WO2020123938A1 - Dimerizing agent regulated immunoreceptor complexes - Google Patents

Dimerizing agent regulated immunoreceptor complexes Download PDF

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Publication number
WO2020123938A1
WO2020123938A1 PCT/US2019/066223 US2019066223W WO2020123938A1 WO 2020123938 A1 WO2020123938 A1 WO 2020123938A1 US 2019066223 W US2019066223 W US 2019066223W WO 2020123938 A1 WO2020123938 A1 WO 2020123938A1
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WIPO (PCT)
Prior art keywords
domain
polypeptide
antibody
binding fragment
antigen binding
Prior art date
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Ceased
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PCT/US2019/066223
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English (en)
French (fr)
Inventor
Jordan JARJOUR
Alexander ASTRAKHAN
Wai-Hang LEUNG
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Bluebird Bio Inc
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Bluebird Bio Inc
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Filing date
Publication date
Priority to US17/312,120 priority Critical patent/US20220031750A1/en
Priority to CN201980091143.9A priority patent/CN113366479A/zh
Priority to AU2019396553A priority patent/AU2019396553A1/en
Priority to KR1020217021631A priority patent/KR20210104772A/ko
Priority to CA3123084A priority patent/CA3123084A1/en
Priority to JP2021533530A priority patent/JP7636650B2/ja
Application filed by Bluebird Bio Inc filed Critical Bluebird Bio Inc
Priority to EP19896568.3A priority patent/EP3895052A4/en
Publication of WO2020123938A1 publication Critical patent/WO2020123938A1/en
Priority to IL283897A priority patent/IL283897A/en
Anticipated expiration legal-status Critical
Priority to JP2024188998A priority patent/JP2025010249A/ja
Ceased legal-status Critical Current

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Definitions

  • the present disclosure relates to improved adoptive cell therapies. More particularly, the disclosure relates to improved chemically regulated signaling molecules, cells, and methods of using the same for modulating spatial and temporal control of cellular signal initiation and downstream responses during adoptive immunotherapy.
  • Cancer The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer related deaths in 2012.
  • the most common cancers are breast cancer, lung and bronchus cancer, prostate cancer, colon and rectum cancer, bladder cancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer.
  • the number of new cancer cases is projected to rise to 23.6 million by 2030.
  • adoptive cell therapies have the potential to execute unique therapeutic tasks owing to their myriad sensory and response programs and increasingly defined
  • cells need to be outfitted with machinery for sensing and integrating chemical and/or biological information associated with local physiological environments.
  • the present disclosure generally relates, in part, to dimerizing agent regulated immunoreceptor complex (DARIC) compositions, polynucleotides, polypeptides and methods of making and using the same.
  • DARIC dimerizing agent regulated immunoreceptor complex
  • a non-natural cell comprises a first polypeptide
  • a 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 first and second multimerization domains.
  • the first and second multimerization domains are different.
  • the first and second costimulatory domains are different.
  • multimerization domain associate with a bridging factor selected from the group consisting of: 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, FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof.
  • a bridging factor selected from the group consisting of: 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, FK506/cyclosporin A (FKCs
  • the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of: FKBP and FKBP-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
  • the first multimerization domain comprises an FKBP polypeptide or variant thereof
  • the second multimerization domain comprises an FRB polypeptide or variant thereof.
  • the first multimerization domain comprises an FRB polypeptide or variant thereof
  • the second multimerization domain comprises an FKBP polypeptide or variant thereof.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the first and second multimerization domains are selected from FRB T2098L and FKBP12; and the bridging factor is sirolimus or AP21967.
  • transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35,
  • APN amnionless
  • PDCD1 programmed cell death 1
  • transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain, a CD8a transmembrane domain, and an AMN transmembrane domain.
  • the first transmembrane domain and the second transmembrane domain are different.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the first and second costimulatory domain are independently selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14,
  • TLR1 Toll-like receptor 1
  • TLR2 TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 caspas
  • TNFRS18 TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
  • the first costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137 and the second costimulatory domain is isolated from CD28, CD278, TNFRS14,
  • TNFRS18 TNFRS25, 0X40 or TNFR2.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from 0X40 or TNFR2.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from 0X40.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from TNFR2.
  • the primary signaling domain is isolated from a O ⁇ 3z polypeptide.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof, a receptor ectodomain, or a ligand.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific
  • the extracellular binding domain comprises a humanized antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises a human antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises an scFv.
  • the extracellular binding domain comprises one or more camelid VHH antibodies.
  • the extracellular binding domain binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the extracellular binding domain binds an antigen selected from the group consisting of: alpha folate receptor (FRa), a n b6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), claudin 6, (CLDN6), claudin 18 isoform 2 (CLDN18.2), C-type lectin-like molecule- 1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T cell lymphoma- associated antigen 1 (CTAGE1), delta like canonical Notch ligand 3 (FRa), B
  • PLAC1 preferentially expressed antigen in melanoma
  • PRAME prostate stem cell antigen
  • PSCA prostate-specific membrane antigen
  • ROR1 receptor tyrosine kinase-like orphan receptor 1
  • SSX2 synovial sarcoma
  • TAG72 tumor associated glycoprotein 72
  • TEM1/CD248 tumor endothelial marker 1
  • TEM7R tumor endothelial marker 7-related
  • TPBG trophoblast glycoprotein
  • ULBP UL 16-binding protein
  • ULBP2 ULBP2
  • ULBP3 ULBP4
  • ULBP5 ULBP6, vascular endothelial growth factor receptor 2
  • WT-1 Wilms tumor 1
  • a non-natural cell comprises a first polypeptide
  • FKBP FK506 binding protein
  • FKBP-rapamycin binding domain polypeptide or variant thereof a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP-rapamycin binding (FRB) multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain; wherein a 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 polypeptides.
  • FKBP FK506 binding protein
  • a non-natural cell comprises a first polypeptide comprising: an FKBP multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain; wherein a 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
  • a non-natural cell comprises a first polypeptide
  • an FRB multimerization domain polypeptide or variant thereof comprising: an FRB multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain; wherein a 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
  • a non-natural cell comprises a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain; wherein a 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
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the FRB multimerization domain is FRB T2098L; the FKBP multimerization domain is FKBP12; and the bridging factor is sirolimus or
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35,
  • transmembrane domain are independently selected from a polypeptide selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DN AX- Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain- Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10
  • caspase recruitment domain family member 11 CD2, CD7, CD27, CD28, CD30
  • transmembrane adaptor 1 (TRATl), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
  • the costimulatory domain is isolated from a
  • costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.
  • the costimulatory domain is isolated from a CD137 costimulatory molecule.
  • the primary signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcRP, CD3y, CD35, CD3e, C/D3 z, CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is isolated from a CD3z polypeptide.
  • the antibody or antigen binding fragment thereof selected is from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • the antibody or antigen binding fragment thereof is human or humanized.
  • the antibody or antigen binding fragment thereof comprises an scFv or one or more camelid VHH antibodies.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: FRa, a n b6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6,
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • the antibody or antigen binding fragment thereof binds BCMA.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD20 or CD22.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD79A.
  • the antibody or antigen binding fragment thereof binds CD79B.
  • the antibody or antigen binding fragment thereof binds EGFRvIII.
  • a non-natural cell comprises a first polypeptide comprising: an FKBP12 multimerization domain polypeptide or variant thereof; a CD8a
  • transmembrane domain a CD 137 costimulatory domain; and a CD3z primary signaling domain
  • a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB T2098L multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and a TNFR2 costimulatory domain; wherein a 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 polypeptides.
  • a non-natural cell comprises a first polypeptide comprising: an FKBP12 multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a O ⁇ 3z primary signaling domain; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB T2098L multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and an 0X40 costimulatory domain; wherein a 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 polypeptides.
  • a non-natural cell comprises a first polypeptide
  • a 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 polypeptides.
  • a non-natural cell comprises a first polypeptide
  • a 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 polypeptides.
  • the bridging factor is AP21967 or sirolimus.
  • the antibody or antigen binding fragment thereof selected is from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific Fab dimer
  • the antibody or antigen binding fragment thereof is human or humanized.
  • the antibody or antigen binding fragment thereof comprises an scFv or one or more camelid VHH antibodies.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: FRa, a n b6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6,
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD20 or CD22.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD79A.
  • the antibody or antigen binding fragment thereof binds CD79B.
  • the antibody or antigen binding fragment thereof binds EGFRvIII, optionally wherein the antibody is EGFR806 or an antigen binding fragment thereof.
  • the multimerization domains localize extracellularly when of the first polypeptide and the second polypeptide are expressed.
  • the cell is a hematopoietic cell.
  • the cell is a T cell.
  • the cell is a CD3+, CD4+, and/or CD8+ cell.
  • the cell is an immune effector cell.
  • the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.
  • CTL cytotoxic T lymphocyte
  • TIL tumor infiltrating lymphocyte
  • helper T cell a helper T cell
  • the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
  • NK natural killer
  • NKT natural killer T
  • the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
  • a fusion polypeptide comprises a first polypeptide comprising: a first multimerization domain polypeptide or variant thereof; a first transmembrane domain; a first costimulatory domain; and/or a primary signaling domain; and a polypeptide cleavage signal; and a second polypeptide comprising: an extracellular binding domain; a second multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a second costimulatory domain.
  • the first and second multimerization domains are different.
  • the first and second costimulatory domains are different.
  • the first multimerization domain and the second multimerization domain associate with a bridging factor selected from the group consisting of: 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, FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof.
  • a bridging factor selected from the group consisting of: 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, FK506/
  • multimerization domain are a pair selected from the group consisting of: FKBP and FKBP-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
  • the first multimerization domain comprises an FKBP polypeptide or variant thereof
  • the second multimerization domain comprises an FRB polypeptide or variant thereof.
  • the first multimerization domain comprises an FRB polypeptide or variant thereof
  • the second multimerization domain comprises an FKBP polypeptide or variant thereof.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the first and second multimerization domains are selected from FRB T2098L and FKBP12; and the bridging factor is sirolimus or AP21967.
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35,
  • APN amnionless
  • PDCD1 programmed cell death 1
  • the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain, a CD8a transmembrane domain, and an AMN transmembrane domain.
  • the first transmembrane domain and the second transmembrane domain are different.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the first and second costimulatory domain are identical to each other.
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARDl l), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14,
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARDl l)
  • TNFRS18 TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
  • the first costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137 and the second costimulatory domain is isolated from CD28, CD278, TNFRS14,
  • TNFRS18 TNFRS25, 0X40 or TNFR2.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from 0X40 or TNFR2.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from 0X40.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from TNFR2.
  • the primary signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcRP, CD3y, CD35, CD3e,
  • the primary signaling domain is isolated from a O ⁇ 3z polypeptide.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof, a receptor ectodomain, or a ligand.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific
  • the extracellular binding domain comprises a humanized antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises a human antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises an scFv.
  • the extracellular binding domain comprises one or more camelid VHH antibodies.
  • the extracellular binding domain binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the extracellular binding domain binds an antigen selected from the group consisting of: FRa, a n b 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS- 1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-lORa, IL-13Ra2, Kappa, LAGE-1A, Lambda, LeY, Ll-CAM, MAGE-A1, MAGE- A3, MAGE-A4, MAGE-A6, MAGE
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4,
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • a fusion polypeptide comprises a first polypeptide comprising: a first multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an extracellular binding domain; a second multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain.
  • a fusion polypeptide comprises a first polypeptide comprising: a first multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an extracellular binding domain; a second multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain.
  • the first and second multimerization domains are the same.
  • the first and second multimerization domains are different.
  • the first multimerization domain and the second multimerization domain associate with a bridging factor selected from the group consisting of: 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, and SLF or a derivative thereof.
  • a bridging factor selected from the group consisting of: 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, and SLF or a derivative thereof.
  • the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of: FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and DHFR, calcineurin and cyclophilin, and PYL1 and ABI1.
  • the first multimerization domain comprises an FKBP polypeptide or variant thereof
  • the second multimerization domain comprises an FRB polypeptide or variant thereof.
  • the first multimerization domain comprises an FRB polypeptide or variant thereof
  • the second multimerization domain comprises an FKBP polypeptide or variant thereof.
  • transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35,
  • transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the first transmembrane domain and the second transmembrane domain are the same.
  • the first transmembrane domain and the second transmembrane domain are different.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRATl, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.
  • a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP
  • the costimulatory domain is isolated from a
  • costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.
  • the costimulatory domain is isolated from a CD137 costimulatory molecule.
  • the primary signaling domain is isolated from a O ⁇ 3z polypeptide.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof, a receptor ectodomain, or a ligand.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific
  • the extracellular binding domain comprises a humanized antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises a human antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises an scFv.
  • the extracellular binding domain comprises one or more camelid VHH antibodies.
  • the extracellular binding domain binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the extracellular binding domain binds an antigen selected from the group consisting of: FRa, a n b 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16,
  • CD 19 CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS- 1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-lORa, IL-13Ra2, Kappa, LAGE-1A, Lambda, LeY, Ll-CAM, MAGE-A1, MAGE- A3, MAGE-A4, MAGE-A6, MAGEA10, Mel an A or MARTI, SLN), MUC1, MUC16, MICA, MICB, NCAM, NY- ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Sur
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4,
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • a fusion polypeptide comprises a first polypeptide comprising: an FKBP multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain.
  • a fusion polypeptide comprises a first polypeptide comprising: an FKBP multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain.
  • a fusion polypeptide comprises a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain.
  • a fusion polypeptide comprises a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain.
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35,
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRATl, TNFR2, TNFRS14, TNFRS18, TNFRS25,and ZAP70.
  • a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP
  • the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.
  • the costimulatory domain is isolated from a CD137 costimulatory molecule.
  • the primary signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcRP, CD3y, CD35, CD3e, C/D3 z, CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is isolated from a CD3z polypeptide.
  • the antibody or antigen binding fragment thereof selected is from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • the antibody or antigen binding fragment thereof is human or humanized.
  • the antibody or antigen binding fragment thereof comprises an scFv or one or more camelid VHH antibodies.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: FRa, a n b6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6,
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • the antibody or antigen binding fragment thereof binds BCMA.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD20 or CD22.
  • the antibody or antigen binding fragment thereof binds B7- H3.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD79A.
  • the antibody or antigen binding fragment thereof binds CD79B.
  • the antibody or antigen binding fragment thereof binds EGFRvIII.
  • a fusion polypeptide comprises a first polypeptide comprising: an FKBP12 multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a O ⁇ 3z primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB T2098L multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and a TNFR2 costimulatory domain.
  • a fusion polypeptide comprises a first polypeptide comprising: an FKBP12 multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a CD3z primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB T2098L multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and an 0X40 costimulatory domain.
  • a fusion polypeptide comprises a first polypeptide comprising: an FRB T2098L multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a CD3z primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP12 multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and a TNFR2 costimulatory domain.
  • a fusion polypeptide comprises a first polypeptide comprising: an FRB T2098L multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a O ⁇ 3z primary signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP12 multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and an 0X40 costimulatory domain.
  • the antibody or antigen binding fragment thereof selected is from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific Fab dimer
  • the antibody or antigen binding fragment thereof is human or humanized.
  • the antibody or antigen binding fragment thereof comprises an scFv or one or more camelid VHH antibodies.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: FRa, a n b6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6,
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • the antibody or antigen binding fragment thereof binds BCMA.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD20 or CD22.
  • the antibody or antigen binding fragment thereof binds B7- H3.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD79A.
  • the antibody or antigen binding fragment thereof binds CD79B.
  • the antibody or antigen binding fragment thereof binds EGFRvIII, optionally wherein the antibody is EGFR806 or an antigen binding fragment thereof.
  • the multimerization domains localize extracellularly when of the first polypeptide and the second polypeptide are expressed.
  • the polypeptide cleavage signal is a viral self-cleaving polypeptide.
  • the polypeptide cleavage signal is a viral self-cleaving 2 A polypeptide.
  • the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus
  • FMDV FMDV
  • F2A F2A peptide
  • E2A equine rhinitis A virus
  • TaV T2A
  • PTV-1 porcine teschovirus-1
  • P2A Theilovirus 2A peptide
  • encephalomyocarditis virus 2A peptide an encephalomyocarditis virus 2A peptide.
  • a polynucleotide encodes a first or a second polypeptide or a fusion polypeptide contemplated herein.
  • a cDNA encodes a first or a second polypeptide or a fusion polypeptide contemplated herein.
  • an RNA encodes a first or a second polypeptide or a fusion polypeptide contemplated herein.
  • a vector comprises a polynucleotide contemplated herein.
  • the vector is an expression vector.
  • the vector is a transposon.
  • the vector is a piggyBAC transposon or a Sleeping Beauty transposon.
  • the vector is a viral vector.
