Modified CCR polypeptides and uses thereof

Abstract

The present disclosure provides improved chimeric costimulatory receptors (CCRs), fusion proteins, genetically modified immune effector cells, and uses of these compositions to treat disease. The disclosure generally relates, in part, to improved CCR polypeptides, fusion proteins, and methods of using the same. In particular, the present invention provides fusion polypeptides comprising a CAR and a modified CCR polypeptide, and their use in treating, preventing, or ameliorating at least one symptom of cancer. More specifically, the CCR comprises a modified hinge region, and the CAR / CCR fusion polypeptide exhibits reduced antigen-independent signaling, e.g., signaling in the absence of a CAR antigen.

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 Application No. 63 / 011,494, filed April 17, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Description The Sequence Listing for this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the Sequence Listing is BLUE-127_PC_ST25.txt. The text file is 27 KB, was created on April 14, 2021, and was submitted electronically via EFS-Web concurrently with the filing of this application. [Background technology]

[0003] The present disclosure relates to improved compositions and methods for treating disease. More particularly, the disclosure relates to improved chimeric costimulatory receptors (CCRs), fusion proteins, genetically modified immune effector cells, and the use of these compositions to treat disease.

[0004] Description of related fields Chimeric antigen receptors (CARs) are receptor proteins that combine both antigen recognition and T cell activation in a single receptor. CARs are complex proteins with several protein domains, such as an antigen-specific binding domain, a hinge or spacer sequence, a transmembrane domain, one or more costimulatory domains, and a signal transduction domain. T cells engineered to express CARs can be redirected to specific cell types (e.g., cancer cells) that express the target antigen. In fact, such CAR T cells have proven useful in inducing T cell-mediated killing of certain leukemias and lymphomas that express the CD19 antigen.

[0005] However, despite the excitement surrounding CAR T technology, designing effective CARs remains a challenging and time-consuming endeavor (see, e.g., Guedan et al., Mol Ther Methods Clin Dev. 2019 Mar 15;12:145-156), and attempts to use CAR T cells have met with limited success. For example, one major obstacle limiting the efficacy of CAR T cell therapy is the recurrence of "antigen-negative" cancers. Furthermore, administration of CAR T cells is known to cause adverse side effects, such as cytokine storm and / or neurotoxicity. Furthermore, the therapeutic efficacy of a given antigen-binding domain used in a CAR can be unpredictable. If the antigen-binding domain is too strong, the CAR T cells may induce massive cytokine release, resulting in a potentially fatal immune response considered a "cytokine storm." If the antigen-binding domain is too weak, the CAR T cells may not exhibit sufficient therapeutic efficacy in eradicating cancer cells. The heterogeneity of antigen expression within tumors also impacts the efficacy of CAR T cell therapy. Successful strategies to improve and / or enhance CAR T cell signaling have yet to be realized, posing a substantial obstacle to advancing CAR T therapy for various cancers. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Guedan et al.,Mol Ther Methods Clin Dev.2019 Mar 15;12:145-156 Summary of the Invention [Means for solving the problem]

[0007] The present disclosure generally relates, in part, to improved CCR polypeptides, fusion proteins, and methods of using same. In particular, the present invention provides fusion polypeptides comprising a CAR and a modified CCR polypeptide, and their use in treating, preventing, or ameliorating at least one symptom of cancer. More specifically, the CCR comprises a modified hinge region, and the CAR / CCR fusion polypeptide exhibits reduced antigen-independent signaling, e.g., signaling in the absence of a CAR antigen.

[0008] In various embodiments, a fusion polypeptide is provided that includes: a) a chimeric antigen receptor (CAR) comprising a first hinge region; b) a polypeptide cleavage signal; and c) a chimeric costimulatory receptor (CCR), the CCR comprising a second hinge region that is modified to reduce CCR antigen-mediated stimulation of T cell signaling in the absence of the CAR antigen compared to a CCR comprising an unmodified hinge region.

[0009] In various embodiments, a fusion polypeptide is provided that includes: a) a chimeric antigen receptor (CAR) that includes a first hinge region; b) a polypeptide cleavage signal; and c) a chimeric costimulatory receptor (CCR) that includes a second hinge region that includes one or more cysteines substituted with another amino acid.

[0010] In certain embodiments, the one or more cysteine ​​substitutions in the second hinge region reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region.

[0011] In certain embodiments, the one or more cysteine ​​substitutions in the second hinge region reduce CAR / CCR association in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region.

[0012] In various embodiments, the first hinge region is modified, and optionally the modification comprises one or more substitutions or deletions. In some embodiments, the modified first hinge region comprises one or more cysteines substituted with different amino acids. In some embodiments, the modified first hinge region reduces CCR antigen-mediated stimulation of T cell signaling in the absence of CAR antigen compared to a CAR comprising an unmodified first hinge region. In some embodiments, the modified first hinge region reduces CAR / CCR association in the absence of CAR antigen compared to a CAR comprising an unmodified first hinge region.

[0013] In various embodiments, one or more cysteine ​​residues in the first and / or second hinge region are substituted with serine or alanine. In some embodiments, the first and second hinge regions are derived from the same protein. In some embodiments, the first and second hinge regions are derived from different proteins.

[0014] In various embodiments, the first and / or second hinge region comprises a hinge region, or functional fragment thereof, selected from the group consisting of: CD8α hinge, CD4 hinge, CD28 hinge, CD7 hinge, CD152 hinge, PD-1 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgG1 hinge / CH2 / CH3, IgG1 hinge-CH3-hinge-M1, IgG1 hinge / CH2 / CH3, IgG1 hinge / CH3 / hinge / M1, IgG4 hinge / CH2 / CH3, and IgG4 hinge / CH2. In a specific embodiment, the first and / or second hinge region is a CD8α hinge region, or functional fragment thereof.

[0015] In some embodiments, the CD8α hinge region comprises an amino acid substitution at position 27 of SEQ ID NO: 2. In some embodiments, the CD8α hinge region comprises an amino acid substitution at position 44 of SEQ ID NO: 2. In some embodiments, the CD8α hinge region comprises amino acid substitutions at positions 27 and 44 of SEQ ID NO: 2.

[0016] In certain embodiments, the first and / or second hinge region comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the first and / or second hinge region comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO: 3.

[0017] In certain embodiments, the first and / or second hinge region comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the first and / or second hinge region comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO: 4.

[0018] In certain embodiments, the first and / or second hinge region comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the first and / or second hinge region comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO: 5.

[0019] In some embodiments, a CD4 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a CD28 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, a CD7 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, a CD152 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, a PD-1 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, an IgG1 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, an IgG2 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity as set forth in SEQ ID NO: 13, or a functional fragment thereof.In some embodiments, an IgG3 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, an IgG4 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, an IgG1 hinge / CH2 / CH3 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, an IgG1 hinge-CH3-hinge-M1 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, an IgG4 hinge / CH2 / CH3 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, an IgG4 hinge / CH2 hinge region, or a functional fragment thereof, comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, a PD-1 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 20, or a functional fragment thereof.

[0020] In various embodiments, the CAR comprises a first antibody or an antigen-specific binding fragment thereof, and the CCR further comprises a second antibody or an antigen-specific binding fragment thereof. In some embodiments, the first and / or second antibody or antigen-specific binding fragment thereof is selected from the group consisting of 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 VHH, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single-domain antibody (sdAb, nanobody), and a camelid antibody (VHH) or fragment thereof. In some embodiments, the first and / or second antibody or antigen-specific binding fragment thereof is an scFv or a VHH.

[0021] In some embodiments, the first and second antibodies or antigen-specific binding fragments bind to the same target antigen. In some embodiments, the first and second antibodies or antigen-specific binding fragments bind to different epitopes on the same target antigen. In some embodiments, the first and second antibodies or antigen-specific binding fragments bind to different target antigens.

[0022] In various embodiments, the first and / or second antibody or antigen-specific binding fragment is selected from the group consisting of alpha folate receptor (FRα), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD135 (fm C-like tyrosine kinase 3 (also known as FLT3), CD138, CD171, carcinoembryonic antigen (CEA), claudin-6 (CLDN6), 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), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 1 (EGFRvIII), epithelial glycoprotein 2 (EGFRvIII), epithelial glycoprotein 1 (EGFRvIII), epithelial glycoprotein 2 (EGFRvIII), epithelial glycoprotein 1 (EGFRvIII), epithelial growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGFRvIII), epithelial glycoprotein 1 (EGFRvIII), epithelial growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 1 (EGFRvIII), epithelial growth factor receptor variant IV (EGFRvIII), epithelial glycoprotein 2 (EGFRvIII), epithelial glycoprotein 1 (EGFRvIII), epithelial growth factor receptor variant IV ... growth factor receptor variant IV (EGFRvIII), epithelial glycoprotein 1 (EGFRvIII), epithelial growth factor receptor variant IV (EGFRvIII), epithelial growth factor receptor variant IV (EGFRvIII), epithelial growth Protein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), 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), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), La Muda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), 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, neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;binds to a target antigen selected from the group consisting of placenta-specific 1 (PLAC1), predominantly 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), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1);

[0023] In various embodiments, the first antibody or antigen-specific binding fragment is directed against a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligands. and the second antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand.

[0024] In various embodiments, the first antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD33, CD79a, CD79b, C-type lectin-like molecule-1 (CLL-1), MAGE-A4, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand, and the second antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), and TAG72.

[0025] In various embodiments, the first antibody or antigen-specific binding fragment binds to B-cell maturation antigen (BCMA) and the second antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), and TAG72.

[0026] In various embodiments, the CAR further comprises a first transmembrane domain and the CCR further comprises a second transmembrane domain. In some embodiments, the first and / or second transmembrane domain is derived from a polypeptide selected from the group consisting of the alpha or beta 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, and PD1. In some embodiments, the first and / or second transmembrane domain is derived from CD8α.

[0027] In various embodiments, the CAR further comprises a first intracellular costimulatory domain and the CCR further comprises a second intracellular costimulatory domain. In some embodiments, the first and / or second costimulatory domains are derived 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, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, tumor necrosis factor receptor 2 (TNFR2), and ZAP70. In some embodiments, the first and second costimulatory domains are derived from CD137 (4-1BB). In some embodiments, the first and second costimulatory domains are derived from CD28.

[0028] In various embodiments, the CAR further comprises a primary signaling domain. In some embodiments, the primary signaling domain is derived from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some embodiments, the primary signaling domain is derived from CD3ζ.

[0029] In various embodiments, the CAR comprises a first antibody or antigen-specific binding fragment, a first hinge region, a first transmembrane domain, a first intracellular costimulatory domain, and a primary signaling domain, and the CCR comprises a second antibody or antigen-specific binding fragment, a second hinge region, a second transmembrane domain, and a second intracellular costimulatory domain.

[0030] In certain embodiments, a fusion polypeptide is provided comprising: a) a chimeric antigen receptor (CAR) comprising a first antibody or antigen-specific binding fragment, a first hinge region, a first transmembrane domain, a first intracellular costimulatory domain, and a primary signaling domain; b) a polypeptide cleavage signal; and c) a chimeric costimulatory receptor (CCR) comprising a second antibody or antigen-specific binding fragment, a second hinge region, a second transmembrane domain, and a second intracellular costimulatory domain, wherein the second hinge region comprises one or more cysteine ​​substitutions or deletions that: i) reduce CCR antigen-mediated stimulation of T cell signaling in the absence of CAR antigen compared to a CCR comprising an unmodified second hinge region; or ii) reduce CAR / CCR association in the absence of CAR antigen compared to a CCR comprising an unmodified second hinge region.

[0031] In certain embodiments, a fusion polypeptide is provided comprising: a) a CAR comprising a first antibody or antigen-specific binding fragment that binds BCMA, a first hinge region derived from CD8α, a first transmembrane domain derived from CD8α, a first intracellular costimulatory domain derived from 4-1BB, and a primary signaling domain derived from CD3ζ; b) a polypeptide cleavage signal; and c) a CCR comprising a second antibody or antigen-specific binding fragment that binds EGFR, a second hinge region derived from CD8α, a second transmembrane domain derived from CD8α, and a second intracellular costimulatory domain derived from CD28, wherein the second hinge region comprises one or more cysteine ​​substitutions or deletions that i) reduce CCR antigen-mediated stimulation of T cell signaling in the absence of CAR antigen compared to a CCR comprising an unmodified second hinge region, or ii) reduce CAR / CCR association in the absence of CAR antigen compared to a CCR comprising an unmodified second hinge region.

[0032] In various embodiments, the modified second hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0033] In various embodiments, the polypeptide cleavage signal is a viral auto-cleaving polypeptide. In some embodiments, the polypeptide cleavage signal is a viral auto-cleaving 2A polypeptide. In some embodiments, the polypeptide cleavage signal is a viral auto-cleaving polypeptide selected from the group consisting of a foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus (TaV) 2A (T2A) peptide, a porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0034] In certain embodiments, a polynucleotide encoding any one of the fusion polypeptides described herein is provided. In certain embodiments, a vector comprising the polynucleotide is provided. In some embodiments, the vector is an expression vector. In some embodiments, the vector is an episomal vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0035] In certain embodiments, a cell is provided that expresses a fusion polypeptide described herein. In some embodiments, the cell comprises a polynucleotide or vector described herein. In some embodiments, the cell is a genetically engineered host cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a hematopoietic stem or progenitor cell. In some embodiments, the cell is a CD34+ hematopoietic stem or progenitor cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD3+, CD4+, and / or CD8+ cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), tumor-infiltrating lymphocyte (TIL), or helper T cell. In some embodiments, the cell is an αβ-T cell. In some embodiments, the cell is a γδ-T cell. In some embodiments, the host cell is a natural killer (NK) cell. In some embodiments, the natural killer cell is a natural killer T (NKT) cell. In some embodiments, the host cell is a macrophage.

[0036] In certain embodiments, a composition is provided comprising the cells described herein and a pharmaceutically acceptable carrier.

[0037] In certain embodiments, methods of treating cancer in a subject in need thereof are provided, hi various embodiments, the methods comprise administering to the subject a therapeutically effective amount of a composition described herein.

[0038] In certain embodiments, methods are provided for improving one or more symptoms associated with cancer in a subject. In various embodiments, the methods include administering to the subject a therapeutically effective amount of a composition described herein sufficient to improve at least one symptom associated with cancer. In some embodiments, the one or more symptoms improved are selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain, bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination.

[0039] In various embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML). In some embodiments, the non-Hodgkin's lymphoma is Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL). In some embodiments, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is MM selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0040] In certain embodiments, methods are provided for generating a cell population that expresses a fusion polypeptide described herein. In various embodiments, the method comprises introducing a polynucleotide or vector described herein into the cell population. In some embodiments, the cell population comprises hematopoietic stem or progenitor cells. In some embodiments, the cell population comprises CD34+ hematopoietic stem or progenitor cells. In some embodiments, the cell population comprises immune effector cells. In some embodiments, the cell population comprises T cells, NK cells, and / or NKT cells. In some embodiments, the cell population comprises T cells.

[0041] In certain embodiments, methods are provided for reducing CCR costimulation of T cell signaling in cells expressing both a CAR and a CCR. In various embodiments, the method includes: a) obtaining a CAR and a CCR polypeptide, each having a hinge domain, optionally wherein the CAR and CCR are expressed as a fusion polypeptide; b) substituting one or more cysteine ​​residues in the CCR hinge domain with another residue, thereby producing a modified CCR; and c) expressing the CAR and modified CCR in the cell.

[0042] In certain embodiments, methods are provided for reducing CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge. In various embodiments, the method includes: a) obtaining a CAR and a CCR polypeptide, each having a hinge domain, optionally wherein the CAR and CCR are expressed as a fusion polypeptide; b) substituting one or more cysteine ​​residues in the CCR hinge domain with another residue, thereby producing a modified CCR; and c) expressing the CAR and modified CCR in a cell.

[0043] In various embodiments, Cysteine ​​residue substitutions within the hinge domain of CCR Antigen-mediated stimulation of T cell signaling in the absence of CAR antigen compared to CCRs containing unmodified hinge regions ReduceIn some embodiments, cysteine ​​residue substitutions in the hinge domain of the CCR reduce association of the CAR with the CAR in the absence of antigen compared to a CCR comprising an unmodified hinge region. In some embodiments, both the CCR and the CAR comprise modified hinge regions. In some embodiments, the modified CAR hinge region comprises one or more cysteines substituted with different amino acids. In some embodiments, one or more cysteines substituted in the CAR hinge region reduce antigen-independent signaling of the CAR compared to a CAR comprising an unmodified hinge region. In some embodiments, one or more cysteines substituted in the CAR hinge region reduce antigen-dependent association with the CCR compared to a CAR comprising an unmodified hinge region. In some embodiments, the CAR and CCR hinge regions are derived from the same protein. In some embodiments, the CAR and CCR hinge regions are derived from different proteins. In some embodiments, one or more cysteine ​​residues in the CCR and / or CAR hinge region are substituted with serine or alanine.

[0044] In various embodiments, the CCR and / or CAR hinge region comprises a hinge region, or functional fragment thereof, selected from the group consisting of a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge (CTLA-4), a PD-1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2. In some embodiments, the CCR and / or CAR hinge region is a CD8α hinge region, or a fragment thereof. In some embodiments, the CCR and / or CAR hinge region is a CD8α hinge region. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises an amino acid substitution at position 27 of SEQ ID NO:2. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises an amino acid substitution at position 44 of SEQ ID NO:2. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises amino acid substitutions at positions 27 and 44 of SEQ ID NO:2. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO:3. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO:4. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO: 5.

