is presented herein.
  • the system includes human gene products with low potential immunogenicity that have been modified to interact with a small molecule dimerizer drug that is capable of causing the selective elimination of transduced T cells expressing the modified gene. Additionally, the inducible
  • Caspase-9 maintains function in T cells overexpressing antiapoptotic molecules.
  • Expression vectors suitable for use as a therapeutic agent were constructed that included a modified human Caspase-9 activity fused to a human FK506 binding protein (FKBP), such as, for example, FKBP12v36.
  • FKBP human FK506 binding protein
  • the Caspase-9/FK506 hybrid activity can be dimerized using a small molecule pharmaceutical.
  • Full length, truncated, and modified versions of the Caspase-9 activity were fused to the ligand binding domain, or multimerizing region, and inserted into the retroviral vector MSCV.IRES.GRP, which also allows expression of the fluorescent marker, GFP.
  • FIG. 1A illustrates the full length, truncated and modified Caspase-9 expression vectors constructed and evaluated as a suicide switch for induction of apoptosis.
  • the full-length inducible Caspase-9 molecule (F'-F-C-Casp9) includes 2, 3, or more FK506 binding proteins (FKBPs— for example, FKBP12v36 variants) linked with a Gly-Ser-Gly-Gly-Gly-Ser linker to the small and large subunit of the Caspase molecule (see FIG. 1 A).
  • FKBPs FK506 binding proteins
  • Full-length inducible Caspase-9 (F'F-C-Casp9.I.GFP) has a full-length Caspase-9, also includes a Caspase recruitment domain (CARD; GenBank NM001 229) linked to 2 12-kDa human FK506 binding proteins
  • FKBP12 GenBank AH002 8178 that contain an F36V mutation
  • F' The amino acid sequence of one or more of the FKBPs (F') was codon-wobbled (e.g., the 3 rd nucleotide of each amino acid codon was altered by a silent mutation that maintained the originally encoded amino acid) to prevent homologous recombination when expressed in a retrovirus.
  • F'F-C-Casp9C3S includes a cysteine to serine mutation at position 287 that disrupts its activation site.
  • F'F-Casp9, F-C-Casp9, and F'-Casp9 either the Caspase activation domain (CARD), one FKBP, or both, were deleted, respectively. All constructs were cloned into MSCV. IRES. GFP as EcoRI- Xhol fragments.
  • 293T cells were transfected with each of these constructs and 48 hours after transduction expression of the marker gene GFP was analyzed by flow cytometry. In addition, 24 hours after transfection, 293T cells were incubated overnight with 100 nM CID and subsequently stained with the apoptosis marker annexin V.
  • the mean and standard deviation of transgene expression level (mean GFP) and number of apoptotic cells before and after exposure to the chemical inducer of dimerization (CID) (% annexin V within GFP- cells) from 4 separate experiments are shown in the second through fifth columns of the table in FIG. 1 A.
  • the expressed gene products of the full length, truncated and modified Caspase-9 were also analyzed by western blot to confirm the Caspase-9 genes were being expressed and the expressed product was the expected size.
  • the results of the western blot are presented in FIG. 1 B.
  • Coexpression of the inducible Caspase-9 constructs of the expected size with the marker gene GFP in transfected 293T cells was demonstrated by Western blot using a Caspase-9 antibody specific for amino acid residues 299-318, present both in the full-length and truncated Caspase molecules as well as a GFP-specific antibody.
  • Western blots were performed as presented herein.
  • Transfected 293T cells were resuspended in lysis buffer (50% Tris/Gly, 10% sodium dodecyl sulfate [SDS], 4% beta-mercaptoethanol, 10% glycerol, 12% water, 4% bromophenol blue at 0.5%) containing aprotinin, leupeptin, and phenylmethylsulfonyl fluoride (Boehringer, Ingelheim,
  • Degradation products for the F'F-C-Casp9 and F'F-Casp9 constructs may not be detected due to a lower expression level of these constructs as a result of their basal activity. Equal loading of each sample was confirmed by the substantially equal amounts of actin shown at the bottom of each lane of the western blot, indicating substantially similar amounts of protein were loaded in each lane.
  • a chimeric polypeptide that may be expressed in the modified cells is provided herein.
  • a single polypeptide is encoded by the nucleic acid vector.
  • the inducible Caspase-9 polypeptide is separated from the CAR polypeptide during translation, due to skipping of a peptide bond. (Donnelly, ML 2001 , J. Gen. Virol. 82:1013-25).
  • CTLs cytotoxic T cells
  • Retrovirus transduction For the transient production of retrovirus, 293T cells were transfected with iCasp9/iFas constructs, along with plasmids encoding gag-pol and RD 1 14 envelope using GeneJuice transfection reagent (Novagen, Madison, Wl). Virus was harvested 48 to 72 hours after transfection, snap frozen, and stored at ⁇ 80°C until use. A stable FLYRD 18-derived retroviral producer line was generated by multiple transductions with VSV-G pseudotyped transient retroviral supernatant. FLYRD18 cells with highest transgene expression were single-cell sorted, and the clone that produced the highest virus titer was expanded and used to produce virus for lymphocyte transduction.
  • transgene expression, function, and retroviral titer of this clone was maintained during continuous culture for more than 8 weeks.
  • a non-tissue-culture-treated 24-well plate (Becton Dickinson, San Jose, CA) was coated with recombinant fibronectin fragment (FN CH- 296; Retronectin; Takara Shuzo, Otsu, Japan; 4 ⁇ g/mL in PBS, overnight at 4°C) and incubated twice with 0.5 mL retrovirus per well for 30 minutes at 37°C.
  • apoptosis CID (AP20187; ARIAD Pharmaceuticals) at indicated concentrations was added to transfected 293T cells or transduced CTLs.
  • Adherent and nonadherent cells were harvested and washed with annexin binding buffer (BD Pharmingen, San Jose, CA). Cells were stained with annexin-V and 7- amino-actinomycin D (7-AAD) for 15 minutes according to the manufacturer's instructions (BD Pharmingen). Within 1 hour after staining, cells were analyzed by flow cytometry using CellQuest software (Becton Dickinson).
  • Target cells included autologous LCLs, human leukocyte antigen (HLA) class l-mismatched LCLs and the lymphokine-activated killer cell-sensitive T-cell lymphoma line HSB-2.
  • Target cells incubated in complete medium or 1 % Triton X-100 (Sigma, St Louis, MO) were used to determine spontaneous and maximum 5 Cr release, respectively.
  • Triton X-100 Sigma, St Louis, MO
  • ANGFR-iFas was detected using anti-NGFR antibody (Chromaprobe, Aptos, CA). Appropriate matched isotype controls (Becton Dickinson) were used in each experiment. Cells were analyzed with a FACSscan flow cytometer (Becton Dickinson).
  • the concentration of interferon- ⁇ (I FN- ⁇ ), IL-2, IL-4, IL-5, IL-10, and tumor necrosis factor- a (TNFa) in CTL culture supernatants was measured using the Human Th1/Th2 cytokine cytometric Bead Array (BD Pharmingen) and the concentration of IL-12 in the culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) according to the instructions of the manufacturer.
  • ELISA enzyme-linked immunosorbent assay
  • Non-obese diabetic severe combined immunodeficient mice 6 to 8 weeks of age, were irradiated (250 rad) and injected subcutaneously in the right flank with 10 x 10 6 to 15 x 10 6 LCLs resuspended in Matrigel (BD Bioscience). Two weeks later mice bearing tumors that were approximately 0.5 cm in diameter were injected into the tail vein with a 1 : 1 mixture of nontransduced and iCasp9.I.GFPhigh-transduced EBV CTLs (total 15 x 10 6 ). At 4 to 6 hours prior and 3 days after CTL infusion, mice were injected intraperitoneal ly with recombinant hlL-2 (2000 U; Proleukin;
  • mice were randomly segregated in 2 groups: 1 group received CID (50 ⁇ g AP20187, intraperitoneally) and 1 group received carrier only (16.7% propanediol, 22.5% PEG400, and 1.25% Tween 80, intraperitoneally). On day 7, all mice were killed. Tumors were homogenized and stained with antihuman CD3 (BD Pharmingen). By FACS analysis, the number of GFP + cells within the gated CD3 + population was evaluated. Tumors from a control group of mice that received only nontransduced CTLs (total 15 x 10 6 ) were used as a negative control in the analysis of CD37GFP + cells.
  • Caspases 3, 7, and 9 were screened for their suitability as inducible safety-switch molecules both in transfected 293T cells and in transduced human T cells. Only inducible Caspase-9 (iCasp9) was expressed at levels sufficient to confer sensitivity to the chosen CID (e.g., chemical inducer of dimerization). An initial screen indicated that the full length iCasp9 could not be maintained stably at high levels in T cells, possibly due to transduced cells being eliminated by the basal activity of the transgene.
  • the CARD domain is involved in physiologic dimerization of Caspase-9 molecules, by a cytochrome C and adenosine triphosphate (ATP)-driven interaction with apoptotic protease- activating factor 1 (Apaf-1). Because of the use of a CID to induce dimerization and activation of the suicide switch, the function of the CARD domain is superfluous in this context and removal of the CARD domain was investigated as a method of reducing basal activity. Given that only dimerization rather than multimerization is required for activation of Caspase-9, a single FKBP12v36 domain also was investigated as a method to effect activation.
  • the activity of the resultant truncated and/or modified forms of Caspase-9 (e.g., the CARD domain, or one of the 2 FKBP domains, or both, are removed) were compared.
  • LTRs retroviral long terminal repeats
  • EBV-CTL EBV-specific CTL lines
  • iCasp9M (mean, 75.3%; range, 71.4%-83.0% in 5 different donors) were obtained after a single transduction with retrovirus.
  • the expression of iCasp9M in EBV-CTLs was stable for at least 4 weeks after transduction without selection or loss of transgene function. iCasp9M does not alter transduced T-cell characteristics
  • transduced and nontransduced CTLs consisted of equal numbers of CD4 + , CD8 + , CD56 + , and TCR ⁇ / ⁇ + cells.
  • production of cytokines including IFN- ⁇ , TNFa, IL-10, IL-4, IL-5, and IL-2 was unaltered by iCasp9 M expression.
  • iCasp9 M -transduced EBV-CTLs specifically lysed autologous LCLs comparable to nontransduced and control-transduced CTLs.
  • Expression of iCasp9M did not affect the growth characteristics of exponentially growing CTLs, and importantly, dependence on antigen and IL-2 for proliferation was preserved.
