Fusion constructs and methods of use thereof

Abstract

Provided herein is a fusion protein comprising an anti-PD1 antibody, or a fragment / variant thereof, and a TGF-β trap, or a composition comprising the fragment or variant thereof. Provided herein is a fusion protein comprising an anti-PD1 antibody, or a fragment / variant thereof, and an ADA2 polypeptide, or a composition comprising the fragment or variant thereof. Also provided herein is a method for using the composition in the treatment of cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 695,623, filed July 9, 2018, U.S. Provisional Patent Application No. 62 / 695,627, filed July 9, 2018, U.S. Provisional Patent Application No. 62 / 863,710, filed June 19, 2019, U.S. Provisional Patent Application No. 62 / 864,367, filed June 20, 2019, and U.S. Provisional Patent Application No. 62 / 866,420, filed June 25, 2019, which are incorporated herein by reference in their entireties. [Background technology]

[0002] In recent years, monoclonal antibody-based cancer immunotherapy, which is based on the interruption of inhibitory signals transmitted to the adaptive immune system, has shown promise in clinical practice. With the FDA approval of CTLA-4 antibody inhibitors (e.g., ipilimumab) and PD-1 inhibitors (e.g., pembrolizumab, nivolumab), more treatment options are now available for treating solid tumors, including lung cancer, renal cell carcinoma, and ovarian cancer. However, in the majority of indications where PD-1 / PD-L1 and TGF-β are co-expressed (e.g., ovarian, gastric, and colorectal), little to no response to immune checkpoint inhibitors has been observed. Therefore, there is a continuing need in the art for safer and more effective cancer treatments.

[0003] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0004] Provided herein are fusion proteins comprising: (a) an antibody that binds to programmed cell death protein 1 (PD-1), or a fragment of said antibody, or a variant of said antibody; and (b) a transforming growth factor beta (TGF-β) cytokine trap, wherein one or more polypeptides of the fusion protein are connected by a linker.

[0005] In one embodiment, the linker comprises (G4S)n, where n is 2, 3, 4, 5, or 6. In one embodiment, the linker comprises (Gly)n, where n is 6, 7, or 8. In one embodiment, the linker comprises (EAAAK)n, where n is 1, 2, 3, 4, 5, or 6. In one embodiment, the linker comprises A(EAAAK)4ALEA(EAAAK)4A. In one embodiment, the linker comprises a sequence set forth in any one of SEQ ID NOs: 17-34. In one embodiment, the TGF-β cytokine trap comprises a transforming growth factor receptor (TGFβR) or a functional fragment thereof, an anti-TGF-β antibody or an antigen-binding fragment thereof, a TGF-β1 inhibitory peptide, or a variant thereof.

[0006] In one embodiment, the TGFβR is transforming growth factor beta receptor II (TGFβRII), or the functional fragment. In one embodiment, the functional fragment of TGFβRII is the extracellular domain (ECD) of TGFβRII. In one embodiment, the ECD binds to TGF-β1. In one embodiment, the ECD binds to TGF-β3. In one embodiment, the ECD binds to TGF-β1 and TGF-β3. In one embodiment, the ECD binds to TGF-β1 and TGF-β3, but not to TGF-β2. In one embodiment, the TGF-β cytokine trap comprises a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 14, SEQ ID NO: 141, or SEQ ID NO: 142. In one embodiment, the TGF-β cytokine trap comprises the sequence set forth in SEQ ID NO: 14, SEQ ID NO: 141, or SEQ ID NO: 142.

[0007] In one embodiment, the TGF-β cytokine trap comprises the sequence set forth in SEQ ID NO: 14. In one embodiment, the antibody (anti-PD1) is an immunoglobulin G (IgG) antibody. In one embodiment, the IgG is IgG1, IgG2, IgG3, or IgG4. In one embodiment, the IgG4 comprises a mutation at position 108 of SEQ ID NO: 146 or SEQ ID NO: 292. In one embodiment, the mutation is an S108P mutation. In one embodiment, the IgG4 is connected to the TGF-β cytokine trap by the linker. In one embodiment, the fragment of the antibody is a Fab, (Fab)2, (Fab')2, Fv, (Fv)2, or scFv.

[0008] In one embodiment, the antibody comprises a heavy chain variable region (V H ) and the variable region of the light chain (V L In one embodiment, the linker comprises the variable region (V H ) to the TGF-β cytokine trap. In one embodiment, the linker connects the variable region of the light chain (V L ) to the TGF-β cytokine trap. In one embodiment, the variable region of the heavy chain (V H ) is linked within the fusion protein by a second linker to the variable region of the light chain (V L In one embodiment, the second linker comprises a sequence set forth in any one of SEQ ID NOs: 17 to 34. In one embodiment, the variable region (V H ) is at least 80% identical to the sequence shown in any one of SEQ ID NOs: 1 to 7 and 149 to 164.

[0009] In one embodiment, the variable region of the light chain (V L ) is at least 80% identical to the sequence set forth in any one of SEQ ID NOs: 8 to 13 and 148. In one embodiment, the variable region (V H) comprises a sequence set forth in any one of SEQ ID NOs: 1 to 7 and 149 to 164. In one embodiment, the variable region (V L ) comprises a sequence set forth in any one of SEQ ID NOs: 8 to 13 and 148. In one embodiment, the variable region (V H ) is at least 90% identical to the sequence set forth in SEQ ID NO: 6, and the variable region of the light chain (V L ) is at least 90% identical to the sequence set forth in SEQ ID NO: 12. In one embodiment, the variable region (V H ) comprises the sequence shown in SEQ ID NO: 6, and the variable region of the light chain (V L ) comprises the sequence shown in SEQ ID NO:12.

[0010] In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 15 and the sequence set forth in SEQ ID NO: 16. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 15 and the sequence set forth in SEQ ID NO: 143. In one embodiment, the variable region (V H ) is at least 90% identical to the sequence set forth in SEQ ID NO: 7, and said variable region of the light chain is at least 90% identical to the sequence set forth in SEQ ID NO: 13. In one embodiment, said variable region of the heavy chain (V H ) comprises the sequence shown in SEQ ID NO: 7, and the variable region of the light chain (V L ) comprises the sequence shown in SEQ ID NO:13.

[0011] In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 145. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 144. In one embodiment, the antibody further comprises a fragment crystallizable region (Fc). In one embodiment, the Fc is human Fc1, human Fc2, human Fc3, human Fc4, or a fragment thereof. In one embodiment, the Fc further comprises one or more mutations. In one embodiment, the antibody comprises the scFv and the Fc fragment. In one embodiment, the TGFβ cytokine trap comprises the anti-TGFβ antibody, or the antigen-binding fragment thereof. In one embodiment, the anti-TGFβ antibody comprises a VH encoded by SEQ ID NO: 166, 168, 169, 171, 173, 175, or 177 and a VL encoded by SEQ ID NO: 165, 167, 170, 172, 174, 176, or 178.

[0012] In one embodiment, the TGFβ cytokine trap comprises the TGFβ inhibitory peptide or a variant thereof. In one embodiment, the TGFβ inhibitory peptide comprises a sequence set forth in any one of SEQ ID NOs: 193-227.

[0013] Provided herein are polynucleotides that encode the fusion proteins disclosed herein.

[0014] The present specification provides an expression vector comprising a polynucleotide encoding the fusion protein disclosed herein, the polynucleotide being operably linked to a promoter. In one embodiment, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In one embodiment, the inducible promoter is a small molecule ligand-inducible, two-polypeptide ecdysone receptor-based gene switch. In one embodiment, the vector is an adenovirus vector.

[0015] Provided herein are pharmaceutical compositions comprising (a) a fusion protein disclosed herein, (b) a polynucleotide encoding a fusion protein disclosed herein, or (c) an expression vector disclosed herein, and (d) a pharmaceutically acceptable excipient.

[0016] Provided herein is a method of treating cancer, comprising contacting a cell with (a) a fusion protein disclosed herein, (b) a polynucleotide encoding the fusion protein disclosed herein, or (c) an expression vector disclosed herein. In one embodiment, the cell is a cancer cell. In one embodiment, the cell is a mammalian cell.

[0017] Provided herein are methods for treating a subject with cancer, the method comprising administering a composition comprising (a) an antibody that binds to programmed cell death protein 1 (PD-1), or a fragment or variant of said antibody, and (b) a fusion protein comprising a transforming growth factor receptor (TGFβR) or a functional fragment thereof, an anti-TGF-β antibody, or an antigen-binding fragment thereof, a TGF-β1 inhibitory peptide, or a variant thereof, wherein one or more polypeptides of the fusion protein are connected by a linker.

[0018] In one embodiment, the linker comprises (G4S)n, where n is 2, 3, 4, 5, or 6. In one embodiment, the linker comprises (Gly)n, where n is 6, 7, or 8. In one embodiment, the linker comprises (EAAAK)n, where n is 1, 2, 3, 4, 5, or 6. In one embodiment, the linker comprises A(EAAAK)4ALEA(EAAAK)4A. In one embodiment, the linker comprises a sequence set forth in any one of SEQ ID NOs: 17-34. In one embodiment, the transforming growth factor receptor protein is TGFβRII. In one embodiment, the functional fragment of TGFβRII is the extracellular domain (ECD) of TGFβRII. In one embodiment, the TGF-β cytokine trap comprises a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 14, SEQ ID NO: 141, or SEQ ID NO: 142. In one embodiment, the TGF-β cytokine trap comprises the sequence set forth in SEQ ID NO:14, SEQ ID NO:141, or SEQ ID NO:142.

[0019] In one embodiment, the antibody is an immunoglobulin G (IgG) antibody. In one embodiment, the IgG is IgG1, IgG2, IgG3, or IgG4. In one embodiment, the IgG4 comprises a mutation at position 108 of SEQ ID NO: 146 or SEQ ID NO: 292. In one embodiment, the mutation is an S108P mutation. In one embodiment, the fragment of the antibody is a Fab, (Fab)2, (Fab')2, Fv, (Fv)2, or scFv of the antibody. In one embodiment, the antibody, or fragment of the antibody, or variant of the antibody comprises a heavy chain variable region (V H ) and the variable region of the light chain (V L In one embodiment, the linker comprises the variable region (V H ) to the TGF-β cytokine trap. In one embodiment, the linker connects the variable region of the light chain (V L ) to the TGF-β cytokine trap.

[0020] In one embodiment, the variable region of the heavy chain (V H ) is connected by a second linker to the variable region of the light chain (V L In one embodiment, the variable region (V H ) is at least 80% identical to the sequence set forth in any one of SEQ ID NOs: 1 to 7 and 149 to 164. In one embodiment, the variable region (V L ) is at least 80% identical to the sequence set forth in any one of SEQ ID NOs: 8 to 13 and 148. In one embodiment, the variable region (V H ) includes any one of SEQ ID NOs: 1 to 7 and 149 to 164.