[0045] In various embodiments, the CAR and CCR are expressed as any one of the fusion polypeptides described herein. In various embodiments, step (c) comprises introducing a polynucleotide or vector encoding the CAR and / or CCR into the cell. In an embodiment of the present invention, for example, the following items are provided: (Item 1) A fusion polypeptide comprising: a) a chimeric antigen receptor (CAR) comprising a first hinge region; b) a polypeptide cleavage signal; and c) a chimeric costimulatory receptor (CCR), wherein the CCR comprises a second hinge region that is modified to reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge region. (Item 2) A fusion polypeptide comprising: a) a chimeric antigen receptor (CAR) comprising a first hinge region; b) a polypeptide cleavage signal; and c) a chimeric costimulatory receptor (CCR) comprising a second hinge region that comprises one or more cysteines substituted with another amino acid. (Item 3) 3. The fusion polypeptide of item 2, wherein the one or more cysteine ​​substitutions in the second hinge region reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region. (Item 4) 4. The fusion polypeptide of item 2 or 3, wherein the one or more cysteine ​​substitutions in the second hinge region reduce CAR / CCR association in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region. (Item 5) 5. The fusion polypeptide of any one of items 1 to 4, wherein the first hinge region is modified, and optionally the modification comprises one or more amino acid substitutions or deletions. (Item 6) 6. The fusion polypeptide of item 5, wherein the modified first hinge region comprises one or more cysteines replaced with different amino acids. (Item 7) 7. The fusion polypeptide of item 6, wherein the modified first hinge region reduces CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CAR comprising an unmodified first hinge region. (Item 8) 7. The fusion polypeptide of item 6, wherein the modified first hinge region reduces CAR / CCR association in the absence of a CAR antigen compared to a CAR comprising an unmodified first hinge region. (Item 9) 9. The fusion polypeptide according to any one of items 2 to 8, wherein the one or more cysteine ​​residues in the first and / or second hinge region are substituted with serine or alanine. (Item 10) Item 10. The fusion polypeptide of any one of the preceding items, wherein the first and second hinge regions are derived from the same protein. (Item 11) Item 11. The fusion polypeptide of any one of the preceding items, wherein the first and second hinge regions are derived from different proteins. (Item 12) the second hinge region consists of a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge, a PD-1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2; Item 11. The fusion polypeptide of any one of the preceding items, comprising a hinge region, or a functional fragment thereof, selected from the group: (Item 13) Item 11. The fusion polypeptide of any one of the preceding items, wherein the first hinge region comprises a hinge region, or a functional fragment thereof, selected from the group consisting of: CD8α hinge, CD4 hinge, CD28 hinge, CD7 hinge, CD152 hinge, PD-1 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgG1 hinge / CH2 / CH3, IgG1 hinge / CH3 / hinge / M1, IgG4 hinge / CH2 / CH3, and IgG4 hinge / CH2. (Item 14) Item 11. The fusion polypeptide of any one of the preceding items, wherein the second hinge region is a CD8α hinge region, or a functional fragment thereof. (Item 15) Item 11. The fusion polypeptide of any one of the preceding items, wherein the first hinge region is a CD8α hinge region, or a functional fragment thereof. (Item 16) Item 11. The fusion polypeptide of any one of the preceding items, wherein the first and second hinge regions are CD8α hinge regions or functional fragments thereof. (Item 17) 17. The fusion polypeptide of any one of items 14 to 16, wherein the CD8α hinge region comprises an amino acid substitution at position 27 of SEQ ID NO:2. (Item 18) 17. The fusion polypeptide of any one of items 14 to 16, wherein the CD8α hinge region comprises an amino acid substitution at position 44 of SEQ ID NO:2. (Item 19) 17. The fusion polypeptide of any one of items 14 to 16, wherein the CD8α hinge region comprises amino acid substitutions at positions 27 and 44 of SEQ ID NO:2. (Item 20) 17. The fusion polypeptide according to any one of items 1 to 16, wherein the first and / or second hinge region comprises the amino acid sequence shown in SEQ ID NO:3. (Item 21) 17. The fusion polypeptide of any one of Items 1 to 16, wherein the first and / or second hinge region comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO: 3. (Item 22) 17. The fusion polypeptide according to any one of items 1 to 16, wherein the first and / or second hinge region comprises the amino acid sequence shown in SEQ ID NO:4. (Item 23) 17. The fusion polypeptide according to any one of Items 1 to 16, wherein the first and / or second hinge region comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO: 4. (Item 24) 17. The fusion polypeptide according to any one of items 1 to 16, wherein the first and / or second hinge region comprises the amino acid sequence shown in SEQ ID NO:5. (Item 25) 17. The fusion polypeptide of any one of Items 1 to 16, wherein the first and / or second hinge region comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO: 5. (Item 26) The first and / or second hinge region has at least 85%, 8, or 9 amino acids identical to those shown in SEQ ID NO: 7. 14. The fusion polypeptide of any one of items 1 to 13, which is a CD4 hinge region comprising an amino acid sequence with 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the CD4 hinge region, or a functional fragment thereof. (Item 27) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first and / or second hinge region is a CD28 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 8, or a functional fragment thereof. (Item 28) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is a CD7 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 9, or a functional fragment thereof. (Item 29) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is a CD152 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 10, or a functional fragment thereof. (Item 30) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is a PD-1 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 11, or a functional fragment thereof. (Item 31) 14. The fusion polypeptide according to any one of Items 1 to 13, wherein the first or second hinge region is an IgG1 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 12, or a functional fragment thereof. (Item 32) 14. The fusion polypeptide according to any one of Items 1 to 13, wherein the first or second hinge region is an IgG2 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13, or a functional fragment thereof. (Item 33) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is an IgG3 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 14, or a functional fragment thereof. (Item 34) the first or second hinge region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 15; 14. The fusion polypeptide according to any one of items 1 to 13, which is a G4 hinge region or a functional fragment thereof. (Item 35) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is an IgG1 hinge / CH2 / CH3 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 16, or a functional fragment thereof. (Item 36) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is an IgG1 hinge-CH3-hinge-M1 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 17, or a functional fragment thereof. (Item 37) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is an IgG4 hinge / CH2 / CH3 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 18, or a functional fragment thereof. (Item 38) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is an IgG4 hinge / CH2 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 19, or a functional fragment thereof. (Item 39) 14. The fusion polypeptide of any one of Items 1 to 13, wherein the first or second hinge region is a PD-1 hinge region comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 20, or a functional fragment thereof. (Item 40) a) the CAR further comprises a first antibody or an antigen-specific binding fragment thereof; b) The fusion polypeptide of any one of the preceding items, wherein the CCR further comprises a second antibody or an antigen-specific binding fragment thereof. (Item 41) The first and / or second antibody or antigen-specific binding fragment thereof may be a Fab' fragment, F(ab') 2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 41. The fusion polypeptide of claim 40, wherein the fusion polypeptide is selected from the group consisting of: a VHH, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody), and a camelid antibody (VHH) or a fragment thereof. (Item 42) 42. The fusion polypeptide of item 41, wherein the first and / or second antibody or antigen-specific binding fragment thereof is an scFv or VHH. (Item 43) 43. The fusion polypeptide of any one of items 40 to 42, wherein the first and second antibodies or antigen-specific binding fragments bind to the same target antigen. (Item 44) 43. The fusion polypeptide according to any one of items 40 to 42, wherein the first and second antibodies or antigen-specific binding fragments bind to different epitopes on the same target antigen. (Item 45) 43. The fusion polypeptide according to any one of items 40 to 42, wherein the first and second antibodies or antigen-specific binding fragments bind to different target antigens. (Item 46) the first antibody or antigen-specific binding fragment binds to any of the following: alpha folate receptor (FRα), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD135 (fmc-like tyrosine kinase 3), Also known as FLT3), CD138, CD171, carcinoembryonic antigen (CEA), claudin-6 (CLDN6), 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), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EG P40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), 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), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, and leukemia antigens. Is-Y (LeY), L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); 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, neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;46. ​​The fusion polypeptide of any one of items 40 to 45, which binds to a target antigen selected from the group consisting of placenta-specific 1 (PLAC1), predominantly melanoma-expressed antigen (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), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1); (Item 47) the second antibody or antigen-specific binding fragment is selected from the group consisting of alpha folate receptor (FRα), αvβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD135 (also known as fmc-like tyrosine kinase 3; FLT3), CD138, CD171, carcinoembryonic antigen (CEA), and / or carcinoembryonic antigen (CEA). Congenital angiotensin-converting antigen (CEA), claudin-6 (CLDN6), 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), 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), 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), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); 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, neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (N 46. ​​The fusion polypeptide of any one of items 40 to 45, which binds to a target antigen selected from the group consisting of polysialic acid; placenta-specific 1 (PLAC1), predominantly 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), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1). (Item 48) a) the first antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand; b) The fusion polypeptide of any one of Aspects 40 to 47, wherein the second antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand. (Item 49) a) the first antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD33, CD79a, CD79b, C-type lectin-like molecule-1 (CLL-1), MAGE-A4, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand; b) The fusion polypeptide of any one of Aspects 40 to 48, wherein the second antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), and TAG72. (Item 50) a) the first antibody or antigen-specific binding fragment binds to B-cell maturation antigen (BCMA); b) The fusion polypeptide of any one of Aspects 40 to 49, wherein the second antibody or antigen-specific binding fragment binds to a target antigen selected from the group consisting of epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), and TAG72. (Item 51) a) the CAR further comprises a first transmembrane domain; b) The fusion polypeptide of any one of the preceding items, wherein the CCR further comprises a second transmembrane domain. (Item 52) 52. The fusion polypeptide of item 51, wherein the first and / or second transmembrane domain is derived from a polypeptide selected from the group consisting of the alpha or beta chain of a 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, and PD1. (Item 53) 52. The fusion polypeptide of item 51, wherein the first and / or second transmembrane domain is derived from CD8α. (Item 54) a) the CAR further comprises a first intracellular costimulatory domain; b) The fusion polypeptide of any one of the preceding items, wherein the CCR further comprises a second intracellular costimulatory domain. (Item 55) 55. The fusion polypeptide of item 54, wherein the first and / or second costimulatory domain is derived 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, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. (Item 56) 55. The fusion polypeptide of item 54, wherein the first and / or second costimulatory domain is derived from CD137(4-1BB). (Item 57) 55. The fusion polypeptide of item 54, wherein the first and / or second costimulatory domain is derived from CD28. (Item 58) Item 11. The fusion polypeptide of any one of the preceding items, wherein the CAR further comprises a primary signaling domain. (Item 59) 59. The fusion polypeptide of paragraph 58, wherein the primary signaling domain is derived from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. (Item 60) 59. The fusion polypeptide of item 58, wherein the primary signaling domain is derived from CD3ζ. (Item 61) A fusion polypeptide comprising: a) a chimeric antigen receptor (CAR) comprising a first antibody or antigen-specific binding fragment, a first hinge region, a first transmembrane domain, a first intracellular costimulatory domain, and a primary signaling domain; b) a polypeptide cleavage signal; and c) a chimeric costimulatory receptor (CCR) comprising a second antibody or antigen-specific binding fragment, a second hinge region, a second transmembrane domain, and a second intracellular costimulatory domain; A fusion polypeptide, wherein the second hinge region comprises one or more cysteine ​​substitutions or deletions that i) reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region, or ii) reduce CAR / CCR association in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region. (Item 62) A fusion polypeptide comprising: a) a CAR comprising a first antibody or antigen-specific binding fragment that binds to BCMA, a first hinge region derived from CD8α, a first transmembrane domain derived from CD8α, a first intracellular costimulatory domain derived from 4-1BB, and a primary signaling domain derived from CD3ζ; b) a polypeptide cleavage signal; and c) a CCR comprising a second antibody or antigen-specific binding fragment that binds to EGFR, a second hinge region derived from CD8α, a second transmembrane domain derived from CD8α, and a second intracellular costimulatory domain derived from CD28; A fusion polypeptide, wherein the second hinge region comprises one or more cysteine ​​substitutions or deletions that i) reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region, or ii) reduce CAR / CCR association in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region. (Item 63) 63. The fusion polypeptide of item 62, wherein the modified second hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. (Item 64) Item 11. The fusion polypeptide of any one of the preceding items, wherein the peptidic cleavage signal is a viral self-cleaving polypeptide. (Item 65) Item 11. The fusion polypeptide of any one of the preceding items, wherein the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide. (Item 66) 10. The fusion polypeptide of any one of the preceding items, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, Thosea asigna virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. (Item 67) A polynucleotide encoding a fusion polypeptide according to any one of the preceding items. (Item 68) 68. A vector comprising the polynucleotide of Item 67. (Item 69) Item 69. The vector according to item 68, wherein the vector is an expression vector. (Item 70) 70. The vector according to item 68 or 69, wherein the vector is an episomal vector. (Item 71) 71. The vector according to any one of items 68 to 70, wherein the vector is a viral vector. (Item 72) 72. Any one of items 68 to 71, wherein the vector is a retroviral vector. The vector described. (Item 73) 73. The vector according to any one of items 68 to 72, wherein the vector is a lentiviral vector. (Item 74) 67. A cell expressing the fusion polypeptide of any one of items 1 to 66. (Item 75) A cell comprising the polynucleotide according to Item 74 or the vector according to any one of Items 52 to 57. (Item 76) 76. The cell of item 72 or 75, wherein the cell is a genetically engineered host cell. (Item 77) 77. The cell according to any one of items 74 to 76, wherein the cell is a hematopoietic cell. (Item 78) 78. The cell according to any one of items 74 to 77, wherein the cell is a hematopoietic stem or progenitor cell. (Item 79) 79. The cell according to any one of items 74 to 78, wherein the cell is a CD34+ hematopoietic stem or progenitor cell. (Item 80) 77. The cell according to any one of items 74 to 76, wherein the cell is an immune effector cell. (Item 81) 77. The cell according to any one of items 74 to 76, wherein the cell is a T cell. (Item 82) The cells are CD3 + , CD4 + , and / or CD8 + 77. The cell according to any one of items 74 to 76, which is a cell. (Item 83) 77. The cell according to any one of items 74 to 76, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 84) 84. The T cell according to any one of items 81 to 83, wherein the T cell is an αβ-T cell. (Item 85) 84. The T cell according to any one of items 81 to 83, wherein the T cell is a γδ-T cell. (Item 86) 77. The cell of any one of items 74 to 76, wherein the host cell is a natural killer (NK) cell. (Item 87) 87. The cell of item 86, wherein the natural killer cell is a natural killer T (NKT) cell. (Item 88) 77. The cell according to any one of items 74 to 76, wherein the host cell is a macrophage. (Item 89) 89. A composition comprising the cells according to any one of items 74 to 88 and a pharmaceutically acceptable carrier. (Item 90) A method of treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of 90. A method comprising administering to the subject an amount of the composition of item 89. (Item 91) 90. A method for improving one or more symptoms associated with cancer in a subject, comprising administering to the subject a therapeutically effective amount of the composition of claim 89 sufficient to improve at least one symptom associated with the cancer. (Item 92) 92. The method of claim 91, wherein the one or more symptoms improved are selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain, bone or joint pain, fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination. (Item 93) 93. The method according to any one of items 90 to 92, wherein the cancer is a solid cancer. (Item 94) 93. The method according to any one of items 90 to 92, wherein the cancer is a liquid cancer. (Item 95) 95. The method of item 94, wherein the cancer is a hematological malignancy. (Item 96) 96. The method of item 94 or 95, wherein the cancer is non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML). (Item 97) 97. The method of item 96, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL). (Item 98) Item 98. The method of item 97, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL). (Item 99) 97. The method of any one of items 94 to 96, wherein the cancer is MM selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma. (Item 100) 72. A method for generating a cell population that expresses the fusion polypeptide according to any one of Items 1 to 66, the method comprising introducing the polynucleotide according to Item 67 or the vector according to any one of Items 68 to 73 into the cell population. (Item 101) 101. The method of claim 100, wherein the cell population comprises hematopoietic stem or progenitor cells. (Item 102) 102. The method of claim 101, wherein the cell population comprises CD34+ hematopoietic stem or progenitor cells. (Item 103) 101. The method of claim 100, wherein the cell population comprises immune effector cells. (Item 104) 101. The method of claim 100, wherein the cell population comprises T cells, NK cells, and / or NKT cells. (Item 105) 101. The method of claim 100, wherein the cell population comprises T cells. (Item 106) 1. A method of reducing CCR costimulation of T cell signaling in a cell that expresses both a CAR and a CCR, comprising: a) obtaining CAR and CCR polypeptides, each having a hinge domain, optionally wherein the CAR and CCR are expressed as fusion polypeptides; b) substituting one or more cysteine ​​residues in the CCR hinge domain with alternative residues, thereby producing a modified CCR; c) expressing the CAR and modified CCR in a cell. (Item 107) 1. A method of reducing CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge, comprising: a) obtaining CAR and CCR polypeptides, each having a hinge domain, optionally wherein the CAR and CCR are expressed as fusion polypeptides; b) substituting one or more cysteine ​​residues in the CCR hinge domain with alternative residues, thereby producing a modified CCR; c) expressing the CAR and modified CCR in a cell. (Item 108) 108. The method of claim 106 or 107, wherein the cysteine ​​residue substitution within the hinge domain of the CCR reduces antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge region. (Item 109) 108. The method of claim 106 or 107, wherein the cysteine ​​residue substitution in the hinge domain of the CCR reduces association with the CAR in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge region. (Item 110) 100. The method of any one of items 106 to 109, wherein both the CCR and CAR comprise a modified hinge region. (Item 111) 111. The method of claim 110, wherein the modified CAR hinge region comprises one or more cysteines replaced with different amino acids. (Item 112) 112. The method of claim 111, wherein the one or more cysteines substituted in the CAR hinge region reduce antigen-independent signaling of the CAR compared to a CAR comprising an unmodified hinge region. (Item 113) 112. The method of claim 111, wherein the one or more cysteines substituted in the CAR hinge region reduce antigen-dependent association with the CCR compared to a CAR comprising an unmodified hinge region. (Item 114) 114. The method of any one of items 106 to 113, wherein the CAR and CCR hinge regions are derived from the same protein. (Item 115) 114. The method of any one of items 106 to 113, wherein the CAR and CCR hinge regions are derived from different proteins. (Item 116) 114. The method of any one of items 106 to 113, wherein the one or more cysteine ​​residues in the CCR and / or CAR hinge region are substituted with serine or alanine. (Item 117) 117. The fusion polypeptide of any one of items 106 to 116, wherein the CCR and / or CAR hinge region comprises a hinge region, or a fragment thereof, selected from the group consisting of a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 (CTLA-4) hinge, a PD-1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2. (Item 118) 118. The method of any one of items 106 to 117, wherein the CCR and / or CAR hinge region is a CD8α hinge region, or a fragment thereof. (Item 119) 119. The method of any one of items 106 to 118, wherein the CCR and / or CAR hinge region is a CD8α hinge region. (Item 120) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises an amino acid substitution at position 27 of SEQ ID NO:2. (Item 121) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises an amino acid substitution at position 44 of SEQ ID NO:2. (Item 122) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises amino acid substitutions at positions 27 and 44 of SEQ ID NO:2. (Item 123) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises the amino acid sequence set forth in SEQ ID NO: 3. (Item 124) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO:3. (Item 125) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises the amino acid sequence set forth in SEQ ID NO:4. (Item 126) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO:4. (Item 127) 120. The method of claim 119, wherein the CD8α hinge region of the CCR and / or CAR comprises the amino acid sequence set forth in SEQ ID NO: 5. (Item 128) 120. The method of item 119, wherein the CD8α hinge region of the CCR and / or CAR comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity to the sequence set forth in SEQ ID NO:5. (Item 129) 108. The method of claim 106 or 107, wherein the CAR and CCR are expressed as a fusion polypeptide according to any one of items 1 to 66. (Item 130) 130. The method of any one of items 92 to 129, wherein step (c) comprises introducing into the cell a polynucleotide or vector encoding the CAR and / or CCR. [Brief explanation of the drawings]

