Multipartite signaling proteins and uses thereof

Abstract

The present disclosure relates to compositions and methods for using cells having chemically-induced fusion protein complexes to spatially and temporally control immune cell signal initiation and downstream responses for treating disease.

What is claimed is: 
     
         1 . A non-natural cell, comprising:
 (a) a first nucleic acid molecule encoding a first fusion protein comprising a first multimerization domain, a hydrophobic domain, and an actuator domain, wherein the first multimerization domain localizes extracellularly when the first fusion protein is expressed; and   (b) a second nucleic acid molecule encoding a second fusion protein comprising a binding domain and a second multimerization domain, wherein the second fusion protein localizes extracellularly when expressed;   wherein a first bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins.   
     
     
         2 . The non-natural cell according to  claim 1 , wherein the first and second multimerization domains are the same or different. 
     
     
         3 . The non-natural cell according to  claim 1  or  claim 2 , wherein the multimerization domains of the first and second fusion proteins associate with a bridging factor selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof. 
     
     
         4 . The non-natural cell according to any one of the preceding claims, wherein the first and second multimerization domains are a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. 
     
     
         5 . The non-natural cell according to any one of the preceding claims, wherein the first multimerization domain comprises a first FKBP polypeptide or variant thereof, and the second multimerization domain comprises a first FRB polypeptide or variant thereof 
     
     
         6 . The non-natural cell according to any one of the preceding claims, wherein the first multimerization domain comprises a first FRB polypeptide or variant thereof, and the second multimerization domain comprises a first FKBP polypeptide or variant thereof 
     
     
         7 . The non-natural cell according to  claim 5  or  6 , wherein the bridging factor is sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. 
     
     
         8 . The non-natural cell according to any one of the preceding claims, wherein the first nucleic acid molecule encodes a first fusion protein further comprising a third multimerization domain. 
     
     
         9 . The non-natural cell according to  claim 8 , wherein the third multimerization domain of the first fusion protein is a binding domain for a bridging factor selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, Tmp-SLF or a derivative thereof, or any combination thereof. 
     
     
         10 . The non-natural cell according to any one of the preceding claims, wherein a second bridging factor promotes the association of at least two first fusion proteins with the bridging factor associated with and disposed between the third multimerization domains of the first fusion proteins. 
     
     
         11 . The non-natural cell according to any one of the preceding claims, wherein the protein complex is a homocomplex comprising at least two first fusion proteins. 
     
     
         12 . The non-natural cell according to any one of the preceding claims, wherein the first fusion protein has at least one multimerization domain of FKBP, DHFR or GyrB. 
     
     
         13 . The non-natural cell according to any one of  claims 1 - 12 , wherein the binding domain of the polypeptide complex specifically binds to a target located on a target cell surface. 
     
     
         14 . The non-natural cell according to  claim 13 , wherein the protein complex is a heterocomplex comprising one or more first fusion proteins and one or more second fusion proteins. 
     
     
         15 . The non-natural cell according to  claim 14 , wherein the binding domain of the protein heterocomplex specifically binds to a target located on a target cell surface. 
     
     
         16 . The non-natural cell according to any one of the preceding claims, wherein the hydrophobic domain is a transmembrane domain. 
     
     
         17 . The non-natural cell according to any one of the preceding claims, wherein the transmembrane domain is a CD4, CD8 or CD28 transmembrane domain. 
     
     
         18 . The non-natural cell according to any one of the preceding claims, wherein the actuator domain comprises a lymphocyte receptor signaling domain. 
     
     
         19 . The non-natural cell according to any one of the preceding claims, wherein the actuator domain comprises one or a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs). 
     
     
         20 . The non-natural cell according to any one of the preceding claims, wherein the actuator domain comprises CD3ε, CD3δ, CD3ζ, pTα, TCRα, TCRβ, FcRα, FcRβ, FcRγ, NKG2D, CD22, CD79A, or CD79B, or any combination thereof. 
     
     
         21 . The non-natural cell according to any one of the preceding claims, wherein the first nucleic acid molecule encodes the first fusion protein further comprising a different actuator domain, a costimulatory domain, an adhesion factor, or any combination thereof. 
     