  • the normal weekly antigenic stimulation with autologous LCLs and I L-2 was continued or discontinued.
  • Disconti nuation of antigen sti mulation resulted i n a steady decli ne of T cells.
  • Inducible iCasp9M proficiency in CTLs was tested by monitoring loss of GFP-expressing cells after administration of CI D; 91.3% (range, 89.5%-92.6% in 5 different donors) of GFP + cells were eliminated after a single 10-nM dose of CID. Similar results were obtained regardless of exposure time to CI D (range, 1 hour-continuous). In all experiments, CTLs that survived CI D treatment had low transgene expression with a 70% (range, 55%-82%) reduction in mean fluorescence intensity of GFP after CI D. No further elimination of the surviving GFP + T cells could be obtained by an antigenic stimulation followed by a second 10-nM dose of CI D.
  • CTLs were sorted for low, intermediate, and high expression of the linked marker gene GFP and mixed 1 : 1 with nontransduced CTLs from the same donor to allow for an accurate quantitation of the number of transduced T cells responding to CI D-induced apoptosis.
  • iCasp9M IRES.GFP-transduced EBV-CTL were selected for low (mean 21), intermediate (mean 80) and high (mean 189) GFP expression.
  • Selected T-cells were incubated overnight with 10 nM CI D and subsequently stained with annexin V and 7-AAD. Indicated are the percentages of annexin V+/7-AAD- and annexin V+/7-AAD+ T-.
  • Selected T-cells were mixed 1 : 1 with non-transduced T-cells and incubated with 10 nM CI D following antigenic stimulation.
  • CI D-induced apoptosis was inhibited by the panCaspase inhibitor zVAD-fmk (100 ⁇ for 1 hour prior to adding CI D. Titration of CI D showed that 1 nM CID was sufficient to obtain the maximal deletion effect.
  • a dose-response curve using the indicated amounts of CI D shows the sensitivity of F-Casp9M .I .GFPhigh to CI D. Survival of GFP + cells is measured on day 7 after administration of the indicated amount of CI D. The mean and standard deviation for each point are given. Similar results were obtained using another chemical inducer of dimerization (CID), AP1903, which was clinically shown to have substantially no adverse effects when administered to healthy volunteers. The dose response remained unchanged for at least 4 weeks after transduction.
  • iCasp9 M is functional in malignant cells that express antiapoptotic molecules
  • Caspase-9 was selected as an inducible proapoptotic molecule for clinical use rather than previously presented iFas and iFADD, because Caspase-9 acts relatively late in apoptosis signaling and therefore is expected to be less susceptible to inhibition by apoptosis inhibitors.
  • suicide function should be preserved not only in malignant, transformed T-cell lines that express antiapoptotic molecules, but also in subpopulations of normal T cells that express elevated antiapoptotic molecules as part of the process to ensure long-term preservation of memory cells.
  • the function of iCasp9M and iFas was first compared in EBV-CTLs.
  • I.GFP-transduced CTLs were sorted for GFPmgh expression and mixed with nontransduced CTLs at a 1 : 1 ratio to obtain cell populations that expressed either iFas or iCasp9ivi at equal proportions and at similar levels.
  • the EBV-CTLs were sorted for high GFP expression and mixed 1 : 1 with nontransduced CTLs as presented. The percentages of
  • ANGFR7GFP + and GFP + T cells are indicated.
  • Elimination of GFP + cells after administration of 10 nM CID was more rapid and more efficient in iCasp9 M than in i Fas-transduced CTLs (99.2% +/- 0.14% of iCasp9 M -transduced cells compared with 89.3% +/- 4.9% of i Fas-transduced cells at day 7 after CID; P ⁇ .05).
  • 10 nM CID was administered, and GFP was measured at the time points indicated to determine the response to CID.
  • Black diamonds represent data for ANGFR-iFas.l.GFP; black squares represent data for iCasp9 M .I.GFP. Mean and standard deviation of 3 experiments are shown.
  • iCasp9M and iFas were also compared in 2 malignant T-cell lines: Jurkat, an apoptosis-sensitive T-cell leukemia line, and MT-2, an apoptosis-resistant T-cell line, due to c-FLIP and bcl-xL expression.
  • Jurkat cells and MT-2 cells were transduced with iFas and iCasp9M with similar efficiencies (92% vs 84% in Jurkat, 76% vs 70% in MT-2) and were cultured in the presence of 10 nM CI D for 8 hours.
  • Annexin-V staining showed that although iFas and iCasp9M induced apoptosis in an equivalent number of Jurkat cells (56.4% +/- 15.6% and 57.2% +/-18.9%, respectively), only activation of iCasp9M resulted in apoptosis of MT-2 cells (19.3% +/- 8.4% and 57.9% +/- 11.9% for iFas and iCasp9M, respectively; see Fig. 5C).
  • the human T-cell lines Jurkat (left) and MT-2 (right) were transduced with ANGFR-iFas.l.GFP or iCasp9ivi .l.GFP.
  • An equal percentage of T cells were transduced with each of the suicide genes: 92% for ANGFR-iFas.l.GFP versus 84% for iCasp9 M .I.GFP in Jurkat, and 76% for ANGFR- iFas.l.GFP versus 70% for iCasp9 M .I.GFP in MT-2.
  • T cells were either nontreated or incubated with 10 nM CID.
  • IL- 12 was undetectable in the supernatant of nontransduced and iCasp9ivi.l ES.GFP-transduced CTLs
  • the supernatant of iCasp9M.I ES. IL-12-transduced cells contained 324 pg/mL to 762 pg/mL IL-12.
  • the IL-12 in the supernatant fell to undetectable levels ( ⁇ 7 . 8 pg/mL).
  • activation of iCasp9 M is sufficient to completely eliminate all T cells producing biologically relevant levels of IL-12.
  • the marker gene GFP in the iCasp9 M .I.GFP constructs was replaced by flexi IL-12, encoding the p40 and p35 subunits of human IL-12.
  • iCasp9 M .I.GFP- and iCasp9 M .I.IL-12-transduced EBV-CTLs were stimulated with LCLs, and then left untreated or exposed to 10 nM CID.
  • the levels of IL-12 in the culture supernatant were measured by IL-12 ELISA (detection limit of this assay is 7.8 pg/mL). The mean and standard deviation of triplicate wells are indicated. Results of 1 of 2 experiments with CTLs from 2 different donors are shown.
  • iCasp9M The function of iCasp9M also was evaluated in transduced EBV-CTLs in vivo.
  • a SCID mouse- human xenograft model was used for adoptive immunotherapy. After intravenous infusion of a 1 :1 mixture of nontransduced and iCasp9M.IRES.GFPhigh-transduced CTLs into SCID mice bearing an autologous LCL xenograft, mice were treated either with a single dose of CID or carrier only. Three days after CID/carrier administration, tumors were analyzed for human CD37GFP + cells.
  • mice treated with CID there was more than a 99% reduction in the number of human CD37GFP + T cells, compared with infused mice treated with carrier alone, demonstrating equally high sensitivity of iCasp9 M -transduced T cells in vivo and in vitro.
  • the function of iCasp9 M in vivo was assayed.
  • NOD/SCI D mice were irradiated and injected subcutaneously with 10 x 10 6 to 15 x 10 6 LCLs.
  • mice bearing tumors of 0.5cm in diameter received a total of 15 x10 6 EBV-CTLs (50% of these cells were nontransduced and 50% were transduced with iCasp9M.I.GFP and sorted for high GFP expression).
  • Human CD3 + T cells isolated from the tumors of a control group of mice that received only nontransduced CTLs (15 x10 6 CTLs; n 4) were used as a negative control for the analysis of CD3 + /GFP + T cells within the tumors.
  • Suicide gene expression vectors presented herein have certain non-limiting advantageous features including stable coexpression in all cells carrying the modifying gene, expression at levels high enough to elicit cell death, low basal activity, high specific activity, and minimal susceptibility to endogenous antiapoptotic molecules.
  • an inducible Caspase-9, iCasp9 M which has low basal activity allowing stable expression for more than 4 weeks in human T cells.
  • a single 10-nM dose of a small molecule chemical inducer of dimerization (CID) is sufficient to kill more than 99% of iCasp9ivrtransduced cells selected for high transgene expression both in vitro and in vivo.
  • iCasp9M when coexpressed with Th1 cytokine IL-12, activation of iCasp9M eliminated all detectable IL-12-producing cells, even without selection for high transgene expression.
  • Caspase- 9 acts downstream of most antiapoptotic molecules, therefore, a high sensitivity to CID is preserved regardless of the presence of increased levels of antiapoptotic molecules of the bcl-2 family.
  • iCasp9ivi also may prove useful for inducing destruction even of transformed T cells and memory T cells that are relatively resistant to apoptosis.
  • proteolysis does not appear sufficient for activation of Caspase-9.
  • Crystallographic and functional data indicate that dimerization of inactive Caspase-9 monomers leads to conformational change-induced activation.
  • the concentration of pro-Caspase-9, in a physiologic setting, is in the range of about 20 nM, well below the threshold needed for dimerization.
  • the energetic barrier to dimerization can be overcome by homophilic interactions between the CARD domains of Apaf-1 and Caspase-9, driven by cytochrome C and ATP.
  • Overexpression of Caspase-9 joined to 2 FKBPs may allow spontaneous dimerization to occur and can account for the observed toxicity of the initial full length Caspase-9 construct.
  • a decrease in toxicity and an increase in gene expression was observed following removal of one FKBP, most likely due to a reduction in toxicity associated with spontaneous dimerization. While multimerization often is involved in activation of surface death receptors, dimerization of Caspase-9 should be sufficient to mediate activation.
  • virus- or bacteria-derived lethal genes such as HSV-TK and cytosine deaminase
  • the persistence and function of virus- or bacteria-derived lethal genes can be impaired by unwanted immune responses against cells expressing the virus or bacteria derived lethal genes.
  • the FKBPs and proapoptotic molecules that form the components of iCasp9 M are human-derived molecules and are therefore less likely to induce an immune response.
  • the linker between FKBP and Caspase-9 and the single point mutation in the FKBP domain introduce novel amino acid sequences, the sequences were not
  • iCasp9M are human-derived molecules, no memory T cells specific for the junction sequences should be present in a recipient, unlike virus-derived proteins such as HSV-TK, thereby reducing the risk of immune response-mediated elimination of iCasp9ivrtransduced T cells.
  • a very small number of resistant residual cells may cause a resurgence of toxicity, a deletion efficiency of up to 2 logs will significantly decrease this possibility.