[0046] [Figure 1A]Shown are IFNγ levels from untransduced T cells (UTD) and CAR T cells expressing anti-BCMA CAR with or without anti-EGFR CCR in the absence or presence of EGFR+ HT-1080 fibrosarcoma cells. [Figure 1B] 1 shows impedance data for calculating % cytotoxicity induced by UTD T cells and CAR T cells expressing anti-BCMA CAR with and without anti-EGFR CCR co-cultured with EGFR+ HT-1080 fibrosarcoma cells at effector:target (E:T) ratios of 20:1, 10:1, and 5:1. [Figure 2] Shown are human A549 tumor volumes in immunodeficient NSG mice after treatment with vehicle, UTD T cells, and anti-BCMA CAR T cells with and without anti-EGFR CCR. [Figure 3] A cartoon depicting several mechanisms by which CCR activation can induce CAR-mediated signaling in the absence of CAR antigen is shown. [Figure 4A] Shown is a cartoon depicting different CAR / CCR construct pairs with mutations in their hinge regions and a graph showing IFNγ levels from UTD T cells and CAR T cells expressing different CAR / CCR pairs in two different cancer cell lines expressing EGFR, with and without exogenous expression of BCMA. [Figure 4A-1] Shown is a cartoon depicting different CAR / CCR construct pairs with mutations in their hinge regions and a graph showing IFNγ levels from UTD T cells and CAR T cells expressing different CAR / CCR pairs in two different cancer cell lines expressing EGFR, with and without exogenous expression of BCMA. [Figure 4B] Shows TNFα levels from UTD T cells and CAR T cells expressing different CAR / CCR pairs in two different cancer cell lines expressing EGFR, with and without exogenous BCMA expression. [Figure 4C]Shows IL-2 levels from UTD T cells and CAR T cells expressing different CAR / CCR pairs in two different cancer cell lines expressing EGFR, with and without exogenous BCMA expression. [Figure 5A] The domains of an exemplary CAR / CCR construct are shown. [Figure 5A-1] The domains of an exemplary CAR / CCR construct are shown. [Figure 5B] FACS expression of CAR and CCR constructs on T cells is shown. [Figure 6] Shows IFNγ levels from UTD T cells and CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without hinge C to S mutations, in two cancer cell lines expressing EGFR but not BCMA. [Figure 7AB] Figure 1 shows IFNγ levels from CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without the hinge C to S mutation, in A549 cells (EGFR+) modified to also express BCMA, and A549 cells (EGFR+). Figure 2 shows IL-2 levels from CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without the hinge C to S mutation, in A549 cells (EGFR+) modified to also express BCMA, and A549 cells (EGFR+). [Figure 7CD] Figure 1 shows IFNγ levels from CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without the hinge C to S mutation, in HT-1080 cells (EGFR+) modified to also express BCMA, and in HT-1080 cells (EGFR+). Figure 2 shows IL-2 levels from CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without the hinge C to S mutation, in HT-1080 cells (EGFR+) modified to also express BCMA, and in HT-1080 cells (EGFR+). [Figure 8AB]Figure 1 shows impedance data for calculating % cytotoxicity induced by UTD T cells and CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without hinge C to S mutation, in EGFR+ HT-1080 fibrosarcoma cells. Figure 1 shows impedance data for calculating % cytotoxicity induced by UTD T cells and CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without hinge C to S mutation, in EGFR+ HT-1080 fibrosarcoma cells modified to also express BCMA. [Figure 8CD] Figure 1 shows impedance data for calculating % cytotoxicity induced by UTD T cells and CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without hinge C to S mutation, in EGFR+ HT-1080 fibrosarcoma cells. Figure 1 shows impedance data for calculating % cytotoxicity induced by UTD T cells and CAR T cells expressing various combinations of anti-BCMA CAR and anti-EGFR CCR, with or without hinge C to S mutation, in EGFR+ HT-1080 fibrosarcoma cells modified to also express BCMA. DETAILED DESCRIPTION OF THE INVENTION

[0047] Brief description of sequence identifiers SEQ ID NO: 1 shows the amino acid sequence of the human CD8α protein (Swiss-Prot accession number P01732).

[0048] SEQ ID NO: 2 shows the amino acids of an exemplary wild-type CD8α hinge region.

[0049] SEQ ID NO: 3 shows the amino acid sequence of an exemplary CD8α hinge containing a cysteine ​​to serine mutation at position 27 (C27S).

[0050] SEQ ID NO: 4 shows the amino acid sequence of an exemplary CD8α hinge containing a cysteine ​​to serine mutation at position 44 (C44S).

[0051] SEQ ID NO: 5 shows the amino acid sequence of an exemplary CD8α hinge containing cysteine ​​to serine mutations at positions 27 and 44 (C27S; C44S).

[0052] SEQ ID NO: 6 shows the amino acid sequence of an exemplary CD8α transmembrane (TM) region.

[0053] SEQ ID NOs: 7 to 20 show further exemplary amino acid sequences of hinge regions.

[0054] SEQ ID NOs: 21 to 31 show the amino acid sequences of various linkers.

[0055] SEQ ID NOs: 32 to 56 show the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0056] In the above sequences, X, if present, refers to any amino acid or the absence of an amino acid.

[0057] A. Overview The present disclosure provides modified CCR polypeptides, fusion polypeptides, and genetically modified cells, wherein the CCR comprises a modified hinge region. In certain embodiments, cells modified to express the CARs and CCRs contemplated herein exhibit reduced antigen-independent signaling in the absence of the CAR target antigen compared to polypeptides, fusion polypeptides, or cells comprising an unmodified CCR hinge region. More specifically, cells modified to express the CARs and CCRs contemplated herein exhibit reduced signaling in the presence of the CCR antigen and in the absence of the CAR antigen.

[0058] In other words, the genetically modified cells described herein are activated against target cells expressing both CAR and CCR antigens, while showing little or no activation against cells expressing only CCR antigens. Without wishing to be bound by any particular theory, it is contemplated that the reduced signaling in the presence of CCR antigens and in the absence of CAR antigens is achieved by reducing the disulfide bond between the CCR and CAR hinge regions.

[0059] In certain embodiments, CCRs are useful for their ability to enhance CAR signaling. Furthermore, CCRs may be designed to target antigens different from the CAR, potentially increasing target cell specificity and / or reducing the occurrence of refractory target cells. Importantly, in its purest form, the dual-antigen approach can utilize Boolean logic (AND, OR, NOT) to provide the ability to target specific subsets of cells and not other subsets. The utility of this approach in cell therapy is underscored by the difficulty of discovering cancer-specific antigens (see, e.g., Newick K. et al., Annu. Rev. Med. 2017;68:139-152). In fact, many antigens show at least some expression on other cell types and / or elsewhere in the body. Therefore, one approach to overcoming this problem is to distinguish between CAR and CCR antigen specificities.

[0060] In certain embodiments, the therapy is designed to target only cells that express both the CAR and CCR antigens. Furthermore, by appropriately adjusting the level of signaling / activation, targeting of cells that express only one of the antigens is excluded. For example, cells that express 1) the CAR antigen and 2) both the CAR antigen and the CCR antigen are targeted, but not cells that express only the CCR antigen. In other embodiments, the therapy is designed to target 1) the CCR antigen and 2) cells that express both the CCR antigen and the CAR antigen, but not cells that express only the CAR antigen. Thus, careful selection and tuning of the CAR / CCR combination and / or fusion polypeptide, and the use of Boolean logic, allows for targeting of specific cell types (e.g., cancer cells) while minimizing off-target effects, such as targeting of cells that express only the CAR or CCR antigen.

[0061] Interestingly, the inventors surprisingly discovered that some CAR / CCR combinations (e.g., fusion polypeptides) exhibit signaling in response to target cells expressing only the CCR antigen, i.e., target cells that do not express the CAR antigen (see, e.g., Figures 1A, 1B, and 2). As discussed above, this is surprising because CCRs do not contain signaling domains and therefore, in theory, should not signal alone. Furthermore, if the CCR antigen is one that is widely expressed on other cell types (e.g., EGFR antigen), activation / signaling in the presence of the CCR antigen significantly reduces the benefit of the dual antigen approach. Furthermore, such signaling may also induce T cell dysfunction and / or killing of off-target cell types.

[0062] Without wishing to be bound by any particular theory, the inventors herein contemplate that certain CCRs can interact with their corresponding CARs via disulfide bonds at cysteine ​​residues between the CAR and the CCR hinge region, inducing CAR-mediated T cell signaling in the absence of the CAR antigen. This CCR antigen-dependent, CAR antigen-independent signaling leads to T cell dysfunction and reduced T cell efficacy. As described and exemplified herein, the inventors surprisingly discovered that the problem of CAR antigen-independent signaling is driven in part by the CCR, and that such signaling can be resolved by mutating specific cysteine ​​residues within the CCR hinge region. In certain embodiments, immune effector cells expressing a CAR and a CCR comprising a modified hinge region contemplated herein are activated against cells expressing both the CAR and CCR antigens, while eliminating / reducing activation against cells expressing only the CCR antigen.

[0063] Thus, the present invention provides, in part, CAR and CCR polypeptides, fusion polypeptides, and genetically modified cells expressing a CAR and a CCR, wherein the CCR comprises a modified hinge region. In particular, the polypeptides, fusion polypeptides, or genetically modified cells described herein exhibit reduced T cell signaling in the absence of a CAR antigen compared to fusion polypeptides or cells comprising an unmodified CCR hinge region. More specifically, the improved polypeptides, fusion polypeptides, or genetically modified cells described herein surprisingly reduce CAR antigen-independent signaling while maintaining or increasing T cell signaling in the presence of both antigens (see, e.g., Figures 7A-7D).

[0064] In various embodiments, a fusion polypeptide is provided that includes a chimeric antigen receptor (CAR) and a chimeric costimulatory receptor (CCR) that includes a polypeptide cleavage signal and a hinge region that has been modified to reduce CCR costimulation of T cell signaling in the absence of the CAR antigen compared to a CCR that includes an unmodified hinge region.

[0065] In various embodiments, a fusion polypeptide is provided that includes a chimeric antigen receptor (CAR) and a chimeric costimulatory receptor (CCR) that includes a polypeptide cleavage signal and a hinge region that includes one or more cysteines substituted with another amino acid. In some embodiments, the one or more cysteine ​​substitutions in the CCR hinge region reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR that includes an unmodified hinge region. In some embodiments, the one or more cysteine ​​substitutions in the CCR hinge region reduce CAR / CCR association in the absence of a CAR antigen compared to a CCR that includes an unmodified hinge region.

[0066] In various embodiments, the CCR and CAR comprise a modified hinge region. In some embodiments, the modified CAR and / or CCR hinge region comprises one or more cysteines substituted with a different amino acid (e.g., serine or alanine). In some embodiments, the one or more cysteines substituted within the CAR and / or CCR hinge region reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CAR and / or CCR comprising an unmodified hinge region. In some embodiments, the one or more cysteine ​​substitutions within the CAR and / or CCR hinge region reduce CAR / CCR association in the absence of a CAR antigen compared to a CAR and / or CCR comprising an unmodified hinge region.

[0067] In various embodiments, the CAR and CCR hinge regions are derived from the same or different proteins. In some embodiments, the hinge region is derived from a CD8α hinge region (e.g., SEQ ID NO: 2). In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises an amino acid substitution at position 27 of SEQ ID NO: 2 (e.g., SEQ ID NO: 3). In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises an amino acid substitution at position 44 of SEQ ID NO: 2 (e.g., SEQ ID NO: 4). In some embodiments, the CD8α hinge region of the CCR and / or CAR comprises amino acid substitutions at positions 27 and 44 of SEQ ID NO: 2 (e.g., SEQ ID NO: 5).

[0068] In various embodiments, a fusion polypeptide is provided that includes a chimeric antigen receptor (CAR), a polypeptide cleavage signal, and a chimeric costimulatory receptor (CCR), wherein the CAR includes a first antibody or antigen-specific binding fragment thereof, a first hinge region, a first transmembrane domain, a first intracellular costimulatory domain, and a primary signaling domain; the CCR includes a second antibody or antigen-specific binding fragment thereof, a second hinge region, a second transmembrane domain, and a second intracellular costimulatory domain; and the second hinge domain of the CCR includes one or more cysteine ​​residues substituted with a different amino acid.

[0069] In various embodiments, polynucleotides encoding the fusion polypeptides described herein are provided. In some embodiments, vectors comprising the polynucleotides described herein are provided. In some embodiments, cells expressing the fusion polypeptides described herein are provided. In some embodiments, the cells are genetically engineered host cells, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells, immune effector cells, T cells, CD3 + cells, CD4 + cells, CD8 + The immunoglobulin is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), a helper T cell, an αβ-T cell, a γδ-T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a macrophage.

[0070] In various embodiments, compositions comprising the cells described herein are provided. In some embodiments, the compositions comprise a pharmaceutically acceptable carrier.

[0071] In some embodiments, provided are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0072] In various embodiments, there is provided a method of generating a cell population that expresses a fusion polypeptide described herein, comprising introducing into the cell population a polynucleotide or vector described herein.

[0073] In various embodiments, methods are provided for reducing CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge region, the methods comprising obtaining CAR and CCR polypeptides, each having a hinge domain, optionally wherein the CAR and CCR are expressed as a fusion polypeptide, substituting one or more cysteine ​​residues in the CCR hinge domain with another residue, thereby producing a modified CCR, and expressing the CAR and modified CCR in a cell. In various embodiments, methods are provided for reducing CCR costimulation of T cell signaling in a cell expressing both a CAR and a CCR, the methods comprising a) obtaining CAR and CCR polypeptides, each having a hinge domain, optionally wherein the CAR and CCR are expressed as a fusion polypeptide, b) substituting one or more cysteine ​​residues in the CCR hinge domain with another residue, thereby producing a modified CCR, and c) expressing the CAR and modified CCR in a cell.

[0074] 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 more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, 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 professional journals such as Advances in Immunology.

[0075] B. Definition Before describing this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred compositions, methods, and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0077] 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 more than one) of the grammatical object of the article. By way of example, "an" means one element or one or more elements.

[0078] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0079] The term "and / or" should be understood to mean either one or both of the alternatives.

[0080] 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.

[0081] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to each number subsumed within the range. For example, in one non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to the expression 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0082] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, 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, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, size, amount, weight, or length that produces an effect, e.g., a physiological effect, that is approximately the same as the reference quantity, level, value, number, frequency, percentage, size, amount, weight, or length.

[0083] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of the specified step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components. "Consisting of" means including and limited to everything that follows the word "consisting of." Thus, the phrase "consisting of" indicates 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 phrase, 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 phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that no other elements are present that materially affect the activity or function of the listed elements.

[0084] 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 these 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 a basis for the exclusion of that feature in certain embodiments.

[0085] Additional definitions are set forth throughout this disclosure.

[0086] C. Chimeric Antigen Receptors (CARs) and Modified Chimeric Costimulatory Receptors (CCRs) In various embodiments, immune effector cells are modified to express a CAR and a CCR to redirect the cytotoxicity of immune effector cells to cancer cells expressing a specific antigen and synergistically increase the efficacy of immune effector cell therapy. CARs are molecules that combine antibody-based specificity for a desired antigen with the primary and costimulatory signaling components of a T cell receptor. Unlike CARs, CCRs are molecules that combine antibody-based specificity for a desired antigen with a T cell receptor costimulatory domain, but lack the primary signaling domain. As used herein, the term "chimera" refers to something composed of different protein or DNA portions derived from different sources.

[0087] In certain embodiments, immune effector cells express a CAR comprising an antigen-binding domain and a CCR comprising a different antigen-binding domain. The CAR comprises an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory domain, and a primary signaling domain, while the CCR comprises an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain, but lacks the primary signaling domain. Binding of the antigen on the surface of the target cell to the antigen-binding domain of the CCR results in clustering of the CCR, delivering a costimulatory signal to the CAR-containing cell. A key feature of the CCR is its ability to further redirect or fine-tune the specificity of immune effector cells in an MHC-independent manner and enhance immune effector cell responses in the presence of the CAR, preferably the CAR antigen.

[0088] In various embodiments, the CCR comprises an extracellular binding domain that includes an antigen-specific binding domain, a modified hinge region, a transmembrane domain, and a costimulatory signaling domain, but not a primary signaling domain.

[0089] In a preferred embodiment, the CCR hinge region comprises one or more cysteine ​​residues substituted with one or more other amino acid residues (eg, one or more serine residues).

[0090] Illustrative examples of CAR and CCR components are further disclosed below.

[0091] 1. Binding Domain In certain embodiments, CARs and CCRs comprise an extracellular binding domain comprising an antibody or antigen-binding fragment thereof that specifically binds to a specific antigen expressed on a target cell, e.g., a cancer cell. As used herein, the terms "binding domain," "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and provide the CAR and CCR with the ability to specifically bind to a target antigen of interest. The binding domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0092] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" describe the binding of an antibody or antigen-binding fragment thereof (or a CAR or CCR comprising the same) to an antigen with a binding affinity higher than background binding. The binding domain (or a CAR or CCR comprising the binding domain or a fusion protein comprising the binding domain) can bind to an antigen with a binding affinity higher than background binding, for example, about 10 5 M -1 In certain embodiments, a binding domain (or fusion protein) "specifically binds" to an antigen if it binds to or associates with the antigen with an affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction, having units of 1 / M) of at least 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 10 13 M -1 A "high affinity" binding domain (or single-chain fusion protein thereof) binds to a target with a Ka of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 The term "binding domains" refers to those binding domains having a Ka of 1 or higher.

[0093] Alternatively, affinity may be expressed in M ​​units (e.g., 10 -5 M~10 -13The affinity of binding domain polypeptides and CAR and CCR proteins according to the present disclosure can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or by using displacement assays using labeled ligands, or using a surface plasmon resonance instrument such as a Biacore T100 (available from Biacore, Inc., Piscataway, NJ), or an optical biosensor such as the EPIC system or EnSpire (available from Corning and Perkin Elmer, respectively) (see also, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660, and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0094] In one embodiment, the affinity of specific binding is about 2-fold greater than background binding, about 5-fold greater than background binding, about 10-fold greater than background binding, about 20-fold greater than background binding, about 50-fold greater than background binding, about 100-fold greater than background binding, or about 1000-fold greater than background binding, or more.

[0095] In certain embodiments, the CAR and CCR extracellular binding domains comprise an antibody or antigen-binding fragment thereof. An "antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide, or antigenic determinant-containing nucleic acid, such as those recognized by immune cells. An "isolated antibody or antigen-binding fragment thereof" is one that has been identified, separated, and / or recovered from a component of its natural environment.

