Fusion Polypeptides for Immunotherapy - Patent application

Abstract

The presently disclosed subject matter provides methods and compositions for enhancing immune responses to tumor and pathogen antigens. The presently disclosed subject matter relates to fusion polypeptides that can be expressed in cells (e.g., immune response cells containing antigen-recognizing receptors) to improve the activity and / or efficiency of the cells. In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory ligand and the intracellular domain of a costimulatory molecule. The presently disclosed subject matter provides fusion polypeptides that can improve the activity and efficacy of immunotherapy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 876,338, filed July 19, 2019, the contents of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing This application was submitted via EFS-Web in ASCII format and contains a Sequence Listing, which is hereby incorporated by reference in its entirety. The ASCII copy was created on July 20, 2020, is named "072734_1102_ST25.txt" and is 126,019 bytes in size.

[0003] 1. Introduction The presently disclosed subject matter provides fusion polypeptides that can improve activity and efficacy for immunotherapy. [Background technology]

[0004] 2. Background of the invention Cell-based immunotherapy is a potentially curative therapy for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens through the introduction of genetic material that encodes natural or modified T cell receptors (TCRs) specific for selected antigens or synthetic receptors for antigens, called chimeric antigen receptors (CARs). Patient engineered CAR T cells have demonstrated remarkable efficacy against a range of liquid and solid malignancies. However, treatment failure and relapse occur in the majority of patients. Thus, improved immunotherapies remain necessary. Summary of the Invention [Means for solving the problem]

[0005] 3. Overview of the Invention The subject matter of the present disclosure provides a fusion polypeptide that can enhance the activity and efficacy of immunotherapy (e.g., T cell immunotherapy).In certain embodiments, the fusion polypeptide comprises a) the extracellular domain and transmembrane domain of a costimulatory ligand, and b) the intracellular domain of a first costimulatory molecule.

[0006] In certain embodiments, the costimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, and immunoglobulin (Ig) superfamily members, and combinations thereof. In certain embodiments, the TNF family members are selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. In certain embodiments, the Ig superfamily members are selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof. In certain embodiments, the costimulatory ligand is CD80. In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:1, or a functional fragment thereof. In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2. In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof. In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0007] In certain embodiments, the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. In certain embodiments, the first costimulatory molecule is 4-1BB. In certain embodiments, the intracellular domain of 4-1BB comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:3. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 or a functional fragment thereof. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3.

[0008] In certain embodiments, the costimulatory ligand is CD80 and the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0009] In certain embodiments, the costimulatory ligand is CD80 and the first costimulatory molecule is 4-1BB. In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:4.

[0010] In certain embodiments, the fusion polypeptide further comprises an intracellular domain of a second costimulatory molecule. In certain embodiments, the second costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. In certain embodiments, the second costimulatory molecule is CD28. In certain embodiments, the first costimulatory molecule is 4-1BB and the second costimulatory molecule is CD28. In certain embodiments, the intracellular domain of CD28 comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:5. In certain embodiments, the intracellular domain of CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5 or a functional fragment thereof. In certain embodiments, the intracellular domain of CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5.

[0011] In certain embodiments, the costimulatory ligand is CD80, the first costimulatory molecule is 4-1BB, and the second costimulatory molecule is CD28. In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:6. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:6.

[0012] In certain embodiments, the fusion polypeptide is capable of stimulating a cell comprising an antigen-recognizing receptor. In certain embodiments, the fusion polypeptide is capable of enhancing an activity of a cell comprising an antigen-recognizing receptor. In certain embodiments, the activity includes cytotoxicity, cell proliferation, cell survival, and combinations thereof.

[0013] In certain embodiments, the fusion polypeptide further comprises a signaling domain of a cytokine receptor. In certain embodiments, the cytokine receptor is selected from the group consisting of CD121a, CDw121b, IL-18Ra, IL18Rb, CD122, CD25, CD132, CD124, CD213a13, CD127, IL-9R, IL15Ra, CDw125, CDw131, CD126, CD130, IL11Ra, Cd114, CD212, CD4, CDw217, CD118, and CDw119.

[0014] The presently disclosed subject matter further provides nucleic acid molecules encoding the fusion polypeptides disclosed herein and vectors comprising the nucleic acid molecules.

[0015] The presently disclosed subject matter also provides a cell comprising a fusion polypeptide, a nucleic acid molecule, or a vector disclosed herein.

[0016] In certain embodiments, the cell further comprises an antigen-recognizing receptor. In certain embodiments, the antigen-recognizing receptor is selected from the group consisting of a chimeric antigen receptor (CAR), a T cell receptor (TCR), and a TCR-like fusion molecule.

[0017] In certain embodiments, the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is CD19, carbonic anhydrase IX (CAlX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell junction antigen (ECA), CD4, CD5, CD6, CD7, CD8, CD9 ...11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, Adhesion molecule (EpCAM), receptor tyrosine protein kinases Erb-B2, Erb-B3, Erb-B4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES0-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME Selected from the group consisting of CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB. In certain embodiments, the tumor antigen is CD19.

[0018] In certain embodiments, the antigen recognition receptor is exogenous or endogenous. In certain embodiments, the antigen recognition receptor is recombinantly expressed. In certain embodiments, the antigen recognition receptor is expressed from a vector.

[0019] In certain embodiments, the cell is a cell of lymphoid lineage. In certain embodiments, the cell is a cell of myeloid lineage. In certain embodiments, the cell of lymphoid lineage is selected from the group consisting of T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is derived from an induced pluripotent stem cell. In certain embodiments, the T cell is CD8 + In certain embodiments, the CD8 + The T cells are CD4-independent. In certain embodiments, the T cells are selected from the group consisting of cytotoxic T lymphocytes (CTLs), regulatory T cells, gamma delta T cells, tumor infiltrating lymphocytes (TILs), and natural killer T (NKT) cells. In certain embodiments, the fusion polypeptide and the antigen-recognizing receptor are integrated at a locus in the genome of the T cells. In certain embodiments, the locus is selected from the group consisting of the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. In certain embodiments, the locus is the TRAC locus. In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). In certain embodiments, the expression of the antigen-recognizing receptor and the fusion polypeptide is under the control of an endogenous promoter. In certain embodiments, the endogenous promoter is selected from the group consisting of the endogenous TRAC promoter, the endogenous TRBC promoter, the endogenous TRDC promoter, and the endogenous TRGC promoter. In certain embodiments, the endogenous promoter is the TRAC promoter.

[0020] In certain embodiments, the fusion polypeptide and the antigen recognition receptor are integrated into a locus that encodes an immune inhibitory molecule. In certain embodiments, the immune inhibitory molecule is selected from the group consisting of CTLA-4, PD-1, LAG3, BTLA, B7-1, B7-H1, B7-H3, B7-H4, TIM3, SHP-1, SHP-2, TIGIT, CD160, and LAIR1.

[0021] In certain embodiments, the cells are autologous. In certain embodiments, the cells are allogeneic.

[0022] The subject of the present disclosure provides a composition comprising the cell disclosed herein.In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma-ceutically acceptable excipient.In certain embodiments, the pharmaceutical composition is for treating and / or preventing neoplasm, autoimmune disease, and / or infectious disease.In certain embodiments, the neoplasm is cancer.

[0023] In certain embodiments, the composition further comprises a regulatory factor capable of regulating or modulating the expression and / or activity of the fusion polypeptide.

[0024] In certain embodiments, the regulatory factor is selected from the group consisting of a promoter capable of controlling expression of the fusion polypeptide, a molecule capable of regulating or modulating the expression and / or activity of a costimulatory ligand, and a molecule capable of regulating or modulating the expression and activity of a costimulatory molecule.

[0025] In certain embodiments, the molecule capable of regulating or modulating the expression and / or activity of a costimulatory ligand is selected from the group consisting of an antibody that binds to a costimulatory ligand and a fusion protein that binds to a costimulatory ligand and regulates or modulates the expression and / or activity of the costimulatory ligand.

[0026] In certain embodiments, the modulator is an anti-CD80 antibody and the fusion polypeptide comprises the extracellular and transmembrane domains of CD80.

[0027] In certain embodiments, the modulator is a fusion protein that binds to CD80 and modulates the activity of CD80, and the fusion polypeptide comprises the extracellular domain and transmembrane domain of CD80. In certain embodiments, the fusion protein comprises a CTLA-4 fragment that binds to CD80. In certain embodiments, the CTLA-4 fragment that binds to CD80 is abatacept or belatacept.

[0028] In certain embodiments, the molecule capable of regulating or modulating the expression and / or activity of a costimulatory molecule is selected from the group consisting of an antibody that binds to a costimulatory molecule and a fusion protein that binds to a costimulatory molecule and regulates or modulates the expression and / or activity of a costimulatory molecule.

[0029] The subject of the present disclosure further provides a composition comprising: a) the fusion polypeptide disclosed herein; and b) an antigen-recognition receptor that binds to antigen.In certain embodiments, the fusion polypeptide is operably linked to a first promoter.In certain embodiments, the antigen-recognition receptor is operably linked to a second promoter.

[0030] In addition, the subject matter of the present disclosure further provides a composition comprising: a) a first polynucleotide that encodes the fusion polypeptide disclosed herein; and b) a second polynucleotide that encodes an antigen-recognition receptor that binds to an antigen.In certain embodiments, the nucleic acid composition further comprises a first promoter that is operably linked to the fusion polypeptide.In certain embodiments, the nucleic acid composition further comprises a second promoter that is operably linked to the antigen-recognition receptor.

[0031] In certain embodiments, one or both of the first and second promoters are endogenous or exogenous.In certain embodiments, the exogenous promoter is selected from the group consisting of elongation factor (EF)-1 promoter, cytomegalovirus immediate early promoter (CMV) promoter, simian virus 40 early promoter (SV40) promoter, phosphoglycerate kinase (PGK) promoter, and metallothionein promoter.In certain embodiments, one or both of the first and second promoters are inducible promoters.In certain embodiments, the inducible promoter is selected from NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

[0032] The subject matter of the present disclosure further provides a cell comprising the composition disclosed herein or the nucleic acid composition disclosed herein. In certain embodiments, the composition or nucleic acid composition is integrated at a locus in the genome of a T cell. In certain embodiments, the locus is selected from the group consisting of the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. In certain embodiments, the composition or nucleic acid composition is integrated at a locus that encodes an immune inhibitory molecule. In certain embodiments, the immune inhibitory molecule is selected from the group consisting of CTLA-4, PD-1, LAG3, BTLA, B7-1, B7-H1, B7-H3, B7-H4, TIM3, SHP-1, SHP-2, TIGIT, CD160, and LAIR1.

[0033] In certain embodiments, the cells, compositions, or nucleic acid compositions disclosed herein are for use in reducing tumor burden, treating and / or preventing a neoplasm, treating and / or preventing an autoimmune disease, treating and / or preventing a pathogen infection (e.g., an infectious disease), and / or treating a subject with recurrence of a neoplasm.

[0034] The presently disclosed subject matter further provides a method for producing a cell. In certain embodiments, the method comprises introducing into the cell a nucleic acid molecule, a nucleic acid composition, or a vector disclosed herein.

[0035] The subject matter of the present disclosure provides a kit comprising the nucleic acid molecule, vector, cell, composition or nucleic acid composition disclosed herein.In certain embodiments, the kit further comprises written instructions for treating and / or preventing neoplasms, pathogen infections (e.g., infectious diseases), and / or autoimmune diseases.

[0036] The subject matter of the present disclosure provides methods of reducing tumor burden in a subject, treating a subject with recurrence of a neoplasm (e.g., cancer), treating and / or preventing a neoplasm (e.g., cancer), treating and / or preventing a pathogen infection (e.g., infectious disease), and / or treating and / or preventing an autoimmune disease. In certain embodiments, the method comprises administering to the subject an effective amount of a cell, composition, or nucleic acid composition disclosed herein. In certain embodiments, the method reduces tumor cell number, reduces tumor size, and / or eradicates a tumor in the subject. In certain embodiments, the neoplasm or tumor is selected from the group consisting of a hematological cancer and a solid tumor. In certain embodiments, the neoplasm or tumor is a solid tumor. In certain embodiments, the hematological cancer is selected from the group consisting of B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

[0037] In certain embodiments, the autoimmune and infectious diseases are selected from the group consisting of rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, ulcerative colitis, psoriasis, psoriatic arthritis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, systemic sclerosis, asthma, transplant organ rejection, transplant-related diseases or conditions, Takayasu's arteritis, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Behcet's syndrome, Wegener's granulomatosis, ANCA-vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, vasculitis of connective tissue disease, Henoch-Schonlein purpura, and / or other inflammatory diseases. purpura), cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, primary vasculitis of the CNS, thromboangiitis obliterans, paraneoplastic arteritis, myelodysplastic syndrome, erythema elevatus diurnalis, amyloidosis, autoimmune myositis, Guillain-Barre syndrome, histiocytosis, atopic dermatitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, Sjögren's syndrome, myelofibrosis, chronic inflammatory demyelinating polyneuropathy autoimmune pulmonary inflammation, autoimmune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atopic thyroiditis, Graves' disease, autoimmune polyglandular syndrome, autoimmune Addison's syndrome, and myasthenia gravis.

[0038] In certain embodiments, the method further comprises administering to the subject a regulator capable of regulating or modulating the expression and / or activity of the fusion polypeptide. In certain embodiments, the regulator is selected from the group consisting of a promoter capable of controlling the expression of the fusion polypeptide, a molecule capable of regulating or modulating the expression and / or activity of a costimulatory ligand, and a molecule capable of regulating or modulating the expression and / or activity of a costimulatory molecule. In certain embodiments, the molecule capable of regulating or modulating the expression and / or activity of a costimulatory ligand is selected from the group consisting of an antibody that binds to a costimulatory ligand, and a fusion protein that binds to a costimulatory ligand and regulates or modulates the expression, activity of the costimulatory ligand. In certain embodiments, the regulator is an anti-CD80 antibody, and the fusion polypeptide comprises the extracellular and transmembrane domains of CD80. In certain embodiments, the regulator is a fusion protein that binds to CD80 and modulates the activity of CD80, and the fusion polypeptide comprises the extracellular and transmembrane domains of CD80. In certain embodiments, the fusion protein is a CTLA4 fragment that binds to CD80. In certain embodiments, the CTLA4 fragment that binds to CD80 is abatacept or belatacept.

[0039] In certain embodiments, the molecule capable of regulating or modulating the expression and / or activity of a costimulatory molecule is selected from the group consisting of an antibody that binds to a costimulatory molecule and a fusion protein that binds to a costimulatory molecule and regulates or modulates the expression and / or activity of a costimulatory molecule.

[0040] In certain embodiments, the regulator can deplete cells. In certain embodiments, the regulator can reduce or eliminate one or more side effects associated with the administration of cells. In certain embodiments, the one or more side effects are selected from the group consisting of off-tumor target effects, cytokine release syndrome, neurotoxicity, and combinations thereof.

[0041] In certain embodiments, the method further comprises administering to the subject a checkpoint immune blockade. In certain embodiments, the checkpoint immune blockade is selected from the group consisting of an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-TIM3 antibody, and combinations thereof. In certain embodiments, the checkpoint immune blockade is an anti-PD-1 antibody.

[0042] The following detailed description, provided by way of example but not intended to limit the subject matter of the present disclosure to the specific embodiments described, can be understood in conjunction with the accompanying drawings. 4. Brief description of the drawings [Brief description of the drawings]

[0043] [Figure 1] FIG. 1 shows a cell according to certain embodiments of the presently disclosed subject matter.

[0044] [Figure 2-1]Figures 2A-2H show a universal CD80-4-1BB fusion polypeptide to boost anti-tumor T cell responses. Figure 2A shows FACS profiles showing expression of 1928z CAR with (right) or without (left) CD80-4-1BB fusion polypeptide. T cells are transduced with gamma retroviral vectors to stably express 1928z and CD80 / 4-1BB fusion polypeptide. Figure 2B shows Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells treated with 105 19-28-z CAR T cells co-expressing exogenous CD80 molecules, exogenous 4-1BBL molecules or CD80 / 4-1BB fusion polypeptide. Figure 2C shows FACS profiles showing expression of 1928z-1xx CAR T cells with (right) or without (left) CD80 / 4-1BB fusion polypeptide. 1928z-1xx and CD80 / 4-1BB fusion polypeptides were targeted to the TRAC locus and expressed under the control of the endogenous TRAC promoter. Figure 2D shows Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells treated with 2x104 19-28-z-1xx CAR T cells with or without co-expression of CD80 / 4-1BB fusion polypeptide. Figure 2E shows FACS profiles showing expression of 19-HIT with (middle and right) or without (left) CD80 / 4-1BB fusion polypeptide. Figure 2E left and Figure 2E middle are for 19-HIT and CD80 / 4-1BB fusion polypeptides targeted to the TRAC locus and expressed under the control of the endogenous TRAC promoter. Figure 2E right is for 19-HIT T cells transduced with SFG gamma-retroviral vector to express high levels of CD80 / 4-1BB fusion polypeptide. Figure 2F shows Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells treated with 105 19-HIT T cells with or without CD80 / 4-1BB fusion polypeptide. Figure 2G shows FACS profiles showing expression of NY-ESO-1 TCR with (right) or without (left) CD80 / 4-1BB fusion polypeptide.Gamma retroviral vectors encoding NY-ESO-1 TCR or CD80 / 4-1BB fusion polypeptides were used. Figure 2H shows Kaplan-Meier survival analysis of mice bearing the SK-MEL-23 melanoma cell line expressing the HLAA2.1 / NYESO complex treated with 2x106 NY-ESO-1 TCR T cells with or without CD80 / 4-1BB fusion polypeptide. P values ​​were determined by the log-rank Mantel-Cox test. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above.

[0045] [Diagram 3] Figures 3A and 3B show the maintenance of antitumor benefit of CD80-4-1BB in the presence of PDL-1 inhibitory ligands. Figure 3A shows Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells overexpressing PDL-1 molecules treated with 5x104 19-CAR T cells with or without CD80 / 4-1BB fusion polypeptide. Figure 3B shows Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells overexpressing PDL-1 molecules treated with 105 HIT T cells with or without CD80 / 4-1BB fusion polypeptide. P values ​​were determined by log-rank Mantel-Cox test.