  • coexpression with a nonimmunogenic selectable marker such as truncated human NGFR, CD20, or CD34 (e.g., instead of GFP) will allow for selection of high transgene-expressing T cells.
  • Coexpression of the suicide switch e.g., ICASP9M
  • a suitable selectable marker e.g., truncated human NGFR, CD20, CD34, the like and combinations thereof
  • IVS internal ribosome entry site
  • 2A posttranslational modification of a fusion protein containing a self-cleaving sequence
  • this selection step may be unnecessary, as tight linkage between iCasp9M and transgene expression enables elimination of substantially all cells expressing biologically relevant levels of the therapeutic transgene.
  • apoptosis inhibitors include c-FLIP, bcl-2 family members and inhibitors of apoptosis proteins (lAPs), which normally regulate the balance between apoptosis and survival.
  • lAPs apoptosis proteins
  • upregulation of c-FLIP and bcl-2 render a subpopulation of T cells, destined to establish the memory pool, resistant to activation-induced cell death in response to cognate target or antigen-presenting cells.
  • T-lymphoid tumors the physiologic balance between apoptosis and survival is disrupted in favor of cell survival.
  • a suicide gene should delete substantially all transduced T cells including memory and malignantly transformed cells. Therefore, the chosen inducible suicide gene should retain a significant portion if not substantially all of its activity in the presence of increased levels of antiapoptotic molecules.
  • the apical location of iFas (or iFADD) in the apoptosis signaling pathway may leave it especially vulnerable to inhibitors of apoptosis, thus making these molecules less well suited to being the key component of an apoptotic safety switch.
  • Caspase 3 or 7 would seem well suited as terminal effector molecules; however neither could be expressed at functional levels in primary human T cells.
  • Caspase-9 was chosen as the suicide gene, because Capsase-9 functions late enough in the apoptosis pathway that it bypasses the inhibitory effects of c-FLIP and antiapoptotic bcl-2 family members, and Caspase-9 also could be expressed stably at functional levels.
  • X-linked inhibitor of apoptosis could in theory reduce spontaneous Caspase-9 activation, the high affinity of AP20187 (or AP1903) for FKBPv36 may displace this noncovalently associated XIAP.
  • iCasp9M remained functional in a transformed T-cell line that overexpresses antiapoptotic molecules, including bcl-xL.
  • iCasp9M can be activated by AP1903 (or analogs), a small chemical inducer of dimerization that has proven safe at the required dose for optimum deletional effect, and unlike ganciclovir or rituximab has no other biologic effects in vivo. Therefore, expression of this suicide gene in T cells for adoptive transfer can increase safety and also may broaden the scope of clinical applications.
  • Example 2 Using the iCasp9 Suicide Gene to Improve the Safety of Allodepleted T Cells after Haploidentical Stem Cell Transplantation
  • CD19 was generated as a safety switch for donor T cells. Even after allodepletion (using anti-CD25 immunotoxin), donor T cells could be efficiently transduced, expanded, and subsequently enriched by CD19 immunomagnetic selection to >90% purity.
  • the engineered cells retained anti-viral specificity and functionality, and contained a subset with regulatory phenotype and function. Activating iCasp9 with a small-molecule dimerizer rapidly produced >90% apoptosis. Although transgene expression was downregulated in quiescent T cells, iCasp9 remained an efficient suicide gene, as expression was rapidly upregulated in activated (alloreactive) T cells.
  • PBMCs peripheral blood mononuclear cells
  • EBV Epstein Barr virus
  • LCL lymphoblastoid cell lines
  • AIM V Invitrogen, Carlsbad, CA
  • activated T cells that expressed CD25 were depleted from the co-culture by overnight incubation in RFT5-SMPT-dgA immunotoxin. Allodepletion was considered adequate if the residual CD3 + CD25 + population was ⁇ 1 % and residual proliferation by 3H- thymidine incorporation was ⁇ 10%.
  • SFG.iCasp9.2A.CD19 consists of inducible Caspase-9 (iCasp9) linked, via a cleavable 2A-like sequence, to truncated human CD19.
  • iCasp9 consists of a human FK5 06-binding protein (FKBP12; GenBank AH002 818) with an F36V mutation, connected via a Ser-Gly-Gly-Gly-Ser linker to human Caspase-9 (CASP9; GenBank NM 001229).
  • the F36V mutation increases the binding affinity of FKBP12 to the synthetic homodimerizer, AP20187 or AP1903.
  • the Caspase recruitment domain (CARD) has been deleted from the human Caspase-9 sequence because its physiological function has been replaced by FKBP12, and its removal increases transgene expression and function.
  • the 2A-like sequence encodes an 20 amino acid peptide from Thosea asigna insect virus, which mediates >99% cleavage between a glycine and terminal proline residue, resulting in 19 extra amino acids in the C terminus of iCasp9, and one extra proline residue in the N terminus of CD19.
  • CD19 consists of full-length CD19 (GenBank NM 001770) truncated at amino acid 333 (TDPTRRF), which shortens the intracytoplasmic domain from 242 to 19 amino acids, and removes all conserved tyrosine residues that are potential sites for phosphorylation.
  • TPTRRF amino acid 333
  • a stable PG13 clone producing Gibbon ape leukemia virus (Gal-V) pseudotyped retrovirus was made by transiently transfecting Phoenix Eco cell line (ATCC product #SD3444; ATCC, Manassas, VA) with SFG.iCasp9.2A.CD19. This produced Eco-pseudotyped retrovirus.
  • the PG13 packaging cell line (ATCC) was transduced three times with Eco-pseudotyped retrovirus to generate a producer line that contained multiple SFG.iCasp9.2A.CD19 proviral integrants per cell. Single cell cloning was performed, and the PG13 clone that produced the highest titer was expanded and used for vector production. Retroviral transduction
  • Culture medium for T cell activation and expansion consisted of 45% RPMI 1640 (Hyclone, Logan, UT), 45% Clicks (Irvine Scientific, Santa Ana, CA) and 10% fetal bovine serum (FBS; Hyclone). Allodepleted cells were activated by immobilized anti-CD3 (OKT3; Ortho Biotech, Bridgewater, NJ) for 48 hours before transduction with retroviral vector. Selective allodepletion was performed by co-culturing donor PBMC with recipient EBV-LCL to activate alloreactive cells: activated cells expressed CD25 and were subsequently eliminated by anti-CD25 immunotoxin. The allodepleted cells were activated by OKT3 and transduced with the retroviral vector 48 hours later. Immunomagnetic selection was performed on day 4 of transduction; the positive fraction was expanded for a further 4 days and cryopreserved.
  • non-tissue culture-treated 24-well plates (Becton Dickinson, San Jose, CA) were coated with OKT3 1 g/ml for 2 to 4 hours at 37°C. Allodepleted cells were added at 1 10 6 cells per well. At 24 hours, 100U/ml of recombinant human interleukin-2 (IL-2) (Proleukin; Chiron, Emeryville, CA) was added. Retroviral transduction was performed 48 hours after activation.
  • IL-2 human interleukin-2
  • Non-tissue culture-treated 24-well plates were coated with 3 ⁇ g/cm 2 recombinant fibronectin fragment (CH-296; Retronectin; Takara Mirus Bio, Madison, Wl) and the wells loaded twice with retroviral vector-containing supernatant at 0.5ml per well for 30 minutes at 37°C, following which OKT3 -activated cells were plated at 5 * 10 5 cells per well in fresh retroviral vector-containing supernatant and T cell culture medium at a ratio of 3: 1 ,
  • IL-2 100U/ml IL-2 was added the next day.
  • retronectin-coated flasks or bags were loaded once with 10ml of retrovirus-containing supernatant for 2 to 3 hours.
  • OKT3- activated T cells were seeded at 1 10 6 cells/ml in fresh retroviral vector-containing medium and T cell culture medium at a ratio of 3: 1 , supplemented with 100U/ml IL-2.
  • Cells were harvested the following morning and expanded in tissue-culture treated T75 or T175 flasks in culture medium supplemented with between about 50 to 100U/ml IL-2 at a seeding density of between about 5x 10 5 cells/ ml to 8x 10 5 cells/ ml.
  • Immunomagnetic selection for CD19 was performed 4 days after transduction.
  • Cells were labeled with paramagnetic microbeads conjugated to monoclonal mouse anti-human CD19 antibodies (Miltenyi Biotech, Auburn, CA) and selected on MS or LS columns in small scale experiments and on a CliniMacs Plus automated selection device in large scale experiments.
  • CD19-selected cells were expanded for a further 4 days and cryopreserved on day 8 post transduction. These cells were referred to as "gene-modified allodepleted cells”. Immunophenotyping and pentamer analysis
  • Flow cytometric analysis was performed using the following antibodies: CD3, CD4, CD8, CD19, CD25, CD27, CD28, CD45RA, CD45RO, CD56 and CD62L.
  • CD19-PE Clone 4G7; Becton Dickinson
  • a Non-transduced control was used to set the negative gate for CD19.
  • An HLA-pentamer, HLA-B8-RAKFKQLL (Proimmune, Springfield, VA) was used to detect T cells recognizing an epitope from EBV lytic antigen (BZLF1 ).
  • HLA-A2-NLVPMVATV pentamer was used to detect T cells recognizing an epitope from CMV-pp65 antigen.
  • Interferon- ELISpot for assessment of responses to EBV, CMV and adenovirus antigens was performed using known methods.
  • Gene-modified allodepleted cells cryopreserved at 8 days post-transduction were thawed and rested overnight in complete medium without IL-2 prior to use as responder cells.
  • Cryopreserved PBMCs from the same donor were used as responder cells.
  • Responder cells were plated in duplicate or triplicate in serial dilutions of 2* 10 5 , 1 x 10 5 , 5 x 10 4 and 2.5 ⁇ 10 4 cells per well. Stimulator cells were plated at 1 ⁇ 10 5 per well.
  • donor-derived EBV-LCLs irradiated at 40Gy were used as stimulators.
  • donor-derived activated monocytes infected with Ad5f35 adenovirus were used.
  • donor PBMCs were plated in X-Vivo 15 (Cambrex, Walkersville, MD) in 24-well plates overnight, harvested the next morning, infected with Ad5f35 at a multiplicity of infection (MOI) of 200 for 2 hours, washed, irradiated at 30Gy, and used as stimulators.
  • MOI multiplicity of infection
  • Ad5f35-pp65 adenovirus encoding the CMV pp65 transgene
  • SFU spot-forming units
  • Suicide gene functionality was assessed by adding a small molecule synthetic homodimerizer, AP20187 (Ariad Pharmaceuticals; Cambridge, MA), at 10nM final concentration the day following CD19 immunomagnetic selection.