[0096] "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 cancer-specific proteins) that are injected into or absorbed into an animal. Antigens react with the products of specific humoral or cellular immunity, including, for example, products induced by heterologous antigens such as the disclosed antigens.

[0097] "Epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent binds. Epitopes can be formed both from contiguous or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically comprises at least three, more usually at least five, about nine, or about eight to ten amino acids in a unique spatial conformation.

[0098] Antibodies include chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific 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.

[0099] As will be understood by those skilled in the art and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y consists of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) joined together, with disulfide bonds (interchain) formed at the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem Ig domains and a hinge region for additional flexibility, while heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm of the Y contains the variable and first constant regions of a single heavy chain bound to the variable and constant regions of a single light chain. The light and heavy chain variable regions are responsible for antigen binding.

[0100] Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." CDRs can be defined or identified by conventional methods, such as by sequence by Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84:2440-2443 (1987) (see Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991, incorporated herein by reference) or by structure by Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987); Chothia, C. et al., Nature, 342:877-883 (1989)).

[0101] Illustrative examples of rules for predicting light chain CDRs include: CDR-L1 begins at about residue 24, is preceded by Cys, is about 10-17 residues, and is followed by Trp (typically Trp-Tyr-Gln, but also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 begins about 16 residues after the end of CDR-L1, is generally preceded by Ile-Tyr, but also Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues; CDR-L3 begins about 33 residues after the end of CDR-L2, is preceded by Cys, is 7-11 residues, and is followed by Phe-Gly-XXX-Gly (XXX is any amino acid) (SEQ ID NO: 58).

[0102] Examples of rules for predicting heavy chain CDRs include: CDR-H1 begins at about residue 26, is preceded by Cys-XXX-XXX-XXX (SEQ ID NO: 59), is 10-12 residues, and is followed by Trp (typically Trp-Val, but also Trp-Ile, Trp-Ala); CDR-H2 begins about 15 residues after the end of CDR-H1, and is generally followed by Leu-Glu-Trp-Ile-Gly (sequence CDR-H3 begins approximately 33 residues after the end of CDR-H2, is preceded by Cys-XXX-XXX (typically Cys-Ala-Arg), is preceded by Cys-XXX-XXX (sequence number 60), or some variations, is 16 to 19 residues long, and is followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala, and is 3 to 25 residues long, and is followed by Trp-Gly-XXX-Gly (sequence number 61).

[0103] In one embodiment, the light chain CDRs and heavy chain CDRs are determined according to the Kabat method.

[0104] In one embodiment, the light chain CDRs and the heavy chain CDR2 and CDR3 are determined according to the Kabat method, and the heavy chain CDR1 is determined according to the AbM method, which is a combination of the Kabat and Clothia methods (see, for example, Whitelegg N & Rees AR, Protein Eng. 2000 Dec; 13(12): 819-24 and Methods Mol Biol. 2004; 248: 51-91). Programs for predicting CDRs are available, for example, at AbYsis (www.bioinf.org.uk / abysis / ).

[0105] The sequences of framework regions of different light or heavy chains are relatively conserved within species, such as humans. The framework region of an antibody is the combined framework region of the constituent light and heavy chains and serves to position and align the CDRs in three-dimensional space. CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, are numbered sequentially from the N-terminus, and are generally identified by the chain in which the particular CDR is located. Thus, CDRs located in the variable domain of an antibody's heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while CDRs located in the variable domain of an antibody's light chain are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs).

[0106] References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment contemplated herein. References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment contemplated herein.

[0107] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of a myeloma cell and an immune spleen cell. Monoclonal antibodies include humanized monoclonal antibodies.

[0108] A "chimeric antibody" has framework residues derived from one species, such as human, and CDRs (which generally confer antigen binding) derived from another species, such as mouse. In certain preferred embodiments, the CAR and / or CCR comprise an antigen-specific binding domain that is a chimeric antibody or antigen-binding fragment thereof.

[0109] In certain embodiments, the antibody is a human antibody (such as a human monoclonal antibody) or a fragment thereof that specifically binds to a human polypeptide. Human antibodies can be constructed by combining Fv clone variable domain sequences selected from a human-derived phage display library with known human constant domain sequences, as described above. Alternatively, human monoclonal antibodies can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147:86 (1991). Furthermore, transgenic animals (e.g., mice) can be used to produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993). Gene shuffling can also be used to derive human antibodies from non-human, e.g., rodent, antibodies, with similar affinities and specificities to the starting non-human antibody. (See PCT WO93 / 06213, published April 1, 1993.) Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin.

[0110] In one embodiment, the CAR and / or CCR comprise a "humanized" antibody. A humanized antibody is an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more. Thus, all portions of a humanized immunoglobulin are substantially identical to corresponding portions of a native human immunoglobulin sequence, except potentially for the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions, which do not substantially affect antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0111] Antibodies include antigen-binding fragments such as 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, nanobody), as well as the portion of a full-length antibody 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, and 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.

[0112] A "heavy chain antibody" refers to an antibody comprising two VH domains and no light chains (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999), WO94 / 04678, WO94 / 25591, U.S. Patent No. 6,005,079). A "camelid antibody" refers to an antibody isolated from a camel, alpaca, or llama comprising two VH domains and no light chains. A "humanized VHH" or "humanized camelid antibody" refers to a non-human VHH or camelid antibody that has undergone humanization to reduce the potential immunogenicity of the antibody in a human recipient.

[0113] "IgNAR," for "immunoglobulin novel antigen receptor," refers to a class of antibodies from the shark immune repertoire consisting of a homodimer of one variable novel antigen receptor (VNAR) domain and five constant novel antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and possess highly stable and efficient binding properties. Their inherent stability can be attributed to both (i) the underlying Ig scaffold, which displays a significant number of charged and hydrophilic surface-exposed residues compared to traditional antibody VH and VL domains found in murine antibodies, and (ii) structural features in the complementarity-determining region (CDR) loops, including inter-loop disulfide bridges and stabilizing patterns of intra-loop hydrogen bonds.

[0114] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0115] An "Fv" is the minimum antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains can associate in a "dimeric" structure similar to that of a two-chain Fv species. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0116] Fab fragments contain heavy and light chain variable domains, as well as the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues in the constant domains bear free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known. A bispecific Fab dimer (Fab2) has two Fab' fragments, each binding to a different antigen. A trispecific Fab trimer (Fab3) has three Fab' fragments, each binding to a different antigen.

[0117] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are more fully described, for example, in EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., PNAS USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0118] A "single domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (Holt, L., et al, Trends in Biotechnology, 21(11):484-490).

[0119] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain and in any orientation (e.g., VL-VH or VH-VL). Generally, scFv polypeptides further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0120] In a preferred embodiment, the antigen-specific binding fragment is an scFv or VHH. In a specific embodiment, the scFv is a mouse, human, or humanized scFv. Single-chain antibodies may be cloned from the V region genes of a hybridoma specific for a desired target. The production of such hybridomas is routine. Techniques that can be used to clone the variable heavy chain (VH) and variable light chain (VL) are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837.

[0121] In various embodiments, the antigen-specific binding domain of the CAR (e.g., first binding domain) and / or CCR (e.g., second binding domain) is selected from the group consisting of alpha folate receptor (FRα), αvβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, and the like. , CD123, CD133, CD135 (also known as fmc-like tyrosine kinase 3; FLT3), CD138, CD171, carcinoembryonic antigen (CEA), claudin-6 (CLDN6), 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), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein Protein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), 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), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAG) E-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); 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, neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;binds to a target antigen selected from the group consisting of placenta-specific 1 (PLAC1), predominantly 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), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1);

[0122] In certain embodiments, the antigen-specific binding domain is an scFv or a VHH.

[0123] In certain embodiments, the antigen-specific binding domain is an scFv that binds to a human BCMA or EGFR polypeptide.

[0124] 2. Linker In certain embodiments, the CAR and / or CCR comprise linker residues between the various domains, e.g., added for proper spacing and structure of the molecule. In certain embodiments, the linker is a sequence that connects the variable regions. A "sequence that connects the variable regions" is defined as a V H and V LThe linker is an amino acid sequence connecting the two sub-binding domains, providing 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 CAR and / or CCR comprises one, two, three, four, or five or more linkers. In certain embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length.

[0125] Illustrative examples of linkers include glycine polymers (G) n and glycine-serine polymers (G 1-5 S 1-5 ) n Flexible linkers include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between domains of fusion proteins such as the CARs or CCRs described herein. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that the design of a CAR or CCR in certain embodiments can include a linker that is fully or partially flexible, whereby the linker can include a flexible linker as well as one or more moieties that confer a less flexible structure to provide the desired CAR or CCR structure.

[0126] Other exemplary linkers include, but are not limited to, the following amino acid sequences: DGGGS (SEQ ID NO: 21); TGEKP (SEQ ID NO: 22) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 23) (Pomerantz et al. 1995, supra); (GGGGS) n (wherein 1, 2, 3, 4, or 5 (SEQ ID NO: 24) (Kim et al., PNAS 93, 1156-1160 (1996)); EGKSSGSGSESKVD (SEQ ID NO: 25) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 26) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 27); LQRDGERP (SEQ ID NO: 28); LRQKDGGGSERP (SEQ ID NO: 29); LRQKD(GGGS)2ERP (SEQ ID NO: 30). Alternatively, flexible linkers can be modeled using a computer program (Desjarlais & Berg, PNAS 93, 1156-1160 (1996)) that can model both the DNA binding site and the peptide itself. 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: 31) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0127] 3. Spacer domain In certain embodiments, the CAR and / or CCR binding domains are followed by one or more "spacer domains," which refer to regions that separate the antigen-binding domains from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). Spacer domains can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is an immunoglobulin portion including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0128] In one embodiment, the spacer domain comprises CH2 and CH3 of IgG1, IgG4, or IgD.

[0129] 4. Hinge domain The terms "hinge," "hinge domain," and "hinge region" are used interchangeably herein and refer to a flexible domain that serves to position the antigen binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation. The binding domain of a CAR and / or CCR is generally followed by one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In some embodiments, the hinge domain comprises a spacer domain.

[0130] As used herein, the terms "altered hinge domain," "modified hinge domain," and "modified hinge domain" refer to (a) a naturally occurring hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region that is at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids) having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a portion of a naturally occurring hinge region that includes the core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length).

[0131] In preferred embodiments, the modified hinge region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to a suitable hinge domain / region described herein and / or known in the art. In some embodiments, the modified hinge region comprises a hinge sequence described herein with no more than four, no more than three, or no more than two amino acid substitutions and / or deletions.

[0132] In certain embodiments, one or more cysteine ​​residues in the naturally occurring hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). In certain embodiments, the modified hinge region comprises a substitution of a proline residue with another amino acid residue (e.g., a serine residue).

[0133] Exemplary hinge domains suitable for use in CARs and / or CCRs contemplated in certain embodiments herein include, but are not limited to, hinge regions derived from the extracellular regions of type 1 membrane proteins, including, for example, CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or may be modified. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge. In another embodiment, the hinge domain comprises a naturally occurring immunoglobin hinge region, e.g., an IgG1, IgG2, IgG3, or IgG4 hinge. In another embodiment, the hinge domain comprises an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, or an IgG4 hinge / CH2.

[0134] In some embodiments, the CD8α hinge region comprises the amino acid sequence set forth in SEQ ID NO: 2, or a functional fragment or modification thereof.

[0135] In some embodiments, the CD4 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 7, or a functional fragment or modification thereof.

[0136] In some embodiments, the CD28 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 8, or a functional fragment or modification thereof.

[0137] In some embodiments, the CD7 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 9, or a functional fragment or modification thereof.

[0138] In some embodiments, the CD152 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 10, or a functional fragment or modification thereof.

[0139] In some embodiments, the PD-1 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 11, or a functional fragment or modification thereof.

[0140] In some embodiments, the IgG1 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 12, or a functional fragment or modification thereof.

[0141] In some embodiments, the IgG2 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 13, or a functional fragment or modification thereof.

[0142] In some embodiments, the IgG3 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 14, or a functional fragment or modification thereof.

[0143] In some embodiments, the IgG4 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 15, or a functional fragment or modification thereof.

[0144] In some embodiments, the IgG1 hinge / CH2 / CH3 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 16, or a functional fragment or modification thereof.

[0145] In some embodiments, the IgG1 hinge-CH3-hinge-M1 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 17, or a functional fragment or modification thereof.

[0146] In some embodiments, the IgG4 hinge / CH2 / CH3 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 18, or a functional fragment or modification thereof.

[0147] In some embodiments, the IgG4 hinge / CH2 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 19, or a functional fragment or modification thereof.

[0148] In some embodiments, the PD-1 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 20, or a functional fragment or modification thereof.

[0149] In various embodiments, the fusion polypeptides described herein comprise a modified first and / or second hinge region comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to any one of the hinge domains / regions described above. In some embodiments, the modified first and / or second hinge region comprises a hinge sequence described above with no more than four, no more than three, or no more than two amino acid substitutions and / or deletions.

[0150] In certain embodiments, one or more cysteine ​​residues in the hinge region / domain may be substituted with one or more other amino acid residues to produce a modified hinge domain. In some embodiments, the modified hinge domain comprises one or more cysteine ​​residues substituted with serine or alanine. In another embodiment, the modified hinge domain comprises one or more cysteine ​​residues substituted with serine. In another embodiment, the modified CD8α hinge domain comprises one or more cysteine ​​residues substituted with alanine. In some embodiments, the CCR hinge domain is modified. In some embodiments, the CAR hinge domain is unmodified. In some embodiments, both the CAR and CCR hinge domains are modified.

[0151] In a preferred embodiment, the hinge domain comprises a CD8α hinge region / domain. In one embodiment, the hinge domain comprises the amino acid residues set forth in SEQ ID NO: 2. In another embodiment, the CAR and / or CCR comprises a modified CD8α hinge domain. In another embodiment, the CCR comprises a modified CD8α hinge domain, and the CAR optionally comprises a wild-type hinge sequence. In another embodiment, the modified CD8α hinge domain comprises one or more cysteine ​​residues substituted with one or more other amino acid residues. In another embodiment, the modified CD8α hinge domain comprises one or more cysteine ​​residues substituted with serine or alanine. In another embodiment, the modified CD8α hinge domain comprises one or more cysteine ​​residues substituted with serine. In another embodiment, the modified CD8α hinge domain comprises one or more cysteine ​​residues substituted with alanine.

[0152] In one embodiment, the modified CD8α hinge domain comprises a cysteine ​​to serine substitution at position 27 of SEQ ID NO: 2. In another embodiment, the modified CD8α hinge domain comprises the amino acid residues set forth in SEQ ID NO: 3. In specific embodiments, the modified CD8α hinge domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence set forth in SEQ ID NO: 3. In specific embodiments, the modified CD8α hinge domain comprises an amino acid sequence having at least 90%, 93%, 95%, or 97% identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the modified CD8α hinge domain comprises no more than four, no more than three, or no more than two amino acid substitutions and / or deletions.

[0153] In one embodiment, the modified CD8α hinge domain comprises a cysteine ​​to serine substitution at position 44 of SEQ ID NO: 2. In another embodiment, the modified CD8α hinge domain comprises the amino acid residues set forth in SEQ ID NO: 4. In specific embodiments, the modified CD8α hinge domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence set forth in SEQ ID NO: 4. In specific embodiments, the modified CD8α hinge domain comprises an amino acid sequence having at least 90%, 93%, 95%, or 97% identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the modified CD8α hinge domain comprises no more than four, no more than three, or no more than two amino acid substitutions and / or deletions.

[0154] In one embodiment, the modified CD8α hinge domain comprises cysteine ​​to serine substitutions at positions 27 and 44 of SEQ ID NO: 2. In another embodiment, the modified CD8α hinge domain comprises the amino acid residues set forth in SEQ ID NO: 5. In specific embodiments, the modified CD8α hinge domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence set forth in SEQ ID NO: 5. In specific embodiments, the modified CD8α hinge domain comprises an amino acid sequence having at least 90%, 93%, 95%, or 97% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the modified CD8α hinge domain comprises no more than four, no more than three, or no more than two amino acid substitutions and / or deletions.

[0155] 5. Transmembrane (TM) domain A "transmembrane domain" is the portion of a CAR and / or CCR that fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAR and / or CCR to the cell membrane of an immune effector cell. TM domains can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. TM domains can be derived from the alpha or beta 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, and PD1 (i.e., comprising at least the transmembrane region of the alpha or beta chain of the T cell receptor). In certain embodiments, the TM domain is synthetic and primarily comprises hydrophobic residues such as leucine and valine.

[0156] In one embodiment, the CAR and / or CCR comprises a TM domain derived from PD1, CD152, CD28, or CD8α. In another embodiment, the CAR and / or CCR comprises a TM domain derived from PD1, CD152, CD28, or CD8α, and optionally a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the TM domain to the intracellular signaling or costimulatory domain of the CAR or CCR. Glycine-serine based linkers provide particularly suitable linkers.

[0157] For example, in one non-limiting and merely exemplary embodiment, the transmembrane domain comprises the CD8α transmembrane domain set forth in SEQ ID NO: 6. In specific embodiments, the CD8α transmembrane domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence set forth in SEQ ID NO: 6.

[0158] 6. Intracellular signaling domains In certain embodiments, a CAR comprises one or more intracellular signaling domains. An "intracellular signaling domain" refers to a portion of a CAR that is involved in transducing the message of effective CAR binding to a human antigen inside an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytokine factors into the CAR-bound target cell or other cellular responses elicited by antigen binding to the extracellular CAR domain.

[0159] The term "effector function" refers to the specialized function of an immune effector cell. The effector function of a T cell can be, for example, cytolytic activity or help or activity, including cytokine secretion. Accordingly, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be employed, it is often 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 can be used in place of the entire domain, so long as it transduces an effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0160] It is known that signals generated by the TCR alone are insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation by the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains, which act in an antigen-dependent manner to provide secondary or costimulatory signals. In a preferred embodiment, the CAR comprises an intracellular signaling domain comprising one or more "costimulatory signaling domains" and "primary signaling domains."

[0161] The primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0162] Illustrative examples of ITAM-containing primary signaling domains useful in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In particularly preferred embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain can be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0163] In certain embodiments, the CAR and / or CCR comprise one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR and CCR receptors. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or an Fc receptor, that, upon binding to an antigen, provides a second signal required for efficient activation and function of T lymphocytes. Illustrative examples of such costimulatory molecules include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, TNFR2, and ZAP70. In one embodiment, the CAR and / or CCR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0164] In another embodiment, the CAR and / or CCR comprises CD28 and CD137 costimulatory signaling domains and a CD3ζ primary signaling domain.