[0046] [Figure 4]Figures 4A and 4B show that CD80-4-1BB expression levels govern antitumor response efficiency. Figure 4A shows FACS profiles showing expression of CD80 / 4-1BB fusion polypeptide under the control of the endogenous TRAC locus or under the control of exogenous EF1a or PGK promoters targeted to the TRBC locus. In all conditions, 1928z-1xx was expressed under the control of the endogenous TRAC promoter. T cells were bead-sorted after transduction based on CD80 expression using an anti-CD80 antibody. The mean fluorescence intensity of CD80 is shown. Figure 4B shows the total flux signal of NALM6 tumor cells expressing firefly luciferase 15 days after CAR T cell injection. P values ​​were determined by Mann-Whitney t-test.

[0047] [Diagram 5] Figure 5 shows Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells treated with 2.5x104 1928z-1xx CAR T cells with CD80 / 4-1BB fusion polypeptide. Endogenous 4-1BB gene knockout was obtained by electroporation of protein CAS9 and specific gRNA. Disruption of 4-1BB was achieved in 68% of injected CAR T cells. TRBC gRNA was used as a control. P values ​​were determined by log-rank Mantel-Cox test.

[0048] [Figure 6] Figure 6 shows Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells treated with 2.5x104 CAR T cells. Endogenous PD-1 gene knockout was obtained by electroporation of the protein CAS9 and specific gRNA. Disruption of PD-1 is achieved in 55% of the injected CAR T cells. TRBC gRNA is used as a control. P values ​​were determined by the log-rank Mantel-Cox test.

[0049] [Figure 7]Figures 7A and 7B show Kaplan-Meier survival analysis of mice bearing NALM6 CD19 leukemia cells treated with 8x105 1928z CAR CD8+ T cells with CD80 / 4-1BB fusion polypeptide (Figure 7A) or 4x105 1928z CAR CD8+ T cells with CD80 / 4-1BB fusion polypeptide (Figure 7B). P values ​​were determined by the log-rank Mantel-Cox test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] 5. Detailed Description of the Invention The subject matter of the present disclosure provides fusion polypeptides that can enhance the activity and / or efficacy of immunotherapy (e.g., T cell immunotherapy). The fusion polypeptides can enhance the activity and / or efficacy of cells (e.g., T cells or NK cells) that contain antigen-recognizing receptors (e.g., CAR, TCR, or TCR-like fusion molecules). The subject matter of the present disclosure also provides methods of using such fusion polypeptides to induce and / or enhance the immune response of cells to target antigens, and / or to treat and / or prevent neoplasms or other diseases / disorders (e.g., autoimmune diseases and infectious diseases), for example, where an increase in antigen-specific immune response is desired. The subject matter of the present disclosure is based, at least in part, on the discovery that the fusion polypeptides disclosed herein can enhance the activity (e.g., cytotoxicity) of cells that contain antigen-recognizing receptors (e.g., CAR, TCR, or TCR-like fusion molecules).

[0051] Non-limiting embodiments of the present disclosure are described herein and in the Examples.

[0052] For purposes of clarity and not limitation of the disclosure, the detailed description is divided into the following subsections: 5.1. Definition; 5.2. Fusion Polypeptides; 5.3.Cell; 5.4. Antigen recognition receptors; 5.5. Compositions and Vectors; 5.6. Polypeptides and analogs; 5.7. Administration; 5.8. Formulation; 5.9. Method of treatment; and 5.10.Kit 5.1.Definition

[0053] Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The following references provide those skilled in the art with general definitions of many of the terms used in the subject matter of this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0054] As used herein, the term "about" or "approximately" refers to within an acceptable error range of a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or above 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, such as up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or 2-fold of a value.

[0055] As used herein, a "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of lymphocytes to an antigen. In certain embodiments, a costimulatory molecule can provide optimal lymphocyte activation.

[0056] As used herein, a "costimulatory ligand" refers to a molecule that, upon binding to its receptor (e.g., a costimulatory molecule), produces a costimulatory response, e.g., an intracellular response that results in stimulation when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen.

[0057] "Immune response cell" refers to a cell or its precursor or progeny that functions in immune response. In certain embodiments, the immune response cell is a cell of lymphoid lineage. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, the immune response cell is a cell of myeloid lineage.

[0058] "Activating immune response cells" refers to the induction of signal transduction or change in protein expression in cells that results in the initiation of an immune response. For example, when CD3 chains assemble in response to binding of a ligand and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade is produced. In certain embodiments, when endogenous TCR or exogenous CAR binds to an antigen, the formation of an immune synapse occurs, which includes the assembly of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the bound receptor. This assembly of membrane-bound signal transduction molecules can phosphorylate the ITAM motifs contained within the CD3 chains. This phosphorylation then initiates the T cell activation pathway, ultimately activating transcription factors, such as NF-κB and AP-1. These transcription factors induce global gene expression in T cells to increase IL-2 production for proliferation and expression of master regulator T cell proteins to initiate a T cell-mediated immune response.

[0059] "Stimulating immune response cells" refers to signals that result in a robust and sustained immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-lBB), OX40, CD40, and ICOS. Receiving multiple stimulatory signals may be important to initiate a robust and long-lasting T cell-mediated immune response. T cells can be rapidly inhibited and become refractory to antigens. The effects of these costimulatory signals may vary, but they generally result in increased gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that robustly respond to antigens for complete and sustained eradication.

[0060] The term "antigen recognition receptor," as used herein, refers to a receptor that is capable of activating an immune or immune response cell (e.g., a T cell) in response to its binding to an antigen.

[0061] As used herein, the term "antibody" refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are routinely used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fe fragment of intact antibodies, are cleared more rapidly from the circulation and may have less non-specific tissue binding of intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983). As used herein, antibodies include whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies. In certain embodiments, antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H) and heavy chain constant (C H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (herein referred to as V L ) and light chain constant C L The light chain constant region consists of one domain, C L It consists of V H and V L The region can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0062] As used herein, "CDR" is defined as the amino acid sequence of the complementarity determining region of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th US Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the antibody to bind to an antigen or epitope. In certain embodiments, the CDR region is represented using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0063] As used herein, the term "single-chain variable fragment" or "scFv" refers to a covalently linked V H ::V L Heterodimeric immunoglobulin heavy chains (V H ) and light chain (V L ) is a fusion protein of the variable region of H and V L are joined directly or by a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids), H N-terminus of V L and the C-terminus of V H The C-terminus of V LThe linker is usually rich in glycine for flexibility and serine or threonine for solubility. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies are synthesized by combining the V and VFv polypeptides as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). H and V L It can be expressed from a nucleic acid containing the coding sequence. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 136(8):2252-61). 2(10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0064] As used herein, the term "affinity" refers to a measure of binding strength. Affinity may depend on the tightness of the stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term "affinity" also includes "avidity", which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody to an antigen are known in the art and include, but are not limited to, various antigen binding experiments, such as functional assays (e.g., flow cytometry assays).

[0065] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule that comprises an extracellular antigen binding domain fused to an intracellular signaling domain and a transmembrane domain that is capable of activating or stimulating an immune cell or immune response cell. In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. The scFv can be derived from a fusion of the variable heavy and light regions of an antibody. Alternatively or additionally, the scFv can be derived from a Fab' (not derived from an antibody, e.g., obtained from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity for the antigen.

[0066] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule that encodes a polypeptide of interest or a fragment thereof. Such a nucleic acid molecule need not be 100% homologous or identical to an endogenous nucleic acid sequence, but may exhibit substantial identity. A polynucleotide that has "substantial identity" or "substantial homology" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to pairing to form a double-stranded molecule with a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0067] For example, stringent salt concentrations are usually less than about 750 mM NaCl and 75 mM trisodium citrate, such as less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, such as at least about 50% formamide. Stringent temperature conditions usually include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are achieved by combining these various conditions as needed. In certain embodiments, hybridization occurs at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In certain embodiments, hybridization occurs at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In certain embodiments, hybridization occurs at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations on these conditions will be readily apparent to those of skill in the art.

[0068] For most applications, the washing step after hybridization also varies in stringency. Stringency conditions of washing can be defined by salt concentration and by temperature. As mentioned above, stringency of washing can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentration for washing step can be less than about 30 mM NaCl and 3 mM trisodium citrate, such as less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for washing step usually include a temperature of at least about 25°C, at least about 42°C, or at least about 68°C. In certain embodiments, washing step occurs at 25°C, 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, washing step occurs at 42°C, 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the washing step occurs at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0069] "Substantially identical" or "substantially homologous" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the amino acid or nucleic acid sequence used for comparison.

[0070] Sequence identity can be measured by using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program may be used with a probability score between e-3 and e-100, which indicates closely related sequences.

[0071] By "analog" is meant a structurally related polypeptide or nucleic acid molecule that has a function of a reference polypeptide or nucleic acid molecule.

[0072] The term "ligand," as used herein, refers to a molecule that binds to a receptor. In certain embodiments, a ligand binds to a receptor on another cell, allowing cell-cell recognition and / or interaction.

[0073] The term "constitutive expression" or "constitutively expressed" as used herein refers to expression or being expressed under all physiological conditions.

[0074] By "disease" is meant any condition, disease, or disorder that damages or interferes with the normal function of a cell, tissue, or organ, such as neoplasms and pathogenic infection of cells.

[0075] By "effective amount" is meant an amount sufficient to have a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to stop, reverse, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0076] By "endogenous" is meant a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0077] "Exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and overexpressed nucleic acid molecules and polypeptides. "Exogenous" nucleic acid refers to a nucleic acid that is not present in a native wild-type cell, for example, an exogenous nucleic acid may differ from its endogenous counterpart in sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or different sequence compared to its native endogenous counterpart, which may be introduced by genetic engineering into the cell itself or its precursor, and may be linked to alternative control sequences, such as non-native promoters or secretion sequences, as necessary.

[0078] By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism, or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0079] By "modulate" is meant to alter, either positively or negatively. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0080] By "increase" is meant a positive alteration of at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0081] By "reduce" is meant to negatively alter by at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0082] The terms "isolated," "purified," or "biologically pure" refer to a material that is free to varying degrees from components that normally accompany it as found in its native state. "Isolated" refers to a degree of separation from its original source or surroundings. "Purified" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or precursor or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications may give rise to different isolated proteins that can be individually purified.

[0083] By "isolated cell" is meant a cell that has been separated from molecules and / or cellular components that naturally accompany the cell.

[0084] The term "antigen-binding domain", as used herein, refers to a domain capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0085] "Linker," as used herein, is intended to mean a functional group (e.g., a chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a "peptide linker" refers to a peptide linker that is used to couple two proteins together (e.g., V H and V LThe linker refers to one or more amino acids used to couple the domains. In certain embodiments, the linker comprises the sequence set forth in GGGGSGGGSGGGGGS [SEQ ID NO: 7].

[0086] "Neoplasm" refers to a disease characterized by pathological proliferation of cells or tissues and subsequent migration or invasion into other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that do not induce or cause the cessation of normal cell replication. Neoplasms can affect a variety of cell types, tissues, or organs, including, but not limited to, the bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, neural tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or organs selected from the tissues or cell types. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In certain embodiments, the neoplasm is cancer.

[0087] By "receptor" is meant a polypeptide or portion thereof present on a cell membrane that selectively binds to one or more ligands.

[0088] "Recognize" means selectively binding to a target. T cells that recognize a tumor can express a receptor (e.g., TCR or CAR) that binds to a tumor antigen.

[0089] "Reference" or "control" refers to a standard for comparison. For example, the level of scFv-antigen binding by a cell expressing a CAR and an scFv can be compared to the level of scFv-antigen binding in a corresponding cell expressing only a CAR.

[0090] By "secreted" is meant a polypeptide that is released from the cell via the secretory pathway through the endoplasmic reticulum, the Golgi apparatus, and as vesicles that transiently fuse with the cell plasma membrane and release the protein extracellularly.

[0091] By "signal sequence" or "leader sequence" is meant a peptide sequence (e.g., 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of newly synthesized proteins that directs them into the secretory pathway. Exemplary leader sequences include, but are not limited to, the human IL-2 signal sequence (e.g., MYRMQLLSCIALSLALVTNS [SEQ ID NO: 8]), mouse IL-2 signal sequence (e.g., MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 9]); human kappa leader sequence (e.g., METPAQLLFLLLLWLPDTTG [SEQ ID NO: 10]), mouse kappa leader sequence (e.g., METDTLLLWVLLLWVPGSTG [SEQ ID NO: 11]); human CD8 leader sequence (e.g., MALPVTALLLPLALLLHAARP [SEQ ID NO: 12]); truncated human CD8 signal peptide (e.g., MALPVTALLLPLALLLHA [SEQ ID NO: 13]); human albumin signal sequence (e.g., MKWVTFISLLFSSAYS [SEQ ID NO: 14]); and human prolactin signal sequence (e.g., MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 15]).

[0092] By "soluble" is meant a polypeptide that is freely diffusible in an aqueous environment (eg, not membrane bound).

[0093] By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds to a biological molecule (e.g., a polypeptide) of interest, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide of the present disclosure.

[0094] The term "tumor antigen" as used herein refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-neoplastic cells. In certain embodiments, tumor antigens include any polypeptide expressed by a tumor that is capable of activating or inducing an immune response via an antigen-recognition receptor (e.g., CD19, MUC-16) or suppressing an immune response via receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0095] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them under U.S. patent law and can mean "includes," "including," etc.

[0096] As used herein, "treatment" refers to clinical intervention in an attempt to change the disease course of the individual or cell being treated, and can be performed either for prevention or during the course of clinical pathology. The therapeutic effect of treatment includes, without limitation, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improving prognosis. By preventing the progression of disease or disorder, treatment can prevent the deterioration of the disorder in subjects who have been affected or diagnosed, or subjects suspected of having the disorder, but treatment can also prevent the occurrence of the disorder or symptoms of the disorder in subjects at risk of the disorder or subjects suspected of having the disorder.

[0097] An "individual" or "subject" herein is a vertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys. The term "immunocompromised" as used herein refers to a subject with an immune deficiency. The subject is highly vulnerable to opportunistic infections, which are infections caused by organisms that do not normally cause disease in people with healthy immune systems, but can affect people with poorly functioning or suppressed immune systems.

[0098] As used herein, a "functional fragment" of a molecule or polypeptide includes fragments of a molecule or polypeptide that retain at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

[0099] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter. 5.2. Fusion Polypeptides

[0100] The subject matter of the present disclosure provides a fusion polypeptide that can enhance the activity and / or efficacy of a cell that comprises an antigen-recognizing receptor (e.g., a CAR, a TCR, or a TCR-like fusion molecule). In certain embodiments, the fusion polypeptide comprises a) the extracellular domain and the transmembrane domain of a costimulatory ligand, and b) the intracellular domain of a first costimulatory molecule. 5.2.1.Co-stimulatory Ligands

[0101] The costimulatory ligand can be selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof. The TNF family members can be selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. The Ig superfamily members can be selected from the group consisting of CD80, CD86, ICOS ligand (ICOSLG (also known as "CD275"), and combinations thereof.

[0102] In certain embodiments, the costimulatory ligand is CD80. In certain embodiments, the costimulatory ligand is human CD80. In certain embodiments, CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_005182 (SEQ ID NO: 16) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 16 is provided below. [ka]

[0103] In certain embodiments, the extracellular domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, or a functional fragment thereof. A functional fragment of SEQ ID NO:1 can be a contiguous portion of SEQ ID NO:1 that is at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200, or at least about 220 amino acids in length. In certain embodiments, a functional fragment of SEQ ID NO:1 retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the extracellular domain of CD80. Non-limiting examples of the primary function of the extracellular domain of CD80 include binding / interacting with CD28, binding / interacting with CTLA-4, binding / interacting with PD-L1, and contributing to CD80 homodimerization. In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:1 (or amino acids 1-242 of SEQ ID NO:16). SEQ ID NO:1 is provided below. [ka]

[0104] In certain embodiments, the transmembrane domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or a fragment thereof. A fragment of SEQ ID NO:2 can be at least about 5, at least about 10, at least about 15, or at least about 20 amino acids in length. In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 (or amino acids 243-263 of SEQ ID NO: 16). SEQ ID NO: 2 is provided below. [ka]

[0105] In certain embodiments, the costimulatory ligand is 4-1BBL. In certain embodiments, the costimulatory ligand is human 4-1BBL. In certain embodiments, 4-1BBL comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 17) having NCBI reference number: NP_003802.1 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 17 is provided below. [ka] [ka]

[0106] In certain embodiments, the costimulatory ligand is OX40L. In certain embodiments, the costimulatory ligand is human OX40L. In certain embodiments, OX40L comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 18) with NCBI reference number: NP_003317 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 18 is provided below. [ka]

[0107] In certain embodiments, the costimulatory ligand is CD70. In certain embodiments, the costimulatory ligand is human CD70. In certain embodiments, CD70 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 19) having NCBI reference number: NP_001243 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 19 is provided below. [ka]

[0108] In certain embodiments, the costimulatory ligand is CD86. In certain embodiments, the costimulatory ligand is human CD86. In certain embodiments, CD86 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 20) having NCBI reference number: NP_787058.5 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 20 is provided below. [ka] [ka]

[0109] In certain embodiments, the costimulatory ligand is GITRL. In certain embodiments, the costimulatory ligand is human GITRL. In certain embodiments, GITRL comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO:21) with NCBI reference number: NP_005083.2 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO:21 is provided below. [ka]

[0110] In certain embodiments, the costimulatory ligand is ICOS ligand (ICOSLG). In certain embodiments, the costimulatory ligand is human ICOSLG. In certain embodiments, ICOSLG comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 22) having NCBI reference number: NP_056074.1 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 22 is provided below. [ka]

[0111] In certain embodiments, the costimulatory ligand is CD40L (also known as "CD154"). In certain embodiments, the costimulatory ligand is human CD40L. In certain embodiments, CD40L comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_000065.1 (SEQ ID NO:23) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO:23 is provided below. [ka]

[0112] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a second costimulatory ligand. In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a third costimulatory ligand. In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a fourth costimulatory ligand. In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a fifth costimulatory ligand. In certain embodiments, the first, second, third, fourth, and fifth costimulatory ligands can be the same or different from each other.