  • the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector.
  • AAV adeno-associated viral
  • the retroviral vector is a lentiviral vector.
  • the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • a composition comprises a non-natural cell, a fusion polypeptide, a polynucleotide, or a vector contemplated herein.
  • a pharmaceutical composition comprises a
  • a method of treating a subject in need thereof comprises administering the subject an effective amount of a composition contemplated herein.
  • a method of treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith comprising administering to the subject an effective amount of a composition contemplated herein.
  • a method of treating a solid cancer comprises administering to the subject an effective amount of a composition contemplated herein.
  • the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
  • the solid cancer is a pancreatic cancer, a lung cancer, or a breast cancer.
  • a method of treating a hematological malignancy comprises administering to the subject an effective amount of a composition contemplated herein.
  • the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.
  • a polypeptide complex comprises a first polypeptide comprising: a first multimerization domain polypeptide or variant thereof; a first transmembrane domain; a first costimulatory domain; and/or a primary signaling domain; and a second polypeptide comprising: an extracellular binding domain; a second multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a second costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • the first and second multimerization domains are different.
  • the first and second costimulatory domains are different.
  • multimerization domain associate with a bridging factor selected from the group consisting of: 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, FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof.
  • a bridging factor selected from the group consisting of: 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, FK506/cyclosporin A (FKCs
  • the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of: FKBP and FKBP-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
  • the first multimerization domain comprises an FKBP polypeptide or variant thereof
  • the second multimerization domain comprises an FRB polypeptide or variant thereof.
  • the first multimerization domain comprises an FRB polypeptide or variant thereof
  • the second multimerization domain comprises an FKBP polypeptide or variant thereof.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the first and second multimerization domains are selected from FRB T2098L and FKBP12; and the bridging factor is sirolimus or AP21967.
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35,
  • APN amnionless
  • PDCD1 programmed cell death 1
  • the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain, a CD8a transmembrane domain, and an AMN transmembrane domain.
  • transmembrane domain are different.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the first and second costimulatory domain are identical to each other.
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARDl l), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14,
  • TLR1 Toll-like receptor 1
  • TLR2 TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARDl l)
  • TNFRS18 TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
  • the first costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137 and the second costimulatory domain is isolated from CD28, CD278, TNFRS14,
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from 0X40 or TNFR2.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from 0X40.
  • the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from TNFR2.
  • the primary signaling domain is isolated from a O ⁇ 3z polypeptide.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof, a receptor ectodomain, or a ligand.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific
  • the extracellular binding domain comprises a humanized antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises a human antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises an scFv.
  • the extracellular binding domain comprises one or more camelid VHH antibodies.
  • the extracellular binding domain binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the extracellular binding domain binds an antigen selected from the group consisting of: FRa, a n b6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL- 1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-lORa, IL-13Ra2, Kappa, LAGE-1A, Lambda, LeY, Ll-CAM, MAGE-A1, MAGE- A3, MAGE-A4, MAGE- A6, MAGEA
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4,
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • a polypeptide complex comprises a first polypeptide comprising: a first multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a second polypeptide comprising: an extracellular binding domain; a second multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a polypeptide complex comprises a first polypeptide comprising: a first multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a second polypeptide comprising: an extracellular binding domain; a second multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • the first and second multimerization domains are the same.
  • the first and second multimerization domains are different.
  • the first multimerization domain and the second multimerization domain associate with a bridging factor selected from the group consisting of: 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, and SLF or a derivative thereof.
  • a bridging factor selected from the group consisting of: 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, and SLF or a derivative thereof.
  • the first multimerization domain and the second multimerization domain are a pair selected from the group consisting of: FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and DHFR, calcineurin and cyclophilin, and PYL1 and ABI1.
  • the first multimerization domain comprises an FKBP polypeptide or variant thereof
  • the second multimerization domain comprises an FRB polypeptide or variant thereof.
  • the first multimerization domain comprises an FRB polypeptide or variant thereof
  • the second multimerization domain comprises an FKBP polypeptide or variant thereof.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the first and second multimerization domains are selected from FRB T2098L and FKBP12; and the bridging factor is sirolimus or AP21967.
  • transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35, CD3e, CD3y, O ⁇ 3z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PDCDl.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35, CD3e, CD3y, O ⁇ 3z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PDCDl.
  • transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • first transmembrane domain and the second transmembrane domain are the same.
  • the first transmembrane domain and the second transmembrane domain are different.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRATl, TNFR2, and ZAP70.
  • a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRATl, TN
  • the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.
  • the costimulatory domain is isolated from a CD137 costimulatory molecule.
  • the primary signaling domain is isolated from a CD3z polypeptide.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof, a receptor ectodomain, or a ligand.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific
  • the extracellular binding domain comprises a humanized antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises a human antibody or antigen binding fragment thereof.
  • the extracellular binding domain comprises an scFv.
  • the extracellular binding domain comprises one or more camelid VHH antibodies.
  • the extracellular binding domain binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the extracellular binding domain binds an antigen selected from the group consisting of: FRa, a n b 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS- 1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-lORa, IL-13Ra2, Kappa, LAGE-1A, Lambda, LeY, Ll-CAM, MAGE-A1, MAGE- A3, MAGE-A4, MAGE-A6, MAGE
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4,
  • the extracellular binding domain binds an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • a polypeptide complex comprises a first polypeptide comprising: an FKBP multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a polypeptide complex comprises a first polypeptide comprising: an FKBP multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a polypeptide complex comprises a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP multimerization domain polypeptide or variant thereof; a second transmembrane domain; and an 0X40 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a polypeptide complex comprises a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a first transmembrane domain; a costimulatory domain; and/or a primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP multimerization domain polypeptide or variant thereof; a second transmembrane domain; and a TNFR2 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the FRB multimerization domain is FRB T2098L; the FKBP multimerization domain is FKBP12; and the bridging factor is sirolimus or
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35, CD3e, CD3y, O ⁇ 3z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PDCDl.
  • a polypeptide selected from the group consisting of: alpha, beta, gamma, or delta chain of the T-cell receptor, CD35, CD3e, CD3y, O ⁇ 3z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PDCDl.
  • the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of: a CD4 transmembrane domain and a CD8a transmembrane domain.
  • the costimulatory domain and/or the primary signaling domain comprise an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM immunoreceptor tyrosine activation motif
  • the costimulatory signaling domaincostimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70.
  • a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DAP10, LAT, SLP
  • the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.
  • the costimulatory domain is isolated from a CD137 costimulatory molecule.
  • the primary signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcR-b, CD3y, CD35, CD3e,
  • CD3C CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain is isolated from a O ⁇ 3z polypeptide.
  • the antibody or antigen binding fragment thereof selected is from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific Fab dimer
  • the antibody or antigen binding fragment thereof is human or humanized.
  • the antibody or antigen binding fragment thereof comprises an scFv or one or more camelid VHH antibodies.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: FRa, a n bd integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6,
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII.
  • the antibody or antigen binding fragment thereof binds BCMA.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD20 or CD22.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD79A.
  • the antibody or antigen binding fragment thereof binds CD79B.
  • the antibody or antigen binding fragment thereof binds EGFRvIII.
  • a polypeptide complex comprises a first polypeptide comprising: an FKBP12 multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a CD3z primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB T2098L multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and a TNFR2 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a polypeptide complex comprises a first polypeptide comprising: an FKBP12 multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a O ⁇ 3z primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FRB T2098L multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and an 0X40 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a polypeptide complex comprises a first polypeptide comprising: an FRB T2098L multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a CD3z primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP12 multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and a TNFR2 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • a polypeptide complex comprises a first polypeptide comprising: an FRB T2098L multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain; a CD 137 costimulatory domain; and a CD3z primary signaling domain; a second polypeptide comprising: an antibody or antigen binding fragment thereof; an FKBP12 multimerization domain polypeptide or variant thereof; a CD4 transmembrane domain; and an 0X40 costimulatory domain; and a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
  • the bridging factor is AP21967 or sirolimus.
  • the antibody or antigen binding fragment thereof selected is from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab dimer
  • Fab3 bispecific Fab dimer
  • the antibody or antigen binding fragment thereof is human or humanized.
  • the antibody or antigen binding fragment thereof comprises an scFv or one or more camelid VHH antibodies.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: tumor associated antigens (TAA), tumor specific antigens (TSA), NKG2D ligands, gd T cell receptor (TCR) ligands, and ab TCR ligands.
  • TAA tumor associated antigens
  • TSA tumor specific antigens
  • NKG2D ligands NKG2D ligands
  • TCR gd T cell receptor
  • ab TCR ligands ab TCR ligands.
  • the antibody or antigen binding fragment thereof binds an antigen selected from the group consisting of: FRa, a n b6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6,
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the antibody or antigen binding fragment thereof binds BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and/or EGFRvIII. In additional embodiments, the antibody or antigen binding fragment thereof binds BCMA.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD20 or CD22.
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds
  • the antibody or antigen binding fragment thereof binds CD79A.
  • the antibody or antigen binding fragment thereof binds CD79B.
  • the antibody or antigen binding fragment thereof binds EGFRvIII, optionally wherein the antibody is EGFR806 or an antigen binding fragment thereof.
  • the multimerization domains localize extracellularly when of the first polypeptide and the second polypeptide are expressed.
  • Figure 1 shows a cartoon of DARIC architectures comprising DARIC binding components with a costimulatory domain.
  • Figure 2 A shows a cartoon of NKG2D DARIC architectures comprising DARIC binding components with various costimulatory domains.
  • Figure 2B shows NKG2D binding domain expression in the CD4 + gate for untransduced T cells, NKG2D DARIC T cells, NKG2D.TNFR2 DARIC T cells,
  • NKG2D.OX40 DARIC T cells NKG2D.CD27 DARIC T cells, NKG2D.HVEM DARIC T cells, NKG2D.DR3 DARIC T cells, and NKG2D.GITR DARIC T cells .
  • Figure 2C shows the growth kinetics of untransduced T cells, NKG2D DARIC T cells, NKG2D . TNFR2 DARIC T cells, NKG2D.OX40 DARIC T cells, NKG2D.CD27 DARIC T cells, NKG2D.HVEM DARIC T cells, NKG2D.DR3 DARIC T cells, and NKG2D.GITR DARIC T cells.
  • Figure 2D shows IFNy, TNFa, and GM-CSF production from culture supernatants of EGFR + NKG2DL + HCT116 cells co-cultured for 24 hrs with untransduced control T cells, NKG2D DARIC T cells, NKG2D.TNFR2 DARIC T cells, NKG2D.OX40 DARIC T cells, NKG2D.CD27 DARIC T cells, NKG2D.HVEM DARIC T cells, NKG2D.DR3 DARIC T cells, or NKG2D.GITR DARIC T cells at a 1 : 1 E:T ratio in rapamycin.
  • Figure 3 A shows IFNy, TNFa, and GM-CSF production from culture supernatants of EGFR + NKG2DL + A549 cells co-cultured for 24 hrs with NKG2D DARIC T cells, NKG2D.OX40 DARIC T cells or NKG2D.TNFR2 DARIC T cells at a 1 : 1 E:T ratio in vehicle, rapamycin or AP21967
  • Figure 3B shows IFNy, TNFa, and GM-CSF production from culture supernatants of EGFR + NKG2DL + A549 cells co-cultured for 24 hrs with NKG2D DARIC T cells, NKG2D.OX40 DARIC T cells or NKG2D TNFR2 DARIC T cells at a 1 : 1 E:T ratio in vehicle, rapamycin or AP21967
  • Figure 3C shows the ratio of cytokine production when T cell co-cultures are treated with AP2167 vs. rapamycin.
  • Anti-EGFR CAR T cells, NKG2D DARIC T cells, NKG2D.TNFR2 DARIC T cells, and NKG2D.OX40 DARIC T cells are co-cultured at a 1 : 1 E:T ratio in rapamycin or AP21967 with either A549 or HCT116 target cells.
  • the ratio of cytokine production from AP2167 cultured divided by cytokine production from rapamycin cultures is shown. Arrows show rapamycin-mediated immunosuppression (>1) or rapamycin-mediated immunoboost ( ⁇ 1).
  • Figure 4 A shows a cartoon of NKG2D DARIC architectures comprising DARIC binding components that have two costimulatory domains.
  • Figure 4B shows IFNy and GM-CSF production from culture supernatants of EGFR + NKG2DL + A549 cells co-cultured for 24 hrs with untransduced control T cells, NKG2D DARIC T cells, NKG2D.DAP10 DARIC T cells, NKG2D.CD28 DARIC T cells, or NKG2D.CD28.DAP10 DARIC T cells at a 1 : 1 E:T ratio in vehicle or rapamycin.
  • Figure 4C shows IFNy and GM-CSF production from culture supernatants of EGFR + NKG2DL + A549 cells co-cultured for 24 hrs with untransduced control T cells, NKG2D DARIC T cells, NKG2D.DAP10 DARIC T cells, NKG2D.DAP 10.0X40 DARIC T cells, or NKG2D.OX40.DAP10 DARIC T cells at a 1 : 1 E:T ratio in vehicle or rapamycin.
  • Figure 5 A shows a cartoon of NKG2D DARIC architectures comprising DARIC binding components that have ICOS-based transmembrane and costimulatory domains.
  • Figure 5B shows IFNy production from culture supernatants of EGFR + NKG2DL + A549 cells co-cultured for 24 hrs with anti-EGFR CAR T cells, NKG2D DARIC T cells, or NKG2D DARIC T cells containing single or dual costimulatory and transmembrane domains derived from ICOS and DAP10 at a 1 : 1 E:T ratio in AP21967.
  • Figure 5C shows GM-CSF production from culture supernatants of
  • Figure 6 A shows a cartoon of a dual targeting DARIC strategy: an NKG2D DARIC comprising a DARIC binding component with a costimulatory domain together with an anti- CD ⁇ DARIC binding component.
  • Figure 6B shows NKG2D binding domain expression in the CD4 + gate for untransduced T cells, NKG2D.TNFR2 DARIC T cells, and NKG2D.TNFR2 DARIC:CD19 DARIC T cells.
  • Figure 6C shows CD19-Fc binding efficiency for untransduced T cells, CD19 DARIC T cells, and NKG2D.TNFR2 DARIC:CD19 DARIC T cells.
  • Figure 6D shows GM-CSF production from culture supernatants of NKG2DL
  • CD 19- A20 cells A20
  • NKG2DL CD 19 + A20 cells A20-hCD19
  • NKG2DL + CD 19 A549 cells A549).
  • Target cells were co-cultured for 24 hrs with untransduced control T cells, CD19 DARIC T cells, NKG2D.TNFR2 DARIC T cells, or NKG2D.TNFR2
  • DARIC:CD19 DARIC T cells at a 1 : 1 E:T ratio in AP21967.
  • Figure 7A shows a cartoon of anti-CD 19 DARIC architecture with costimulatory domains.
  • Figure 7B shows CD19-Fc binding efficiency for untransduced T cells, anti-CD19 CAR T cells, CD19 DARIC T cells, CD19.OX40 DARIC T cells and CD19.TNFR2 DARIC T cells.
  • Figure 7C shows IFNy, GM-CSF and TNFa production from culture supernatants of CD19 + Nalm6 cells co-cultured for 24 hrs with anti-CD 19 CAR T cells, CD 19 DARIC T cells, CD 19.0X40 DARIC T cells, and CD19.TNFR2 DARIC T cells at a 1 : 1 E:T ratio in vehicle or Rapamycin.
  • Figure 7D shows IFNy, GM-CSF and TNFa production from culture supernatants of CD19 + Jeko-1 cells co-cultured for 24 hrs with anti-CD 19 CAR T cells, CD 19 DARIC T cells, CD 19.0X40 DARIC T cells, and CD19.TNFR2 DARIC T cells at a 1 : 1 E:T ratio in vehicle or Rapamycin.
  • Figure 8A shows a cartoon of anti-CD33 DARIC architecture with costimulatory domains.
  • Figure 8B shows CD33-Fc binding efficiency for untransduced T cells, anti-CD33 CAR T cells, anti-CD33 DARIC T cells, CD33.OX40 DARIC T cells and CD33.TNFR2 DARIC T cells. Two different anti-CD33 scFvs are shown.
  • Figure 8C shows IFNy production from culture supernatants of CD33 + THP-1 or CD33 + Molm-1 cells co-cultured for 24 hrs with ant-CD33 CAR T cells, CD33 DARIC T cells, CD33.OX40 DARIC T cells, and CD33.TNFR2 DARIC T cells at a 1 : 1 E:T ratio in Vehicle or Rapamycin. Two different anti-CD33 scFvs are shown.
  • SEQ ID NO: 1 sets forth the amino acid sequence for an FRB T2098L-CD8a TM- E0137-E03z DARIC signaling component.