[0165] In yet another embodiment, the CAR and / or CCR comprises CD28 and CD134 costimulatory signaling domains and a CD3ζ primary signaling domain.

[0166] In one embodiment, the CAR and / or CCR comprises CD137 and CD134 costimulatory signaling domains and a CD3ζ primary signaling domain.

[0167] In one embodiment, the CAR and / or CCR comprises a CD137 costimulatory signaling domain and a CD3ζ primary signaling domain.

[0168] In one embodiment, the CAR and / or CCR comprises a CD134 costimulatory signaling domain and a CD3ζ primary signaling domain.

[0169] In one embodiment, the CAR and / or CCR comprises a CD28 costimulatory signaling domain and a CD3ζ primary signaling domain.

[0170] D. CAR and CCR Exemplary Embodiments In one embodiment, the CAR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen, a hinge region, a transmembrane domain, one or more intracellular costimulatory signaling domains from a costimulatory molecule, and a primary signaling domain.

[0171] In one embodiment, the CAR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen expressed on a cancer cell, a hinge region, a transmembrane domain derived from a polypeptide selected from the group consisting of the alpha or beta chain of a T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN1, and PD1, and a polypeptide selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15, TLR16, TLR17, TLR18, TLR19, TLR20, TLR21, TLR22, TLR23, TLR24, TLR25, TLR26, TLR27, TLR28, TLR29, TLR30, TLR31, TLR32, TLR33, TLR34, TLR35, TLR36, TLR37, TLR38, TLR39, TLR40, TLR41, TLR42, TLR43, TLR44, TLR45, TLR46, TLR47, TLR48, TLR49 ... , TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70, and a primary signaling domain derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0172] In one embodiment, the CAR comprises a hinge domain selected from the group consisting of a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge, a PD1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2.

[0173] In one embodiment, the CAR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen expressed in cancer and a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge, a PD1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2. a hinge domain selected from the group consisting of the alpha or beta chain of a T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN1, and PD1; and a transmembrane domain derived from a polypeptide selected from the group consisting of a T A short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, links the M domain to the intracellular signaling domain of the CAR, and a linker that links the M domain to the intracellular signaling domain of the CAR. The antibody comprises one or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of D134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70, and primary signaling domains derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0174] In certain embodiments, the CAR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen expressed in cancer, a hinge domain comprising a CD8α polypeptide, a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids (e.g., a tail-CD8 linker), a CD137 (4-1BB) intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0175] In certain embodiments, the CAR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen expressed in cancer, a hinge domain comprising a CD8α polypeptide, a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids (e.g., a tail-CD8 linker), a CD134 intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0176] In certain embodiments, the CAR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen expressed in cancer, a hinge domain comprising a CD8α polypeptide, a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids (e.g., a tail-CD8 linker), a CD28 intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0177] In various embodiments, the CAR antibody or antigen-specific binding fragment thereof is an scFv or a VHH. In some embodiments, the CAR antibody or antigen-specific binding fragment thereof is an scFv. In some embodiments, the CAR antibody or antigen-specific binding fragment thereof is a VHH.

[0178] In any one of the embodiments described herein, the CAR hinge region can be modified as described herein to reduce antigen-independent T cell signaling. In any one of the embodiments described herein, the hinge region can be modified to reduce association with a CCR in the absence of a CAR antigen, compared to a CAR comprising an unmodified hinge region.

[0179] In one embodiment, the modified CAR hinge region comprises one or more cysteines substituted with a different amino acid. In another embodiment, one or more cysteine ​​residues are substituted with serine or alanine. In a preferred embodiment, the modified hinge region is a CD8α hinge region. In another embodiment, the modified CD8α hinge region is derived from SEQ ID NO:2. In another embodiment, the modified CD8α hinge region comprises an amino acid substitution at position 27 of SEQ ID NO:2 (i.e., see SEQ ID NO:3). In another embodiment, the modified CD8α hinge region comprises an amino acid substitution at position 44 of SEQ ID NO:2 (i.e., see SEQ ID NO:4). In another embodiment, the modified CD8α hinge region comprises amino acid substitutions at positions 27 and 44 of SEQ ID NO:2 (i.e., see SEQ ID NO:5).

[0180] In any of the above embodiments, the one or more cysteine ​​substitutions in the CAR hinge region reduce CCR costimulation of T cell signaling in the absence of a CAR antigen compared to a CAR comprising an unmodified hinge region. In any one of the above embodiments, the one or more cysteine ​​substitutions in the CAR hinge region reduce association with a CCR in the absence of a CAR antigen compared to a CAR comprising an unmodified hinge region.

[0181] In one embodiment, the CCR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen, a hinge region, a transmembrane domain, and one or more intracellular costimulatory signaling domains from a costimulatory molecule.

[0182] In one embodiment, the CCR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen expressed in a cancer, a modified hinge region comprising one or more cysteines replaced with different amino acids, and a polypeptide selected from the group consisting of the alpha or beta chain of a 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, AMN1, and PD1. and one or more intracellular costimulatory signaling domains 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, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0183] In one embodiment, the CCR comprises a modified hinge region selected from the group consisting of a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge, a PD1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2.

[0184] In one embodiment, the CCR comprises an antibody or antigen-specific binding fragment thereof that binds to an antigen expressed in cancer and a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge, a PD1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, or an IgG4 hinge / CH2 / CH3. , and IgG4 hinge / CH2, and a modified hinge region selected from the group consisting of the alpha or beta 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, AMN1, and PD a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the TM domain to the intracellular signaling domain of the CAR; and one or more intracellular costimulatory signaling domains from costimulatory molecules 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, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, TNFR2, and ZAP70.

[0185] In certain embodiments, the CCR comprises an scFv or VHH that binds to an antigen expressed on a cancer, a modified hinge region comprising a CD8α polypeptide described herein, a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids (e.g., a tail-CD8 linker), and a CD137 intracellular costimulatory signaling domain.

[0186] In certain embodiments, the CCR comprises an scFv or VHH that binds to an antigen expressed on a cancer, a modified hinge region comprising a CD8α polypeptide described herein, a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids (e.g., a tail-CD8 linker), and a CD134 intracellular costimulatory signaling domain.

[0187] In certain embodiments, the CCR comprises an scFv or VHH that binds to an antigen expressed on a cancer, a modified hinge region comprising a CD8α polypeptide described herein, a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids (e.g., a tail-CD8 linker), and a CD28 intracellular costimulatory signaling domain.

[0188] In various embodiments, the CCR antibody or antigen-specific binding fragment thereof is an scFv or a VHH. In some embodiments, the CCR antibody or antigen-specific binding fragment thereof is an scFv. In some embodiments, the CCR antibody or antigen-specific binding fragment thereof is a VHH.

[0189] In any one of the embodiments described herein, the CCR hinge region can be modified as described herein to reduce antigen-independent T cell signaling. In any one of the embodiments described herein, the CCR hinge region can be modified to reduce association with the CAR in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge region.

[0190] In one embodiment, the modified CCR hinge region comprises one or more cysteines substituted with a different amino acid. In another embodiment, one or more cysteine ​​residues are substituted with serine or alanine. In a preferred embodiment, the modified hinge region is a CD8α hinge region. In another embodiment, the modified CD8α hinge region is derived from SEQ ID NO:2. In another embodiment, the modified CD8α hinge region comprises an amino acid substitution at position 27 of SEQ ID NO:2 (i.e., see SEQ ID NO:3). In another embodiment, the modified CD8α hinge region comprises an amino acid substitution at position 44 of SEQ ID NO:2 (i.e., see SEQ ID NO:4). In another embodiment, the modified CD8α hinge region comprises amino acid substitutions at positions 27 and 44 of SEQ ID NO:2 (i.e., see SEQ ID NO:5).

[0191] In any of the above embodiments, the one or more cysteine ​​substitutions in the CCR hinge region reduce CCR costimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge region. In any one of the above embodiments, the one or more cysteine ​​substitutions in the CCR hinge region reduce association with a CAR in the absence of a CAR antigen compared to a CCR comprising an unmodified hinge region.

[0192] In various embodiments, the antigen-specific binding domain of the CAR and / or CCR is alpha folate receptor (FRα), α vβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD135 (also known as fmc-like tyrosine kinase 3; FLT3), CD138, CD171, and carcinoma Childhood antigen (CEA), claudin-6 (CLDN6), 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), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCA) M), ephrin type A receptor 2 (EPHA2), 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), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY) , L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), 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, neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;The antigen-specific binding domain binds to a target antigen selected from the group consisting of placenta-specific 1 (PLAC1), predominantly melanoma-expressed antigen (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), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1). In certain embodiments, the antigen-specific binding domain is an scFv or VHH. In certain embodiments, the antigen-specific binding domain is an scFv. In certain embodiments, the antigen-specific binding domain is a VHH. In certain embodiments, the antigen-specific binding domain is an scFv that binds to a human BCMA or EGFR polypeptide;

[0193] In various embodiments, the antigen-specific binding domain of the CAR is alpha folate receptor (FRα), α vβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD135 (also known as fmc-like tyrosine kinase 3; FLT3), CD138, CD171, and carcinoma Childhood antigen (CEA), claudin-6 (CLDN6), 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), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCA) M), ephrin type A receptor 2 (EPHA2), 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), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY) , L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), 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, neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;binds to a target antigen selected from the group consisting of placenta-specific 1 (PLAC1), predominantly 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), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1);

[0194] In various embodiments, the antigen-specific binding domain of the CAR binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligands.

[0195] In various embodiments, the antigen-specific binding domain of the CAR binds to a human BCMA polypeptide.

[0196] In various embodiments, the antigen-specific binding domain of a CCR is alpha folate receptor (FRα), α vβ6 integrin, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CCR1, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD135 (also known as fmc-like tyrosine kinase 3; FLT3), CD138, CD171, and carcinoma Childhood antigen (CEA), claudin-6 (CLDN6), 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), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCA) M), ephrin type A receptor 2 (EPHA2), 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), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY) , L1 cell adhesion molecule (L1-CAM), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), 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, neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;The antibody binds to a target antigen selected from the group consisting of placenta-specific 1 (PLAC1), predominantly 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), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), NKG2D ligand, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1). In various embodiments, the antigen-specific binding domain of the CCR binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand;

[0197] In various embodiments, the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide, an EGFRvIII polypeptide, a TAG72 polypeptide, or a CD20 polypeptide.

[0198] In various embodiments, the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide. In various embodiments, the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide. In various embodiments, the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide. In various embodiments, the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0199] In certain embodiments, the antigen-specific binding domain of the CCR is an scFv or a VHH.

[0200] In various embodiments, the CAR comprises a first antibody or antigen-specific binding fragment thereof, and the CCR further comprises a second antibody or antigen-specific binding fragment thereof. In some embodiments, the first and second antibodies or antigen-specific binding fragments bind to the same target antigen. In some embodiments, the first and second antibodies or antigen-specific binding fragments bind to different epitopes on the same target antigen. In some embodiments, the first and second antibodies or antigen-specific binding fragments bind to different target antigens.

[0201] In various embodiments, the antigen-specific binding domain of the CAR is a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand. and the antigen-specific binding domain of the CCR binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand.

[0202] In various embodiments, the antigen-specific binding domain of the CAR is selected from the group consisting of B-cell maturation antigen (BCMA), CD20, CD33, CD70, CD79a, CD79b, CD123, CD133, C-type lectin-like molecule-1 (CLL-1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR family including ErbB2 (HER2), MAGE-A4, MUC1, MUC16, cancer / testis antigen 1 (NY-ESO-1), melanoma The CCR binds to a target antigen selected from the group consisting of predominantly expressed antigen (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand, and the antigen-specific binding domain of the CCR binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), and TAG72.

[0203] In various embodiments, the antigen-specific binding domain of the CAR binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD33, CD79a, CD79b, C-type lectin-like molecule-1 (CLL-1), MAGE-A4, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and NKG2D ligand, and the antigen-specific binding domain of the CCR binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), and TAG72.

[0204] In various embodiments, the antigen-specific binding domain of the CAR binds to a target antigen that is B-cell maturation antigen (BCMA), CD33, CD79a, CD79b, C-type lectin-like molecule-1 (CLL-1), MAGE-A4, MUC16, cancer / testis antigen 1 (NY-ESO-1), predominantly expressed antigen in melanoma (PRAME), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tumor-associated glycoprotein 72 (TAG72), and an NKG2D ligand, and the antigen-specific binding domain of the CCR binds to a target antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD20, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), and TAG72.

[0205] In various embodiments, the antigen-specific binding domain of the CAR binds to a human BCMA polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0206] In various embodiments, the antigen-specific binding domain of the CAR binds to a human BCMA polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0207] In various embodiments, the antigen-specific binding domain of the CAR binds to a human BCMA polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0208] In various embodiments, the antigen-specific binding domain of the CAR binds to a human BCMA polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0209] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD33 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0210] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD33 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0211] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD33 polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0212] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD33 polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0213] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79a polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0214] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79a polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0215] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79a polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0216] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79a polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0217] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79b polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0218] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79b polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0219] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79b polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0220] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CD79b polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0221] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CLL-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0222] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CLL-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0223] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CLL-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0224] In various embodiments, the antigen-specific binding domain of the CAR binds to a human CLL-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0225] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MAGE-A4 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0226] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MAGE-A4 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0227] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MAGE-A4 polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0228] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MAGE-A4 polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0229] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MUC16 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0230] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MUC16 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0231] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MUC16 polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0232] In various embodiments, the antigen-specific binding domain of the CAR binds to a human MUC16 polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0233] In various embodiments, the antigen-specific binding domain of the CAR binds to a human NY-ESO-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0234] In various embodiments, the antigen-specific binding domain binds to a human NY-ESO-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0235] In various embodiments, the antigen-specific binding domain of the CAR binds to a human NY-ESO-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0236] In various embodiments, the antigen-specific binding domain of the CAR binds to a human NY-ESO-1 polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0237] In various embodiments, the antigen-specific binding domain of the CAR binds to a human PRAME polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0238] In various embodiments, the antigen-specific binding domain of the CAR binds to a human PRAME polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0239] In various embodiments, the antigen-specific binding domain of the CAR binds to a human PRAME polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0240] In various embodiments, the antigen-specific binding domain of the CAR binds to a human PRAME polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0241] In various embodiments, the antigen-specific binding domain of the CAR binds to a human ROR1 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0242] In various embodiments, the antigen-specific binding domain of the CAR binds to a human ROR1 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0243] In various embodiments, the antigen-specific binding domain of the CAR binds to a human ROR1 polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0244] In various embodiments, the antigen-specific binding domain of the CAR binds to a human ROR1 polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0245] In various embodiments, the antigen-specific binding domain of the CAR binds to a human TAG72 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0246] In various embodiments, the antigen-specific binding domain of the CAR binds to a human TAG72 polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0247] In various embodiments, the antigen-specific binding domain of the CAR binds to a human TAG72 polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0248] In various embodiments, the antigen-specific binding domain of the CAR binds to a human NKG2D ligand polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFR polypeptide.

[0249] In various embodiments, the antigen-specific binding domain of the CAR binds to a human NKG2D ligand polypeptide and the antigen-specific binding domain of the CCR binds to a human EGFRvIII polypeptide.

[0250] In various embodiments, the antigen-specific binding domain of the CAR binds to a human NKG2D ligand polypeptide and the antigen-specific binding domain of the CCR binds to a human CD20 polypeptide.

[0251] In various embodiments, the antigen-specific binding domain of the CAR binds to a human NKG2D ligand polypeptide and the antigen-specific binding domain of the CCR binds to a human TAG72 polypeptide.

[0252] The CAR and CCR designs contemplated in certain embodiments allow for improved expansion, long-term persistence, and cytotoxic properties in T cells expressing CARs and CCRs compared to unmodified T cells or T cells modified to express other CARs.

[0253] E. Polypeptides Various polypeptides, fusion polypeptides, and polypeptide variants are contemplated herein, including, but not limited to, CAR polypeptides, CCR polypeptides, fusion polypeptides, and fragments thereof. In certain embodiments, exemplary polypeptides contemplated herein include CCRs comprising modified hinge regions and associated fusion polypeptides. In particular, the polypeptides described herein exhibit reduced T cell signaling in the absence of CAR antigen compared to polypeptides or cells comprising unmodified CCR hinge regions. More specifically, the improved polypeptides described herein surprisingly reduce CCR-mediated CAR antigen-independent signaling while increasing T cell signaling in the presence of both CAR and CCR antigens.

[0254] "Polypeptide," "peptide," and "protein" are used interchangeably and follow their conventional meaning, i.e., as a sequence of amino acids, unless specified to the contrary. A polypeptide is not limited to a particular length, e.g., a polypeptide may include a full-length polypeptide or a polypeptide fragment, and a polypeptide may include one or more post-translational modifications of a polypeptide, such as, for example, glycosylation, acetylation, phosphorylation, and other modifications, both naturally occurring and non-naturally occurring, known in the art.

[0255] As used herein, "isolated polypeptide" and the like refers to the in vitro synthesis, 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., not substantially associated with substances in vivo. In certain embodiments, an isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0256] 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 naturally occurring or may be 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 the CAR and / or CCR by introducing one or more substitutions, deletions, additions, and / or insertions into the binding domain, hinge, TM domain, costimulatory signaling domain, or, if present, the primary signaling domain. 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, 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 cytolytic activity.

[0257] Polypeptides include "polypeptide fragments." Polypeptide fragments may be monomeric or multimeric and refer to polypeptides having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions or substitutions in naturally occurring or recombinantly produced polypeptides. Illustrative examples of biologically active polypeptide fragments include antibody fragments. As used herein, the terms "biologically active fragment" or "minimal biologically active fragment" refer 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 a naturally occurring polypeptide. In preferred embodiments, the biological activity is binding affinity for an epitope. In certain embodiments, a polypeptide fragment may comprise an amino acid chain at least 5 to about 500 amino acids in length. In certain embodiments, fragments are 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, or 450 amino acids in length. Particularly useful polypeptide fragments contain functional domains, including antigen-binding domains or fragments of antibodies.

[0258] The polypeptides may also be fused in-frame or attached to linkers or other sequences for ease of synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.

[0259] As noted above, in certain embodiments, polypeptides may be modified in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be generated by mutations in the DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, e.g., 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 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).