     
         22 . The non-natural cell according to  claim 18 , wherein the costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any combination thereof. 
     
     
         23 . The non-natural cell according to any one of the preceding claims, wherein the actuator domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein. 
     
     
         24 . The non-natural cell according to  claim 23 , wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein comprising a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs), a costimulatory domain, an adhesion factor, or any combination thereof. 
     
     
         25 . The non-natural cell according to  claim 24 , wherein the lymphocyte receptor or signaling domain thereof is CD3ε, CD3δ, CD3ζ, pTα, TCRα, TCRβ, FcRα, FcRβ, FcRγ, NKG2D, CD22, CD79A, or CD79B, or any combination thereof. 
     
     
         26 . The non-natural cell according to  claim 24 , wherein the costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any combination thereof. 
     
     
         27 . The non-natural cell according to any one of the preceding claims, further overexpressing a costimulatory factor, an immunomodulatoy factor, an agonist for a costimulatory factor, an agonist for an immunomodulatoy factor, or any combination thereof. 
     
     
         28 . The non-natural cell according to any one of the preceding claims, wherein the second nucleic acid molecule further encodes a secretion signal such that the second fusion protein is secreted from the non-natural cell when expressed, and optionally further encodes an anchor domain. 
     
     
         29 . The non-natural cell according to any one of the preceding claims, wherein the binding domain of the second fusion protein is a single chain antibody variable region, a receptor ectodomain, or a ligand. 
     
     
         30 . The non-natural cell according to  claim 29 , wherein the single chain antibody variable region is a domain antibody, sFv, scFv, F(ab′) 2 , or Fab. 
     
     
         31 . The non-natural cell according to any one of the preceding claims, wherein the binding domain of the second fusion protein is amino terminal to the multimerization domain. 
     
     
         32 . The non-natural cell according to any one of the preceding claims, wherein the binding domain of the second fusion protein is carboxy terminal to the multimerization domain. 
     
     
         33 . The non-natural cell according to any one of the preceding claims, wherein the second nucleic acid molecule encoding the second fusion protein further comprises a sequence encoding a linker disposed between the binding domain and the second multimerization domain. 
     
     
         34 . The non-natural cell according to any one of the preceding claims, wherein the cell further comprises a third nucleic acid molecule encoding a third fusion protein comprising a binding domain and a second multimerization domain, wherein the third fusion protein localizes extracellularly when expressed. 
     
     
         35 . The non-natural cell according to any one of the preceding claims, wherein the fusion proteins comprising a binding domain have one, two, three, or four binding domains. 
     
     
         36 . The non-natural cell according to any one of the preceding claims, wherein the one, two, three, or four binding domains are specific for one target or up to four different targets. 
     
     
         37 . The non-natural cell according to any one of the preceding claims, wherein the binding domain is specific for a target that is an antigen associated with a cancer, an inflammatory disease, an autoimmune disease, or a graft versus host disease. 
     
     
         38 . The non-natural cell according to  claim 37 , wherein the cancer is a solid malignancy or a hematologic malignancy. 
     
     
         39 . The non-natural cell according to  claim 38 , wherein the hematologic malignancy associated antigen target is CD19, CD20, CD22, CD33, or CD37. 
     
     
         40 . The non-natural cell according to any one of the preceding claims, wherein the binding domain specifically binds to a target selected from a-folate receptor, α v β 6  integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11Rα, IL13R-α2, KDR, κ light chain, λ light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notch1, Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2, and ROR1. 
     
     
         41 . The non-natural cell according to any one of the preceding claims, wherein the first bridging factor is rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, or Tmp-SLF or a derivative thereof. 
     
     
         42 . The non-natural cell according to  claim 10 , wherein the second bridging factor is rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, or Tmp-SLF or a derivative thereof. 
     