  • SEQ ID NO: 2 sets forth the amino acid sequence for an FRB T2098L-CD8a TM- 0 ⁇ 134-0 ⁇ 3z ⁇ AM0 signaling component.
  • SEQ ID NO: 3 sets forth the amino acid sequence for an FRB T2098L-CD8a TM- CD28-CD z DARIC signaling component.
  • SEQ ID NO: 4 sets forth the amino acid sequence for an anti-BCMA-FKBP12-
  • CD4 TM DARIC. TNFR2 binding component CD4 TM DARIC. TNFR2 binding component.
  • SEQ ID NO: 5 sets forth the amino acid sequence for an anti-BCMA-FKBP12- AMN TM DARIC. TNFR2 binding component
  • SEQ ID NO: 6 sets forth the amino acid sequence for an anti-CD 19-FKBP12-CD4 TM DARIC. TNFR2 binding component.
  • SEQ ID NO: 7 sets forth the amino acid sequence for an anti-CD 19-FKBP12-CD4 TM DARIC.0X40 binding component.
  • SEQ ID NO: 8 sets forth the amino acid sequence for an anti-B7-H3-FKBP12-CD4 TM DARIC.0X40 binding component.
  • SEQ ID NO: 9 sets forth the amino acid sequence for an anti-B7-H3-FKBP12-CD4
  • SEQ ID NO: 10 sets forth the amino acid sequence for an anti-CD20-FKBP12- CD4 TM DARIC.0X40 binding component.
  • SEQ ID NO: 11 sets forth the amino acid sequence for an anti-CD20-FKBP12- CD4 TM DARIC. TNFR2 binding component.
  • SEQ ID NO: 12 sets forth the amino acid sequence for an anti-CD22-FKBP12- CD4 TM DARIC.0X40 binding component.
  • SEQ ID NO: 13 sets forth the amino acid sequence for an anti-CD22-FKBP12- CD4 TM DARIC. TNFR2 binding component.
  • SEQ ID NO: 14 sets forth the amino acid sequence for an anti-EGFRvIII-FKBP12-
  • CD4 TM DARIC.0X40 binding component CD4 TM DARIC.0X40 binding component.
  • SEQ ID NO: 15 sets forth the amino acid sequence for an anti-EGFRvIII-FKBP12- CD4 TM DARIC. TNFR2 binding component.
  • SEQ ID NO: 16 sets forth the amino acid sequence for an anti-CD33-FKBP12- CD4 TM DARIC.OX40 binding component.
  • SEQ ID NO: 17 sets forth the amino acid sequence for an anti-CD33-FKBP12- CD4 TM DARIC.TNFR2 binding component.
  • SEQ ID NO: 18 sets forth the amino acid sequence for an anti-CD33-FKBP12-
  • CD4 TM DARIC.OX40 binding component CD4 TM DARIC.OX40 binding component.
  • SEQ ID NO: 19 sets forth the amino acid sequence for an anti-CD33-FKBP12- CD4 TM DARIC.TNFR2 binding component.
  • SEQ ID NO: 20 sets forth the amino acid sequence for an NKG2D-FKBP12-CD4 TM DARJC.OX40 binding component.
  • SEQ ID NO: 21 sets forth the amino acid sequence for an NKG2D-FKBP12-CD4 TM DARJC.TNFR2 binding component.
  • SEQ ID NO: 22 sets forth the amino acid sequence for an NKG2D DARIC polyprotein.
  • SEQ ID NO: 23 sets forth the amino acid sequence for a CD 19 DARIC polyprotein.
  • SEQ ID NO: 24 sets forth the amino acid sequence for a B7-H3 DARIC polyprotein.
  • SEQ ID NO: 25 sets forth the amino acid sequence for a CD20 DARIC polyprotein.
  • SEQ ID NO: 26 sets forth the amino acid sequence for a CD22 DARIC polyprotein.
  • SEQ ID NO: 27 sets forth the amino acid sequence for an EGFRvIII DARIC polyprotein.
  • SEQ ID NO: 28 sets forth the amino acid sequence for a CD33 DARIC-1 polyprotein.
  • SEQ ID NO: 29 sets forth the amino acid sequence for a CD33 DARIC-2 polyprotein.
  • SEQ ID NOs: 30-40 set forth the amino acid sequences of various linkers.
  • SEQ ID NOs: 41-65 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.
  • CAR T cell therapy is the lack of spatial and temporal control of the CAR T cell activity. Lack of control over CAR T cell activity can trigger a range of side effects, many of which begin subtly but can rapidly worsen.
  • a particularly severe complication is cytokine release syndrome (CRS) or“cytokine storm” where CAR T cells induce massive and potentially fatal cytokine release.
  • CRS cytokine release syndrome
  • cytokine storm cytokine storm
  • CRS can produce dangerously high fevers, extreme fatigue, difficulty breathing, and a sharp drop in blood pressure.
  • CRS can also produce a second wave of side effects that involve the nervous system, including neurotoxicity, tremors, headaches, confusion, loss of balance, trouble speaking, seizures, and
  • compositions and methods contemplated herein offer solutions to these and other problems plaguing adoptive cell therapies.
  • the disclosure generally relates to improved compositions and methods for regulating the spatial and temporal control of adoptive cell therapies using costimulatory dimerizing agent regulated immunoreceptor complexes (DARIC).
  • a DARIC comprises one or more DARIC binding components and/or one or more DARIC signaling
  • DARIC compositions and methods contemplated herein provide numerous advantages over CAR T cell therapies existing in the art, including but not limited to, both spatial and temporal control over immune effector cell signal transduction binding and signaling activities.
  • DARIC temporal control primes the DARIC machinery for signaling through bridging factor mediated association of a DARIC binding component to a DARIC signaling component.
  • DARIC spatial control engages the signaling machinery through target antigen recognition by the binding domain on the DARIC binding component. In this manner, DARIC immune effector cells become activated when both a target antigen and a bridging factor are present.
  • DARICs comprising two or more binding components directed to different target antigens enable dual or multiplex targeting of target cells and may be advantageous in enhancing efficacy, tumor clearance, and safety; and in decreasing relapse, antigen escape, on-target off-tumor cell lysis.
  • the disclosure contemplates improved DARIC
  • DARIC binding components comprising an intracellular signaling domain increase the potency of DARIC immune effector cells by, for example, increasing inflammatory cytokine secretion and increasing antigen dependent cytotoxicity against target cells.
  • DARIC binding components comprising an intracellular signaling domain increase the potency of DARIC immune effector cells by, for example, increasing inflammatory cytokine secretion and increasing antigen dependent cytotoxicity against target cells.
  • an immunosuppressive bridging factor molecule e.g., rapamycin
  • a DARIC binding component comprising an intracellular signaling domain, e.g, a costimulatory domain, surprisingly reduces or eliminates the immunosuppressive activity associated with the bridging factor.
  • a DARIC includes a polypeptide (DARIC signaling component) that comprises a multimerization domain polypeptide or variant thereof, a transmembrane domain, and one or more intracellular signaling domains; and a polypeptide (DARIC binding component) that comprises a binding domain, a multimerization domain polypeptide or variant thereof, a transmembrane domain; and one or more intracellular signaling domains.
  • DARIC signaling component that comprises a multimerization domain polypeptide or variant thereof, a transmembrane domain
  • DARIC binding component that comprises a binding domain, a multimerization domain polypeptide or variant thereof, a transmembrane domain
  • the one or more intracellular signaling domains in the DARIC signaling component are different than the one or more intracellular signaling domains in the DARIC binding component.
  • a DARIC includes a polypeptide (DARIC signaling component) that comprises a multimerization domain polypeptide or variant thereof, a transmembrane domain, a costimulatory domain; and/or a primary signaling domain; and a polypeptide (DARIC binding component) that comprises a binding domain, a
  • the costimulatory domain in the DARIC signaling component is different than the costimulatory domain in the DARIC binding component.
  • the two different costimulatory domains synergistically enhance the cytokine secretion profile and cytotoxicity of DARIC T cells directed to target cells.
  • the DARIC binding and signaling components associate with one another through the bridging factor to form a functionally active DARIC.
  • the disclosure contemplates DARIC components that generate an anti-cancer response against cancers that express two or more target antigens expressed on one or more target cells.
  • a DARIC signaling component that comprises a multimerization domain polypeptide or variant thereof, a transmembrane domain, a costimulatory domain; and/or a primary signaling domain; a first DARIC binding component that comprises a binding domain that binds a first target antigen, a
  • the DARIC binding components each associate with the DARIC signaling component through the bridging factor to form functionally active DARICs.
  • the multimerization domains of the DARIC binding and DARIC signaling components are positioned extracellularly.
  • Extracellular position of the multimerization domains provides numerous advantages over intracellular positioning including, but not limited to, more efficient positioning of the binding domain, higher temporal sensitivity to bridging factor regulation, and less toxicity due to ability to use non immunosuppressive doses of particular bridging factors.
  • DARIC protein complexes DARIC fusion proteins
  • cells comprising polynucleotides encoding DARICs, DARIC binding components, and DARIC signaling components and/or expressing the same; and methods of using the same to treat an immune disorder are also contemplated herein.
  • Techniques for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation ( e.g ., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology as cited and discussed throughout the present specification. See , e.g.
  • the term“and/or” should be understood to mean either one, or both of the alternatives.
  • the term“about” or“approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term“about” or“approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
  • the range“1 to 5” is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.
  • the term“substantially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • “substantially the same” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • An“antigen (Ag)” refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions (such as one that includes a cancer-specific protein) that are injected or absorbed into an animal.
  • exemplary antigens include but are not limited to lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens.
  • A“target antigen” or“target antigen of interest” is an antigen that a binding domain contemplated herein, is designed to bind.
  • one or more target antigens are selected from the group consisting of: alpha folate receptor (FRa), a n b 6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD 123, CD 133, CD 138, CD171, carcinoembryonic antigen (CEA), claudin 6, (CLDN6), claudin 18 isoform 2 (CLDN18.2), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (
  • MHC class I chain related proteins A MHC class I chain related proteins A
  • MHC class I chain related proteins B MHC class I chain related proteins B
  • NCAM neural cell adhesion molecule
  • NY-ESO-1 cancer/testis antigen 1
  • PLAC1 poly sialic acid
  • PRAME preferentially expressed antigen in melanoma
  • PSCA prostate stem cell antigen
  • PSMA receptor tyrosine kinase-like orphan receptor 1
  • ROR1 receptor tyrosine kinase-like orphan receptor 1
  • SSX2 synovial sarcoma
  • Survivin tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL 16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1).
  • TAG72 tumor associated glycoprotein 72
  • TEM1/CD248 tumor endothelial marker 1
  • TEM7R tumor endothelial marker 7-related
  • TPBG trophoblast glycoprotein
  • ULBP UL 16-binding protein
  • ULBP2 ULBP2
  • ULBP3 ULBP
  • the antigen is an MHC -peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.
  • NKG2D ligand refers to a polypeptide that is recognized and/or bound by a natural-killer group 2, member D (NKG2D) receptor.
  • NKG2D ligands Two families of NKG2D ligands have been identified in humans: MHC class I chain related proteins A (MICA) and B (MICB) and HCMV UL16-binding proteins, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • MICA and MICB each have an al, a2, a3, and transmembrane domain; ULBP1, ULBP2, ULBP3, and ULBP6 each have an al and a2 domain and are
  • glycosylphosphatidylinositol GPI-linked to the cell membrane
  • ULBP4 and ULBP 5 each have an al and a2 domain and a transmembrane domain.
  • NKG2D ligands are expressed, in various combinations, on many human cancer cells and immunosuppressive cells (T-regs and myeloid derived suppressor cells (MDSCs) within tumor
  • Cancers expressing one or more NKG2D ligands include, but are not limited to, carcinomas (ovarian, bladder, breast, lung, liver, colon, kidney, prostate, melanoma, Ewing’s sarcoma, glioma, and neuroblastoma), leukemias (AML, CML, CLL), lymphomas, and multiple myeloma.
  • NKG2D ligands can also be induced at sites of chronic inflammation, transiently after some infections, following local irradiation, and after treatment with particular drugs, e.g., HD AC inhibitors and bortezomib.
  • NKG2D receptor binding domain or NKG2D ligand binding portion thereof refers to the NKG2D receptor or a portion thereof necessary or sufficient to bind one or more NKG2D ligands.
  • NK natural killer
  • NKG2D is expressed on NK cells, CD8 + T cells, subsets of CD4 + T cells, and subsets of gd T cells as a costimulatory receptor.
  • NKG2D receptor binding domain or NKG2D ligand binding portion thereof binds one or more NKG2D ligands including, but not limited to MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • binding domain As used herein, the terms,“binding domain,”“extracellular domain,”“antigen binding domain,”“extracellular binding domain,”“extracellular antigen binding domain,” “antigen-specific binding domain,” and“extracellular antigen specific binding domain,” are used interchangeably and refer to a polypeptide with the ability to specifically and/or selectively bind to the target antigen of interest.
  • the binding domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • binding domain or“specifically bound” or“specific binding” or“specifically targets” as used herein, describe binding of binding domain to a target antigen at greater binding affinity than background binding.
  • a binding domain“specifically binds” to a target antigen if it binds to or associates with the antigen with an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) of, for example, greater than or equal to about 10 5 M 1 .
  • a binding domain (or a fusion protein comprising the same) binds to a target with a K a greater than or equal to about 10 6 M 1 , 10 7 M 1 , 10 8 M 1 , 10 9 M 1 , 10 10 M l , 10 11 M 1 , 10 12 M 1 , or 10 13 M 1 .
  • “High affinity” binding domains (or single chain fusion proteins thereof) refer to those binding domains with a K a of at least 10 7 M 1 , at least 10 8 M 1 , at least 10 9 M 1 , at least 10 10 M 1 , at least 10 11 M 1 , at least 10 12 M 1 , at least 10 13 M 1 , or greater.
  • an“antibody” refers to a binding agent that is a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell.
  • an antigen such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell.
  • An“epitope” or“antigenic determinant” refers to the region of an antigen to which a binding agent binds.
  • the polypeptide is intracellular and the epitope is a short oligopeptide (about 2 to about 20 amino acids) displayed in complex with an MHC.
  • Antibodies include antigen binding fragments thereof, such as a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis- scFv, (SCFV)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody) and portions of full length antibodies responsible for antigen binding.
  • a Camel Ig such as a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific
  • Antibodies also include: polyclonal and monoclonal antibodies and antigen binding fragments thereof; murine antibodies, camelid antibodies, and human antibodies, and antigen binding fragments thereof; and chimeric antibodies, heteroconjugate antibodies, and humanized antibodies, and antigen binding fragments thereof. See also , Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman &
  • A“linker” refers to a plurality of amino acid residues between the various polypeptide domains added for appropriate spacing and conformation of the molecule.
  • the linker is a variable region linking sequence.
  • A“variable region linking sequence,” is an amino acid sequence that connects the VH and VL domains and provides a spacer function compatible with interaction of the two sub binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions.
  • a linker separates one or more heavy or light chain variable domains, hinge domains, multimerization domains, transmembrane domains, costimulatory domains, and/or primary signaling domains.
  • linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 40) (Cooper et al., Blood , 101(4): 1637-1644 (2003)).
  • a spacer domain moves an antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation (Patel et al. , Gene Therapy , 1999; 6: 412-419).
  • a spacer domain separates one or more binding domains, multimerization domains, transmembrane domains, costimulatory domains, and/or primary signaling domains.
  • the spacer domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3.
  • the spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • polypeptides refers to a polypeptide that plays a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation.
  • polypeptides may comprise one or more hinge domains between the binding domain and the multimerization domain, between the binding domain and the transmembrane domain (TM), or between the multimerization domain and the transmembrane domain.
  • the hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • the hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • A“multimerization domain,” as used herein, refers to a polypeptide that preferentially interacts or associates with another different polypeptide directly or via a bridging molecule, e.g, a chemically inducible dimerizer, wherein the interaction of different multimerization domains substantially contributes to or efficiently promotes multimerization (i.e., the formation of a dimer, trimer, or multipartite complex, which may be a homodimer, heterodimer, homotrimer, heterotrimer, homomultimer, heteromultimer).
  • a multimerization domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • multimerization domains suitable for use in particular embodiments contemplated herein include an FK506 binding protein (FKBP) polypeptide or variants thereof, an FKBP-rapamycin binding (FRB) polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variants thereof, a PYRl-like 1 (PYL1) polypeptide or variants thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or variants thereof, a GIB1 polypeptide or variants thereof, or a GAI polypeptide or variants thereof.
  • FKBP FK506 binding protein
  • a calcineurin polypeptide or variants thereof a cyclophilin polypeptide or variants thereof
  • DHFR bacterial dihydrofolate reductase
  • the term“FKBP-rapamycin binding polypeptide” refers to an FRB polypeptide.
  • the FRB polypeptide is an FKBP12-rapamycin binding polypeptide.