[0260] In certain embodiments, polypeptide variants contain 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. Modifications can be made to the polynucleotide and polypeptide structures contemplated in certain embodiments while still obtaining functional molecules that encode 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 of the codons in the encoding DNA sequence, for example, according to Table 1. [Table 1]

[0261] 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, Mac Vector, 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. Suitable conservative substitutions of amino acids in peptides or proteins 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, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0262] As discussed above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0263] Polypeptide variants also include glycosylated forms, aggregate conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., PEGylated molecules). Covalent variants can be prepared by linking functionalities to groups present in the amino acid chain or in the N- or C-terminal residues, as is known in the art. Variants also include allelic variants, species variants, and mutant proteins. Truncation or deletion of regions that do not affect the functional activity of the protein are also variants.

[0264] In certain embodiments, expression of the CAR and CCR in the same cell is desired. The polynucleotide sequences encoding the CAR and CCR may be separated by an IRES sequence, as discussed elsewhere herein.

[0265] In a preferred embodiment, fusion polypeptides are contemplated herein.

[0266] In certain preferred embodiments, the CAR and CCR may be expressed as a fusion polypeptide comprising one or more self-cleaving polypeptide sequences separating the CAR and CCR.

[0267] 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. Fusion polypeptides are typically linked C-terminus to N-terminus, but they may also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of a fusion protein may be in any order or a specified order. In one embodiment, the fusion protein comprises a CAR, a polypeptide cleavage signal, and a CCR. In another embodiment, the fusion protein comprises a CCR, a polypeptide cleavage signal, and a CAR.

[0268] In one embodiment, the fusion protein comprises a CAR, a polypeptide cleavage signal, and a CCR. In another embodiment, the fusion protein comprises a CCR, a polypeptide cleavage signal, and a CAR.

[0269] In certain embodiments, the fusion protein comprises a CAR comprising an scFv that binds to an antigen expressed in cancer, a hinge region, a transmembrane domain, a costimulatory domain, and a primary signaling domain, and a CCR comprising a polypeptide cleavage signal, an scFv that binds to the antigen, a modified hinge region, a transmembrane domain, and a costimulatory domain as described herein. Exemplary domains / regions may be selected from those described herein.

[0270] In a preferred embodiment, the fusion protein comprises a CAR comprising an scFv that binds to an antigen expressed in cancer, a CD8α hinge, a transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, and a CCR comprising a polypeptide cleavage signal, an scFv that binds to the antigen, a modified CD8α hinge, a transmembrane domain, and a CD28 costimulatory domain as described herein.

[0271] Examples of polypeptide cleavage signals include polypeptide cleavage recognition sites such as, for example, 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).

[0272] 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, biovirus 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 (Rice Tungro Spherical Virus) 3C-like protease, PYVF (Parsnip Yellow Virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, TEV (Tobacco Etch Virus) protease cleavage sites are preferred in one embodiment, e.g., EXXYXQ(G / S) (SEQ ID NO: 32), e.g., ENLYFQG (SEQ ID NO: 33) and ENLYFQS (SEQ ID NO: 34) (X represents any amino acid) (cleavage by TEV occurs between Q and G or Q and S).

[0273] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0274] Illustrative examples of 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.

[0275] 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 zosea signavirus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0276] Illustrative examples of 2A sites are provided in Table 2. [Table 2]

[0277] In a preferred embodiment, the fusion polypeptide comprises a CAR described herein, a T2A self-cleaving polypeptide, and a CCR described herein.

[0278] F. Polynucleotides In a preferred embodiment, a polynucleotide encoding one or more CAR polypeptides, CCR polypeptides, or fusion polypeptides comprising a CAR, a 2A peptide, and a CCR is provided. As used herein, the term "polynucleotide" or "nucleic acid" refers 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, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotide refers to a polymeric form of nucleotides 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 10,000, or at least 15,000 or more nucleotides in length, 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, e.g., 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.

[0279] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been created by 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.

[0280] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein, hi certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0281] In certain embodiments, a polynucleotide may be codon-optimized. As used herein, the term "codon optimization" refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of 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 tRNA; (v) variation of codons with the GC % either across the entire triplet or at one position in the triplet; (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 underlying the design of the codon substitution set; (x) synthetic variation of the codon set for each amino acid; and / or (xi) isolated removal of incorrect translation start positions.

[0282] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence or that hybridizes to a reference sequence under stringent conditions as defined herein. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides, compared to the reference polynucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, and the modified polynucleotide can retain the biological function or activity of the reference polynucleotide.

[0283] Polynucleotide variants include polynucleotide fragments that encode biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a polynucleotide fragment that encodes a polypeptide 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 a naturally occurring polypeptide. "A" refers to a polynucleotide fragment having a length of 8, 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 nucleotides. A polynucleotide fragment refers to a polynucleotide that encodes a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, a carboxy-terminal deletion, and / or an internal deletion or substitution of one or more amino acids of a naturally occurring or recombinantly produced polypeptide.

[0284] 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" may 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 to calculate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to calculate the percentage of sequence identity. Included are nucleotides and 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 described herein; typically, the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0285] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" comprises nucleotides and amino acid residues that are at least 12 monomer units in length, often 15-18 monomer units in length, and often at least 25 monomer units in length. Two polynucleotides may each contain (1) sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) sequence that diverges between the two polynucleotides. Sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least six contiguous positions, typically about 50 to about 100, more commonly about 100 to about 150, in which a sequence is compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window may be performed by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology across the comparison window) generated by any of a variety of selected methods. Reference may also be made to the BLAST family of programs, for example, as disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0286] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the 5' to 3' orientation or the 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the "sense," "plus," or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except uracil (U) in RNA replaces thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated the "template," "antisense," "minus," or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in the 3' to 5' direction or a 3' to 5' sequence written in the 5' to 3' direction.

[0287] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG3' is 3'TCAGTAC5'. The latter sequence is often written as the reverse complement, 5'CATGACT 3', with the 5' end to the left and the 3' end to the right. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," where only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids.

[0288] Furthermore, those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the polypeptides described herein or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are contemplated, and in certain embodiments, polynucleotides optimized for, for example, human and / or primate codon preferences are contemplated. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions, and / or substitutions of nucleotides.

[0289] 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 includes a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette includes 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 can include 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 into 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. In a preferred embodiment, the nucleic acid cassette encodes a CAR and / or a CCR. The cassette can be removed and inserted as a single unit into a plasmid or viral vector.

[0290] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide encoding a polypeptide, polypeptide variant, or fusion polypeptide. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, a polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or injury. Polynucleotides of interest, and the polypeptides encoded therefrom, include both polynucleotides encoding wild-type polypeptides and functional variants and fragments. In certain embodiments, functional variants have at least 80%, at least 90%, at least 95%, or at least 99% identity to a corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, functional variants or fragments have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

[0291] Polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences disclosed elsewhere herein or known in the art, 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., such that their overall length may vary considerably. Thus, polynucleotide fragments of almost any length may be used in particular embodiments, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0292] Polynucleotides may be prepared, manipulated, and / or expressed 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.

[0293] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as piggyBac, Sleeping Beauty, Mos1, Tc1 / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and derivatives thereof.

[0294] Additional illustrative 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.

[0295] Illustrative 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 viruses), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40).

[0296] Illustrative examples of expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, 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 of the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0297] 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 integrating into the host chromosomal DNA and without being gradually lost with the division of the host cell, and also means that the vector replicates extrachromosomally or episomally.

[0298] "Control elements" or "control 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 that interact with host cellular proteins to carry out transcription and translation. Such elements can 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.

[0299] In certain embodiments, vectors, including but not limited to expression vectors and viral vectors, contain exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous" regulatory sequence is a sequence that is naturally linked to a given gene in the genome. An "exogenous" regulatory sequence is one that is placed in juxtaposition to a gene by means of genetic engineering (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" regulatory sequence is an exogenous sequence that originates from a species different from the cell being genetically engineered.

[0300] 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 and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, promoters operated in mammalian cells include an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or another sequence found 70-80 bases upstream from the start of transcription is a CNCAAT region, where N can be any nucleotide.

[0301] The term "enhancer" refers to a DNA segment containing sequences that can provide transcriptional enhancement, and in some cases can function regardless of orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing sequences that can provide both promoter and enhancer function.

[0302] The term "operably linked" refers to a juxtaposition wherein the components described 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 (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0303] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that permits continuous or sequential transcription of an operably linked sequence. A constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a restricted variety of cell and tissue types, respectively.

[0304] Exemplary 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), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the 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 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 beta member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA These include 26 loci (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-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).

[0305] In one embodiment, the vector comprises the MNDU3 promoter.

[0306] In one embodiment, the vector comprises the EF1a promoter including the first intron of the human EF1a gene.

[0307] In one embodiment, the vector comprises the EF1a promoter lacking the first intron of the human EF1a gene.

[0308] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide, for example, conditional expression of a polynucleotide of interest, where expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide, or that causes increased or decreased expression of a polynucleotide encoded by the polynucleotide of interest.

[0309] Illustrative 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 hormones), 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 (WO 2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0310] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, a vector contains at least one (typically two) site for recombination mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes exclusive or integrative proteins, enzymes, cofactors, or associated proteins involved in a recombination reaction involving one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), and may be a wild-type protein (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or a mutant, derivative (e.g., a fusion protein comprising the recombination protein sequence or a fragment thereof), fragment, and variants thereof. Illustrative examples of recombinases suitable for use in certain embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0311] A vector may contain one or more recombination sites for any of a wide range of site-specific recombinases. It should be understood that target sites for site-specific recombinases are in addition to any sites required for integration of the vector, e.g., retroviral or lentiviral vectors. As used herein, the terms "recombination sequence," "recombination site," or "site-specific recombination site" refer to specific nucleic acid sequences that a recombinase recognizes and binds to.

[0312] For example, one recombination site for Cre recombinase is loxP, a 34-base pair sequence containing two 13-base pair inverted repeats (which remain as recombinase binding sites) flanking an 8-base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0313] Suitable recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0314] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ integrase, e.g., phi-c31. The phi-c31 SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). attB and attP are named for the binding sites for phage integrase on the bacterial and phage genomes, respectively, and both contain imperfect inverted repeats that can be bound by phi-c31 homodimers (Groth et al., 2000). The product sites, attL and attR, are also effectively inactive to phi-c31-mediated recombination (Belteki et al., 2003), rendering the reaction irreversible. To catalyze insertion, it has been found that attB-bearing DNA inserts more easily into genomic attP sites than attP sites insert into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy involves positioning an attP-bearing "docking site" at a defined locus by homologous recombination, which is then joined with an attB-bearing incoming sequence for insertion.

[0315] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates 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, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83 and Jackson and Kaminski, 1995. RNA 1(10):985-1000. In certain embodiments, a vector comprises one or more polynucleotides of interest encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0316] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes initial binding of mRNA to the small ribosomal subunit, increasing translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 57), 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). In certain embodiments, a vector comprises a polynucleotide having a consensus Kozak sequence encoding a desired polypeptide, e.g., a CAR.

[0317] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' of a polynucleotide encoding an expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNAs has two recognition elements flanking the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable element 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 SV40 poly(A), 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.

[0318] 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 aspects, the suicide gene is not immunogenic to the host or cells harboring the polynucleotide. Certain 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).

[0319] G. Vector In certain embodiments, one or more polynucleotides encoding a CAR and a CCR are introduced into a cell (e.g., an immune effector cell) by a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encoding a CAR and a CCR is introduced into a cell by a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encoding a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and an anti-CCR is introduced into a cell by a non-viral or viral vector.

[0320] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally inserted, for example, into a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication within a 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. In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a polycistronic message encoding a CAR and a CCR.

[0321] In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR.

[0322] Illustrative examples of non-viral vectors include, but are not limited to, mRNA, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0323] Illustrative examples of non-viral delivery of polynucleotides or vectors 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.

[0324] Illustrative 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.

[0325] In various embodiments, the polynucleotide is an mRNA that is introduced into a cell to transiently express a desired polypeptide. As used herein, "transient" refers to expression of a non-integrated transgene over a period of hours, days, or weeks, which is shorter than the period of expression of a polynucleotide when integrated into the genome of the cell or contained within a stable plasmid replicon.

[0326] In certain embodiments, the mRNA encoding the polypeptide is in vitro transcribed mRNA.As used herein, " in vitro transcribed RNA " refers to in vitro synthesized RNA, preferably mRNA.Generally, in vitro transcribed RNA is produced from an in vitro transcription vector.The in vitro transcription vector comprises the template used to produce in vitro transcribed RNA.

[0327] In certain embodiments, an mRNA may further comprise a 5' cap or modified 5' cap, and / or a poly(A) sequence. As used herein, a 5' cap (also referred to as an RNA cap, an RNA 7-methylguanosine cap, or an RNA m 7G A 5' cap (also called a cap) is a modified guanine nucleotide added to the "front" or 5' end of a eukaryotic messenger RNA immediately after transcription initiation. The 5' cap is linked to the first transcribed nucleotide and contains a terminal group that is recognized by the ribosome and protects against RNases. The capping moiety can be modified to modulate mRNA function, such as translation stability or efficiency. In certain embodiments, an mRNA contains a poly(A) sequence of about 50 to about 5,000 adenines. In one embodiment, an mRNA contains a poly(A) sequence of about 100 to about 1,000 bases, about 200 to about 500 bases, or about 300 to about 400 bases. In one embodiment, an mRNA contains a poly(A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1,000 bases or more. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functions such as localization, stability, or translation efficiency.

[0328] Viral vectors comprising polynucleotides contemplated in certain embodiments 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.

[0329] In one embodiment, a viral vector comprising a polynucleotide encoding an anti-CAR and a CCR is administered directly to an organism for in vivo cell transduction. In one embodiment, a viral vector comprising a polynucleotide encoding a CAR, a 2A self-cleaving polypeptide, and a CCR is administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA may be administered. Administration is by any route typically used to introduce molecules into final contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and while a particular composition can be administered using more than one route, certain routes may often provide a more immediate and effective response than other routes.

[0330] Illustrative examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0331] In various embodiments, one or more polynucleotides encoding a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR, are introduced into immune effector cells, e.g., T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) comprising the one or more polynucleotides.

[0332] AAV is a small (approximately 26 nm), replication-deficient, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of a 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, rAAV comprises ITR and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0333] 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 capsid sequences from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, an 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.

[0334] In some embodiments, methods of engineering and selection can be applied to AAV capsids to increase the likelihood that they will transduce cells of a subject.

[0335] The construction of rAAV vectors, their production, and purification are disclosed, for example, in U.S. Pat. Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058, and 8,784,799, each of which is incorporated by reference herein in its entirety.

[0336] In various embodiments, one or more polynucleotides encoding a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR, are introduced into immune effector cells by transducing the cells with a retrovirus, e.g., a lentivirus containing the one or more polynucleotides.

[0337] 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 the genomic DNA into the host genome. Exemplary 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 lentiviruses.

[0338] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV types 1 and 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.

[0339] In various embodiments, lentiviral vectors contemplated herein include 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 may optionally include a WPRE or HPRE, an insulator factor, a selectable marker, and a cell suicide gene, as otherwise discussed herein.

[0340] In certain embodiments, the lentiviral vectors contemplated herein may be integrative, non-integrative, or integration-defective lentiviruses. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that has an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. Integration-incompetent viral vectors are described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0341] Exemplary mutations in the HIV-1 pol gene that are suitable for reducing integrase activity include, but are not limited to, H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K15 9E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

[0342] In one embodiment, the HIV-1 integrase defective pol gene comprises D64V, D116I, D116A, E152G, or E152A mutations, D64V, D116I and E152G mutations, or D64V, D116A and E152A mutations.

[0343] In one embodiment, the HIV-1 integrase defective pol gene comprises a D64V mutation.

[0344] 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 includes the U3, R, and U5 regions.

[0345] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. In another embodiment, a lentiviral vector comprises a FLAP element with one or more mutations in the cPPT and / or CTS element. In yet another embodiment, a lentiviral vector comprises either a cPPT or a CTS element. In yet another embodiment, a lentiviral vector does not comprise a cPPT or a CTS element.

[0346] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located in the retroviral genome that is required for the insertion of viral RNA into the viral capsid or particle. See, e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0347] The term "export element" 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 elements 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.

[0348] In certain embodiments, expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vector. Various posttranscriptional regulatory elements, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886), the posttranscriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864), and similar (Liu et al., 1995, Genes Dev., 9:1766), can increase expression of heterologous nucleic acids in proteins.

[0349] Lentiviral vectors preferably include several safety enhancements as a result of modifications to the LTR. A "self-inactivating" (SIN) vector refers to a replication-deficient vector, e.g., in which the right (3') LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to inhibit viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter that directs transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0350] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus having a viral envelope protein substituted with a viral envelope protein of another virus having favorable properties. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells, because the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4 + This is to make them presentation cells.

[0351] In certain embodiments, 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.

[0352] According to certain embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from a lentivirus, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or that numerous combined substitutions and modifications of a particular lentiviral sequence can be accommodated without impairing the ability of the transfer vector 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 which can be adapted to produce the viral vectors or transfer plasmids contemplated herein.

[0353] In various embodiments, one or more polynucleotides encoding a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR, are introduced into immune effector cells by transducing the cells with an adenovirus containing the one or more polynucleotides.

[0354] Adenovirus-based vectors are capable of extremely high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. These vectors can be produced in large quantities using a relatively simple system. Most adenovirus vectors are engineered so that a transgene replaces the Ad E1a, E1b, and / or E3 genes, and the replication-deficient vector is then propagated in human 293 cells, which supply the deleted gene function in trans. Ad vectors can transduce multiple tissue types in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle. Traditional Ad vectors have a high carrying capacity.

[0355] The generation and propagation of current replication-deficient adenoviral vectors can utilize a unique helper cell line called 293, which was transformed from human embryonic kidney cells with Ad5 DNA fragments and constitutively expresses the E1 protein (Graham et al., 1977). Because the E3 region is unnecessary for the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors utilize 293 cells to deliver foreign DNA in either the E1, D3, or both regions (Graham & Prevec, 1991). Adenoviral vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies of administering recombinant adenoviruses to different tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). An example of the use of Ad vectors in clinical trials involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0356] In various embodiments, one or more polynucleotides encoding a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR, are introduced into immune effector cells by transducing the cells with a herpes simplex virus, e.g., HSV-1, HSV-2, containing one or more polynucleotides.

[0357] 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 is defective in one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-deficient. Most replication-deficient HSV vectors contain deletions to remove one or more immediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may be defective in an immediate-early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. Advantages of HSV vectors are their ability to enter latency, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate up to 25 kb of exogenous DNA. 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, each of which is incorporated herein by reference in its entirety.

[0358] H. Genetically modified cells In various embodiments, cells are provided that are genetically modified to express the CARs and CCRs contemplated herein for use in treating cancer. 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. As used herein, the term "gene therapy" refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell to restore, correct, or modify the expression of a gene or to express a fusion protein encoding a CAR and CCR, or a CAR, a 2A self-cleaving polypeptide, and a CCR.