[0113] In certain embodiments, the fusion polypeptide comprises the extracellular and transmembrane domains of CD80. 5.2.2. Co-stimulatory molecules

[0114] Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0115] In certain embodiments, the costimulatory molecule is 4-1BB. 4-1BB can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. In certain embodiments, the costimulatory molecule is human 4-1BB. In certain embodiments, 4-1BB comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 24) having NCBI reference number: NP_001552 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 24 is provided below. [ka]

[0116] In certain embodiments, the intracellular domain of 4-1BB comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:3 (or amino acids 214-255 of SEQ ID NO:24) or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, or a functional fragment thereof. A functional fragment of SEQ ID NO:3 may be a contiguous portion of SEQ ID NO:3 that is at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 amino acids in length. In certain embodiments, the functional fragment of SEQ ID NO:3 retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the intracellular domain of 4-1BB. Non-limiting examples of the primary function of the intracellular domain of 4-1BB include providing costimulatory signaling for the activation and proliferation of immune response cells (e.g., T cells), and interacting with and activating downstream adaptors (e.g., TRAFs). In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3. SEQ ID NO:3 is provided below: [ka]

[0117] In certain embodiments, the costimulatory molecule is CD28. In certain embodiments, the costimulatory molecule is human CD28. In certain embodiments, CD28 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 25) having NCBI reference number: NP_006130 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 25 is provided below. [ka]

[0118] In certain embodiments, the intracellular domain of CD28 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:5 (or amino acids 180-219 of SEQ ID NO:25) or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, or a functional fragment thereof. A functional fragment of SEQ ID NO:5 can be a contiguous portion of SEQ ID NO:5 that is at least about 20, at least about 25, at least about 30, or at least about 35 amino acids in length. In certain embodiments, the functional fragment of SEQ ID NO:5 retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the intracellular domain of CD28. Non-limiting examples of the primary function of the intracellular domain of CD28 include providing costimulatory signaling for the activation and proliferation of immune response cells (e.g., T cells) and interacting with protein adaptors (e.g., PI3K, GRB2, and LCK). In certain embodiments, the intracellular domain of CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5. SEQ ID NO:5 is provided below: [ka]

[0119] In certain embodiments, the costimulatory molecule is OX40.In certain embodiments, the costimulatory molecule is human OX40.In certain embodiments, OX40 comprises or consists of the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO:26) with NCBI reference number: NP_003318.1 or its fragment, and / or can comprise up to 1, or up to 2, or up to 3 conservative amino acid substitutions as required. In certain embodiments, the intracellular domain of OX40 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 236-277 of SEQ ID NO:26 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of OX40 comprises or consists of amino acids 236-277 of SEQ ID NO:26 or a fragment thereof. In certain embodiments, the intracellular domain of OX40 comprises or consists of amino acids 236-277 of SEQ ID NO:26. SEQ ID NO:26 is provided below. [ka]

[0120] In certain embodiments, the costimulatory molecule is ICOS.In certain embodiments, the costimulatory molecule is human ICOS.In certain embodiments, ICOS comprises or consists of the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical and / or may contain up to one, or up to two, or up to three conservative amino acid substitutions as required with the amino acid sequence (SEQ ID NO:27) with NCBI reference number: NP_036224.1 or a fragment thereof. In certain embodiments, the intracellular domain of ICOS comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 162-199 of SEQ ID NO: 27 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of ICOS comprises or consists of amino acids 162-199 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the intracellular domain of ICOS comprises or consists of amino acids 162-199 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below. [ka]

[0121] In certain embodiments, the costimulatory molecule is DAP-10. In certain embodiments, the costimulatory molecule is human DAP-10. In certain embodiments, DAP-10 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO:28) having NCBI reference number: NP_001007470.1 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of DAP10 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 70-92 of SEQ ID NO:28 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of DAP10 comprises or consists of amino acids 70-92 of SEQ ID NO:28 or a fragment thereof. In certain embodiments, the intracellular domain of DAP10 comprises or consists of amino acids 70-92 of SEQ ID NO:28. SEQ ID NO:28 is provided below. [ka]

[0122] In certain embodiments, the costimulatory molecule is CD27. In certain embodiments, the costimulatory molecule is human CD27. In certain embodiments, CD27 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical and homologous to the amino acid sequence (SEQ ID NO:29) having NCBI reference number: NP_001233.1 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD27 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 213-260 of SEQ ID NO:29 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD27 comprises or consists of amino acids 213-260 of SEQ ID NO:29 or a fragment thereof. In certain embodiments, the intracellular domain of CD27 comprises or consists of amino acids 213-260 of SEQ ID NO:29. SEQ ID NO:29 is provided below. [ka]

[0123] In certain embodiments, the costimulatory molecule is CD40. In certain embodiments, the costimulatory molecule is human CD40. In certain embodiments, CD40 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_001241.1 (SEQ ID NO: 30) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD40 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 216-277 of SEQ ID NO: 30, or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD40 comprises or consists of amino acids 216-277 of SEQ ID NO: 30, or a fragment thereof. In certain embodiments, the intracellular domain of CD40 comprises or consists of amino acids 216-277 of SEQ ID NO: 30. SEQ ID NO: 30 is provided below. [ka]

[0124] In certain embodiments, the costimulatory molecule is NKG2D.In certain embodiments, the costimulatory molecule is human NKG2D.In certain embodiments, NKG2D comprises or consists of the amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical and homologous to the amino acid sequence (SEQ ID NO:31) with NCBI reference number: NP_031386.2 or a fragment thereof, and / or can comprise up to one, or up to two, or up to three conservative amino acid substitutions as required. In certain embodiments, the intracellular domain of NKG2D comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 73-216 of SEQ ID NO: 31 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of NKG2D comprises or consists of amino acids 73-216 of SEQ ID NO: 31 or a fragment thereof. In certain embodiments, the intracellular domain of NKG2D comprises or consists of amino acids 73-216 of SEQ ID NO: 31. SEQ ID NO: 31 is provided below. [ka]

[0125] In certain embodiments, the costimulatory molecule is CD2. In certain embodiments, the costimulatory molecule is human CD2. In certain embodiments, CD2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 32) having NCBI reference number: NP_001315538 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 236-351 of SEQ ID NO: 32, or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD2 comprises or consists of amino acids 236-351 of SEQ ID NO: 32, or a fragment thereof. In certain embodiments, the intracellular domain of CD2 comprises or consists of amino acids 236-351 of SEQ ID NO: 32. SEQ ID NO: 32 is provided below. [ka]

[0126] In certain embodiments, the fusion polypeptide comprises the intracellular domain of the second costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of the third costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of the fourth costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of the fifth costimulatory molecule. In certain embodiments, the first, second, third, fourth and fifth costimulatory molecules can be the same or different from each other.

[0127] In certain embodiments, the fusion polypeptide comprises the intracellular domain of a first costimulatory molecule that is 4-1BB and the intracellular domain of a second costimulatory molecule that is CD28. Cytokine receptors

[0128] In certain embodiments, the fusion polypeptide further comprises a cytokine receptor. In certain embodiments, the fusion polypeptide further comprises a signaling domain of a cytokine receptor. The cytokine receptor or the signaling domain of the cytokine receptor can be located at the N-terminus or C-terminus of the fusion polypeptide. In certain embodiments, the cytokine receptor or the signaling domain of the cytokine receptor is located at the C-terminus of the fusion polypeptide. In certain embodiments, the cytokine receptor or the signaling domain of the cytokine receptor is located at the C-terminus of a costimulatory ligand (e.g., CD80, 4-1BBL, OX40L, CD70, GITRL, CD40L, CD86, or ICOSLG). Non-limiting examples of cytokine receptors include CD121a, CDw121b, IL-18Ra, IL18Rb, CD122, CD25, CD132, CD124, CD213a13, CD127, IL-9R, IL15Ra, CDw125, CDw131, CD126, CD130, IL11Ra, Cd114, CD212, CD4, CDw217, CD118, and CDw119. 5.2.4. Exemplary Fusion Polypeptides

[0129] In certain embodiments, the fusion polypeptide comprises: a) the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80; and b) the intracellular domain of a first costimulatory molecule selected from the group consisting of 4-1BB, CD28, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. CD80 / 4-1BB Fusion Polypeptides

[0130] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand that is CD80; and b) the intracellular domain of a first costimulatory molecule that is 4-1BB.

[0131] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0132] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0133] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0134] In certain embodiments, 4-1BB comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:24.

[0135] In certain embodiments, the intracellular domain of 4-1BB comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:3 (or amino acids 214-255 of SEQ ID NO:24). In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 (or amino acids 214-255 of SEQ ID NO:24).

[0136] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO: 4 is provided below.

[0137] [ka] CD80 / 4-1BB-CD28 Fusion Polypeptides

[0138] In certain embodiments, the fusion polypeptide comprises: a) the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80; b) the intracellular domain of a first costimulatory molecule that is 4-1BB; and c) the intracellular domain of a second costimulatory molecule that is CD28.

[0139] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0140] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0141] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0142] In certain embodiments, 4-1BB comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:24.

[0143] In certain embodiments, the intracellular domain of 4-1BB comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO:3 (or amino acids 214-255 of SEQ ID NO:24). In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 (or amino acids 214-255 of SEQ ID NO:24).

[0144] In certain embodiments, CD28 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:25.

[0145] In certain embodiments, the intracellular domain of CD28 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:5 (or amino acids 180-219 of SEQ ID NO:25). In certain embodiments, the intracellular domain of CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5 (or amino acids 180-219 of SEQ ID NO:25).

[0146] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:6. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:6. SEQ ID NO:6 is provided below. [ka] CD80 / CD28 Fusion Polypeptides

[0147] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand, which is CD80; and b) the intracellular domain of a first costimulatory molecule, which is CD28.

[0148] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0149] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0150] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0151] In certain embodiments, CD28 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:25.

[0152] In certain embodiments, the intracellular domain of CD28 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:5 (or amino acids 180-219 of SEQ ID NO:25). In certain embodiments, the intracellular domain of CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5 (or amino acids 180-219 of SEQ ID NO:25). CD80 / OX40 Fusion Polypeptides

[0153] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand, which is CD80; and b) the intracellular domain of a first costimulatory molecule, which is OX40.

[0154] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0155] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0156] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0157] In certain embodiments, OX40 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 26 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of OX40 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 236-277 of SEQ ID NO: 26 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of OX40 comprises or consists of amino acids 236-277 of SEQ ID NO:26, or a fragment thereof. In certain embodiments, the intracellular domain of OX40 comprises or consists of amino acids 236-277 of SEQ ID NO:26. CD80 / ICOS Fusion Polypeptides

[0158] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand, which is CD80; and b) the intracellular domain of a first costimulatory molecule, which is ICOS.

[0159] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0160] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0161] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0162] In certain embodiments, ICOS comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 27 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of ICOS comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 162-199 of SEQ ID NO: 27 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of ICOS comprises or consists of amino acids 162 to 199 of SEQ ID NO: 27, or a fragment thereof. In certain embodiments, the intracellular domain of ICOS comprises or consists of amino acids 162 to 199 of SEQ ID NO: 27. CD80 / DAP-10 Fusion Polypeptides

[0163] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand, which is CD80; and b) the intracellular domain of a first costimulatory molecule, which is DAP-10.

[0164] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0165] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0166] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0167] In certain embodiments, DAP-10 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 28 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of DAP10 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 70-92 of SEQ ID NO: 28 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of DAP10 comprises or consists of amino acids 70 to 92 of SEQ ID NO:28, or a fragment thereof. In certain embodiments, the intracellular domain of DAP10 comprises or consists of amino acids 70 to 92 of SEQ ID NO:28. 5.2.4.7. CD80 / CD27 Fusion Polypeptides

[0168] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand, which is CD80; and b) the intracellular domain of a first costimulatory molecule, which is CD27.

[0169] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0170] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0171] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0172] In certain embodiments, CD27 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 29 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD27 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 213-260 of SEQ ID NO: 29 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD27 comprises or consists of amino acids 213-260 of SEQ ID NO:29, or a fragment thereof. In certain embodiments, the intracellular domain of CD27 comprises or consists of amino acids 213-260 of SEQ ID NO:29. 5.2.4.8. CD80 / CD40 Fusion Polypeptides

[0173] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand that is CD80; and b) the intracellular domain of a first costimulatory molecule that is CD40.

[0174] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0175] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0176] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0177] In certain embodiments, CD40 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 30 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD40 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 216-277 of SEQ ID NO: 30 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD40 comprises or consists of amino acids 216-277 of SEQ ID NO: 30, or a fragment thereof. In certain embodiments, the intracellular domain of CD40 comprises or consists of amino acids 216-277 of SEQ ID NO: 30. CD80 / NKG2D Fusion Polypeptides

[0178] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand that is CD80; and b) the intracellular domain of a first costimulatory molecule that is NKG2D.

[0179] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0180] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0181] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0182] In certain embodiments, NKG2D comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 31 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of NKG2D comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 73-216 of SEQ ID NO: 31 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of NKG2D comprises or consists of amino acids 73-216 of SEQ ID NO: 31, or a fragment thereof. In certain embodiments, the intracellular domain of NKG2D comprises or consists of amino acids 73-216 of SEQ ID NO: 31. 5.2.4.9. CD80 / CD2 Fusion Polypeptides

[0183] In certain embodiments, the fusion polypeptide comprises: a) the extracellular and transmembrane domains of a costimulatory ligand, which is CD80; and b) the intracellular domain of a first costimulatory molecule, which is CD2.

[0184] In certain embodiments, CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0185] In certain embodiments, the extracellular domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16). In certain embodiments, the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 (or amino acids 1-242 of SEQ ID NO: 16).

[0186] In certain embodiments, the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16). In certain embodiments, the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 (or amino acids 243-263 of SEQ ID NO:16).

[0187] In certain embodiments, CD2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 32 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD2 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to amino acids 236-351 of SEQ ID NO: 32 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD2 comprises or consists of amino acids 236 to 351 of SEQ ID NO: 32, or a fragment thereof. In certain embodiments, the intracellular domain of CD2 comprises or consists of amino acids 236 to 351 of SEQ ID NO: 32. 5.2.5. Activity of the Fusion Polypeptide

[0188] The fusion polypeptides of the present disclosure are capable of stimulating cells that contain an antigen-recognizing receptor (e.g., a CAR, a TCR, or a TCR-like molecule). In certain embodiments, the fusion polypeptides provide stimulation to T cells in cis and / or trans.

[0189] In certain embodiments, the costimulatory ligand included in the fusion polypeptide of the present disclosure is CD80. CD80 can interact with CD28, CTLA-4, and PD-L1. CD80 is the ligand of the CD28 molecule, which is the main costimulatory molecule expressed by T cells. Upon activation, T cells express CTLA-4 and PD-L1, which negatively regulate downstream signaling of the T cell receptor ("TCR") and CD28. CTLA-4 functions as a competitor of CD28 by interacting with CD80 (the affinity of the CTLA-4 / CD80 interaction is usually higher than that of CD80 / CD28). PD-L1, the receptor for PDL1, functions as a T cell inhibitor by dephosphorylating the downstream signalosome of TCR and CD28. Similar to CTLA-4, PD-L1 can also interact with CD80 under certain conditions.

[0190] The fusion polypeptides of the present disclosure (e.g., fusion polypeptides comprising the extracellular and transmembrane domains of CD80) can ligate three different receptors: CD28, CTLA-4, and PD-L1. By doing so, these ligations provide (i) signaling through the intracellular domains of costimulatory molecules (e.g., 4-1BB, CD28, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, or CD2), and (ii) competition with CTLA4 / CD80 and PDL-1 / PD-1 ligation, thereby shifting inhibitory signaling to activating signaling for T cells. Constitutive expression (e.g., at high levels) of CD80 on T cells provides strong costimulation upon interaction with endogenous CD28. By interacting with CTLA-4 and PD-L1, a fusion polypeptide comprising the extracellular domain and transmembrane domain of CD80 can compete with endogenous CD80 / CTLA4 and PD-1 / PD-L1 (e.g., expressed at low levels) to provide positive signaling to T cells. CTLA-4 is expressed on engineered bystander T cells. PD-L1 is expressed on different cell types (mainly antigen-presenting cells and tumor cells) and can also be found on engineered T cells. By exchanging the intracellular domain of CD80 with a costimulatory derived signaling domain (e.g., the intracellular domain of a costimulatory molecule (e.g., CD28, and / or 4-1BB, etc.)), CD80 signaling is shifted from negative to positive.

[0191] The fusion polypeptide of the present disclosure (e.g., fusion polypeptide comprising the extracellular domain and transmembrane domain of CD80) provides stimulation of T cells in cis and trans through engagement with CD28 molecule, and delivery of signaling through the intracellular domain of one or more costimulatory molecules (e.g., 4-1BB and / or CD28). Moreover, the fusion polypeptide comprising the extracellular domain and transmembrane domain of CD80 can bind to CTLA-4 and PD-L1, two inhibitory molecules known to be upregulated in tumor microenvironment. These interactions shift T cell inhibition to T cell activation. Thus, the fusion polypeptide of the present disclosure (e.g., fusion polypeptide comprising the extracellular domain and transmembrane domain of CD80) provides costimulation to T cells, ensuring that they benefit from T cell inhibitory receptors expressed in unfavorable tumor microenvironment. 5.2.6. Regulatability of Fusion Polypeptides

[0192] In certain embodiments, the fusion polypeptide can be regulated, e.g., regulatable. The regulator can regulate or modulate the expression and / or activity of the fusion polypeptide. The regulator can switch on or switch off the expression and / or activity of the fusion polypeptide. The fusion polypeptide can be regulated by controlling its expression level (i.e., constitutively high, constitutively low, or inducible) or by administering an exogenous molecule (e.g., antibody, Ig fusion protein). These approaches serve the purpose of switching on and off the function of the fusion polypeptide.