  • Cells were stained with annexin V and 7-amino- actinomycin (7-AAD)(BD Pharmingen) at 24 hours and analyzed by flow cytometry.
  • Cells negative for both annexin V and 7-AAD were considered viable, cells that were annexin V positive were apoptotic, and cells that were both annexin V and 7-AAD positive were necrotic.
  • Cells were maintained in T cell medium containing 50U/ml IL-2 until 22 days after transduction. A portion of cells was reactivated on 24-well plates coated with 1 g/ml OKT3 and ⁇ g/ml anti- CD28 (Clone CD28.2, BD Pharmingen, San Jose, CA) for 48 to 72 hours. CD19 expression and suicide gene function in both reactivated and non-reactivated cells were measured on day 24 or 25 post transduction. In some experiments, cells also were cultured for 3 weeks post transduction and stimulated with 30G-irradiated allogeneic PBMC at a responder: stimulator ratio of 1 : 1. After 4 days of co- culture, a portion of cells was treated with 10nM AP20187. Killing was measured by annexin V/7-AAD staining at 24 hours, and the effect of dimerizer on bystander virus-specific T cells was assessed by pentamer analysis on AP20187-treated and untreated cells.
  • CD4 CD25 and Foxp3 expression was analyzed in gene-modified allodepleted cells using flow cytometry.
  • human Foxp3 staining the eBioscience (San Diego, CA) staining set was used with an appropriate rat lgG2a isotype control. These cells were co-stained with surface CD25- FITC and CD4-PE. Functional analysis was performed by co-culturing CD4 + 25 + cells selected after allodepletion and gene modification with carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled autologous PBMC.
  • CFSE carboxyfluorescein diacetate N-succinimidyl ester
  • CD4 + 25 + selection was performed by first depleting CD8 + cells using anti-CD 8 microbeads (Miltenyi Biotec, Auburn, CA), followed by positive selection using anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA).
  • CFSE-labeling was performed by incubating autologous PBMC at 2x 10 7 /ml in phosphate buffered saline containing 1.5 ⁇ CFSE for 10 minutes. The reaction was stopped by adding an equivalent volume of FBS and incubating for 10 minutes at 37°C. Cells were washed twice before use.
  • CFSE-labeled PBMCs were stimulated with OKT3 500ng/ml and 40G-irradiated allogeneic PBMC feeders at a
  • the cells were then cultured with or without an equal number of autologous CD4 + 25 + gene-modified allodepleted cells. After 5 days of culture, cell division was analyzed by flow cytometry; CD19 was used to gate out non-CFSE-labeled CD4 + CD25 + gene-modified T cells.
  • Selectively allodepleted T cells can be efficiently transduced with iCasp9 and expanded Selective allodepletion was performed in accordance with clinical protocol procedures. Briefly, 3/6 to 5/6 HLA-mismatched PBMC and lymphoblastoid cell lines (LCL) were co-cultured.
  • RFT5- SMPT-dgA immunotoxin was applied after 72 hours of co-culture and reliably produced allodepleted cells with ⁇ 10% residual proliferation (mean 4.5 ⁇ 2.8%; range 0.74 to 9.1 %; 10 experiments) and containing ⁇ 1 % residual CD3 + CD25 + cells (mean 0.23 ⁇ 0.20%; range 0.06 to 0.73%; 10 experiments), thereby fulfilling the release criteria for selective allodepletion, and serving as starting materials for subsequent manipulation.
  • Allodepleted cells activated on immobilized OKT3 for 48 hours could be efficiently transduced with Gal-V pseudotyped retrovirus vector encoding SFG.iCasp9.2A.CD19.
  • Transduction efficiency assessed by FACS analysis for CD19 expression 2 to 4 days after transduction was about 53% ⁇ 8%, with comparable results for small-scale (24-well plates) and large-scale (T75 flasks) transduction (about 55 ⁇ 8% versus about 50% ⁇ 10% in 6 and 4 experiments, respectively).
  • Cell numbers contracted in the first 2 days following OKT3 activation such that only about 61 % ⁇ 12% (range of about 45% to 80%) of allodepleted cells were recovered on the day of
  • ACD19 enables efficient and selective enrichment of transduced cells on immunomagnetic columns
  • the efficiency of suicide gene activation sometimes depends on the functionality of the suicide gene itself, and sometimes on the selection system used to enrich for gene-modified cells.
  • the use of CD19 as a selectable marker was investigated to determine if CD19 selection enabled the selection of gene-modified cells with sufficient purity and yield, and whether selection had any deleterious effects on subsequent cell growth. Small-scale selection was performed according to manufacturer's instruction; however, it was determined that large-scale selection was optimum when 101 of CD 19 microbeads was used per 1.3 ⁇ 10 7 cells. FACS analysis was performed at 24 hours after immunomagnetic selection to minimize interference from anti-CD19 microbeads.
  • the absolute yield of small- and large-scale selections were about 31 % ⁇ 11 % and about 28% ⁇ 6%, respectively; after correction for transduction efficiency.
  • the mean recovery of transduced cells was about 54% ⁇ 14 % in small-scale and about 72% ⁇ 18 % in large-scale selections. The selection process did not have any discernable deleterious effect on subsequent cell expansion.
  • the final cell product (gene-modified allodepleted cells that had been cryopreserved 8 days after transduction) was immunophenotyped and was found to contain both CD4 and CD8 cells, with CD8 cells predominant, at 62% ⁇ 1 1 % CD8 + versus 23% ⁇ 8% CD4 + , as shown in the table below.
  • CD45RO + The majorities of cells were CD45RO + and had the surface immunophenotype of effector memory T cells.
  • end-product cells to mediate antiviral immunity was assessed by interferon- ELISpot, cytotoxicity assay, and pentamer analysis.
  • the cryopreserved gene-modified allodepleted cells were used in all analyses, since they were representative of the product currently being evaluated for use in a clinical study.
  • Interferon- ⁇ secretion in response to adenovirus, CMV or EBV antigens presented by donor cells was preserved although there was a trend towards reduced anti-EBV response in gene-modified allodepleted cells versus
  • unmanipulated PBMC unmanipulated PBMC.
  • the response to viral antigens was assessed by ELISpot in 4 pairs of unmanipulated PBMC and gene-modified allodepleted cells (GMAC).
  • GMAC gene-modified allodepleted cells
  • Adenovirus and CMV antigens were presented by donor-derived activated monocytes through infection with Ad5f35 null vector and Ad5f35-pp65 vector, respectively.
  • EBV antigens were presented by donor EBV- LCL.
  • SFU spot-forming units
  • Cytotoxicity was assessed using donor-derived EBV-LCL as targets.
  • Gene-modified allodepleted cells that had undergone 2 or 3 rounds of stimulation with donor-derived EBV-LCL could efficiently lyse virus-infected autologous target cells
  • Gene-modified allodepleted cells were stimulated with donor EBV-LCL for 2 or 3 cycles.
  • 5 Cr release assay was performed using donor-derived EBV-LCL and donor OKT3 blasts as targets.
  • NK activity was blocked with 30-fold excess cold K562.
  • the left panel shows results from 5 independent experiments using totally or partially mismatched donor-recipient pairs.
  • the right panel shows results from 3 experiments using unrelated HLA haploidentical donor-recipient pairs. Error bars indicate standard deviation.
  • EBV-LCLs were used as antigen-presenting cells during selective allodepletion, therefore it was possible that EBV-specific T cells could be significantly depleted when the donor and recipient were haploidentical.
  • three experiments using unrelated HLA- haploidentical donor-recipient pairs were included, and the results showed that cytotoxicity against donor-derived EBV-LCL was retained.
  • the results were corroborated by pentamer analysis for T cells recognizing HLA-B8-RAKFKQLL, an EBV lytic antigen (BZLF1) epitope, in two informative donors following allodepletion against HLA-B8 negative haploidentical recipients. Unmanipulated PBMC were used as comparators.
  • the RAK-pentamer positive population was retained in gene-modified allodepleted cells and could be expanded following several rounds of in vitro stimulation with donor-derived EBV-LCL. Together, these results indicate that gene-modified allodepleted cells retained significant anti-viral functionality. Regulatory T cells in the Gene-modified allodepleted cell population
  • CD4 + CD25 + enriched fraction demonstrated suppressor function when co-cultured with CFSE-labeled autologous PBMC in the presence of OKT3 and allogeneic feeders.
  • Donor-derived PBMC was labeled with CFSE and stimulated with OKT3 and allogeneic feeders.
  • CD4 + CD25 + cells were immunomagnetically selected from the gene-modified cell population and added at 1 : 1 ratio to test wells. Flow cytometry was performed after 5 days. Gene-modified T cells were gated out by CD19 expression. The addition of CD4 + CD25 + gene-modified cells (bottom panel) significantly reduced cell
  • allodepleted T cells may reacquire regulatory phenotype even after exposure to a CD25 depleting immunotoxin.
  • transgene expression and function were maintained in T cell culture medium and low dose IL-2 (50U/ml) until 24 days after transduction. A portion of cells was then reactivated with OKT3/ anti-CD28. CD19 expression was analyzed by flow cytometry 48 to 72 hours later, and suicide gene function was assessed by treatment with 10nM AP20187. The obtained are for cells from day 5 post transduction (ie, 1 day after CD 19 selection) and day 24 post transduction, with or without 48-72 hours of reactivation (5 experiments). In 2 experiments, CD25 selection was performed after OKT3/aCD28 activation to further enrich activated cells.
  • suicide gene function also increased significantly from about a 63.1 % ⁇ 6.2% killing efficiency to about a 84.6% ⁇ 8.0% (p ⁇ 0.01) killing efficiency.
  • killing efficiency was completely restored if the cells were immunomagnetically sorted for the activation marker CD25: killing efficiency of CD25 positive cells was about 93%.2 ⁇ 1.2%, which was the same as killing efficiency on day 5 post transduction (93.1 ⁇ 3.5%). Killing of the CD25 negative fraction was 78.6 ⁇ 9.1 %.
  • virus-specific T cells were partially and preferentially retained.
  • Neomycin phosphotransferase encodes a potentially immunogenic foreign protein and requires a 7-day culture in selection medium, which not only increases the complexity of the system, but is also potentially damaging to virus-specific T cells.
  • a widely used surface selection marker, LNGFR has recently had concerns raised, regarding its oncogenic potential and potential correlation with leukemia, in a mouse model, despite its apparent clinical safety.