     
         43 . The non-natural cell according to any one of the preceding claims, wherein the encoded first fusion protein comprises a first multimerization domain of FRB T2098L, a transmembrane domain, a costimulatory domain of 4-1BB, and actuator domain of CD3ζ;
 wherein the second encoded fusion protein comprises a binding domain of an scFv specific for CD19 and a second multimerization domain of FKBP12; and wherein the first bridging factor that promotes the formation of a polypeptide complex on the non-natural cell surface is rapalog AP21967. 
 
     
     
         44 . The non-natural cell according to  claim 43 , wherein the first fusion protein has an amino acid sequence as set forth in SEQ ID NO.:15 and the second fusion protein has an amino acid sequence as set forth in SEQ ID NO.:1. 
     
     
         45 . A method for treating a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease, comprising:
 (a) administering a recombinant cell comprising a first and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a first fusion protein comprising a first multimerization domain, a hydrophobic domain, and an actuator domain, wherein the first multimerization domain localizes extracellularly when the first fusion protein is expressed, and the second nucleic acid molecule encodes a second fusion protein comprising a binding domain and a second multimerization domain, wherein the second fusion protein localizes extracellularly when expressed; and   (b) administering a bridging factor, wherein the bridging factor promotes the formation of a polypeptide complex on the recombinant cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins;   wherein the binding domain of the polypeptide complex specifically binds a cell surface target on a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease cell to promote an immunomodulatory response and thereby treats the hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease.   
     
     
         46 . A method for treating a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease, comprising:
 (a) administering a non-natural cell comprising a first nucleic acid molecule encoding a first fusion protein comprising a first multimerization domain, a hydrophobic domain, and an actuator domain, wherein the first multimerization domain localizes extracellularly when the first fusion protein is expressed;   (b) administering a second fusion protein comprising a binding domain and a second multimerization domain; and   (c) administering a bridging factor, wherein the bridging factor promotes the formation of a polypeptide complex on the recombinant cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins;   wherein the binding domain of the polypeptide complex specifically binds a cell surface target on a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease cell to promote an immunomodulatory response and thereby treats the hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease.   
     
     
         47 . The method according to  claim 45  or  46 , wherein the first and second multimerization domains are the same or different. 
     
     
         48 . The method according to any one of  claims 45 - 47 , wherein the multimerization domains of the first and second fusion proteins associate with a bridging factor selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof. 
     
     
         49 . The method according to any one of  claims 45 - 48 , wherein the first and second multimerization domains are a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. 
     
     
         50 . The method according to any one of  claims 45 - 49 , wherein the first multimerization domain comprises a first FKBP polypeptide or variant thereof, and the second multimerization domain comprises a first FRB polypeptide or variant thereof. 
     
     
         51 . The method according to any one of  claims 45 - 50 , wherein the first multimerization domain comprises a first FRB polypeptide or variant thereof, and the second multimerization domain comprises a first FKBP polypeptide or variant thereof. 
     
     
         52 . The method according to  claim 50  or  51 , wherein the bridging factor is sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. 
     
     
         53 . The method according to any one of  claims 45 - 52 , wherein the first nucleic acid molecule encodes a first fusion protein further comprising a third multimerization domain. 
     
     
         54 . The method according to  claim 53 , wherein the third multimerization domain of the first fusion protein is a binding domain for a bridging factor selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, Tmp-SLF or a derivative thereof, or any combination thereof. 
     
     
         55 . The method according to any one of  claims 45 - 54 , wherein a second bridging factor promotes the association of at least two first fusion proteins with the bridging factor associated with and disposed between the third multimerization domains of the first fusion proteins. 
     
     
         56 . The method according to any one of  claims 45 - 55 , wherein the protein complex is a homocomplex comprising at least two first fusion proteins. 
     
     
         57 . The method according to any one of  claims 45 - 56 , wherein the first fusion protein has at least one multimerization domain of FKBP, DHFR or GyrB. 
     
     
         58 . The method according to any one of  claims 45 - 57 , wherein the binding domain of the polypeptide complex specifically binds to a target located on a target hyperproliferative disease cell surface. 
     
     
         59 . The method according to  claim 58 , wherein the protein complex is a heterocomplex comprising one or more first fusion proteins and one or more second fusion proteins. 
     