[0193] Non-limiting examples of regulators include promoters (e.g., inducible promoters) capable of controlling expression of a fusion polypeptide, molecules capable of regulating or modulating the expression and / or activity of a costimulatory ligand contained in the fusion polypeptide, and molecules capable of regulating or modulating the expression and / or activity of a costimulatory molecule contained in the fusion polypeptide.

[0194] Molecules capable of regulating or modulating the expression and / or activity of the costimulatory ligand contained in the fusion polypeptide include antibodies that bind to the costimulatory ligand and fusion proteins that bind to the costimulatory ligand and modulate the expression and / or activity of the costimulatory ligand. In certain embodiments, the modulator is an antibody that binds to the costimulatory ligand.

[0195] Molecules capable of regulating or modulating the expression and / or activity of a costimulatory molecule contained in a fusion polypeptide include antibodies that bind to a costimulatory molecule and fusion proteins that bind to a costimulatory molecule and modulate the expression and / or activity of a costimulatory molecule. In certain embodiments, the modulator is an antibody that binds to a costimulatory molecule.

[0196] In certain embodiments, the fusion polypeptide comprises the extracellular and transmembrane domains of CD80, and the modulator is an anti-CD80 antibody.

[0197] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of CD80, and the modulator is a fusion protein that binds to CD80 and modulates the activity of CD80. In certain embodiments, the fusion protein comprises a CTLA4 fragment that binds to CD80. In certain embodiments, the CTLA4 fragment is selected from the group consisting of abatacept and belatacept. 5.2.7. Nucleic Acid Molecules Encoding Fusion Polypeptides

[0198] The subject of the present disclosure further provides a nucleic acid molecule that encodes the fusion polypeptide disclosed herein.In addition, the subject of the present disclosure provides a vector that comprises the nucleic acid molecule described herein.The vector can be a viral vector or a non-viral vector.In certain embodiments, the vector is a viral vector.In certain embodiments, the viral vector is a retroviral vector, such as a gamma retroviral vector or a lentiviral vector. 5.3.Cells

[0199] The subject matter of the present disclosure provides a cell comprising the fusion polypeptide disclosed herein. In certain embodiments, the fusion protein can promote the anti-tumor effect of the cell. In certain embodiments, the cell is selected from the group consisting of a cell of lymphoid lineage and a cell of myeloid lineage. In certain embodiments, the cell is an immune response cell. In certain embodiments, the immune response cell is a cell of lymphoid lineage.

[0200] In certain embodiments, the cell is a lymphoid lineage cell. The lymphoid lineage cell can provide antibody production, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell).

[0201] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of the present disclosure include helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., T EM Cells and T EMRA The T cells may be of any type, including but not limited to, T-cells, regulatory T cells (also known as suppressor T cells), tumor infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. The patient's own T cells may be genetically modified to target specific antigens through the introduction of an antigen recognition receptor, e.g., a CAR or TCR. T cells are CD4 +T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + In certain embodiments, the CD8 + The T cells are CD4-independent. In certain embodiments, the T cells are derived from induced pluripotent stem cells (iPSCs). In certain embodiments, the T cells are CD8 + T cells, CD8 + The T cells are derived from iPSCs.

[0202] In certain embodiments, the cell is a NK cell. Natural killer (NK) cell is a lymphocyte that is part of cellular immunity and acts during natural immune response. NK cell does not require prior activation to carry out its cytotoxic effect on target cell.

[0203] Types of human lymphocytes of the subject matter of the present disclosure include peripheral donor lymphocytes, such as those described in Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes containing α and β heterodimers), Panelli, MC, et al. 2000 J Immunol 164:495-504; Panelli, MC, et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res. 65:5417-5427; Papanicolaou, GA, et al. 2003 Blood 102:2498-2505 (disclosing selective in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPC) or pulsed dendritic cells).

[0204] The cells (eg, T cells) can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0205] In certain embodiments, the cells of the present disclosure are capable of modulating the tumor microenvironment. Tumors have an unfavorable microenvironment for the host immune response, involving a series of mechanisms by malignant cells to protect tumors from immune recognition and elimination. This "unfavorable tumor microenvironment" is characterized by the infiltrating regulatory CD4 +The tumor microenvironment contains a variety of immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and expression of ligands targeted to immunoinhibitory receptors (CTLA-4 and PD-1) expressed by activated T cells. These mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inappropriate immune responses, but within the tumor microenvironment, these mechanisms prevent effective anti-tumor immune responses. Collectively, these immunosuppressive factors can induce either significant anergy or apoptosis of adoptively transferred CAR-modified T cells upon encounter with targeted tumor cells.

[0206] In certain embodiments, the cell is a cell of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and stem cells from which cells of the myeloid lineage can differentiate.

[0207] In certain embodiments, the stem cells are pluripotent stem cells (eg, embryonic stem cells or induced pluripotent stem cells).

[0208] In certain embodiments, the cell further comprises an antigen recognition receptor. In certain embodiments, the antigen recognition receptor is capable of activating the cell. The cell can be transduced with the antigen recognition receptor and the fusion polypeptide such that the cell co-expresses the antigen recognition receptor and the fusion polypeptide.

[0209] The fusion polypeptide may be operably linked to a first promoter. The antigen recognition receptor may be operably linked to a second promoter. The first promoter may be the same as the second promoter. Alternatively, the first promoter is different from the second promoter. The first and second promoters may be endogenous or exogenous. Non-limiting examples of exogenous promoters include elongation factor (EF)-1 promoter, cytomegalovirus immediate early promoter (CMV) promoter, simian virus 40 early promoter (SV40) promoter, phosphoglycerate kinase (PGK) promoter, and metallothionein promoter. In certain embodiments, one or both of the first and second promoters are inducible promoters. In certain embodiments, the inducible promoter is selected from NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

[0210] In certain embodiments, the cell is a T cell, and the fusion polypeptide and the antigen recognition receptor are integrated at a locus in the genome of the T cell.Non-limiting examples of the locus include the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus.In certain embodiments, the locus is the TRAC locus or the TRBC locus.The method of targeting CAR to a site in the genome of a T cell is disclosed in WO2017180989 and Eyquem et al., Nature. (2017 Mar 2); 543(7643): 113-117, both of which are incorporated by reference in their entirety.

[0211] In certain embodiments, the fusion polypeptide and the antigen recognition receptor are integrated at a locus that encodes an immune inhibitory molecule.Non-limiting examples of immune inhibitory molecules include CTLA-4, PD-1, LAG3, BTLA, B7-1, B7-H1, B7-H3, B7-H4, TIM3, SHP-1, SHP-2, TIGIT, CD160, and LAIR1. 5.4. Antigen Recognition Receptors

[0212] In certain embodiments, the antigen-recognizing receptor binds to an antigen of interest. In certain embodiments, the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). In certain embodiments, the antigen-recognizing receptor is a T cell receptor (TCR). In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule. 5.4.1. Antigen

[0213] In certain embodiments, the antigen-recognizing receptor binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, cancer proteins. Antigens can be expressed as peptides or as intact proteins or portions thereof. The intact proteins or portions thereof can be native or mutagenized.Non-limiting examples of tumor antigens include CD19, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion. Molecule (EpCAM), receptor tyrosine protein kinase Erb-B2, Erb-B3, Erb-B4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES0-1, carcinoembryonic antigen These include IFN-γ-γ (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, and ERBB.

[0214] In certain embodiments, the antigen recognition receptor binds to CD19. In certain embodiments, the antigen recognition receptor binds to a mouse CD19 polypeptide. In certain embodiments, the mouse CD19 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33. SEQ ID NO: 33 is provided below. [ka]

[0215] In certain embodiments, the antigen recognition receptor binds to a human CD19 polypeptide. In certain embodiments, the human CD19 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 34. SEQ ID NO: 34 is provided below. [ka]

[0216] In certain embodiments, the antigen recognition receptor binds to the extracellular domain of the CD19 protein.

[0217] In certain embodiments, the antigen recognition receptor binds to a pathogen antigen, for use in treating and / or preventing, for example, a pathogen infection or other infectious disease, for example in an immunocompromised subject. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0218] Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 (also referred to as HDLV-III, LAVE, or HTLV-III / LAV, or HIV-III; and other isolates, e.g., HIV-LP; Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantavirus, Bunga virus, Phlebovirus, and Naira virus); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B viruses); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex viruses (HSV) types 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes viruses; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever viruses); and nontypeable viruses (e.g., the agents of hepatitis delta (thought to be a defective satellite of hepatitis B virus), agents of non-A non-B hepatitis (class 1 = internal transmission; class 2 = parenteral transmission (i.e., hepatitis C);Norwalk and related viruses, and Astroviruses;

[0219] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include Helicobacter pyloris, Borelia burgdorferi, Legionella, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, M. leprae), Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A streptococci), Streptococcus agalactiae (group B streptococci), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp., Campylobacter jejuni, Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium spp., Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli, Mycoplasma, Pseudomonas aeruginosa, Pseudomonas fluorescens, Corynobacteria diphtheriae, Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia, shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis, Listeria monocytogenes, Mycoplasma spp., Vibrio cholerae, Borrelia, Francisella, Brucella melitensis, Proteus mirabilis, and Proteus.

[0220] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus. T cell receptor (TCR)

[0221] In certain embodiments, the antigen recognition receptor is a TCR. A TCR is a disulfide-bonded heterodimeric protein consisting of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. A TCR is found on the surface of a T cell and is responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, a TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, a TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0222] Each chain of the TCR is composed of two extracellular domains: the variable (V) region and the constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity determining regions (CDRs).

[0223] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. When the TCR complex binds to its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0224] In certain embodiments, the antigen-recognizing receptor is an endogenous TCR. In certain embodiments, the antigen-recognizing receptor is a naturally occurring TCR.

[0225] In certain embodiments, the antigen-recognizing receptor is an exogenous TCR. In certain embodiments, the antigen-recognizing receptor is a recombinant TCR. In certain embodiments, the antigen-recognizing receptor is a non-naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more amino acid residues. In certain embodiments, the non-naturally occurring TCR is modified by at least one amino acid residue from a naturally occurring TCR. In certain embodiments, a non-naturally occurring TCR has at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more amino acid residues altered from a naturally occurring TCR. Chimeric Antigen Receptors (CARs)

[0226] In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR).CAR is an engineered receptor that grafts or gives immune effector cells the specificity of interest.CAR can be used to graft the specificity of monoclonal antibodies into T cells, and the transfer of its coding sequence is facilitated by retroviral vectors.

[0227] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen-binding domain (e.g., scFv) that is fused to a transmembrane domain that is fused to a cytoplasmic / intracellular signaling domain. "First generation" CARs provide de novo antigen recognition and bind to CD4 through their CD3 ζ chain signaling domain in a single fusion molecule independent of HLA-mediated antigen presentation. + and CD8 +The "second generation" CARs can cause both activation of T cells and activation of T cells. The "second generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40, and NKG2D) to the cytoplasmic tail of the CAR to provide additional signals to T cells. The "second generation" CARs include CARs that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). The "third generation" CARs include CARs that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the CAR is a second generation CAR. In certain embodiments, the CAR comprises an extracellular antigen binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the CAR further comprises a hinge / spacer region.

[0228] In certain non-limiting embodiments, the extracellular antigen binding domain of the CAR (e.g., an scFv or analog thereof) is about 2×10 -7 M or less dissociation constant (K d In certain embodiments, K d is about 2 x 10 -7 M or less, approximately 1 x 10 -7 M or less, approx. 9 x 10 -8 M or less, approximately 1 x 10 -8 M or less, approx. 9 x 10 -9 M or less, about 5 x 10 -9 M or less, about 4 x 10 -9 M or less, about 3 x 10 -9 or less, about 2 × 10 -9 M or less, approximately 1 x 10 -9 M or less, approximately 1 x 10 -10 M or less, or about 1 x 10 -11 In certain non-limiting embodiments, K d is about 1 x 10 -8M or less. In certain non-limiting embodiments, K d is about 1 x 10 -9 M or less. In certain non-limiting embodiments, K d is about 1 x 10 -9 M ~ approx. 1×10 -7 It's M.

[0229] The binding of the extracellular antigen-binding domain (e.g., in scFv or its analog) can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFv can be radioactively labeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioactive isotopes can be detected by such means as using a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). 5.4.3.1. Extracellular Antigen-Binding Domain of the CAR

[0230] In certain embodiments, the extracellular antigen binding domain specifically binds to an antigen. In certain embodiments, the extracellular antigen binding domain is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the extracellular antigen binding domain is an optionally cross-linked Fab. In certain embodiments, the extracellular antigen binding domain is a F(ab)2. In certain embodiments, any of the aforementioned molecules may be included in a fusion protein with a heterologous sequence to form the extracellular antigen binding domain. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein. The scFv is a human V L and / or V H The scFv may be derived from a mouse carrying the gene. The scFv may also be replaced by a camelid heavy chain (e.g., VHH from camel, llama, etc.) or a partial natural ligand of a cell surface receptor. In certain embodiments, the antigen is a tumor antigen, such as an antigen disclosed herein. In certain embodiments, the antigen is a pathogen antigen, such as an antigen disclosed herein.

[0231] In certain embodiments, the extracellular antigen-binding domain is a mouse scFv. In certain embodiments, the extracellular antigen-binding domain is a mouse scFv that binds to a human CD19 polypeptide. In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 35 and specifically binds to a human CD19 polypeptide (e.g., a human CD19 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34 or a portion thereof). In certain embodiments, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35 is set forth in SEQ ID NO: 36. In certain embodiments, the scFv is derived from a clone designated as "SJ25C1".

[0232] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO: 37, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof H CDR2, and V comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof H In certain embodiments, the extracellular antigen-binding domain comprises a V CDR3 comprising the amino acid sequence set forth in SEQ ID NO:37. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:38 H CDR2, and V comprising the amino acid sequence set forth in SEQ ID NO:39 H In certain embodiments, the extracellular antigen-binding domain comprises a light chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO: 40, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof L In certain embodiments, the extracellular antigen-binding domain comprises a V CDR3 comprising the amino acid sequence set forth in SEQ ID NO:40. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:41 L CDR2, and V comprising the amino acid sequence set forth in SEQ ID NO:42 L Includes CDR3.

[0233] In certain embodiments, the extracellular antigen-binding domain comprises a V sequence comprising the amino acid sequence set forth in SEQ ID NO: 37, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:38 or a conservative modification thereof H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:39 or a conservative modification thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereofL In certain embodiments, the extracellular antigen-binding domain comprises a V CDR3 comprising an amino acid sequence having the sequence set forth in SEQ ID NO:37. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:38 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:39 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:40 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:41 L CDR2, and V comprising the amino acid sequence set forth in SEQ ID NO:42 L Includes CDR3.

[0234] In certain embodiments, the extracellular antigen-binding domain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:43. H For example, the extracellular antigen-binding domain comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:43. H In certain embodiments, the extracellular antigen-binding domain comprises a V H Includes.

[0235] In certain embodiments, the extracellular antigen-binding domain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:44. LFor example, the extracellular antigen-binding domain comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:44. L In certain embodiments, the extracellular antigen-binding domain comprises a V L Includes.

[0236] In certain embodiments, the extracellular antigen-binding domain comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:43. H and V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:44. L Includes.

[0237] In certain embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:43. H In certain embodiments, the extracellular antigen-binding domain comprises a V L In certain embodiments, the extracellular antigen-binding domain comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:44 L and, if necessary, (iii) V H and V L In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:7.

[0238] SEQ ID NOs:35-44 are provided in Table 1 below. Table 1 [Table 1-1] [Table 1-2]

[0239] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of a CAR of the present disclosure (e.g., an extracellular antigen-binding domain of a CAR) that contains the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of a CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar) glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retention of function (i.e., the functions described in (c) to (l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues are altered in a specified sequence or CDR region.

[0240] A V that has at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a particular sequence (e.g., SEQ ID NOs: 43 and 44). H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the designated sequence, but retain the ability to bind to a target antigen (e.g., CD19). In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in a particular sequence (e.g., SEQ ID NOs: 43 and 44). In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs of the extracellular antigen-binding domain (e.g., in the FRs). In certain embodiments, the extracellular antigen-binding domain comprises a V-terminal sequence selected from SEQ ID NOs: 43 and 44, including post-translational modifications of the sequences (SEQ ID NOs: 43 and 44). H and / or V L Contains arrays.

[0241] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between two sequences.The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology=number of identical positions / total number of positions×100).Comparing sequences between two sequences and determining percent identity can be achieved using mathematical algorithms.

[0242] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0243] Additionally or alternatively, the amino acid sequences of the disclosed subject matter can be used as a "query sequence" to perform searches against public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. 5.4.3.2. CAR Transmembrane Domain

[0244] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptor assembles and a signal is transmitted to the cell. In accordance with the subject matter of the present disclosure, the transmembrane domain of the CAR can comprise a native or modified transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 peptide, an NKG2D peptide, a synthetic polypeptide (not based on a protein associated with immune response), or a combination thereof. CD8

[0245] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 45) having the NCBI reference number NP_001139345.1 provided below (homology herein may be determined using standard software such as BLAST or FASTA) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 45 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, or 200-235 of SEQ ID NO: 45. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137-209 of SEQ ID NO: 45. SEQ ID NO: 45 is provided below. [ka]

[0246] In certain embodiments, a CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence having GenBank No: AAA92533.1 (SEQ ID NO: 46) provided below (homology herein may be determined using standard software such as BLAST or FASTA), or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, a CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 46 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 46. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151-219 of SEQ ID NO: 46. SEQ ID NO: 46 is provided below. [ka]

[0247] In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:47, provided below: [ka]

[0248] In accordance with the subject matter of this disclosure, a "CD8 nucleic acid molecule" refers to a polynucleotide that encodes a CD8 polypeptide.