[0359] In certain embodiments, fusion proteins encoding the CAR and CCR, or the CAR, 2A self-cleaving polypeptide, and CCR contemplated herein, are introduced into and expressed in immune effector cells to redirect their specificity to a target antigen of interest. 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 (HTL; CD4+ T cells). In certain embodiments, the cells comprise αβ T cells. In certain embodiments, the cells comprise γδ T cells. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0360] Immune effector cells may be autologous or non-autologous (non-self, e.g., allogeneic, syngeneic, or xenogeneic). 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 preferred embodiments, the cells are autologous.

[0361] Exemplary immune effector cells for use with CARs and CCRs contemplated in certain embodiments include T lymphocytes. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+ CD8+ T cells, CD4- CD8- T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells (TN), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF).

[0362] As will be appreciated by those skilled in the art, other cells can also be used as immune effector cells bearing the CARs and CCRs contemplated herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, and 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, for example, 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.

[0363] As used herein, the term "CD34+ cells" refers to cells that express the 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 T cell entry into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSCs), which, when administered to a patient, differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0364] Methods for generating immune effector cells expressing a CAR and a CCR contemplated herein are provided in certain embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a polycistronic message encoding the CAR and CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR contemplated herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells may then be readministered directly to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express the CAR and CCR. In this regard, the immune effector cells may be cultured before and / or after genetic modification (i.e., transduced or transfected to express the CAR and CCR contemplated herein).

[0365] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of immune effector cells as described herein, hi certain embodiments, the modified immune effector cells comprise T cells.

[0366] In certain embodiments, PBMCs may be directly genetically modified to express a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR, using the methods contemplated herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be preserved into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0367] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated, in the case of progenitor cells) in vitro before being genetically modified. In certain embodiments, immune effector cells, such as T cells, are genetically modified with a chimeric antigen receptor contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR), and then activated and expanded in vitro. In various embodiments, the T cells can be activated and expanded before or after genetic modification using methods such as those described in, for example, 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, 6,867,041, and U.S. Patent Application Publication No. 2006 / 0121005.

[0368] In one embodiment, CD34+ cells are transduced with a nucleic acid construct contemplated herein. In certain embodiments, the transduced CD34+ cells are differentiated in vivo into mature immune effector cells after administration to a subject, generally the subject from whom the cells were originally isolated. In another embodiment, the CD34+ cells may be stimulated in vitro before exposure to or after being genetically modified with one or more of the following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF), megakaryocyte growth and differentiation factor (TPO), IL-3, and IL-6, according to previously described methods (Asheuer et al., 2004; Imren, et al., 2004).

[0369] In certain embodiments, a population of modified immune effector cells for cancer treatment comprises a CAR and a CCR as contemplated herein. For example, the population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient (autologous donor) diagnosed with a B-cell malignancy as described herein. The PBMCs form a heterogeneous population of T lymphocytes that can be CD4+, CD8+, or CD4+ and CD8+.

[0370] PBMCs may also contain other cytotoxic lymphocytes, such as NK cells or NKT cells. Expression vectors carrying coding sequences for CARs and CCRs contemplated in certain embodiments are introduced into a human donor T cell, NK cell, or NKT cell population. In certain embodiments, successfully transduced T cells carrying the expression vectors are sorted using flow cytometry to isolate CD3-positive T cells, which can then be further expanded to increase the number of these CAR- and CCR-expressing T cells, in addition to cell activation using anti-CD3 and / or anti-CD28 antibodies and IL-2, or any other method known in the art as described elsewhere herein. Standard procedures are used to cryopreserve T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal-derived products, such as fetal calf serum and fetal bovine serum. Because a heterogeneous population of PBMCs is genetically modified, the resulting transduced cells are a heterogeneous population of modified cells that contain polynucleotides encoding a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR as contemplated herein.

[0371] In further embodiments, a mixture of, for example, one, two, three, four, five, or more different expression vectors can be used in genetically modifying a donor population of immune effector cells, each vector encoding a different chimeric antigen receptor protein contemplated herein, and the resulting modified immune effector cells form a mixed population of modified cells.

[0372] IT cell manufacturing method In various embodiments, the genetically modified T cells are expanded by contact with an agent that stimulates a CD3 TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.

[0373] In certain embodiments, PBMCs, or isolated T cells, are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies bound to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15.

[0374] In certain embodiments, PBMCs, or isolated T cells, are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies bound to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15, and / or a PI3K inhibitor.

[0375] In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell manufacturing methods contemplated herein. PBMCs form a heterogeneous population of T lymphocytes, which can be CD4+, CD8+, or CD4+ and CD8+, and may include other mononuclear cells such as monocytes, B cells, NK cells, and NKT cells. An expression vector containing a polynucleotide encoding a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR as contemplated in certain embodiments, is introduced into a population of human donor T cells, NK cells, or NKT cells. In certain embodiments, successfully transduced T cells carrying the expression vector are sorted using flow cytometry to isolate CD3-positive T cells, which can then be further expanded to expand the number of these modified T cells in addition to cell activation using anti-CD3 and / or anti-CD28 antibodies, and IL-2, IL-7, and / or IL-15.

[0376] To achieve a sufficient therapeutic dose of a T cell composition, the T cells often undergo one or more rounds of stimulation, activation, and / or expansion. T cells can generally be activated and expanded using methods as described, for example, in U.S. Pat. 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, each of which is incorporated herein by reference in its entirety.

[0377] In preferred embodiments, T cells produced by the methods contemplated herein provide improved adoptive immunotherapy compositions. Without wishing to be bound by any particular theory, it is believed that T cell compositions produced by the methods of certain embodiments contemplated herein have superior properties, including increased survival, proliferation in the relative absence of differentiation, and persistence in vivo. In one embodiment, a method of producing T cells includes contacting the cells with one or more agents that modulate the PI3K cell signaling pathway.

[0378] In certain embodiments, T cells are produced by stimulating T cells to activate and proliferate in the presence of one or more stimulatory signals and optionally a PI3K inhibitor.

[0379] Then, the T cells 、C AR and CCR Polycistronic message encodingor a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR. In one embodiment, T cells are modified by transducing the T cells with a viral vector comprising a polycistronic message encoding a CAR and a CCR, or a fusion protein encoding a CAR, a 2A self-cleaving polypeptide, and a CCR as contemplated herein. In certain embodiments, T cells are modified prior to stimulation and activation. In other embodiments, T cells are modified after stimulation and activation. In certain embodiments, T cells are modified within 12 hours, 24 hours, 36 hours, or 48 hours of stimulation and activation. In certain embodiments, T cells are activated, stimulated, and / or modified in the presence or absence of a PI3K inhibitor.

[0380] After the T cells are transduced and / or edited, the cells are expanded in culture. The T cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion. In certain embodiments, the T cells are expanded in culture.

[0381] In various embodiments, the T cell composition is produced in the presence of a PI3K inhibitor. Without wishing to be bound by any particular theory, treating or contacting T cells with one or more inhibitors of the PI3K pathway during any one, any combination, or all of the stimulation, activation, and / or expansion phases of the production process will preferentially expand young T cells, thereby producing a superior therapeutic T cell composition.

[0382] As used herein, the term "PI3K inhibitor" refers to a nucleic acid, peptide, or small organic molecule that binds to and inhibits at least one activity of PI3K. PI3K proteins can be divided into three classes: class 1 PI3K, class 2 PI3K, and class 3 PI3K. Class 1 PI3K exists as a heterodimer consisting of one of four p110 catalytic subunits (p110α, p110β, p110δ, and p110γ) and one of two regulatory subunit families. PI3K inhibitors preferably target class 1 PI3K inhibitors. In one embodiment, the PI3K inhibitor exhibits selectivity for one or more isoforms of class 1 PI3K inhibitors (i.e., selectivity for p110α, p110β, p110δ, and p110γ or one or more of p110α, p110β, p110δ, and p110γ). In another embodiment, the PI3K inhibitor does not exhibit isoform selectivity and is considered a “pan-PI3K inhibitor.” In one embodiment, the PI3K inhibitor competes with ATP for binding to the PI3K catalytic domain.

[0383] In certain embodiments, PI3K inhibitors can target, for example, PI3K as well as additional proteins in the PI3K-AKT-mTOR pathway. In certain embodiments, PI3K inhibitors that target both mTOR and PI3K may be referred to as either mTOR inhibitors or PI3K inhibitors. PI3K inhibitors that target only PI3K may be referred to as selective PI3K inhibitors. In one embodiment, a selective PI3K inhibitor may be understood to refer to an agent that exhibits a 50% inhibitory concentration with respect to PI3K that is at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more lower than the IC50 of the inhibitor with respect to mTOR and / or other proteins in the pathway.

[0384] In certain embodiments, exemplary PI3K inhibitors inhibit PI3K with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, or 1 μM or less. In one embodiment, the PI3K inhibitor inhibits PI3K with an IC50 of about 2 nM to about 100 nM, more preferably about 2 nM to about 50 nM, even more preferably about 2 nM to about 15 nM.

[0385] Examples of PI3K inhibitors suitable for use in the T cell manufacturing methods contemplated in certain embodiments include, but are not limited to, BKM120 (Class 1 PI3K inhibitor, Novartis), XL147 (Class 1 PI3K inhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline), and PX-866 (Class 1 PI3K inhibitor, p110α, p110β, and p110γ isoforms, Oncothyreon).

[0386] Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145.

[0387] Further illustrative examples of pan-PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

[0388] In a preferred embodiment, the PI3K inhibitor is ZSTK474.

[0389] In certain embodiments, methods are provided for increasing the proliferation of T cells expressing an engineered T cell receptor. Such methods may include, for example, harvesting a source of T cells from a subject, stimulating and activating the T cells, modifying the T cells to express a CAR and a CCR, and expanding the T cells in culture, wherein the T cells are produced in the presence of one or more PI3K inhibitors at any one of more steps in the production process.

[0390] The manufacturing methods contemplated herein may further include cryopreserving the modified T cells for storage and / or preparation for use in a human subject. In one embodiment, the method of preserving genetically modified immune effector cells includes cryopreserving the immune effector cells such that the cells remain viable upon thawing. The T cells are cryopreserved such that the cells remain viable upon thawing. If desired, the cryopreserved, transformed cells can be thawed, propagated, and expanded to larger numbers of such cells. As used herein, "cryopreservation" refers to preserving cells by cooling to a subzero temperature, such as (typically) 77 K or -196°C (the boiling point of liquid nitrogen). Cryopreservatives are often used at subzero temperatures to prevent damage to the preserved cells from freezing at low temperatures or heating to room temperature. The cryopreservative and optimal cooling rate can protect against cell damage. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183:1394-1395; Ashwood-Smith, Nature, 1961; 190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85:576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196:48). The preferred cooling rate is 1°C to 3°C per minute. After at least 2 hours, the T cells will reach a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, such as in a long-term cryogenic storage vessel.

[0391] J. Compositions and Formulations In certain embodiments, formulations of pharmaceutically acceptable carrier solutions are well known in the art, as are the development of suitable 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 skilled in the art will understand that certain embodiments contemplated herein may include other formulations, such as, for example, formulations that are well known in the pharmaceutical arts and are described in, for example, Remington: The Science and Practice of Pharmacy, volume I and volume II, 22nd Edition. Edited by Loyd V. Allen Jr., Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0392] Compositions contemplated herein may comprise one or more of the CAR polypeptides, CCR polypeptides, polynucleotides, vectors comprising the same, genetically modified immune effector cells, etc. contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. In preferred embodiments, the composition comprises one or more cells modified to express a CAR and a CCR; a fusion protein encoding a CAR, a self-cleaving polypeptide, and a CCR; or a fusion protein encoding a CAR, a self-cleaving polypeptide, and a CCR.

[0393] 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 conjunction with other agents as well, 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. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient and one or more cells modified to express a CAR and a CCR, or a fusion protein encoding a CAR and a CCR.

[0394] As used herein, the phrase "pharmaceutically acceptable" is employed to refer to those compounds, materials, 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.

[0395] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for human or veterinary use. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and any other compatible substance used in pharmaceutical formulations.

[0396] In certain embodiments, the compositions comprise an amount of CAR- and CCR-expressing immune effector cells contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective quantity" of genetically modified therapeutic cells, such as T cells, to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0397] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0398] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition 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.

[0399] Generally, it can be said that pharmaceutical compositions containing T cells described herein can be administered at doses of 10 to 10 cells / kg body weight, preferably 10 to 10 cells / kg body weight, including all integers within that range. The number of cells will depend on the intended end use of the composition and the type of cells included therein. For the uses presented herein, cells are generally administered in sub-liter volumes, which may be 500 mL or less, or even 250 mL or less, or 100 mL or less. Thus, desired cell densities are often greater than 10 cells / ml, typically greater than 10 cells / ml, and typically greater than 10 cells / ml. Clinically relevant immune cell numbers may be divided into multiple infusions, cumulatively resulting in 10, 10, 10, 10, 10, 10, 10, or 10 cells or more. In some embodiments, small numbers of cells may be administered, in the range of 10 per kilogram (10 to 10 per patient), particularly because all infused cells are redirected to a specific target antigen. The composition may be administered multiple times at doses within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy. 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.

[0400] Generally, compositions comprising activated and expanded cells as described herein may be utilized for the treatment and prevention of diseases occurring in immunocompromised individuals. In certain embodiments, compositions comprising immune effector cells modified to express a CAR and a CCR, or fusion proteins encoding a CAR and a CCR as contemplated herein, are used to treat cancer. The modified immune effector cells may be administered alone or in a pharmaceutical composition in combination with a carrier, diluent, excipient, and / or other components, such as IL-2 or other cytokines or other cell populations. In certain embodiments, a pharmaceutical composition comprises an amount of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0401] Pharmaceutical compositions comprising fusion proteins encoding CARs and CCRs, such as immune effector cell populations or T cells modified to express CARs and CCRs, can include a buffer such as neutral buffered saline or phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions are preferably formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0402] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic saline, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0403] In one embodiment, the T cell composition contemplated herein is formulated in a pharmaceutically acceptable cell culture medium. Such a composition is suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0404] Serum-free media have several advantages over serum-containing media, including a simpler and more defined composition, reduced contaminant load, elimination of potential sources of infectious agents, and reduced cost. In various embodiments, serum-free media may be 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.

[0405] 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.

[0406] In a preferred embodiment, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising PlasmaLyte A.

[0407] In another preferred embodiment, the compositions comprising immune effector cells contemplated herein are formulated in a solution comprising 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 certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0408] In a more preferred embodiment, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.

[0409] In certain embodiments, the compositions comprise an effective amount of immune effector cells modified to express a CAR and a CCR, or a fusion protein encoding a CAR and a CCR, alone or in combination with one or more therapeutic agents. Thus, the CAR-expressing immune effector cell compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, or photodynamic therapy. The compositions may also be administered in combination with antibiotics. Such therapeutic agents may be art-recognized as standard treatments for certain disease states described herein, such as certain cancers. Examples of contemplated therapeutic agents include, in certain embodiments, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active adjunctive agents.

[0410] In certain embodiments, compositions comprising immune effector cells modified to express a CAR and a CCR, or fusion proteins encoding a CAR and a CCR, can be administered in conjunction with any number of chemotherapeutic agents.

[0411] A variety of other therapeutic agents may be used in conjunction with the compositions described herein, hi one embodiment, a composition comprising immune effector cells, a CAR and a CCR, or a fusion protein encoding a CAR and a CCR is administered with an anti-inflammatory agent.

[0412] In one embodiment, the composition comprising immune effector cells, CARs and CCRs, or fusion proteins encoding them, is administered in conjunction with a therapeutic antibody. Illustrative examples of therapeutic antibodies suitable for use in combination with CAR-modified T cells contemplated in certain embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, crizotinib, daratumumab, duligotumab, dacetuzumab, and dalotuzumab. , durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0413] K. Treatment method The genetically modified immune effector cells expressing the CARs and CCRs contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of cancer, or for use in the prevention, treatment, or amelioration of at least one symptom associated with cancer.

[0414] In various embodiments, the genetically modified immune effector cells contemplated herein provide improved methods of adoptive immunotherapy used to increase cytotoxicity at cancer cells in a subject or to reduce the number of cancer cells in a subject.

[0415] In certain embodiments, the specificity of primary immune effector cells is redirected to cells that express a particular antigen, e.g., cancer cells, by genetically modifying the primary immune effector cells with a CAR and / or CCR as contemplated herein. In various embodiments, viral vectors are used to genetically modify immune effector cells with specific polynucleotides encoding the CAR and CCR.

[0416] In one embodiment, a type of cell therapy is provided in which T cells are genetically modified to express CARs and / or CCRs that target specific antigens expressed on cancer cells, and the T cells are then infused into a recipient in need thereof. The infused cells can kill disease-causing cells in the recipient. Unlike antibody therapy, T cell therapy can replicate in vivo, resulting in long-term persistence that can provide sustained cancer treatment.

[0417] In one embodiment, T cells expressing a CAR and a CCR can undergo robust in vivo T cell expansion and can persist for long periods of time, hi another embodiment, T cells expressing a CAR and a CCR evolve into specific memory T cells or stem cell memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

[0418] In certain embodiments, compositions comprising immune effector cells expressing CARs and CCRs contemplated herein are used to treat condition-associated specific antigen-expressing cancer cells or cancer stem cells.

[0419] Illustrative examples of conditions that can be treated, prevented, or ameliorated using immune effector cells expressing CARs and CCRs are contemplated in certain embodiments.

[0420] In certain embodiments, compositions comprising T cells expressing a CAR and a CCR contemplated herein are used to treat osteosarcoma or Ewing's sarcoma.

[0421] In certain embodiments, compositions comprising T cells expressing a CAR and a CCR contemplated herein are used to treat liquid or hematological cancers.

[0422] In certain embodiments, the liquid or hematological cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

[0423] In certain embodiments, the liquid or hematological cancer is selected from 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 lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, and leukemia. The tumor is selected from the group consisting of: diffuse large B-cell lymphoma (SLL), follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary 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.

[0424] In certain embodiments, the liquid or hematological cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

[0425] In a preferred embodiment, the liquid or blood cancer is DLBCL.

[0426] In a preferred embodiment, the liquid or hematological cancer is relapsed / refractory DLBCL.

[0427] In certain embodiments, methods are provided that include administering a therapeutically effective amount of immune effector cells expressing a CAR and CCR contemplated herein, or a composition comprising same, to a patient in need thereof, alone or in combination with one or more therapeutic agents. In certain embodiments, the cells are used to treat a patient at risk of developing a pathology associated with cancer cells. Thus, in certain embodiments, a method of treating or preventing or ameliorating at least one symptom of cancer includes administering a therapeutically effective amount of modified T cells expressing a CAR and CCR contemplated herein to a subject in need thereof.

