Dimerizer-regulated immune receptor complexes

Abstract

The present disclosure provides improved adoptive T cell therapy compositions for treating, preventing, or ameliorating at least one symptom of, or a condition associated with, cancer, an infectious disease, an autoimmune disease, an inflammatory disease, and an immune disorder.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 62 / 835,659, filed April 18, 2019, and U.S. Provisional Patent Application No. 62 / 779,971, filed December 14, 2018, which are incorporated herein by reference in their entireties.

[0002] Sequence Listing Statement The sequence listing for this application has been submitted in text format instead of paper and is incorporated herein by reference. The text file containing the sequence listing is named BLBD_113_02WO_ST25.txt. The text file is 157 KB, was created on December 11, 2019, and will be submitted electronically via EFS-Web simultaneously with the filing of this application.

[0003] The present disclosure relates to adoptive cell therapy, and more particularly to improved chemically controlled signaling molecules, cells, and methods of their use for modulating the spatial and temporal control of cell signaling initiation and downstream responses during adoptive immunotherapy. [Background technology]

[0004] Description of related fields Cancer incidence doubled worldwide between 1975 and 2000. It 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 bronchial cancer, prostate cancer, colon and rectum cancer, bladder cancer, melanoma of the skin, non-Hodgkin's lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. It is estimated that there will be 23.6 million new cancer cases by 2030.

[0005] Adoptive cell therapy has emerged as a powerful paradigm for transducing complex biological signals to treat cancer. In contrast to small molecule or biopharmaceutical drugs, adoptive cell therapy has the potential to exert unique therapeutic tasks through diverse sensory and responsive programs and more defined gene regulatory mechanisms. To realize such therapeutic value, cells must be equipped with mechanisms to understand and integrate chemical and / or biological information associated with their local physiological environment. Summary of the Invention [Means for solving the problem]

[0006] Generally, the present disclosure relates in part to dimerizing agent regulated immunoreceptor complex (DARIC) compositions, polynucleotides, polypeptides, and methods of making and using them.

[0007] In various embodiments, the non-naturally occurring cell 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, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-naturally occurring cell, wherein the cross-linking agent is associated with and disposed between the first and second multimerization domains.

[0008] In certain embodiments, the first and second multimerization domains are different.

[0009] In certain embodiments, the first and second costimulatory domains are different.

[0010] In further embodiments, the first multimerization domain and the second multimerization domain are associated with a cross-linking agent 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 a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof.

[0011] In various embodiments, 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 PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

[0012] In some embodiments, the first multimerization domain comprises an FKBP polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof.

[0013] In certain embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP polypeptide or a variant thereof.

[0014] In additional embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0015] In various embodiments, the first and second multimerization domains are selected from FRB T2098L and FKBP12, and the cross-linking agent is sirolimus or AP21967.

[0016] In certain embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1).

[0017] In a further embodiment, the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain.

[0018] In certain embodiments, the first transmembrane domain and the second transmembrane domain are different.

[0019] In some embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0020] In additional embodiments, the first and second costimulatory domains 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 (OX40), 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), and T cell receptor associated transmembrane adaptor. 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

[0021] In various embodiments, 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, OX40 or TNFR2.

[0022] In a specific embodiment, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from OX40 or TNFR2.

[0023] In a further embodiment, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from OX40.

[0024] In a specific embodiment, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from TNFR2.

[0025] In certain embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0026] In some embodiments, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0027] In additional embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.

[0028] In various embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single-chain Fv protein (scFv), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), and a single-domain antibody (sdAb, camelid VHH, nanobody).

[0029] In a further embodiment, the extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.

[0030] In certain embodiments, the extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.

[0031] In additional embodiments, the extracellular binding domain comprises an scFv.

[0032] In certain embodiments, the extracellular binding domain comprises one or more Camelid VHH antibodies.

[0033] In certain embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma delta T cell receptor (TCR) ligand, and an alpha beta TCR ligand.

[0034] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: alpha folate receptor (FRα), α vβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase type IX (CAIX), CD16, CD19, 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 (DLL3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), HER2 p95, IL-10Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A),Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain related proteins A (MICA), MHC class I chain related proteins B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1).

[0035] In further embodiments, the non-naturally occurring cell comprises a first polypeptide comprising an FK506 binding protein (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 FKBP-rapamycin binding (FRB) multimerization domain polypeptide or variant thereof, a second transmembrane domain, and an OX40 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-naturally occurring cell, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0036] In various embodiments, the non-naturally occurring cell comprises a first polypeptide comprising an FKBP multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and a TNFR2 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-naturally occurring cell, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0037] In one embodiment, the non-naturally occurring cell comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and an OX40 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-naturally occurring cell, and the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0038] In various embodiments, the non-naturally occurring cell comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and a TNFR2 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-naturally occurring cell, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0039] In additional embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0040] In a further embodiment, the FRB multimerization domain is FRB T2098L, the FKBP multimerization domain is FKBP12, and the cross-linking agent is sirolimus or AP21967.

[0041] In certain embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1.

[0042] In various embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from polypeptides selected from the group consisting of: a CD4 transmembrane domain and a CD8α transmembrane domain.

[0043] In some embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0044] In certain embodiments, 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 (OX40), 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), and T cell receptor associated transmembrane adaptor. 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

[0045] In additional embodiments, the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

[0046] In various embodiments, the costimulatory domain is isolated from a CD137 costimulatory molecule.

[0047] In certain embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0048] In certain embodiments, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0050] In various embodiments, the antibody or antigen-binding fragment thereof is human or humanized.

[0051] In various embodiments, the antibody or antigen-binding fragment thereof comprises an scFv or one or more Camelid VHH antibodies.

[0052] In further embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma-delta T cell receptor (TCR) ligand, and an alpha-beta TCR ligand.

[0053] In certain embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0054] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0055] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0056] In some embodiments, the antibody or antigen-binding fragment thereof binds to BCMA.

[0057] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD19.

[0058] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD20 or CD22.

[0059] In various embodiments, the antibody or antigen-binding fragment thereof binds to B7-H3.

[0060] In a further embodiment, the antibody or antigen-binding fragment thereof binds to CD33.

[0061] In additional embodiments, the antibody or antigen-binding fragment thereof binds to CD79A.

[0062] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD79B.

[0063] In certain embodiments, the antibody or antigen-binding fragment thereof binds to EGFRvIII.

[0064] In some embodiments, the non-naturally occurring cell comprises a first polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain, and a second polypeptide comprising an antibody or antigen-binding fragment thereof, an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and a TNFR2 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-naturally occurring cell, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0065] In various embodiments, the non-native cell comprises a first polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain, and a second polypeptide comprising an antibody or antigen-binding fragment thereof, an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and an OX40 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-native cell, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0066] In one embodiment, the non-native cell comprises a first polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain, and a second polypeptide comprising an antibody or antigen-binding fragment thereof, an FKBP12 multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and a TNFR2 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-native cell, and the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0067] In various embodiments, the non-native cell comprises a first polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ primary signaling domain, and a second polypeptide comprising an antibody or antigen-binding fragment thereof, an FKBP12 multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and an OX40 costimulatory domain, wherein a cross-linking agent promotes formation of a polypeptide complex on the surface of the non-native cell, and wherein the cross-linking agent is associated with and disposed between the multimerization domains of the first and second polypeptides.

[0068] In a further embodiment, the cross-linking agent is AP21967 or sirolimus.

[0069] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof is human or humanized.

[0071] In a further embodiment, the antibody or antigen-binding fragment thereof comprises an scFv or one or more Camelid VHH antibodies.

[0072] In various embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma-delta T cell receptor (TCR) ligand, and an alpha-beta TCR ligand.

[0073] In certain embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: FRα, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0075] In certain embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0076] In certain embodiments, the antibody or antigen-binding fragment thereof binds to BCMA.

[0077] In a further embodiment, the antibody or antigen-binding fragment thereof binds to CD19.

[0078] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD20 or CD22.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof binds to B7H3.

[0080] In additional embodiments, the antibody or antigen-binding fragment thereof binds to CD33.

[0081] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD79A.

[0082] In some embodiments, the antibody or antigen-binding fragment thereof binds to CD79B.

[0083] In a further embodiment, the antibody or antigen-binding fragment thereof binds to EGFRvIII, optionally in which case the antibody is EGFR806 or an antigen-binding fragment thereof.

[0084] In certain embodiments, the multimerization domain is localized extracellularly when the first polypeptide and the second polypeptide are expressed.

[0085] In various embodiments, the cells are hematopoietic cells.

[0086] In additional embodiments, the cell is a T cell.

[0087] In various embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0088] In certain embodiments, the cell is an immune effector cell.

[0089] In further embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

[0090] In certain embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0091] In additional embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

[0092] In various embodiments, the fusion polypeptide comprises a first polypeptide comprising a first multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and a second costimulatory domain.

[0093] In some embodiments, the first and second multimerization domains are different.

[0094] In some embodiments, the first and second costimulatory domains are different.

[0095] In certain embodiments, the first multimerization domain and the second multimerization domain are associated with a cross-linking agent 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 a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof.

[0096] In further embodiments, 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 PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

[0097] In various embodiments, the first multimerization domain comprises an FKBP polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof.

[0098] In additional embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP polypeptide or a variant thereof.

[0099] In certain embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0100] In various embodiments, the first and second multimerization domains are selected from FRB T2098L and FKBP12, and the cross-linking agent is sirolimus or AP21967.

[0101] In further embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1).

[0102] In certain embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain.

[0103] In additional embodiments, the first transmembrane domain and the second transmembrane domain are different.

[0104] In certain embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0105] In various embodiments, the first and second costimulatory domains 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 (OX40), 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), and T cell receptor associated transmembrane adaptor. 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

[0106] In specific embodiments, 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, OX40 or TNFR2.

[0107] In some embodiments, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from OX40 or TNFR2.

[0108] In a further embodiment, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from OX40.

[0109] In a specific embodiment, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from TNFR2.

[0110] In additional embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0111] In certain embodiments, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0112] In various embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.

[0113] In additional embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0114] In a further embodiment, the extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.

[0115] In various embodiments, the extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.

[0116] In some embodiments, the extracellular binding domain comprises an scFv.

[0117] In one embodiment, the extracellular binding domain comprises one or more Camelid VHH antibodies.

[0118] In additional embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma delta T cell receptor (TCR) ligand, and an alpha beta TCR ligand.

[0119] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: FRα, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0120] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0121] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0122] In additional embodiments, the 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 OX40 costimulatory domain.

[0123] In certain embodiments, the fusion polypeptide comprises a first polypeptide comprising a first multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and a TNFR2 costimulatory domain.

[0124] In a further embodiment, the first and second multimerization domains are identical.

[0125] In certain embodiments, the first and second multimerization domains are different.

[0126] In various embodiments, the first multimerization domain and the second multimerization domain are associated with a cross-linking agent 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.

[0127] In certain embodiments, 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.

[0128] In additional embodiments, the first multimerization domain comprises an FKBP polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof.

[0129] In certain embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP polypeptide or a variant thereof.

[0130] In some embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1.

[0131] In a further embodiment, the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8α transmembrane domain.

[0132] In certain embodiments, the first transmembrane domain and the second transmembrane domain are identical.

[0133] In additional embodiments, the first transmembrane domain and the second transmembrane domain are different.

[0134] In various embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0135] In further embodiments, 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 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.

[0136] In certain embodiments, the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

[0137] In some embodiments, the costimulatory domain is isolated from a CD137 costimulatory molecule.

[0138] In certain embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0139] In additional embodiments, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0140] In various embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.

[0141] In certain embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein (dsFv), and single-domain antibody (sdAb, camelid VHH, nanobody).

[0142] In certain embodiments, the extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.

[0143] In a further embodiment, the extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.

[0144] In certain embodiments, the extracellular binding domain comprises an scFv.

[0145] In a further embodiment, the extracellular binding domain comprises one or more Camelid VHH antibodies.

[0146] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma delta T cell receptor (TCR) ligand, and an alpha beta TCR ligand.

[0147] In a further embodiment, the extracellular binding domain binds to an antigen selected from the group consisting of: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0148] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0149] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0150] In certain embodiments, the fusion polypeptide comprises a first polypeptide comprising an FKBP multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and an OX40 costimulatory domain.

[0151] In some embodiments, the fusion polypeptide comprises a first polypeptide comprising an FKBP multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and a TNFR2 costimulatory domain.

[0152] In various embodiments, the fusion polypeptide comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a first transmembrane domain, a costimulatory domain, and / or a primary signaling domain, an antibody or antigen-binding fragment thereof, an FKBP multimerization domain polypeptide or a variant thereof, a second transmembrane domain, and an OX40 costimulatory domain.

[0153] In additional embodiments, the fusion polypeptide comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and a TNFR2 costimulatory domain.

[0154] In certain embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1.

[0155] In various embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from polypeptides selected from the group consisting of: a CD4 transmembrane domain and a CD8α transmembrane domain.

[0156] In further embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0157] In certain embodiments, 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 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.

[0158] In various embodiments, the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

[0159] In an additional embodiment, the costimulatory domain is isolated from a CD137 costimulatory molecule.

[0160] In some embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0161] In a further embodiment, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0162] In various embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0163] In certain embodiments, the antibody or antigen-binding fragment thereof is human or humanized.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof comprises an scFv or one or more Camelid VHH antibodies.

[0165] In certain embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma-delta T cell receptor (TCR) ligand, and an alpha-beta TCR ligand.

[0166] In various embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0167] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0168] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0169] In certain embodiments, the antibody or antigen-binding fragment thereof binds to BCMA.

[0170] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD19.

[0171] In a further embodiment, the antibody or antigen-binding fragment thereof binds to CD20 or CD22.

[0172] In certain embodiments, the antibody or antigen-binding fragment thereof binds to B7-H3.

[0173] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD33.

[0174] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD79A.

[0175] In additional embodiments, the antibody or antigen-binding fragment thereof binds to CD79B.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof binds to EGFRvIII.

[0177] In various embodiments, the fusion polypeptide comprises a first polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ 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 a variant thereof, a CD4 transmembrane domain, and a TNFR2 costimulatory domain.

[0178] In certain embodiments, the fusion polypeptide comprises a first polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ 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 a variant thereof, a CD4 transmembrane domain, and an OX40 costimulatory domain.

[0179] In a further embodiment, the fusion polypeptide comprises a first polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ 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 a variant thereof, a CD4 transmembrane domain, and a TNFR2 costimulatory domain.

[0180] In various embodiments, the fusion polypeptide comprises a first polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and / or a CD3ζ 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 a variant thereof, a CD4 transmembrane domain, and an OX40 costimulatory domain.

[0181] In additional embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0182] In certain embodiments, the antibody or antigen-binding fragment thereof is human or humanized.

[0183] In a further embodiment, the antibody or antigen-binding fragment thereof comprises an scFv or one or more Camelid VHH antibodies.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma-delta T cell receptor (TCR) ligand, and an alpha-beta TCR ligand.

[0185] In various embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: FRα, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0186] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0187] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof binds to BCMA.

[0189] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD19.

[0190] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD20 or CD22.

[0191] In a further embodiment, the antibody or antigen-binding fragment thereof binds to B7-H3.

[0192] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD33.

[0193] In additional embodiments, the antibody or antigen-binding fragment thereof binds to CD79A.

[0194] In some embodiments, the antibody or antigen-binding fragment thereof binds to CD79B.

[0195] In various embodiments, the antibody or antigen-binding fragment thereof binds to EGFRvIII, optionally in which case the antibody is EGFR806 or an antigen-binding fragment thereof.

[0196] In a further embodiment, the multimerization domain is localized extracellularly when the first polypeptide and the second polypeptide are expressed.

[0197] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide.

[0198] In certain embodiments, the polypeptide cleavage signal is a viral auto-cleaving 2A polypeptide.

[0199] In various embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0200] In some embodiments, the polynucleotide encodes a first or second polypeptide or a fusion polypeptide as contemplated herein.

[0201] In certain embodiments, the cDNA encodes a first or second polypeptide or a fusion polypeptide contemplated herein.

[0202] In additional embodiments, the RNA encodes a first or second polypeptide or a fusion polypeptide as contemplated herein.

[0203] In various embodiments, the vector contains a polynucleotide as contemplated herein.

[0204] In certain embodiments, the vector is an expression vector.

[0205] In additional embodiments, the vector is a transposon.

[0206] In one embodiment, the vector is a piggyBAC transposon or a Sleeping Beauty transposon.

[0207] In a further embodiment, the vector is a viral vector.

[0208] In various embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a vaccinia viral vector, or a retroviral vector.

[0209] In some embodiments, the retroviral vector is a lentiviral vector.

[0210] In some embodiments, 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); caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0211] In certain embodiments, the composition contains a non-native cell, fusion polypeptide, polynucleotide, or vector as contemplated herein.

[0212] In various embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a non-native cell, fusion polypeptide, polynucleotide, or vector contemplated herein.

[0213] In additional embodiments, a method of treating a subject in need thereof comprises administering to the subject an effective amount of a composition contemplated herein.

[0214] In a further embodiment, a method for treating, preventing, or ameliorating at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, and an immune disorder, or a condition associated therewith, comprises administering to a subject an effective amount of a composition contemplated herein.