[0021] In one embodiment, the variable region of the light chain (V L ) comprises a sequence set forth in any one of SEQ ID NOs: 8 to 13 and 148. In one embodiment, the variable region (V H ) is at least 90% identical to the sequence set forth in SEQ ID NO: 6, and the variable region of the light chain (V L ) is at least 90% identical to the sequence set forth in SEQ ID NO: 12. In one embodiment, the variable region (V H ) comprises the sequence shown in SEQ ID NO: 6, and the variable region of the light chain (V L ) comprises the sequence set forth in SEQ ID NO: 12. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 15 and the sequence set forth in SEQ ID NO: 16.

[0022] In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 15 (VL5 Igg4) and the sequence set forth in SEQ ID NO: 143. In one embodiment, the variable region of the heavy chain (VH) is at least 90% identical to the sequence set forth in SEQ ID NO: 7, and the variable region of the light chain (V L) is at least 90% identical to the sequence set forth in SEQ ID NO: 13. In one embodiment, the variable region of the heavy chain comprises the sequence set forth in SEQ ID NO: 7 and the variable region of the light chain comprises the sequence set forth in SEQ ID NO: 13. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 145. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 144.

[0023] In one embodiment, the antibody further comprises a fragment crystallizable region (Fc). In one embodiment, the Fc is human Fc1, human Fc2, human Fc3, human Fc4, or a fragment thereof. In one embodiment, the Fc further comprises one or more mutations. In one embodiment, the antibody comprises the scFv and the Fc fragment. In one embodiment, the cancer is a refractory cancer. In one embodiment, the subject is non-responsive to treatment with a PD-1 antibody or a CTLA-4 antibody. In one embodiment, the method further comprises administering one or more additional anti-cancer agents. In one embodiment, the additional anti-cancer agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody, or a fragment or variant thereof.

[0024] In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody, or a fragment or variant thereof. In one embodiment, the method further comprises administering one or more cytokines. In one embodiment, the subject is a mammalian subject. In one embodiment, the subject is human. In one embodiment, the cancer is mesothelioma, glioblastoma, endometrial cancer, colorectal cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, stomach cancer, bladder cancer, liver cancer, Hodgkin's lymphoma, lung cancer, skin cancer, renal cancer, or head and neck cancer.

[0025] In one embodiment, the skin cancer is cutaneous squamous cell carcinoma, melanoma, or basal cell carcinoma. In one embodiment, the lung cancer is non-small cell lung cancer (NSLC) or small cell lung cancer (SCLC). In one embodiment, the breast cancer is triple-negative breast cancer (TNBC). In one embodiment, the method further comprises administering an effective amount of T cells engineered to express an exogenous receptor. In one embodiment, the exogenous receptor is a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor is an engineered T cell receptor.

[0026] In one embodiment, the chimeric antigen receptor comprises an antigen-binding domain that binds to an epitope in CD19, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor α, MUC-1, MUC-4, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2. In one embodiment, the antigen-binding domain comprises a sequence selected from SEQ ID NOs: 37-56. In one embodiment, the antigen-binding domain comprises a sequence selected from SEQ ID NOs: 35-36.

[0027] In one embodiment, an effective amount of engineered T cells is at least 10 cells per kg. 2 In one embodiment, an effective amount of engineered T cells is at least 10 cells per kg. 4 In one embodiment, an effective amount of engineered T cells is at least 10 cells per kg. 5 In one embodiment, the engineered T cells further express a cytokine. In one embodiment, the cytokine is a fusion protein comprising IL-15 and IL-15Rα.

[0028] Provided herein are methods of treating cancer in a subject in need thereof, the methods comprising: (a) administering a composition comprising an antibody that binds to programmed cell death protein 1 (PD-1), or a fragment of said antibody, or a variant of said antibody; and a fusion protein comprising a transforming growth factor receptor (TGFβR) protein or functional fragment thereof, wherein one or more polypeptides of the fusion protein are connected by a linker; and (b) administering to the subject one or more doses of an effective amount of engineered T cells, wherein the engineered T cells comprise a chimeric receptor and membrane-bound IL-15.

[0029] In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 15 and the sequence set forth in SEQ ID NO: 294. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 295. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 15 and the sequence set forth in SEQ ID NO: 294. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 13 and the sequence set forth in SEQ ID NO: 295.

[0030] Provided herein, inter alia, are fusion proteins comprising: (a) an antibody that binds to programmed cell death protein 1 (PD-1), or a fragment or variant of said antibody; and (b) an adenosine deaminase (ADA) protein, or a functional fragment thereof, wherein one or more polypeptides of the fusion protein are connected by a linker. In one embodiment, the linker comprises (G4S)n, where n is 2, 3, 4, 5, or 6. In some embodiments, the linker comprises (Gly)n, where n is 6, 7, or 8. In other embodiments, the linker comprises (EAAAK)n, where n is 1, 2, 3, 4, 5, or 6. In certain embodiments, the linker comprises A(EAAAK)4ALEA(EAAAK)4A. In certain embodiments, the linker comprises the sequence set forth in any one of SEQ ID NOs: 17-34.

[0031] In one embodiment, the adenosine deaminase protein is adenosine deaminase 2 (ADA2) or a mutant or variant thereof. In another embodiment, the adenosine deaminase (ADA) protein comprises any one of ADA2 mutant 1 (SEQ ID NO: 273), ADA2 mutant 2 (SEQ ID NO: 274), ADA2 mutant 3 (SEQ ID NO: 275), ADA2 mutant 4 (SEQ ID NO: 276), ADA2 mutant 5 (SEQ ID NO: 277), or ADA2 mutant 6 (SEQ ID NO: 278), ADA2 mutant 7 (SEQ ID NO: 279), or wild-type ADA2 (SEQ ID NO: 284). In other embodiments, the adenosine deaminase (ADA) protein comprises a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 284 or any one of SEQ ID NOs: 273-279. In certain embodiments, the adenosine deaminase (ADA) protein comprises the sequence set forth in SEQ ID NO: 284 or any one of SEQ ID NOs: 273-279.

[0032] In one embodiment, the antibody is an immunoglobulin G (IgG) antibody. In certain embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4. In one embodiment, the IgG4 comprises a mutation at position 108 of SEQ ID NO: 146 or 292. In some embodiments, the mutation is an S108P mutation. In some examples, the antibody fragment is a Fab, (Fab)2, (Fab')2, Fv, (Fv)2, or scFv of the antibody. In one embodiment, the antibody, or antibody fragment or antibody variant, comprises a heavy chain variable region (V H ) and the variable region of the light chain (V L In one embodiment, the linker comprises a heavy chain variable region (V H ) to adenosine deaminase 2 (ADA2), or a mutant or variant thereof. In another embodiment, the linker connects the variable region (V L ) is connected to adenosine deaminase 2 (ADA2) or a mutant or variant thereof.

[0033] In one example, the heavy chain variable region (V H ) is connected by a second linker to the light chain variable region (V L In one embodiment, the variable region of the heavy chain (V H ) is at least 80% identical to any one of the sequences set forth in SEQ ID NOS: 1 to 7 and 149 to 164. In another example, the variable region (V L ) is at least 80% identical to the sequence set forth in any one of SEQ ID NOs: 8-13 and 148. In a further embodiment, the variable region (V H ) comprises a sequence set forth in any one of SEQ ID NOs: 1 to 7 and 149 to 164. In a further embodiment, the variable region of the light chain (V L ) comprises a sequence set forth in any one of SEQ ID NOS: 8-13 and 148. In one embodiment, the variable region of the heavy chain (VH) is at least 90% identical to the sequence set forth in SEQ ID NOS: 6 (VH6), and the variable region of the light chain (VL) is at least 90% identical to the sequence set forth in SEQ ID NOS: 12 (VL5). In one embodiment, the variable region of the heavy chain (V H ) comprises the sequence shown in SEQ ID NO: 6, and the variable region of the light chain (V L ) comprises the sequence shown in SEQ ID NO:12.

[0034] In one example, the fusion protein comprises the sequence set forth in SEQ ID NO: 12 (VL5) and the sequence set forth in SEQ ID NO: 280 (VH6 IgG4(mut)-ADA2 wt). In another example, the fusion protein comprises the sequence set forth in SEQ ID NO: 12 (VL5) and the sequence set forth in SEQ ID NO: 281 (VH6 igG4(mut)ADA2 mut 7). In one example, the variable region of the heavy chain (V H ) is at least 90% identical to the sequence shown in SEQ ID NO: 7 (VH7), and the variable region of the light chain (V L ) is at least 90% identical to the sequence set forth in SEQ ID NO: 13 (VL6). In one example, the variable region of the heavy chain (VH) comprises the sequence set forth in SEQ ID NO: 7 (VH7), and the variable region of the light chain (V L) comprises the sequence set forth in SEQ ID NO: 13 (VL6). In another example, the fusion protein comprises the sequence set forth in SEQ ID NO: 13 (VL6) and the sequence set forth in SEQ ID NO: 282 (VH7 igG4(mut)ADA2 wt). In one example, the fusion protein comprises the sequence set forth in SEQ ID NO: 13 (VL6) and the sequence set forth in SEQ ID NO: 283 (VH7 igG4(mut)ADA2 mut 7).

[0035] The present specification provides a polynucleotide encoding a fusion protein.The present specification also provides an expression vector, comprising a polynucleotide encoding a fusion protein according to any one of the above aspects, wherein the polynucleotide is operably linked to a promoter.In some embodiments, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.In some embodiments, the inducible promoter is a small molecule ligand-induced, two-polypeptide, ecdysone receptor-based gene switch.In some embodiments, the vector is an adenovirus vector.

[0036] Provided herein are methods for treating cancer, comprising contacting a cell with a fusion protein, a polynucleotide encoding the fusion protein, or an expression vector. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a mammalian cell.

[0037] Further provided herein is a pharmaceutical composition comprising the fusion protein; or a polynucleotide encoding the fusion protein; or an expression vector, and a pharmaceutically acceptable excipient.

[0038] Provided herein is a method for treating a subject with cancer, comprising administering a composition comprising an antibody that binds to programmed cell death protein 1 (PD-1), or a fragment of the antibody, or a variant of the antibody; and an adenosine deaminase protein or a functional fragment thereof, wherein one or more polypeptides of the fusion protein are connected by a linker. In certain cases, the adenosine deaminase protein is adenosine deaminase 2 (ADA2). In other embodiments, the adenosine deaminase (ADA) protein comprises any one of ADA2 mutant 1 (SEQ ID NO: 273), ADA2 mutant 2 (SEQ ID NO: 274), ADA2 mutant 3 (SEQ ID NO: 275), ADA2 mutant 4 (SEQ ID NO: 276), ADA2 mutant 5 (SEQ ID NO: 277), ADA2 mutant 6 (SEQ ID NO: 278), ADA2 mutant 7 (SEQ ID NO: 279), or wild-type ADA2 (SEQ ID NO: 284).