     
         60 . The method according to  claim 59 , wherein the binding domain of the protein heterocomplex specifically binds to a target located on a target hyperproliferative disease cell surface. 
     
     
         61 . The method according to any one of  claims 45 - 60 , wherein the hydrophobic domain is a transmembrane domain. 
     
     
         62 . The method according to any one of  claims 45 - 61 , wherein the transmembrane domain is a CD4, CD8 or CD28 transmembrane domain. 
     
     
         63 . The method according to any one of  claims 45 - 62 , wherein the actuator domain comprises a lymphocyte receptor signaling domain. 
     
     
         64 . The method according to any one of  claims 45 - 63 , wherein the actuator domain comprises a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs). 
     
     
         65 . The method according to any one of  claims 45 - 64 , wherein the actuator domain comprises CD3ε, CD3δ, CD3ζ, pTα, TCRα, TCRβ, FcRα, FcRβ, FcRγ, NKG2D, CD22, CD79A, or CD79B, or any combination thereof. 
     
     
         66 . The method according to any one of  claims 45 - 65 , wherein the first nucleic acid molecule encodes the first fusion protein further comprising a different actuator domain, a costimulatory domain, an adhesion factor, or any combination thereof. 
     
     
         67 . The method according to  claim 66 , wherein the costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any combination thereof. 
     
     
         68 . The method according to any one of  claims 45 - 67 , wherein the actuator domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein. 
     
     
         69 . The method according to  claim 68 , wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein comprising one or a plurality of immunoreceptor tyrosine-based activation motifs (ITAMs), a costimulatory domain, an adhesion factor, or any combination thereof. 
     
     
         70 . The method according to  claim 69 , wherein the lymphocyte receptor or signaling domain thereof is CD3ε, CD3,δ, CD3ζ, pTα, TCRα, TCRβ, FcRα, FcRβ, FcRγNKG2D, CD22, CD79A, or CD79B, or any combination thereof. 
     
     
         71 . The method according to  claim 69 , wherein the costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any combination thereof. 
     
     
         72 . The method according to  claim 69 , wherein the cytoplasmic signaling protein is combination of CD3ζ and 4-1BB or a combination of CD3t and OX40. 
     
     
         73 . The method according to any one of  claims 45 - 72 , wherein the non-natural cell is further overexpressing a costimulatory factor, an immunomodulatoy factor, an agonist for a costimulatory factor, an agonist for an immunomodulatoy factor, or any combination thereof. 
     
     
         74 . The method according to any one of  claims 45 - 73 , wherein the binding domain of the second fusion protein is a single chain antibody variable region, a receptor ectodomain, or a ligand. 
     
     
         75 . The method according to  claim 74 , wherein the single chain antibody variable region is a domain antibody, sFv, scFv, F(ab′)2, or Fab. 
     
     
         76 . The method according to any one of  claims 45 - 75 , wherein the binding domain of the second fusion protein is amino terminal to the multimerization domain. 
     
     
         77 . The method according to any one of  claims 45 - 76 , wherein the binding domain of the second fusion protein is carboxy terminal to the multimerization domain. 
     
     
         78 . The method according to any one of  claims 45 - 77 , wherein the second fusion protein further comprises a linker disposed between the binding domain and the second multimerization domain. 
     
     
         79 . The method according to any one of  claims 45 - 78 , wherein the cell further comprises a third nucleic acid molecule encoding a third fusion protein comprising a binding domain and a second multimerization domain, wherein the third fusion protein localizes extracellularly when expressed. 
     
     
         80 . The method according to any one of  claims 45 - 76 , wherein the fusion proteins comprising a binding domain have one, two, three, or four binding domains. 
     
     
         81 . The method according to any one of  claims 45 - 80 , wherein the one, two, three, or four binding domains are specific for one target or up to four different targets. 
     
     
         82 . The method according to any one of  claims 45 - 81 , wherein the binding domain is specific for a target that is an antigen associated with a cancer. 
     
     
         83 . The method according to  claim 82 , wherein the cancer is a solid malignancy or a hematologic malignancy. 
     