  • FRB polypeptides suitable for use in particular embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues.
  • the FRB polypeptide comprises a 93 amino acid sequence Ile- 2021 through Lys-2113 and a mutation of T2098L, with reference to GenBank Accession No. L34075.1.
  • An FRB polypeptide contemplated herein binds to an FKBP polypeptide through a bridging factor, thereby forming a ternary complex.
  • the term“FK506 binding protein” refers to an FKBP polypeptide.
  • the FKBP polypeptide is an FKBP 12 polypeptide or an FKBP12 polypeptide comprising an F36V mutation.
  • an FKBP domain may also be referred to as a“rapamycin binding domain”.
  • Information concerning the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, e.g., Staendart el al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J 374:361, 1996).
  • An FKBP polypeptide contemplated herein binds to an FRB
  • polypeptide through a bridging factor, thereby forming a ternary complex.
  • A“bridging factor” refers to a molecule that associates with and that is disposed between two or more multimerization domains.
  • A“bridging factor” refers to a molecule that associates with and that is disposed between two or more multimerization domains.
  • multimerization domains substantially contribute to or efficiently promote formation of a polypeptide complex only in the presence of a bridging factor.
  • multimerization domains do not contribute to or do not efficiently promote formation of a polypeptide complex in the absence of a bridging factor.
  • bridging factors suitable for use in particular embodiments contemplated herein include, but are not limited to AP21967, rapamycin (sirolimus) 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.
  • AP21967 rapamycin (sirolimus) 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)
  • Rapamycin analogs include but are not limited to those disclosed in U.S. Pat. No. 6,649,595, which rapalog structures are incorporated herein by reference in their entirety.
  • a bridging factor is a rapalog with substantially reduced immunosuppressive effect as compared to rapamycin.
  • rapalogs suitable for use in particular embodiments contemplated herein include, but are not limited to, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • A“substantially reduced immunosuppressive effect” refers to at least less than 0.1 to 0.005 times the immunosuppressive effect observed or expected for the same dose measured either clinically or in an appropriate in vitro (e.g., inhibition of T cell
  • A“transmembrane domain” or“TM domain” is a domain that anchors a polypeptide to the plasma membrane of a cell.
  • the TM domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • effector function refers to a specialized function of an immune effector cell. Effector function includes, but is not limited to, activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses elicited with antigen binding to the receptor expressed on the immune effector cell.
  • An“intracellular signaling domain” or“endodomain” refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain.
  • intracellular signaling domain is meant to include any truncated portion of an intracellular signaling domain necessary or sufficient to transduce an effector function signal.
  • T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g a TCR/CD3 complex) and costimulatory domains that act in an antigen-independent manner to provide a secondary or costimulatory signal.
  • primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g a TCR/CD3 complex)
  • costimulatory domains that act in an antigen-independent manner to provide a secondary or costimulatory signal.
  • A“primary signaling domain” refers to an intracellular signaling domain that regulates the primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM containing primary signaling domains include, but are not limited to those derived from FcRy, FcRp, CD3y, CD35, CD3e, CD3C, CD22, CD79a, CD79b, and CD66d.
  • costimulatory domain refers to an intracellular signaling domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Illustrative examples of such costimulatory molecules from which
  • costimulatory domains may be isolated include, but are not limited to: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, TLR1, TLR2, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30,
  • LAT T cell receptor associated transmembrane adaptor 1
  • TFRS14; HVEM TNF receptor superfamily member 14
  • TNF receptor superfamily member 18 TNF receptor superfamily member 18
  • GITR TNF receptor superfamily member 25
  • ZAP70 zeta chain of T
  • an“immune disorder” refers to a disease that evokes a response from the immune system.
  • the term“immune disorder” refers to a cancer, an autoimmune disease, or an immunodeficiency.
  • immune disorders encompass infectious disease.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.
  • the term“malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth ( i.e ., division beyond normal limits), invasion ⁇ i.e., intrusion on and destruction of adjacent tissues), and metastasis ⁇ i.e., spread to other locations in the body via lymph or blood).
  • the term“metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a“metastatic tumor” or a“metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor.
  • Benign or“non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • A“cancer cell” refers to an individual cell of a cancerous growth or tissue. Cancer cells include both solid cancers and liquid cancers. A“tumor” or“tumor cell” refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers, e.g., leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of a tumor in an individual is the“tumor burden” which can be measured as the number, volume, or weight of the tumor.
  • relapse refers to the diagnosis of return, or signs and symptoms of return, of a cancer after a period of improvement or remission.
  • Remission is also referred to as“clinical remission,” and includes both partial and complete remission. In partial remission, some, but not all, signs and symptoms of cancer have disappeared. In complete remission, all signs and symptoms of cancer have disappeared, although cancer still may be in the body.
  • Refractory refers to a cancer that is resistant to, or non-responsive to, therapy with a particular therapeutic agent.
  • a cancer can be refractory from the onset of treatment (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period or during a subsequent treatment period.
  • Antigen negative refers to a cell that does not express antigen or expresses a negligible amount of antigen that is undetectable. In one embodiment, antigen negative cells do not bind receptors directed to the antigen. In one embodiment, antigen negative cells do not substantially bind receptors directed to the antigen.
  • An“autoimmune disease” refers to a disease in which the body produces an immunogenic (i.e ., immune system) response to some constituent of its own tissue. In other words, the immune system loses its ability to recognize some tissue or system within the body as“self’ and targets and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which predominantly one organ is affected (e.g ., hemolytic anemia and anti-immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (e.g., systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths that surround the nerve fibers of the brain and spinal cord.
  • Autoimmune diseases include, for instance, Hashimoto’s thyroiditis, Grave’s disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.
  • An“immunodeficiency” means the state of a patient whose immune system has been compromised by disease or by administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance.
  • Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, and diabetes.
  • An“infectious disease” refers to a disease that can be transmitted from person to person or from organism to organism and is caused by a microbial or viral agent (e.g ., common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
  • a microbial or viral agent e.g ., common cold.
  • Infectious diseases include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
  • the terms“individual” and“subject” are often used interchangeably and refer to any animal that exhibits a symptom of cancer or other immune disorder that can be treated with the compositions and methods contemplated elsewhere herein.
  • Suitable subjects e.g, patients
  • laboratory animals such as mouse, rat, rabbit, or guinea pig
  • farm animals such as a cat or dog
  • domestic animals or pets such as a cat or dog
  • Non-human primates and, preferably, human patients are included.
  • Typical subjects include human patients that have, have been diagnosed with, or are at risk or having, cancer or another immune disorder.
  • the term“patient” refers to a subject that has been diagnosed with cancer or another immune disorder that can be treated with the compositions and methods disclosed elsewhere herein.
  • treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction of the disease or condition, or the delaying of the progression of the disease or condition, e.g., delaying tumor outgrowth.“Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • “prevent,” and similar words such as“prevented,”“preventing” etc. indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein,“prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
  • the phrase“ameliorating at least one symptom of’ refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated.
  • the disease or condition being treated is a cancer, wherein the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).
  • By“enhance” or“promote,” or“increase” or“expand” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a measurable physiological response may include an increase in T cell expansion, activation, persistence, cytokine secretion, and/or an increase in cancer cell killing ability, among others apparent from the
  • An“increased” or“enhanced” amount is typically a“statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.
  • By“decrease” or“lower,” or“lessen,” or“reduce,” or“abate” refers generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • A“decrease” or“reduced” amount is typically a“statistically significant” amount, and may include a decrease that is 1.1, 1.2,
  • By“maintain,” or“preserve,” or“maintenance,” or“no change,” or“no substantial change,” or“no substantial decrease” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a substantially similar or comparable physiological response (i.e., downstream effects) in a cell, as compared to the response caused by either vehicle, a control molecule/composition, or the response in a particular cell lineage.
  • a comparable response is one that is not significantly different or measurable different from the reference response.
  • one or more costimulatory DARICs redirect cytotoxicity of an immune effector cell to a cancer cell that expresses at least one or more target antigens.
  • the term“DARIC” refers to a dimerizing agent regulated immunoreceptor complex.
  • a DARIC comprises one or more non-naturally occurring polypeptides that transduces an immunostimulatory signal in an immune effector cell upon exposure to a multimerizing agent or bridging factor, e.g, stimulating immune effector cell activity and function, increasing production and/or secretion of proinflammatory cytokines.
  • a DARIC is a multi-chain chimeric receptor comprising one or more DARIC signaling components that each comprise a multimerization domain polypeptide or variant thereof, a transmembrane domain, and one or more intracellular signaling domains and one or more DARIC binding components that each comprise a binding domain, a
  • the multimerization domain polypeptide or variant thereof a transmembrane domain, and one or more intracellular signaling domains.
  • the one or more intracellular signaling domains in the DARIC signaling component are different than the one or more intracellular signaling domains in the DARIC binding component.
  • a DARIC signaling component and a DARIC binding component are expressed from one or more polynucleotides in the same cell. In another embodiment, a DARIC signaling component and a DARIC binding component are expressed from a polycistronic polynucleotide in the same cell.
  • A“DARIC signaling component” or“DARIC signaling polypeptide” refers to a polypeptide comprising one or more multimerization domains, a transmembrane domain, and one or more intracellular signaling domains.
  • the DARIC signaling component comprises a multimerization domain, a transmembrane domain, a costimulatory domain and/or a primary signaling domain.
  • the DARIC signaling component comprises a first multimerization domain, a first
  • transmembrane domain a first costimulatory domain and/or a primary signaling domain.
  • a DARIC signaling component comprises one or more multimerization domains.
  • multimerization domains suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to, an FK506 binding protein (FKBP) polypeptide or variants thereof, an FKBP-rapamycin binding (FRB) polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variants thereof, a PYRl-like 1 (PYL1) polypeptide or variants thereof and an abscisic acid insensitive 1 (ABI1) polypeptide or variants thereof.
  • FKBP FK506 binding protein
  • a calcineurin polypeptide or variants thereof a cyclophilin polypeptide or variants thereof
  • DHFR bacterial dihydrofolate reductase
  • ABSI1 abscisic acid insensitive 1
  • a DARIC signaling component comprises an FRB polypeptide.
  • a DARIC signaling component comprises an FRB polypeptide comprising a T2098L mutation, or variant thereof.
  • a DARIC signaling component comprises an FKBP polypeptide or variant thereof.
  • a DARIC signaling component comprises an FK506-binding protein 12 (FKBP12) polypeptide, or variant thereof.
  • a DARIC signaling component comprises a
  • transmembrane domains suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to, the transmembrane region(s) of the alpha, beta, gamma, or delta chain of a T-cell receptor, CD3e, CD3C, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, amnionless (AMN), programmed cell death 1 (PDCD1), NKG2A, NKG2B, NKG2C, and NKG2D.
  • a DARIC signaling component comprises a CD4 transmembrane domain.
  • a DARIC signaling component comprises a CD8a transmembrane domain.
  • a DARIC signaling component comprises a linker that links the C-terminus of the transmembrane domain to the N-terminus of an intracellular signaling domain.
  • a short oligo- or poly-peptide linker preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length links the transmembrane domain and an intracellular signaling domain.
  • a glycine-serine based linker provides a particularly suitable linker.
  • DARIC signaling components contemplated in particular embodiments herein comprise one or more intracellular signaling domains.
  • a DARIC signaling component comprises one or more costimulatory domains and/or a primary signaling domain.
  • the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif (IT AM).
  • IT AM containing primary signaling domains that are suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to those derived from FcRy, FcR-b, CD3y, CD35, CD3e, O ⁇ 3z, CD22, CD79a, CD79b, and CD66d.
  • a DARIC signaling component comprises a O ⁇ 3z primary signaling domain and one or more costimulatory domains.
  • the primary signaling and costimulatory domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
  • costimulatory domains suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to those domains isolated from the following costimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DN AX- Activation Protein 10 (DAPIO), Linker for activation of T-cells family member 1 (LAT), SH2 Domain- Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated
  • transmembrane adaptor 1 (TRATl), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
  • a DARIC signaling component contemplated herein comprises a signal peptide, e.g., secretion signal peptide, and do not comprise a transmembrane domain.
  • signal peptides suitable for use in particular DARIC signaling components include but are not limited to an IgGl heavy chain signal polypeptide, an IgK light chain signal polypeptide, a CD8a signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide.
  • a DARIC signaling component comprises a CD8a signal polypeptide.
  • a DARIC signaling component comprises one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134.
  • a DARIC signaling component comprises one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134, and a O ⁇ 3z primary signaling domain.
  • a DARIC signaling component comprises a CD137 costimulatory domain and a O ⁇ 3z primary signaling domain.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain.
  • A“DARIC binding component” or“DARIC binding polypeptide” refers to a polypeptide comprising a binding domain that binds a target antigen, one or more multimerization domains, a transmembrane domain, and one or more intracellular signaling domains.
  • the DARIC binding component comprises a binding domain that binds a target antigen, a multimerization domain, a transmembrane domain, and a costimulatory domain.
  • the DARIC binding component comprises a binding domain that binds a target antigen, a second multimerization domain, a second transmembrane domain, and a second costimulatory domain.
  • a DARIC comprises two or more DARIC binding components that each comprise a binding domain that binds to a different antigen, a multimerization domain, a transmembrane domain, and a costimulatory domain.
  • a DARIC comprises two or more DARIC binding components that each comprise a binding domain that binds to a different antigen, a second or third multimerization domain, a second or third transmembrane domain, and a second or third costimulatory domain.
  • two or more DARIC binding components comprise different binding domains but comprise the same multimerization
  • binding domains suitable for use in particular DARIC binding components include, but are not limited to, antibodies or antigen binding fragments thereof.
  • antibodies and antigen binding fragments thereof suitable for use in particular DARIC binding components include, but are not limited to, murine antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, or human antibodies.
  • the antibody or antigen binding fragment thereof is derived from a monoclonal antibody.
  • Illustrative examples of antibodies and antigen binding fragments thereof suitable for use in particular DARIC binding components include, but are not limited to, a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (SCFV)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
  • a Camel Ig a Llama Ig, an Alpaca Ig, Ig NAR
  • Fab' fragment fragment
  • F(ab')2 fragment fragment
  • Fab2 bispecific Fab
  • the binding domain comprises an scFv.
  • the binding domain comprises one or more camelid VHH antibodies.
  • a DARIC binding component comprises a binding domain that binds a tumor associated antigen (TAA), a tumor specific antigen (TSA), an NKG2D ligand, a gd T cell receptor (y5TCR) ligand, or an aPTCR ligand.
  • TAA tumor associated antigen
  • TSA tumor specific antigen
  • y5TCR gd T cell receptor
  • a DARIC binding component comprises a binding domain that binds a target antigen selected from the group consisting of: FRa, a n b 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD 123, CD 133, CD 138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-lORa, IL-13Ra2, Kappa, LAGE-1A, Lambda, LeY, Ll- CAM, MAGE-A1, MAGE- A
  • a DARIC binding component comprises a binding domain that binds BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, a multimerization domain, a transmembrane domain, and one or more intracellular signaling domains.
  • a DARIC binding component comprises one or more multimerization domains.
  • multimerization domains suitable for use in particular DARIC binding components contemplated herein include, but are not limited to, an FKBP polypeptide or variants thereof, an FRB polypeptide or variants thereof, a calcineurin polypeptide or variants thereof, a cyclophilin polypeptide or variants thereof, a DHFR polypeptide or variants thereof, a PYL1 polypeptide or variants thereof and an ABI1 polypeptide or variants thereof.
  • a DARIC binding component comprises an FRB polypeptide or variant thereof and a DARIC signaling component comprises an FKBP polypeptide or variant thereof.
  • a DARIC binding component comprises an FRB polypeptide comprising a T2098L mutation, or variant thereof and a DARIC signaling component comprises an FKBP12 polypeptide or variant thereof.
  • a DARIC binding component comprises an FKBP polypeptide or variant thereof and a DARIC signaling component comprises an FRB polypeptide, or variant thereof.
  • a DARIC binding component comprises an FKBP12 polypeptide, or variant thereof and a DARIC signaling component comprises an FRB polypeptide comprising a T2098L mutation, or variant thereof.
  • a DARIC binding component comprises a binding domain, an FKBP polypeptide or variant thereof, a transmembrane domain, and one or more intracellular signaling domains; and a DARIC signaling component comprises an FRB polypeptide or variant thereof, a transmembrane domain, and one or more
  • a DARIC binding component comprises a binding domain, an FKBP12 polypeptide or variant thereof, a transmembrane domain, and a costimulatory domain; and a DARIC signaling component comprises an FRB polypeptide comprising a T2098L mutation or variant thereof, a transmembrane domain, a costimulatory domain, and a primary signaling domain.
  • a DARIC binding component comprises a
  • the transmembrane domain may be the same as the transmembrane domain used in the DARIC signaling component. In one embodiment, the transmembrane domain may be different from the transmembrane domain used in the DARIC signaling component.