[0039] In certain instances, the linker comprises (G4S)n, where n is 2, 3, 4, 5, or 6. In some embodiments, the linker is (Gly)n, where n is 6, 7, or 8. In some embodiments, the linker comprises (EAAAK)n, where n is 1, 2, 3, 4, 5, or 6. In some embodiments, the linker comprises A(EAAAK)4ALEA(EAAAK)4A. In some instances, the linker comprises a sequence set forth in any one of SEQ ID NOs: 17-34.

[0040] This paper provides a method for treating a subject with cancer.In some cases, cancer is mesothelioma, glioblastoma, endometrial cancer, colorectal cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, gastric cancer, bladder cancer, liver cancer, Hodgkin's lymphoma, lung cancer, skin cancer, renal cancer, or head and neck cancer.In some cases, skin cancer is cutaneous squamous cell carcinoma, melanoma, or basal cell carcinoma.In other cases, lung cancer is non-small cell lung cancer (NSLC) or small cell lung cancer (SCLC).In some cases, breast cancer is triple-negative breast cancer (TNBC).

[0041] In another embodiment, a method of treating a subject with cancer is provided, comprising administering a composition comprising a fusion protein comprising an antibody that binds to programmed cell death protein 1 (PD-1), or a fragment of said antibody, or a variant of said antibody; and an adenosine deaminase protein, or a functional fragment thereof, wherein one or more polypeptides of the fusion protein are connected by a linker. In a further embodiment, the method of treating a subject with cancer further comprises administering an effective amount of T cells engineered to express an exogenous receptor.

[0042] In some cases, the exogenous receptor is a chimeric antigen receptor. In other cases, the chimeric antigen receptor is an engineered T cell receptor. In one case, the chimeric antigen receptor comprises an antigen-binding domain that binds to an epitope in CD19, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor α, MUC-1, MUC-4, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2. In some embodiments, the antigen-binding domain comprises a sequence selected from SEQ ID NOs: 37-56; in other embodiments, the antigen-binding domain comprises a sequence selected from SEQ ID NOs: 35-36.

[0043] In one embodiment, an effective amount of engineered T cells is at least 10 cells per kg. 2 In another embodiment, the effective amount of engineered T cells is at least 10 cells per kg. 4 In a further embodiment, the effective amount of engineered T cells is at least 10 cells per kg. 5 There are individuals.

[0044] In a further embodiment, the engineered T cells further express a cytokine, hi another embodiment, the cytokine is a fusion protein comprising IL-15 and IL-15Rα.

[0045] Provided herein are methods of treating cancer in a subject in need thereof, the method comprising: administering a composition comprising an antibody that binds to programmed cell death protein 1 (PD-1), or a fragment of said antibody, or a variant of said antibody; and a fusion protein comprising an adenosine deaminase protein, or a functional fragment thereof, wherein one or more polypeptides of the fusion protein are connected by a linker; and administering to the subject one or more doses of an effective amount of engineered T cells, wherein the engineered T cells comprise a chimeric receptor and membrane-bound IL-15.

[0046] The features of the present disclosure are pointed out with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments in which the principles of the disclosure are utilized. [Brief explanation of the drawings]

[0047] [Figure 1] FIG. 1 shows an overview of PD-1 / PD-L1 in immunosuppression. [Figure 2] FIG. 1 shows an overview of TGF-β in immunosuppression. [Figure 3]FIG. 1 shows a cluster of TGF-β-related genes (enriched in stage III / IV) that correlates with metastatic disease and poor prognosis in a subset of ovarian cancer patients. [Figure 4] 4A, 4B, and 4C show the schematic design for an anti-PD1-TGFRII fusion protein. In another exemplary embodiment, ADA2 may be fused to anti-PD1. [Figure 5] 1 is a graph showing blockade of PD-1 / PD-L1 interaction by anti-PD1(VH6-VL5) IgG1-TGFβRII and anti-PD1(VH6-VL5) IgG4-TGFβRII. [Figure 6] Graph showing neutralization of TGF-β1 isoform signaling by anti-PD1(VH6-VL5) IgG1-TGFβRII and anti-PD1(VH6-VL5) IgG4-TGFβRII. [Figure 7] 1 is a graph showing neutralization of TGF-β2 isoforms by anti-PD1(VH6-VL5) IgG1-TGFβRII and anti-PD1(VH6-VL5) IgG4-TGFβRII. [Figure 8] Graph showing neutralization of TGF-β3 isoform signaling by anti-PD1(VH6-VL5) IgG1-TGFβRII and anti-PD1(VH6-VL5) IgG4-TGFβRII. [Figure 9-1] Figures 9A, 9B, and 9C are graphs showing dose-dependent enhancement of proliferation and IFN-γ production by PBMCs stimulated in the presence of anti-PD1-TGFRII fusion protein compared to anti-PD1 antibody or control antibody. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Figures 9D and 9E are graphs showing PD1 receptor occupancy on CD8+ T cells and IFN-γ production, respectively, when anti-PD1-TGFRII fusion protein was added to cocultures of PBMCs and colorectal cancer (colorectal adenocarcinoma) cell lines. [Figure 9-5]Figures 9F and 9G are graphs showing PD1 receptor occupancy on CD8+ T cells and IFN-γ production, respectively, when anti-PD1-TGFRII fusion protein was added to cocultures of PBMCs and head and neck cancer (laryngeal cancer) cell lines. [Figure 9-6] Figures 9H and 9I are graphs depicting the concentrations of TGF-β1 and TGF-β2, respectively, in the supernatants of co-cultures of PBMCs with colorectal cancer (colorectal adenocarcinoma) cells in the presence of anti-PD1-TGFRII fusion protein. [Figure 9-7] Figures 9J and 9K are graphs depicting the concentrations of TGF-β1 and TGF-β2, respectively, in the supernatants of co-cultures of PBCM with head and neck cancer (laryngeal cancer) cells in the presence of anti-PD1-TGFRII fusion protein. [Figure 9-8] Figures 9L and 9M are graphs depicting IFN-γ production in 3D spheroid cultures of colorectal cancer (colorectal adenocarcinoma) cells or head and neck cancer (laryngeal carcinoma) cells and PBMCs, respectively, in the presence of anti-PD1-TGFRII fusion protein compared to anti-PD1 alone. [Figure 10] 10A-10D show the effect of anti-PD1-TGFRII fusion protein treatment on T cell proliferation and activation in the presence of recombinant TGF-β1. [Figure 11-1] 11A-11F show the expression of various cytokines by PBMCs in the presence of recombinant TGF-β1 and in the presence of anti-PD1, anti-PD1-TGFRII fusion protein, or control antibody. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 12-1] FIG. 12A shows the effect of anti-PD1-TGFRII fusion protein compared to anti-PD1 alone on tumor growth in a humanized mouse model of colorectal cancer. [Figure 12-2]Figure 12B shows that treatment with anti-PD1-TGFRII fusion protein significantly increases the ratio of CD8+ T cells to Tregs within tumors in a humanized mouse model of colorectal cancer. [Figure 12-3] Figure 12C shows the effect of anti-PD1-TGFRII fusion protein treatment compared to anti-PD1 treatment on perforin expression levels in a humanized mouse model of colorectal cancer. Figures 12D and 12E show the effect of anti-PD1-TGFRII fusion protein treatment compared to anti-PD1 treatment on TGF-β1 and TGF-β2 concentrations, respectively, in a humanized mouse model of colorectal cancer. [Figure 13-1] Figure 13A shows that treatment with anti-PD1-TGFRII fusion protein significantly improved IFNγ production compared to anti-PD1 treatment in an in vitro model of head and neck cancer. Figures 13B-13G show that treatment with anti-PD1-TGFRII fusion protein significantly enhanced T cell function, as evidenced by expression analysis of various pathway genes. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 14-1] FIG. 14A shows the effect of anti-PD1-TGFRII fusion protein compared to anti-PD1 alone on tumor growth in a humanized mouse model of head and neck cancer. [Figure 14-2] Figure 14B shows the survival of tumor-bearing mice treated with anti-PD1-TGFRII fusion protein compared to anti-PD1 alone or isotype control in a humanized mouse model of head and neck cancer. Figure 14C shows the ratio of CD8+ T cells to regulatory T cells in tumors of mice treated with anti-PD1-TGFRII fusion protein in a humanized mouse model of head and neck cancer. [Figure 14-3]Figures 14D and 14E show the effect of anti-PD1-TGFRII fusion protein compared to anti-PD1 alone on TGF-β1 and TGF-β2 concentrations in a humanized mouse model of head and neck cancer. Figure 14F shows the effect of anti-PD1-TGFRII fusion protein on IFN-γ production in a humanized mouse model of head and neck cancer. [Figure 15-1] 15A-15B show the effect of anti-PD1(VH7 / VL6)-TGFRII fusion protein on IFN-γ production and TGF-β1 concentrations in primary colorectal cancer patient samples. [Figure 15-2] Figure 15C shows gene expression analysis of primary colorectal cancer patient samples co-cultured with anti-PD1(VH7 / VL6)-TGFRII fusion protein. [Figure 16] Figure 1 shows a graph depicting the results of a cytotoxicity assay for anti-PD1(VH7 / VL6)-TGFRII fusion protein compared to anti-PD-L1-TGFRII fusion protein. [Figure 17] Graph depicting the results of a cytotoxicity assay comparing anti-PD1 (VH7 / VL6)-TGFRII fusion protein in combination with chimeric antigen receptor (CAR) T cells, anti-PD1 in combination with CAR T cells, and CAR T cells alone. [Figure 18] Figures 18A and 18B are graphs depicting the results of a cytotoxicity assay for an anti-PD1(VH6 / VL5)-TGFRII fusion protein in combination with CD33 CAR-T, and a cytotoxicity assay for an anti-PD1(VH7 / VL6)-TGFRII fusion protein in combination with CD33 CAR-T, respectively. [Figure 19] Figures 19A and 19B are graphs depicting tumor cell lysis using anti-PD1 (VH6 / VL5)-TGFRII fusion protein and anti-PD1 (VH7 / VL6)-TGFRII fusion protein, respectively, when co-cultured with NK cells. [Figure 20]1 is a graph depicting Biacore analysis of simultaneous binding to TGF-b1 and PD1 by anti-PD1(VH6 / VL5)-TGFRII fusion proteins using different linkers. [Figure 21] 1 is a graph showing blockade of PD-1 / PD-L1 interaction by anti-PD1 IgG4-ADA2. [Figure 22] 1 is a graph showing the enzymatic activity of ADA2 measured for anti-PD1 hIgG1-ADA2 and anti-PD1 hIgG4-ADA2. [Figure 23-1] Figures 23A-23C are graphs showing the effect of various mutants of anti-PD1 and anti-PD1-ADA2 fusion proteins on PD-L1 / PD-1 interaction. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 24-1] 24A-24F are graphs showing the enzymatic activity of various variants of anti-PD1-ADA2 fusion proteins, as measured by ADA enzymatic activity. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above. [Figure 25] Graph showing the enzymatic activity of anti-PD1-mutADA2 compared to anti-PD1-wtADA2, as measured by ADA enzymatic activity. [Figure 26-1] 26A-26D show graphs depicting the effect of anti-PD1-wtADA2 mutants on T cell proliferation. [Figure 26-2] Same as above. [Figure 26-3] Figure 26E is a graph depicting IFNγ production by anti-PD1-wtADA2 compared to anti-PD1 or isotype control. [Figure 27-1] 27A-27B show graphs depicting the efficacy of wtADA2 and mutADA2 in reversing adenosine-mediated suppression of T cell proliferation. [Figure 27-2]Same as above. [Figure 28] 1 is a graph depicting the effect of mutants of the anti-PD1-ADA2 fusion protein on blocking the PD1-PDL1 interaction. [Figure 29] 1 is a graph showing the enzymatic activity of anti-PD1-ADA2-scFv-Fc, as measured by ADA enzymatic activity. [Figure 30] 1 is a bar graph showing the enzymatic activity of anti-PD1-ADA2 variants as measured by ADA enzymatic activity. [Figure 31] 31A-31B show graphs depicting the effect of anti-PD1-wtADA2 on the production of IFN-γ and proliferation of tumor-infiltrating lymphocytes (TILs) in tumors of primary CRC patients. [Figure 32] 1 is a graph depicting the effect of anti-PD1 versus anti-PD1-wtADA2 on tumor volume in a humanized mouse model of lung cancer. [Figure 33] 33A-33C show the schematic design for anti-PD1-adenosine deaminase 2 (ADA2): anti-PD1-ADA2. DETAILED DESCRIPTION OF THE INVENTION