     
         84 . The method according to  claim 83 , wherein the hematologic malignancy associated antigen target is CD19, CD20, CD22, CD33, or CD37. 
     
     
         85 . The method according to any one of  claims 45 - 84 , wherein the binding domain specifically binds to a target selected from α-folate receptor, α v β 6  integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11Rα, IL13R-α2, KDR, κ light chain, λ light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notchl, Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2, and ROR1. 
     
     
         86 . The method according to any one of  claims 45 - 85 , wherein the first bridging factor is rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, or Tmp-SLF or a derivative thereof. 
     
     
         87 . The method according to  claim 55 , wherein the second bridging factor is rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, or Tmp-SLF or a derivative thereof. 
     
     
         88 . The method according to any one of  claims 45 - 87 , wherein the first fusion protein comprises a first multimerization domain of FRB T2098L, a transmembrane domain, a costimulatory domain of 4-1BB, and actuator domain of CD3ζ;wherein the second fusion protein comprises a binding domain of an scFv specific for CD19 and a second multimerization domain of FKBP12; and wherein the first bridging factor that promotes the formation of a polypeptide complex on the non-natural cell surface is rapalog AP21967. 
     
     
         89 . The method according to  claim 88 , wherein the first fusion protein has an amino acid sequence as set forth in SEQ ID NO.:15 and the second fusion protein has an amino acid sequence as set forth in SEQ ID NO.:1. 
     
     
         90 . The method according to any one of  claims 45 - 89 , wherein the method further comprises administering an agent that antagonizes or blocks an inhibitor of T-cell activation. 
     
     
         91 . The method according to  claim 90 , wherein the agent antagonizes or blocks a T-cell ligand. 
     
     
         92 . The method according to  claim 90 , wherein the agent antagonizes or blocks a T-cell receptor. 
     
     
         93 . The method according to any one of  claims 90 - 92 , wherein the agent that antagonizes or blocks an inhibitor of T-cell activation is an anti-PD1 antibody or antigen binding fragment thereof, anti-PD-L1 antibody or antigen binding fragment thereof, or an anti-CTLA4 antibody or antigen binding fragment thereof or an engineered homing endonuclease that targets PD-1. 
     
     
         94 . The method according to any one of  claims 45 - 93 , wherein the method further comprises administering a cytokine agonist. 
     
     
         95 . A fusion polypeptide heterocomplex, comprising:
 (a) a first fusion protein comprising a first multimerization domain, a hydrophobic domain, and an actuator domain;   (b) a second fusion protein comprising an extracellular binding domain and second multimerization domain; and   (c) a bridging factor;   wherein the first fusion protein, second fusion protein, and bridging factor associate to form a polypeptide heterocomplex with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins.   
     
     
         96 . The polypeptide heterocomplex according to  claim 95 , wherein the binding domain is a single chain antibody variable region, a receptor ectodomain, or a ligand. 
     
     
         97 . The polypeptide heterocomplex according to  claim 96 , wherein the single chain antibody variable region is a domain antibody, sFv, scFv, F(ab′) 2 , or Fab. 
     
     
         98 . The polypeptide heterocomplex according to any one of  claims 95 - 97 , wherein the binding domain is amino terminal to the multimerization domain. 
     
     
         99 . The polypeptide heterocomplex according to any one of  claims 95 - 97 , wherein the binding domain is carboxy terminal to the multimerization domain. 
     
     
         100 . The polypeptide heterocomplex according to any one of  claims 95 - 99 , wherein the first multimerization domain comprises a first FKBP polypeptide or variant thereof, and the second multimerization domain comprises a first FRB polypeptide or variant thereof. 
     
     
         101 . The polypeptide heterocomplex according to any one of  claims 95 - 99 , wherein the first multimerization domain comprises a first FRB polypeptide or variant thereof, and the second multimerization domain comprises a first FKBP polypeptide or variant thereof. 
     
     
         102 . The polypeptide heterocomplex according to any one of  claims 95 - 101 , wherein the hydrophobic domain is a transmembrane domain. 
     
     
         103 . The polypeptide heterocomplex according to any one of  claims 95 - 102 , wherein the actuator domain comprises a lymphocyte receptor chain. 
     