[0249] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:47 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:48, provided below. [ka] CD28

[0250] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a CD28 polypeptide. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:25 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:25 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 153-179, or 200-220 of SEQ ID NO:25. In certain embodiments, the CAR comprises the transmembrane domain of CD28 (e.g., human CD28), or a portion thereof. In certain embodiments, the transmembrane domain of CD28, or a portion thereof, comprises or consists of amino acids 153-179 of SEQ ID NO:25. In certain embodiments, the CAR comprises a CD28 polypeptide comprising or consists of amino acids 153-179 of SEQ ID NO:25.

[0251] An exemplary nucleic acid sequence encoding amino acids 153-179 of SEQ ID NO:25 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:49, provided below. [ka] CD84

[0252] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a native or modified transmembrane domain of a CD84 polypeptide. In certain embodiments, the CD84 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_001171808.1 (SEQ ID NO: 50) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD84 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 50 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 345 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD84 polypeptide comprises or consists of an amino acid sequence of amino acids 1-345, 1-50, 50-100, 100-150, 150-200, 200-250, 226-250, 250-300, or 300-345 of SEQ ID NO: 50. In certain embodiments, the transmembrane domain of the CAR comprises a CD84 polypeptide comprising or consisting of amino acids 226-250 of SEQ ID NO:50.

[0253] SEQ ID NO:50 is provided below: [ka]

[0254] In accordance with the subject matter of this disclosure, a "CD84 nucleic acid molecule" refers to a polynucleotide that encodes a CD84 polypeptide. An exemplary nucleic acid sequence encoding amino acids 226-250 of SEQ ID NO:50 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:51, provided below. [ka] CD166

[0255] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a native or modified transmembrane domain of a CD166 polypeptide. In certain embodiments, the CD166 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_001618.2 (SEQ ID NO: 52) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD166 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 52 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 583 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD166 polypeptide comprises or consists of an amino acid sequence of amino acids 1-583, 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 528-549, 528-553, or 550-583 of SEQ ID NO: 52. In certain embodiments, the transmembrane domain of the CAR comprises a CD166 polypeptide comprising or consisting of amino acids 528-553 of SEQ ID NO: 52. In certain embodiments, the transmembrane domain of the CAR comprises a CD166 polypeptide comprising or consisting of amino acids 528-549 of SEQ ID NO: 52.

[0256] SEQ ID NO:52 is provided below: [ka]

[0257] In accordance with the subject matter of the present disclosure, a "CD166 nucleic acid molecule" refers to a polynucleotide that encodes a CD166 polypeptide. An exemplary nucleic acid sequence encoding amino acids 528-553 of SEQ ID NO:52 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:53, provided below. [ka] CD8a

[0258] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a native or modified transmembrane domain of a CD8a polypeptide. In certain embodiments, the CD8a polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_001139345.1 (SEQ ID NO: 54) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8a polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 54 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8a polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 183-207, or 200-235 of SEQ ID NO: 54. In certain embodiments, the transmembrane domain of the CAR comprises a CD8a polypeptide comprising or consisting of amino acids 183-207 of SEQ ID NO:54.

[0259] SEQ ID NO:54 is provided below: [ka]

[0260] In accordance with the subject matter of this disclosure, a "CD8a nucleic acid molecule" refers to a polynucleotide that encodes a CD8a polypeptide. An exemplary nucleic acid sequence encoding amino acids 183-207 of SEQ ID NO:54 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:55, provided below. [ka] CD8b

[0261] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a native or modified transmembrane domain of a CD8b polypeptide. In certain embodiments, the CD8b polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_742099.1 (SEQ ID NO: 56) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8b polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 56 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 221 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8b polypeptide comprises or consists of the amino acid sequence of amino acids 1-221, 1-50, 50-100, 100-150, 114-220, 150-200, 171-195, or 200-221 of SEQ ID NO: 56. In certain embodiments, the transmembrane domain of the CAR comprises a CD8b polypeptide comprising or consisting of amino acids 171-195 of SEQ ID NO:56.

[0262] SEQ ID NO:56 is provided below: [ka]

[0263] In accordance with the subject matter of the present disclosure, a "CD8b nucleic acid molecule" refers to a polynucleotide that encodes a CD8b polypeptide. An exemplary nucleic acid sequence encoding amino acids 171-195 of SEQ ID NO:56 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:57, provided below. [ka] ICOS

[0264] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a native or modified transmembrane domain of an ICOS polypeptide. In certain embodiments, the ICOS polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:27 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the ICOS polypeptide comprises or consists of an amino acid sequence that is a continuous portion of SEQ ID NO:27 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 199 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the ICOS polypeptide comprises or consists of the amino acid sequence of amino acids 1-199, 1-50, 50-100, 100-150, 141-165, or 150-199 of SEQ ID NO: 27. In certain embodiments, the transmembrane domain of CAR comprises an ICOS polypeptide comprising or consisting of amino acids 141-165 of SEQ ID NO:27.

[0265] In accordance with the subject matter of the present disclosure, an "ICOS nucleic acid molecule" refers to a polynucleotide that encodes an ICOS polypeptide. An exemplary nucleic acid sequence encoding amino acids 141-165 of SEQ ID NO:27 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:58, provided below. [ka] CTLA-4

[0266] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a native or modified transmembrane domain of a CTLA-4 polypeptide. In certain embodiments, the CTLA-4 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO:59) having NCBI reference number: NP_005205.2 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CTLA-4 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO:59 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 223 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CTLA-4 polypeptide comprises or consists of amino acids 1-223, 1-50, 50-100, 100-150, 150-200, 162-186, or 200-223 of SEQ ID NO: 59. In certain embodiments, the transmembrane domain of the CAR comprises a CTLA-4 polypeptide comprising or consisting of amino acids 162-186 of SEQ ID NO:59.

[0267] SEQ ID NO:59 is provided below: [ka]

[0268] In accordance with the subject matter of the present disclosure, a "CTLA-4 nucleic acid molecule" refers to a polynucleotide that encodes a CTLA-4 polypeptide. An exemplary nucleic acid sequence encoding amino acids 162-186 of SEQ ID NO:59 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:60, provided below. [ka] ICAM-1

[0269] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a native or modified transmembrane domain of an ICAM-1 polypeptide. In certain embodiments, the ICAM-1 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 61) having NCBI reference number: NP_000192.2 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the ICAM-1 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 61 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 532 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the ICAM-1 polypeptide comprises or consists of the amino acid sequence of amino acids 1-532, 1-50, 50-100, 100-150, 150-200, 200-225, 250-300, 300-350, 350-400, 400-450, 481-507, 450-500, or 500-532 of SEQ ID NO: 61. In certain embodiments, the transmembrane domain of the CAR comprises an ICAM-1 polypeptide comprising or consisting of amino acids 481-507 of SEQ ID NO: 61.

[0270] SEQ ID NO:61 is provided below: [ka]

[0271] In accordance with the subject matter of the present disclosure, an "ICAM-1 nucleic acid molecule" refers to a polynucleotide that encodes an ICAM-1 polypeptide. An exemplary nucleic acid sequence encoding amino acids 481-507 of SEQ ID NO:61 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:62, provided below. [ka] Hinge / Spacer Region

[0272] In certain embodiments, the CAR further comprises a hinge / spacer region that connects the extracellular antigen-binding domain to the transmembrane domain. The hinge / spacer region can be sufficiently flexible so that the antigen-binding domain can be oriented in different directions to facilitate antigen recognition. In certain embodiments, the hinge / spacer region of the CAR can comprise a native or modified hinge region of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 peptide, an NKG2D peptide, a synthetic polypeptide (not based on a protein related to immune response), or a combination thereof. The hinge / spacer region can be a hinge region from IgG1, or an immunoglobulin CH2CH3 region, and a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 25), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 45, or a portion of SEQ ID NO: 46), a variation of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence. CD28

[0273] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of a CD28 polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises a CD28 polypeptide comprising or consisting of amino acids 114-152 of SEQ ID NO: 25. An exemplary nucleic acid sequence encoding amino acids 114-152 of SEQ ID NO: 25 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 63, provided below. [ka] CD84

[0274] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of a CD84 polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises a CD84 polypeptide comprising or consisting of the amino acid sequence of amino acids 187-225 of SEQ ID NO: 50. An exemplary nucleic acid sequence encoding amino acids 187-225 of SEQ ID NO: 50 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 64, provided below. [ka] CD166

[0275] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of a CD166 polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises a CD166 polypeptide comprising or consisting of amino acids 489-527 of SEQ ID NO: 52. An exemplary nucleic acid sequence encoding amino acids 489-527 of SEQ ID NO: 52 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 65, provided below. [ka]

[0276] In certain embodiments, the CD166 polypeptide comprised in the hinge / spacer region of a CAR of the disclosure comprises or consists of amino acids 484-527 of SEQ ID NO: 52. In certain embodiments, the hinge / spacer region of a CAR comprises a CD166 that comprises or consists of amino acids 506-527 of SEQ ID NO: 52. In certain embodiments, the hinge / spacer region of a CAR comprises a CD166 polypeptide that comprises or consists of amino acids 517-527 of SEQ ID NO: 52. In certain embodiments, the hinge / spacer region of a CAR comprises a CD166 polypeptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66 or SEQ ID NO: 67. [ka]

[0277] In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region and transmembrane domain of a CAR of the disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74. [ka] [ka] CD8a

[0278] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of a CD8a polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises a CD8a polypeptide comprising or consisting of amino acids 137-182 of SEQ ID NO: 54. An exemplary nucleic acid sequence encoding amino acids 137-182 of SEQ ID NO: 54 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 75, provided below. [ka] CD8b

[0279] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of a CD8b polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises a CD8b polypeptide comprising or consisting of amino acids 132-170 of SEQ ID NO: 56. An exemplary nucleic acid sequence encoding amino acids 132-170 of SEQ ID NO: 56 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 76, provided below. [ka] ICOS

[0280] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of an ICOS polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises an ICOS polypeptide comprising or consisting of amino acids 102-140 of SEQ ID NO: 27. An exemplary nucleic acid sequence encoding amino acids 102-140 of SEQ ID NO: 27 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 77, provided below. [ka] CTLA-4

[0281] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of a CTLA-4 polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises a CTLA-4 polypeptide comprising or consisting of amino acids 123-161 of SEQ ID NO: 59. An exemplary nucleic acid sequence encoding amino acids 123-161 of SEQ ID NO: 59 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 78, provided below. [ka] ICAM-1

[0282] In certain embodiments, the hinge / spacer region of a CAR of the disclosure comprises a native or modified hinge region of an ICAM-1 polypeptide described herein. In certain embodiments, the hinge / spacer region of a CAR comprises an ICAM-1 polypeptide comprising or consisting of amino acids 442-480 of SEQ ID NO:61. An exemplary nucleic acid sequence encoding amino acids 442-480 of SEQ ID NO:61 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:79, provided below. [ka]

[0283] In certain embodiments, the hinge / spacer region is located between the extracellular antigen binding domain and the transmembrane domain.In certain embodiments, the hinge / spacer region comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 peptide, an NKG2D peptide, a synthetic polypeptide (not based on a protein related to immune response), or a combination thereof.In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 peptide, an NKG2D peptide, a synthetic polypeptide (not based on a protein related to immune response), or a combination thereof.

[0284] In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from the same molecule. In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region of the CAR comprises a CD28 polypeptide, and the transmembrane domain of the CAR comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD28 polypeptide, and the transmembrane domain of the CAR comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD84 polypeptide, and the transmembrane domain of the CAR comprises a CD84 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD166 polypeptide, and the transmembrane domain of the CAR comprises a CD166 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD8a polypeptide, and the transmembrane domain of the CAR comprises a CD8a polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD8b polypeptide, and the transmembrane domain of the CAR comprises a CD8b polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD28 polypeptide and the transmembrane domain of the CAR comprises an ICOS polypeptide. 5.4.3.4. CAR Intracellular Signaling Domain A.CD3ζ

[0285] In certain non-limiting embodiments, the CAR comprises an intracellular signaling domain. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that can activate or stimulate a cell (e.g., a cell of lymphoid lineage, e.g., a T cell). Wild-type ("native") CD3ζ comprises three immunoreceptor tyrosine-based activation motifs ("ITAMs") (e.g., ITAM1, ITAM2, and ITAM3), three base-rich stretch (BRS) regions (BRS1, BRS2, ​​and BRS3), and transmits an activation signal to a cell (e.g., a cell of lymphoid lineage, e.g., a T cell) after antigen binding. The intracellular signaling domain of the native CD3ζ chain is the primary transmitter of a signal from an endogenous TCR. The CD3ζ used in the embodiments herein is not a native CD3ζ, but a modified CD3ζ. In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI reference number: NP_932170 (SEQ ID NO: 80) or a fragment thereof. In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 80 that is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 110, or at least 113, and up to 164 amino acids in length. Alternatively or additionally, in certain embodiments, the modified CD3 ζ polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 50, 50 to 100, 100 to 150, 50 to 164, 52 to 164, 55 to 164, or 150 to 164 of SEQ ID NO: 80. In certain embodiments, the modified CD3 ζ polypeptide comprises or consists of amino acids 52 to 164 of SEQ ID NO:80.

[0286] SEQ ID NO:80 is provided below: [ka]

[0287] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified human CD3 zeta polypeptide. In certain embodiments, the modified human CD3 zeta polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO: 81 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 81 is provided below: [ka]

[0288] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:81 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:82, provided below. [ka]

[0289] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified human CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to SEQ ID NO: 83 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 83 is provided below: [ka]

[0290] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:83 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:84, provided below. [ka] Immunoreceptor tyrosine-based activation motifs (ITAMs)

[0291] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising or consisting of one, two, or three ITAMs. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:85. [ka]

[0292] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:85 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:86, provided below. [ka]

[0293] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises or consists of a mutation of a tyrosine residue in ITAM1. In certain embodiments, the ITAM1 variant (e.g., a variant consisting of two loss-of-function mutations) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87 provided below. [ka]

[0294] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:87 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:88, provided below. [ka]

[0295] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:89, provided below. [ka]

[0296] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:89 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:90, provided below. [ka]

[0297] In certain embodiments, the modified CD3 zeta polypeptide comprises an ITAM2 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3 zeta polypeptide comprises an ITAM2 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the ITAM2 variant (e.g., a variant consisting of two loss-of-function mutations) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 91, as provided below. [ka]

[0298] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:91 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:92, provided below. [ka]

[0299] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM3 comprising the amino acid sequence set forth in SEQ ID NO:93, provided below. [ka]

[0300] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:93 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:94, provided below. [ka]

[0301] In certain embodiments, the modified CD3 ζ polypeptide comprises an ITAM3 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3 ζ polypeptide comprises an ITAM3 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant (e.g., a variant consisting of two loss-of-function mutations) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95 provided below. [ka]

[0302] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:95 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:96, provided below. [ka]

[0303] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises, essentially consists of, or consists of an ITAM1 variant comprising one or more loss-of-function mutations, an ITAM2 variant comprising one or more loss-of-function mutations, an ITAM3 variant comprising one or more loss-of-function mutations, or a combination thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises an ITAM2 variant comprising one or more (e.g., two) loss-of-function mutations and an ITAM3 variant comprising one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises a native ITAM1, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and an ITAM3 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:85, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:91, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:95 (e.g., a construct designated "1XX"). In certain embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:83.

[0304] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant comprising one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant comprising two loss-of-function mutations, a native ITAM2, and an ITAM3 variant comprising two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant consisting of the amino acid sequence set forth in SEQ ID NO:87, a native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO:89, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:95 (e.g., a construct designated "X2X").

[0305] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant comprising one or more (e.g., two) loss-of-function mutations, and an ITAM2 variant comprising one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises an modified CD3ζ polypeptide comprising an ITAM1 variant comprising or consisting of two loss-of-function mutations, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises an modified CD3ζ polypeptide comprising an ITAM1 variant consisting of the amino acid sequence set forth in SEQ ID NO:87, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:91, and a native ITAM3 consisting of the amino acid sequence set forth in SEQ ID NO:93 (e.g., a construct designated "XX3").

[0306] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant comprising one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant comprising or consisting of two loss-of-function mutations, a native ITAM2, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant consisting of the amino acid sequence set forth in SEQ ID NO:87, a native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO:89, and a native ITAM3 consisting of the amino acid sequence set forth in SEQ ID NO:93 (e.g., a construct designated "X23").

[0307] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, a native ITAM2, and an ITAM3 variant comprising one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, a native ITAM2, and an ITAM1 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:85, a native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO:89, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:95 (e.g., a construct designated "12X").

[0308] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant comprising one or more (e.g., two) loss-of-function mutations, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:85, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:91, and a native ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:93 (e.g., a construct designated "1X3").

[0309] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising one or two ITAM deletions. In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM1 and ITAM2, for example, the modified CD3ζ polypeptide comprises a native ITAM3 or an ITAM3 variant, and does not comprise ITAM1 or ITAM2. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM3 consisting of the amino acid sequence set forth in SEQ ID NO:93, and does not comprise ITAM1 (native or modified) or ITAM2 (native or modified) (e.g., the construct designated "D12").

[0310] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM2 and ITAM3, e.g., the modified CD3ζ polypeptide comprises a native ITAM1 or an ITAM1 variant, and does not comprise ITAM2 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:85, and does not comprise ITAM2 (native or modified) or ITAM3 (native or modified) (e.g., a construct designated "D23").

[0311] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM1 and ITAM3, e.g., the modified CD3ζ polypeptide comprises a native ITAM2 or an ITAM2 variant, and does not comprise ITAM1 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO:89, and does not comprise ITAM1 (native or modified) or ITAM3 (native or modified) (e.g., a construct designated "D13").

[0312] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM1, for example, the modified CD3ζ polypeptide comprises native ITAM2 or an ITAM2 variant, and native ITAM3 or an ITAM3 variant, and does not comprise ITAM1 (native or modified).

[0313] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM2, for example, the modified CD3ζ polypeptide comprises native ITAM1 or an ITAM1 variant, and native ITAM3 or an ITAM3 variant, and does not comprise ITAM2 (native or modified).