  • CD34 has been well-studied in vitro, but the steps required to optimize a system configured primarily for selection of rare hematopoietic progenitors, and more critically, the potential for altered in vivo T cell homing, make CD34 sub-optimal for use as a selectable marker for a suicide switch expression construct.
  • CD19 was chosen as an alternative selectable marker, since clinical grade CD19 selection is readily available as a method for B-cell depletion of stem cell autografts. The results presented herein demonstrated that CD19 enrichment could be performed with high purity and yield and, furthermore, the selection process had no discernable effect on subsequent cell growth and functionality.
  • Optimal culture conditions for maintaining the immunological competence of suicide gene- modified T cells must be determined and defined for each combination of safety switch, selectable marker and cell type, since phenotype, repertoire and functionality can all be affected by the stimulation used for polyclonal T cell activation, the method for selection of transduced cells, and duration of culture.
  • the addition of CD28 co-stimulation and the use of cell-sized paramagnetic beads to generate gene modified-cells that more closely resemble unmanipulated PBMC in terms of CD4:CD8 ratio, and expression of memory subset markers including lymph node homing molecules CD62L and CCR7, may improve the in vivo functionality of gene- modified T cells.
  • CD28 co-stimulation also may increase the efficiency of retroviral
  • iCasp9-modified allodepleted cells retained significant anti-viral functionality, and approximately one fourth retained CD62L expression. Regeneration of CD4 + CD25 + Foxp3 + regulatory T cells was also seen.
  • the allodepleted cells used as the starting material for T cell activation and transduction may have been less sensitive to the addition of anti-CD28 antibody as co-stimulation.
  • CD25-depleted PBMC / EBV-LCL co-cultures contained T cells and B cells that already express CD86 at significantly higher level than unmanipulated PBMCs and may they provide co-stimulation. Depletion of CD25 + regulatory T cells prior to polyclonal T cell activation with anti-CD3 has been reported to enhance the immunological competence of the final T cell product. In order to minimize the effect of in vitro culture and expansion on functional competence, a relatively brief culture period was used in some experiments presented herein, whereby cells were expanded for a total of 8 days post-transduction with CD19-selection being performed on day 4.
  • allodepleted cells can be activated and transduced at 4x 10 7 cells per flask, and a minimum of 8-fold return of CD19- selected final cell product can be obtained on day 8 post-transduction, to produce at least 3* 10 8 allodepleted gene-modified cells per original flask.
  • the increased culture volume is readily accommodated in additional flasks or bags.
  • the allodepletion and iCasp9-modification presented herein may significantly improve the safety of adding back T cells, particularly after haploidentical stem cell allografts.
  • Example 3 CASPALLO - Phase 1 Clinical Trial of Allodepleted T Cells Transduced with Inducible Caspase-9 Suicide Gene after Haploidentical Stem Cell Transplantation
  • This example presents results of a phase 1 clinical trial using the alternative suicide gene strategy illustrated in FIG. 2. Briefly, donor peripheral blood mononuclear cells were co-cultured with recipient irradiated EBV-transformed lymphoblastoid cells (40:1) for 72 hrs, allodepleted with a CD25 immunotoxin and then transduced with a retroviral supernatant carrying the iCasp9 suicide gene and a selection marker ( ⁇ CD19); ⁇ CD19 allowed enrichment to >90% purity via
  • a leukopheresis was performed to isolate sufficient T cells. 10cc-30cc of blood also was drawn from the recipient and was used to generate the Epstein Barr virus (EBV)- transformed lymphoblastoid cell line used as stimulator cells.
  • EBV Epstein Barr virus
  • the LCLs were generated using appropriate 1st degree relative (e.g., parent, sibling, or offspring) peripheral blood mononuclear cells.
  • Allodepleted cells were generated from the transplant donors as presented herein.
  • Peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with irradiated recipient Epstein Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) at responder-to-stimulator ratio of 40: 1 in serum-free medium (AIM V; Invitrogen, Carlsbad, CA).
  • EBV Epstein Barr virus
  • LCD lymphoblastoid cell lines
  • AIM V Invitrogen, Carlsbad, CA
  • activated T cells that express CD25 were depleted from the co-culture by overnight incubation in RFT5-SMPT-dgA immunotoxin. Allodepletion is considered adequate if the residual CD3 + CD25 + population was ⁇ 1 % and residual proliferation by 3 H-thymidine incorporation was ⁇ 10%.
  • a retroviral producer line clone was generated for the iCasp9-CD19 construct.
  • a master cell-bank of the producer also was generated. Testing of the master-cell bank was performed to exclude generation of replication competent retrovirus and infection by Mycoplasma, HIV, HBV, HCV and the like.
  • the producer line was grown to confluency, supernatant harvested, filtered, aliquoted and rapidly frozen and stored at -80°C. Additional testing was performed on all batches of retroviral supernatant to exclude Replication Competent Retrovirus (RCR) and issued with a certificate of analysis, as per protocol.
  • RCR Replication Competent Retrovirus
  • Allodepleted T-lymphocytes were transduced using Fibronectin. Plates or bags were coated with recombinant Fibronectin fragment CH-296 (RetronectinTM, Takara Shuzo, Otsu, Japan). Virus was attached to retronectin by incubating producer supernatant in coated plates or bags. Cells were then transferred to virus coated plates or bags. After transduction allodepleted T cells were expanded, feeding them with IL-2 twice a week to reach the sufficient number of cells as per protocol.
  • CD 19 Immunomagnetic Selection Immunomagnetic selection for CD19 was performed 4 days after transduction. Cells are labeled with paramagnetic microbeads conjugated to monoclonal mouse anti-human CD19 antibodies (Miltenyi Biotech, Auburn, CA) and selected on a CliniMacs Plus automated selection device. Depending upon the number of cells required for clinical infusion cells were either cryopreserved after the CliniMacs selection or further expanded with IL-2 and cryopreserved on day 6 or day 8 post transduction.
  • RFT5-SMPT-dgA is a murine lgG1 anti-CD25 (IL-2 receptor alpha chain) conjugated via a hetero- bifunctional crosslinker [N-succinimidyloxycarbonyl-alpha-methyl-d- (2-pyridylthio) toluene]
  • RFT5-SMPT-dgA chemically deglycosylated ricin A chain
  • AP1903-inducible cell death is achieved by expressing a chimeric protein comprising the intracellular portion of the human (Caspase-9 protein) receptor, which signals apoptotic cell death, fused to a drug-binding domain derived from human FK506- binding protein (FKBP). This chimeric protein remains quiescent inside cells until
  • AP1903 which cross-links the FKBP domains, initiating Caspase signaling and apoptosis.
  • AP1903 has been evaluated as an Investigational New Drug (IND) by the FDA and has successfully completed a phase 1 clinical safety study. No significant adverse effects were noted when API 903 was administered over a 0.01 mg/kg to LOmglkg dose range.
  • IND Investigational New Drug
  • Pharmacology/Pharmacokinetics Patients received 0.4 mg/kg of AP1903 as a 2 h infusion - based on published Pk data which show plasma concentrations of 10 ng/mL - 1275 ng/mL over the 0.01 mg/kg to 1.0 mg/kg dose range with plasma levels falling to 18% and 7% of maximum at 0.5 and 2 hrs post dose.
  • GVHD patients developing grade 1 GVHD were treated with 0.4 mg/kg AP1903 as a 2-hour infusion. Protocols for administration of AP1903 to patients grade 1 GVHD were established as follows. Patients developing GvHD after infusion of allodepleted T cells are biopsied to confirm the diagnosis and receive 0.4 mg/kg of AP1903 as a 2 h infusion. Patients with Grade I GVHD received no other therapy initially, however if they showed progression of GvHD conventional GvHD therapy was administered as per institutional guidelines. Patients developing grades 2- 4 GVHD were administered standard systemic immunosuppressive therapy per institutional guidelines, in addition to the AP1903 dimerizer drug.
  • AP1903 for injection is obtained as a concentrated solution of 2.33 ml in a 3-ml vial, at a concentration of 5 mg/ml, (i.e., 11.66 mg per vial).
  • AP1903 may also be provided, for example, at 8ml per vial, at 5mg/ml. Prior to administration, the calculated dose was diluted to 100 ml_ in 0.9% normal saline for infusion. AP1903 for injection (0.4 mg/kg) in a volume of 100 ml was administered via IV infusion over 2 hours, using a non-DEHP, non-ethylene oxide sterilized infusion set and infusion pump.
  • the iCasp9 suicide gene expression construct (e.g., SFG.iCasp9.2A. ⁇ CD19), shown in FIG. 24 consists of inducible Caspase-9 (iCasp9) linked, via a cleavable 2A-like sequence, to truncated human CD19 ( ⁇ CD19).
  • iCasp9 includes a human FK506-binding protein (FKBP12; GenBank AH002 818) with an F36V mutation, connected via a Ser-Gly-Gly-Gly-Ser-Gly linker to human Caspase-9 (CASP9; GenBank NM 001229).
  • the F36V mutation may increase the binding affinity of FKBP12 to the synthetic homodimerizer, AP20187 or AP1903.
  • the Caspase recruitment domain (CARD) has been deleted from the human Caspase-9 sequence and its physiological function has been replaced by FKBP12. The replacement of CARD with FKBP12 increases transgene expression and function.
  • the 2A-like sequence encodes an 18 amino acid peptide from Thosea Asigna insect virus, which mediates >99% cleavage between a glycine and terminal proline residue, resulting in 17 extra amino acids in the C terminus of iCasp9, and one extra proline residue in the N terminus of CD19.
  • ⁇ CD19 consists of full length CD19 (GenBank NM 001770) truncated at amino acid 333 (TDPTRRF), which shortens the intracytoplasmic domain from 242 to 19 amino acids, and removes all conserved tyrosine residues that are potential sites for phosphorylation.
  • SCT haplo-CD34 + stem cell transplantation
  • Table 2 Characteristics of the patients and clinical outcome.
  • Infused T cells were detected in vivo by flow cytometry (CD3 + ⁇ CD19 + ) or qPCR as early as day 7 after infusion, with a maximum fold expansion of 170 ⁇ 5 (day 29 ⁇ 9 after infusion), as illustrated in FIGS. 27, 28, and 29.
  • Two patients developed grade l/ll aGVHD (see FIGS. 31-32) and AP1903 administration caused >90% ablation of CD3 + ⁇ CD19 + cells, within 30 minutes of infusion (see
  • FIGS. 30, 33, and 34 with a further log reduction within 24 hours, and resolution of skin and liver aGvHD within 24hrs, showing that iCasp9 transgene was functional in vivo.