[0215] In certain embodiments, the method of treating a solid tumor comprises administering to a subject an effective amount of a composition contemplated herein.

[0216] In various embodiments, 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.

[0217] In additional embodiments, the solid cancer is pancreatic cancer, lung cancer, or breast cancer.

[0218] In certain embodiments, a method for treating a hematological malignancy comprises administering to a subject an effective amount of a composition contemplated herein.

[0219] In various embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma.

[0220] In some embodiments, the polypeptide complex comprises a first polypeptide comprising a first multimerization domain polypeptide or a variant thereof, a first transmembrane domain, a first costimulatory domain, and / or a primary signaling domain; a second polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or a variant thereof, a second transmembrane domain, and a second costimulatory domain; and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0221] In certain embodiments, the first and second multimerization domains are different.

[0222] In various embodiments, the first and second costimulatory domains are different.

[0223] In further embodiments, the first multimerization domain and the second multimerization domain are associated with a cross-linking agent 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 a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof.

[0224] In additional embodiments, 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 PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

[0225] In various embodiments, the first multimerization domain comprises an FKBP polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof.

[0226] In certain embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP polypeptide or a variant thereof.

[0227] In certain embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0228] In some embodiments, the first and second multimerization domains are selected from FRB T2098L and FKBP12, and the cross-linking agent is sirolimus or AP21967.

[0229] In further embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1).

[0230] In various embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain.

[0231] In some embodiments, the first transmembrane domain and the second transmembrane domain are different.

[0232] In additional embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0233] In further embodiments, the first and second costimulatory domains 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 (OX40), 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), and T cell receptor associated transmembrane adaptor. 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

[0234] In specific embodiments, 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, OX40 or TNFR2.

[0235] In additional embodiments, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from OX40 or TNFR2.

[0236] In various embodiments, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from OX40.

[0237] In one embodiment, the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from TNFR2.

[0238] In further embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0239] In a further embodiment, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0240] In various embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.

[0241] In some embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0242] In additional embodiments, the extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.

[0243] In certain embodiments, the extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.

[0244] In various embodiments, the extracellular binding domain comprises an scFv.

[0245] In various embodiments, the extracellular binding domain comprises one or more Camelid VHH antibodies.

[0246] In additional embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma delta T cell receptor (TCR) ligand, and an alpha beta TCR ligand.

[0247] In certain embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: FRα, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0248] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0249] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0250] In various embodiments, the polypeptide complex comprises a first polypeptide comprising a first multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and an OX40 costimulatory domain, and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0251] In some embodiments, the polypeptide complex comprises a first polypeptide comprising a first multimerization domain polypeptide or a 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 a variant thereof, a second transmembrane domain, and a TNFR2 costimulatory domain, and a cross-linking agent associated with and disposed between the multimerization domains of the first polypeptide and the second polypeptide.

[0252] In certain embodiments, the first and second multimerization domains are the same.

[0253] In a further embodiment, the first and second multimerization domains are different.

[0254] In various embodiments, the first multimerization domain and the second multimerization domain are associated with a cross-linking agent 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.

[0255] In certain embodiments, 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.

[0256] In some embodiments, the first multimerization domain comprises an FKBP polypeptide or a variant thereof, and the second multimerization domain comprises an FRB polypeptide or a variant thereof.

[0257] In certain embodiments, the first multimerization domain comprises an FRB polypeptide or a variant thereof, and the second multimerization domain comprises an FKBP polypeptide or a variant thereof.

[0258] In additional embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0259] In various embodiments, the first and second multimerization domains are selected from FRB T2098L and FKBP12, and the cross-linking agent is sirolimus or AP21967.

[0260] In further embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1.

[0261] In some embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of: a CD4 transmembrane domain and a CD8α transmembrane domain.

[0262] In additional embodiments, the first transmembrane domain and the second transmembrane domain are the same.

[0263] In certain embodiments, the first transmembrane domain and the second transmembrane domain are different.

[0264] In various embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0265] In further embodiments, 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 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70.

[0266] In certain embodiments, the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

[0267] In an additional embodiment, the costimulatory domain is isolated from a CD137 costimulatory molecule.

[0268] In various embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0269] In a further embodiment, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0270] In certain embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.

[0271] In some embodiments, the extracellular binding domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0272] In some embodiments, the extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.

[0273] In certain embodiments, the extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.

[0274] In various embodiments, the extracellular binding domain comprises an scFv.

[0275] In a further embodiment, the extracellular binding domain comprises one or more Camelid VHH antibodies.

[0276] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma delta T cell receptor (TCR) ligand, and an alpha beta TCR ligand.

[0277] In some embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: FRα, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0278] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0279] In various embodiments, the extracellular binding domain binds to an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0280] In certain embodiments, the polypeptide complex comprises a first polypeptide comprising an FKBP multimerization domain polypeptide or a variant thereof, a first transmembrane domain, a costimulatory domain, and / or a primary signaling domain, an antibody or antigen-binding fragment thereof, a FRB multimerization domain polypeptide or a variant thereof, a second polypeptide comprising a second transmembrane domain, and an OX40 costimulatory domain, and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0281] In various embodiments, the polypeptide complex comprises a first polypeptide comprising an FKBP multimerization domain polypeptide or a variant thereof, a first transmembrane domain, a costimulatory domain, and / or a primary signaling domain, an antibody or antigen-binding fragment thereof, a FRB multimerization domain polypeptide or a variant thereof, a second polypeptide comprising a second transmembrane domain, and a TNFR2 costimulatory domain, and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0282] In certain embodiments, the polypeptide complex comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a first transmembrane domain, a costimulatory domain, and / or a primary signaling domain, an antibody or antigen-binding fragment thereof, a second polypeptide comprising an FKBP multimerization domain polypeptide or a variant thereof, a second transmembrane domain, and an OX40 costimulatory domain, and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0283] In a further embodiment, the polypeptide complex comprises a first polypeptide comprising an FRB multimerization domain polypeptide or a variant thereof, a first transmembrane domain, a costimulatory domain, and / or a primary signaling domain, an antibody or antigen-binding fragment thereof, a second polypeptide comprising an FKBP multimerization domain polypeptide or a variant thereof, a second transmembrane domain, and a TNFR2 costimulatory domain, and a cross-linking agent associated with and positioned between the multimerization domains of the first and second polypeptides.

[0284] In various embodiments, the cross-linking agent is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0285] In a specific embodiment, the FRB multimerization domain is FRB T2098L, the FKBP multimerization domain is FKBP12, and the cross-linking agent is sirolimus or AP21967.

[0286] In one embodiment, the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma, or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1.

[0287] In additional embodiments, the first transmembrane domain and the second transmembrane domain are independently selected from polypeptides selected from the group consisting of: a CD4 transmembrane domain and a CD8α transmembrane domain.

[0288] In some embodiments, the costimulatory domain and / or the primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0289] In further embodiments, the costimulatory signaling 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 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70.

[0290] In various embodiments, the costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

[0291] In one embodiment, the costimulatory domain is isolated from a CD137 costimulatory molecule.

[0292] In further embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0293] In certain embodiments, the primary signaling domain is isolated from a CD3ζ polypeptide.

[0294] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0295] In various embodiments, the antibody or antigen-binding fragment thereof is human or humanized.

[0296] In a further embodiment, the antibody or antigen-binding fragment thereof comprises an scFv or one or more Camelid VHH antibodies.

[0297] In further embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma-delta T cell receptor (TCR) ligand, and an alpha-beta TCR ligand.

[0298] In various embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0299] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0300] In some embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0301] In certain embodiments, the antibody or antigen-binding fragment thereof binds to BCMA.

[0302] In additional embodiments, the antibody or antigen-binding fragment thereof binds to CD19.

[0303] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD20 or CD22.

[0304] In various embodiments, the antibody or antigen-binding fragment thereof binds to B7-H3.

[0305] In a further embodiment, the antibody or antigen-binding fragment thereof binds to CD33.

[0306] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD79A.

[0307] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD79B.

[0308] In various embodiments, the antibody or antigen-binding fragment thereof binds to EGFRvIII.

[0309] In some embodiments, the polypeptide complex comprises a first polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain; an antibody or antigen-binding fragment thereof; a second polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and a TNFR2 costimulatory domain; and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0310] In one embodiment, the polypeptide complex comprises a first polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain; an antibody or antigen-binding fragment thereof; a second polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and an OX40 costimulatory domain; and a cross-linking agent associated with and disposed between the multimerization domains of the first polypeptide and the second polypeptide.

[0311] In various embodiments, the polypeptide complex comprises a first polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain; an antibody or antigen-binding fragment thereof; a second polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and a TNFR2 costimulatory domain; and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0312] In certain embodiments, the polypeptide complex comprises a first polypeptide comprising an FRB T2098L multimerization domain polypeptide or a variant thereof, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain; an antibody or antigen-binding fragment thereof; a second polypeptide comprising an FKBP12 multimerization domain polypeptide or a variant thereof, a CD4 transmembrane domain, and an OX40 costimulatory domain; and a cross-linking agent associated with and disposed between the multimerization domains of the first and second polypeptides.

[0313] In some embodiments, the cross-linking agent is AP21967 or sirolimus.

[0314] In various embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody).

[0315] In additional embodiments, the antibody or antigen-binding fragment thereof is human or humanized.

[0316] In a further embodiment, the antibody or antigen-binding fragment thereof comprises an scFv or one or more Camelid VHH antibodies.

[0317] In various embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma-delta T cell receptor (TCR) ligand, and an alpha-beta TCR ligand.

[0318] In certain embodiments, the antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0319] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CLDN6, CLDN18.2, DLL3, ERBB4, HER2, HER2 p95, MUC16, MICA, MICB, TAG72, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0320] In certain embodiments, the antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, and / or EGFRvIII.

[0321] In additional embodiments, the antibody or antigen-binding fragment thereof binds to BCMA.

[0322] In a further embodiment, the antibody or antigen-binding fragment thereof binds to CD19.

[0323] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD20 or CD22.

[0324] In various embodiments, the antibody or antigen-binding fragment thereof binds to B7-H3.

[0325] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD33.

[0326] In some embodiments, the antibody or antigen-binding fragment thereof binds to CD79A.

[0327] In various embodiments, the antibody or antigen-binding fragment thereof binds to CD79B.

[0328] In certain embodiments, the antibody or antigen-binding fragment thereof binds to EGFRvIII, optionally in which case the antibody is EGFR806 or an antigen-binding fragment thereof.

[0329] In various embodiments, the multimerization domain is localized extracellularly when the first polypeptide and the second polypeptide are expressed. [Brief explanation of the drawings]

[0330] [Figure 1] FIG. 1 shows a diagram of the DARIC structure, which includes the DARIC binding component along with the costimulatory domain. [Figure 2-1] Figure 2A shows a diagram of the NKG2D DARIC structure, which includes the DARIC-binding component along with various costimulatory domains. Figure 2B shows the expression of the NKG2D-binding domain 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 2-2]Figure 2C shows the proliferation rates 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 2-3] Figure 2D shows the production of IFNγ, TNFα, and GM-CSF from the culture supernatant of EGFR+NKG2DL+ HCT116 cells cocultured 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 for 24 hours in the presence of rapamycin. [Figure 3-1] Figure 3A shows the production of IFNγ, TNFα, and GM-CSF from the culture supernatant of EGFR+NKG2DL+ A549 cells cocultured with NKG2D DARIC T cells, NKG2D.OX40 DARIC T cells, or NKG2D.TNFR2 DARIC T cells at a 1:1 E:T ratio for 24 hours in the presence of vehicle, rapamycin, or AP21967. [Figure 3-2] Figure 3B shows the production of IFNγ, TNFα, and GM-CSF from the culture supernatant of EGFR+NKG2DL+ A549 cells cocultured with NKG2D DARIC T cells, NKG2D.OX40 DARIC T cells, or NKG2D.TNFR2 DARIC T cells at a 1:1 E:T ratio for 24 hours in the presence of vehicle, rapamycin, or AP21967. [Figure 3-3]Figure 3C shows the ratio of cytokine production in T cell cocultures compared with AP2167 treatment and rapamycin treatment. Anti-EGFR CAR T cells, NKG2D DARIC T cells, NKG2D.TNFR2 DARIC T cells, and NKG2D.OX40 DARIC T cells were cocultured with either A549 or HCT116 target cells at a 1:1 E:T ratio in the presence of rapamycin or AP2167. The ratio of cytokine production from AP2167 cultures to that from rapamycin cultures is shown. Arrows indicate rapamycin-mediated immunosuppression (>1) or rapamycin-mediated immunoenhancement (<1). [Figure 4-1] FIG. 4A shows a diagram of the NKG2D DARIC structure, which contains a DARIC-binding component with two costimulatory domains. [Figure 4-2] Figure 4B shows IFNγ and GM-CSF production from culture supernatants of EGFR+NKG2DL+ A549 cells cocultured 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 for 24 hours in the presence of vehicle or rapamycin. [Figure 4-3] Figure 4C shows IFNγ and GM-CSF production from culture supernatants of EGFR+NKG2DL+ A549 cells cocultured with untransduced control T cells, NKG2D DARIC T cells, NKG2D.DAP10 DARIC T cells, NKG2D.DAP10.OX40 DARIC T cells, or NKG2D.OX40.DAP10 DARIC T cells at a 1:1 E:T ratio for 24 hours in the presence of vehicle or rapamycin. [Figure 5-1] FIG. 5A shows a diagram of the NKG2D DARIC structure, which contains a DARIC-binding component with ICOS-based transmembrane and costimulatory domains. [Figure 5-2]Figure 5B shows IFNγ production from culture supernatants of EGFR+NKG2DL+ A549 cells co-cultured 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 for 24 hours in the presence of AP21967. [Figure 5-3] Figure 5C shows GM-CSF production from culture supernatants of EGFR+NKG2DL+ A549 cells co-cultured 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 for 24 hours in the presence of AP21967. [Figure 6-1] Figure 6A shows a diagram of the dual-targeting DARIC strategy. NKG2D DARIC contains a DARIC binding component that includes a costimulatory domain along with an anti-CD19 DARIC binding component. [Figure 6-2] FIG. 6B shows the expression of the NKG2D binding domain in the CD4+ gate of untransduced T cells, NKG2D.TNFR2 DARIC T cells, and NKG2D.TNFR2 DARIC:CD19 DARIC T cells. [Figure 6-3] FIG. 6C shows the CD19-Fc binding efficiency of untransduced T cells, CD19 DARIC T cells, and NKG2D.TNFR2 DARIC:CD19 DARIC T cells. [Figure 6-4] Figure 6D shows GM-CSF production from the culture supernatant of NKG2DL-CD19+ A20 cells (A20), NKG2DL-CD19+ A20 cells (A20-hCD19), and NKG2DL+CD19- A549 cells (A549). Target cells were cocultured 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 for 24 hours in the presence of AP21967. [Figure 7-1] FIG. 7A shows a diagram of the anti-CD19 DARIC structure with a costimulatory domain. [Figure 7-2] Figure 7B shows the CD19-Fc binding efficiency of untransduced T cells, anti-CD19 CAR T cells, CD19 DARIC T cells, CD19.OX40 DARIC T cells, and CD19.TNFR2 DARIC T cells. [Figure 7-3] Figure 7C shows the production of IFNγ, GM-CSF, and TNFα from the culture supernatant of CD19+ Nalm6 cells cocultured with anti-CD19 CAR T cells, CD19 DARIC T cells, CD19.OX40 DARIC T cells, and CD19.TNFR2 DARIC T cells at a 1:1 E:T ratio for 24 hours in the presence of vehicle or rapamycin. [Figure 7-4] Figure 7D shows the production of IFNγ, GM-CSF, and TNFα from the culture supernatant of CD19+ Jeko-1 cells co-cultured with anti-CD19 CAR T cells, CD19 DARIC T cells, CD19.OX40 DARIC T cells, and CD19.TNFR2 DARIC T cells at a 1:1 E:T ratio for 24 hours in the presence of vehicle or rapamycin. [Figure 8-1] FIG. 8A shows a diagram of the anti-CD33 DARIC structure with a costimulatory domain. [Figure 8-2] Figure 8B shows the CD33-Fc binding efficiency of 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 8-3] Figure 8C shows IFNγ production from culture supernatants of CD33+ THP-1 or CD33+ Molm-1 cells co-cultured with anti-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 for 24 hours in the presence of vehicle or rapamycin. Two different anti-CD33 scFvs are shown. DETAILED DESCRIPTION OF THE INVENTION