[0314] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM3, for example, the modified CD3ζ polypeptide comprises native ITAM1 or an ITAM1 variant, and native ITAM2 or an ITAM2 variant, and does not comprise ITAM3 (native or modified). B. Costimulatory Signaling Regions

[0315] In certain embodiments, the intracellular signaling domain of the CAR further comprises at least a costimulatory signaling region.In certain embodiments, at least one costimulatory signaling region comprises the intracellular domain of a costimulatory molecule (e.g., a costimulatory molecule disclosed in Section 5.2.2).In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, ICOS, DAP-10, CD2, and NKG2D.

[0316] At least one costimulatory signaling region may comprise a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a CD2 polypeptide, an NKG2D polypeptide, or a combination thereof. At least one costimulatory signaling region may comprise an intracellular domain or a portion thereof of CD28, an intracellular domain or a portion thereof of 4-1BB, an intracellular domain or a portion thereof of OX40, an intracellular domain or a portion thereof of ICOS, an intracellular domain or a portion thereof of DAP-10, an intracellular domain or a portion thereof of CD27, an intracellular domain or a portion thereof of CD40, an intracellular domain or a portion thereof of CD2, an intracellular domain or a portion thereof of NKG2D, or a combination thereof.

[0317] The costimulatory molecule can bind to a costimulatory ligand (e.g., a costimulatory ligand disclosed in Section 5.2.1). As an example, 4-1BB ligand (i.e., 4-1BBL) binds to a CAR in combination with a CAR signal. +It can bind to 4-1BB (also known as "CD137") to provide an intracellular signal that induces effector cell function of T cells. CARs that contain an intracellular signaling domain that includes a costimulatory signaling region that includes a 4-1BB polypeptide, an ICOS polypeptide, or a DAP-10 polypeptide are disclosed in US 7,446,190, the entirety of which is incorporated herein by reference.

[0318] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of CD28). In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:25 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:25 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 180-220, 180-219, or 200-220 of SEQ ID NO:25. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO:25. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of amino acids 180-219 of SEQ ID NO:25.

[0319] In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to SEQ ID NO: 101 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 101 (or amino acids 180-220 of SEQ ID NO: 25). SEQ ID NO: 101 is provided below: [ka]

[0320] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:101 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:102, provided below. [ka]

[0321] In certain embodiments, the intracellular signaling domain of the CAR comprises a deimmunized intracellular domain of human CD28 or a portion thereof. In certain embodiments, the deimmunized intracellular domain of human CD28 or a portion thereof comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to SEQ ID NO: 103 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 103. SEQ ID NO: 103 is provided below: [ka]

[0322] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence (SEQ ID NO: 97) having NCBI reference number: NP_031668.3 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 97 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, a CD28 polypeptide comprises or consists of an amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 114-220, 150-200, 178-218, or 200-218 of SEQ ID NO: 97. In certain embodiments, a costimulatory signaling region of a CAR of the disclosure comprises a CD28 polypeptide comprising or consisting of amino acids 178-218 of SEQ ID NO:97.

[0323] SEQ ID NO:97 is provided below: [ka]

[0324] An exemplary nucleic acid sequence encoding amino acids 178-218 of SEQ ID NO:97 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:98, provided below. [ka]

[0325] In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO:99 or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:99. SEQ ID NO:99 is provided below: [ka]

[0326] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:99 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:100, provided below. [ka]

[0327] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises two costimulatory molecules, for example, the costimulatory signaling region of CD28 and 4-1BB, or the costimulatory signaling region of CD28 and OX40.

[0328] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:24 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions.

[0329] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of 4-1BB or a portion thereof comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to SEQ ID NO:3 or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3.

[0330] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:3 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:104, provided below. [ka]

[0331] The OX40 polypeptide may comprise or have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:26 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions.

[0332] An ICOS polypeptide may comprise or have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:27 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions.

[0333] In certain embodiments, the CAR of the present disclosure further comprises an inducible promoter for expressing the nucleic acid sequence in a human cell. The promoter for use in expressing the CAR gene can be a constitutive promoter, such as the ubiquitin C (UbiC) promoter.

[0334] In certain embodiments, mutation sites and / or junctions between domains / motifs / regions of CARs derived from different proteins are deimmunized. The immunogenicity of the junctions between different CAR parts can be predicted using the NetMHC 4.0 server. For each peptide containing at least one amino acid from the following parts, the binding affinity to HLA A, B, and C can be predicted for all alleles. A score of immunogenicity of each peptide can be assigned for each peptide. The immunogenicity score is calculated as: Immunogenicity score = [(50-binding affinity) * HLA frequency] n The formula can be used to calculate the predicted number of peptides: 5.4.3.5. Example CAR 1928z CAR

[0335] In certain embodiments, the cell comprises a CAR comprising a fusion polypeptide of the disclosure and an extracellular antigen-binding domain that binds CD19 (e.g., human CD19), a transmembrane domain comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., a transmembrane domain of CD28 (e.g., human CD28) or a portion thereof), an intracellular signaling domain comprising a CD3ζ polypeptide (e.g., a human CD3ζ polypeptide), and a costimulatory signaling domain comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., an intracellular domain of CD28 (e.g., human CD28) or a portion thereof). In certain embodiments, the CAR is represented as "1928z". In certain embodiments, the CAR (e.g., 1928z) comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 105, provided below. In certain embodiments, a CAR (e.g., 1928z) comprises the amino acid sequence set forth in SEQ ID NO: 105. A CAR comprising the amino acid sequence set forth in SEQ ID NO: 105 can bind to CD19 (e.g., human CD19). [ka]

[0336] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:105 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:106, provided below. [ka] [ka]

[0337] In certain embodiments, the CAR further comprises a CD8 leader. In certain embodiments, the CD8 leader comprises or has the amino acid sequence set forth in SEQ ID NO: 13.

[0338] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:13 is set forth in SEQ ID NO:107, provided below. [ka] 1928z-1xx CAR

[0339] In certain embodiments, a CAR of the disclosure comprises an extracellular antigen-binding domain that binds a CD19 polypeptide (e.g., a human CD19 polypeptide), a transmembrane domain comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., the transmembrane domain of CD28 (e.g., human CD28) or a portion thereof), a hinge / spacer region derived from a CD28 polypeptide (e.g., human CD28), an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide) comprising a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations, and a costimulatory signaling region comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., the intracellular domain of CD28 (e.g., human CD28) or a portion thereof). In certain embodiments, a CAR is designated as "1928z-1xx." In certain embodiments, a CAR (e.g., 1928z-1xx) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 109 provided below. In certain embodiments, a CAR (e.g., 1928z-1xx) comprises the amino acid sequence set forth in SEQ ID NO: 109. A CAR comprising the amino acid sequence set forth in SEQ ID NO: 109 can bind to CD19 (e.g., human CD19). [ka] [ka]

[0340] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:109 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:110, provided below. [ka]

[0341] In certain embodiments, the CAR further comprises a CD8 leader. In certain embodiments, the CD8 leader comprises or has the amino acid sequence set forth in SEQ ID NO: 13.

[0342] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:13 comprises or consists of the nucleotide sequence set forth in SEQ ID NO:107. 5.4.4. TCR-like fusion molecules

[0343] In certain embodiments, antigen-recognizing receptor is a TCR-like fusion molecule.Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptor (also known as "HIT-CAR", for example, as disclosed in International Patent Application No. PCT / US19 / 017525, the entirety of which is incorporated herein by reference) and T cell receptor fusion construct (TRuC) (for example, as disclosed in Baeuerle et al., "Synthetic TRuC receptors engage the complete T cell receptor for potent anti-tumor response", Nature Communications volume 10, Article number: 2087 (2019), the entirety of which is incorporated herein by reference).

[0344] In certain embodiments, the TCR-like fusion molecule that binds to an antigen in an HLA-independent manner comprises an antigen-binding chain that comprises an extracellular antigen-binding domain and a constant domain. In certain embodiments, the constant domain comprises a T cell receptor constant region selected from the group consisting of a native or modified TRAC peptide, a native or modified TRBC peptide, a native or modified TRDC peptide, a native or modified TRGC peptide, and any variant or functional fragment thereof. In certain embodiments, the constant domain comprises a native or modified TRAC peptide. In certain embodiments, the constant domain comprises a native or modified TRBC peptide. In certain embodiments, the constant domain is capable of forming a homodimer or heterodimer with another constant domain. In certain embodiments, the antigen-binding chain is capable of associating with a CD3ζ polypeptide. In certain embodiments, the antigen-binding chain is capable of activating the CD3ζ polypeptide associated with the antigen-binding chain upon binding to an antigen. In certain embodiments, the activation of the CD3ζ polypeptide is capable of activating an immune response cell. In certain embodiments, the TCR-like fusion molecule is capable of associating with the CD3 complex and providing HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding portion of an antibody or fragment thereof, or an antigen-binding portion of a TCR. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises one or two immunoglobulin variable regions. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a heavy chain variable region (V) of an antibody. H In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the light chain variable region of an antibody (V LIn certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. H Including V H is the V of the antibody L In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain comprising the V of an antibody to form a variable fragment (Fv). L Including V L is the V of the antibody H and can dimerize with another extracellular antigen-binding domain comprising the variable fragment (Fv).

[0345] The TCR-like fusion molecule may bind to a tumor antigen or a pathogen antigen. In certain embodiments, the TCR-like fusion molecule binds to a tumor antigen. In certain embodiments, the TCR-like fusion molecule binds to CD19. In certain embodiments, the TCR-like fusion molecule binds to human CD19. In certain embodiments, the TCR-like fusion molecule is designated as "19-HIT". In certain embodiments, the TCR-like fusion molecule (e.g., 19-HIT) comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 111 provided below. In certain embodiments, the TCR-like fusion molecule (e.g., 19-HIT) comprises an amino acid sequence set forth in SEQ ID NO: 111. The TCR fusion molecule comprising the amino acid sequence set forth in SEQ ID NO: 111 can bind to CD19 (e.g., human CD19). [ka]

[0346] In certain embodiments, the cells comprise a TCR-like fusion molecule and a fusion polypeptide of the present disclosure. In certain embodiments, the cells express high levels of the fusion polypeptide. 5.5. Compositions and Vectors

[0347] The present disclosure subject matter provides compositions comprising a fusion polypeptide of the present disclosure (e.g., as disclosed in Section 5.2) and an antigen recognition receptor of the present disclosure (e.g., as disclosed in Section 5.4). Cells comprising such compositions are also provided.

[0348] In certain embodiments, the fusion polypeptide is operably linked to a first promoter. In certain embodiments, the antigen recognition receptor is operably linked to a second promoter.

[0349] Additionally, the present disclosures subject matter provides nucleic acid compositions comprising a first polynucleotide encoding a fusion polypeptide of the present disclosure (e.g., as disclosed in Section 5.2) and a second polynucleotide encoding an antigen recognition receptor of the present disclosure (e.g., as disclosed in Section 5.4). Cells comprising such nucleic acid compositions are also provided.

[0350] In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the fusion polypeptide. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the antigen recognition receptor.

[0351] In certain embodiments, one or both of the first and second promoters are endogenous or exogenous.

[0352] In certain embodiments, the exogenous promoter is selected from the group consisting of elongation factor (EF)-1 promoter, CMV promoter, SV40 promoter, PGK promoter, and metallothionein promoter.In certain embodiments, one or both of the first and second promoters are inducible promoters.In certain embodiments, the inducible promoter is selected from the group consisting of NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

[0353] In certain embodiments, the antigen recognition receptor is a TCR, and the fusion polypeptide is operably linked to a first promoter capable of inducing stable high expression levels of the fusion polypeptide.

[0354] In certain embodiments, the antigen recognition receptor is a TCR-like fusion molecule (e.g., HIT CAR), and the fusion polypeptide is operably linked to a first promoter capable of inducing stable high expression levels of the fusion polypeptide.

[0355] In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor, and the expression of the chimeric antigen receptor and the fusion polypeptide is under the control of endogenous promoter.Non-limiting examples of endogenous promoter include endogenous TRAC promoter, endogenous TRBC promoter, endogenous TRDC promoter, and endogenous TRGC promoter.In certain embodiments, the endogenous promoter is endogenous TRAC promoter.

[0356] The compositions and nucleic acid compositions can be administered to a subject or delivered into cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be achieved by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, retroviral vectors (either gamma-retroviruses or lentiviruses) are used for the introduction of nucleic acid compositions into cells. For example, a first polynucleotide encoding a fusion polypeptide and a second polynucleotide encoding an antigen-recognizing receptor can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from the long terminal repeat of the retrovirus, or from a promoter specific to the target cell type of interest. Non-viral vectors can be used as well.

[0357] For the initial genetic modification of cells to contain an antigen-recognizing receptor (e.g., CAR, TCR, or TCR-like fusion molecule), retroviral vectors are generally used for transduction, but any other suitable viral vector or non-viral delivery system may be used. The antigen-recognizing receptor and the fusion polypeptide may be constructed in a single multicistronic expression cassette, in multiple expression cassettes in a single vector, or in multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis Virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Also suitable is the combination of a retroviral vector and an appropriate packaging system, where the capsid protein is functional to infect human cells. A variety of amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114 or GALV envelopes, and any others known in the art, are also suitable. In certain embodiments, the P2A peptide comprises or has the amino acid sequence set forth in SEQ ID NO: 108, provided below: [ka]

[0358] Possible methods of transduction include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culture with viral supernatant alone or concentrated vector stocks, with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0359] Other transduction virus vectors can be used to modify cells.In certain embodiments, the selected vector shows high efficiency infection and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997).Other viral vectors that may be used include, for example, adenovirus, lentivirus, and adeno-associated virus vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S- 83S, 1995). Retroviral vectors have been particularly well developed and used in clinical practice (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0360] Non-viral approaches can also be used to genetically modify cells. For example, nucleic acid molecules can be administered in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987;Ono et al., Neuroscience Letters 17:259, 1990;Brigham et al., Am. J. Med. Sci. 298:278, 1989;Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988;Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for delivery of DNA into cells. Transplantation of normal genes into diseased tissues of subjects can also be achieved by ex vivo transfer of normal nucleic acid into culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into targeted tissues or systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0361] Any targeted genome editing method can also be used to deliver the fusion polypeptide and / or antigen recognition receptor of the present disclosure to cell or subject.In certain embodiments, CRISPR system is used to deliver the fusion polypeptide and / or antigen recognition receptor of the present disclosure.In certain embodiments, zinc finger nuclease is used to deliver the fusion polypeptide and / or antigen recognition receptor of the present disclosure.In certain embodiments, TALEN system is used to deliver the fusion polypeptide and / or antigen recognition receptor of the present disclosure.

[0362] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, this system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct section of host DNA, along with a region that binds to tracrRNA (usually in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cell repair process, allowing the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid to transfect target cells. The crRNA needs to be designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell. A CAR expression cassette carrying the repair template also needs to be designed for each application, since it must overlap the sequences on either side of the cut and must code for the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be spliced ​​together with the Cas9 gene and made into a plasmid to be transfected into cells.

[0363] Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are generated by combining zinc finger DNA binding domains with DNA cleavage domains. Zinc finger domains can be engineered to target specific DNA sequences, allowing zinc finger nucleases to target desired sequences in the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating new zinc finger domains is to combine zinc finger "modules" of lesser known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type II restriction endonuclease FokI. Using endogenous homologous recombination (HR) machinery and a CAR expression cassette carrying a homologous DNA template, ZFNs can be used to insert a CAR expression cassette into a genome. Once the targeted sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template and then copies the sequence of the template between the two cut ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0364] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems work on much the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a repeating motif of 33-34 amino acids with two variable positions that have strong recognition for specific nucleotides. By assembling an array of these TALEs, the DNA binding domain of the TALEs can be engineered to bind to a desired DNA sequence, thereby guiding the nuclease to cleave at a specific location in the genome. cDNA expression for use in polynucleotide therapy can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of nucleic acid.The enhancers used can include, without limitation, those characterized as tissue-specific or cell-specific enhancers.Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by homologous regulatory sequences, or, if desired, by regulatory sequences derived from heterologous sources that contain any of the above promoters or regulatory elements.

[0365] The method for delivering genome editing agent / system can vary depending on the need. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered via viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, envelope protein pseudotype of viral vector, cis- and trans-acting elements of replication-competent vector, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0366] The composition or nucleic acid composition of the present disclosure can be placed anywhere in genome.In certain embodiments, the composition or nucleic acid composition is placed in a locus in the genome of T cell, including but not limited to TRAC locus, TRBC locus, TRDC locus, and / or TRGC locus.In certain embodiments, the composition or nucleic acid composition is placed to disrupt the expression of endogenous T cell receptor.In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor (CAR).

[0367] In certain embodiments, the composition or nucleic acid composition is integrated into a locus encoding an immune inhibitory molecule, non-limiting examples of which include CTLA-4, PD-1, LAG3, BTLA, B7-1, B7-H1, B7-H3, B7-H4, TIM3, SHP-1, SHP-2, TIGIT, CD160, and LAIR1. 5.6. Polypeptides and Analogs

[0368] The subject matter of the present disclosure also includes polypeptides of the present disclosure (e.g., CD19, 4-1BB, CD80, CD28, CD3zeta, etc., or fragments thereof) that have been modified in a manner for a desired purpose, for example, to enhance their anti-neoplastic and / or anti-tumor activity when expressed in cells. The subject matter of the present disclosure provides methods for optimizing amino acid or nucleic acid sequences by generating changes in the sequence, as well as modified amino acid and nucleic acid sequences. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure further includes analogs of any of the polypeptides of the present disclosure. Analogs may differ from the polypeptides of the present disclosure by amino acid sequence differences, by post-translational modifications, or both. Analogs may exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homologous to all or a portion of an amino acid sequence of the subject matter of the present disclosure. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, e.g., at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Again, an exemplary approach for determining the degree of identity can use the BLAST program, e.g., -3 and e -100A probability score between indicates a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of the polypeptide, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogs may also differ from polypeptides by changes in the primary sequence. These include both natural and induced (e.g., resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate, or by site-directed mutagenesis, as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra) genetic variants. Also included are cyclized peptides, molecules, and analogs that contain residues other than L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids, such as β- or γ-amino acids.