[0048] Detailed Description of the Invention The following description and examples further illustrate embodiments of the present disclosure.

[0049] It is to be understood that the present disclosure is not limited to the particular embodiments described herein, and as such may vary. Those skilled in the art will recognize that variations and modifications to the present disclosure are possible and are encompassed within its scope.

[0050] All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0052] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be presented separately or in any suitable combination. Conversely, although the present disclosure may, for clarity, be described herein in the context of separate embodiments, the present disclosure can also be practiced in a single embodiment.

[0053] The following definitions complement those in the art and are directed to this application, and are not to be construed as being related or unrelated to any matter, for example, any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the tests of the present disclosure, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0054] definition In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0055] In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the terms "and / or" and "any combination thereof," and their grammatical equivalents, can be used interchangeably. These terms may convey that any combination is specifically contemplated. By way of example only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C." Unless the context dictates otherwise, the term "or" can be used conjunctively or disjunctively.

[0056] Furthermore, use of the term "including" as well as other forms such as "includes," "including," and "included" is non-limiting.

[0057] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.

[0058] As used in the specification and claims, the words "comprising" (any form of "comprising," such as "comprise" and "comprises"), "having" (any form of "having," such as "have" and "has"), "including" (any form of "including," such as "include" and "includes"), or "containing" (any form of "containing," such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0059] As used herein, with respect to a reference numerical value, the term "about" and its grammatical equivalents may include the numerical value itself as well as values ​​in a range of ±10% from that numerical value.

[0060] The term "about" or "approximately" refers to an acceptable error range for a particular value, as determined by one of ordinary skill in the art, that 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 one standard deviation or more than one standard deviation, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes any of the amounts 10 and 9-11. In yet another example, the term "about" in relation to a reference numerical value also includes a value plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold. When particular values ​​are described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0061] As used herein, "polynucleotide" or "oligonucleotide" refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double-stranded and single-stranded DNA, triplex DNA, as well as double-stranded and single-stranded RNA. The term can also include modified forms of polynucleotides, for example, by methylation and / or capping, as well as unmodified forms of polynucleotides. The term is also intended to include molecules containing non-naturally occurring or synthetic nucleotides, as well as nucleotide analogs.

[0062] As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. The polynucleotide sequences and vectors disclosed or contemplated herein can be introduced into a cell, for example, by transfection, transformation, or transduction. Many transfection methods are known in the art, including, for example, calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted gene gun (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after propagating infectious particles in suitable packaging cells, many of which are commercially available.

[0063] As used herein, "polypeptide," "peptide," and their grammatical equivalents refer to a polymer of amino acid residues. A polypeptide may optionally include glycosylation or other modifications typical of a given protein in a given intracellular environment. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycerin ... Lysine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The present disclosure further contemplates that expression of a polypeptide or protein described herein in an engineered cell may be associated with post-translational modification of one or more amino acids of the polypeptide or protein. Non-limiting examples of post-translational modifications include phosphorylation, acylation, including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation, including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypionation, lipoylation, and iodination.

[0064] As used herein, in the context of two nucleic acid or amino acid sequences of polypeptides, the terms "identical" or "sequence identity" and grammatical equivalents refer to residues in two sequences that are the same when aligned for maximum correspondence over a designated comparison window. As used herein, a "comparison window" refers to a segment of at least about 20, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions within which a sequence may be compared to a reference sequence over the same number of contiguous positions after optimal alignment of the two sequences. Sequence alignment methods for comparison are well known in the art.Optimal sequence alignment for comparison can be achieved by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci. USA, 85:2444 (1988); computerized implementations of these algorithms (including, but not limited to, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the PC / Gene program by Intelligentics, Mountain View, Calif., Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA), and the CLUSTAL program is described in Higgins and Sharp, Gene, 73:237-244. (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by visual and manual alignment. In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or fragment thereof, as determined, for example, by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.Similarly, nucleic acids can also be described in terms of a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference nucleic acid or fragment thereof, e.g., as determined by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have a certain percentage of sequence identity to a larger molecule, this means that if the two molecules are optimally aligned, the percentage of residues in the smaller molecule will find matching residues in the larger molecule, according to the order in which the two molecules are optimally aligned.

[0065] The term "substantially identical" and its grammatical equivalents, as applied to a nucleic acid sequence or an amino acid sequence, means that the nucleic acid sequence or amino acid sequence includes a sequence having at least 90% sequence identity, or more, at least 95%, at least 98%, and at least 99%, when compared to a reference sequence using the programs described above, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). Percent sequence identity is determined by comparing two optimally aligned sequences over a comparison window; portions of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage sequence identity. In some embodiments, substantial identity exists over a region of the sequences at least about 50 residues in length, over a region of at least about 100 residues, and in some embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequences are substantially identical over the entire length of the coding regions.

[0066] "Homology" is generally inferred from the sequence identity between two or more nucleic acids or proteins (or sequences thereof). The exact percentage of identity between sequences useful for establishing homology varies with the nucleic acid and protein in question, but at least 25% sequence identity is used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining percent sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are publicly available. Nucleic acids and / or nucleic acid sequences are "homologous" if they are derived from a common ancestral nucleic acid or ancestral nucleic acid sequence, whether natural or artificial. Proteins and / or protein sequences are "homologous" if their encoding DNA is derived from a common ancestral nucleic acid or ancestral nucleic acid sequence, whether natural or artificial. Homologous molecules may be referred to as "homologues." For example, any naturally occurring protein may be modified by any available mutagenesis method. Upon expression, this mutant nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid.

[0067] As used herein, the term "isolated" and its grammatical equivalents refer to the removal of a nucleic acid from its natural environment. As used herein, the term "purified" and its grammatical equivalents refer to a molecule or composition that has been removed from nature (including genomic DNA and mRNA), synthesized (including cDNA), and / or amplified under laboratory conditions, and in this case, "purity" is a relative term and not "absolute purity." On the other hand, it should be understood that nucleic acids and proteins may be formulated with a diluent or adjuvant and, for practical purposes, be considered isolated. For example, when used for introduction into a cell, nucleic acids are typically mixed with an acceptable carrier or diluent. As used herein, the term "substantially purified" and its grammatical equivalents refer to a nucleic acid sequence, polypeptide, protein, or other compound that is essentially free, i.e., greater than about 50% free, greater than about 70% free, or greater than about 90% free, of polynucleotides, proteins, polypeptides, and other molecules with which it is naturally associated.

[0068] An "expression vector" or "vector" is any genetic element, e.g., a plasmid, chromosome, virus, or transposon, to which another polynucleotide segment has been attached so as to bring about replication and / or expression of the attached segment, that either behaves as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replication under its own control) or is capable of replication by insertion into a host cell chromosome. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and cosmids. A vector may contain polynucleotide sequences necessary for ligation or insertion of the vector into a desired host cell and for expression of the attached segment. Such sequences vary depending on the host organism and include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosome binding site sequences, and transcription-translation termination sequences. Alternatively, an expression vector may be capable of directly expressing the product of a nucleic acid sequence encoded therein, without ligation or integration of the vector into a host cell's DNA sequence. In some embodiments, the vector is an "episomal expression vector" or "episome" that is capable of replicating in a host cell and, under appropriate selective pressure, persists as an extrachromosomal segment of DNA within the host cell (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative, commercially available episomal expression vectors include, but are not limited to, episomal plasmids that use the Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (LaJolla, Calif.) represent non-limiting examples of episomal vectors that use T antigen and the SV40 origin of replication instead of EBNA1 and oriP. The vector may also contain a selectable marker gene.

[0069] As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected in the presence of a corresponding selection agent, either forward or backward. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Applications WO 1992 / 08796 and WO 1994 / 28143, Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980), O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527 (1981), Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981), Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981), Santerre et al., Gene, 30: 147 (1984), Kent et al., Science, 237: 901-903 (1987), Wigler et al., Cell, 11: 223 (1977), Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026 (1962), Lowy et al., Cell, 22: 817 (1980), and U.S. Pat. Nos. 5,122,464 and 5,770,359.

[0070] As used herein, "coding sequence" refers to a segment of a polynucleotide that encodes a protein or polypeptide. The region or sequence is bounded near the 5' end by an initiation codon and near the 3' end by a termination codon. A coding sequence may also be referred to as an open reading frame.

[0071] As used herein, "operably linked" refers to the physical and / or functional linkage of a DNA segment to another DNA segment, allowing the segments to function in their intended manner. A DNA sequence encoding a gene product is operably linked to a regulatory sequence, such as a promoter, enhancer, and / or silencer, when it is linked to the regulatory sequence in a manner that allows it to directly or indirectly modulate the transcription of the DNA sequence. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream of the transcription start site of the promoter, in the correct reading frame with respect to the transcription start site, allowing transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when it is ligated to the DNA sequence so as to increase or decrease the transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream, downstream, or embedded in the coding region of a DNA sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if the signal sequence is expressed as a preprotein that participates in the secretion of the polypeptide. Linkage of a DNA sequence to a regulatory sequence is typically achieved via adapters or linkers inserted within the sequence by ligation at appropriate restriction sites or using restriction endonucleases known to those skilled in the art.