  • transmembrane domains suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to, the transmembrane region(s) of the alpha, beta, gamma, or delta chain of a T-cell receptor, CD3e, CD3C, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, amnionless (AMN), programmed cell death 1 (PDCD1), NKG2A, NKG2B, NKG2C, and NKG2D.
  • a DARIC binding component comprises a CD8a transmembrane domain.
  • a DARIC binding component comprises a CD4 transmembrane domain.
  • a short oligo- or poly-peptide linker preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length links the transmembrane domain and the intracellular signaling domain.
  • a glycine-serine based linker provides a particularly suitable linker.
  • DARIC binding components contemplated in particular embodiments herein comprise one or more intracellular signaling domains.
  • a DARIC binding component comprises a costimulatory domain.
  • costimulatory domains suitable for use in particular DARIC signaling components contemplated herein include, but are not limited to those domains isolated from the following costimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DN AX- Activation Protein 10 (DAPIO), Linker for activation of T-cells family member 1 (LAT), SH2 Domain- Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated
  • transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
  • a DARIC binding component comprises a CD27 or CD28 costimulatory domain.
  • a DARIC binding component comprises a TNFRS14, TNFRS18, or TNFRS25 costimulatory domain.
  • a DARIC binding component comprises an 0X40 costimulatory domain.
  • a DARIC binding component comprises a TNFR2 costimulatory domain.
  • a DARIC binding component contemplated herein comprises a signal peptide, e.g., secretion signal peptide, and do not comprise a transmembrane domain.
  • signal peptides suitable for use in particular DARIC binding components include but are not limited to an IgGl heavy chain signal polypeptide, an IgK light chain signal polypeptide, a CD8a signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide.
  • a DARIC binding component comprises a CD8a signal polypeptide.
  • a DARIC binding component comprises a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B,
  • CD 123, CLL-1, or EGFRvIII an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, 0X40 or TNFR2 costimulatory domain.
  • a DARIC binding component comprises a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B,
  • CD 123 CD 123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and a CD27 or CD28
  • a DARIC binding component comprises a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B,
  • CD 123, CLL-1, or EGFRvIII an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and a TNFRS14, TNFRS18, or TNFRS25 costimulatory domain.
  • a DARIC binding component comprises a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4
  • transmembrane domain or AMN transmembrane domain, and an 0X40 costimulatory domain.
  • a DARIC binding component comprises a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4
  • transmembrane domain or AMN transmembrane domain, and an TNFR2 costimulatory domain.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain and a DARIC binding component comprises a binding domain that binds BCMA, CD 19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, 0X40 or TNFR2 costimulatory domain.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain and a DARIC binding component comprises a binding domain that binds BCMA, CD 19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and a CD27 or CD28 costimulatory domain.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a O ⁇ 3z primary signaling domain and a DARIC binding component comprises a binding domain that binds BCMA, CD 19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and a TNFRS14,
  • TNFRS18 or TNFRS25 costimulatory domain.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain and a DARIC binding component comprises a binding domain that binds BCMA, CD 19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and an 0X40 costimulatory domain.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain and a DARIC binding component comprises a binding domain that binds BCMA, CD 19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or AMN transmembrane domain, and a TNFR2 costimulatory domain.
  • Bridging factors contemplated in particular embodiments herein mediate or promote the association of one or more DARIC signaling components with one or more DARIC binding components through multimerization domains in the respective components.
  • a bridging factor associates with and is disposed between the multimerization domains to promote association of a DARIC signaling component and a DARIC binding component.
  • the DARIC binding component and the DARIC signaling component associate and initiate immune effector cell activity against a target cell when the DARIC binding polypeptide is bound to a target antigen on the target cell.
  • the DARIC binding component does not associate with the DARIC signaling component and the DARIC is inactive.
  • a DARIC signaling component and a DARIC binding component comprise a cognate pair of multimerization domains selected from the group consisting of: FKBP and FKBP-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin, and PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
  • the multimerization domains of DARIC signaling and binding components associate with a bridging factor selected from the group consisting of: 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, FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or a derivative thereof.
  • a bridging factor selected from the group consisting of: 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, FK50
  • a DARIC signaling component and a DARIC binding component comprise one or more FRB and/or FKBP multimerization domains or variants thereof.
  • a DARIC signaling component comprises an FRB multimerization domain or variant thereof and a DARIC binding component comprises an FKBP multimerization domain or variant thereof.
  • a DARIC signaling component comprises an FRB T2098L multimerization domain or variant thereof and a DARIC binding component comprises an FKBP 12 or FKBP 12 F36V multimerization domains or variant thereof.
  • bridging factors suitable for use in particular embodiments contemplated herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylindolerapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
  • the bridging factor is AP21967.
  • the bridging factor is a non-immunosuppressive dose of sirolimus (rapamycin).
  • a cell is engineered or modified to express one or more DARIC binding and/or signaling components and an engineered antigen receptor.
  • a nucleic acid or vector encodes a fusion polypeptide comprising an engineered receptor and a DARIC binding component and/or DARIC signaling component, and one or more polypeptide cleavage signals interspersed between the receptor and the components.
  • a polynucleotide or vector encoding a DARIC is introduced into an immune effector cell that comprises an engineered antigen receptor.
  • any suitable mechanism known in the art may be used to introduce and co-express an engineered antigen receptor and a DARIC in the same immune effector cell or population of cells to the efficiency, potency, and durability of the immune effector cell response.
  • the intracellular signaling domains, e.g., costimulatory domains, of the engineered antigen receptor and the DARIC binding and/or DARIC signaling domains will be different from each other.
  • immune effector cells contemplated herein comprise an engineered antigen receptor and one or more components of a DARIC.
  • the engineered antigen receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), or a zetakine.
  • immune effector cells contemplated herein comprise an engineered TCR and one or more components of a DARIC.
  • T cells are engineered by introducing a polynucleotide or vector encoding an engineered TCR and one or more components of a DARIC separated by one or more polypeptide cleavage signals.
  • T cells are engineered by introducing a polynucleotide or vector encoding an engineered TCR and a polynucleotide or vector encoding one or more components of a DARIC.
  • T cells engineered to express an engineered TCR are further engineered by introducing a polynucleotide or vector encoding one or more components of a DARIC.
  • Naturally occurring T cell receptors comprise two subunits, an alpha chain and a beta chain subunit (a.pTCR), or a gamma chain and a delta chain subunit (ybTCR), each of which is a unique protein produced by recombination event in each T cell’s genome.
  • a.pTCR alpha chain and a beta chain subunit
  • ybTCR gamma chain and a delta chain subunit
  • TCRs may be screened for their selectivity to particular target antigens. In this manner, natural TCRs, which have a high-avidity and reactivity toward target antigens may be selected, cloned, and subsequently introduced into a population of T cells used for adoptive immunotherapy.
  • the TCR is an a.pTCR. In one embodiment, the TCR is a ybTCR.
  • T cells are modified by introducing a TCR subunit that has the ability to form TCRs that confer specificity to T cells for tumor cells expressing a target antigen.
  • the subunits have one or more amino acid
  • the engineered TCRs preferably also bind target cells displaying the relevant tumor-associated peptide with high avidity, and optionally mediate efficient killing of target cells presenting the relevant peptide in vivo.
  • the nucleic acids encoding engineered TCRs are preferably isolated from their natural context in a (naturally-occurring) chromosome of a T cell and can be incorporated into suitable vectors as described elsewhere herein. Both the nucleic acids and the vectors comprising them can be transferred into a cell, preferably a T cell in particular
  • the modified T cells are then able to express one or more chains of a TCR encoded by the transduced nucleic acid or nucleic acids.
  • the engineered TCR is an exogenous TCR because it is introduced into T cells that do not normally express the particular TCR.
  • the essential aspect of the engineered TCRs is that it has high avidity for a tumor antigen presented by a major histocompatibility complex (MHC) or similar immunological component.
  • MHC major histocompatibility complex
  • CARs are engineered to bind target antigens in an MHC independent manner.
  • the TCR can be expressed with additional polypeptides attached to the amino- terminal or carboxyl -terminal portion of the alpha chain or beta chain of a TCR, or of the gamma chain or delta chain of a TCR so long as the attached additional polypeptide does not interfere with the ability of the alpha chain or beta chain to form a functional T cell receptor and the MHC dependent antigen recognition.
  • Antigens that are recognized by the engineered TCRs contemplated in particular embodiments include, but are not limited to cancer antigens, including antigens on both hematological cancers and solid tumors.
  • Illustrative antigens include, but are not limited to FRa, a.vp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD 123, CD 133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-lORa, IL-13Ra2, Kap
  • the target antigen is expressed on one or more cells of a cancer and is selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII.
  • immune effector cells contemplated herein comprise a CAR and one or more components of a DARIC.
  • Chimeric antigen receptors are molecules that combine antibody-based specificity for a target antigen (e.g, tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity.
  • target antigen e.g, tumor antigen
  • T cell receptor-activating intracellular domain e.g, T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity.
  • the term,“chimeric,” describes being composed of parts of different proteins or DNAs from different origins.
  • T cells are engineered by introducing a polynucleotide or vector encoding a CAR and one or more DARIC components separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding a CAR and a polynucleotide or vector encoding one or more DARIC components. In one embodiment, T cells that are engineered to express a CAR are further engineered by introducing a polynucleotide or vector encoding one or more DARIC components.
  • a CAR comprises an extracellular domain that binds to a specific target antigen (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain and one or more intracellular signaling domains.
  • a specific target antigen also referred to as a binding domain or antigen-specific binding domain
  • the main characteristic of CARs is their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific coreceptors.
  • MHC major histocompatibility
  • CARs comprise an extracellular binding domain that specifically binds to a target polypeptide.
  • a CAR binds a target polypeptide that is different than the target polypeptide(s) bound by a DARIC binding component.
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.
  • the extracellular binding domain comprises an antibody or antigen binding fragment thereof.
  • the binding domain comprises an scFv.
  • the binding domain comprises one or more camelid antibodies.
  • a CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: FRa, a n b6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD 16, CD 19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2,
  • a CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII.
  • the CARs comprise an extracellular binding domain, e.g., antibody or antigen binding fragment thereof that binds an antigen, wherein the antigen is an MHC -peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.
  • an extracellular binding domain e.g., antibody or antigen binding fragment thereof that binds an antigen
  • the antigen is an MHC -peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.
  • the spacer domain comprises the CH2 and CH3 of IgGl, IgG4, or
  • hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, and CD4, which may be wild-type hinge regions from these molecules or may be altered.
  • the hinge domain comprises a CD8a hinge region.
  • the hinge is a PD-1 hinge or CD 152 hinge.
  • the transmembrane (TM) domain of the CAR fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell.
  • the TM domain may be derived either from a natural, synthetic, semi synthetic, or recombinant source.
  • Illustrative TM domains may be derived from (i.e., comprise at least the
  • transmembrane region(s) of the alpha, beta, gamma, or delta chain of a T-cell receptor CD3e, O ⁇ 3z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, PDCD1, NKG2A, NKG2B, NKG2C, and NKG2D.
  • a CAR comprises a TM domain derived from CD8a.
  • a CAR contemplated herein comprises a TM domain derived from CD8a and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that links the TM domain and the intracellular signaling domain of the CAR.
  • a glycine-serine linker provides a particularly suitable linker.
  • a CAR comprises an intracellular signaling domain that comprises one or more costimulatory domains and a primary signaling domain.
  • Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • a CAR comprises a CD3z primary signaling domain and one or more costimulatory domains.
  • the intracellular primary signaling and costimulatory domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
  • a CAR comprises one or more costimulatory domains to enhance the efficacy and expansion of T cells expressing CAR receptors.
  • costimulatory molecules suitable for use in CARs contemplated in particular embodiments include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28,
  • a CAR comprises one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134, and a CD3z primary signaling domain.
  • the CAR comprises: an extracellular domain that binds an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII; a CD4, CD8a or CD28 transmembrane domain; one or more intracellular costimulatory domains isolated from a polypeptide selected from the group consisting of: CD28, CD134, and CD137; and a O ⁇ 3z primary signaling domain.
  • immune effector cells contemplated herein comprise one or more chains of a zetakine receptor and one or more DARIC components.
  • Zetakines are chimeric transmembrane immunoreceptors that comprise an extracellular domain comprising a soluble receptor ligand linked to a support region capable of tethering the extracellular domain to a cell surface, a transmembrane region and an intracellular signaling domain.
  • Zetakines when expressed on the surface of T lymphocytes, direct T cell activity to those cells expressing a receptor for which the soluble receptor ligand is specific.
  • Zetakine chimeric transmembrane immunoreceptors that comprise an extracellular domain comprising a soluble receptor ligand linked to a support region capable of tethering the extracellular domain to a cell surface, a transmembrane region and an intracellular signaling domain.
  • Zetakines when expressed on the surface of T lymphocytes, direct T cell activity to those cells expressing a receptor for which the soluble
  • immunoreceptors redirect the antigen specificity of T cells, with application to treatment of a variety of cancers, particularly via the autocrine/paracrine cytokine systems utilized by human malignancy.
  • T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a zetakine receptor and one or more DARIC components separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a zetakine receptor and a polynucleotide or vector encoding one or more DARIC components.
  • T cells are engineered to express one or more chains of a zetakine receptor are further engineered by introducing a polynucleotide or vector encoding one or more DARIC components.
  • the zetakine comprises an immunosuppressive cytokine or cytokine receptor binding variant thereof, a linker, a transmembrane domain, and an intracellular signaling domain.
  • the cytokine or cytokine receptor binding variant thereof is selected from the group consisting of: interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 10 (IL-10), and interleukin- 13 (IL-13).
  • the linker comprises a CH2CH3 domain, hinge domain, or the like.
  • a linker comprises the CH2 and CH3 domains of IgGl, IgG4, or IgD.
  • a linker comprises a CD8a or CD4 hinge domain.
  • the transmembrane domain is selected from the group consisting of: the alpha, beta, gamma, or delta chain of the T-cell receptor, CD35, CD3e,
  • CD3y O ⁇ 3z CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD-1.
  • the intracellular signaling domain is selected from the group consisting of: an ITAM containing primary signaling domain and/or a costimulatory domain.
  • the intracellular signaling domain is selected from the group consisting of: FcRy, FcRp, CD3y, CD35, CD3e, CD3 , CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain is selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARDl l, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (0X40), CD137 (4- IBB), CD278 (ICOS), DAP10, LAT, SLP76, TRATl, TNFR2, and ZAP70.
  • a chimeric cytokine receptor comprises one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134, and a CD3z primary signaling domain.
  • polypeptides are contemplated herein, including, but not limited to, DARIC binding components, DARIC signaling components, engineered TCRs, CARs, zetakines, fusion proteins comprising the foregoing polypeptides and fragments thereof.
  • a polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-29.
  • “Polypeptide,”“peptide” and“protein” are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids.
  • a“polypeptide” includes fusion polypeptides and other variants.
  • Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence, a fragment of a full-length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In particular preferred embodiments, fusion polypeptides, polypeptides, fragments and other variants thereof are prepared, obtained, or isolated from one or more human polypeptides.
  • an“isolated peptide” or an“isolated polypeptide” and the like refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
  • an isolated polypeptide is a synthetic polypeptide, a semi -synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.
  • Polypeptides include“polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the binding affinity and/or other biological properties of a polypeptide by introducing one or more substitutions, deletions, additions and/or insertions the polypeptide.
  • polypeptides include polypeptides having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
  • the biological activity is binding affinity.
  • the biological activity is enzymatic activity.
  • a DARIC comprises a polypeptide complex comprising (i) a first polypeptide, e.g., first fusion polypeptide, having a first multimerization domain and (ii) second polypeptide, e.g, second fusion polypeptide, having a second multimerization domain.
  • the multimerization domains are the same; in certain embodiments, the first multimerization domain is different than the second multimerization domain.
  • the first and second multimerization domains substantially contribute to or efficiently promote formation of the polypeptide complex in the presence of a bridging factor.
  • the interaction(s) between the first and second multimerization domains substantially contributes to or efficiently promotes the multimerization of the first and second fusion polypeptides if there is a statistically significant reduction in the association between the first and second fusion polypeptides in the absence of the first multimerization domain, the second multimerization domain, or the bridging factor.
  • the first and second fusion polypeptides when the first and second fusion polypeptides are co-expressed, at least about 60%, for instance, at least about 60% to about 70%, at least about 70% to about 80%, at least about 80% to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second single chain polypeptides form multimers with each other in the presence of a bridging factor.
  • Polypeptides variants include biologically active“polypeptide fragments.”
  • biologically active polypeptide fragments include binding domains, intracellular signaling domains, and the like.
  • biologically active fragment or“minimal biologically active fragment” refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity.