[0369] In addition to full-length polypeptides, the subject matter of the present disclosure also provides fragments of any one of the polypeptides of the present disclosure. As used herein, the term "fragment" means at least 5, 10, 13, or 15 amino acids. In certain embodiments, the fragment comprises at least 20 consecutive amino acids, at least 30 consecutive amino acids, or at least 50 consecutive amino acids. In certain embodiments, the fragment comprises at least 60-80, 100, 200, 300, or more consecutive amino acids. Fragments can be generated by methods known to those of skill in the art, or can result from normal protein processing (e.g., removal of amino acids not required for biological activity from a nascent polypeptide, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0370] Non-protein analogs have chemical structures designed to mimic the functional activity of the proteins (e.g., fusion polypeptides) of the present disclosure. Such analogs may exceed the biological activity of the original polypeptide. Methods for analog design are well known in the art, and the synthesis of analogs can be carried out according to such methods by modifying the chemical structure so that the resulting analog increases the antineoplastic activity of the original polypeptide when expressed in cells. These chemical modifications include, but are not limited to, the substitution of alternative R groups and the variation of the degree of saturation at certain carbon atoms of the reference polypeptide. In certain embodiments, protein analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the following examples. Administration

[0371] The composition comprising the cells of the present disclosure can be provided systemically or directly to a subject to induce and / or enhance immune response to an antigen and / or treat and / or prevent neoplasms (e.g., cancer), pathogen infection, or infectious disease. In certain embodiments, the cells, compositions, or nucleic acid compositions of the present disclosure are directly injected into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the cells, compositions, or nucleic acid compositions of the present disclosure are provided indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., tumor vasculature). Expansion and differentiation agents can be provided before, during, or after administration of the cells, compositions, or nucleic acid compositions to increase the production of cells (e.g., T cells (e.g., CTL cells) or NK cells) in vitro or in vivo.

[0372] The cells, compositions, or nucleic acid compositions of the present disclosure may be administered by any suitable route, including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, or intraperitoneal. Typically, a final concentration of about 1×10 10 or more, reaching at least about 1 × 10 5A total of 100 cells are administered. The cells of the present disclosure may constitute a purified population of cells. One of skill in the art can easily determine the percentage of cells of the present disclosure in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable ranges of purity in a population containing cells of the present disclosure are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be readily adjusted by one of skill in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc. The composition of the present disclosure can be a pharmaceutical composition comprising the cells of the present disclosure or their precursors and a pharma- ceutically acceptable carrier. Administration can be autologous or heterologous. For example, cells, or precursors, can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood derived cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering the therapeutic composition of the subject of the present disclosure (e.g., pharmaceutical composition comprising the cells of the present disclosure), it can be formulated in an injectable unit dosage form (solution, suspension, emulsion).

[0373] In certain embodiments, the pharmaceutical composition further comprises a regulatory factor capable of regulating or modulating the expression and / or activity of a fusion polypeptide (eg, those disclosed in Section 5.2.4). 5.8. Formulation

[0374] The composition comprising the cells of the present disclosure can be conveniently provided as a sterile liquid preparation, for example, an isotonic aqueous solution, a suspension, an emulsion, a dispersion, or a viscous composition, which can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0375] Sterile injectable solutions can be prepared by incorporating the genetically modified cells in the required amount of an appropriate solvent, with various amounts of other ingredients as desired. Such compositions can be mixed with suitable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, and the like. The compositions can also be lyophilized. Depending on the desired route of administration and preparation, the compositions can contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, pigments, and the like. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is hereby incorporated by reference, can be consulted to prepare suitable preparations without undue experimentation.

[0376] Various additives that enhance the stability and sterility of the composition can be added, including antibacterial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used must be compatible with genetically modified cells or their precursors.

[0377] The composition may be isotonic, i.e., have the same osmotic pressure as blood and tears. The desired isotonicity of the composition may be achieved using sodium chloride or other pharma- ceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride may be particularly for buffers that contain sodium ions.

[0378] The viscosity of the composition can be maintained at a selected level using a pharma- ceutically acceptable thickening agent if desired. For example, methylcellulose is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickener can depend on the agent selected. The important point is to use an amount that achieves the selected viscosity. Obviously, the selection of suitable carriers and other additives depends on the exact route of administration and the nature of the particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or another liquid form, for example, a sustained release form or a liquid-filled form).

[0379] The amount of cells administered will vary depending on the subject being treated. In certain embodiments, about 10 4 pcs and about 10 10 Between pieces, about 105 pcs and about 10 9 Between or about 10 4 pcs and about 10 8 Between about 10 and 20 cells of the present disclosure are administered to a human subject. In certain embodiments, between about 10 and 20 cells of the present disclosure are administered to a human subject. 4 pcs and about 10 7 Between about 1×10 cells of the present disclosure are administered to a human subject. More effective cells may be administered in even smaller numbers. In certain embodiments, at least about 1×10 4 The cells of the present disclosure are administered to a human subject. In certain embodiments, at least about 1×10 5 The cells of the present disclosure are administered to human subjects.The exact determination of what is considered to be an effective dose can be based on individual factors for each subject, including their size, age, sex, weight, and the condition of a particular subject.Dosage can be easily ascertained by those skilled in the art from this disclosure and knowledge in the field.

[0380] One skilled in the art can readily determine the amount of cells and optional additives, vehicles, and / or carriers in the compositions and administered in the methods. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of 0.001 to 50% (by weight) solution in phosphate buffered saline, and the active ingredient is present on the order of micrograms to milligrams, e.g., about 0.0001 to about 5 wt%, about 0.0001 to about 1 wt%, about 0.0001 to about 0.05 wt%, or about 0.001 to about 20 wt%, about 0.01 to about 10 wt%, or about 0.05 to about 5 wt%. For any composition administered to animals or humans, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice; the dosage of the composition, the concentration of the components therein, and the timing of administering the composition that elicits an appropriate response. Such determinations do not require undue experimentation from the knowledge of those skilled in the art, this disclosure, and documents cited herein. And the time for sequential administration can be ascertained without undue experimentation. 5.9. Treatment Method

[0381] The subject matter of the present disclosure provides a method for inducing and / or increasing an immune response in a subject in need thereof. The cells, compositions, and nucleic acid compositions of the present disclosure may be used in therapy or medicine. The cells of the present disclosure and compositions comprising the same may be used to treat and / or prevent a neoplasm in a subject. The cells, compositions, and nucleic acid compositions of the present disclosure may be used to prolong the survival of a subject suffering from a neoplasm. The cells, compositions, and nucleic acid compositions of the present disclosure may also be used to treat and / or prevent a neoplasm in a subject. In certain embodiments, the neoplasm is cancer. The cells, compositions, and nucleic acid compositions of the present disclosure may also be used to treat and / or prevent a pathogen infection or other infectious disease in a subject, for example, an immunocompromised human subject. The cells, compositions, and nucleic acid compositions of the present disclosure may also be used to treat and / or prevent an autoimmune disease in a subject. Such methods include administering the cells of the present disclosure, the compositions of the present disclosure (e.g., pharmaceutical compositions), or the nucleic acid compositions of the present disclosure in an effective amount to achieve the desired effect, which is the alleviation of an existing condition or the prevention of recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be provided in a single dose or a series of doses. An effective amount can be provided in a bolus or by continuous perfusion.

[0382] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce beneficial or desired clinical results upon treatment. An effective amount may be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or slow the progression of a disease, or to otherwise reduce the pathological consequences of a disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of a person of ordinary skill in the art. Typically, several factors are considered when determining the appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells administered.

[0383] For adoptive immunotherapy using antigen-specific T cells, approximately 6 ~10 10 range (e.g., about 10 9 A cell dose of approximately 10 ...

[0384] The subject matter of the present disclosure provides a method for treating and / or preventing a neoplasm in a subject, which may include administering to a subject having a neoplasm an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure.

[0385] Non-limiting examples of neoplasms include blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, throat cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and miscellaneous carcinomas (including prostate cancer and small cell lung cancer). Suitable cancers include astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovium, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, and pulmonary carcinoma. The tumor types include, but are not limited to, any of those known in the field of oncology, including, but not limited to, follicular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors, such as ductal and lobular adenocarcinoma, squamous cell and adenocarcinoma of the cervix, uterine and ovarian epithelial carcinoma, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphoma (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma. In certain embodiments, the neoplasm is cancer. In certain embodiments, the neoplasm is selected from the group consisting of blood cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and throat cancer.In certain embodiments, the cells, compositions, and nucleic acid compositions of the present disclosure may be used to treat and / or prevent hematological cancers (e.g., leukemia, lymphoma, and myeloma) or ovarian cancers that are not amenable to conventional therapeutic intervention. In certain embodiments, the cells, compositions, and nucleic acid compositions of the present disclosure may be used to treat and / or prevent solid tumors.

[0386] The subject matter of the present disclosure provides a method for treating and / or preventing a viral infection in a subject. The method may include administering an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure to a subject having a viral infection. Non-limiting examples of viral infections include viral infections caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis A, B, C, D, E, F or G, human immunodeficiency virus (HIV), adenovirus, BK polyomavirus, coronavirus, coxsackievirus, poliovirus, herpes simplex type 1, herpes simplex type 2, human cytomegalovirus, human herpes virus type 8, varicella zoster virus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, papillomavirus, rabies virus, and rubella virus.Other viral targets include Paramyxoviridae (e.g., Pneumoviruses, Morbilliviruses, Metapneumoviruses, Respiroviruses, or Rubulaviruses), Adenoviridae (e.g., Adenoviruses), Arenaviridae (e.g., Arenaviruses, e.g., Lymphocytic Choriomeningitis Virus), Arteriviridae (e.g., Porcine Reproductive and Respiratory Syndrome Virus or Equine Arteritis Virus), Bunyaviridae (e.g., Phleboviruses or Hantaviruses), Caliciviridae (e.g., Norwalk Virus), Coronaviridae (e.g., Coronaviruses or Toroviruses), Filoviridae (e.g., Ebola-like viruses), Flaviviridae (e.g., Hepaciviruses or Flaviviruses), Herpesviridae (e.g., simplexvirus, varicellovirus, cytomegalovirus, roseolovirus, or lymphocryptovirus), Orthomyxoviridae (e.g., influenza virus or thogotovirus), Parvoviridae (e.g., parvovirus), Picomaviridae (e.g., enterovirus or hepatovirus), Poxviridae (e.g., orthopoxvirus, avipoxvirus, or leporipoxvirus), Retroviridae (e.g., lentivirus or spumavirus), Reoviridae (e.g., rotavirus), Rhabdoviridae (e.g., lyssavirus, novirhabdovirus, or vesiculovirus), and Togaviridae (e.g., alphavirus or rubivirus). In certain embodiments, the viral infection includes human respiratory coronavirus, influenza viruses A-C, hepatitis A-G, and herpes simplex viruses 1-9. In certain embodiments, the subject has an immunodeficiency.

[0387] The subject matter of the present disclosure provides a method for treating and / or preventing a bacterial infection in a subject. The method may include administering an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure to a subject having a bacterial infection. Bacterial infections include Mycobacteria, Rickettsia, Mycoplasma, Neisseria meningitides, Neisseria gonorrheoeae, Legionella, Vibrio cholerae, Streptococci, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Corynobacteria diphtheriae, Clostridium spp., enterotoxigenic Escherichia coli, Bacillus anthracis, Rickettsia, Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia, Mycobacterium leprae, Salmonella, shigella, Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacterium tuberculosis;Listeria monocytogenes;Mycoplasma spp., Pseudomonas fluorescens, Vibrio cholerae, Haemophilus influenzae, Bacillus anthracis, Treponema pallidum, Leptospira, Borrelia, Corynebacterium diphtheriae, Francisella, Brucella melitensis, Campylobacter jejuni, Enterobacter, Proteus mirabilis, Proteus, and Klebsiella pneumoniae.

[0388] The subject matter of the present disclosure provides a method for treating and / or preventing an autoimmune disease in a subject. The method may include administering an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure to a subject having an autoimmune disease.

[0389] The subject matter of the present disclosure provides a method for treating and / or preventing an autoimmune disease in a subject, which may include administering an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure to a subject having an infectious disease.

[0390] Non-limiting examples of autoimmune and inflammatory diseases or conditions thereof include arthritis, e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, ulcerative colitis, psoriasis, psoriatic arthritis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, systemic sclerosis, asthma, organ transplant rejection, transplant-related diseases or conditions, Takayasu's arteritis, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Behcet's syndrome, Wegener's granulomatosis, ANCA-vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, vasculitis of connective tissue diseases, Henoch-Schönlein purpura, cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, primary vasculitis of the CNS. , thromboangiitis obliterans, paraneoplastic arteritis, myelodysplastic syndrome, erythema elevatus, amyloidosis, autoimmune myositis, Guillain-Barré syndrome, histiocytosis, atopic dermatitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, Sjögren's syndrome, myelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, idiopathic bleomycin-induced pulmonary inflammation, cytarabine-induced pulmonary inflammation, autoimmune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atopic thyroiditis, Graves' disease, autoimmune polyendocrine syndrome, autoimmune Addison's syndrome, and / or myasthenia gravis. In accordance with the presently disclosed subject matter, the various methods can include administering a checkpoint immune blockade agent to the subject.

[0391] In certain embodiments, the checkpoint immune blockade is selected from the group consisting of anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-LAG3 antibody, anti-B7-H3 antibody, anti-TIM3 antibody, and combinations thereof. In certain embodiments, the checkpoint immune blockade is an anti-PD-L1 antibody or an anti-PD-1 antibody. In certain embodiments, the checkpoint immune blockade is an anti-PD-1 antibody.

[0392] According to the subject matter of the present disclosure, the various methods may include administering to a subject a regulator capable of regulating or modulating the expression, activity of the fusion polypeptide.

[0393] In certain embodiments, the regulatory factor is selected from the group consisting of a promoter capable of controlling expression of the fusion polypeptide, a molecule capable of regulating or modulating the expression and / or activity of a costimulatory ligand, and a molecule capable of regulating or modulating the expression and / or activity of a costimulatory molecule.

[0394] In certain embodiments, the molecule capable of regulating or modulating the expression and / or activity of a costimulatory ligand is selected from the group consisting of an antibody that binds to a costimulatory ligand and a fusion protein that binds to a costimulatory ligand and regulates or modulates the expression and / or activity of the costimulatory ligand.

[0395] In certain embodiments, the modulator is an anti-CD80 antibody and the fusion polypeptide comprises the extracellular and transmembrane domains of CD80.

[0396] In certain embodiments, the modulator is a fusion protein that binds to CD80 and modulates the activity of CD80, and the fusion polypeptide comprises the extracellular domain and transmembrane domain of CD80. In certain embodiments, the fusion protein is a CTLA-4 fragment that binds to CD80. In certain embodiments, the CTLA-4 fragment that binds to CD80 is abatacept or belatacept.

[0397] In certain embodiments, the molecule capable of regulating or modulating the expression or activity of a costimulatory molecule is selected from the group consisting of an antibody that binds to a costimulatory molecule, a fusion protein that binds to a costimulatory molecule and regulates or modulates the expression or activity of a costimulatory molecule.

[0398] In certain embodiments, the regulator can deplete cells. In certain embodiments, the regulator can reduce or eliminate one or more side effects associated with the administration of cells. In certain embodiments, the one or more side effects are selected from the group consisting of off-tumor target effects, cytokine release syndrome, neurotoxicity, and combinations thereof. In certain embodiments, the subject is a human.

[0399] The subject may have an active form of the disease, in which case the treatment goals may include reducing or reversing disease progression and / or ameliorating side effects. The subject may have a history of a condition that has already been treated, in which case the treatment goals typically include reducing or delaying the risk of recurrence.

[0400] Human subjects suitable for treatment typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are subjects that carry clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on tumor burden (e.g., by palpation, CAT scan, sonogram, mammogram, or X-ray; positive biochemical or histopathological markers alone are insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to elicit an anti-tumor response, with the goal of alleviating their condition. Ideally, a reduction in tumor burden results, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduction in the risk or rate of tumor progression, or a reduction in the pathological consequences of tumors.

[0401] The second group of suitable subjects is known in the art as the "adjuvant group". These are individuals who have a history of neoplasms but have been responsive to other forms of treatment. Previous treatments may include, but are not limited to, surgical resection, radiation therapy, and conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk for disease progression near the original tumor site or by metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on features observed before or after initial treatment. These features are known in the clinical field and are appropriately defined for each different neoplasm. Typical features for the high-risk subgroup are those in which the tumor invades adjacent tissues or those that show lymph node involvement.

[0402] Another group has a genetic predisposition to neoplasia, but has not yet demonstrated clinical signs of neoplasia.For example, a woman who tests positive for a genetic mutation associated with breast cancer, but is still of childbearing age, may wish to receive one or more of the cells described herein in a prophylactic treatment to prevent the development of neoplasia until it is appropriate to carry out prophylactic surgery.

[0403] As a result of the expression of antigen-recognition receptors that bind to tumor antigens and fusion polypeptides that enhance the antitumor effect of the cells, the adoptively transferred cells are endowed with improved and selective cytolytic activity at the tumor site. Furthermore, following their localization to the tumor or viral infection and their proliferation, T cells transform the tumor or viral infection site into a highly conductive environment for a wide range of immune cells (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages) that participate in the physiological antitumor or antiviral response.

[0404] Furthermore, the subject matter of the present disclosure provides a method for treating and / or preventing a pathogen infection (e.g., a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoan infection) in a subject, for example, in an immunocompromised subject. The method may include administering an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure to a subject having a pathogen infection. Exemplary viral infections susceptible to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infection.