[0072] As used herein, the terms "induce," "induction," and grammatical equivalents refer to an increase in transcription of a nucleic acid sequence, promoter activity, and / or promoter expression brought about by a transcriptional regulator, relative to some basal transcription level.

[0073] The term "transcriptional regulator" refers to a biochemical element that acts to prevent or inhibit transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., a repressor protein or nuclear inhibitory protein), or a biochemical element that acts to enable or stimulate transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or enhancer).

[0074] The term "enhancer," as used herein, refers to a DNA sequence that, for example, increases the transcription of a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, changes in DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a variety of different sources are known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as the ATCC, as well as other commercial or personal sources). Many polynucleotides that contain a promoter (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream of, within, or downstream of a coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region mapped within the immunoglobulin (Ig) locus, including, for example, the heavy chain (mu) 5' enhancer, the light chain (kappa) 5' enhancer, the kappa and mu intronic enhancers, and the 3' enhancer (see generally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363, and U.S. Pat. No. 5,885,827).

[0075] A "promoter" refers to a region of a polynucleotide that induces transcription of a coding sequence. A promoter is located near and upstream (toward the 5' region of the sense strand) of the transcription start site of a gene, on the same strand of DNA. Some promoters are constitutive, as they are active under all conditions within a cell, whereas other promoters, such as inducible promoters, are regulated and active in response to specific stimuli. As used herein, the term "promoter activity" and its grammatical equivalents refer to the degree of expression of a nucleotide sequence operably linked to a promoter whose activity is measured. Promoter activity can be measured directly by determining the amount of RNA transcript produced, for example, by Northern blot analysis, or indirectly by determining the amount of a product encoded by a linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0076] As used herein, "inducible promoter" refers to a promoter that is induced to be active by the presence or absence of a transcriptional regulatory factor, e.g., a biotic or abiotic factor. Inducible promoters are useful because the expression of a gene operably linked to them can be turned on or off at certain stages of the development of an organism or, in particular, a tissue. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenic mechanism-regulated promoters, temperature-regulated promoters, and light-regulated promoters. An inducible promoter can be part of a gene switch or a gene switch.

[0077] As used herein, a "T cell" or "T lymphocyte" is a type of lymphocyte that plays a central role in cell-mediated immunity. "T cells" or "T lymphocytes" can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface.

[0078] As used herein, the term "antibody," also known as immunoglobulin (Ig), can be a monoclonal or polyclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B cells and that binds to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells and that bind to different epitopes of the same antigen. The antibody can be from any animal. The antibody can be IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. In some embodiments, the antibody can be a whole antibody, including a single-chain whole antibody. In some embodiments, the antibody can be Fab, Fab', F(ab'), Fd(V), or other fragments. H and CH1), Fv fragment (V H and V L (scFv), single chain antibody, disulfide-linked variable fragment (dsFv), and V L Domain or V HThe fragments may be antibody fragments, including, but not limited to, fragments containing domains. Whole antibodies typically consist of four polypeptides: two identical copies of heavy (H) chain polypeptides and two identical copies of light (L) chain polypeptides. Each heavy chain contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. The VH and VL regions share a common general structure, each containing four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for binding to an antigen. These specific regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entireties, where the definitions include overlapping amino acid residues or subsets of amino acid residues when compared against each other. Preferably, the term "CDR" refers to the CDRs defined by Kabat based on sequence comparison. CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0079] The terms "antibody fragment," "antibody fragment," "antibody fragment," "antigen-binding portion," or grammatical equivalents thereof, are used interchangeably herein to refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Antibody fragments desirably include, for example, one or more CDRs, a variable region (or portion thereof), a constant region (or portion thereof), or a combination thereof. Non-limiting examples of antibody fragments include: (1) a Fab fragment, which is a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain; (2) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the stalk region; (3) an Fv fragment, which is composed of the VL and VH domains of a single arm of an antibody; and (4) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment joined by a linker that allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778). (1998); and (5) diabodies, which are dimers of polypeptide chains in which each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to create a dimeric molecule with two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent No. 8,603,950.

[0080] An "antigen recognition moiety," "antigen recognition domain," "antigen-binding domain," or "antigen-binding region" refers to a molecule or portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, antibody-like molecule, or fragment thereof.

[0081] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Following such an analysis, it is possible to define groups of amino acids in which amino acids within a group are preferentially exchanged with each other and are therefore most similar to each other in their effect on overall protein structure (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, such as substitution of arginine with lysine, and vice versa, to maintain a positive charge; substitution of aspartic acid with glutamic acid, and vice versa, to maintain a negative charge; substitution of threonine with serine, to maintain a free -OH; and substitution of asparagine with glutamine, to maintain a free -NH2. Alternatively, or in addition, a functional variant may contain the amino acid sequence of the reference protein with at least one non-conservative amino acid substitution.

[0082] The term "non-conservative mutation" involves amino acid substitutions between different groups, such as substitution of tryptophan with lysine or substitution of serine with phenylalanine. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent protein.

[0083] As referred to herein, "proliferative disease" refers to the unifying concept that excessive cell proliferation and / or excessive cell-matrix turnover significantly contribute to the pathogenesis of diseases, including cancer. In some embodiments, the proliferative disease is cancer.

[0084] As used herein, "patient" or "subject" refers to a mammalian subject diagnosed with or suspected of having or developing a proliferative disorder, such as cancer. In some embodiments, the term "patient" refers to a mammalian subject who has a higher than average likelihood of developing a proliferative disorder, such as cancer. Exemplary patients can be humans, apes, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female. As used herein, a "patient in need thereof" or a "subject in need thereof" is considered to be a patient who has been diagnosed with or is suspected of having a disease or disorder, for example, but not limited to, cancer.

[0085] As used herein, "administering" refers to administering one or more compositions described herein to a patient or subject. By way of example and not limitation, administration, e.g., injection, of a composition can be performed by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be utilized. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by oral route. In addition, administration can also be by bolus or cell pellet depot surgery or placement of a medical device. In certain embodiments, a composition of the present disclosure can include engineered or host cells expressing a nucleic acid sequence described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount effective to treat or prevent a proliferative disorder. Pharmaceutical compositions may comprise the target cell populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, amino acids such as polypeptides or glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0086] As used herein, the terms "treatment," "treating," or their grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptoms attributable to the disease. In some embodiments, the term "treating" can include "preventing" a disease or condition.

[0087] As used herein, a "treatment interval" refers to a treatment cycle, e.g., a course of administration of a therapeutic agent, which may be repeated on a regular schedule. In embodiments, a dosage regimen may have one or more periods during the treatment interval during which no therapeutic agent is administered. For example, a treatment interval may include a single administration of a fusion protein administered in combination with (before, concurrently with, or after) administration of a second therapeutic agent, e.g., CAR-T cells.

[0088] As used herein, the terms "administered in combination" or "co-administration" or "co-administering" or "administering together" mean that two (or more) different treatments are delivered to a subject during the course of the subject's illness, for example, after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before treatment is discontinued for other reasons. In some embodiments, the delivery of one treatment occurs even as the delivery of a second treatment begins, such that there is overlap in administration. Herein, this is sometimes referred to as "simultaneous delivery" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In either case, in some embodiments, the treatments are more effective due to the combined administration. For example, the second treatment may be more effective, e.g., a lower amount of the second treatment may have a comparable effect, or the second treatment may reduce symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation would be seen with the first treatment. In some embodiments, delivery is performed such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery may be performed such that the effect of the first treatment is still detectable at the time the second treatment is delivered.

[0089] In some embodiments, the first treatment and the second treatment can be administered simultaneously (for example, at the same time) or sequentially by the same or separate compositions.Sequential administration refers to the administration of one treatment before the administration of another treatment, for example, a second treatment (for example, less than 5, 10, 15, 30, 45, 60 minutes, or immediately before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 hours or more; 4, 5, 6, 7, 8, 9 days or more; 1, 2, 3, 4, 5, 6, 7, 8 weeks or more).The order of administration of the first treatment and the second treatment can also be reversed.

[0090] The terms "therapeutically effective amount," "therapeutic amount," "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibiting effective amount," or their grammatical equivalents refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compositions described herein to elicit a desired response in one or more subjects. The exact amount of the composition of the present disclosure to be administered can be determined by a physician, taking into account the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and individual differences in condition.

[0091] Alternatively, the pharmacological and / or physiological effect of administering one or more compositions described herein to a patient or subject may be "prophylactic," i.e., the effect may prevent, completely or partially, a disease or its symptoms. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset). Programmed cell death proteins and other checkpoint inhibitors

[0092] Programmed cell death protein 1, also known as PD-1 or CD279 (cluster of differentiation 279), is an immune checkpoint protein. The PD-1 / PD-L1 signaling axis can promote tumor-mediated immune evasion. In some cases, PDL-1 can be overexpressed by tumor cells and accessory cells within the tumor microenvironment, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and antigen-presenting cells (APCs). In some cases, PD-1 can be upregulated by "exhausted" T cells, and upon binding to its ligands (PDL-1, PDL2, and CD80), it can signal to suppress effector T cell function. Blockade of the PD-1 / PD-L1 pathway with anti-PD-1 or anti-PD-L1 can restore exhausted T cell function and promote tumor cell killing (Figure 1).

[0093] In some embodiments, the fusion protein comprising a PD-1 inhibitor and a TGF-β trap can include, but is not limited to, full-length nivolumab (anti-PD-1), MK-3945 (anti-PD-1), pembrolizumab (anti-PD-1), pidilizumab (anti-PD-1), REGN2810 (anti-PD-1), AMP-224 (anti-PD-1), MEDI0680 (anti-PD-1), PDR001 (anti-PD-1), CT-001 (anti-PD-1), or a functional fragment or variant thereof. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab.

[0094] fusion proteins In some embodiments, the fusion proteins provided herein, or fragments or variants thereof, comprise a PD-1 inhibitor or PD-1 antibody fused to a cytokine trap via a linker.

[0095] In some embodiments, the PD-1 inhibitor may be an antibody, antibody fragment, or antibody variant that targets PD-1. In some embodiments, a fusion protein comprising pembrolizumab may be fused to a cytokine trap (e.g., a TGF-β trap). In some embodiments, a fusion protein comprising nivolumab may be fused to a cytokine trap (e.g., a TGF-β trap).

[0096] In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise the cytokine trap described above and an antibody, antibody fragment, or variant that targets an immune checkpoint gene. In some embodiments, antibodies, antibody fragments, or antibody variants that target immune checkpoints, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PDL1), can be fused to the TGF-β trap molecule via a linker. In some embodiments, the PD-L1 inhibitor is atezolizumab.