[0331] A brief explanation of sequence numbers SEQ ID NO: 1 sets forth the amino acid sequence of the FRB T2098L-CD8α TM-CD137-CD3ζ DARIC signaling component. SEQ ID NO:2 sets forth the amino acid sequence of the FRB T2098L-CD8α TM-CD134-CD3ζ DARIC signaling component. SEQ ID NO:3 sets forth the amino acid sequence of the FRB T2098L-CD8α TM-CD28-CD ζ DARIC signaling component. SEQ ID NO: 4 sets forth the amino acid sequence of the anti-BCMA-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 5 sets forth the amino acid sequence of the anti-BCMA-FKBP12-AMN™ DARIC.TNFR2 binding component. SEQ ID NO: 6 sets forth the amino acid sequence of the anti-CD19-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 7 sets forth the amino acid sequence of the anti-CD19-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 8 sets forth the amino acid sequence of the anti-B7-H3-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 9 sets forth the amino acid sequence of the anti-B7-H3-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 10 sets forth the amino acid sequence of the anti-CD20-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 11 sets forth the amino acid sequence of the anti-CD20-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 12 sets forth the amino acid sequence of the anti-CD22-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 13 sets forth the amino acid sequence of the anti-CD22-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 14 sets forth the amino acid sequence of the anti-EGFRvIII-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 15 sets forth the amino acid sequence of the anti-EGFRvIII-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 16 sets forth the amino acid sequence of the anti-CD33-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 17 sets forth the amino acid sequence of the anti-CD33-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 18 sets forth the amino acid sequence of the anti-CD33-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 19 sets forth the amino acid sequence of the anti-CD33-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 20 sets forth the amino acid sequence of the NKG2D-FKBP12-CD4™ DARIC.OX40 binding component. SEQ ID NO: 21 sets forth the amino acid sequence of the NKG2D-FKBP12-CD4™ DARIC.TNFR2 binding component. SEQ ID NO: 22 sets forth the amino acid sequence of the NKG2D DARIC polyprotein. SEQ ID NO: 23 sets forth the amino acid sequence of the CD19 DARIC polyprotein. SEQ ID NO:24 sets forth the amino acid sequence of the B7-H3 DARIC polyprotein. SEQ ID NO: 25 sets forth the amino acid sequence of the CD20 DARIC polyprotein. SEQ ID NO: 26 sets forth the amino acid sequence of the CD22 DARIC polyprotein. SEQ ID NO: 27 sets forth the amino acid sequence of the EGFRvIII DARIC polyprotein. SEQ ID NO: 28 sets forth the amino acid sequence of the CD33 DARIC-1 polyprotein. SEQ ID NO: 29 sets forth the amino acid sequence of the CD33 DARIC-2 polyprotein. SEQ ID NOs: 30 to 40 list the amino acid sequences of various linkers. SEQ ID NOs: 41 to 65 describe the amino acid sequences of the protease cleavage site and the cleavage site of the self-cleaving polypeptide. (Mode for Carrying Out the Invention)

[0332] A. Overview Cancer is one of the world's leading causes of death. In recent years, oncologists have introduced genetic approaches as a potential means of enhancing immune recognition and elimination of cancer cells. One promising strategy is adoptive cellular immunotherapy, which uses immune effector cells genetically engineered to express chimeric antigen receptors (CARs), which redirect the cytotoxic activity of these CAR T cells toward cancer cells. A major drawback of CAR T cell therapy is the inability to spatially and temporally control CAR T cell activity. This inability to control CAR T cell activity can induce a wide range of side effects, many of which begin silently and can rapidly worsen. A particularly serious complication is cytokine release syndrome (CRS), or "cytokine storm," in which CAR T cells induce the release of massive, even fatal, cytokines. CRS can result in dangerously high fever, extreme fatigue, difficulty breathing, and a sudden drop in blood pressure. CRS can also produce a second wave of side effects, which affect the nervous system, including neurotoxicity, tremors, headache, confusion, loss of balance, difficulty speaking, seizures, and hallucinations. The compositions and methods contemplated herein provide solutions to these and other problems plaguing adoptive cell therapy.

[0333] The present disclosure generally relates to improved compositions and methods for modulating the spatial and temporal control of adoptive cell therapy using a costimulatory dimerizing agent-regulated immunoreceptor complex (DARIC). DARIC contains one or more DARIC binding components and / or one or more DARIC signaling components. Without wishing to be bound by any particular theory, the DARIC compositions and methods contemplated herein offer numerous benefits over existing CAR T cell therapies in the art, including, but not limited to, both spatial and temporal control over the signal-transducing binding and signaling activity of immune effector cells. Temporal control of DARIC triggers the DARIC mechanism of signal transduction through crosslinker-mediated association of the DARIC binding component with the DARIC signaling component. Spatial control of DARIC engages the signaling mechanism through target antigen recognition by a binding domain on the DARIC binding component. In this way, DARIC immune effector cells become activated when both the target antigen and the crosslinker are present. Furthermore, DARICs containing two or more binding components directed against different target antigens allow dual or multiple targeting of target cells, which may be beneficial in enhancing efficacy, tumor clearance and safety, as well as reducing recurrence, antigen escape, and on-target off-tumor cell lysis.

[0334] In various embodiments, the present disclosure contemplates improved DARIC components. Without wishing to be bound by any particular theory, the inventors have unexpectedly discovered that DARIC binding components comprising an intracellular signaling domain increase the potency of DARIC immune effector cells, for example, by increasing the secretion of inflammatory cytokines and increasing antigen-dependent cytotoxicity against target cells. In further specific embodiments, DARIC multimerization depends on an immunosuppressive cross-linking factor molecule, e.g., rapamycin, a DARIC binding component comprising an intracellular signaling domain, e.g., a costimulatory domain, and surprisingly reduces or eliminates the immunosuppressive activity associated with the cross-linking factor.

[0335] In certain embodiments, DARIC comprises a polypeptide comprising a multimerization domain polypeptide or a variant thereof, a transmembrane domain, and one or more intracellular signaling domains (DARIC signaling component), and a polypeptide comprising a binding domain, a multimerization domain polypeptide or a variant thereof, a transmembrane domain, and one or more intracellular signaling domains (DARIC binding component). In preferred embodiments, one or more intracellular signaling domains in the DARIC signaling component are different from one or more intracellular signaling domains in the DARIC binding component. In the presence of a cross-linking agent, the DARIC binding component and the DARIC signaling component associate with each other through the cross-linking agent to form functionally active DARIC.

[0336] In certain embodiments, DARIC comprises a polypeptide comprising a multimerization domain polypeptide or a variant thereof, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain (DARIC signaling component), and a polypeptide comprising a binding domain, a multimerization domain polypeptide or a variant thereof, a transmembrane domain, and a costimulatory domain (DARIC binding component). In a preferred embodiment, the costimulatory domain in the DARIC signaling component is different from the costimulatory domain in the DARIC binding component. Without wishing to be bound by any particular theory, it is believed that the two different costimulatory domains synergistically enhance the cytokine secretion profile and cytotoxic activity of DARIC T cells directed against target cells. In the presence of a cross-linking agent, the DARIC binding component and the DARIC signaling component associate with each other through the cross-linking agent to form a functionally active DARIC.

[0337] In various embodiments, the present disclosure contemplates DARIC components that generate anti-cancer responses against cancers that express two or more target antigens that are expressed on one or more target cells.

[0338] In certain embodiments, a DARIC signaling component includes a multimerization domain polypeptide or a variant thereof, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain; a first DARIC binding component includes a binding domain that binds to a first target antigen, a multimerization domain polypeptide or a variant thereof, a transmembrane domain, and a costimulatory domain; and a second DARIC binding component includes a binding domain that binds to a second target antigen, a multimerization domain polypeptide or a variant thereof, a transmembrane domain, and optionally a costimulatory domain. In the presence of a cross-linking agent, each of the DARIC binding components associates with the DARIC signaling component through the cross-linking agent to form a functionally active DARIC.

[0339] In a preferred embodiment, the DARIC binding component and the DARIC signal transduction component multimerization domain are placed extracellularly. The extracellular placement of the multimerization domain provides many advantages over intracellular placement, including, but not limited to, more efficient placement of the binding domain, high temporal sensitivity to cross-linking agent control, and low toxicity due to the use of non-immunosuppressive doses of certain cross-linking agents.

[0340] Also contemplated herein are polynucleotides encoding DARIC, DARIC binding components, and DARIC signaling components; DARIC binding components, DARIC signaling components, DARIC protein complexes, DARIC fusion proteins; cells containing and / or expressing polynucleotides encoding DARIC, DARIC binding components, and DARIC signaling components; and methods of using them to treat immune disorders.

[0341] 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 generally be performed as described in various general and specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, as cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning:A Practical Approach,vol.I & II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid The Hybridization(B.Hames & S.Higgins,Eds.,1985);Transcription and Translation(B.Hames & S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q.E. Coligigan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research articles in journals such as, for example, Advances in Immunology.

[0342] B. Definition Before describing the present disclosure in detail, it may be helpful to an understanding thereof to provide definitions of certain terms that will be used herein.

[0343] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test particular embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0344] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0345] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0346] The term "and / or" should be understood to mean either one or both of the alternatives.

[0347] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0348] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to each value subsumed within the range. For example, in one non-limiting, merely exemplary embodiment, the range "1 to 5" is equivalent to: 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.

[0349] As used herein, 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 more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces approximately the same effect, e.g., a physiological effect, as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0350] Throughout this specification, unless otherwise required, the terms "comprise" and "comprising" should be understood to imply the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including, but limited to, what follows the words "consisting of." Thus, the words "consisting of" indicate that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the words, and any elements limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the words "consisting essentially of" indicate that the listed elements are necessary or mandatory, but that there are no other elements that materially affect the activity or function of the listed elements.

[0351] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, it should be understood that the affirmative recitation of a feature in one embodiment serves as grounds for the exclusion of that feature in certain embodiments.

[0352] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions (e.g., compositions containing a cancer-specific protein) that are injected into or absorbed into an animal. Examples of antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the antigens disclosed herein.

[0353] A "target antigen" or "target antigen of interest" is an antigen to which a binding domain contemplated herein is designed to bind. In certain embodiments, one or more target antigens is the alpha folate receptor (FRα), α vβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase type IX (CAIX), CD16, CD19, 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 (DLL3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), HER2 p95, IL-10Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A),Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain related proteins A (MICA), MHC class I chain related proteins B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1).

[0354] In one embodiment, the antigen is an MHC-peptide complex, such as, for example, a class I MHC-peptide complex or a class II MHC-peptide complex.

[0355] "NKG2D ligand" refers to a polypeptide recognized and / or bound by the natural-killer group 2, member D (NKG2D) receptor. Two families of NKG2D ligands have been identified in humans: MHC class I chain related proteins A (MICA) and B (MICB), and the HCMV UL16-binding proteins, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. MICA and MICB have α1, α2, α3, and transmembrane domains, respectively. ULBP1, ULBP2, ULBP3, and ULBP6 have α1 and α2 domains, respectively, and are glycosylphosphatidylinositol (GPI)-linked to the cell membrane. ULBP4 and ULBP5 have α1 and α2 domains, as well as a transmembrane domain, respectively. NKG2D ligands are expressed in various combinations on many human cancer cells and immunosuppressive cells (T-regs and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment). Cancers that express one or more NKG2D ligands include, but are not limited to, carcinomas (ovarian cancer, bladder cancer, breast cancer, lung cancer, liver cancer, colon cancer, renal cancer, prostate cancer, melanoma, Ewing's sarcoma, glioma, and neuroblastoma), leukemias (AML, CML, CLL), lymphoma, and multiple myeloma. NKG2D ligands can also be induced at sites of chronic inflammation, transiently after certain infections, after local irradiation, and after treatment with certain agents, such as HDAC inhibitors and bortezomib.

[0356] "NKG2D receptor binding domain, or NKG2D ligand binding portion thereof" refers to the NKG2D receptor or a portion thereof that is necessary or sufficient to bind to one or more NKG2D ligands. Natural-killer group 2, member D (NKG2D), also known as Klrk1, is a C-type lectin-like receptor that was first identified on natural killer (NK) cells as an activating immunoreceptor. In humans, NKG2D binds to NK cells, CD8 +T cells, CD4 + It is expressed as a costimulatory receptor on T cell subsets and γδ T cell subsets. The NKG2D receptor-binding domain, or an NKG2D ligand-binding portion thereof, binds to one or more NKG2D ligands, including, but not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0357] 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 that has the ability to specifically and / or selectively bind to a target antigen of interest. Binding domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0358] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targeted" describe the binding of a binding domain to a target antigen with a binding affinity higher than background binding. A binding domain may have a binding affinity of, for example, about 10 5 M -1 Affinity or K a (i.e., the equilibrium binding constant for a particular binding interaction, having units of 1 / M). In one embodiment, a binding domain (or a fusion protein comprising the binding domain) binds to a target with a binding affinity of about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 1013 M -1 More than K a A "high affinity" binding domain (or single-chain fusion protein thereof) is one that binds with 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 higher K a refers to a binding domain having the following structure:

[0359] The terms "selectively bind" or "selectively bound" or "selective binding" or "selectively target" describe the preferential binding of one molecule to a target molecule (on-target binding) in the presence of multiple off-target molecules.

[0360] "Antibody" refers to a binding agent that is a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, or nucleic acid, containing an antigenic determinant recognized by an immune cell.

[0361] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds. In certain embodiments, the polypeptide is intracellular and the epitope is a short oligopeptide (about 2 to about 20 amino acids) presented in a complex with MHC.

[0362] Antibodies include, for example, camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins (scFv), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single-domain antibodies (sdAb, camelid VHH, nanobodies), as well as antigen-binding fragments thereof, such as the portion of a full-length antibody that is responsible for antigen binding. Antibodies also include polyclonal and monoclonal antibodies and antigen-binding fragments thereof, murine, camelid, and human antibodies and antigen-binding fragments thereof, as well as chimeric, heteroconjugate, 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 & Co., New York, 1997.

[0363] "Linker" refers to a number of amino acid residues between various polypeptide domains, which are added for proper spacing and conformation of the molecule. In certain embodiments, the linker is a sequence that connects the variable regions. The "sequence that connects the variable regions" refers to a V H and V L and a linker, which provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In certain embodiments, the linker separates one or more heavy or light chain variable domains, hinge domains, multimerization domains, transmembrane domains, costimulatory domains, and / or primary signaling domains.

[0364] Exemplary linkers suitable for use in certain embodiments contemplated herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 30); TGEKP (SEQ ID NO: 31) (e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 32) (Pomerantz et al. 1995, supra); (GGGGS) n , where n=1, 2, 3, 4, or 5 (SEQ ID NO: 33) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 34) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSSEQLAQFRSLD (SEQ ID NO: 35) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 36); LQRDGERP (SEQ ID NO: 37); LRQKDGGGSERP (SEQ ID NO: 38); LRQKD(GGGS)2ERP (SEQ ID NO: 39). Alternatively, flexible linkers can be modeled using a computer program (Desjarlais & Berg, PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 34) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSSEQLAQFRSLD (SEQ ID NO: 35) (Bird et al., 1988, Science 242:423-426). 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or rationally designed by phage display. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 40) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0365] A "spacer domain" refers to a polypeptide that separates two domains. In one embodiment, the spacer domain separates the antigen-binding domain from the surface of the effector cell, allowing for proper cell-to-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). In certain embodiments, the 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 from natural, synthetic, semisynthetic, or recombinant sources. In certain embodiments, the spacer domain is an immunoglobulin portion containing one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may comprise the amino acid sequence of a native immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0366] "Hinge domain" refers to a polypeptide that positions an antigen-binding domain away from the surface of an effector cell and plays an important role in proper cell-to-cell contact, antigen binding, and activation. In certain embodiments, a polypeptide 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 from any natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may comprise the amino acid sequence of a native immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0367] As used herein, a "multimerization domain" refers to a polypeptide that preferentially interacts or associates with another distinct polypeptide, either directly or via a bridging molecule, such as a chemically induced dimerization agent, where the interaction of the distinct multimerization domains significantly contributes to or effectively promotes multimerization (i.e., the formation of dimers, trimers, or multimerized complexes, which may be homodimers, heterodimers, homotrimers, heterotrimers, homomultimers, or heteromultimers). Multimerization domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0368] Exemplary multimerization domains suitable for use in certain embodiments contemplated herein include an FK506-binding protein (FKBP) polypeptide or variant thereof, an FKBP-rapamycin-binding (FRB) polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, a PYR1-like 1 (PYL1) polypeptide or variant thereof, an abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof, a GIB1 polypeptide or variant thereof, or a GAI polypeptide or variant thereof.

[0369] As used herein, the term "FKBP-rapamycin binding polypeptide" refers to an FRB polypeptide. In certain embodiments, the FRB polypeptide is an FKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for use in certain embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, the FRB polypeptide comprises the 93-amino acid sequence from Ile-2021 to Lys-2113, as defined by GenBank Accession No. L34075.1, and includes the T2098L mutation. The FRB polypeptides contemplated herein bind to FKBP polypeptides via a cross-linking agent, thereby forming a ternary complex.

[0370] As used herein, the term "FK506-binding protein" refers to an FKBP polypeptide. In certain embodiments, the FKBP polypeptide is an FKBP12 polypeptide or an FKBP12 polypeptide containing an F36V mutation. In some embodiments, the FKBP domain may also be referred to as a "rapamycin-binding domain." Information regarding the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, for example, Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). The FKBP polypeptide contemplated herein binds to an FRB polypeptide via a cross-linking agent, thereby forming a ternary complex.

[0371] A "cross-linking agent" refers to a molecule that associates with and is positioned between two or more multimerization domains. In certain embodiments, a multimerization domain significantly contributes to or efficiently promotes the formation of a polypeptide complex only in the presence of a cross-linking agent. In certain embodiments, a multimerization domain does not contribute to or efficiently promote the formation of a polypeptide complex in the absence of a cross-linking agent. Examples of cross-linking agents suitable for use in certain 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)-FKBP synthetic ligand (SLF) or a derivative thereof, or any combination thereof.