  • a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • polypeptides set forth herein may comprise one or more amino acids denoted as“X.”“X” if present in an amino acid SEQ ID NO, refers to any one or more amino acids.
  • SEQ ID NOs denoting a fusion protein comprise a sequence of continuous X residues that cumulatively represent any amino acid sequence.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See , for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al, ( 1987, Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J. D.
  • a polypeptide variant comprises one or more conservative substitutions.
  • A“conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
  • polypeptide sequences encoding them can be separated by an IRES sequence as disclosed elsewhere herein.
  • Polypeptides contemplated in particular embodiments include fusion polypeptides.
  • fusion polypeptides and polynucleotides encoding fusion polypeptides are provided.
  • Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
  • a fusion polypeptide comprises one or more DARIC components.
  • the fusion polypeptide comprises one or more DARICs.
  • two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving peptide sequences between the
  • Fusion polypeptides can comprise one or more polypeptide domains or segments including, but are not limited to signal peptides, cell permeable peptide domains (CPP), binding domains, signaling domains, etc ., epitope tags (e.g, maltose binding protein (“MBP”), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals.
  • Fusion polypeptides are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C- terminus, N-terminus to N-terminus, or N-terminus to C-terminus.
  • the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques. Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.
  • Fusion polypeptides may optionally comprise one or more linkers that can be used to link the one or more polypeptides or domains within a polypeptide.
  • a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions.
  • Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence.
  • Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al ., Gene 40:39-46, 1985; Murphy et al. , Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S.
  • Linker sequences are not required when a particular fusion polypeptide segment contains non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • preferred linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein.
  • Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between.
  • Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites ( e.g ., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides ( see deFelipe and Ryan, 2004. Traffic , 5(8); 616-26).
  • polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g ., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides ( see deFelipe and Ryan, 2004. Traffic , 5(8); 616-26).
  • Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan et al. , 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).
  • Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus NIa proteases (e.g, tobacco etch virus protease), poty virus HC proteases, potyvirus PI (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2 A proteases, picoma 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.
  • potyvirus NIa proteases e.g, tobacco etch virus protease
  • potyvirus PI (P35) proteases by
  • TEV tobacco etch virus protease cleavage sites
  • EXXYXQ(G/S) SEQ ID NO: 41
  • ENLYFQG SEQ ID NO: 42
  • ENLYFQS SEQ ID NO: 43
  • the polypeptide cleavage signal is a viral self-cleaving peptide or ribosomal skipping sequence.
  • ribosomal skipping sequences include but are not limited to: a 2A or 2A-like site, sequence or domain (Donnelly et ah, 2001. J. Gen. Virol. 82: 1027- 1041).
  • the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
  • the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis A virus
  • TaV Thosea asigna virus
  • PTV-1 porcine teschovirus-1
  • Theilovirus 2A peptide a Theilovirus 2A peptide
  • an encephalomyocarditis virus 2A peptide Illustrative examples of 2A sites are provided in Table 2.
  • a polypeptide or fusion polypeptide comprises one or more DARIC components or DARJCs.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain; a viral self- cleaving 2A polypeptide; and a DARIC binding component comprising a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4
  • transmembrane domain or AMN transmembrane domain and a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, 0X40 or TNFR2 costimulatory domain.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain; a viral self cleaving 2A polypeptide; and a DARIC binding component comprising a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4
  • transmembrane domain or AMN transmembrane domain or AMN transmembrane domain, and a CD27 or CD28 costimulatory domain.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain; a viral self cleaving 2A polypeptide; and a DARIC binding component comprising a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4
  • transmembrane domain or AMN transmembrane domain a transmembrane domain or AMN transmembrane domain, and a TNFRS14, TNFRS18, or TNFRS25 costimulatory domain.
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a CD3z primary signaling domain; a viral self cleaving 2A polypeptide; and a DARIC binding component comprising a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4
  • a fusion polypeptide comprises a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8a transmembrane domain, a CD 137 costimulatory domain and a O ⁇ 3z primary signaling domain; a viral self cleaving 2A polypeptide; and a DARIC binding component comprising a binding domain that binds BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4
  • transmembrane domain or AMN transmembrane domain or AMN transmembrane domain, and a TNFR2 costimulatory domain.
  • polynucleotides encoding one or more DARIC components engineered TCRs, CARs, zetakines, fusion proteins comprising the foregoing polypeptides and fragments thereof are provided.
  • DARIC components engineered TCRs, CARs, zetakines, fusion proteins comprising the foregoing polypeptides and fragments thereof.
  • polynucleotide or“nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotides may be single-stranded or double- stranded and either recombinant, synthetic, or isolated.
  • Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
  • pre-mRNA pre-messenger RNA
  • mRNA messenger RNA
  • RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • ribozymes genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sg
  • Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or
  • deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths means any length between the quoted values, such as 6, 7, 8, 9, etc ., 101, 102, 103, etc .; 151, 152, 153, etc .; 201, 202, 203, etc.
  • polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%,
  • isolated polynucleotide refers to a polynucleotide that has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • An“isolated polynucleotide” also refers to a complementary DNA (cDNA), a recombinant DNA, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
  • an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.
  • a polynucleotide comprises an mRNA encoding a polypeptide contemplated herein.
  • the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.
  • polynucleotides encoding one or more DARJC components may be codon-optimized.
  • the term“codon-optimized” refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
  • Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated removal of spurious translation initiation sites.
  • nucleotide sequences that encode a polypeptide, or fragment of variant thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection. In particular embodiments, the polynucleotides are codon optimized for expression and/or stability.
  • nucleotide refers to a heterocyclic nitrogenous base in N- glycosidic linkage with a phosphorylated sugar.
  • Nucleotides are understood to include natural bases, and a wide variety of art-recognized modified bases. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety.
  • Nucleotides generally comprise a base, sugar and a phosphate group.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • deoxyribose i.e., a sugar lacking a hydroxyl group that is present in ribose.
  • Exemplary natural nitrogenous bases include the purines, adenosine (A) and guanidine (G), and the pyrimidines, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)).
  • the C-l atom of deoxyribose is bonded to N-l of a pyrimidine or N-9 of a purine.
  • Nucleotides are usually mono, di- or triphosphates. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety,
  • nucleic acid bases are summarized by Limbach et ah, (1994, Nucleic Acids Res. 22, 2183-2196).
  • a nucleotide may also be regarded as a phosphate ester of a nucleoside, with esterification occurring on the hydroxyl group attached to C-5 of the sugar.
  • the term“nucleoside” refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, and also to include well known modified bases. Such bases are generally located at the 1 ' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group.
  • the nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides).
  • modified nucleic acid bases are summarized by Limbach et al ., (1994, Nucleic Acids Res. 22, 2183-2196).
  • polynucleotides include, but are not limited to,
  • polynucleotides encoding polypeptides set forth in SEQ ID NOs: 1-29.
  • polynucleotides contemplated herein include, but are not limited to polynucleotides encoding one or more DARIC components, DARJCs, engineered antigen receptors, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein.
  • polynucleotide variant and“variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and“variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
  • the recitations“sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide- by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a“percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g A, T, C, G, I) or the identical amino acid residue (e.g Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are polynucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
  • nucleic acid cassette or“expression cassette” as used herein refers to genetic sequences within the vector which can express an RNA, and subsequently a polypeptide.
  • the nucleic acid cassette contains a gene(s)-of-interest, e.g ., a polynucleotide(s)-of-interest.
  • nucleic acid cassette contains one or more expression control sequences, e.g. , a promoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g, a polynucleotide(s)-of-interest.
  • Vectors may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes.
  • the nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into
  • the cassette has its 3' and 5' ends adapted for ready insertion into a vector, e.g. , it has restriction endonuclease sites at each end.
  • the cassette can be removed and inserted into a plasmid or viral vector as a single unit.
  • Polynucleotides include polynucleotide(s)-of-interest.
  • polynucleotide-of-interest refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for the transcription of an inhibitory polynucleotide, as contemplated herein.
  • polynucleotides contemplated herein may be combined with other DNA sequences, e.g, expression control sequences such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g ., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • expression control sequences such as promoters and/or enhancers, untranslated regions (UTRs
  • Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art.
  • a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
  • vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac.
  • vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC)
  • bacteriophages such as lambda phage or M13 phage
  • animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or PI -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g, SV40).
  • retrovirus including lentivirus
  • adenovirus e.g, adeno-associated virus
  • herpesvirus e.g, herpes simplex virus
  • poxvirus baculovirus
  • papillomavirus papillomavirus
  • papovavirus e.g, SV40
  • expression vectors include, but are not limited to, pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5- DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
  • the vector is an episomal vector or a vector that is maintained extrachromosomally.
  • episomal vector refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates
  • “Expression control sequences,”“control elements,” or“regulatory sequences” present in an expression vector are those non-translated regions of the vector including an origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions, all of which interact with host cellular proteins to carry out transcription and translation.
  • Such elements may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including ubiquitous promoters and inducible promoters may be used.
  • a polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors.
  • a vector may comprise one or more exogenous, endogenous, or heterologous control sequences such as promoters and/or enhancers.
  • An“endogenous control sequence” is one which is naturally linked with a given gene in the genome.
  • An“exogenous control sequence” is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
  • A“heterologous control sequence” is an exogenous sequence that is from a different species than the cell being genetically manipulated.
  • A“synthetic” control sequence may comprise elements of one more endogenous and/or exogenous sequences, and/or sequences determined in vitro or in silico that provide optimal promoter and/or enhancer activity for the particular therapy.
  • promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds.
  • An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter.
  • promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
  • enhancer refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
  • An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
  • promoter/enhancer refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • constitutive expression control sequence refers to a promoter, enhancer, or promoter/enhancer that continually or continuously allows for transcription of an operably linked sequence.
  • a constitutive expression control sequence may be a“ubiquitous” promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types or a“cell specific,”“cell type specific,”“cell lineage specific,” or“tissue specific” promoter, enhancer, or promoter/enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
  • Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and PI 1 promoters from vaccinia virus, an elongation factor 1 -alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5
  • CMV cytomegalovirus
  • HSPA5 heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), b-kinesin (b-KIN), the human ROSA 26 locus (Irions et al. , Nature
  • Ubiquitin C promoter Ubiquitin C promoter
  • PGK phosphoglycerate kinase-1
  • CAG cytomegalovirus enhancer/chicken b-actin
  • MND myeloproliferative sarcoma virus enhancer
  • a vector comprises an MNDTJ3 promoter.
  • a vector comprises an EFla promoter comprising the first intron of the human EFla gene.
  • a vector comprises an EFla promoter that lacks the first intron of the human EFla gene.
  • a cell, cell type, cell lineage or tissue specific expression control sequence may be desirable to use to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).
  • condition expression may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression;
  • Certain embodiments provide conditional expression of a polynucleotide-of-interest, e.g, expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.
  • inducible prom oters/sy stems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone),
  • Inducer agents include, but are not limited to glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.
  • an“internal ribosome entry site” or“IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al. , 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736.
  • IRES immunoglobulin heavy-chain binding protein
  • VEGF vascular endothelial growth factor
  • IRES have also been reported in viral genomes of Picornaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV).
  • the IRES used in polynucleotides contemplated herein is an EMCV IRES.
  • the polynucleotides comprise a consensus Kozak sequence.
  • Kozak sequence refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation.
  • the consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 66), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20): 8125-48).
  • heterologous nucleic acid transcripts increases heterologous gene expression.
  • Transcription termination signals are generally found downstream of the polyadenylation signal.
  • vectors comprise a polyadenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed.
  • the term“polyA site” or“polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation are directed by a poly(A) sequence in the RNA.
  • the core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage- polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product.
  • the core poly(A) sequence is an ideal polyA sequence (e.g ., AATAAA, ATT AAA, AGTAAA).
  • the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit b-globin polyA sequence (rPgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.
  • the poly(A) sequence is synthetic.
  • a polynucleotide or cell harboring the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and/or uncontrolled proliferation.
  • the suicide gene is not immunogenic to the host harboring the polynucleotide or cell.
  • a certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).
  • polynucleotides encoding one or more polypeptides, or fusion polypeptides may be introduced into immune effector cells, e.g., T cells, by both non-viral and viral methods.
  • delivery of one or more polynucleotides may be provided by the same method or by different methods, and/or by the same vector or by different vectors.
  • vector is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to, e.g, inserted into, the vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA.
  • non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell.
  • non-viral vectors include, but are not limited to plasmids (e.g, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.
  • Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock.
  • polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc.
  • Lipofection reagents are sold commercially (e.g, TransfectamTM and LipofectinTM). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10: 180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011 : 1-12.
  • Antibody-targeted, bacterially derived, non-living nanocell-based delivery is also contemplated in particular embodiments.
  • Viral vectors comprising polynucleotides contemplated in particular embodiments can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g, intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo , such as cells explanted from an individual patient (e.g, mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient.
  • viral vectors comprising polynucleotides contemplated herein are administered directly to an organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • embodiments contemplated in particular embodiments include, but are not limited to, adeno- associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • herpes simplex virus adenovirus
  • vaccinia virus vectors vaccinia virus vectors.
  • one or more polynucleotides encoding one or more DARIC components and/or other polypeptides contemplated herein are introduced into an immune effector cell, e.g, T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV), comprising the one or more polynucleotides.
  • rAAV adeno-associated virus
  • AAV is a small ( ⁇ 26 nm) replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.
  • Recombinant AAV are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).
  • the ITR sequences are about 145 bp in length.
  • the rAAV comprises ITRs and capsid sequences isolated from AAVl, AAV2, AAV3,
  • AAV4 AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • a chimeric rAAV is used the ITR sequences are isolated from one AAV serotype and the capsid sequences are isolated from a different AAV serotype.
  • a rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2/AAV6.
  • the rAAV vector may comprise ITRs from AAV2, and capsid proteins from any one of AAVl, AAV2,
  • the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV2.
  • engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.
  • one or more polynucleotides encoding one or more DARIC components and/or other polypeptides contemplated herein are introduced into an immune effector cell, e.g, T cell, by transducing the cell with a retrovirus, e.g, lentivirus, comprising the one or more polynucleotides.
  • an immune effector cell e.g, T cell
  • a retrovirus e.g, lentivirus
  • retrovirus refers to an RNA virus that reverse transcribes its genomic RNA into a linear double- stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome.
  • retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M- MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus
  • HaMuSV murine mammary tumor virus
  • MoMTV gibbon ape leukemia virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • BSV Rous Sarcoma Virus
  • lentivirus refers to a group (or genus) of complex retroviruses.
  • Illustrative lentiviruses include, but are not limited to, HIV (human
  • HIV based vector backbones i.e HIV cis-acting sequence elements are preferred.
  • a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT/FLAP, a Psi (Y) packaging signal, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
  • lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus.
  • integration defective lentivirus or“IDLV” refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells.
  • HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71 A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253
  • FLAP element refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2.
  • cPPT and CTS central polypurine tract and central termination sequences
  • Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, etal, 2000, Cell , 101 : 173.
  • the term“packaging signal” or“packaging sequence” refers to psi [Y] sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle, see e.g, Clever etal, 1995. ./. of Virology, Vol. 69, No.
  • RNA export element refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
  • Examples of RNA export elements include, but are not limited to, the human
  • HIV immunodeficiency virus
  • RRE immunodeficiency virus rev response element
  • HPRE hepatitis B virus post- transcriptional regulatory element
  • expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors.
  • posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g, woodchuck hepatitis vims posttranscriptional regulatory element (WPRE; Zufferey et al, 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B vims (HPRE) (Huang el al, Mol. Cell. Biol., 5:3864); and the like (Liu el al, 1995, Genes Dev., 9: 1766).
  • WPRE woodchuck hepatitis vims posttranscriptional regulatory element
  • HPRE posttranscriptional regulatory element present in hepatitis B vims
  • Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs.
  • “Self-inactivating” (SIN) vectors refers to replication-defective vectors, e.g, retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g, by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
  • Self-inactivation is preferably achieved through in the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to produce the vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA.
  • heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g, immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.
  • SV40 viral simian virus 40
  • CMV cytomegalovirus
  • MoMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • HSV herpes simplex virus
  • pseudotype or“pseudotyping” as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing preferable characteristics.
  • HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4 + presenting cells.
  • VSV-G vesicular stomatitis virus G-protein
  • lentiviral vectors are produced according to known methods. See e.g., Kutner et al, BMC Biotechnol. 2009;9: 10. doi: 10.1186/1472-6750-9- 10; Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22.
  • most or all of the viral vector backbone sequences are derived from a lentivirus, e.g, HIV-1.
  • a lentivirus e.g, HIV-1.
  • many different sources of retroviral and/or lentiviral sequences can be used or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein.