[0097] In some embodiments, the fusion proteins provided herein, or fragments or variants thereof, comprise a PD1 inhibitor or PD1 antibody fused to an adenosine deaminase (e.g., ADA2) described herein, or a functional variant or derivative thereof.

[0098] Cytokine Trap Cytokines affect many biological processes. Inhibition of cytokines can have clinical benefits, for example, in cancer. Several cytokines have been shown to be causative agents in various diseases. Such cytokines include, but are not limited to, IL-1, IL-4, IL-6, TNF-α, TGF-beta, and their various isoforms. As used herein, the term "cytokine trap" refers to a blocker or neutralizer of cytokine action. Examples of such cytokine traps include, but are not limited to, the extracellular domain of a cytokine receptor, an antibody that binds to the cytokine, and a peptide (e.g., an inhibitory peptide) that binds to the cytokine. In one embodiment, the cytokine is TGF-β. In one embodiment, the cytokine is TGF-β1. In one embodiment, the cytokine is TGF-β3. In one embodiment, the cytokines are TGF-β1 and TGF-β3. In further embodiments, a cytokine trap (e.g., a TGF-β trap) that targets TGF-β may include the extracellular domain of TGF-βRII, or a variant thereof (e.g., SEQ ID NOs: 141 and 142), an anti-TGF-β antibody, and an inhibitory peptide of TGF-β1, TGF-β2, and / or TGF-β3.

[0099] Transforming Growth Factor Transforming growth factor-β (TGF-β) is a multifunctional set of peptides that can regulate proliferation, differentiation, and other functions in many cell types. TGF-β can act synergistically with TGF-α to induce transformation. TGF-β can also act as a negative autocrine growth factor. Dysregulation of TGF-β activation and signaling can result in apoptosis. Many cells can synthesize TGF-β, and almost all of them have specific receptors for this peptide. TGF-β1, TGF-β2, and TGF-β3 can all function through the same receptor signaling pathway. TGF-β1 may play an important role in regulating the immune system and may exhibit different activities on different cell types or cells at different developmental stages. Most immune cells (or leukocytes) can secrete TGF-β1. TGF-β1 is a 112-amino acid peptide derived by proteolytic cleavage from the C-terminus of a precursor protein. TGF-β, a small secreted polypeptide, can signal through a type II serine / threonine kinase dimeric receptor (TGFβRII), which can recruit and phosphorylate a type I dimeric receptor (TGFβRI). TGFβRI can phosphorylate and activate SMADs, which can be transcription factors that regulate genes involved in cell proliferation, differentiation, apoptosis, and growth. Many advanced cancers are known to overexpress both TGF-β and TGFβR, promoting invasive tumor formation. Inhibition of the TGFβ signaling pathway may be a key therapeutic strategy for cancer treatment.

[0100] Some T cells (e.g., regulatory T cells) can release TGF-β1 to inhibit the action of other T cells. IL-1 / IL-2-dependent proliferation of activated T cells and activation of dormant helper T cells and cytotoxic T cells can be prevented by the activity of TGF-β1. Similarly, TGF-β1 can inhibit the secretion and activity of many other cytokines, including, but not limited to, interferon-γ, tumor necrosis factor alpha (TNF-α), and various interleukins. TGF-β1 can also downregulate the activity of immune cells by reducing the expression levels of cytokine receptors, such as the IL-2 receptor. However, TGF-β1 can also increase the expression of certain cytokines in T cells, promoting their proliferation, especially when the cells are immature (Figure 2).

[0101] TGF-β1 may have similar effects on B cells, which may vary depending on the differentiation state of the cells. TGF-β1 may inhibit B cell proliferation, stimulate apoptosis, and play a role in regulating the expression of antibodies, transferrin, and MHC class II proteins on immature and mature B cells.

[0102] The effects of TGF-β1 on macrophages and monocytes may be primarily inhibitory; this cytokine inhibits the proliferation of these cells and their production of reactive oxygen species (e.g., superoxide (O2)). - ) and nitrogen (e.g., nitric oxide (NO)) intermediates. However, as with other cell types, TGF-β1 can also have adverse effects on bone marrow-derived cells. For example, TGF-β1 can act as a chemoattractant, directing the immune response toward some pathogens; macrophages and monocytes can respond chemotactically to low levels of TGF-β1. Furthermore, expression of monocytic cytokines (including IL-1α, IL-1β, and TNF-α) and phagocytic killing by macrophages can be increased by the action of TGF-β1 (Figure 2).

[0103] Transforming growth factor-beta III (TGF-β3), a subset of the cytokine family, is responsible for a wide variety of functions, including cell proliferation, embryogenesis, immune system regulation, and differentiation.

[0104] Transforming growth factor β receptor II (TGFβRII) TGF-β receptors (TGFβRs) are single-pass transmembrane serine / threonine kinase receptors. TGFβRs can exist in several different isoforms, which can be homodimeric or heterodimeric. The number of characterized ligands within the TGF-β superfamily far exceeds the number of known receptors, suggesting that ligand-receptor interactions are promiscuous. Three TGF-β superfamily receptors (TGFβRs) specific for TGF-β can be distinguished by their structural and functional properties. TGFβRI (ALK5) and TGFβRII have similar ligand-binding affinities and can be distinguished from each other by peptide mapping alone. Both TGFβRI and TGFβRII can have high affinity for TGF-β1 and low affinity for TGF-β2. TGFβRIII (β-glycan) has high affinity for both the homodimers TGF-β1 and TGF-β2, and may also have high affinity for the heterodimer TGF-β1,2. The TGFβ receptor can also bind to TGF-β3. "TGFβRII" or "TGFβ receptor II" refers to a polypeptide having a wild-type human type 2 TGFβ receptor isoform A sequence (e.g., the amino acid sequence of NCBI Reference Sequence (RefSeq) Accession No. NP_001020018 (SEQ ID NO: 289)), or a polypeptide having a wild-type human type 2 TGFβ receptor isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq Accession No. NP_003233 (SEQ ID NO: 290)), or a sequence having substantial sequence identity to the amino acid sequence of SEQ ID NO: 289 or SEQ ID NO: 290. TGFβRII can retain at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99% of the TGFβ binding activity of the wild-type sequence. Expressed TGFβRII polypeptides lack a signal sequence.

[0105] TGF-β1 may reduce the efficacy of MHC II in stellate cells and dendritic cells, which may reduce the activation of appropriate helper T cell populations. TGF-β1 may promote tumor growth as cancer progresses and, in some embodiments, may not suppress inflammatory cell responses but may promote regulatory T cell function. TGF-β1 may be produced by tumor cells, tumor-associated fibroblasts, regulatory T cells, and immature myeloid cells. TGF-β1 may inhibit T cell priming and promote an exhaustion phenotype. TGF-β1 may suppress the anti-tumor activity of innate immune cell populations, including natural killer cells, macrophages, and dendritic cells. TGF-β receptor II may be upregulated by tumor-associated myeloid cells and may promote metastasis.

[0106] TGF-β trap fusion protein, or a fragment or variant thereof Provided herein are fusion proteins, or fragments or variants thereof, comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor or PD-1 antibody, and a cytokine trap capable of neutralizing a cytokine (e.g., TGF-β). In certain instances, the cytokine trap may be a TGF-β trap (also referred to as TGF-βRII, or a fragment or variant thereof), comprising SEQ ID NO: 142. Examples of TGF-β traps may include, but are not limited to, the extracellular domain (ECD) of a receptor (e.g., TGF-βRII) or a functional variant or derivative thereof, a TGF-β inhibitory peptide (e.g., SEQ ID NOs: 193-227), or an anti-TGF-β antibody. In some embodiments, the anti-TGF-β antibody has a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to a sequence selected from any one of SEQ ID NOs: 166, 168, 169, 171, 173, 175, or 177. HIn some embodiments, the anti-TGF-β antibody comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to a sequence selected from any one of SEQ ID NOs: 165, 167, 170, 172, 174, 176, or 178. L ). In certain embodiments, the TGF-β trap may specifically bind to or have high affinity for TGF-β1 or TGF-β2 or TGF-β3. In other embodiments, the TGF-β trap may specifically bind to or have high affinity for TGF-β1, TGF-β2, and TGF-β3. In other embodiments, the TGF-β trap may specifically bind to or have high affinity for TGF-β1 and TGF-β3. In further embodiments, the TGF-β trap may have low affinity for or not bind to TGF-β2.

[0107] The fusion proteins provided herein, or fragments or variants thereof (e.g., PD-1 inhibitors or PD-1 antibodies fused to cytokine traps such as TGF-β traps), can elicit a synergistic antitumor effect, resulting, for example, from the simultaneous blockade of the interaction between PD-L1 on tumor cells and PD-1 on immune cells, and the neutralization of TGF-β, for example, within the tumor microenvironment. Without being bound by theory, this effect results from the simultaneous blockade of two major immune evasion mechanisms by a single molecular entity and the targeted depletion of TGF-β within the tumor microenvironment. This depletion can be achieved by one or more of the following: (1) targeting tumor cells with anti-PD-1; (2) binding of TGF-β within the tumor microenvironment by a TGF-β trap (e.g., TGFbRII); and / or (3) disruption of TGF-β binding via PD-L1 receptor-mediated endocytosis. The fusion proteins provided herein, or fragments or variants thereof (e.g., a PD-1 inhibitor or PD-1 antibody fused to a cytokine trap, such as a TGF-β trap), may also promote natural killer cell-mediated killing of tumor cells.

[0108] (i) a TGFβRII trap fusion protein, or a fragment or variant thereof In some embodiments, the fusion protein, or a fragment or variant thereof, also comprises a cytokine trap and a PD-1 inhibitor or an anti-PD-1 antibody, or a fragment or variant thereof. In some embodiments, the fusion protein comprising a cytokine trap (e.g., a TGF-β trap), or a fragment or variant thereof, is optionally fused to a PD-1 inhibitor via a cleavable or non-cleavable linker. In some embodiments, the cytokine trap (e.g., a TGF-β trap) is a cytokine receptor (e.g., TGFβRII). In some embodiments, the cytokine receptor sequence in the fusion proteins described herein comprises the extracellular domain (ECD) of the receptor (e.g., TGFβRII), or a functional variant or derivative thereof. In some embodiments, the extracellular domain (ECD) of TGFβRII comprises the polypeptide sequence set forth in SEQ ID NO: 14. In some embodiments, the cytokine receptor sequence in the fusion proteins, or fragments or variants thereof, described herein comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 14. In some embodiments, the cytokine receptor sequence in the fusion proteins described herein comprises the extracellular domain (ECD) of a receptor (e.g., TGFβRII), or a functional variant or derivative thereof. In some embodiments, the extracellular domain (ECD) of TGFβRII comprises the polypeptide sequence set forth in SEQ ID NO: 141. In some embodiments, the cytokine receptor sequence in the fusion protein, or fragment or variant thereof, described herein comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 141.In some embodiments, the cytokine receptor sequence in the fusion proteins described herein comprises the extracellular domain (ECD) of a receptor (e.g., TGFβRII), or a functional variant or derivative thereof. In some embodiments, the extracellular domain (ECD) of TGFβRII comprises the polypeptide sequence set forth in SEQ ID NO: 142. In some embodiments, the cytokine receptor sequence in the fusion proteins, or fragments or variants thereof, described herein comprises a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 142. In some embodiments, the cytokine receptor sequence in the fusion proteins, or fragments or variants thereof, described herein, binds to TGF-β1 and / or TGF-β3, but does not bind to TGF-β2. In certain embodiments, the cytokine receptor sequence in the fusion proteins, or fragments or variants thereof, described herein binds only to TGF-β1. In certain embodiments, the cytokine receptor sequence in the fusion proteins, or fragments or variants thereof, described herein binds only to TGF-β3. In certain embodiments, the cytokine receptor sequence in the fusion proteins, or fragments or variants thereof, described herein binds only to TGF-β1 and / or TGF-β3, and has low or no affinity for TGF-β2.