[0428] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting symptoms of a disease, injury, or condition that can be treated with the gene therapy vectors, cell-based therapeutic agents, and methods contemplated elsewhere herein. In preferred embodiments, a subject includes any animal exhibiting symptoms of a cancer-related disease, injury, or condition that can be treated with the gene therapy vectors, cell-based therapeutic agents, and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory 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. Exemplary subjects include human patients with cancer or diagnosed with, at risk for, or having cancer.

[0429] As used herein, the term "patient" refers to a subject diagnosed with a particular disease, injury, or condition that can be treated using the gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein.

[0430] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include either a reduction in the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0431] As used herein, "prevent" and similar terms, e.g., "prevented," "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 prior to the onset or recurrence of the disease or condition.

[0432] As used herein, "amelioration of at least one symptom of" refers to a decrease in one or more symptoms of the disease or condition for which the subject is being treated. In certain embodiments, the disease or condition for which treatment is 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).

[0433] "Enhance," or "promote," or "increase," or "expand" generally refer to the ability of the compositions contemplated herein, e.g., genetically modified T cells expressing CARs and CCRs, to produce, induce, 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, and / or cancer cell killing capacity, and are 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 in 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.

[0434] "Decrease," or "lower," or "reduce," or "reduce," or "attenuate" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a physiological response (i.e., a downstream effect) that is smaller than the response caused by a vehicle or control molecule / composition. A "decrease" or "reduced" amount 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 caused by the vehicle, a control composition, or a response in a particular cell line.

[0435] "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 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.

[0436] In one embodiment, a method for treating cancer in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising a genetically modified immune effector cell as contemplated herein. The amount and frequency of administration will depend on factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined through clinical trials.

[0437] In one embodiment, the amount of immune effector cells, e.g., T cells expressing a CAR and a CCR, in the composition administered to a subject is at least 0.1 x 10 5 cells, at least 0.5 x 10 5 cells, at least 1 x 10 5 Cells, at least 5 x 10 5 cells, at least 1 x 10 6 cells, at least 0.5 x 10 7 cells, at least 1 x 10 7 cells, at least 0.5 x 10 8 cells, at least 1 x 10 8 cells, at least 0.5 x 10 9 cells, at least 1 x 10 9 Cells, at least 2 x 10 9 Cells, at least 3 x 10 9 Cells, at least 4 x 10 9 Cells, at least 5 x 10 9 cells, or at least 1 x 10 10 It is a cell.

[0438] In certain embodiments, about 1×107 T cells ~ approx. 1 x 10 9 T cells, approximately 2 x 10 7 T cells ~ approx. 0.9×10 9 T cells, approximately 3 x 10 7 T cells ~ approx. 0.8×10 9 T cells, approximately 4 x 10 7 T cells ~ approx. 0.7×10 9 T cells, approximately 5 x 10 7 T cells ~ approx. 0.6×10 9 T cells, or approximately 5 × 10 7 T cells ~ approx. 0.5×10 9 The T cells are administered to the subject.

[0439] In one embodiment, the amount of immune effector cells, e.g., T cells expressing a CAR and a CCR, in the composition administered to a subject is at least 0.1 x 10 4 At least 0.5 × 10 cells / kg body weight 4 At least 1 x 10 cells / kg body weight 4 At least 5 x 10 cells / kg body weight 4 At least 1 x 10 cells / kg body weight 5 At least 0.5 × 10 cells / kg body weight 6 At least 1 x 10 cells / kg body weight 6 At least 0.5 × 10 cells / kg body weight 7 At least 1 x 10 cells / kg body weight 7 At least 0.5 × 10 cells / kg body weight 8 At least 1 x 10 cells / kg body weight 8 At least 2 x 10 cells / kg body weight 8 cells / kg body weight, at least 3 x 10 cells / kg body weight, at least 4 x 10 8 At least 5 x 10 cells / kg body weight 8 At least 1 x 10 cells / kg body weight 9 cells / kg body weight.

[0440] In certain embodiments, about 1×10 6 T cells / kg body weight ~1×10 8 T cells / kg body weight, approximately 2×10 6 T cells / kg body weight ~0.9×10 8T cells / kg body weight, 3×10 6 T cells / kg body weight ~ approx. 0.8×10 8 T cells / kg body weight, approximately 4×10 6 T cells / kg body weight ~ approx. 0.7×10 8 T cells / kg body weight, approximately 5×10 6 T cells / kg body weight ~ approx. 0.6×10 8 T cells / kg body weight, or approximately 5 x 10 6 T cells / kg body weight ~ approx. 0.5×10 8 T cells / kg body weight are administered to the subject.

[0441] Those skilled in the art will recognize that multiple administrations of the compositions contemplated herein may be required 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.

[0442] In certain embodiments, it may be desirable to administer activated immune effector cells to a subject, then withdraw blood (or perform apheresis) from the subject, activate the immune effector cells therefrom, and reinfuse these activated and expanded immune effector cells back into the patient. This process may be performed multiple times, every few weeks. In certain embodiments, immune effector cells may be activated from a blood withdrawal of 10 cc to 400 cc. In certain embodiments, immune effector cells are activated from a blood withdrawal 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 a multiple blood withdrawal / multiple reinfusion protocol may aid in the selection of certain populations of immune effector cells.

[0443] Administration of the compositions contemplated herein can be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, transplantation, or implantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, typically by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0444] In one embodiment, an effective amount of the composition is administered to a subject in need thereof to increase a cellular immune response to a B cell-related condition in the subject. The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. A humoral immune response, mediated primarily by helper T cells, which can activate B cells and thus lead to antibody production, may also be elicited. Various techniques can be used to analyze the type of immune response induced by the composition, and are fully explained in the art, for example, in *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.

[0445] In one embodiment, a method is provided for treating a subject diagnosed with cancer, comprising removing immune effector cells from the subject, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a CAR and CCR as contemplated herein, thereby producing 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.

[0446] In certain embodiments, a method of stimulating an immune effector cell-mediated immunomodulatory response against a target cell population in a subject is provided, the method comprising administering to the subject a population of immune effector cells that express a nucleic acid construct encoding a CAR and a CCR.

[0447] Methods of administering cell compositions contemplated in certain embodiments include any method effective to result in the reintroduction of ex vivo genetically modified immune effector cells, either directly expressing a CAR and CCR in the subject, or reintroducing genetically modified precursors of immune effector cells that differentiate into mature immune effector cells expressing a CAR and CCR when introduced into the subject. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct contemplated herein and returning the transduced cells to the subject.

[0448] L. Sequence Listing [Table 3-1] [Table 3-2]

[0449] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0450] 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 offered for purposes of illustration only, and not for purposes of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results. [Example]

[0451] Example 1 Activation of T cell signaling in the presence of CCR antigen alone T cells were modified using lentiviral transduction to deliver constructs encoding either CAR molecules or CAR and CCR (CAR / CCR) molecules. The CAR construct expressed an anti-BCMA scFv fused to 4-1BB and CD3z signaling domains (BBz). The CAR+CCR construct expressed a BBz anti-BCMA CAR and an anti-EGFR scFv fused to the CD28 signaling domain CCR, which formed a CCR. The CAR and CCR sequences within the CAR+CCR construct were separated using a 2A ribosomal skipping element.

[0452] The modified T cells were then co-cultured with the EGFR+ HT-1080 tumor cell line, which lacks endogenous BCMA expression. In Figure 1A, culture supernatants were collected after 24 hours, and IFNγ production was assessed by Luminex assay. As shown in Figure 1A, unmodified T cells (UTD) or anti-BCMA CAR T cells failed to secrete IFNγ in the absence of antigen exposure. On the other hand, CAR+ CCR T cells produced detectable amounts of IFNγ despite being exposed only to EGFR.

[0453] Similarly, in Figure 1B, only CAR+ CCR T cells were able to kill EGFR+ tumor targets in a dose-dependent manner during a standard 4-hour in vitro killing / cytotoxicity assay.

[0454] Example 2 CCR-dependent signaling reduces tumor volume Immunodeficient NSG mice were subcutaneously implanted with human A549 tumor cells, which endogenously express EGFR but lack BCMA expression. Approximately 20 days after implantation, the mice were treated with unmodified T cells (UTD), anti-BCMA CAR T cells, or anti-BCMA CAR + anti-EGFR CCR T cells, and tumor volume was assessed. As shown in Figure 2, neither untransduced nor anti-BCMA CAR T cells were able to control tumor growth, and the mice eventually succumbed to tumor formation. Alternatively, mice treated with anti-BCMA CAR + anti-EGFR CCR T cells showed complete tumor regression over the course of the study.

[0455] Without wishing to be bound by any particular theory, Figure 3 shows three exemplary, non-limiting models that explain how T cells endowed with a CAR and a CCR can respond to tumor target cells expressing the CCR antigen while lacking the CAR antigen. The CCR can bind to the CCR antigen and then transduce signals independent of the CAR molecule (Figure 3, left panel). Antigen-independent basal CAR signaling via CD3z is enhanced by the CCR costimulatory signal to a level that is now detectable by standard in vitro experiments and appears as a tumor control in vivo (Figure 3, left panel). Molecular interactions between the CAR molecule and the CCR molecule exist such that binding of the CCR via the CCR antigen initiates signaling and induces T cell activation.

[0456] Example 3 CAR and CCR hinge mutations regulate CAR T cell signaling The experiment was designed to test whether any one of the four cysteines in the CD8a hinge region of the CAR and CCR (two in the CAR and two in the CCR) is involved in the molecular interaction between the two different proteins. Six different CAR+CCR constructs that varied in their use of cysteines and serine at positions 289 and 306 (corresponding to positions 27 and 44 of SEQ ID NO:2) of the CD8a hinge of either the CAR or CCR were generated and transduced into T cells. C1 is the parental CAR+CCR construct that utilizes cysteines at all four positions. C2 contains two serines in the CAR and two cysteines in the CCR. C3 contains two cysteines in the CAR and two serines in the CCR. C4 contains a serine at position 27 of SEQ ID NO:2 and a cysteine ​​at position 44 of SEQ ID NO:2 in the CAR, and a cysteine ​​at position 27 of SEQ ID NO:2 and a serine at position 44 of SEQ ID NO:2 in the CCR. C5 contains a cysteine ​​at position 27 of SEQ ID NO:2 and a serine at position 44 of SEQ ID NO:2 for CAR, and a serine at position 27 of SEQ ID NO:2 and a cysteine ​​at position 44 of SEQ ID NO:2 for CCR. C6 utilizes serine at all four positions.

[0457] T cells were modified with the six constructs described above, as well as two controls: an anti-BCMA BBz CAR and an anti-BCMA CAR variant in which cysteine ​​was replaced with serine at positions 27 and 44 of SEQ ID NO:2. Each T cell condition was then co-cultured with various tumor cells expressing either EGFR alone (A549 and HT-1080) or both BCMA and EGFR (A549.BCMA and HT-1080.BCMA). IFNγ production was assessed 24 hours later. As shown in Figures 4A-4C, control anti-BCMA CAR T cells produced cytokines only when cultured in the presence of BCMA-expressing tumor cells. Introducing serine at positions 27 and 44 of SEQ ID NO:2 in the anti-BCMA CAR resulted in reduced IFNγ production when cultured with BCMA-expressing tumor cells. T cells engineered to express the parental CAR+CCR construct (C1) responded to tumor cells expressing either BCMA and EGFR, or EGFR alone. None of the other CAR+CCR variants used in these experiments were able to retain full reactivity against tumor cells expressing EGFR alone. Interestingly, construct C4 was the only variant that exhibited partial reactivity against EGFR-expressing tumor cells. Together, these data demonstrate the importance of the hinge cysteine ​​in mediating the CAR / CCR molecular interaction.

[0458] Example 4 Design and expression of CAR and CCR fusion polypeptides. Figure 5A shows the modular protein domains used in the construction of an anti-EGFR CCR, an anti-EGFR CCR containing two cysteine-to-serine mutations, and the corresponding CAR+CCR constructs (C1 and C3, respectively). C3 is a construct encoding an anti-BCMA CAR and an anti-EGFR CCR with serine instead of the cysteines in the CD8a hinge corresponding to positions 27 and 44 of SEQ ID NO: 2. C1 is the parental CAR+CCR construct with all four cysteines present in the CAR and CCR.

[0459] Each of these constructs was transduced into T cells from three different donors, and flow cytometry was used to assess cell surface expression of both the CAR and CCR, compared to a UTD control that does not express either the CAR or CCR. As shown in Figure 5B, the presence of serine at positions 27 and 44 of SEQ ID NO:2 does not affect the overall transduction efficiency of the CCR-only construct. Similarly, T cells transduced with the C1 or C3 constructs result in expression of both the CAR and CCR on the cell surface. Despite the presence of serine at both positions in the CCR, there is no effect on transduction efficiency.

[0460] Example 5 CCR hinge double mutation reduces T cell activation in the absence of CAR antigen T cells transduced with the constructs described in Example 4 were co-cultured with A549 or HT-1080 tumor cells expressing only antigens against CCR, EGFR, and IFNγ production was assessed 24 hours later. Figure 6 shows the responsiveness of CAR+CCR T cells to tumor cells expressing EGFR but lacking BCMA, suggesting that these T cells targeted not only BCMA+ tumors but also tumors expressing only EGFR. Introducing serine at positions 27 and 44 of SEQ ID NO:2 in the CCR reduced this reactivity to levels consistent with the negative control, indicating that cysteines within the CCR hinge are important in mediating reactivity to EGFR-single-positive tumor cells.

[0461] These results are further supported by the data in Figures 7A-7D and 8A-8D, which show that CAR+CCR T cells targeting BCMA and E...

Claims

1. 1. A fusion polypeptide comprising: a) a chimeric antigen receptor (CAR) comprising a first hinge region; b) a polypeptide cleavage signal; and c) a chimeric costimulatory receptor (CCR) comprising a second hinge region; a fusion polypeptide, wherein the first hinge region and / or the second hinge region comprise one or more cysteine ​​substitutions with another amino acid, and the one or more cysteine ​​substitutions in the first hinge region and / or the second hinge region reduce CAR / CCR association in the absence of a CAR antigen compared to a CCR comprising a wild-type second hinge region and / or a CAR comprising a wild-type first hinge region.

2. 2. The fusion polypeptide of claim 1, wherein the one or more cysteine ​​substitutions in the first hinge region and / or the second hinge region eliminate CAR / CCR association in the presence of the CCR antigen and the absence of a CAR antigen, compared to a CCR comprising a wild-type second hinge region and a CAR comprising a wild-type first hinge region.

3. 3. The fusion polypeptide of claim 1 or 2, wherein the one or more cysteine ​​substitutions in the second hinge region further reduce CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising an unmodified second hinge region.

4. 4. The fusion polypeptide of any one of claims 1 to 3, wherein the first hinge region further reduces CCR antigen-mediated stimulation of T cell signaling in the absence of CAR antigen compared to a CAR comprising an unmodified second hinge region.

5. The fusion polypeptide of any one of claims 1 to 4, wherein the one or more cysteine ​​residues in the first and / or second hinge region are substituted with serine or alanine.

6. (i) the first hinge region comprises a hinge region, or a functional fragment thereof, selected from the group consisting of a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge, a PD-1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2; and / or (ii) the second hinge region comprises a hinge region, or a functional fragment thereof, selected from the group consisting of a CD8α hinge, a CD4 hinge, a CD28 hinge, a CD7 hinge, a CD152 hinge, a PD-1 hinge, an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, an IgG4 hinge, an IgG1 hinge / CH2 / CH3, an IgG1 hinge / CH3 / hinge / M1, an IgG4 hinge / CH2 / CH3, and an IgG4 hinge / CH2.

6. The fusion polypeptide of claim 1, wherein the hinge region comprises a hinge region, or a functional fragment thereof, selected from the group consisting of CD8α hinge, CD4 hinge, CD28 hinge, CD7 hinge, CD152 hinge, PD-1 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, IgG4 hinge, IgG1 hinge / CH2 / CH3, IgG1 hinge / CH3 / hinge / M1, IgG4 hinge / CH2 / CH3, and IgG4 hinge / CH2.

7. The fusion polypeptide according to any one of claims 1 to 6, wherein the first and second hinge regions are CD8α hinge regions or functional fragments thereof.

8. (a) the first or second CD8α hinge region comprises an amino acid substitution at position 27 and / or 44 of SEQ ID NO: 2; (b) the first and / or second hinge region comprises an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity thereto; (c) the first and / or second hinge region comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity thereto; and / or (d) the first and / or second hinge region comprises an amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% identity thereto; A fusion polypeptide according to any one of claims 1 to 7.

9. a) the CAR is i) a first antibody or an antigen-specific binding fragment thereof; ii) a first transmembrane domain; iii) a first intracellular costimulatory domain, and / or iv) Primary Signaling Domain Further comprising: b) the CCR is i) a second antibody or antigen-specific binding fragment thereof; ii) a second transmembrane domain, and iii) a second intracellular costimulatory domain The fusion polypeptide of any one of claims 1 to 8, further comprising:

10. 10. The fusion polypeptide of claim 1, wherein the second hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:

5.

11. A polynucleotide encoding the fusion polypeptide of any one of claims 1 to 10.

12. A vector comprising the polynucleotide of claim 11.

13. The vector of claim 12 , wherein the vector is a lentiviral vector.

14. A cell expressing (i) the fusion polypeptide or (ii) the CAR and CCR according to any one of claims 1 to 10.

15. The cells (a) immune effector cells; (b) T cells, (c) CD3 ＋ , CD4 ＋ , and / or CD8 ＋ cell, (d) cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or helper T cells; (e) αβ-T cells or γδ-T cells, (f) natural killer (NK) cells or natural killer T (NKT) cells, or (g) macrophages The cell of claim 14,

16. 15. A composition comprising the cells of claim 14 and a pharmaceutically acceptable carrier.

17. 17. The composition of claim 16 for use in treating cancer in a subject in need thereof.

18. 18. The composition of claim 17, wherein the cancer is a solid cancer or a hematological malignancy.

19. 19. The composition of claim 17 or claim 18, wherein the cancer is non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

20. 20. The composition of claim 19, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL).

21. 17. The composition of claim 16, for use in reducing CCR antigen-mediated stimulation of T cell signaling in the absence of a CAR antigen compared to a CCR comprising a hinge domain without the substitution, said use comprising: a) obtaining a CAR and a CCR polypeptide, each having a hinge domain; b) substituting one or more cysteine ​​residues in the CCR hinge domain with alternative residues, thereby producing a modified CCR; c) expressing the CAR and modified CCR in a cell. composition.

22. 22. The composition of claim 21 , wherein the CAR hinge region comprises one or more cysteines substituted with a different amino acid.

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