  • the disappearance of skin rash within 24 hours post treatment was observed.
  • Table 3 Patients with GvHD (dose level 1)
  • T cell subsets CD3, CD4, CD8, CD16, CD19, CD27, CD28, CD44, CD62L, CCR7, CD56,
  • CD45RA CD45RO
  • alpha/beta CD45RO
  • gamma/delta T cell receptors CD45RA, CD45RO, alpha/beta and gamma/delta T cell receptors
  • T regulatory cell markers such as CD41 , CD251 , and FoxP3 also are analyzed. Approximately 10-60 ml of patient blood is taken, when possible, 4 hours after infusion, weekly for 1 month, monthly x 9 months, and then at 1 and 2 years.
  • amount of blood taken is dependent on the size of the recipient and does not exceed 1-2 cc/kg in total (allowing for blood taken for clinical care and study evaluation) at any one blood draw.
  • Phenotype by flow cytometry to detect the presence of transgenic cells RCR testing by PCR.
  • Quantitative real-time PCR for detecting retroviral integrants.
  • RCR testing by PCR is performed pre study, at 3, 6, and 12 months, and then yearly for a total of 15 years. Tissue, cell, and serum samples are archived for use in future studies for RCR as required by the FDA.
  • the MTD is defined to be the dose which causes grade lll/IV acute GVHD in at most 25% of eligible cases.
  • the determination is based on a modified continual reassessment method (CRM) using a logistic model with a cohort of size 2.
  • CCM continual reassessment method
  • Three dose groups are being evaluated namely, 1x10 6 , 3x10 6 , 1x10 7 with prior probabilities of toxicity estimated at 10%, 15%, and 30%, respectively.
  • the proposed CRM design employs modifications to the original CRM by accruing more than one subject in each cohort, limiting dose escalation to no more than one dose level, and starting patient enrollment at the lowest dose level shown to be safe for non-transduced cells. Toxicity outcome in the lowest dose cohort is used to update the dose-toxicity curve.
  • the next patient cohort is assigned to the dose level with an associated probability of toxicity closest to the target probability of 25%. This process continues until at least 10 patients have been accrued into this dose-escalation study. Depending on patient availability, at most 18 patients may be enrolled into the Phase 1 trial or until 6 patients have been treated at the current MTD. The final MTD will be the dose with probability closest to the target toxicity rate at these termination points.
  • Simulations were performed to determine the operating characteristics of the proposed design and compared this with a standard 3+3 dose-escalation design.
  • the proposed design delivers better estimates of the MTD based on a higher probability of declaring the appropriate dose level as the MTD, afforded smaller number of patients accrued at lower and likely ineffective dose levels, and maintained a lower average total number of patients required for the trial.
  • a shallow dose-toxicity curve is expected over the range of doses proposed herein and therefore accelerated dose- escalations can be conducted without comprising patient safety.
  • the simulations performed indicate that the modified CRM design does not incur a larger average number of total toxicities when compared to the standard design (total toxicities equal to 1.9 and 2.1 , respectively.).
  • Grade lll/IV GVHD that occurs within 45 days after initial infusion of allodepleted T cells will be factored into the CRM calculations to determine the recommended dose for the subsequent cohort.
  • Real-time monitoring of patient toxicity outcome is performed during the study in order to implement estimation of the dose-toxicity curve and determine dose level for the next patient cohort using one of the pre-specified dose levels.
  • Treatment limiting toxicities will include: grade 4 reactions related to infusion,
  • graft failure defined as a subsequent decline in the ANC to ⁇ 500/mm 3 for three consecutive measurements on different days, unresponsive to growth factor therapy that persists for at least 14 days.) occurring within 30 days after infusion of TC-T
  • GVHD rates are summarized using descriptive statistics along with other measures of safety and toxicity. Likewise, descriptive statistics will be calculated to summarize the clinical and biologic response in patients who receive AP1903 due to great than Grade 1 GVHD.
  • T regulatory cell markers such as CD3, CD4, CDS, CD16, CD19, CD27, CD44, CD62L, CCR7, CD56, CD45RA, CD45RO, alpha/beta and gamma/delta T cell receptors.
  • CD4/CD25/FoxP3 will also be analyzed. Each subject will be measured pre-infusion and at multiple time points post-infusion as presented above.
  • Longitudinal analysis of each repeatedly-measured immune reconstitution parameter using the random coefficients model will be performed. Longitudinal analysis allows construction of model patterns of immune reconstitution per patient while allowing for varying intercepts and slopes within a patient. Dose level as an independent variable in the model to account for the different dose levels received by the patients will also be used. Testing whether there is a significant
  • a strategy similar to the one presented above can be employed to assess kinetics of T cell survival, expansion and persistence.
  • the ratio of the absolute T cell numbers with the number of marker gene positive cells will be determined and modeled longitudinally over time. A positive estimate of the slope will indicate increasing contribution of T cells for immune recovery.
  • Virus- specific immunity of the iCasp9 T cells will be evaluated by analysis of the number of T cells releasing I FN gamma based on ex-vivo stimulation virus-specific CTLs using longitudinal models. Separate models will be generated for analysis of EBV, CMV and adenovirus evaluations of immunity.
  • the protocols provided in Examples 1-3 may also be modified to provide for in vivo T cell allodepletion.
  • the protocol may be simplified, by providing for an in vivo method of T cell depletion.
  • EBV-transformed lymphoblastoid cell lines are first prepared from the recipient, which then act as alloantigen presenting cells. This procedure can take up to 8 weeks, and may fail in extensively pre-treated subjects with malignancy, particularly if they have received rituximab as a component of their initial therapy.
  • the donor T cells are co-cultured with recipient EBV-LCL, and the alloreactive T cells (which express the activation antigen CD25) are then treated with CD25- ricin conjugated monoclonal antibody. This procedure may take many additional days of laboratory work for each subject.
  • the process may be simplified by using an in vivo method of allodepletion, building on the observed rapid in vivo depletion of alloreactive T cells by dimerizer drug and the sparing of unstimulated but virus /fungus reactive T cells.
  • a single dose of dimerizer drug is administered, for example at a dose of 0.4 mg/kg of AP1903 as a 2-hour intravenous infusion. Up to 3 additional doses of dimerizer drug may be administered at 48 hour intervals if acute GvHD persists. In subjects with Grade II or greater acute GvHD, these additional doses of dimerizer drug may be combined with steroids. For patients with persistent GVHD who cannot receive additional doses of the dimerizer due to a Grade III or IV reaction to the dimerizer, the patient may be treated with steroids alone, after either 0 or 1 doses of the dimerizer.
  • peripheral blood Up to 240 ml (in 2 collections) of peripheral blood is obtained from the transplant donor according to the procurement consent. If necessary, a leukapheresis is used to obtain sufficient T cells; (either prior to stem cell mobilization or seven days after the last dose of G-CSF). An extra 10-30 mis of blood may also be collected to test for infectious diseases such as hepatitis and HIV.
  • Peripheral blood mononuclear cells are be activated using anti-human CD3 antibody (e.g. from Orthotech or Miltenyi) on day 0 and expanded in the presence of recombinant human interleukin-2 (rhlL-2) on day 2.
  • CD3 antibody-activated T cells are transduced by the iCaspase-9 retroviral vector on flasks or plates coated with recombinant Fibronectin fragment CH-296 (RetronectinTM, Takara Shuzo, Otsu, Japan). Virus is attached to retronectin by incubating producer supernatant in retronectin coated plates or flasks. Cells are then transferred to virus coated tissue culture devices.
  • T cells are expanded by feeding them with rhlL-2 twice a week to reach the sufficient number of cells as per protocol.
  • a selectable marker truncated human CD19 ( ⁇ CD19) and a commercial selection device, may be used to select the transduced cells to >90% purity.
  • Immunomagnetic selection for CD19 may be performed 4 days after transduction. Cells are labeled with paramagnetic microbeads conjugated to monoclonal mouse anti-human CD19 antibodies (Miltenyi Biotech, Auburn, CA) and selected on a CliniMacs Plus automated selection device.
  • cells might either be cryopreserved after the CliniMacs selection or further expanded with IL-2 and cryopreserved as soon as sufficient cells have expanded (up to day 14 from product initiation). Aliquots of cells may be removed for testing of transduction efficiency, identity, phenotype, autonomous growth and microbiological examination as required for final release testing by the FDA. The cells are cryopreserved prior to administration.
  • the transduced T cells are administered to patients from, for example, between 30 and 120 days following stem cell transplantation.
  • the cryopreserved T cells are thawed and infused through a catheter line with normal saline.
  • premedications are dosed by weight.
  • Doses of cells may range from, for example, from about 1 x 10 4 cells/kg to 1 x 10 8 cells/kg, for example from about 1 x 10 5 cells/kg to 1 x 10 7 cells/kg, from about 1 x 10 6 cells/kg to 5 x 10 6 cells/kg, from about 1 x 10 4 cells/kg to 5 x 10 6 cells/kg, for example, about 1 x 10 4 , about 1 x 10 5 , about 2 x 10 5 , about 3 x 10 5 , about 5 x 10 5 , 6 x 10 5 , about 7 x 10 5 , about 8 x 10 5 , about 9 x 10 5 , about 1 x 10 6 , about 2 x 10 6 , about 3 x 10 6 , about 4 x 10 6 , or about 5 x 10 6 cells/kg.
  • Treatment of GvHD Treatment of GvHD
  • AP1903 for injection may be provided, for example, as a concentrated solution of 2.33 ml in a 3 ml vial, at a concentration of 5 mg/ml, (i.e 11.66 mg per vial). AP1903 may also provided in different sized vials, for example, 8 ml at 5mg/ml may be provided. Prior to administration, the calculated dose will be diluted to 100 ml_ in 0.9% normal saline for infusion. AP1903 for Injection (0.4 mg/kg) in a volume of 100 ml may be administered via IV infusion over 2 hours, using a non- DEHP, non-ethylene oxide sterilized infusion set and an infusion pump. Table 4: Sample treatment schedule
  • Example 5 Using the iCasp9 Suicide Gene to Improve the Safety of Mesenchymal Stromal Cell Therapies
  • MSCs Mesenchymal stromal cells
  • the long term side effects are not known due to limited follow-up and a relatively short time since MSCs have been used in treatment of disease.
  • Several animal models have indicated that there exists the potential for side effects, and therefore a system allowing control over the growth and survival of MSCs used therapeutically is desirable.