[0109] In some embodiments, the PD-1 antibody is fused to TGFβRII or a fragment thereof (e.g., the ECD of TGFβRII). In some embodiments, the PD-1 antibody moiety is fused to TGFβRII or a fragment thereof (e.g., the ECD of TGFβRII) via a linker. In some embodiments, the PD-1 antibody moiety is fused to at least one extracellular domain of TGFβRII. In some embodiments, the PD-1 antibody moiety is fused to at least one extracellular domain of TGFβRII via a linker.

[0110] In some embodiments, the PD-1 antibody fragment or variant is a Fab, Fab2, (Fab')2, Fv, (Fv)2, scFv, scFv-Fc, Fc, diabody, triabody, or minibody of a PD-1 antibody. In some embodiments, the PD-1 antibody fragment is a single domain antibody of a PD-1 antibody. In some embodiments, the single domain antibody is a V NAR Fragment or V H It is the H fragment.

[0111] Non-limiting, exemplary fusion proteins are illustrated in Figures 4A-4C. In some embodiments, fusion proteins comprising an anti-PD-1 antibody, or fragment or variant thereof, fused to a TGF-β trap can elicit a synergistic anti-tumor effect resulting from the simultaneous blockade of the interaction between PD-L1 on tumor cells and PD-1 on immune cells and the neutralization of TGF-β within the tumor microenvironment. Without being bound by theory, this effect results from the simultaneous blockade of two major immune evasion mechanisms and targeted depletion of TGF-β within the tumor microenvironment by a single molecular entity. This depletion is achieved by (1) targeting of tumor cells by PD-1; (2) binding of TGF-β within the tumor microenvironment by a TGF-β trap (e.g., TGFβRII); and (3) disruption of TGF-β binding via PD-L1 receptor-mediated endocytosis.

[0112] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antibody, or fragment / variant thereof, comprising a heavy chain variable region (V H ) of the heavy chain of a PD-1 antibody, or a fragment / variant thereof (e.g., Figure 4A). In other embodiments, the TGF-β trap is fused to an IgG of a PD-1 antibody (e.g., Figure 4A). In certain aspects, the IgG is an IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the IgG is an IgG4. In another embodiment, the IgG4 is SEQ ID NO: 146 (wild type), SEQ ID NO: 291, SEQ ID NO: 292, or SEQ ID NO: 147 (S108P). In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the variable region (V) of the heavy chain of a PD-1 antibody, or a fragment / variant thereof, via a linker. H In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. H The V of a PD-1 antibody, or a fragment or variant thereof, is fused to the V H Exemplary sequences include, but are not limited to, SEQ ID NOS: 1-7 and 149-164. L Exemplary sequences include, but are not limited to, SEQ ID NOs: 8-13 and 148. In some embodiments, a TGF-β trap (e.g., TGFβRII) is a PD-1 antibody, or fragment / variant thereof, comprising a variable region (V L In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. L In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the variable region of the light chain (V) of the PD-1 antibody, or fragment / variant thereof, via a linker. L In one embodiment, the TGF-β trap (e.g., TGFβRII) is fused via a linker to the N-terminus or C-terminus of the VL chain or VH chain, or fragment / variant thereof.

[0113] The terms "anti-PD1(VL / VH)-TGFβRII" or "anti-PD1(VH / VL)-TGFβRII" are used interchangeably and refer to the specific VL or VH used within the fusion protein. In one embodiment, the terms "anti-PD1(VL / VH)-TGFβRII" or "anti-PD1(VH / VL)-TGFβRII" refer to a TGF-β trap (e.g., TGFβRII) fused to the constant region of the heavy chain of anti-PD1, or alternatively, to a TGF-β trap (e.g., TGFβRII) fused to the constant region of the light chain of anti-PD1.

[0114] In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the Fab of the PD-1 antibody, or fragment / variant thereof. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the Fab of the PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the Fab2 of the PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the (Fab')2 of the PD-1 antibody, or fragment / variant thereof. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the (Fab')2 of the PD-1 antibody, or fragment / variant thereof, via a linker. In one embodiment, a TGF-β trap (e.g., TGFβRII) is fused via a linker to the N-terminus or C-terminus of the Fab or (Fab')2 of the PD-1 antibody, or fragment / variant thereof.

[0115] In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the Fv of a PD-1 antibody, or fragment / variant thereof. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the Fv of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the (Fv)2 of a PD-1 antibody, or fragment / variant thereof, via a linker. In one aspect, the TGF-β trap (e.g., TGFβRII) is fused to the N-terminus or C-terminus of the Fv or (Fv)2 of a PD-1 antibody, or fragment / variant thereof, via a linker.

[0116] In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the scFv of a PD-1 antibody, or fragment / variant thereof. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the scFv of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the scFv-Fc of a PD-1 antibody, or fragment / variant thereof, via a linker. In one aspect, the TGF-β trap (e.g., TGFβRII) is fused to the N-terminus or C-terminus of the scFv or scFv-Fc of a PD-1 antibody, or fragment / variant thereof, via a linker.

[0117] In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the Fc of a PD-1 antibody, or fragment / variant thereof. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the Fc of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, the TGF-β trap (e.g., TGFβRII) is linked to the C-terminal Fc of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, the TGF-β trap (e.g., TGFβRII) is linked to the N-terminal Fc of a PD-1 antibody, or fragment / variant thereof, via a linker.

[0118] In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a PD-1 antibody, or a diabody of a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a PD-1 antibody, or a diabody of a fragment / variant thereof via a linker. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a PD-1 antibody, or a triabody of a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a PD-1 antibody, or a triabody of a fragment / variant thereof via a linker. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a PD-1 antibody, or a minibody of a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a PD-1 antibody, or a minibody of a fragment / variant thereof via a linker. In one embodiment, a TGF-β trap (e.g., TGFβRII) is fused to the N-terminus or C-terminus of a PD-1 antibody, or fragment / variant thereof, minibody via a linker.

[0119] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antibody or a fragment / variant thereof. NAR In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. NAR In all embodiments described, the TGF-β trap (e.g., TGFβRII) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. NAR It is fused to the N-terminus or C-terminus of

[0120] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antibody or a fragment / variant thereof. HIn some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. H In all embodiments described, the TGF-β trap (e.g., TGFβRII) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. H It is fused to the N-terminus or C-terminus of H.

[0121] In some embodiments, the PD-1 antibody portion is fused to TGFβRII, or a fragment or variant thereof (e.g., the ECD of TGFβRII). In some embodiments, the PD-1 antibody portion is fused to TGFβRII, or a fragment or variant thereof (e.g., the ECD of TGFβRII) via a linker. In some embodiments, the PD-1 antibody portion is fused to at least one extracellular domain of TGFβRII. In some embodiments, the PD-1 antibody portion is fused to at least one extracellular domain of TGFβRII via a linker. Non-limiting exemplary fusion proteins are illustrated in Figures 4A-4C.

[0122] In some embodiments, the PD-1 antibody fragment is a Fab, Fab2, (Fab')2, Fv, (Fv)2, scFv, scFv-Fc, Fc, diabody, triabody, or minibody of a PD-1 antibody. In some embodiments, the PD-1 antibody fragment is a single domain antibody of a PD-1 antibody. In some embodiments, the single domain antibody is a V NAR Fragment or V H It is the H fragment.

[0123] In some embodiments, the variable region of the heavy chain (V H ) comprises one or more polypeptide sequences set forth in any one of SEQ ID NOs: 1-7. In some embodiments, the variable region (V) of the heavy chain of the PD-1 antibody, or fragment / variant thereof, H) comprises one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to any one of the polypeptide sequences set forth in any one of SEQ ID NOs: 1-7 and 149-164.

[0124] In some embodiments, the variable region of the heavy chain (V L ) comprises one or more polypeptide sequences set forth in any one of SEQ ID NOs: 8-13 and 148. In some embodiments, the variable region (V) of the heavy chain of the PD-1 antibody, or fragment / variant thereof, L ) comprises one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to any one of the polypeptide sequences set forth in SEQ ID NOs: 8-13 and 148.

[0125] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:6. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 12. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:6. H) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12. L ) and

[0126] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 15, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 16.

[0127] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:15 and the sequence set forth in SEQ ID NO:16.

[0128] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 15 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 143.

[0129] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:15 and the sequence set forth in SEQ ID NO:143.

[0130] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 15 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:294.

[0131] In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO:15 and the sequence set forth in SEQ ID NO:294.

[0132] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:7. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 13. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:7. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 13. L ) and

[0133] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 296 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 145.

[0134] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:296 and the sequence set forth in SEQ ID NO:145.

[0135] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 296 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 144.

[0136] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:296 and the sequence set forth in SEQ ID NO:144.

[0137] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:296 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:295.

[0138] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:296 and the sequence set forth in SEQ ID NO:295.

[0139] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 16.

[0140] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 143.

[0141] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 13, and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 145. In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 145.

[0142] In some embodiments, the fusion protein comprises a linker and a variable region of a heavy chain (V H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 15. LIn some embodiments, the fusion protein comprises a linker and a variable region of a heavy chain (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 16. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 15. L ) and

[0143] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 1. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:1. H ) is included.

[0144] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:2. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:2. H ) is included.

[0145] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:3. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:3. H ) is included.

[0146] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:4. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:4. H ) is included.

[0147] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:5. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:5. H ) is included.

[0148] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:6. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:6. H ) is included.

[0149] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:7. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:7. H ) is included.

[0150] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:5. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:5. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0151] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 149. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0152] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 157. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0153] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0154] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:5. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:5. H ) is included.

[0155] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 149. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149. H ) is included.

[0156] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 157. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157. H ) is included.

[0157] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) is included.

[0158] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) is included.

[0159] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:8. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) is included.