[0372] Rapamycin analogs (rapalogs) include, but are not limited to, those described in U.S. Patent No. 6,649,595. Rapalog structures are incorporated herein by reference in their entirety. In certain embodiments, the cross-linking agent is a rapalog that has significantly reduced immunosuppressive effects compared to rapamycin. In preferred embodiments, the rapalog is AP21967 (also known as C-6-(S)-7-methylindole rapamycin, IC 50 =10 nM, a chemically modified, non-immunosuppressive rapamycin analog). Other examples of rapalogs suitable for use in certain embodiments contemplated herein include, but are not limited to, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

[0373] A "significantly reduced immunosuppressive effect" refers to an immunosuppressive effect that is at least 0.1 to 0.005 times less than that observed or predicted for the same dose, as measured clinically or appropriately in vitro (e.g., inhibition of T-cell proliferation), or as measured in an in vivo surrogate of human immunosuppressive activity.

[0374] A "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to a cell membrane. TM domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0375] The term "effector function" or "effector cell function" refers to the specialized functions of immune effector cells. Effector functions include, but are not limited to, activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors, or other cellular responses triggered by antigen binding to receptors expressed on immune effector cells.

[0376] "Intracellular signaling domain" or "endodomain" refers to the portion of a protein that transmits an effector function signal, instructing the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be employed, although in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the entire domain, so long as the effector function signal is transmitted. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is necessary or sufficient to transmit the effector function signal.

[0377] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct intracellular signaling domains. The primary signaling domain initiates antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex). The costimulatory domain then acts in an antigen-dependent manner to generate secondary or costimulatory signals.

[0378] A "primary signaling domain" refers to an intracellular signaling domain that controls the primary activation of the TCR complex, either stimulatory or inhibitory. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing primary signaling domains suitable for use in certain embodiments include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0379] As used herein, the term "costimulatory domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, generates a secondary signal required for efficient activation and function of T lymphocytes. Examples of costimulatory molecules from which costimulatory domains can 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, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), 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, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3), and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

[0380] "Immune disorder" refers to a disease that provokes a response from the immune system. In certain embodiments, the term "immune disorder" refers to cancer, an autoimmune disease, or an immunodeficiency. In one embodiment, an immune disorder includes an infectious disease.

[0381] As used herein, the term "cancer" generally relates to certain diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues.

[0382] As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other parts of the body via the lymph or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by spread cells is called a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to those of the original tumor (primary tumor).

[0383] As used herein, the terms "benign" or "non-malignant" refer to tumors that can grow large but do not spread to other parts of the body. Benign tumors are self-limited and often do not invade or metastasize.

[0384] "Cancer cell" refers to an individual cell of a cancerous growth or cancerous tissue. Cancer cells include both solid and liquid cancers. "Tumor" or "tumor cell" generally refers to an expansion due to abnormal cell proliferation or a lesion formed by abnormal cell proliferation, which may be benign, precancerous, or malignant. Most cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. For cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0385] The term "recurrence" refers to the diagnosis of a return of cancer, or the diagnosis of signs and symptoms of a return of cancer, after a period of improvement or remission.

[0386] "Remission," also called "clinical remission," includes both partial and complete remission. In a partial remission, some, but not all, signs and symptoms of cancer disappear. In a complete remission, all signs and symptoms of cancer disappear, although cancer may still be present in the body.

[0387] "Refractory" refers to a cancer that is resistant or unresponsive to therapy with a particular therapeutic agent. A cancer may be refractory from the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent) or may become refractory as a result of developing resistance to the therapeutic agent either during the initial treatment period or during subsequent treatment periods.

[0388] "Antigen-negative" refers to cells that do not express the antigen or that express such small amounts of the antigen that they are undetectable. In one embodiment, antigen-negative cells do not bind to the target receptor for the antigen. In one embodiment, antigen-negative cells do not substantially bind to the target receptor for the antigen.

[0389] An "autoimmune disease" refers to a condition in which the body mounts an immunogenic (i.e., immune system) response against some of the body's own tissue components. In other words, the immune system loses the ability to recognize some of the body's tissues or systems as "self" and attacks them as foreign. Autoimmune diseases can be classified as those that primarily affect one organ (e.g., hemolytic anemia and autoimmune thyroiditis) or those in which the autoimmune disease process spreads to many tissues (e.g., systemic lupus erythematosus). Multiple sclerosis, for example, is thought to arise when T cells attack the sheaths surrounding nerve fibers in the brain and spinal cord. This results in loss of coordination, weakness, and vision problems. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus erythematosus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.

[0390] "Immunodeficiency" refers to a condition in a patient in which the immune system is impaired by disease or chemical exposure. This condition results in the immune system being deficient in the number and types of blood cells needed to defend against foreign agents. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, and diabetes.

[0391] "Infectious disease" refers to a disease that can be transmitted from person to person or from organism to organism and is caused by a microorganism or virus (e.g., the 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.

[0392] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of cancer or other immune disorders that can be treated with the compositions and methods otherwise contemplated herein. Suitable subjects (e.g., patients) include experimental animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Typical subjects include human patients who have, are at risk of, or have been diagnosed with cancer or another immune disorder.

[0393] As used herein, the term "patient" refers to a subject diagnosed with cancer or another immune disorder that can be treated with the compositions and methods otherwise disclosed herein.

[0394] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal decrease in one or more measurable markers of the disease or condition being treated. Treatment may optionally include a decrease in the disease or condition, or a delay in the progression of the disease or condition, e.g., a delay in tumor growth. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0395] As used herein, "prevent" and similar terms, such as "prevented" and "preventing," refer to an approach aimed at preventing, inhibiting, or reducing the likelihood of 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 onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0396] As used herein, "amelioration of at least one symptom of" refers to a reduction in one or more symptoms of the disease or condition being treated in a subject. In certain embodiments, the disease or condition being treated is cancer, in which case the one or more symptoms that are improved include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (caused by distended abdominal organs), bone or joint pain, broken bones, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0397] "Enhance," "promote," "increase," or "expand" generally refer to the ability of a composition contemplated herein to produce, elicit, or generate a greater physiological response (i.e., downstream effect) compared to the response induced by a vehicle or control molecule / composition. Measurable physiological responses include, among others, increased T cell expansion, activation, persistence, cytokine secretion, and / or cancer cell killing capacity, as would be apparent from an understanding of the art and the present disclosure. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response induced by a vehicle or control composition.

[0398] "Decrease," or "lower," or "reduce," or "diminish," or "attenuate" generally refers to the ability of a composition contemplated herein to produce, elicit, or generate a physiological response (i.e., a downstream effect) that is smaller than the response induced by a vehicle or control molecule / composition. The amount of "reduction" or "decreased" is typically a "statistically significant" amount and can include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response induced by a vehicle, a control composition, or a response in a particular cell line.

[0399] "Maintain," or "preserve," or "maintain," or "no change," or "no substantial change," or "no substantial decrease" generally refer to the ability of a composition contemplated herein to produce, elicit, or produce a substantially similar or equivalent physiological response (i.e., downstream effect) in a cell compared to the response produced by a vehicle, a control molecule / composition, or the response in a particular cell line. An equivalent response is one that is not substantially different, or not measurably different, from the reference response.

[0400] Additional definitions are set forth throughout this disclosure.

[0401] C.DARIC In certain embodiments, one or more costimulatory DARICs redirect the cytotoxic activity of immune effector cells toward cancer cells expressing at least one or more target antigens. As used herein, the term "DARIC" refers to a dimerization agent-regulated immune receptor complex. DARICs comprise one or more non-naturally occurring polypeptides that, upon exposure to a multimerization agent or cross-linking agent, transmit an immunostimulatory signal in immune effector cells, such as increasing the production and / or secretion of pro-inflammatory cytokines, which stimulate the activity and function of immune effector cells.

[0402] In certain embodiments, the DARIC contemplated herein reduces the immunosuppressive effect of certain cross-linking agents and improves the spatial and temporal control of immunostimulatory signals in immune effectors. In a preferred embodiment, DARIC is a multi-chain chimeric receptor comprising one or more DARIC signaling components, each comprising a multimerization domain polypeptide or its variant, a transmembrane domain, and one or more intracellular signaling domains, and one or more DARIC binding components, each comprising a binding domain, a multimerization domain polypeptide or its variant, a transmembrane domain, and one or more intracellular signaling domains. In a preferred embodiment, one or more intracellular signaling domains in the DARIC signaling component are different from one or more intracellular signaling domains in the DARIC binding component.

[0403] In one embodiment, the DARIC signaling component and the DARIC binding component are expressed from one or more polynucleotides in the same cell, hi another embodiment, the DARIC signaling component and the DARIC binding component are expressed from a polycistronic polynucleotide in the same cell.

[0404] 1.DARIC signaling components A "DARIC signaling component" or "DARIC signaling polypeptide" refers to a polypeptide containing one or more multimerization domains, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, a DARIC signaling component contains a multimerization domain, a transmembrane domain, a costimulatory domain, and / or a primary signaling domain. In certain embodiments, a DARIC signaling component contains a first multimerization domain, a first transmembrane domain, a first costimulatory domain, and / or a primary signaling domain.

[0405] In certain embodiments, the DARIC signaling component comprises one or more multimerization domains.

[0406] Examples of multimerization domains suitable for use in certain DARIC signaling components contemplated herein include, but are not limited to, an FK506-binding protein (FKBP) polypeptide or variant thereof, an FKBP-rapamycin-binding (FRB) polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a bacterial dihydrofolate reductase (DHFR) polypeptide or variant thereof, a PYR1-like 1 (PYL1) polypeptide or variant thereof, and an abscisic acid insensitive 1 (ABI1) polypeptide or variant thereof.

[0407] In certain embodiments, the DARIC signaling component comprises an FRB polypeptide. In preferred embodiments, the DARIC signaling component comprises an FRB polypeptide comprising a T2098L mutation or a variant thereof.

[0408] In certain embodiments, the DARIC signaling component comprises an FKBP polypeptide or a variant thereof. In preferred embodiments, the DARIC signaling component comprises an FK506-binding protein 12 (FKBP12) polypeptide or a variant thereof.

[0409] In certain embodiments, the DARIC signaling component comprises a transmembrane domain.

[0410] Examples of transmembrane domains suitable for use in specific DARIC signaling components contemplated herein include, but are not limited to, the transmembrane regions of the alpha, beta, gamma, or delta chains of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, amnionless (AMN), programmed cell death 1 (PDCD1), NKG2A, NKG2B, NKG2C, and NKG2D. In a preferred embodiment, the DARIC signaling component comprises a CD4 transmembrane domain. In a preferred embodiment, the DARIC signaling component comprises a CD8α transmembrane domain.

[0411] In certain embodiments, the DARIC signaling component includes a linker connecting the C-terminus of the transmembrane domain and the N-terminus of the intracellular signaling domain. In various preferred embodiments, a short oligopeptide or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, connects the transmembrane domain and the intracellular signaling domain. Glycine-serine based linkers provide particularly suitable linkers.

[0412] In certain embodiments of the present disclosure, the DARIC signaling component contemplated herein contains one or more intracellular signaling domains. In one embodiment, the DARIC signaling component contains one or more costimulatory domains and / or a primary signaling domain. In one embodiment, the intracellular signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).

[0413] Examples of ITAM-containing primary signaling domains suitable for use in specific DARIC signaling components contemplated herein include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In preferred embodiments, the DARIC signaling component contains a CD3ζ primary signaling domain and one or more costimulatory domains. The primary signaling domain and costimulatory domain may be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0414] Examples of costimulatory domains suitable for use in certain DARIC signaling components contemplated herein include, but are not limited to, 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 (OX40), 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), and T cell receptor associated transmembrane adaptor. 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

[0415] In certain embodiments, the DARIC signaling components contemplated herein contain a signal peptide, such as a secretory signal peptide, and do not contain a transmembrane domain. Examples of signal peptides suitable for use in particular DARIC signaling components include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. In various preferred embodiments, the DARIC signaling component contains a CD8α signal polypeptide.

[0416] In certain embodiments, the DARIC signaling component contains one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134. In certain embodiments, the DARIC signaling component contains one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain. In certain embodiments, the DARIC signaling component comprises a CD137 costimulatory domain and a CD3ζ primary signaling domain.

[0417] In a preferred embodiment, the DARIC signaling component contains the FRB T2098L multimerization domain, the CD8α transmembrane domain, the CD137 costimulatory domain, and the CD3ζ primary signaling domain.

[0418] 2.DARIC combination component "DARIC-binding component" or "DARIC-binding polypeptide" refers to a polypeptide containing a binding domain that binds to a target antigen, one or more multimerization domains, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, the DARIC-binding component contains a binding domain that binds to a target antigen, a multimerization domain, a transmembrane domain, and a costimulatory domain. In certain embodiments, the DARIC-binding component contains a binding domain that binds to a target antigen, a second multimerization domain, a second transmembrane domain, and a second costimulatory domain.

[0419] In certain embodiments, DARIC comprises two or more DARIC binding components, each of which comprises a binding domain that binds to a different antigen, a multimerization domain, a transmembrane domain, and a costimulatory domain. In certain embodiments, DARIC comprises two or more DARIC binding components, each of which comprises 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. In some embodiments, the two or more DARIC binding components comprise different binding domains but the same multimerization domain, transmembrane domain, and / or intracellular signaling domain.

[0420] Examples of binding domains suitable for use in specific DARIC binding entities include, but are not limited to, antibodies or antigen-binding fragments thereof.

[0421] In certain embodiments, antibodies and antigen-binding fragments thereof suitable for use with particular DARIC binding entities include, but are not limited to, murine, camelid, chimeric, humanized, or human antibodies. In preferred embodiments, the antibody or antigen-binding fragment thereof is derived from a monoclonal antibody.

[0422] Examples of antibodies or antigen-binding fragments thereof suitable for use in specific DARIC binding components include, but are not limited to, camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins (scFv), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single-domain antibodies (sdAb, camelid VHH, nanobodies).

[0423] In a preferred embodiment, the binding domain comprises an scFv.

[0424] In a preferred embodiment, the binding domain comprises one or more Camelid VHH antibodies.

[0425] In certain embodiments, the DARIC binding entity comprises a binding domain that binds to a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a gamma delta T cell receptor (gamma delta TCR) ligand, or an alpha beta TCR ligand.

[0426] In certain embodiments, the DARIC binding component comprises a binding domain that binds to a target antigen selected from the group consisting of: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0427] In certain embodiments, the DARIC binding component comprises a binding domain that binds to 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.

[0428] In certain embodiments, the DARIC binding entity comprises one or more multimerization domains.

[0429] Examples of multimerization domains suitable for use in specific DARIC binding components contemplated herein include, but are not limited to, an FKBP polypeptide or variant thereof, an FRB polypeptide or variant thereof, a calcineurin polypeptide or variant thereof, a cyclophilin polypeptide or variant thereof, a DHFR polypeptide or variant thereof, a PYL1 polypeptide or variant thereof, and an ABI1 polypeptide or variant thereof.

[0430] In certain embodiments, the DARIC binding component comprises an FRB polypeptide or a variant thereof, and the DARIC signaling component comprises an FKBP polypeptide or a variant thereof. In preferred embodiments, the DARIC binding component comprises an FRB polypeptide or a variant thereof comprising a T2098L mutation, and the DARIC signaling component comprises an FKBP12 polypeptide or a variant thereof.

[0431] In certain embodiments, the DARIC binding component comprises an FKBP polypeptide or a variant thereof, and the DARIC signaling component comprises an FRB polypeptide or a variant thereof. In preferred embodiments, the DARIC binding component comprises an FKBP12 polypeptide or a variant thereof, and the DARIC signaling component comprises an FRB polypeptide or a variant thereof comprising a T2098L mutation.

[0432] In certain embodiments, the DARIC binding component comprises a binding domain, an FKBP polypeptide or a variant thereof, a transmembrane domain, and one or more intracellular signaling domains, and the DARIC signaling component comprises an FRB polypeptide or a variant thereof, a transmembrane domain, and one or more intracellular signaling domains. In preferred embodiments, the DARIC binding component comprises a binding domain, an FKBP12 polypeptide or a variant thereof, a transmembrane domain, and a costimulatory domain, and the DARIC signaling component comprises an FRB polypeptide or a variant thereof comprising a T2098L mutation, a transmembrane domain, a costimulatory domain, and a primary signaling domain.

[0433] In certain embodiments, the DARIC binding component comprises a transmembrane domain. In one embodiment, 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.

[0434] Examples of transmembrane domains suitable for use in specific DARIC signaling components contemplated herein include, but are not limited to, the transmembrane regions of the alpha, beta, gamma, or delta chains of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, amnionless (AMN), programmed cell death 1 (PDCD1), NKG2A, NKG2B, NKG2C, and NKG2D. In certain embodiments, the DARIC binding component comprises a CD8α transmembrane domain. In preferred embodiments, the DARIC binding component comprises a CD4 transmembrane domain.

[0435] In various preferred embodiments, a short oligopeptide or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, connects the transmembrane domain and the intracellular signaling domain. Glycine-serine based linkers provide particularly suitable linkers.