  • the inducible Caspase-9 suicide switch expression vector construct presented herein was investigated as a method of eliminating MSCs in vivo and in vitro. Materials and Methods MSC isolation
  • MSCs were isolated from healthy donors. Briefly, post-infusion discarded healthy donor bone marrow collection bags and filters were washed with RPMI 1640 (HyClone, Logan, UT) and plated on tissue culture flasks in DMEM (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS), 2 mM alanyl-glutamine (Glutamax, Invitrogen), 100 units/mL penicillin and 100 ⁇ g/mL streptomycin (Invitrogen).
  • RPMI 1640 HyClone, Logan, UT
  • FBS fetal bovine serum
  • Glutamax Glutamax
  • Invitrogen 100 units/mL penicillin and 100 ⁇ g/mL streptomycin
  • CCM complete culture medium
  • Immunophenotyping Phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP) or allophycocyanin (APC)-conjugated CD14, CD34, CD45, CD73, CD90, CD105 and CD133 monoclonal antibodies were used to stain MSCs. All antibodies were from Becton Dickinson- Pharmingen (San Diego, CA), except where indicated. Control samples labeled with an appropriate isotype-matched antibody were included in each experiment. Cells were analyzed by fluorescence-activated cell sorting FACScan (Becton Dickinson) equipped with a filter set for 4 fluorescence signals.
  • FACScan Fluorescence-activated cell sorting FACScan
  • AdipoDiff Medium (Miltenyi Biotech, Auburn, CA). Medium was changed every third day for 21 days. Cells were stained with Oil Red O solution (obtained by diluting 0.5% w/v Oil Red O in isopropanol with water at a 3:2 ratio), after fixation with 4% formaldehyde in phosphate buffered saline (PBS).
  • Oil Red O solution obtained by diluting 0.5% w/v Oil Red O in isopropanol with water at a 3:2 ratio
  • PBS phosphate buffered saline
  • MSCs (4.5x 10 4 cells) were plated in 6-well plates in NH OsteoDiff Medium (Miltenyi Biotech). Medium was changed every third day for 10 days. Cells were stained for alkaline phosphatase activity using Sigma Fast BCIP/NBT substrate (Sigma-Aldrich, St. Louis, MO) as per manufacturer instructions, after fixation with cold methanol.
  • MSC pellets containing 2.5x 10 5 to 5 ⁇ 10 5 cells were obtained by centrifugation in 15 mL or 1.5 ml_ polypropylene conical tubes and cultured in NH ChondroDiff Medium (Miltenyi Biotech). Medium was changed every third day for a total of 24 days.
  • Cell pellets were fixed in 4% formalin in PBS and processed for routine paraffin sectioning. Sections were stained with alcian blue or using indirect immunofluorescence for type II collagen (mouse anti-collagen type II monoclonal antibody MAB8887, Millipore, Billerica, MA) after antigen retrieval with pepsin (Thermo Scientific, Fremont, CA).
  • the SFG.iCasp9.2A. ⁇ CD19 (iCasp- ⁇ CD19) retrovirus consists of iCasp9 linked, via a cleavable 2A-like sequence, to truncated human CD19 ( ⁇ CD19).
  • iCasp9 is a human FK506-binding protein (FKBP12) with an F36V mutation, which increases the binding affinity of the protein to a synthetic homodimerizer (AP20187 or AP1903), connected via a Ser-Gly-Gly-Gly-Ser- Gly linker to human Caspase-9, whose recruitment domain (CARD) has been deleted, its function replaced by FKBP12.
  • the 2A-like sequence encodes a 20 amino acid peptide from Thosea Asigna insect virus, which mediates more than 99% cleavage between a glycine and terminal proline residue, to ensure separation of iCasp9 and ⁇ CD19 upon translation.
  • ⁇ CD19 consists of human CD19 truncated at amino acid 333, which removes all conserved intracytoplasmic tyrosine residues that are potential sites for phosphorylation.
  • a stable PG13 clone producing Gibbon ape leukemia virus (Gal-V) pseudotyped retrovirus was made by transiently transfecting Phoenix Eco cell line (ATCC product #SD3444; ATCC, Manassas, VA) with SFG.iCasp9.2A. ⁇ CD19, which yielded Eco-pseudotyped retrovirus.
  • the PG13 packaging cell line (ATCC) was transduced 3 times with Eco-pseudotyped retrovirus to generate a producer line that contained multiple SFG.iCasp9.2A. ⁇ CD19 proviral integrants per cell.
  • Retroviral supernatant was obtained via culture of the producer cell lines in IMDM (Invitrogen) with 10% FBS, 2 mM alanyl-glutamine, 100 units/mL penicillin and 100 ⁇ g/mL streptomycin. Supernatant containing the retrovirus was collected 48 and 72 hours after initial culture. For transduction, approximately 2* 10 4 MSCs/cm 2 were plated in CM in 6-well plates, T75 or T175 flasks. After 24 hours, medium was replaced by viral supernatant diluted 10-fold together with polybrene (final concentration 5 ⁇ g/mL) and the cells were incubated at 37°C in 5% CO 2 for 48 hours, after which cells were maintained in complete medium.
  • iCasp9-transduced MSCs were maintained in culture without exposure to CID.
  • Differentiated MSCs Transduced MSCs were differentiated as presented above. At the end of the differentiation period, CID was added to the differentiation media at 50 nM.
  • tissue were stained appropriately for the tissue being studied, as presented above, and a contrast stain (methylene azur or methylene blue) was used to evaluate the nuclear and cytoplasmic morphology.
  • tissue were processed for terminal deoxynucleotidyl-transferase dUTP nick end labeling (TUN EL) assay as per manufacturer instructions (In Situ Cell Death Detection Kit, Roche Diagnostics, Mannheim, Germany). For each time point, four random fields were photographed at a final magnification of 40* and the images were analyzed with ImageJ software version 1.43o (NIH, Bethesda, MD). Cell density was calculated as the number of nuclei (DAPI positivity) per unit of surface area (in mm 2 ). The percentage of apoptotic cells was determined as the ratio of the number of nuclei with positive TUNEL signal (FITC positivity) to the total number of nuclei.
  • TUN EL terminal deoxynucleotidyl-transferase
  • MSCs were transduced with retroviruses coding for the enhanced green fluorescent protein-firefly luciferase (eGFP-FFLuc) gene alone or together with the iCasp9- ⁇ CD19 gene.
  • eGFP-FFLuc enhanced green fluorescent protein-firefly luciferase
  • Cells were sorted for eGFP positivity by fluorescence activated cell sorting using a MoFlo flow cytometer (Beckman Coulter, Fullerton, CA).
  • Total luminescence (a measurement proportional to the total labeled MSCs deposited) at each time point was calculated by automatically defining regions-of-interest (ROIs) over the MSC implantation sites. These ROIs included all areas with luminescence signals at least 5% above background. Total photon counts were integrated for each ROI and an average value calculated. Results were normalized so that time zero would correspond to 100% signal.
  • ROIs regions-of-interest
  • a mixture of 2.5x 10 6 eGFP-FFLuc-labeled MSCs and 2.5x 10 6 eGFP-FFLuc-labeled, iCasp9- ⁇ CD19-transduced MSCs was injected subcutaneously in the right flank, and the mice received two intraperitoneal injections of 50 ⁇ g of CID 24 h apart starting 7 days later.
  • the subcutaneous pellet of MSCs was harvested using tissue luminescence to identify and collect the whole human specimen and to minimize mouse tissue contamination. Genomic DNA was then isolated using QIAmp® DNA Mini (Qiagen, Valencia, CA).
  • N iCasp9 N eGFP (k/g) [C/(C+G)], i.e., the ratio between iCasp9 copy number and eGFP copy number is proportional to the fraction of doubly transduced (iCasp9-positive) cells among all eGFP positive cells.
  • the absolute values of N iCasp9 and N eGFP will decrease with increasing contamination by murine cells in each MSC explant, for each time point the ratio will be constant regardless of the amount of murine tissue included, since both types of human cells are physically mixed.
  • MSCs are readily transduced with iCasp9- ⁇ CD19 and maintain their basic phenotype
  • MSCs from 3 healthy donors showed they were uniformly positive for CD73, CD90 and CD105 and negative for the hematopoietic markers CD45, CD14, CD133 and CD34.
  • the mononuclear adherent fraction isolated from bone marrow was homogenously positive for CD73, CD90 and CD105 and negative for hematopoietic markers.
  • the differentiation potential, of isolated MSCs, into adipocytes, osteoblasts and chondroblasts was confirmed in specific assays, demonstrating that these cells are bona fide MSCs.
  • the phenotype of the iCasp9-CD19-positive cells was otherwise substantially identical to that of untransduced cells, with virtually all cells positive for CD73, CD90 and CD105 and negative for hematopoietic markers, confirming that the genetic manipulation of MSCs did not modify their basic characteristics.
  • iCasp9-ACD19 transduced MSCs undergo selective apoptosis after exposure to CID in vitro
  • the proapoptotic gene product iCasp9 can activated by a small chemical inducer of dimerization (CID), AP20187, an analogue of tacrolimus that binds the FK506-binding domain present in the iCasp9 product.
  • CID chemical inducer of dimerization
  • AP20187 an analogue of tacrolimus that binds the FK506-binding domain present in the iCasp9 product.
  • Magnetic immunoselection of iCap9-CD19-positive cells can be achieved to high degree of purity. More than 95% of the selected cells become apoptotic after exposure to CID. Analysis of a highly purified iCasp9-positive population at later time points after a single exposure to CID shows that the small fraction of iCasp9-negative cells expands and that a population of iCasp9-positive cells remains, but that the latter can be killed by re-exposure to CID. Thus, no iCasp9-positive population resistant to further killing by CID was detected. A population of iCasp9- CD19-negative MSCs emerges as early as 24 hours after CID introduction.
  • iCasp9-CD19-negative MSCs A population of iCasp9-CD19-negative MSCs is expected since achieving a population with 100% purity is unrealistic and because the MSCs are being cultured in conditions that favor their rapid expansion in vitro.
  • a fraction of iCasp9-CD19-positive population persists, as predicted by the fact that killing is not 100% efficient (assuming, for example, 99% killing of a 99% pure population, the resulting population would have 49.7% iCasp9-positive and 50.3% iCasp9-negative cells).
  • the surviving cells however, can be killed at later time points by re-exposure to CID.
  • iCasp9-ACD19 transduced MSCs maintain the differentiation potential of unmodified MSCs and their progeny is killed by exposure to CID
  • immunomagnetic selection for CD19 was used to increase the purity of the modified population (>90% after one round of selection.