     
         104 . The polypeptide heterocomplex according to any one of  claims 95 - 103 , wherein the bridging factor is rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, or Tmp-SLF or a derivative thereof. 
     
     
         105 . The polypeptide heterocomplex according to any one of  claims 95 - 104 , wherein the second fusion protein further comprises an anchor domain. 
     
     
         106 . The polypeptide heterocomplex according to  claim 105 , wherein the anchor domain is a transmembrane domain. 
     
     
         107 . The polypeptide heterocomplex according to  claim 105  or  106 , wherein the second fusion protein further comprises a sub-threshold signaling domain. 
     
     
         108 . The polypeptide heterocomplex according to  claim 105 , wherein the anchor domain is a GPI signal sequence. 
     
     
         109 . The polypeptide heterocomplex according to  claim 105 , wherein the GPI signal sequence has been altered and the second fusion protein further comprises a GPI molecule. 
     
     
         110 . The polypeptide heterocomplex according to any one of  claims 95 - 109 , wherein the binding domain is specific for a target that is an antigen associated with a cancer, an inflammatory disease, an autoimmune disease, or a graft versus host disease. 
     
     
         111 . The polypeptide heterocomplex according to  claim 110 , wherein the cancer is a hematologic malignancy having an antigen target of CD19, CD20, CD22, CD33, or CD37. 
     
     
         112 . A nucleic acid molecule encoding any one or more of the fusion proteins according to any one of  claim 1 - 44  or  95 - 109 . 
     
     
         113 . The nucleic acid molecule of  claim 112 , wherein the nucleic acid molecule is disposed between 5′ and 3′ polynucleotide sequences homologous to a genomic locus. 
     
     
         114 . An expression vector containing a nucleic acid according to  claim 112  or  claim 113 . 
     
     
         115 . The expression vector according to  claim 114 , wherein the first and second fusion proteins are encoded as a polycistronic message or as a single protein separated by a 2A peptide. 
     
     
         116 . The expression vector according to  claim 115 , wherein the polycistronic message comprises an internal ribosome entry site (IRES) between the nucleotide sequences that encode the fusion proteins. 
     
     
         117 . A non-natural cell, comprising:
 (a) a first nucleic acid molecule encoding a first fusion protein comprising a binding domain that binds a receptor on a T cell and a first multimerization domain, wherein the first fusion protein is secreted from the cell; and   (b) a second nucleic acid molecule encoding a second fusion protein comprising a binding domain that binds a target located on a target cell surface and a second multimerization domain, wherein the second fusion protein is secreted from the cell;   wherein a bridging factor promotes the formation of a polypeptide complex with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins.   
     
     
         118 . The non-natural cell according to  claim 117 , wherein the first and second multimerization domains are the same or different. 
     
     
         119 . The non-natural cell according to  claim 117  or  claim 118 , wherein the multimerization domains of the first and second fusion proteins associate with a bridging factor selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof. 
     
     
         120 . The non-natural cell according to any one of  claims 117 - 119 , wherein the first and second multimerization domains are a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. 
     
     
         121 . The non-natural cell according to any one of  claims 117 - 120 , wherein the first multimerization domain comprises a first FKBP polypeptide or variant thereof, and the second multimerization domain comprises a first FRB polypeptide or variant thereof. 
     
     
         122 . The non-natural cell according to any one of  claims 117 - 120 , wherein the first multimerization domain comprises a first FRB polypeptide or variant thereof, and the second multimerization domain comprises a first FKBP polypeptide or variant thereof. 
     
     
         123 . The non-natural cell according to  claim 121  or  claim 122 , wherein the bridging factor is sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. 
     
     
         124 . The non-natural cell according to any one of  claims 117 - 123 , further overexpressing a costimulatory factor, an immunomodulatoy factor, an agonist for a costimulatory factor, an agonist for an immunomodulatoy factor, or any combination thereof. 
     
     
         125 . The non-natural cell according to any one of  claims 117 - 124 , wherein the binding domain of the first fusion protein and the binding domain of the second fusion protein are each independently selected from the group consisting of: a single chain antibody variable region, a receptor ectodomain, or a ligand. 
     