[0436] DARIC-binding entities contemplated in certain embodiments herein contain one or more intracellular signaling domains. In one embodiment, the DARIC-binding entity comprises a costimulatory domain.

[0437] Examples of costimulatory domains suitable for use in certain DARIC signaling components contemplated herein include, but are not limited to, 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 (OX40), 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), and T cell receptor associated transmembrane adaptor. 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNFRS25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

[0438] In certain embodiments, the DARIC binding component comprises a CD27 or CD28 costimulatory domain.

[0439] In certain embodiments, the DARIC binding component comprises a TNFRS14, TNFRS18, or TNFRS25 costimulatory domain.

[0440] In certain embodiments, the DARIC binding component comprises an OX40 costimulatory domain.

[0441] In some preferred embodiments, the DARIC binding entity comprises a TNFR2 costimulatory domain.

[0442] In certain embodiments, the DARIC binding components contemplated herein contain a signal peptide, such as a secretory signal peptide, and do not contain a transmembrane domain. Examples of signal peptides suitable for use in certain DARIC binding components include, but are not limited to, an IgG1 heavy chain signal polypeptide, an Igκ light chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. In various preferred embodiments, the DARIC binding component contains a CD8α signal polypeptide.

[0443] In certain embodiments, the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1 or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, OX40 or TNFR2 costimulatory domain.

[0444] In certain embodiments, the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a CD27 or CD28 costimulatory domain.

[0445] In certain embodiments, the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a TNFRS14, TNFRS18, or TNFRS25 costimulatory domain.

[0446] In one embodiment, the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and an OX40 costimulatory domain.

[0447] In another embodiment, the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a TNFR2 costimulatory domain.

[0448] In certain embodiments, the DARIC signaling component comprises a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, and the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, OX40, or TNFR2 costimulatory domain.

[0449] In certain embodiments, the DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, and the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a CD27 or CD28 costimulatory domain.

[0450] In certain embodiments, the DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, and the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a TNFRS14, TNFRS18, or TNFRS25 costimulatory domain.

[0451] In certain embodiments, the DARIC signaling component comprises a FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, and the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and an OX40 costimulatory domain.

[0452] In certain embodiments, the DARIC signaling component comprises an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, and the DARIC binding component comprises a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a TNFR2 costimulatory domain.

[0453] 3. Crosslinking factor In certain embodiments of the present specification, a cross-linking agent is contemplated to mediate or promote the association of one or more DARIC signaling components with one or more DARIC binding components through the multimerization domains in each component. The cross-linking agent is associated with and positioned between the multimerization domains to promote the association of the DARIC signaling component with the DARIC binding component. In the presence of the cross-linking agent, the DARIC binding component and the DARIC signaling component associate, initiating immune effector cell activity against the target cell when the DARIC binding polypeptide binds to a target antigen on the target cell. In the absence of the cross-linking agent, the DARIC binding component does not associate with the DARIC signaling component, and DARIC is inactive.

[0454] In certain embodiments, the DARIC signaling component and the DARIC binding component comprise cognate pair 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 PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

[0455] In certain embodiments, the multimerization domains of the DARIC signaling component and the DARIC binding component are associated with a cross-linking agent 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 a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof.

[0456] In certain embodiments, the DARIC signaling component and the DARIC binding component contain one or more FRB and / or FKBP multimerization domains or variants thereof. In certain embodiments, the DARIC signaling component contains an FRB multimerization domain or variant thereof, and the DARIC binding component contains an FKBP multimerization domain or variant thereof. In certain preferred embodiments, the DARIC signaling component contains an FRB T2098L multimerization domain or variant thereof, and the DARIC binding component contains an FKBP12 or FKBP12 F36V multimerization domain or variant thereof.

[0457] Examples of cross-linking agents suitable for use in certain embodiments contemplated herein include, but are not limited to, AP1903, AP20187, AP21967 (also known as C-16-(S)-7-methylindole rapamycin), everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. In certain preferred embodiments, the cross-linking agent is AP21967. In certain preferred embodiments, the cross-linking agent is a non-immunosuppressive dose of sirolimus (rapamycin).

[0458] D. Engineered Antigen Receptors In certain embodiments, cells are engineered or modified to express one or more DARIC-binding and / or DARIC-signaling components and an engineered antigen receptor. In certain embodiments, the nucleic acid or vector encodes a fusion polypeptide, which contains an engineered receptor, a DARIC-binding and / or DARIC-signaling component, and one or more polypeptide cleavage signals interspersed between the receptor and the component. In other specific embodiments, a polynucleotide or vector encoding DARIC is introduced into immune effector cells containing an engineered antigen receptor. While not wishing to be bound by any particular theory, it is anticipated that in certain embodiments, an engineered antigen receptor and DARIC can be introduced and co-expressed in the same immune effector cell or cells using any suitable mechanism known in the art to enhance the efficiency, potency, and durability of the immune effector cell response. In preferred embodiments, the intracellular signaling domain, e.g., the costimulatory domain, of the engineered antigen receptor, and the DARIC-binding and / or DARIC-signaling domain are different from each other.

[0459] In certain embodiments, immune effector cells contemplated herein comprise an engineered antigen receptor and one or more components of DARIC, hi certain embodiments, the engineered antigen receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), or a zetakine.

[0460] 1. Engineered TCR In certain embodiments, immune effector cells contemplated herein comprise an engineered TCR and one or more components of DARIC. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding the engineered TCR and one or more components of DARIC, separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding the engineered TCR and a polynucleotide or vector encoding one or more components of DARIC. In one embodiment, T cells are engineered to express an engineered TCR and are further engineered by introducing a polynucleotide or vector encoding one or more components of DARIC.

[0461] Natural T cell receptors contain two subunits: an alpha and beta chain subunit (αβTCR) or a gamma and delta chain subunit (γδTCR), each of which is a unique protein generated by a recombination event in each T cell genome. Libraries of TCRs may be screened for selectivity for a particular target antigen. In this manner, natural TCRs with high avidity and reactivity to the target antigen are selected, cloned, and then introduced into a T cell population to be used for adoptive immunotherapy. In one embodiment, the TCR is an αβTCR. In one embodiment, the TCR is a δγTCR.

[0462] In one embodiment, T cells are modified by introducing a TCR subunit capable of forming a TCR that confers specificity to the T cell for tumor cells expressing a target antigen. In a specific embodiment, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to the native subunit, provided that the subunit retains the ability to form a TCR, confer the transfected T cell the ability to home to target cells, and participate in immunologically relevant cytokine signaling. The engineered TCR also preferably binds with high avidity to target cells presenting the relevant tumor-associated peptide, and optionally mediates efficient killing of target cells presenting the relevant peptide in vivo.

[0463] The nucleic acid encoding the engineered TCR is preferably isolated from its original context in the (natural) chromosome of the T cell and may be introduced into a suitable vector as described elsewhere herein. Both the nucleic acid and the vector containing the nucleic acid may be transferred into a cell, preferably a T cell in certain embodiments. The modified T cell can then express one or more chains of the TCR encoded by the transduced nucleic acid. In a preferred embodiment, the engineered TCR is an exogenous TCR in that it is introduced into a T cell that does not normally express the specific TCR. An essential aspect of an engineered TCR is that it has high avidity for tumor antigens presented by the major histocompatibility complex (MHC) or similar immune components. In contrast to engineered TCRs, CARs are engineered to bind to target antigens in an MHC-independent manner.

[0464] The TCR may be expressed with additional polypeptides attached to the amino- or carboxyl-terminal portions of the alpha or beta chains, or the gamma or delta chains of the TCR, provided that the added additional polypeptides do not interfere with the ability of the alpha or beta chains to form a functional T cell receptor and to recognize antigen in an MHC-dependent manner.

[0465] In certain embodiments, antigens recognized by the engineered TCRs contemplated include, but are not limited to, cancer antigens, including antigens in both hematological cancers and solid tumors. Exemplary antigens include, but are not limited to, FRα, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0466] In a preferred embodiment, the target antigen is expressed on one or more cells of the cancer and is selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII.

[0467] 2. Chimeric Antigen Receptor In certain embodiments, the immune effector cells contemplated herein comprise one or more components of a CAR and a DARIC. A chimeric antigen receptor (CAR) is a molecule that combines the specificity of an antibody system for a target antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell-mediated immune activity. As used herein, the term "chimera" refers to something that is composed of different protein or DNA portions derived from different sources.

[0468] In one embodiment, 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 engineered to express a CAR are further engineered by introducing a polynucleotide or vector encoding one or more DARIC components.

[0469] In various embodiments, CARs contain an extracellular domain (also referred to as a binding domain or antigen-specific binding domain) that binds to a specific target antigen, a transmembrane domain, and one or more intracellular signaling domains. A key feature of CARs is their ability to redirect the specificity of immune effector cells, thereby eliciting the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, eliciting the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0470] In certain embodiments, the CAR contains an extracellular binding domain that specifically binds to a target polypeptide. In a preferred embodiment, the CAR binds to a target polypeptide that is different from the target polypeptide bound by the DARIC binding component. The binding domain contains any natural, synthetic, semi-synthetic, or recombinantly produced binding partner for the target biological molecule.

[0471] In certain embodiments, the extracellular binding domain comprises an antibody or an antigen-binding fragment thereof.

[0472] In one preferred embodiment, the binding domain comprises an scFv.

[0473] In another preferred embodiment, the binding domain comprises one or more camelid antibodies.

[0474] In certain embodiments, the CAR comprises an extracellular domain that binds to an antigen selected from the group consisting of: FRα, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD1 71, 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-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN), MUC1, MUC16, MICA, MIC B, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1 / CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, VEGFR2, and WT-1.

[0475] In a preferred embodiment, the CAR comprises an extracellular domain that binds to an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII.

[0476] In certain embodiments, the CAR contains an extracellular binding domain, such as an antibody or an antigen-binding fragment thereof, that binds to an antigen, where the antigen is an MHC-peptide complex, such as a class I MHC-peptide complex, or a class II MHC-peptide complex.

[0477] In one embodiment, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4 or IgD.

[0478] Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α and CD4, which may be the wild-type hinge regions of these molecules or may be modified. In another embodiment, the hinge domain contains a CD8α hinge region.

[0479] In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge.

[0480] The transmembrane (TM) domain of the CAR connects the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the cell membrane of the immune effector cell. The TM domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0481] Exemplary TM domains may be derived from (i.e., contain) at least the transmembrane regions of the alpha, beta, gamma, or delta chains of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD134, CD137, CD152, CD154, AMN, PDCD1, NKG2A, NKG2B, NKG2C, and NKG2D.

[0482] In one embodiment, the CAR contains a TM domain derived from CD8α. In another embodiment, the CARs contemplated herein contain a TM domain derived from CD8α and a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, which connects the TM domain of the CAR to the intracellular signaling domain. A glycine-serine linker provides a particularly suitable linker.

[0483] In a preferred embodiment, the CAR contains one or more costimulatory domains and an intracellular signaling domain comprising a primary signaling domain.

[0484] The primary stimulatory signaling domain may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0485] Exemplary ITAM-containing primary signaling domains suitable for use in CARs contemplated in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In certain preferred embodiments, the CAR contains a CD3ζ primary signaling domain and one or more costimulatory domains. The intracellular primary signaling domain and costimulatory domain may be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0486] In certain embodiments, the CAR contains one or more costimulatory domains to enhance the efficacy and expansion of T cells expressing the CAR receptor.

[0487] Exemplary costimulatory molecules suitable for use in CARs contemplated in certain embodiments include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70. In one embodiment, the CAR contains one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0488] In various embodiments, the CAR comprises an extracellular domain that binds to an antigen selected from the group consisting of: BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B, CD123, CLL-1, or EGFRvIII; a CD4, CD8α, 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 CD3ζ primary signaling domain.

[0489] 3. Zetakine In various embodiments, immune effector cells contemplated herein contain one or more chains of a zetakine receptor and one or more DARIC components. Zetakines are chimeric transmembrane immunoreceptors, containing an extracellular domain with a soluble receptor ligand linked to a support region, which can connect the extracellular domain to the cell surface, a transmembrane region, and an intracellular signaling domain. When zetakines are expressed on the surface of T lymphocytes, they direct T cell activity toward cells expressing receptors specific for the soluble receptor ligand. Zetakine chimeric immunoreceptors redirect the antigen specificity of T cells via an autocrine / paracrine cytokine system, particularly exploited in human malignancies, and are applicable to the treatment of various cancers.

[0490] In one embodiment, 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. In one embodiment, T cells are engineered to express one or more chains of a zetakine receptor and are further engineered by introducing a polynucleotide or vector encoding one or more DARIC components.

[0491] In certain embodiments, the zetakine contains an immunosuppressive cytokine or cytokine receptor-binding variant thereof, a linker, a transmembrane domain, and an intracellular signaling domain.

[0492] In certain embodiments, 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).

[0493] In certain embodiments, the linker contains a CH2CH3 domain, a hinge domain, etc. In one embodiment, the linker contains the CH2 and CH3 domains of IgG1, IgG4, or IgD. In one embodiment, the linker contains the hinge domain of CD8α or CD4.

[0494] In certain embodiments, the transmembrane domain is selected from the group consisting of the alpha, beta, gamma or delta chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD-1.

[0495] In certain embodiments, the intracellular signaling domain is selected from the group consisting of an ITAM-containing primary signaling domain and / or a costimulatory domain.

[0496] In specific embodiments, the intracellular signaling domain is selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0497] In certain embodiments, the intracellular signaling domain is 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 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70.

[0498] In one embodiment, the chimeric cytokine receptor contains one or more costimulatory domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0499] E. Polypeptides Various polypeptides are contemplated herein, including, but not limited to, DARIC binding components, DARIC signaling components, engineered TCRs, CARs, zetakines, and fusion proteins containing the aforementioned polypeptides and fragments thereof. In preferred embodiments, the polypeptide contains the amino acid sequence set forth in any one of SEQ ID NOS: 1-29. "Polypeptide," "peptide," and "protein" are used interchangeably and follow their conventional meanings unless otherwise specified, i.e., as used in reference to amino acid sequences. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of known recombinant and / or synthetic techniques. Polypeptides are not limited to a particular length. For example, polypeptides may contain full-length protein sequences, fragments of full-length proteins, or fusion proteins. Polypeptides may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and other natural and non-natural modifications known in the art. In certain preferred embodiments, fusion polypeptides, polypeptides, fragments, and other variants thereof are prepared, obtained, or isolated from one or more human polypeptides.

[0500] As used herein, "isolated peptide" or "isolated polypeptide" or the like refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., substantially free from association with in vivo substances. In certain embodiments, the isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0501] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may be natural or synthetically produced, for example, by modifying one or more of the above-described polypeptide sequences. For example, in certain 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 into the polypeptide. In certain embodiments, polypeptides include those 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%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, and typically, such variants retain at least one biological activity of the reference sequence. In certain embodiments, the biological activity is binding affinity. In certain embodiments, the biological activity is enzymatic activity.

[0502] In some embodiments, the DARIC contains a polypeptide complex containing (i) a first polypeptide, e.g., a first fusion polypeptide having a first multimerization domain, and (ii) a second polypeptide, e.g., a second fusion polypeptide having a second multimerization domain. In certain embodiments, the multimerization domains are identical. In some embodiments, the first multimerization domain is different from the second multimerization domain. The first and second multimerization domains significantly contribute to or efficiently promote the formation of the polypeptide complex in the presence of a cross-linking agent. The interaction between the first multimerization domain and the second multimerization domain significantly contributes to or efficiently promotes the multimerization of the first fusion polypeptide and the second fusion polypeptide if, in the absence of the first multimerization domain, the second multimerization domain, or the cross-linking agent, the association between the first fusion polypeptide and the second fusion polypeptide is statistically significantly reduced. In certain embodiments, when the first fusion polypeptide and the second fusion polypeptide are co-expressed, at least about 60%, e.g., 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 single-chain polypeptide and the second single-chain polypeptide form multimers with each other in the presence of a cross-linking agent.

[0503] Polypeptide variants include biologically active "polypeptide fragments." Examples of biologically active polypeptide fragments include binding domains, intracellular signaling domains, and the like. As used herein, the term "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 activity of the native polypeptide. In certain embodiments, a polypeptide fragment may contain an amino acid chain of at least 5 to about 1700 amino acids in length. In certain embodiments, the fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or more amino acids in length.

[0504] In certain embodiments, the polypeptides described herein may contain one or more amino acids designated "X." When present in an amino acid SEQ ID NO:, "X" refers to any one or more amino acids. In certain embodiments, a SEQ ID NO representing a fusion protein contains a sequence of consecutive X residues that cumulatively represent any amino acid sequence.

[0505] As mentioned above, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation and insertion. Methods for such manipulation are generally known in the art. For example, amino acid sequence variants of reference polypeptides can be created by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. For example, see Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, J.D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0506] In some embodiments, a polypeptide variant contains one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid with similar properties, and one skilled in the art of peptide chemistry would predict that such a substitution would not substantially alter the secondary structure and hydrophilic properties of the polypeptide. In certain embodiments, modifications may be made to the expected polynucleotide and polypeptide structures while still obtaining functional molecules encoding variant or derivative polypeptides with desired characteristics. If it is desired to alter the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art can change one or more codons in the encoding DNA sequence, for example, according to Table 1. [Table 1-1] [Table 1-2]

[0507] Guidance for determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs known in the art, such as DNASTAR, DNA Strider, Geneious, MacVector, or Vector NTI software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids that are related by their side chains. Natural amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0508] In one embodiment, when expression of more than one polypeptide is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence, as otherwise disclosed herein.