  • the iCasp9-positive cells thus selected were able to differentiate in vivo into all connective tissue lineages studied (see FIGS. 19A-19Q).
  • Human MSCs were immunomagnetically selected for CD19 (thus iCasp9) expression, with a purity greater than 91 %.
  • iCasp9-positive cells After culture in specific differentiation media, iCasp9-positive cells were able to give rise to adipocytic (A, oil red and methylene azur), osteoblastic (B, alkaline phosphatase-BCIP/NBT and methylene blue) and chondroblastic lineages (C, alcian blue and nuclear red) lineages. These differentiated tissues are driven to apoptosis by exposure to 50 nM CID (D-N).
  • iCasp9 remained functional even after MSC differentiation, and its activation results in the death of the differentiated progeny.
  • iCasp9-ACD19 transduced MSCs undergo selective apoptosis after in vivo exposure to CID
  • MSCs intravenously injected MSC
  • eGFP-FFLuc previously presented
  • iCasp9- ⁇ CD19 genes previously presented
  • MSCs were also singly transduced with eGFP- FFLuc.
  • the eGFP-positive (and CD19-positive, where applicable) fractions were isolated by fluorescence activated cell sorting, with a purity > 95%.
  • Each animal was injected subcutaneously with iCasp9-positive and control MSCs (both eGFP-FFLuc-positive) in opposite flanks. Localization of the MSCs was evaluated using the Xenogen-IVIS Imaging System.
  • a 1 : 1 mixture of singly and doubly transduced MSCs was injected subcutaneously in the right flank and the mice received CID as above.
  • the subcutaneous pellet of MSCs was harvested at different time points, genomic DNA was isolated and qPCR was used to determine copy numbers of the eGFP-FFLuc and iCasp9- ⁇ CD19 genes.
  • the ratio of the iCasp9 to eGFP gene copy numbers is proportional to the fraction of iCasp9-positive cells among total human cells (see Methods above for details). The ratios were normalized so that time zero corresponds to 100% of iCasp9-positive cells.
  • Serial examination of animals after subcutaneous inoculation of MSCs (prior to CID injection) shows evidence of spontaneous apoptosis in both cell populations (as demonstrated by a fall in the overall luminescence signal to -20% of the baseline). This has been previously observed after systemic and local delivery of MSCs in xenogeneic models.
  • the luminescence data showed a substantial loss of human MSCs over the first 96 h after local delivery of MSCs, even before administration of CID, with only approximately 20% cells surviving after one week. From that time point onward, however, there were significant differences between the survival of icasp9-positive MSCs with and without dimerizer drug.
  • Seven days after MSC implantation animals were given two injections of 50 ⁇ g of CID, 24 hours apart. MSCs transduced with iCasp9 were quickly killed by the drug, as demonstrated by the disappearance of their luminescence signal. Cells negative for iCasp9 were not affected by the drug. Animals not injected with the drug showed persistence of signal in both populations up to a month after MSC implantation.
  • qPCR assays were developed to measure copy numbers of the eGFP-FFLuc and iCasp9- ⁇ CD19 genes.
  • Mice were injected subcutaneously with a 1 :1 mixture of doubly and singly transduced MSCs and administered CID as above, one week after MSC implantation.
  • MSCs explants were collected at several time points, genomic DNA isolated from the samples and qPCR assays performed on substantially identical amounts of DNA. Under these conditions (see Methods), at any time point, the ratio of iCasp9- ⁇ CD19 to eGFP- FFLuc copy numbers is proportional to the fraction of viable iCasp9-positive cells.
  • transduced genes are stable both in MSCs and their differentiated progeny, and does not evidently alter their phenotype or potential for differentiation.
  • These transduced cells can be killed in vitro and in vivo when exposed to the appropriate small molecule chemical inducer of dimerization that binds to the iCasp9.
  • transplanted cells must survive the period between their harvest and their ultimate in vivo clinical application.
  • a safe cell based therapy also should include the ability to control the unwanted growth and activity of successfully transplanted cells.
  • MSCs have been administered to many patients without notable side effects
  • additional protections such as the safety switch presented herein
  • Subjects receiving MSCs that have been genetically modified to release biologically active proteins might particularly benefit from the added safety provided by a suicide gene.
  • the suicide system presented herein offers several potential advantages over other known suicide systems.
  • nucleoside analogues such as those combining Herpes Simplex Virus thymidine kinase (HSV-tk) with gancyclovir (GCV) and bacterial or yeast cytosine deaminase (CD) with 5-fluoro-cytosine (5-FC), are cell-cycle dependent and are unlikely to be effective in the post-mitotic tissues that may be formed during the application of MSCs to regenerative medicine. Moreover, even in proliferating tissues the mitotic fraction does not comprise all cells, and a significant portion of the graft may survive and remain dysfunctional.
  • HSV-tk Herpes Simplex Virus thymidine kinase
  • GCV gancyclovir
  • CD bacterial or yeast cytosine deaminase
  • 5-FC 5-fluoro-cytosine
  • the prodrugs required for suicide may themselves have therapeutic uses that are therefore excluded (e.g., GCV), or may be toxic (e.g., 5-FC), either as a result of their metabolism by non-target organs (e.g., many cytochrome P450 substrates), or due to diffusion to neighboring tissues after activation by target cells (e.g., CB1954, a substrate for bacterial nitroreductase).
  • GCV GCV
  • 5-FC cytochrome P450 substrates
  • target cells e.g., CB1954, a substrate for bacterial nitroreductase
  • the small molecule chemical inducers of dimerization presented herein have shown no evidence of toxicities even at doses ten fold higher than those required to activate the iCasp9.
  • nonhuman enzymatic systems such as HSV-tk and DC, carry a high risk of destructive immune responses against transduced cells.
  • Both the iCasp9 suicide gene and the selection marker CD19 are of human origin, and thus should be less likely to induce unwanted immune responses.
  • linkage of expression of the selectable marker to the suicide gene by a 2A-like cleavable peptide of nonhuman origin could pose problems, the 2A-like linker is 20 amino acids long, and is likely less immunogenic than a nonhuman protein.
  • the effectiveness of suicide gene activation in iCasp9-positive cells compares favorably to killing of cells expressing other suicide systems, with 90% or more of iCasp9-modified T cells eliminated after a single dose of dimerizer, a level that is likely to be clinically efficacious.
  • the iCasp9 system presented herein also may avoid additional limitations seen with other cell based and/or suicide switch based therapies. Loss of expression due to silencing of the transduced construct is frequently observed after retroviral transduction of mammalian cells. The expression constructs presented herein showed no evidence of such an effect. No decrease in expression or induced death was evident, even after one month in culture.
  • a potential limitation specific to the system presented herein may be spontaneous dimerization of iCasp9, which in turn could cause unwanted cell death and poor persistence. This effect has been observed in certain other inducible systems that utilize Fas. The observation of low spontaneous death rate in transduced cells and long term persistence of transgenic cells in vivo indicate this possibility is not a significant consideration when using iCasp9 based expression constructs.
  • Integration events deriving from retroviral transduction of MSCs may potentially drive deleterious mutagenesis, especially when there are multiple insertions of the retroviral vector, causing unwanted copy number effects and/or other undesirable effects. These unwanted effects could offset the benefit of a retrovirally transduced suicide system. These effects often can be minimized using clinical grade retroviral supernatant obtained from stable producer cell lines and similar culture conditions to transduce T lymphocytes.
  • the T cells transduced and evaluated herein contain in the range of about 1 to 3 integrants (the supernatant containing in the range of about 1 10 6 viral particles/mL).
  • the substitution of lentiviral for retroviral vectors could further reduce the risk of genotoxicity, especially in cells with high self-renewal and differentiation potential.
  • the CD19 molecule which is physiologically expressed by B lymphocytes, was chosen as the selectable marker for transduced cells, because of its potential advantages over other available selection systems, such as neomycin phosphotransferase (neo) and truncated low affinity nerve growth factor receptor ( ⁇ LNGFR).
  • neo neomycin phosphotransferase
  • ⁇ LNGFR truncated low affinity nerve growth factor receptor
  • ALNGFR expression should allow for isolation strategies similar to other surface markers, but these are not widely available for clinical use and a lingering concern remains about the oncogenic potential of ALNGFR.
  • magnetic selection of iCasp9-positive cells by CD19 expression using a clinical grade device is readily available and has shown no notable effects on subsequent cell growth or differentiation.
  • the procedure used for preparation and administration of mesenchymal stromal cells comprising the Caspase-9 safety switch may also be used for the preparation of embryonic stem cells and inducible pluripotent stem cells.
  • embryonic stem cells or inducible pluripotent stem cells may be substituted for the mesenchymal stromal cells provided in the example.
  • retroviral and lentiviral vectors may be used, with, for example, CMV promoters, or the ronin promoter.
  • Example 6 Modified Caspase-9 Polypeptides with Lower Basal Activity and Minimal Loss of Ligand IC50
  • Basal signaling signaling in the absence of agonist or activating agent, is prevalent in a multitude of biomolecules. For example, it has been observed in more than 60 wild-type G protein coupled receptors (GPCRs) from multiple subfamilies [1], kinases, such as ERK and abl [2], surface immunoglobulins [3], and proteases. Basal signaling has been hypothesized to contribute to a vast variety of biological events, from maintenance of embryonic stem cell pluripotency, B cell development and differentiation [4-6], T cell differentiation [2, 7], thymocyte development [8], endocytosis and drug tolerance [9], autoimmunity [10], to plant growth and development [11].
  • GPCRs G protein coupled receptors
  • PCR-based site directed mutagenesis [31] was done with mutation-containing oligos and Kapa (Kapa Biosystems, Woburn, MA). After 18 cycles of amplification, parental plasmid was removed with methylation-dependent Dpnl restriction enzyme that leaves the PCR products intact. 2 ⁇ of resulting reaction was used to chemically transform XL1-blue or DH5a. Positive mutants were subsequently identified via sequencing (SeqWright, Houston, TX).
  • HEK293T/16 cells ATCC, Manassas, VA
  • IMDM IMDM
  • GlutaMAXTM Life Technologies, Carlsbad, CA
  • FBS fetal bovine serum
  • penicillin 100 U/mL
  • streptomycin 100 U/mL
  • Cells in logarithmic-phase growth were transiently transfected with 800 ng to 2 ⁇ g of expression plasmid encoding iCasp9 mutants and 500 ng of an expression plasmid encoding SRa promoter driven SEAP per million cells in 15-mL conical tubes.

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