     
         126 . The non-natural cell according to  claim 125 , wherein the single chain antibody variable region is a domain antibody, sFv, scFv, F(ab′) 2 , or Fab. 
     
     
         127 . The non-natural cell according to any one of  claims 117 - 126 , wherein the binding domain of the first fusion protein is amino terminal to the first multimerization domain. 
     
     
         128 . The non-natural cell according to any one of  claims 117 - 126 , wherein the binding domain of the first fusion protein is carboxy terminal to the first multimerization domain. 
     
     
         129 . The non-natural cell according to any one of  claims 117 - 126 , wherein the binding domain of the second fusion protein is amino terminal to the second multimerization domain. 
     
     
         130 . The non-natural cell according to any one of  claims 117 - 126 , wherein the binding domain of the second fusion protein is carboxy terminal to the second multimerization domain. 
     
     
         131 . The non-natural cell according to any one of  claims 117 - 130 , wherein the first nucleic acid molecule encoding the first fusion protein further comprises a sequence encoding a linker disposed between the binding domain and the first multimerization domain. 
     
     
         132 . The non-natural cell according to any one of  claims 117 - 130 , wherein the second nucleic acid molecule encoding the second fusion protein further comprises a sequence encoding a linker disposed between the binding domain and the second multimerization domain. 
     
     
         133 . The non-natural cell according to any one of  claims 117 - 132 , wherein the binding domain of the second nucleic acid molecule is specific for a target that is an antigen associated with a cancer, an inflammatory disease, an autoimmune disease, or a graft versus host disease. 
     
     
         134 . The non-natural cell according to  claim 133 , wherein the cancer is a solid malignancy or a hematologic malignancy. 
     
     
         135 . The non-natural cell according to  claim 134 , wherein the hematologic malignancy associated antigen target is CD19, CD20, CD22, CD33, or CD37. 
     
     
         136 . The non-natural cell according to any one of  claims 117 - 132 , wherein the binding domain of the second nucleic acid molecule specifically binds to a target selected from a-folate receptor, α v β 6  integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11Rα, IL13R-α2, KDR, κ light chain, λ light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notchl, Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2, and ROR1. 
     
     
         137 . The non-natural cell according to any one of  claims 117 - 136 , wherein the bridging factor is rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, or Tmp-SLF or a derivative thereof. 
     
     
         138 . The non-natural cell according to any one of  claims 117 - 137 , wherein the first nucleic acid encodes a first fusion protein comprising a binding domain of an scFv specific for CD3 and a first multimerization domain of FRB T2098L; wherein the second nucleic acid encodes a second fusion protein comprising a binding domain of an scFv specific for CD19 and a second multimerization domain of FKBP12; and wherein the bridging factor that promotes the formation of a polypeptide complex is rapalog AP21967. 
     
     
         139 . The non-natural cell according to any one of  claims 117 - 137 , wherein the first nucleic acid encodes a first fusion protein comprising a binding domain of an scFv specific for CD3 and a first multimerization domain of FRB T2098L; wherein the second nucleic acid encodes a second fusion protein comprising a binding domain of an scFv specific for BCMA and a second multimerization domain of FKBP12; and wherein the bridging factor that promotes the formation of a polypeptide complex is rapalog AP21967. 
     
     
         140 . A method for treating a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease, comprising administering a non-natural cell according to any one of  claims 117 - 139  and administering a bridging factor, wherein the bridging factor promotes the formation of a polypeptide complex with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins; wherein the binding domain of the second fusion polypeptide specifically binds a cell surface target on a hyperproliferative disease cell to promote an immunomodulatory response and thereby treats the hyperproliferative disease. 
     
     
         141 . A method for treating a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease, comprising:
 (a) administering a first fusion protein comprising a binding domain that binds a receptor on a T cell and a first multimerization domain; and a second fusion protein comprising a binding domain that binds a cell surface target on a hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease cell and a second multimerization domain; and   (b) administering a bridging factor that promotes the formation of a polypeptide complex with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins;   thereby treating the hyperproliferative, inflammatory, autoimmune, or graft-versus-host disease.   
     