[0509] In certain embodiments, contemplated polypeptides include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding the fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments. In preferred embodiments, the fusion polypeptide contains one or more DARIC components. In other preferred embodiments, the fusion polypeptide contains one or more DARIC components.

[0510] In another embodiment, two or more polypeptides may be expressed as a fusion protein containing one or more self-cleaving polypeptide sequences between the polypeptides as otherwise disclosed herein.

[0511] Fusion polypeptides may contain one or more polypeptide domains or segments, including, but not limited to, signal peptides, cell permeable peptide domains (CPPs), binding domains, signaling domains, 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-terminally to N-terminally, but can also be linked C-terminally to C-terminally, N-terminally to N-terminally, or M-terminally to C-terminally. In certain embodiments, the polypeptides of the fusion protein may be in any order. Fusion polypeptides or fusion proteins may include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, provided that the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be made by chemical synthesis or by chemical conjugation between two moieties, or may be generally prepared using other standard methods. The ligated DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements as otherwise disclosed herein.

[0512] Fusion polypeptides may optionally contain one or more linkers, which can be used to link 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 allow each polypeptide to fold into its appropriate secondary and tertiary structure, thereby enabling the polypeptide domain to perform its desired function. Such peptide linker sequences are incorporated into fusion polypeptides using standard techniques in the art. An appropriate peptide linker sequence may be selected based on the following factors: (1) the ability to adopt a flexible, extended conformation; (2) the ability to avoid secondary structures that could interact with functional epitopes on the first and second polypeptides; and (3) the absence of hydrophobic or charged residues that could interact with the functional epitopes of the polypeptides. In certain embodiments, preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other closely related neutral amino acids, such as Thr and Ala, may also be used in linker sequences. Amino acid sequences that can be usefully employed as linkers include those described in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. A linker sequence is not necessary if a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate the functional domains and prevent steric interference. In certain embodiments, preferred linkers are typically flexible amino acid subsequences that are synthesized as part of the recombinant fusion protein. Linker polypeptides can be 1 to 200 amino acids in length, 1 to 100 amino acids in length, or 1 to 50 amino acids in length, including all integer values ​​between those values.

[0513] Examples of 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).

[0514] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, in one embodiment TEV (tobacco etch virus) protease cleavage sites, e.g., EXXYXQ(G / S) (SEQ ID NO: 41), such as ENLYFQG (SEQ ID NO: 42) and ENLYFQS (SEQ ID NO: 43), are preferred, where X represents any amino acid (TEV cleavage occurs between Q and G or between Q and S).

[0515] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0516] Exemplary ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0517] In one embodiment, 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 tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0518] Examples of 2A sites are provided in Table 2. [Table 2-1] [Table 2-2]

[0519] In a preferred embodiment, the polypeptide or fusion polypeptide contains one or more DARIC components or DARICs.

[0520] In certain embodiments, the fusion polypeptide contains a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain, a viral self-cleaving 2A polypeptide, and a DARIC binding component comprising a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1 or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a CD27, CD28, TNFRS14, TNFRS18, TNFRS25, OX40 or TNFR2 costimulatory domain.

[0521] In certain embodiments, the fusion polypeptide contains a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain, a viral self-cleaving 2A polypeptide, and a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1 or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a CD27 or CD28 costimulatory domain.

[0522] In certain embodiments, the fusion polypeptide contains a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain, a viral self-cleaving 2A polypeptide, and a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1 or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a TNFRS14, TNFRS18 or TNFRS25 costimulatory domain.

[0523] In certain embodiments, the fusion polypeptide contains a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain, a viral self-cleaving 2A polypeptide, and a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1 or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and an OX40 costimulatory domain.

[0524] In certain embodiments, the fusion polypeptide contains a DARIC signaling component comprising an FRB T2098L multimerization domain, a CD8α transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain, a viral self-cleaving 2A polypeptide, and a binding domain that binds to BCMA, CD19, CD20, CD22, CD33, B7H3, CD33, CD79A, CD79B, CD123, CLL-1 or EGFRvIII, an FKBP12 multimerization domain polypeptide, a CD4 transmembrane domain or an AMN transmembrane domain, and a TNFR2 costimulatory domain.

[0525] F. Polynucleotides In certain embodiments, polynucleotides are provided that encode one or more DARIC components, engineered TCRs, CARs, zetakines, or fusion proteins comprising the aforementioned polypeptides and fragments thereof. As used herein, the terms "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 may be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-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. Polynucleotide refers to a polymeric form of nucleotides having a length 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, including ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the recited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc.In certain embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.

[0526] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from sequences that normally flank it. An "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been made by the hand of man. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0527] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein, hi some embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0528] In certain embodiments, a polynucleotide encoding one or more DARICs may be codon-optimized. As used herein, the term "codon-optimized" refers to the substitution of codons in a polynucleotide encoding a polypeptide to improve expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias 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 with their context; (iv) variation of codons with their decoding tRNAs; (v) variation of codons with GC % either across triplets or at a single position in the triplets; (vi) variation in similarity to a reference sequence, e.g., a natural sequence; (vii) variation in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence upon which the codon substitution set is designed; (x) synthetic variation of the codon set for each amino acid; and / or (xi) isolated elimination of incorrect translation start sites. Furthermore, one of skill in the art will recognize that, as a result of the degeneracy of the genetic code, numerous nucleotide sequences exist that encode the polypeptides described herein, or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any natural gene. However, polynucleotides that vary due to differences in codon usage are contemplated, and in certain embodiments, polynucleotides that are optimized for, for example, human and / or primate codon preferences are contemplated, hi certain embodiments, the polynucleotides are codon optimized for expression and / or stability.

[0529] As used herein, the term "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 modified bases recognized in the art. Such bases are typically located at the 1' position of the nucleotide sugar moiety. Nucleotides generally contain a base, sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose. That is, deoxyribose is a sugar lacking the hydroxyl group present in ribose. Examples of natural nitrogenous bases include the purines adenosine (A) and guanidine (G) and the pyrimidines cytidine (C) and thymidine (T) (or, in the case of RNA, uracil (U)). The C-1 atom of deoxyribose is linked to the N-1 atom of a pyrimidine or the N-9 atom of a purine. Nucleotides are typically mono-, di-, or triphosphates. Nucleotides can be unmodified or modified at the sugar, phosphate, and / or base moieties (interchangeably referred to as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see, e.g., WO 92 / 07065 and WO 93 / 15187). Examples of modified nucleobases are summarized in Limbach et al. (1994, Nucleic Acids Res. 22, 2183-2196).

[0530] Nucleotides can also be considered as phosphate esters of nucleosides, with esterification occurring at the hydroxyl group attached to C-5 of the sugar. As used herein, 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 known modified bases. Such bases are generally located at the 1' position of the nucleoside sugar moiety. Nucleosides generally contain a base and a sugar group. Nucleosides can be unmodified or modified in the sugar and / or base moieties (interchangeably referred to as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). As mentioned above, examples of modified nucleobases are summarized in Limbach et al. (1994, Nucleic Acids Res. 22, 2183-2196).

[0531] Examples of polynucleotides include, but are not limited to, polynucleotides that encode the polypeptides set forth in SEQ ID NOs: 1-29.

[0532] In various embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding one or more DARIC components, DARIC, engineered antigen receptors, fusion polypeptides, as well as expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein.

[0533] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with reference polynucleotide sequence, or hybridizes with reference sequence under stringent conditions as defined herein.These terms also include polynucleotides that are distinguished from reference polynucleotides by the addition, deletion, substitution or modification of at least one nucleotide.Therefore, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides are added, deleted, or modified, or replaced with another nucleotide.In this regard, it is understood in the art that certain modifications, including mutations, additions, deletions and substitutions, can be made to reference polynucleotides, and modified polynucleotides can retain the biological function or biological activity of reference polynucleotides.

[0534] As used herein, "sequence identity," or phrases such as "a sequence 50% identical to," refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percentage of sequence identity" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present in both sequences; calculating the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size); and multiplying the result by 100 to calculate the percentage of sequence identity. Contained are polynucleotides or polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences contemplated herein.

[0535] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence in a vector capable of expressing RNA and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid cassettes. The nucleic acid cassettes are positionally and sequentially oriented within the vector, allowing the nucleic acid in the cassette to be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in transformed cells, and targeted to an appropriate intracellular compartment for translocation to a compartment suitable for biological activity or secreted into an extracellular compartment. The cassette preferably has 3' and 5' ends adapted for immediate insertion into a vector, e.g., restriction endonuclease sites at each end. The cassette can be excised and inserted as a single unit into a plasmid or viral vector.

[0536] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide, or serves as a template for transcription of an inhibitory polynucleotide, as contemplated herein.

[0537] The polynucleotides contemplated herein may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., regardless of the length of the coding sequence itself, as described elsewhere herein or as known in the art. Thus, it is contemplated that polynucleotide fragments of almost any length may be employed, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0538] Polynucleotides may be prepared, manipulated, expressed, and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide may be inserted into an appropriate vector.

[0539] Examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as Sleeping Beauty and PiggyBac.

[0540] Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.

[0541] Examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40).

[0542] Exemplary expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0543] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the chromosomal DNA of a host, and is not gradually reduced with the division of the host cell, which also means that the vector replicates extrachromosomally or episomally.

[0544] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are untranslated regions of the vector, including origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, and 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. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, may be used.

[0545] In certain embodiments, the polynucleotide comprises a vector, including, but not limited to, an expression vector and a viral vector. The vector may contain one or more exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous regulatory sequence" is a sequence naturally linked to a given gene in the genome. An "exogenous regulatory sequence" is a sequence placed in juxtaposition to a gene by genetic engineering (i.e., molecular biological techniques), such that transcription of the gene is induced by the linked enhancer / promoter. A "heterologous regulatory sequence" is an exogenous sequence derived from a species different from the cell being genetically engineered. A "synthetic" regulatory sequence may contain one or more endogenous and / or exogenous sequence elements, and / or sequence elements determined in vitro or in silico, to provide optimal promoter and / or enhancer activity for a particular therapy.

[0546] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates transcription of a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells contain an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or a separate sequence, a CNCAAT region, located 70-80 bases upstream from the transcription start site, where N can be any nucleotide.

[0547] The term "enhancer" refers to a DNA segment containing a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoter elements and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing a sequence that can provide both promoter and enhancer functions.

[0548] The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0549] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that permits continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in restricted cell and tissue types, respectively.

[0550] Examples of ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney Murine Leukemia Virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa, and the like. beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003;77(17):9439-9450).

[0551] In one embodiment, the vector contains the MNDU3 promoter.

[0552] In one embodiment, the vector contains the EF1a promoter including the first intron of the human EF1a gene.

[0553] In one embodiment, the vector contains the EF1a promoter lacking the first intron of the human EF1a gene.

[0554] In certain embodiments, it may be desirable to use cell-, cell type-, cell line-, or tissue-specific expression control sequences to achieve cell-type-, cell line-, or tissue-specific expression of a desired polynucleotide sequence (e.g., expressing a nucleic acid encoding a particular polypeptide only in a subset of cell types, cell lines, or tissues, or only at a particular stage of development).

[0555] In certain embodiments, it may be desirable to express the polynucleotide from a T cell specific promoter.

[0556] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, and expression in cells or tissues that are in a particular physiological, biological, or disease state. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression controlled by exposing a cell, tissue, or organism to a treatment or condition that results in expression of the polynucleotide or that increases or decreases expression of a polynucleotide encoded by the polynucleotide of interest.

[0557] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducers include, but are not limited to, glucocorticoids, estrogen, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0558] As used herein, "internal ribosome entry site" or "IRES" refers to a factor that promotes direct entry of an internal ribosome into an initiation codon, such as ATG, of a cistron (protein-coding region), resulting in cap-independent gene translation. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83 and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly employed by those skilled in the art include those described in U.S. Pat. No. 6,692,736. Further examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picornaviruses (Jackson et al., 1990), and IRES obtainable from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), the translation initiation factor eIF4G, and the yeast transcription factors TFIID and HAP4, the encephalomyocarditis virus (EMCV) IRES available from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9), and the VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRESes have also been reported in viral genomes of species from the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0559] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0560] In certain embodiments, the polynucleotide comprises a consensus Kozak sequence. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that significantly promotes the initial binding of mRNA to the small ribosomal subunit, increasing 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).

[0561] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally located downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed. As used herein, the terms "polyA site" or "polyA sequence" refer to a DNA sequence that induces both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thereby contributing to enhanced translation efficiency. Cleavage and polyadenylation are induced by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNAs has two recognition factors flanking the cleavage-polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable factor rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, adding up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a poly(A) sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 poly(A) sequence, bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art. In certain embodiments, the poly(A) sequence is synthetic.

[0562] In some embodiments, the polynucleotide or cells harboring the polynucleotide utilize a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled amplification. In certain embodiments, the suicide gene is not immunogenic to the host or cells harboring the polynucleotide. Examples of suicide genes that can be used are caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0563] In certain embodiments, polynucleotides encoding one or more polypeptides or fusion polypeptides may be introduced into immune effector cells, such as T cells, by both non-viral and viral methods. In certain embodiments, delivery of one or more polynucleotides may be provided by the same or different methods and / or on the same or different vectors.

[0564] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The nucleic acid to be transferred is generally linked, e.g., inserted, into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in the cell or may contain sequences sufficient to allow integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to T cells.

[0565] Examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0566] Non-viral methods of polynucleotide delivery contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0567] Examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids 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 delivery, bacteria-guided delivery, and non-biological nanocell-based delivery are also contemplated in certain embodiments.

[0568] In certain embodiments, viral vectors containing contemplated polynucleotides can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or local application as described below. Alternatively, vectors can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or hematopoietic stem cells of a universal donor, followed by reimplantation of the cells into the patient.

[0569] In one embodiment, a viral vector containing a polynucleotide contemplated herein is administered directly to an organism and transduced into cells in vivo. Alternatively, naked DNA may be administered. Administration may be by any of the routes commonly used to introduce molecules into blood or tissue cells for ultimate contact, including, but not limited to, injection, infusion, topical application, and electroporation. While suitable methods for administering such nucleic acids are available and known to those skilled in the art, multiple routes may be used to administer a particular composition, and certain routes can often provide a more immediate and effective response than other routes.

[0570] Examples of viral vector systems suitable for use in certain contemplated embodiments include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0571] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells, such as T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) containing the one or more polynucleotides.

[0572] AAV is a small (approximately 26 nm), replication-incompetent, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may integrate its genome into the genome of the host cell. Recombinant AAV (rAAV) typically consists, at a minimum, of a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are approximately 145 bp in length. In a specific embodiment, the rAAV contains ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0573] In some embodiments, chimeric rAAVs are used. The ITR sequences are isolated from one AAV serotype, and the capsid sequence is isolated from another AAV serotype. For example, an rAAV containing ITR sequences from AAV2 and a capsid sequence from AAV6 is called AAV2 / AAV6. In certain embodiments, the rAAV vector may contain ITRs from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV contains ITR sequences from AAV2 and a capsid sequence from AAV6. In a preferred embodiment, the rAAV contains ITR sequences from AAV2 and a capsid sequence from AAV2.

[0574] In some embodiments, AAV capsids may be subjected to methods of engineering and selection to increase the probability that they will transduce cells of interest.

[0575] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Patent Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058, and 8,784,799, which are incorporated herein by reference in their entireties.

[0576] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells, e.g., T cells, by transducing the cells with a retrovirus, e.g., a lentivirus, containing the one or more polynucleotides.

[0577] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Examples of retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus.

[0578] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Examples of lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2), Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0579] In various embodiments, lentiviral vectors contemplated herein contain one or more LTRs and one or more or all of the following accessory elements: cPPT / FLAP, Psi (Ψ) packaging signal, export element, poly(A) sequence, and optionally, a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as otherwise discussed herein.

[0580] In certain embodiments, the lentiviral vector contemplated herein can be an integrative, or non-integrative, or integration-defective lentivirus.As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that has an integrase that is deficient in the ability to integrate viral genome into host cell genome.Integration-defective viral vectors are described in patent application WO 2006 / 010834, and this application is incorporated herein by reference in its entirety.

[0581] Examples of mutations in the HIV-1 pol gene that are suitable for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0582] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA; in the native sequence, the LTR is a direct repeat and contains the U3, R and U5 regions.

[0583] As used herein, the term "FLAP element," or "cPPT / FLAP," refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of retroviruses, such as HIV-1 and HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173.

[0584] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located in the retroviral genome, which is required for the insertion of viral RNA into the viral capsid or viral particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0585] The term "export factor" refers to a cis-acting post-transcriptional regulatory element that controls the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export factors include, but are not limited to, the rev-responsive element (RRE) of human immunodeficiency virus (HIV) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the post-transcriptional regulatory element (HPRE) of hepatitis B virus.