  • a variety of lentiviral vectors are known in the art, see Naldini et al, (1996a, 1996b, and 1998); Zufferey et al, (1997); Dull et ah, 1998, U.S. Pat. Nos. 6,013,516; and 5,994, 136, many of which may be adapted to produce a viral vector or transfer plasmid contemplated herein.
  • one or more polynucleotides encoding one or more DARIC components and/or other polypeptides contemplated herein are introduced into an immune effector cell, by transducing the cell with an adenovirus comprising the one or more polynucleotides.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo , including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • Generation and propagation of the current adenovirus vectors may utilize a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al ., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991).
  • a unique helper cell line designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al ., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham & Prevec, 1991
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al. , 1991; Gomez-Foix et al. , 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992).
  • Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al. , 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al. , 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al. , 1993).
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman etal, Hum. Gene Ther. 7:1083-9 (1998)).
  • one or more polynucleotides encoding one or more DARIC components and/or other polypeptides contemplated herein are introduced into an immune effector cell by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides.
  • a herpes simplex virus e.g., HSV-1, HSV-2
  • the mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb.
  • the HSV based viral vector is deficient in one or more essential or non- essential HSV genes.
  • the HSV based viral vector is replication deficient. Most replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication.
  • the HSV vector may be deficient in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof.
  • HSV vectors are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb.
  • HSV- based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, each of which are incorporated by reference herein in its entirety.
  • cells are modified to express one or more DARIC components, DARICs, engineered TCRs, CARs, zetakines, and/or fusion proteins contemplated herein, for use in the treatment of cancer.
  • Cells may be non-genetically modified to express one or more of the polypeptides contemplated herein, or in particular preferred embodiments, cells may be genetically modified to express one or more of the polypeptides contemplated herein.
  • the term“genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell.
  • the terms,“genetically modified cells,” “modified cells,” and“redirected cells,” are used interchangeably in particular
  • one or more DARIC components contemplated herein are introduced and expressed in immune effector cells to improve the efficacy of the immune effector cells.
  • a dual targeting immune effector cell is contemplated where the target cell expresses an antigen recognized by a first DARIC binding component and another antigen recognized by a second DARIC binding component.
  • one or more DARIC components are introduced and expressed in immune effector cells that have been redirected to a target cell by virtue of co-expressing an engineered antigen receptor, e.g., a CAR, in the cell.
  • a dual targeting immune effector cell is contemplated where the target cell expresses an antigen recognized by the engineered antigen receptor and a different antigen recognized by a DARIC.
  • An“immune effector cell,” is any cell of the immune system that has one or more effector functions (e.g, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the illustrative immune effector cells contemplated herein are T lymphocytes, including but not limited to cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + T cells).
  • the cells comprise ab T cells.
  • the cells comprise gd T cells.
  • immune effector cells include natural killer (NK) cells.
  • immune effector cells include natural killer T (NKT) cells.
  • Immune effector cells can be autologous/autogeneic (“self’) or non-autologous (“non-self,” e.g, allogeneic, syngeneic or xenogeneic).
  • “Autologous,” as used herein, refers to cells from the same subject.“Allogeneic,” as used herein, refers to cells of the same species that differ genetically to the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison.“Xenogeneic,” as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells are human autologous immune effector cells.
  • Illustrative immune effector cells suitable for introducing one or more DARIC components or a DARIC contemplated herein include T lymphocytes.
  • T cell or“T lymphocyte” are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • a T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
  • the T cell can be a helper T cell (HTL; CD4 + T cell) CD4 + T cell, a cytotoxic T cell (CTL; CD8 + T cell), CD4 + CD8 + T cell, CD4 CD8- T cell, or any other subset of T cells.
  • helper T cell HTL; CD4 + T cell
  • CTL cytotoxic T cell
  • CD4 + CD8 + T cell CD4 + CD8 + T cell
  • CD4 CD8- T cell CD4 CD8- T cell
  • immune effector cells comprising one or more DARIC components or DARIC s contemplated herein.
  • immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages.
  • Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro.
  • immune effector cells include progenitors of immune effectors cells such as hematopoietic stem cells (HSCs) contained within the CD34 + population of cells derived from cord blood, bone marrow or mobilized peripheral blood which upon administration in a subject differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells.
  • HSCs hematopoietic stem cells
  • 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.
  • the CD34 + cell population contains hematopoietic stem cells (HSC), which upon administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils and cells of the monocyte/macrophage lineage.
  • HSC hematopoietic stem cells
  • the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells with one or more nucleic acids and/or vectors or combination thereof comprising one or more DARIC components contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more DARIC components and engineered antigen receptors contemplated herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified. In this regard, the immune effector cells may be cultured before and/or after being genetically modified.
  • the source of cells is obtained from a subject.
  • the modified immune effector cells comprise T cells.
  • T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLLTM separation.
  • an isolated or purified population of T cells is used.
  • both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • an isolated or purified population of T cells expresses one or more of the markers including, but not limited to a CD3 + , CD4 + , CD8 + , or a combination thereof
  • the T cells are isolated from an individual and first activated and stimulated to proliferate in vitro prior to being modified to express one or more DARIC components.
  • T cells are often subjected to one or more rounds of stimulation, activation and/or expansion.
  • T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964;
  • T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours or about 24 hours prior to introduction of vectors or polynucleotides encoding one or more DARIC components, optionally in combination with an engineered antigen receptor contemplated herein.
  • T cells are activated at the same time that they are modified.
  • a method of generating an immune effector cell comprises activating a population of cells comprising T cells and expanding the population of T cells.
  • T cell activation can be accomplished by providing a primary stimulation signal through the T cell TCR/CD3 complex and by providing a secondary costimulation signal through an accessory molecule, e.g., CD28.
  • the TCR/CD3 complex may be stimulated by contacting the T cell with a suitable CD3 binding agent, e.g, a CD3 ligand or an anti-CD3 monoclonal antibody.
  • a suitable CD3 binding agent e.g, a CD3 ligand or an anti-CD3 monoclonal antibody.
  • CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.
  • CD28 binding agent can be used to provide a costimulatory signal.
  • CD28 binding agents include but are not limited to: natural CD 28 ligands, e.g., a natural ligand for CD28 (e.g.
  • B7-1(CD80) and B7-2 (CD86); and anti-CD28 monoclonal antibody or fragment thereof capable of crosslinking the CD28 molecule e.g, monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.
  • the molecule providing the primary stimulation signal for example a molecule which provides stimulation through the TCR/CD3 complex and the costimulatory molecule are coupled to the same surface.
  • binding agents that provide stimulatory and costimulatory signals are localized on the surface of a cell. This can be accomplished by transfecting or transducing a cell with a nucleic acid encoding the binding agent in a form suitable for its expression on the cell surface or alternatively by coupling a binding agent to the cell surface.
  • the molecule providing the primary stimulation signal for example a molecule which provides stimulation through the TCR/CD3 complex and the costimulatory molecule are displayed on antigen presenting cells.
  • the molecule providing the primary stimulation signal for example a molecule which provides stimulation through the TCR/CD3 complex and the costimulatory molecule are provided on separate surfaces.
  • one of the binding agents that provides stimulatory and costimulatory signals is soluble (provided in solution) and the other agent(s) is provided on one or more surfaces.
  • the binding agents that provide stimulatory and costimulatory signals are both provided in a soluble form (provided in solution).
  • the methods for making T cells contemplated herein comprise activating T cells with anti-CD3 and anti-CD28 antibodies.
  • expanding T cells activated by the methods contemplated herein further comprises culturing a population of cells comprising T cells for several hours (about 3 hours) to about 7 days to about 28 days or any hourly integer value in between.
  • the T cell composition may be cultured for 14 days.
  • T cells are cultured for about 21 days.
  • the T cell compositions are cultured for about 2-3 days.
  • stimulation/activation/expansion may also be desired such that culture time of T cells can be 60 days or more.
  • conditions appropriate for T cell culture include an appropriate media (e.g ., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability including, but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives suitable for the growth of cells known to the skilled artisan.
  • serum e.g., fetal bovine or human serum
  • IL-2 interleukin-2
  • insulin IFN-g
  • IL-4 interleukin-2
  • IFN-g interleukin-2
  • IL-7 interleukin-2
  • IL-21 interleukin-21
  • GM-CSF IL-10
  • IL-12 IL-15
  • TGFp TNF-a or any other additive
  • cell culture media include, but are not limited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g, penicillin and streptomycin
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g, 37° C) and atmosphere (e.g, air plus 5% C02).
  • PBMCs or isolated T cells are contacted with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2, IL-7, and/or IL-15.
  • a stimulatory agent and costimulatory agent such as anti-CD3 and anti-CD28 antibodies
  • cytokines such as IL-2, IL-7, and/or IL-15.
  • artificial APC made by engineering K562, U937, 721.221, T2, and C1R cells to direct the stable expression and secretion, of a variety of costimulatory molecules and cytokines.
  • K32 or U32 aAPCs are used to direct the display of one or more antibody -based stimulatory molecules on the AAPC cell surface.
  • Populations of T cells can be expanded by aAPCs expressing a variety of costimulatory molecules including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and/or CD80 or CD86.
  • the aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8 T cells.
  • aAPCs provided in WO 03/057171 and US2003/0147869 are hereby incorporated by reference in their entirety.
  • a polynucleotide encoding one or more DARIC components is introduced into the population of T cells.
  • a polynucleotide encoding one or more DARIC components is introduced into a population of T cells that express an engineered antigen receptor.
  • the polynucleotides may be introduced into the T cells by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transfer, and the like.
  • polynucleotides are introduced into a T cell by viral transduction.
  • polynucleotide into an immune effector cell or CD34 + cell include but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • herpes simplex virus adenovirus
  • vaccinia virus vectors for gene transfer include but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.
  • polynucleotides are introduced into a T cell by AAV transduction.
  • polynucleotides are introduced into a T cell by retroviral transduction.
  • polynucleotides are introduced into a T cell by lentiviral transduction.
  • polynucleotides are introduced into a T cell by adenovirus transduction.
  • polynucleotides are introduced into a T cell by herpes simplex virus transduction. In one embodiment, polynucleotides are introduced into a T cell by vaccinia virus transduction.
  • compositions contemplated herein may comprise one or more DARIC polypeptides, polynucleotides encoding DARIC polypeptides, vectors comprising same, genetically modified immune effector cells, bridging factors, etc.
  • Compositions include, but are not limited to, pharmaceutical compositions.
  • A“pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically - acceptable solutions for administration to a cell or an animal, either alone, or in
  • compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents.
  • agents such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents.
  • additional agents do not adversely affect the ability of the composition to deliver the intended therapy.
  • phrases“pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which the bridging factors, polypeptides, polynucleotides, vectors comprising same, or genetically modified immune effector cells are administered.
  • pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject.
  • a pharmaceutically acceptable carrier is suitable for administration to a subject.
  • composition comprising a carrier is suitable for parenteral administration, e.g .,
  • a composition comprising a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal, or intrathecal administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions.
  • sterile aqueous solutions include sterile aqueous solutions, cell culture media, or dispersions.
  • 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
  • compositions contemplated herein comprise T cells genetically modified to express one or more DARIC components and/or engineered antigen receptors and a pharmaceutically acceptable carrier.
  • a composition comprising a cell-based composition contemplated herein can be administered separately by enteral or parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals.
  • compositions contemplated herein comprise a bridging factor and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition.
  • the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the composition.
  • the pharmaceutically acceptable carrier can be, without limitation, a binding agent (e.g ., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g, sodium lauryl sulfate, etc.).
  • a binding agent e.g ., pregelatinized maize starch, poly
  • compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like.
  • Such carrier solutions also can contain buffers, diluents and other suitable additives.
  • buffer refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
  • buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
  • the pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7.
  • the composition has a pH in a range from about 6.8 to about 7.4, e.g, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4.
  • the composition has a pH of about 7.4.
  • compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium.
  • the compositions may be a suspension.
  • the term“suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish.
  • compositions contemplated herein are formulated in a suspension, where the T cells modified with one or more polynucleotides encoding one or more DARIC components and/or engineered antigen receptors are dispersed within an acceptable liquid medium or solution, e.g, saline or serum-free medium, in an intravenous (IV) bag or the like.
  • acceptable liquid medium or solution e.g, saline or serum-free medium
  • IV intravenous
  • a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of modified T cells.
  • the therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.
  • compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects.
  • the pharmaceutically acceptable cell culture medium is a serum free medium.
  • Serum-free medium has several advantages over serum containing medium, including a simplified and better-defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost.
  • the serum-free medium is animal-free, and may optionally be protein-free.
  • the medium may contain biopharmaceutically acceptable recombinant proteins.
  • “Animal-free” medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal- free medium and the nutrients are obtained from synthetic, plant or microbial sources.
  • Protein-free in contrast, is defined as substantially free of protein.
  • serum-free media used in particular compositions includes, but is not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.
  • compositions comprising modified T cells are formulated in PlasmaLyte.
  • compositions comprising modified T cells are formulated in a cryopreservation medium.
  • cryopreservation agents may be used to maintain a high cell viability outcome post-thaw.
  • Illustrative examples of cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.
  • compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.
  • the composition is substantially free of mycoplasma, endotoxin, and microbial contamination.
  • substantially free with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells.
  • compositions contemplated herein contain about 0.5 EU/mL to about 5.0 EU/mL, or about 0.5 EU/mL, 1.0 EU/mL, 1.5 EU/mL, 2.0 EU/mL, 2.5 EU/mL, 3.0 EU/mL, 3.5 EU/mL, 4.0 EU/mL, 4.5 EU/mL, or 5.0 EU/mL.
  • formulation of pharmaceutically-acceptable carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g ., enteral and parenteral, e.g, intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • enteral and parenteral e.g, intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • enteral and parenteral e.g, intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
  • particular embodiments contemplated herein may comprise
  • compositions comprise an amount of immune effector cells that express one or more DARIC components contemplated herein.
  • compositions comprise an amount of immune effector cells that express an engineered antigen receptor and one or more DARIC components contemplated herein.
  • the term“amount” refers to“an amount effective” or“an effective amount” of cells comprising one or more DARIC components contemplated herein, etc., to achieve a beneficial or desired prophylactic or therapeutic result in the presence of a bridging factor, including clinical results.
  • A“prophylactically effective amount” refers to an amount of cells comprising one or more DARIC components contemplated herein, etc ., effective to achieve the desired prophylactic result in the presence of a bridging factor. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • A“therapeutically effective amount” refers to an amount of cells comprising one or more DARIC components contemplated herein that is effective to“treat” a subject (e.g ., a patient) in the presence of a bridging factor.
  • a therapeutic amount is indicated, the precise amount of the compositions to be administered, cells, bridging factor, etc, can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • a pharmaceutical composition comprising the immune effector cells described herein may be administered at a dosage of 10 2 to
  • 10 10 cells/kg body weight preferably 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges.
  • the number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein.
  • the cells are generally in a volume of a liter or less, can be 500 mLs or less, even 250 mLs or 100 mLs or less.
  • the density of the desired cells is typically greater than 10 6 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells.
  • lower numbers of cells in the range of 10 6 /kilogram (10 6 -10 u per patient) may be administered.
  • the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g, IFN-g, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIRIa, etc.) as described herein to enhance induction of the immune response.
  • mitogens e.g., PHA
  • lymphokines e.g., lymphokines, cytokines, and/or chemokines (e.g, IFN-g, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIRIa, etc.)
  • mitogens e.g., PHA
  • lymphokines e.g., lymphokines, e.
  • compositions comprise an amount of genetically modified T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions comprise an amount of bridging factor, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions comprise an effective amount of immune effector cells comprising one or more DARIC components contemplated herein, alone or in combination with a bridging factor and/or one or more therapeutic agents, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc.
  • therapeutic agents such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc.
  • the compositions may also be administered in combination with antibiotics.
  • therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer.
  • Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti inflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents.
  • compositions comprising an effective amount of immune effector cells comprising one or more DARIC components contemplated herein are administered to a subject, and a composition comprising an effective amount of a bridging factor is administered to the subject, before, during, in combination with or subsequently to the cellular composition, and optionally repetitively administered to the subject.
  • compositions comprising immune effector cells comprising one or more DARIC components contemplated herein may be administered in conjunction with any number of chemotherapeutic agents.
  • compositions described herein comprising immune effector cells comprising one or more DARIC components contemplated herein is administered with an anti-inflammatory agent.
  • Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone,
  • NS AIDS nonsteroidal anti inflammatory drugs
  • aspirin ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
  • therapeutic antibodies suitable for combination treatment with the modified T cells comprising one or more DARIC components contemplated herein include but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab
  • bivatuzumab blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab, dalotuzumab, durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.
  • compositions described herein are administered in conjunction with a cytokine.
  • cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • Immune effector cells modified to express a DARIC and/or an engineered antigen receptor contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of, or for preventing, treating, or ameliorating at least one symptom associated with, a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
  • immune effector cells modified to express a DARIC provide improved methods of adoptive immunotherapy to fine-tune the safety and efficacy of a cytotoxic response against target cells, e.g., tumor cells, expressing target antigens while decreasing the risk of on-target antigen, off-target cell cytotoxicity (recognizing the target antigen on a normal, non-target cell).
  • target cells e.g., tumor cells, expressing target antigens while decreasing the risk of on-target antigen, off-target cell cytotoxicity (recognizing the target antigen on a normal, non-target cell).
  • a method of preventing, treating, or ameliorating at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency comprises administering the subject an effective amount of modified immune effector cells or T cells comprising one or more components of a DARIC and an engineered TCR, CAR, or other therapeutic transgene to redirect the cells to a target cell.
  • the genetically modified cells are a more efficacious and safe cellular immunotherapy by virtue of transducing a chemically regulatable
  • one or more immune effector cells are modified to express both a DARIC binding component and a DARIC signaling component.
  • the modified cells are administered to a subject in need thereof and home to the target cells via the interaction of the DARIC binding component expressed on the immune effector cell and the target antigen expressed on the target cell.
  • a bridging factor is administered to the subject before the modified cells, about the same time as the modified cells, or after the modified cells have been administered to the subject.
  • a ternary complex forms between the DARIC binding component, the bridging factor, and the DARIC signaling component.
  • the DARIC transduces an immunostimulatory signal to the immune effector cell that in turn, elicits a cytotoxic response from the immune effector cell against the target cell.
  • immune effector cells comprising a DARIC and/or an engineered antigen receptor fine-tune the safety and efficacy of a cytotoxic response against target cells using a dual targeting strategy wherein one or more target cells express one or more target antigens recognized by the engineered antigen receptor and one or more target antigens recognized by the DARIC.
  • one or more immune effector cells are modified to express both the DARIC binding component and the DARIC signaling component and an engineered antigen receptor, e.g, a CAR.
  • the modified cells are administered to a subject in need thereof and home to the target cells via the interaction of the DARIC binding component and the CAR, both of which are expressed on the immune effector cell, and the target antigens expressed on the target cell. Interaction of the CAR with a target antigen on the target cell may elicit a cytotoxic response from the immune effector cell against the target cell.
  • a bridging factor is administered to the subject before the modified cells, about the same time as the modified cells, or after the modified cells have been administered to the subject.
  • a ternary complex forms between the DARIC binding component, the bridging factor, and the DARIC signaling component.
  • the DARIC transduces an immunostimulatory signal to the immune effector cell that in turn, elicits or augments a cytotoxic response from the immune effector cell against the target cell.
  • DARIC activation can be induced in cases where remission or regression is incomplete and the condition relapses or becomes refractory to treatment.
  • the specificity of a primary T cell is redirected to tumor or cancer cells that express one or more target antigens by genetically modifying a T cell, e.g, a primary T cell, with one or more DARIC components.
  • the specificity of a primary T cell is redirected to tumor or cancer cells that express a target antigen recognized by an engineered antigen receptor and a target antigen recognized by a DARIC.
  • the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers.
  • the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical
  • teratoid/rhabdoid tumor basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer,
  • craniopharyngioma ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing’s sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors,
  • DCIS ductal carcinoma in situ
  • GIST gastrointestinal stromal tumor
  • germ cell tumors glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, menangioma, melanoma, Merkel cell carcinoma, midline tract carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer,
  • nasopharyngeal cancer neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine
  • the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
  • the modified immune effector cells contemplated herein are used in the treatment of various cancers including but not limited to pancreatic, bladder, and lung. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of liquid cancers or hematological cancers.
  • the modified immune effector cells contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma.
  • the modified immune effector cells contemplated herein are used in the treatment of liquid cancers including, but not limited to leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymph
  • ALL acute
  • Preferred cells for use in the methods contemplated herein include
  • autologous/autogeneic (“self’) cells preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.
  • a method comprises administering a therapeutically effective amount of modified immune effector cells that express one or more DARIC components, and optionally an engineered antigen receptor, or a composition comprising the same, to a patient in need thereof, and also administering a bridging factor to the subject.
  • the cells are used in the treatment of patients at risk for developing a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
  • particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency comprising administering to a subject in need thereof, a therapeutically effective amount of the modified immune effector cells contemplated herein and a bridging factor.
  • a method comprises administering a therapeutically effective amount of modified immune effector cells that express a DARIC signaling component, and optionally an engineered antigen receptor, or a composition comprising the same, to a patient in need thereof, and also administering a DARIC binding component and a bridging factor, optionally where the DARIC binding component is bound to the bridging factor prior to administration, to the subject.
  • the cells are used in the treatment of patients at risk for developing a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.
  • particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency comprising administering to a subject in need thereof, a therapeutically effective amount of the modified immune effector cells that express an DARIC signaling component and optionally an engineered antigen receptor, a DARIC binding component, and a bridging factor.
  • modified immune effector cells DARIC binding components, and/or bridging factor
  • the quantity and frequency of administration of modified immune effector cells, DARIC binding components, and/or bridging factor will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages and dose schedules may be determined by clinical trials.
  • the effective amount of modified immune effector cells provided to a subject is at least 2 x 10 6 cells/kg, at least 3 x 10 6 cells/kg, at least 4 x 10 6 cells/kg, at least 5 x 10 6 cells/kg, at least 6 x 10 6 cells/kg, at least 7 x 10 6 cells/kg, at least 8 x 10 6 cells/kg, at least 9 x 10 6 cells/kg, or at least 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of modified immune effector cells provided to a subject is about 2 x 10 6 cells/kg, about 3 x 10 6 cells/kg, about 4 x 10 6 cells/kg, about 5 x 10 6 cells/kg, about 6 x 10 6 cells/kg, about 7 x 10 6 cells/kg, about 8 x 10 6 cells/kg, about 9 x 10 6 cells/kg, or about 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of modified immune effector cells provided to a subject is from about 2 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 3 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 4 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 5 x 10 6 cells/kg to about 10 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells
  • compositions contemplated in particular embodiments may be required to effect the desired therapy.
  • a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.
  • Modified immune effector cells, DARIC components, and bridging factor may be administered in the same or different compositions; in one or more compositions at the same time; or more than one composition at different times.
  • Modified immune effector cells, DARIC components, and bridging factor may be administered through the same route of administration or different routes.
  • T cells can be activated from blood draws of from lOcc to 400cc.
  • T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, lOOcc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more.
  • using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells.
  • a method of treating a subject diagnosed with a cancer comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing one or more vectors encoding one or more DARIC components into the cell and producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject.
  • the immune effector cells comprise T cells.
  • a method of treating a subject diagnosed with a cancer comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing one or more vectors encoding an engineered antigen receptor and one or more DARIC components into the cell and producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject.
  • the immune effector cells comprise T cells.
  • the methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo modified immune effector cells or reintroduction of modified progenitors of immune effector cells that upon introduction into a subject differentiate into mature immune effector cells.
  • One method comprises modifying peripheral blood T cells ex vivo by introducing one or more vectors encoding an engineered antigen receptor and one or more DARIC components and returning the transduced cells into the subject.
  • the methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo modified immune effector cells or reintroduction of modified progenitors of immune effector cells that upon introduction into a subject differentiate into mature immune effector cells.
  • One method comprises modifying peripheral blood T cells ex vivo by introducing one or more vectors encoding one or more DARIC components and returning the transduced cells into the subject.
  • Lenti viral vectors comprising NKG2D DARIC s that have DARIC binding components comprising various costimulatory signaling domains were designed, constructed, and verified.
  • Figure 2A The costimulatory domains were obtained from TNFR2, 0X40, CD27, HVEM (TNFRS14), GITR (TNFRS18) and DR3 (TNFRS25) proteins. See, e.g., SEQ ID NOs: 1 and 20-22.
  • Human PBMCs (1 x 10 6 cells/mL) were activated with soluble anti-CD3 and anti- CD28 antibodies (50 ng/mL) on day 0. After 24hrs. incubation, 1 x 10 6 cells were transduced with LVV encoding the DARICs. An additional untransduced sample was included as a control (UTD). The cells were washed and resuspended at 0.3 x 10 6 cells/mL) on day 3. The cells were cultured for expansion 7 additional days in T cell growth medium containing IL-2 (250 IU/mL). Medium was changed every other day.
  • the cells were counted and split to a defined density with every media exchange. After the 10-day expansion period, the T cells were counted and phenotyped using CD4 and CD8 antibody staining.
  • NKG2D.OX40 DARIC T cells NKG2D.CD27 DARIC T cells, NKG2D.HVEM DARIC T cells, NKG2D.DR3 DARIC T cells, and NKG2D.GITR DARIC T cells displayed similar rates of ex vivo expansion.
  • Figure 2B The T cells were stained with anti-NKG2D antibody and DARIC binding component expression was quantified on CD4 + T cells. Expression was comparable among the different NKG2D DARIC binding components.
  • Figure 2C Together, the data suggest that a DARIC binding component comprising a costimulatory domain does not alter ex vivo T cell expansion or expression.
  • NKG2D.OX40 DARIC T cells, NKG2D.CD27 DARIC T cells, NKG2D.HVEM DARIC T cells, NKG2D.DR3 DARIC T cells, and NKG2D.GITR DARIC T cells were co-cultured with NKG2DL + HCT116 cells for 24 hrs in the presence or absence of rapamycin at 1 : 1 E:T ratio and cytokine production was analyzed by Qbead PlexScreen.
  • DARIC binding domains comprising a costimulatory domain consistently boosted cytokine production when T cells were cultured with tumor cells in the presence of rapamycin.
  • Human PBMCs were activated, transduced and expanded as described in Example 1.
  • Anti-EGFR CAR T cells, NKG2D DARIC T cells, NKG2D.TNFR2 DARIC T cells, and NKG2D.OX40 DARIC T cells were co-cultured with NKG2DL + A549 cells or NKG2DL + HT1080 cells at a 1 :1 ratio, in vehicle, rapamycin, or the non
  • NKG2D DARIC T cells did not produce cytokines when co-cultured with tumor cells in the absence of dimerization drug.
  • Figure 3 A and 3B There was robust cytokine production when NKG2D DARIC T cells were co-cultured with tumor cells in the presence of rapamycin and AP21967. Id. As expected, addition of rapamycin resulted in suppressed T cell activation and reduced cytokine production from anti-EGFR CAR T cells. Id. Similar immunosuppressive effects were observed for NKG2D DARIC T cells and NKG2D.OX40 DARIC T cells when comparing cytokine production in rapamycin and AP21967 co-cultures.
  • NKG2D.TNFR2 DARIC T cells were resistant to immunosuppression when cultured in rapamycin.
  • the cytokine production data was normalized using a ratio of AP21967 to rapamycin.
  • Figure 3C Using ratiometric analysis, rapamycin-mediated
  • Lenti viral vectors encoding NKG2D DARIC binding components comprising single or dual costimulatory signaling domains were designed, constructed, and verified.
  • Figure 4A The costimulatory domains used for the DARIC binding components used in this Example were obtained from CD28, DAP 10, 0X40, or a combination of these domains.
  • Human PBMCs were activated, transduced and expanded as described in Example 1.
  • Anti-EGFR CAR T cells NKG2D DARIC T cells, NKG2D.DAP10 DARIC T cells, NKG2D.CD28 DARIC T cells, NKG2D.CD28.DAP10 DARIC T cells,
  • NKG2D.DAP10.0X40 DARIC T cells displayed similar rates of ex vivo expansion and the NKG2D DARICs had comparable expression levels compared to the parental NKG2D DARIC.
  • Anti-EGFR CAR T cells NKG2D DARIC T cells, NKG2D.DAP10 DARIC T cells, NKG2D.CD28 DARIC T cells, NKG2D.CD28.DAP10 DARIC T cells,
  • NKG2D.DAP 10.0X40 DARIC T cells, and NKG2D.OX40.DAP10 DARIC T cells were co-cultured with NKG2DL + A549 cells for 24 hrs in the presence or absence of rapamycin at a 1 : 1 E:T ratio and cytokine production was analyzed by Qbead PI exScreen.
  • DARIC binding components comprising a CD28 costimulatory domain, DAP 10 costimulatory domain, or CD28 costimulatory domain and DAP 10 costimulatory domain had minimal impact on cytokine production.
  • Figure 4B In addition, DARIC binding components comprising a DAP 10 costimulatory domain with or without an 0X40 costimulatory domain (in either orientation) did not result in altered cytokine production.
  • Figure 4C
  • DmrA is FKBP12
  • DmrB is FKBP12 F36V
  • DmrC is FRB (2021- 2113) T2098L.
  • Human PBMCs were activated, transduced and expanded as described in Example 1.
  • Anti-EGFR CAR T cells, NKG2D DARIC T cells were used as controls.
  • the various groups of DARIC T cells displayed similar rates of ex vivo expansion, similar CD4:CD8 ratios, and had comparable expression levels compared to the parental NKG2D DARIC.
  • Anti-EGFR CAR T cells and DARIC T cells were co-cultured with NKG2DL + A549 cells at a 1 : 1 E:T ratio for 24 hrs. in the presence or absence of AP21967 and cytokine production was analyzed by Qbead PlexScreen.
  • DARIC binding components comprising an ICOS transmembrane domain or costimulatory domain alone or in combination with DAP 10 had minimal impact on cytokine production.
  • DARIC signaling components comprising an ICOS transmembrane domain or costimulatory domain significantly reduced cytokine production compared to the NKG2D DARIC control T cells.
  • Figure 5B and Figure 5C Figure 5B and Figure 5C.
  • a lentiviral vector comprising a DARIC signaling component (FRB T2098L-CD8a TM-CD137-CD3C), an NKG2D.TNFR2 DARIC binding component, and a CD19 DARIC binding component (anti-CD19 scFV-FKBP12-CD4 TM) was designed, constructed, and verified.
  • Figure 6A A lentiviral vector comprising a DARIC signaling component (FRB T2098L-CD8a TM-CD137-CD3C), an NKG2D.TNFR2 DARIC binding component, and a CD19 DARIC binding component (anti-CD19 scFV-FKBP12-CD4 TM) was designed, constructed, and verified.
  • Figure 6A A lentiviral vector comprising a DARIC signaling component (FRB T2098L-CD8a TM-CD137-CD3C), an NKG2D.TNFR2 DARIC binding component, and a CD19 DARIC binding component
  • Human PBMCs were activated, transduced and expanded as described in Example 4.
  • NKG2D/CD19 DARIC dual targeting T cells were stained with either anti-NKG2D antibodies or recombinant CD19-Fc protein.
  • the NKG2D DARIC binding component and CD 19 DARIC binding component had similar expression levels in both DARIC single targeting and DARIC dual targeting T cells.
  • NKG2D/CD19 DARIC dual targeting T cells were co-cultured with NKG2DL + A549 cells, an NKG2DL neg mouse B cell line A20, and A20 cells stably expressing CD 19 (A20- hCD19) at 1 : 1 E:T ratio for 24 hrs. with or without AP21967. Cytokine production was measured from culture supernatants using a Qbead assay kit. Negligible cytokine production was observed in the absence of AP21967 or rapamycin. NKG2D/CD19- DARIC dual targeting T cells produced GM-CSF when cultured with both A549 and A20- CD19 cells. NKG2D.TNFR2 DARIC T cells and CD19 DARIC T cells produced cytokines when co-cultured with target cells expressing the cognate ligand. Figure 6D.
  • CD 19 DARIC BINDING COMPONENTS CONTAINING A COSTIMULATORY DOMAIN
  • CD 19 DARIC binding and signaling components were designed, constructed, and verified.
  • a CD 19 DARIC lentiviral vector was constructed comprising an MNDU3 promoter operably linked to a polynucleotide encoding: a DARIC signaling component (CD8a-signal peptide, an FRB variant (T82L), a CD8a transmembrane domain, an intracellular 4-1BB costimulatory domain, and a CD3zsignaling domain); a P2A sequence; and a DARIC binding component (an IgK-signal peptide, an anti-CD 19 scFv binding domain, a G4S linker, an FKBP12 domain, a CD4 transmembrane domain and an 0X40 costimulatory domain or TNFR2 costimulatory domain).
  • a DARIC signaling component CD8a-signal peptide, an FRB variant (T82L), a CD8a transmembrane domain, an intracellular 4
  • FIG. 7A SEQ ID NO: 23. Human PBMCs were activated, transduced and expanded as described in Example 1. UTD T cells, anti-CD 19 CAR T cells, CD 19 DARIC T cells, CD 19.0X40 DARIC T cells, and CD19.TNFR2 DARIC T cells displayed similar rates of ex vivo expansion. The T cells were stained with recombinant CD19-Fc protein and anti-CD19 scFv expression was quantified on CD4 + T cells. Expression was comparable among the different CD 19 CAR and DARIC binding components. Figure 7B.

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