     
         142 . A fusion polypeptide heterocomplex, comprising:
 (a) a first fusion protein comprising a binding domain that binds a receptor on a T cell and a first multimerization domain;   (b) a second fusion protein comprising a binding domain that binds a cell surface target on a target cell; and   (c) a bridging factor;   wherein the first fusion protein, second fusion protein, and bridging factor associate to form a polypeptide heterocomplex with the bridging factor associated with and disposed between the multimerization domains of the first and second fusion proteins.   
     
     
         143 . The fusion polypeptide heterocomplex according to  claim 142 , wherein the first and second multimerization domains are the same or different. 
     
     
         144 . The fusion polypeptide heterocomplex according to  claim 142  or  claim 143 , wherein the multimerization domains of the first and second fusion proteins associate with a bridging factor selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or any combination thereof. 
     
     
         145 . The fusion polypeptide heterocomplex according to any one of  claims 142 - 144 , wherein the first and second multimerization domains are a pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. 
     
     
         146 . The fusion polypeptide heterocomplex according to any one of  claims 142 - 145 , wherein the first multimerization domain comprises a first FKBP polypeptide or variant thereof, and the second multimerization domain comprises a first FRB polypeptide or variant thereof. 
     
     
         147 . The fusion polypeptide heterocomplex according to any one of  claims 142 - 145 , wherein the first multimerization domain comprises a first FRB polypeptide or variant thereof, and the second multimerization domain comprises a first FKBP polypeptide or variant thereof. 
     
     
         148 . The fusion polypeptide heterocomplex according to any one of  claims 146  or  claim 147 , wherein the bridging factor is sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus. 
     
     
         149 . The fusion polypeptide heterocomplex according to any one of  claims 142 - 148 , wherein the binding domain of the first fusion protein and the binding domain of the second fusion protein are each independently selected from the group consisting of: a single chain antibody variable region, a receptor ectodomain, or a ligand. 
     
     
         150 . The fusion polypeptide heterocomplex according to  claim 149 , wherein the single chain antibody variable region is a domain antibody, sFv, scFv, F(ab′) 2 , or Fab. 
     
     
         151 . The fusion polypeptide heterocomplex according to any one of  claims 142 - 150 , wherein the binding domain of the second fusion polypeptide specifically binds to a target selected from a-folate receptor, 46 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11Rα, IL13R-α2, KDR, κ light chain, λ light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notchl, Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2, and ROR1. 
     
     
         152 . The fusion polypeptide heterocomplex according to any one of  claims 141 - 150 , wherein the first fusion protein comprises a binding domain of an scFv specific for CD3 and a first multimerization domain of FRB T2098L; wherein the second fusion protein comprises a binding domain of an scFv specific for CD19 and a second multimerization domain of FKBP12;
 and wherein the bridging factor is rapalog AP21967.   
     
     
         153 . The fusion polypeptide heterocomplex according to any one of  claims 141 - 150 , wherein the first fusion protein comprises a binding domain of an scFv specific for CD3 and a first multimerization domain of FRB T2098L; wherein the second fusion protein comprises a binding domain of an scFv specific for BCMA and a second multimerization domain of FKBP12; and wherein the bridging factor is rapalog AP21967. 
     
     
         154 . A nucleic acid molecule encoding any one or more of the fusion proteins according to any one of  claims 117 - 153 . 
     
     
         155 . The nucleic acid molecule of  claim 154 , wherein the nucleic acid molecule is disposed between 5′ and 3′ polynucleotide sequences homologous to a genomic locus. 
     
     
         156 . An expression vector containing a nucleic acid according to  claim 154  or  claim 155 . 
     
     
         157 . The expression vector according to  claim 156 , wherein the first and second fusion proteins are encoded as a polycistronic message or as a single protein separated by a 2A peptide. 
     
     
         158 . The expression vector according to  claim 157 , wherein the polycistronic message comprises an internal ribosome entry site (IRES) between the nucleotide sequences that encode the fusion proteins.