[0586] In certain embodiments, the expression of heterologous sequences in viral vectors can be increased by incorporating post-transcriptional regulators, efficient polyadenylation sites, and optionally transcription termination signals into the vector.Various post-transcriptional regulators can increase the expression of heterologous nucleic acids in protein.For example, woodchuck hepatitis virus post-transcriptional regulator (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); post-transcriptional regulator (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); etc. (Liu et al., 1995, Genes Dev., 9:1766).

[0587] Lentiviral vectors preferably contain several safety enhancements as a result of the LTR modifications. A "self-inactivating" (SIN) vector refers to a vector lacking replication competence, such as a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to inhibit viral transcription beyond the first round of viral replication. Self-inactivation is preferably achieved by introducing a deletion into the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to generate vector RNA. This deletion is then transferred to the 5' LTR of the proviral DNA during reverse transcription. In certain embodiments, it is desirable to remove the U3 sequence sufficiently to greatly reduce or completely abolish the transcriptional activity of the LTR, thereby greatly reducing or abolishing the production of full-length vector RNA in transduced cells. In the case of HIV-based lentivectors, it has been shown that such vectors can tolerate large U3 deletions, including removal of the LTR TATA box (e.g., deletion of -418 to -18), without significant loss of vector titer.

[0588] Additional safety enhancements are provided by replacing the U3 region of the 5'LTR with a heterologous promoter that drives transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include 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.

[0589] As used herein, the term "pseudotype" or "pseudotyped" refers to a virus whose viral envelope protein has been replaced with the envelope of another virus that has favorable properties. For example, the HIV envelope protein (encoded by the env gene) is normally encoded by the CD4 + Although targeting the virus to presentation cells, pseudotyping HIV with the vesicular stomatitis virus G-protein (VSV-G) envelope protein allows HIV to infect a broad range of cells.

[0590] In one embodiment, lentiviral vectors are produced by 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.

[0591] According to certain embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it should be understood that retroviral and / or lentiviral sequences from many different sources may be used or combined, and numerous substitutions and modifications in a particular lentiviral sequence may be accommodated without impairing the transfer vector's ability to perform the functions described herein. Furthermore, a variety of lentiviral vectors are known in the art. See Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998; U.S. Patent Nos. 6,013,516 and 5,994,136. Many of these can be adapted to produce the viral vectors or transfer plasmids contemplated herein.

[0592] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells by transducing the cells with an adenovirus containing the one or more polynucleotides.

[0593] Adenovirus-based vectors can transduce many cell types with very high efficiency and do not require cell division. Such vectors can achieve high titers and high levels of expression. They can be produced in large quantities using a relatively simple system. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes. The resulting replication-deficient vectors are amplified in human 293 cells, which then supply the missing gene function in trans. Ad vectors can transduce multiple tissue types in vivo, including non-dividing and differentiated cells, such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0594] Current replication-deficient adenovirus vectors are sometimes generated and amplified using a unique helper cell line called 293. This cell line was derived from human embryonic kidney cells transformed with Ad5 DNA fragments and constitutively expresses the E1 protein (Graham et al., 1977). Because the E3 region of the adenovirus genome is nonessential (Jones & Shenk, 1978), current adenovirus vectors rely on the help of 293 cells to carry foreign DNA in the E1, D3, or both regions (Graham & Prevec, 1991). Adenovirus vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Experiments using recombinant adenoviruses to administer them to various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Clinical trials using Ad vectors include polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0595] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into immune effector cells by transducing the cells with a herpes simplex virus, e.g., HSV-1 or HSV-2, containing the one or more polynucleotides.

[0596] Mature HSV virions consist of an enveloped icosahedral capsid containing a viral genome consisting of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions that remove one or more intermediate-early, early, or late HSV genes, inhibiting replication. For example, the HSV vector may lack an immediate-early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter late stages and generate long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA inserts of up to 25 kb. HSV-based vectors are described, for example, in 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, which are incorporated herein by reference in their entireties.

[0597] G. Genetically Modified Cells In various embodiments, cells are modified to express one or more DARIC components, DARIC, engineered TCRs, CARs, zetakines, and / or fusion proteins contemplated herein for use in cancer therapy. Cells may be non-genetically modified to express one or more of the polypeptides contemplated herein, or in certain preferred embodiments, cells may be genetically modified to express one or more of the polypeptides contemplated herein. As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably in certain embodiments.

[0598] In certain embodiments, one or more DARIC components contemplated herein are introduced into and expressed in immune effector cells to improve the efficacy of the immune effector cells. In certain embodiments, dual-targeted immune effector cells are contemplated when target cells express an antigen recognized by a first DARIC binding component and another antigen recognized by a second DARIC binding component. In certain embodiments, one or more DARIC components are introduced into and expressed in immune effector cells that have been redirected to target cells by co-expressing an engineered antigen receptor, such as a CAR, in the cells. In certain embodiments, dual-targeted immune effector cells are contemplated when target cells express an antigen recognized by an engineered antigen receptor and a different antigen recognized by DARIC.

[0599] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.). Exemplary immune effector cells contemplated herein are T lymphocytes, including, but not limited to, cytotoxic T cells (CTL; ​​CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + In certain embodiments, the cells include αβ T cells. In certain embodiments, the cells include γδ T cells. In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells. Immune effector cells may be self or non-self (e.g., allogeneic, syngeneic, or xenogeneic).

[0600] As used herein, "autologous" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cells. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the compared cells. As used herein, "xenogeneic" refers to cells of a different species from the compared cells. In a preferred embodiment, the cells are human autoimmune effector cells.

[0601] Examples of immune effector cells suitable for the introduction of one or more DARIC components or DARICs contemplated herein include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, such as T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells may be helper T cells (HT1; CD4 + T cell) CD4 + T cells, cytotoxic T cells (CTL:CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The T cells may be T cells, or any other subset of T cells. Other examples of T cell populations suitable for use in certain embodiments include naive T cells and memory T cells.

[0602] As will be understood by those skilled in the art, other cells can also be used as immune effector cells comprising one or more DARIC components or DARICs contemplated herein. In certain embodiments, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, where such precursor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject or may be induced to differentiate into mature immune effector cells in vitro.

[0603] As used herein, the term "CD34+ cells" refers to cells that express CD34 protein on their cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes. CD34+ cell populations contain hematopoietic stem cells (HSCs), which, when administered to patients, differentiate and give rise to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0604] Methods for generating immune effector cells expressing one or more DARIC components contemplated herein are provided in certain embodiments. In one embodiment, the method involves transfecting or transducing immune effector cells isolated from an individual, such that the immune effector cells, comprising one or more nucleic acids and / or vectors, or a combination thereof, contain one or more DARIC components contemplated herein. In one embodiment, the method involves transfecting or transducing immune effector cells isolated from an individual, such that the immune effector cells express one or more DARIC components contemplated herein and an engineered antigen receptor. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells may then be directly re-administered to the individual. In a further embodiment, immune effector cells are first activated in vitro and stimulated to proliferate, followed by genetic modification. In this regard, the immune effector cells may be cultured before and / or after genetic modification.

[0605] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein, hi certain embodiments, the modified immune effector cells comprise T cells.

[0606] T cells can be obtained from many sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as sedimentation, for example, FICOLL™ separation.

[0607] In other embodiments, isolated or purified T cell populations are used. In some embodiments, after isolation of PBMCs, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subpopulations before or after activation, expansion, and / or genetic modification.

[0608] In one embodiment, the isolated or purified T cell population includes, but is not limited to, CD3 + , CD4 + , CD8 + or express one or more of the markers, including a combination thereof.

[0609] In one embodiment, T cells are isolated from an individual and first activated and stimulated in vitro to expand and then modified to express one or more DARIC components.

[0610] To obtain a sufficient therapeutic dose of a T cell composition, T cells often undergo one or more rounds of stimulation, activation, and / or expansion. In certain embodiments, T cells may generally be activated and expanded using methods described, for example, in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041. These patents are incorporated herein by reference in their entirety. In certain embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours, after which a vector or polynucleotide encoding one or more DARIC components is introduced, optionally in combination with an engineered antigen receptor as contemplated herein.

[0611] In one embodiment, the T cells are simultaneously activated when they are modified.

[0612] In various embodiments, methods for generating immune effector cells include activating a population of cells comprising T cells and expanding the T cell population. T cell activation can be achieved by providing a primary stimulatory signal via the TCR / CD3 complex of the T cells and a secondary costimulatory signal via an accessory molecule such as CD28.

[0613] The TCR / CD3 complex may be stimulated by contacting the T cell with a suitable CD3-binding agent, such as a CD3 ligand or an anti-CD3 monoclonal antibody, including, but not limited to, OKT3, G19-4, BC3, and 64.1.

[0614] In addition to the primary stimulatory signal provided via the TCR / CD3 complex, induction of a T cell response requires a secondary costimulatory signal. In certain embodiments, a CD28 binding agent may be used to provide the costimulatory signal. Exemplary CD28 binding agents include, but are not limited to, natural CD28 ligands, such as natural ligands for CD28 (e.g., members of the B7 family of proteins, e.g., B7-1 (CD80) and B7-2 (CD86)); and anti-CD28 monoclonal antibodies, or fragments thereof capable of cross-linking the CD28 molecule, such as monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0615] In one embodiment, the molecule that provides the primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and the costimulatory molecule are linked to the same surface.

[0616] In certain embodiments, the binding agents that provide the stimulatory and costimulatory signals are localized on the cell surface, which can be achieved by transfecting or transducing the cell with a nucleic acid encoding the binding agent in a form suitable for expression on the cell surface, or by linking the binding agent to the cell surface.

[0617] In another embodiment, a molecule that provides a primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and a costimulatory molecule are presented on an antigen-presenting cell.

[0618] In one embodiment, the molecule that provides the primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and the costimulatory molecule are displayed on separate surfaces.

[0619] In certain embodiments, one of the binding agents providing the stimulatory and costimulatory signals is soluble (provided in solution) and the other agent is provided on one or more surfaces.

[0620] In certain embodiments, the binding agents that provide the stimulatory signal and the costimulatory signal are both provided in soluble form (provided in solution).

[0621] In various embodiments, the methods of generating T cells contemplated herein include activating T cells with anti-CD3 and anti-CD28 antibodies.

[0622] In one embodiment, expansion of T cells activated by the methods contemplated herein further comprises culturing the population of cells comprising the T cells for a period of time ranging from several hours (about 3 hours) to about 7 days to about 28 days, or any integer number of hours therebetween. In another embodiment, the T cell composition may be cultured for 14 days. In a specific embodiment, the T cells are cultured for about 21 days. In another embodiment, the T cell composition is cultured for about 2-3 days. Multiple cycles of stimulation / activation / expansion may be desirable, potentially extending the T cell culture period to 60 days or more.

[0623] In certain embodiments, conditions suitable for T cell culture include an appropriate medium (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for growth and activity, including, but not limited to, serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other supplement suitable for cell growth known to one of skill in the art.

[0624] Further exemplary 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, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a combination of predetermined hormones and / or cytokines in amounts sufficient for T cell growth and expansion.

[0625] Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures and not in the cell cultures intended for injection into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0626] In certain embodiments, PBMCs, or isolated T cells, are contacted with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15.

[0627] In other embodiments, artificial APCs (aAPCs) are generated by engineering K562, U937, 721.221, T2, and C1R cells to induce stable expression and secretion of various costimulatory molecules and cytokines. In specific embodiments, K32 or U32 aAPCs are used to induce the presentation of one or more antibody-based stimulatory molecules on the aAPC cell surface. T cell populations may be expanded with aAPCs expressing various costimulatory molecules, including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. Finally, aAPCs provide an efficient platform for the expansion of genetically modified T cells and the maintenance of CD28 expression on CD8 T cells. The aAPCs presented in WO03 / 057171 and US2003 / 0147869 are incorporated herein by reference in their entireties.

[0628] In certain embodiments, a polynucleotide encoding one or more DARIC components is introduced into a population of T cells. In certain embodiments, a polynucleotide encoding one or more DARIC components is introduced into a population of T cells expressing an engineered antigen receptor. The polynucleotide may be introduced into the T cells by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran mediated transfer, or the like.

[0629] In a preferred embodiment, the polynucleotide is introduced into the T cell by viral transduction.

[0630] Immune effector cells or CD34 + Examples of viral vector systems suitable for introducing polynucleotides into cells include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors for gene transfer.

[0631] In one embodiment, the polynucleotide is introduced into the T cell by AAV transduction.

[0632] In one embodiment, the polynucleotide is introduced into the T cell by retroviral transduction.

[0633] In one embodiment, the polynucleotide is introduced into the T cells by lentiviral transduction.

[0634] In one embodiment, the polynucleotide is introduced into the T cell by adenoviral transduction.

[0635] In one embodiment, the polynucleotide is introduced into the T cell by herpes simplex virus transduction.

[0636] In one embodiment, the polynucleotide is introduced into the T cell by vaccinia virus transduction.

[0637] H. Compositions and Formulations Compositions contemplated herein may include one or more DARIC polypeptides, polynucleotides encoding DARIC polypeptides, vectors containing them, genetically modified immune effector cells, cross-linking agents, etc. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in a pharmaceutically or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the composition may be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There is virtually no limit to the other components that may be included in the composition, provided that the added agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0638] As used herein, the phrase "pharmaceutically acceptable" is employed to refer to those compounds, substances, compositions and / or dosage forms that 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.

[0639] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a cross-linking agent, polypeptide, polynucleotide, vector containing the same, or genetically modified immune effector cells is administered. Examples of pharmaceutical carriers may 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, and sesame oil. Saline solutions and dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients in certain embodiments 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.

[0640] In one embodiment, the composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In certain embodiments, the composition comprising a carrier is suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration. In certain embodiments, the composition comprising a pharmaceutically acceptable carrier is suitable for intracerebroventricular, intrathecal, or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the cross-linking agent, polypeptide, polynucleotide, vector containing same, or genetically modified immune effector cells, its use in the pharmaceutical composition is contemplated.

[0641] In certain embodiments, 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. Compositions containing the cell-based compositions contemplated herein may be administered enterally or parenterally, separately, or in combination with other appropriate compounds to achieve a desired therapeutic purpose.

[0642] In certain embodiments, compositions contemplated herein comprise a cross-linking agent and a pharmaceutically acceptable carrier.

[0643] Pharmaceutically acceptable carriers must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the human subject being treated. They must also maintain or enhance the stability of the composition. Pharmaceutically acceptable carriers may be liquid or solid and are selected to provide the desired volume and concentration when mixed with the other components of the composition, taking into account the intended mode of administration. For example, pharmaceutically acceptable carriers may be, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), or wetting agents (e.g., sodium lauryl sulfate, etc.). Other pharmaceutically acceptable carriers suitable for the compositions contemplated herein include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0644] Such carrier solutions may contain buffers, diluents, and other suitable additives. As used herein, the term "buffer" refers to a solution or liquid whose chemical composition neutralizes acids or bases without significantly changing the pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate-buffered saline (PBS), Ringer's solution, 5% dextrose solution (D5W), and normal / physiological saline (0.9% NaCl).

[0645] The pharmaceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the composition has a pH in the range of about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In yet another embodiment, the composition has a pH of about 7.4.

[0646] The composition anticipated herein may contain a non-toxic pharmaceutically acceptable medium.The composition may be a suspension.As used herein, the term "suspension" refers to a non-adherent state in which cells do not bind to a solid support.For example, cells maintained as a suspension may be stirred or agitated, and do not adhere to a support such as a culture dish.

[0647] In certain embodiments, compositions contemplated herein are formulated in a suspension, in which T cells modified with one or more DARIC components and / or one or more polynucleotides encoding an engineered antigen receptor are dispersed in an acceptable liquid medium or solution, such as saline or serum-free medium, in an intravenous (IV) bag, etc. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride solution (saline), serum-free cell culture medium, and medium suitable for cryogenic storage, such as Cryostor® medium.

[0648] In certain embodiments, the pharmaceutically acceptable carrier is substantially free of naturally occurring proteins of human or animal origin and is suitable for storing compositions comprising engineered T cell populations. Therapeutic compositions are intended for administration to human patients and therefore are substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.

[0649] In some embodiments, the composition is formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0650] Serum-free media have several advantages over serum-containing media, including a simpler and more transparent composition, reduced contaminant load, elimination of potential sources of infectious agents, and reduced cost. In various embodiments, serum-free media are animal-free and optionally protein-free. Optionally, the media may contain biopharmaceutical-acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and the nutrients are obtained from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0651] Examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0652] In one embodiment, the composition comprising the modified T cells is formulated in PlasmaLyte.

[0653] In various embodiments, compositions comprising modified T cells are formulated in a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative may be used to maintain high cell viability after thawing. Exemplary cryopreservation media for use in particular compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0654] In one embodiment, the composition is formulated in a solution comprising 50:50 PlasmaLyte A and CryoStor CS10.

[0655] In certain embodiments, the compositions are substantially free of mycoplasma, endotoxin, and microbial contamination. "Substantially free," with respect to endotoxin, means lower endotoxin per cell dose than permitted by the FDA for biologics, which is 5 EU / kg body weight total endotoxin per day, or 350 EU per total cell dose for an average 70 kg human. In certain embodiments, the 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.