[0405] Further modifications can be introduced into the cells (e.g., T cells) of the present disclosure to avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or outcomes similar to GvHD when healthy tissue expresses the same target antigen as tumor cells. A potential solution to this problem is to engineer a suicide gene into the cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can allow for T cell ablation by administering anti-EGFR monoclonal antibodies (e.g., cetuximab). The EGFRt can be covalently joined upstream of the antigen recognition receptor of the CAR of the present disclosure. The suicide gene can be included in a vector that includes a nucleic acid encoding the CAR of the present disclosure. In this approach, administration of a prodrug (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9) designed to activate a suicide gene during malignant T cell transformation (e.g., GVHD) induces apoptosis in CAR-expressing T cells in which the suicide gene has been activated. Incorporation of a suicide gene into the CAR of the present disclosure confers an additional level of safety with the ability to eliminate the majority of CAR T cells within a very short period of time. Cells (e.g., T cells) of the present disclosure that have an incorporated suicide gene can be preemptively eliminated at a given time point after CAR T cell infusion or eradicated at the earliest sign of toxicity.

[0406] Additionally, as disclosed in Section 5.2.4, expression and / or activity of the fusion polypeptide may be regulated or modulated by a regulator (e.g., those disclosed in Section 5.2.4). In certain embodiments, the regulator may deplete the cells of the disclosure, e.g., to reduce or eliminate side effect(s) associated with administration of the cells of the disclosure, e.g., off-tumor target effects, cytokine release syndrome, and / or neurotoxicity.

[0407] In certain embodiments, the methods or uses described herein further comprise administering a regulator capable of regulating or modulating the expression and / or activity of the fusion polypeptide (e.g., those disclosed in section 5.2.4). The regulator may be administered prior to, simultaneously with, or after administration (e.g., initial administration) of the cells of the present disclosure or compositions comprising the same. In certain embodiments, the methods or uses described herein further comprise administering an anti-CD80 antibody, and the fusion polypeptide comprised in the cells comprises the extracellular and transmembrane domains of CD80. In certain embodiments, the methods or uses described herein further comprise administering a fusion protein that binds to CD80 and modulates the activity of CD80, and the fusion polypeptide comprised in the cells comprises the extracellular and transmembrane domains of CD80. In certain embodiments, the fusion protein is, for example, a CTLA-4 fragment that binds to CD80. 5.10.Kit

[0408] The subject matter of the present disclosure provides a kit for inducing and / or enhancing immune response and / or treating and / or preventing neoplasm or pathogen infection in a subject.In certain embodiments, the kit comprises an effective amount of the cell of the present disclosure, the composition of the present disclosure, or the nucleic acid composition of the present disclosure.In certain embodiments, the kit comprises a sterile container; such container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such container can be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding medicine.In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule that encodes an antigen-recognizing receptor (e.g., CAR or TCR) for an antigen of interest, and an isolated nucleic acid molecule that encodes a fusion polypeptide in an expressible form, which can be optionally contained in the same or different vectors.

[0409] If desired, the cell, composition, or nucleic acid composition is provided with instructions for administering the cell, composition, or nucleic acid composition to a subject having or at risk of developing a neoplasm (e.g., cancer), or a pathogen infection (e.g., an infectious disease), or an immune disorder (e.g., an autoimmune disease). The instructions generally include information about the use of the cell, composition, or nucleic acid composition for the treatment and / or prevention of a neoplasm, or a pathogen infection (e.g., an infectious disease), or an immune disorder (e.g., an autoimmune disease). In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosing schedule and administration for the treatment or prevention of a neoplasm, a pathogen infection (e.g., an infectious disease), or an immune disorder (e.g., an autoimmune disease) or a symptom thereof; precautions for use; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. EXAMPLES

[0410] 6. Working Example The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the skill of those in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present disclosure and therefore may be considered in making and practicing the subject matter of the present disclosure. Particularly useful techniques for particular embodiments are discussed in the following sections.

[0411] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed cells and compositions, and are not intended to limit the scope of what the inventors regard as their invention. Example 1 CD80 / 4-1BB costimulatory molecule

[0412] The fusion polypeptide of the present disclosure was generated. The fusion polypeptide comprises the extracellular and transmembrane domains of human CD80 and the intracellular domain of human 4-1BB (represented as "CD80 / 4-1BB fusion polypeptide"). The CD80 / 4-1BB fusion polypeptide was transfected into T cells comprising a CAR targeting human CD19 ("1928z" or "1928z-1xx"), a CD19-targeting TCR-like fusion molecule that is an HLA-independent TCR receptor (HIT) (represented as "19-HIT"), or a NY-ESO-1 TCR. The expression of 1928z CAR with CD80 / 4-1BB fusion polypeptide, 1928z CAR without CD80 / 4-1BB fusion polypeptide, 1928z-1xx CAR with CD80 / 4-1BB fusion polypeptide, 1928z-1xx CAR without CD80 / 4-1BB fusion polypeptide, 19-HIT with CD80 / 4-1BB fusion polypeptide, 19-HIT without CD80 / 4-1BB fusion polypeptide, and NY-ESO-1 TCR with CD80 / 4-1BB fusion polypeptide or without CD80 / 4-1BB was measured. The FACS expression results are shown in Figures 2A, 2C, 2E and 2G. US Patent No. 9,220,728 discloses that exogenous costimulatory ligands (e.g., CD80, 4-1BBL) can enhance the cytotoxicity of CAR T cells. To compare the cytotoxicity of CAR T cells containing exogenous costimulatory ligands, mice bearing NALM6 CD19 leukemia cells were treated with (a) 10 CAR T cells containing 1928z and this CD80 / 4-1BB fusion polypeptide ("1928z-CD80 / 4-1BB"). 5 (b) 10 T cells containing 1928z and exogenous 4-1BBL molecules ("1928z-4-1BBL"). 5 or (c) 10 T cells containing 1928z and an exogenous CD80 molecule ("1928z-CD80"). 5The mice were treated with 1928z-CD80 / 4-1BB T cells. The survival rates of these mice were measured, and the results are shown in FIG. 2B. As shown in FIG. 2B, the CD80 / 4-1BB fusion polypeptide enhanced the cytotoxicity and / or antitumor activity of the T cells (e.g., increased the survival rate of mice treated with 1928z-CD80 / 4-1BB expressing T cells compared to mice treated with 1928z-4-1BBL expressing T cells or 1928z-CD80 expressing T cells).

[0413] Additionally, NALM6 CD19 leukemia cells were cultured in a 2×10 4-well plate containing (a) 19-28z-1xx and this CD80 / 4-1BB fusion polypeptide ("19-28z-1xx-CD80 / 4-1BB"); 4 (b) 2 × 10 T cells containing 19-28z-1xx 4 (c) 10 T cells containing 19-HIT 5 or (d) 10 T cells comprising 19-HIT and this CD80 / 4-1BB fusion polypeptide ("19-HIT-CD80 / 4-1BB"). 5 The survival rates of these mice were measured and the results are shown in Figures 2D and 2F. Additionally, mice bearing the SK-MEL-23 melanoma cell line expressing the HLAA2.1 / NYESO complex were treated with 2 x 10 T cells without the CD80 / 4-1BB fusion polypeptide. 6 10 NY-ESO-1 TCR T cells or 10 6 The mice were treated with NY-ESO-1 TCR T cells. The survival rates of these mice were measured and the results are shown in Figure 2H.

[0414] As shown in Figures 2D, 2F, and 2H, CD80 / 4-1BB fusion polypeptide improved survival rates compared to untreated mice and mice treated with T cells containing 19-28z-1xx CAR alone or T cells containing 19-HIT alone. The survival rates of mice treated with 19-HIT-CD80 / 4-1BB, in which 19-HIT and CD80 / 4-1BB fusion polypeptides were targeted to the TRAC locus and expressed under the control of the endogenous TRAC promoter, were likely due to low expression of the CD80 / 4-1BB fusion polypeptide. All results presented herein support the broad use of the CD80 / 4-1BB fusion polypeptides of the present disclosure to improve anti-tumor T cell responses. Example 2

[0415] Mice bearing NALM6 CD19 leukemia cells overexpressing the PDL-1 molecule were cultured in vitro with 5 × 10 4 T cells, 5 × 10 containing 19-28z-1xx with CD80 / 4-1BB fusion polypeptide 4 T cells, 10 containing 19-HIT without CD80 / 4-1BB fusion polypeptide 5 T cells or 10 containing 19-HIT with CD80 / 4-1BB fusion polypeptide 5 The mice were treated with 100 T cells. The survival rates of these mice were measured and the results are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, the CD80 / 4-1BB fusion polypeptide of the present disclosure can preserve T cell anti-tumor responses despite the presence of inhibitory molecules such as PD-1 present in the tumor microenvironment. Example 3

[0416] We investigated whether the expression level of CD80 / 4-1BB fusion polypeptide governs antitumor response efficiency. The data shown in Figures 4A and 4B highlight the fine-tuning regulation of T cell antitumor response, determined by the level of expression of CD80-41-BB. HIT and TCR-specific T cells benefit from high expression of CD80-4-1BB, whereas CAR T cells confer improved antitumor response when CD80-4-1BB is expressed under the endogenous TRAC promoter. Example 4

[0417] Whether the CD80 / 4-1BB fusion polypeptides of the present disclosure can compensate for the lack of endogenous 4-1BB was investigated. Mice bearing NALM6 CD19 leukemia cells were cultured with 2.5×10 4 T cells or 2.5 × 10 4 100 T cells were treated with 100 T cells. Endogenous 4-1BB gene knockout was obtained by electroporation of the protein CAS9 and specific gRNA. Disruption of endogenous 4-1BB was achieved in 68% of the injected CAR T cells. TRBC gRNA was used as a control. The survival rate of these mice was measured and the results are shown in FIG. 5. As shown in FIG. 5, although the combination of exogenous CD80 and exogenous 4-1BBL as well as the CD80 / 4-1BB fusion polypeptide provided a comparable antitumor effect of CAR T cells to the CD80 / 4-1BB fusion polypeptide, which provided better survival, the former (combination of exogenous CD80 and exogenous 4-1BBL) depends on the endogenous expression of the 4-1BB molecule, which is upregulated upon T cell activation, in contrast to the CD80 / 4-1BB fusion polypeptide. In the absence of endogenous 4-1BB, the CD80 / 4-1BB fusion polypeptide improved CAR T cell anti-tumor responses to levels similar to the CD80-4-1BBL TRBC knockout. Example 5

[0418] Next, we investigated whether disruption of the inhibitory molecule PD-1 would enhance anti-tumor responses in the presence of CD80-4-1BB. Mice bearing NALM6 CD19 leukemia cells were cultured with 2.5×10 4 100 T cells were treated with 100 T cells. Endogenous PD-1 gene knockout was obtained by electroporation of the protein CAS9 and specific gRNA. Disruption of PD-1 was achieved in 55% of the injected CAR T cells. TRBC gRNA was used as a control. The survival rate of these mice was measured and the results are shown in Figure 6. The data shown in Figure 6 demonstrate the feasibility of multiplexing genetic manipulation and highlight the benefit of KO PD-1 molecule in the context T cells expressing the CD80 / 4-1BB fusion polypeptide of the present disclosure. Example 6

[0419] CD4 + CAR T cells are CD8 + CAR T cell responses are known to be maintained. However, + To reduce side effects resulting from the presence of T cells, the inventors have demonstrated that the CD80 / 4-1BB fusion polypeptides of the present disclosure inhibit the expression of CD4 + We investigated whether it was possible to compensate for the absence of CAR T cells.

[0420] Mice bearing NALM6 CD19 leukemia cells were cultured with (a) 4×10 5 CD8 + T cells, (b) 4 × 10 containing 1928z and exogenous 4-1BBL molecules (“1928z-4-1BBL”). 5 CD8 + T cells, 8 × 10 containing (c)1928z-CD80 / 4-1BB 5 CD8 + T cells, or (d) 8 × 10 5 CD8+ The survival rates of these mice were measured and the results are shown in Figures 7A and 7B. As shown in Figures 7A and 7B, the CD80 / 4-1BB fusion polypeptide of the present disclosure exhibited optimal CD8 + By providing the costimulation needed for CAR T cell responses, CD4 + Compensated for the absence of T cells. Embodiments of the Subject Matter of the Disclosure

[0421] From the above description, it is apparent that changes and modifications may be made to the subject matter of the present disclosure to adapt it to various uses and conditions, such embodiments also falling within the scope of the following claims.

[0422] The recitation of an index of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0423] All patents and publications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference into this specification. In certain embodiments, for example, the following are provided: (Item 1) a) the extracellular and transmembrane domains of a costimulatory ligand, and b) the intracellular domain of the first costimulatory molecule A fusion polypeptide comprising: (Item 2) 2. The fusion polypeptide of item 1, wherein the costimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof. (Item 3) 3. The fusion polypeptide of item 2, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. (Item 4) 3. The fusion polypeptide of item 2, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof. (Item 5) 5. The fusion polypeptide of any one of items 1, 2, and 4, wherein the costimulatory ligand is CD80. (Item 6) 6. The fusion polypeptide of item 5, wherein the extracellular domain of CD80 comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:1. (Item 7) 7. The fusion polypeptide according to item 5 or 6, wherein the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or a functional fragment thereof. (Item 8) 8. The fusion polypeptide of any one of items 5 to 7, wherein the transmembrane domain of CD80 comprises an amino acid sequence that is at least about 85% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2. (Item 9) 9. The fusion polypeptide according to any one of items 5 to 8, wherein the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof. (Item 10) 10. The fusion polypeptide of any one of items 1 to 9, wherein the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. ...

Claims

1. a) the extracellular and transmembrane domains of CD80, and b) The intracellular domain of 4-1BB A fusion polypeptide comprising:

2. a) the extracellular domain of CD80 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1; b) the extracellular domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or a functional fragment thereof; c) the transmembrane domain of CD80 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2; d) the transmembrane domain of CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or a fragment thereof; e) the intracellular domain of 4-1BB comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3; and / or f) The fusion polypeptide of claim 1, wherein the intracellular domain of 4-1BB comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 or a functional fragment thereof.

3. a) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4; or b) A fusion polypeptide according to claim 2, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

4.

4. The fusion polypeptide according to any one of claims 1 to 3, further comprising the intracellular domain of CD28.

5. a) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6; or b) A fusion polypeptide according to claim 4, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

6.

6. 6. The fusion polypeptide of any one of claims 1 to 5, further comprising a signaling domain of a cytokine receptor selected from the group consisting of CD121a, CDw121b, IL-18Ra, IL18Rb, CD122, CD25, CD132, CD124, CD213a13, CD127, IL-9R, IL15Ra, CDw125, CDw131, CD126, CD130, IL11Ra, Cd114, CD212, CD4, CDw217, CD118, and CDw119.

7. A nucleic acid encoding a fusion polypeptide according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid of claim 7.

9. A cell comprising the fusion polypeptide according to any one of claims 1 to 6, the nucleic acid according to claim 7, or the vector according to claim 8.

10. The cell of claim 9 , further comprising an antigen recognition receptor that binds to an antigen.

11. The cell of claim 10 , wherein the antigen is a tumor antigen or a pathogen antigen.

12. The tumor antigen is CD19, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (Ep CAM), receptor tyrosine protein kinase Erb-B2, 3, 4 (erb-B2, 3, 4), folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES 12. The cell of claim 11, wherein the cell is selected from O-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, tumor associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB.

13. The cells, a) cells of lymphoid or myeloid lineage; b) T cells; c) T cells derived from induced pluripotent stem cells; d) CD8 ＋ T cells; e) CD8, which is CD4-independent ＋ T cells; or f) The cell according to any one of claims 9 to 12, which is a T cell selected from the group consisting of cytotoxic T lymphocytes (CTL), γδ T cells, tumor infiltrating lymphocytes (TIL), regulatory T cells, and natural killer T (NKT) cells.

14. The cell according to any one of claims 9 to 13, which is autologous or allogeneic.

15. A pharmaceutical composition comprising an effective amount of the cells according to any one of claims 9 to 14, further comprising a pharma- ceutically acceptable excipient.

16. a regulatory factor capable of regulating or modulating the expression and / or activity of said fusion polypeptide, The regulator is selected from the group consisting of a promoter capable of controlling the expression of the fusion polypeptide, a molecule capable of regulating or modulating the expression and / or activity of a costimulatory ligand, and a molecule capable of regulating or modulating the expression and / or activity of a costimulatory molecule. the modulator is an anti-CD80 antibody and the fusion polypeptide comprises the extracellular and transmembrane domains of CD80; and / or The pharmaceutical composition of claim 15, wherein the modulator is a fusion protein that binds to CD80 and modulates an activity of CD80, the fusion polypeptide comprising the extracellular and transmembrane domains of CD80.

17. the molecule capable of regulating or modulating expression and / or activity of the costimulatory ligand is selected from the group consisting of an antibody that binds to the costimulatory ligand, and a fusion protein that binds to the costimulatory ligand and regulates or modulates expression and / or activity of the costimulatory ligand; and / or 17. The pharmaceutical composition of claim 16, wherein the molecule capable of regulating or modulating expression and / or activity of the costimulatory molecule is selected from the group consisting of an antibody that binds to the costimulatory molecule and a fusion protein that binds to the costimulatory molecule and regulates or modulates expression and / or activity of the costimulatory molecule.

18. The pharmaceutical composition of claim 17, wherein the fusion protein comprises a CTLA-4 fragment that binds to CD80.

19. A nucleic acid composition comprising: a) a first polynucleotide encoding a fusion polypeptide according to any one of claims 1 to 6; and b) a second polynucleotide encoding an antigen-recognizing receptor that binds to an antigen.

20. 20. The nucleic acid composition of claim 19, further comprising a first promoter operably linked to the fusion polypeptide and / or a second promoter operably linked to the antigen recognition receptor.

21. A cell comprising the nucleic acid composition of claim 19 or 20.

22. A method for producing a cell, the method comprising introducing into the cell a nucleic acid according to claim 7 or a vector according to claim 8.

23. A kit comprising the nucleic acid according to claim 7, the vector according to claim 8, the cell according to any one of claims 9 to 14 and 21, or the nucleic acid composition according to claim 19 or 20.

24. 24. The pharmaceutical composition according to any one of claims 15 to 23 for use in reducing tumor burden, treating and / or preventing a neoplasm and / or treating a subject with recurrence of a neoplasm and / or treating and / or preventing an autoimmune or infectious disease.

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