[0656] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is known in the art, as is the development of appropriate dosing and treatment regimens for use of certain compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, intrathecal, and intramedullary administration and formulations. Those of skill in the art will appreciate that certain embodiments contemplated herein are known, for example, in the pharmaceutical arts, and can be readily understood by reference to, for example, Remington: The Science and Practice of Pharmacy, volume I and volume II.22 nd It will be understood that the present invention may include other formulations such as those described in "Therapeutic Agents for the Treatment of Acute Acetaminophen," 1999 Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0657] In certain embodiments, the composition comprises an amount of immune effector cells expressing one or more DARIC components contemplated herein. In certain embodiments, the composition comprises an amount of immune effector cells expressing an engineered antigen receptor and one or more DARIC components contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective dose" of cells containing one or more DARIC components contemplated herein that, in the presence of a crosslinking agent, achieves a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0658] A "prophylactically effective amount" refers to an amount of cells containing one or more DARIC components contemplated herein effective in the presence of a crosslinking agent to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount will be less than a therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an early stage of disease.

[0659] A "therapeutically effective amount" refers to an amount of cells containing one or more DARIC components as contemplated herein that, in the presence of a cross-linking agent, is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of composition, cells, cross-linking agent, etc. to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and condition.

[0660] Generally, pharmaceutical compositions comprising the immune effector cells described herein are administered in a 10 2 ~10 10 cells / kg body weight, preferably 10 5 ~10 6 It can be said that a dose of cells / kg body weight can be administered, including all integer values ​​within the range. The number of cells depends on the intended end use of the composition and the type of cells contained in the composition. For the uses presented herein, the cells are generally in a volume of liter or less, and can be 500 mL or less, or even 250 mL or 100 mL or less. Therefore, the desired cell density is often 106 cells / ml, typically greater than 10 7 cells / ml, typically >10 8 Clinically relevant immune cell counts are greater than 10 cells / ml, which may be divided into multiple infusions and cumulatively reach 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, all infused cells are specifically redirected to a specific target antigen, resulting in more than 10 6 / kilogram (10 per patient) 6 -10 11 ) may be administered.

[0661] If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the induction of an immune response.

[0662] Generally, compositions comprising cells activated and expanded as described herein may be utilized for the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, the compositions contemplated herein are used to treat cancer. In certain embodiments, immune effector cells may be administered alone or in a pharmaceutical composition with a carrier, diluent, excipient, and / or other components, such as IL-2 or other cytokines or other cells.

[0663] In certain embodiments, the pharmaceutical composition comprises a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0664] In certain embodiments, a pharmaceutical composition contains an amount of a cross-linking agent in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0665] In certain embodiments, compositions contain an effective amount of immune effector cells comprising one or more DARIC components contemplated herein, alone or in combination with a crosslinking agent and / or one or more therapeutic agents, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics. Such therapeutic agents may be art-accepted as standard treatments for certain disease states described herein, such as certain cancers. Examples of contemplated therapeutic agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active adjunctive agents.

[0666] In certain embodiments, a composition comprising an effective amount of immune effector cells comprising one or more DARIC components as contemplated herein is administered to a subject, and a composition comprising an effective amount of a cross-linking agent is administered to the subject prior to, during, in combination with, or following the cell composition, and optionally repeatedly administered to the subject.

[0667] In certain embodiments, compositions comprising immune effector cells comprising one or more DARIC components contemplated herein may be administered in conjunction with any number of chemotherapeutic agents.

[0668] Various other therapeutic agents may be used in conjunction with the compositions described herein. In one embodiment, a composition comprising immune effector cells comprising one or more DARIC components as contemplated herein is administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide, and nonsteroidal anti-inflammatory drugs (NSAIDs), including mycophenolate.

[0669] Examples of therapeutic antibodies suitable for combination therapy with engineered T cells containing one or more DARIC components contemplated herein include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, durigotumab, dacetuzumab, darotuzumab, and darotuzumab. These include ib, durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0670] In some embodiments, the compositions described herein are administered in combination with cytokines. As used herein, the term "cytokine" refers to a general term for proteins released by one cell group that act as intercellular mediators on another cell group. Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones.

[0671] I. Treatment method The immune effector cells modified to express the DARIC and / or engineered antigen receptors contemplated herein provide adoptive immunotherapy for use in the prevention, treatment and amelioration of at least one symptom associated with cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, or immune deficiency, or improved adoptive immunotherapy for preventing, treating, or ameliorating the symptoms.

[0672] In certain embodiments, immune effector cells engineered to express DARIC provide an improvement in adoptive immunotherapy to stabilize the safety and efficacy of cytotoxic responses against target cells, e.g., tumor cells, that express the target antigen, while reducing the risk of on-target, off-target cytotoxic activity (recognizing the target antigen on normal, non-target cells).

[0673] In certain embodiments, a method for preventing, treating, or ameliorating at least one symptom of cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune disorder comprises administering to a subject an effective amount of modified immune effector cells or T cells comprising DARIC and one or more components of an engineered TCR, CAR, or other therapeutic transgene for redirecting cells to target cells. Genetically modified cells are a highly effective and safe cellular immunotherapy in that they deliver chemically controllable immunostimulatory signals.

[0674] In certain embodiments, one or more immune effector cells, such as T cells, are modified to express both a DARIC binding component and a DARIC signaling component. The modified cells are then administered to a subject in need thereof and directed to target cells via interaction between the DARIC binding component expressed on the immune effector cells and a target antigen expressed on the target cells. A cross-linking agent is administered to the subject before, at about the same time as, or after the modified cells are administered to the subject. In the presence of the cross-linking agent, a ternary complex is formed between the DARIC binding component, the cross-linking agent, and the DARIC signaling component. Upon formation of the ternary complex, DARIC transmits an immunostimulatory signal to the immune effector cells, which then initiate a cytotoxic response against the target cells.

[0675] In various embodiments, DARIC and / or immune effector cells comprising engineered antigen receptors stabilize the safety and efficacy of cytotoxic responses against target cells using a dual targeting strategy, in which one or more target cells express one or more antigens recognized by the engineered antigen receptor and one or more target antigens recognized by DARIC.

[0676] In certain embodiments, one or more immune effector cells, such as T cells, are modified to express both a DARIC binding component and a DARIC signaling component, as well as an engineered antigen receptor, such as a CAR. In this case, the modified cells are administered to a subject in need thereof and directed to the target cells through interaction of the DARIC binding component and the CAR with a target antigen expressed on the target cells, and both the DARIC binding component and the CAR are expressed on the immune effector cells. The interaction of the CAR with the target antigen on the target cells can elicit a cytotoxic response from the immune effector cells against the target cells. A cross-linking agent is administered to the subject before, at about the same time as, or after the modified cells are administered to the subject. In the presence of the cross-linking agent, a ternary complex is formed between the DARIC binding component, the cross-linking agent, and the DARIC signaling component. Upon formation of the ternary complex, DARIC transmits an immunostimulatory signal to the immune effector cells, which then elicits or enhances a cytotoxic response from the immune effector cells against the target cells. In certain embodiments, DARIC activation can be induced when remission or regression is incomplete and the condition relapses or becomes refractory to treatment.

[0677] In certain preferred embodiments, the specificity of early T cells is redirected against tumor or cancer cells expressing one or more target antigens by genetically modifying T cells, e.g., early T cells, using one or more DARIC components.

[0678] In a particularly preferred embodiment, the specificity of primary T cells is redirected to tumor or cancer cells expressing a target antigen recognized by an engineered antigen receptor and a target antigen recognized by DARIC.

[0679] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat solid tumors or cancers.

[0680] In certain embodiments, the engineered immune effector cells contemplated herein are selected from, but are not limited to, adrenal carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid tumor / atypical rhabdomyoid tumor, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), endometrial cancer, epithelial carcinoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, cranial Extragonadal germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous tissue sarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, tongue cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, embryonal carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline carcinoma, oral cancer Used to treat solid tumors or cancers, including uterine cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer (oral cavity cancer), oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal carcinoma, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary pleurima, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, cancer of the renal pelvis and ureter, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovial tumor, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor

[0681] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat solid tumors or cancers, including but not limited to liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.

[0682] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat various cancers, including, but not limited to, pancreatic, bladder, and lung cancer.

[0683] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat liquid or hematological cancers.

[0684] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat B-cell malignancies, including but not limited to, leukemia, lymphoma, and multiple myeloma.

[0685] In certain embodiments, the engineered immune effector cells contemplated herein are directed against leukemias, lymphomas, and multiple myeloma, including, but not limited to, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, It is used to treat liquid cancers including Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0686] Preferred cells for use in the methods contemplated herein include self cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.

[0687] In certain embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of engineered immune effector cells expressing one or more DARIC components and, optionally, an engineered antigen receptor, or a composition comprising same, and also comprises administering to the subject a cross-linking agent. In certain embodiments, the cells are used to treat a patient at risk of developing cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune deficiency. Thus, certain embodiments include treating or preventing or ameliorating at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune deficiency, comprising administering to a subject in need thereof a therapeutically effective amount of engineered immune effector cells and a cross-linking agent as contemplated herein.

[0688] In certain embodiments, the method includes administering to a patient in need thereof a therapeutically effective amount of engineered immune effector cells expressing a DARIC signaling component and, optionally, an engineered antigen receptor, or a therapeutically effective amount of a composition comprising same, and also administering to the subject a DARIC binding component and a cross-linking agent, wherein the DARIC binding component is conjugated to the cross-linking agent prior to administration to the subject. In certain embodiments, the cells are used to treat a patient at risk of developing cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune deficiency. Thus, certain embodiments include treating or preventing or ameliorating at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune deficiency, comprising administering to a subject in need thereof a therapeutically effective amount of engineered immune effector cells expressing a DARIC signaling component and, optionally, an engineered antigen receptor, a DARIC binding component, and a cross-linking agent.

[0689] The amount and frequency of administration of the modified immune effector cells, DARIC binding components, and / or cross-linking agents will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate doses and administration schedules may be determined through clinical trials.

[0690] In one exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is at least 2 x 10 6 Cells / kg, at least 3x10 6 cells / kg, at least 4x10 6 Cells / kg, at least 5x10 6 cells / kg, at least 6x10 6 Cells / kg, at least 7x10 6 Cells / kg, at least 8x10 6 Cells / kg, at least 9x10 6 cells / kg, or at least 10x10 6 cells / kg, or more, including all cell doses in between.

[0691] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2x10 6 cells / kg, approximately 3x10 6 cells / kg, approximately 4x10 6 cells / kg, approximately 5x10 6 cells / kg, approximately 6x10 6 cells / kg, approximately 7x10 6 cells / kg, approximately 8x10 6 cells / kg, approximately 9x10 6 cells / kg, or approximately 10x10 6 cells / kg, or more, including all cell doses in between.

[0692] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 3x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 4x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 5x10 6 cells / kg ~ approx. 10x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 7x106 cells / kg, 2x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 8x10 6 cells / kg, or 6x10 6 cells / kg ~ approx. 8x10 6 cells / kg and includes all cell doses in between.

[0693] Those skilled in the art will recognize that multiple administrations of the contemplated compositions may be required in certain embodiments to achieve the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period 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. The engineered immune effector cells, DARIC components, and cross-linking agents may be administered in the same or different compositions, simultaneously in one or more compositions, or in multiple compositions at different times. The engineered immune effector cells, DARIC components, and cross-linking agents may be administered via the same or different routes of administration.

[0694] In some embodiments, it may be desirable to administer activated T cells to a subject, then withdraw blood from the subject (or perform apheresis), activate the T cells derived therefrom, and reinfuse these activated and expanded T cells back into the patient. This process may be performed multiple times, every few weeks. In some embodiments, T cells may be activated from a blood draw of 10 cc to 400 cc. In some embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 cc or more. Without being bound by theory, the use of multiple blood draws / multiple reinfusion protocols may aid in the selection of certain populations of T cells.

[0695] In one embodiment, a method of treating a subject diagnosed with cancer comprises collecting immune effector cells from the subject, modifying the immune effector cells by introducing into the cells one or more vectors encoding one or more DARIC components, generating a population of modified immune effector cells, and administering to the subject the population of modified immune effector cells. In a preferred embodiment, the immune effector cells comprise T cells.

[0696] In one embodiment, a method of treating a subject diagnosed with cancer comprises harvesting immune effector cells from the subject, modifying the immune effector cells by introducing into the cells one or more vectors encoding an engineered antigen receptor and one or more DARIC components, generating a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0697] In certain embodiments, contemplated methods of administering cell compositions include any method effective to result in the reintroduction of modified immune effector cells ex vivo, or any method effective to result in the reintroduction of modified precursors of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method involves 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 to the subject.

[0698] In certain embodiments, contemplated methods of administering cell compositions include any method effective to result in the reintroduction of engineered immune effector cells ex vivo, or any method effective to result in the reintroduction of engineered precursors of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method involves modifying peripheral blood T cells ex vivo by introducing one or more vectors encoding one or more DARIC components, and returning the transduced cells to the subject.

[0699] All publications, patent applications, and issued patents cited herein are hereby incorporated by reference as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0700] Although the foregoing embodiments have been described in detail in the figures and examples for purposes of clarity and understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified in specific embodiments to yield essentially similar results. [Example]

[0701] Example 1 NKG2D DARIC-binding component containing a costimulatory domain We designed, constructed, and demonstrated lentiviral vectors containing NKG2D DARIC with DARIC-binding components containing various costimulatory signaling domains (Figure 2A). The costimulatory domains were obtained from TNFR2, OX40, CD27, HVEM (TNFRS14), GITR (TNFRS18), and DR3 (TNFRS25) proteins. See, for example, SEQ ID NO: 1 and SEQ ID NOs: 20-22.

[0702] 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 24 hours of incubation, 1 × 10 6 Cells were transduced with LVV encoding DARIC. An additional untransduced sample was included as a control (UTD). On day 3, cells were washed and 0.3x10 6 Cells were resuspended at 1000 cells / mL. For expansion, cells were cultured for an additional 7 days in T cell expansion medium containing IL-2 (250 IU / mL). The medium was changed every other day.

[0703] Cells were counted and split to a defined density at each medium change. After a 10-day expansion period, T cells were counted and phenotyped using CD4 and CD8 antibody staining.

[0704] ...

Claims

1. A non-naturally occurring cell, (a) a first polypeptide comprising a first multimerization domain polypeptide, a first transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain; and (b) a second polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide, a second transmembrane domain, and a TNFR2 costimulatory domain; the first multimerization domain comprises an FKBP polypeptide and the second multimerization domain comprises an FRB polypeptide; or the first multimerization domain comprises a FRB polypeptide and the second multimerization domain comprises a FKBP polypeptide; a cross-linking agent promotes formation of a polypeptide complex on the non-native cell surface, said cross-linking agent being associated with and positioned between said first and second multimerization domains; The non-native cell, wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

2. The non-naturally occurring cell of claim 1 , wherein the first polypeptide comprises a CD137 costimulatory domain and a CD3ζ primary signaling domain.

3. 3. The non-naturally occurring cell of any one of claims 1 to 2, wherein the first and second multimerization domains are selected from FRB T2098L and FKBP12, and the cross-linking agent is sirolimus or AP21967.

4. 4. The non-naturally occurring cell of any one of claims 1-3, wherein the first transmembrane domain and the second transmembrane domain are independently selected from a polypeptide selected from the group consisting of the alpha, beta, gamma or delta chain of the T cell receptor, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1).

5. 5. The non-naturally occurring cell of any one of claims 1-4, wherein the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain.

6. The non-naturally occurring cell of any one of claims 1 to 5, wherein the first transmembrane domain and the second transmembrane domain are different.

7. The non-naturally occurring cell of any one of claims 1 to 6, wherein the extracellular binding domain comprises an antibody or an antigen-binding fragment thereof, an extracellular domain of a receptor, or a ligand.

8. The extracellular binding domains include camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab') ２ Fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins (scFv), bis-scFv, (scFv) ２ 8. The non-naturally occurring cell of any one of claims 1 to 7, comprising an antibody or antigen-binding fragment thereof selected from the group consisting of: a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), and a single domain antibody (sdAb, camelid VHH, nanobody).

9. The non-naturally occurring cell of any one of claims 1 to 8, wherein the extracellular binding domain comprises one or more Camelidae VHH antibodies.

10. The extracellular binding domain is the alpha folate receptor (FRα), α ｖ β ６ Integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase type IX (CAIX), CD16, CD19, 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 (DLL3), epithelial growth factor receptor (EGFR), epithelial growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activation protein (FAP), Fc Receptor Like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), HER2 p95, IL-10Rα, IL-13Rα2, Kappa, cancer / testis antigen 2 (LAGE-1A), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen recognized by Tcells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain related proteins A (MICA), MHC class I chain related proteins B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate stem cell prostate specific membrane antigen (PSCA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding 10. The non-naturally occurring cell of any one of claims 1 to 9, which binds to an antigen selected from the group consisting of ULBP 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1).

11. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and the non-naturally occurring cell of any one of claims 1-10.

12. 12. A pharmaceutical composition according to claim 11 for use in the treatment, prevention or amelioration of at least one symptom of cancer or an autoimmune disease.

13. 13. The pharmaceutical composition of claim 12, wherein the 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, renal cancer, or skin cancer.

14. 12. The pharmaceutical composition of claim 11 for use in the treatment of malignant hematological disorders.

15. The pharmaceutical composition of claim 14, wherein the malignant hematological disease is leukemia, lymphoma or multiple myeloma.

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