[0160] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 149. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149. H ) is included.

[0161] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 150. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 150. H ) is included.

[0162] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 151. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 151. H ) is included.

[0163] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 152. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 152. H ) is included.

[0164] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 153. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 153. H ) is included.

[0165] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 154. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 154. H ) is included.

[0166] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 155. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 155. H ) is included.

[0167] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 156. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 156. H ) is included.

[0168] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 157. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157. H ) is included.

[0169] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) is included.

[0170] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 159. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 159. H ) is included.

[0171] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 160. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 160. H ) is included.

[0172] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 161. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 161. H ) is included.

[0173] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 162. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 162. H ) is included.

[0174] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 163. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 163. H ) is included.

[0175] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 164. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 164. H ) is included.

[0176] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:8. LIn some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) is included.

[0177] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:9. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:9. L ) is included.

[0178] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 10. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 10. L ) is included.

[0179] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:11. LIn some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:11. L ) is included.

[0180] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 12. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12. L ) is included.

[0181] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 13. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 13. L ) is included.

[0182] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 148. LIn some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 148. L ) is included.

[0183] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 15. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 15.

[0184] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 16. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 16.

[0185] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 143. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 143.

[0186] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 144. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 144.

[0187] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 145. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 145.

[0188] (ii) an anti-TGFβ antibody fusion protein, or a fragment or variant thereof; In other embodiments, the cytokine trap is an antibody, antibody fragment, or antibody variant against TGF-β. In such embodiments, such antibodies may include anti-TGFβ pan-neutralizing antibodies or anti-receptor antibodies that block receptor binding to TGFβ1, 2, and / or 3. In certain embodiments, the antibody fragment or variant is a Fab, (Fab)2, (Fab')2, Fv, (Fv)2, scFv, scFv-Fc, Fc, diabody, triabody, or minibody of a TGFβ antibody. In one embodiment, the anti-TGF-β antibody, or fragment or variant thereof, binds to TGF-β1, TGF-β2, and TGF-β3. In certain embodiments, the anti-TGF-β antibody, or fragment or variant thereof, binds to TGF-β1. In certain embodiments, the anti-TGF-β antibody, or fragment or variant thereof, binds to TGF-β3. In certain embodiments, the anti-TGF-β antibody, or fragment or variant thereof, binds to TGF-β1 and TGF-β2. In certain embodiments, the anti-TGF-β antibody, or fragment or variant thereof, binds to TGF-β1 and TGF-β3. Exemplary VH sequences of TGF-β antibodies, or fragments or variants thereof, include, but are not limited to, SEQ ID NOs: 166, 168, 169, 171, 173, 175, and 177. Exemplary VL sequences of TGF-β antibodies, or fragments or variants thereof, include, but are not limited to, SEQ ID NOs: 165, 167, 170, 172, 174, 176, and 178. In some embodiments, the anti-TGF-β antibody has a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to a sequence selected from any one of SEQ ID NOs: 166, 168, 169, 171, 173, 175, or 177. HIn some embodiments, the anti-TGF-β antibody comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to a sequence selected from any one of SEQ ID NOs: 165, 167, 170, 172, 174, 176, or 178. L ) is included.

[0189] (iii) a TGF-β antagonistic peptide fusion protein, or a fragment or variant thereof In certain embodiments, the cytokine trap may comprise a TGF-β antagonistic peptide or a TGF-β inhibitory peptide. Such peptides may be generated de novo using phage display. In one embodiment, such peptides may be derived from a segment of a TGF-β isoform(s) or a TGF-β receptor(s), and examples of TGF-β antagonistic peptides may include, but are not limited to, SEQ ID NOs: 193-227. Examples of TGF-β inhibitory peptides may include, but are not limited to, SEQ ID NOs: 193-227. In one embodiment, the peptide is linked to the V domain of a PD-1 antibody, or a fragment / variant thereof, via a linker. H and / or V L In another embodiment, one peptide may be fused to a PD-1 antibody, or fragment / variant thereof. In a further embodiment, more than one peptide may be fused to a PD-1 antibody, or fragment / variant thereof. When more than one peptide is used, the peptides may be fused with or without a linker to form a concatamer. When more than one peptide is used, the same peptide may be used to form the concatamer. Alternatively, two or more different peptide combinations may be used to form the concatamer.

[0190] Adenosine: Adenosine is a key immunomodulator in the tumor microenvironment. Extracellular adenosine suppresses inflammatory responses when it binds to the A2A adenosine receptor (A2AR), a subtype predominantly expressed in most immune cells. Some tumors express high levels of CD39 and CD73, ectonucleotidases that convert ATP and ADP to AMP and AMP to adenosine, respectively. Therefore, adenosine promotes the suppressive activity of regulatory T cells by inducing the expression of Foxp3, CD39, and CD73. In addition, hypoxia also induces the accumulation of extracellular adenosine in the tumor microenvironment through the induction of CD39 and CD73. Additionally, high levels of extracellular adenosine in the tumor microenvironment are maintained by hypoxia-inducible factor (HIF)-dependent inhibition of the nucleotide transporter ENT-1 and inhibition of adenosine kinase, which prevents adenosine from relocating into the intracellular lumen and preventing the formation of AMP. Therefore, targeting the reduction of extracellular adenosine in the tumor microenvironment is understood to enhance immune cell function and promote tumor cell killing.

[0191] Presented herein, in certain embodiments, is a fusion protein comprising an adenosine deaminase (e.g., ADA2) that targets the reduction of extracellular adenosine within the tumor microenvironment.

[0192] Adenosine deaminase Adenosine deaminase (also known as adenosine aminohydrolase or ADA) irreversibly deaminates adenosine, converting it to the related nucleoside, inosine, through replacement of the amino group with a ketone group. Inosine is then deribosylated (removed from ribose) by another enzyme called purine nucleoside phosphorylase (PNP), which converts it to hypoxanthine. ADA is required for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues. In humans, its primary function is the development and maintenance of the immune system. However, ADA has also been observed to be associated with epithelial cell differentiation, neurotransmission, and the maintenance of pregnancy. Two isoforms of ADA exist: ADA1 and ADA2.

[0193] ADA1 is found in most somatic cells, particularly lymphocytes and macrophages, where it exists not only in the cytosol and nucleus, but also as an extracellular form on the cell membrane conjugated to dipeptidyl peptidase 4 (also known as CD26). ADA1 is largely responsible for intracellular activity and exists in both small (monomer) and large (dimer) forms. The interconversion of the small to large form is regulated by a "conversion factor" in the lung.

[0194] ADA2 was first identified in human spleen. It was subsequently found in other tissues, including macrophages, where it coexists with ADA1. The two isoforms regulate the ratio of adenosine to deoxyadenosine. ADA2 is primarily found in human plasma and serum and exists primarily as a homodimer. ADA2 is the predominant form present in human plasma and is elevated in many diseases, particularly those related to the immune system, such as rheumatoid arthritis, psoriasis, and sarcoidosis. Plasma ADA2 isoforms are also elevated in most cancers. ADA2 is not ubiquitous and coexists with ADA1 in monocyte-macrophages.

[0195] ADA2 fusion protein, or a fragment or variant thereof Provided herein is a fusion protein, or a fragment or variant thereof, comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor or PD-1 antibody, and an adenosine deaminase (e.g., ADA2) that can neutralize adenosine. The fusion proteins provided herein, or fragments or variants thereof, such as PD-1 inhibitors or PD-1 antibodies fused to an adenosine deaminase (e.g., ADA2), can elicit a synergistic anti-tumor effect, resulting from, for example, the simultaneous blockade of the interaction between PD-L1 on tumor cells and PD-1 on immune cells, and the neutralization of adenosine, for example, within the tumor microenvironment. Without being bound by theory, this effect results from the simultaneous blockade of two major immune evasion mechanisms by a single molecular entity and the targeted depletion of adenosine within the tumor microenvironment. This depletion may be achieved by one or more of the following: (1) targeting of tumor cells with anti-PD-1; (2) binding of adenosine within the tumor microenvironment by adenosine deaminase (e.g., ADA2); and (3) disruption of adenosine binding via PD-L1 receptor-mediated endocytosis.

[0196] In some embodiments, the adenosine deaminase (e.g., ADA2) is part of a fusion protein, or fragment or variant thereof, that also includes a PD-1 inhibitor or PD-1 antibody, or a fragment or variant thereof. In some embodiments, the fusion protein including adenosine deaminase (e.g., ADA2), or a fragment or variant thereof, is optionally fused to the PD-1 inhibitor via a cleavable or non-cleavable linker. In some embodiments, the adenosine deaminase is adenosine deaminase 2 (ADA2). In some embodiments, the fusion proteins described herein include an adenosine deaminase (e.g., ADA2), or a functional variant or derivative thereof, as described herein. Examples of ADA2 and variants are described in WO2016061286, which is incorporated herein by reference in its entirety. In some embodiments, the TGF-β cytokine trap comprises any one of ADA2 mutant 1, ADA2 mutant 2, ADA2 mutant 3, ADA2 mutant 4, ADA2 mutant 5, ADA2 mutant 6, or ADA2 mutant 7. In some embodiments, the fusion proteins provided herein, or fragments or variants thereof, comprise a PD-1 inhibitor or PD-1 antibody fused to an ADA protein, or functional fragment thereof, via a linker. In some embodiments, the PD-1 inhibitor can be an antibody, or antibody fragment or variant, that targets PD-1.

[0197] In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 284. In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 273. In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 274. In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 37275.In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 276. In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 277. In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 278. In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise a polypeptide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to the polypeptide sequence of SEQ ID NO: 279.

[0198] In some embodiments, the fusion proteins, or fragments or variants thereof, described herein comprise an adenosine deaminase (e.g., ADA2) and an antibody, antibody fragment, or variant that targets an immune checkpoint gene, as described above. In some embodiments, antibodies, antibody fragments, or variants that target immune checkpoints, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), may be fused to the adenosine deaminase (e.g., ADA2) molecule via a linker.

[0199] In some embodiments, the PD-1 antibody portion is fused to an adenosine deaminase (e.g., ADA2), or a fragment thereof. In some embodiments, the PD-1 antibody portion is fused to an adenosine deaminase (e.g., ADA2) via a linker. In some embodiments, the PD-1 antibody portion is fused to at least one domain of ADA2. In some embodiments, the PD-1 antibody portion is fused to at least one domain of ADA2 via a linker.

[0200] In some embodiments, the PD-1 antibody fragment or variant is a Fab, (Fab)2, (Fab')2, Fv, (Fv)2, scFv, scFv-Fc, Fc, diabody, triabody, or minibody of a PD-1 antibody. In some embodiments, the PD-1 antibody fragment is a single domain antibody of a PD-1 antibody. In some embodiments, the single domain antibody is a V NAR Fragment or V H It is the H fragment.

[0201] Non-limiting, exemplary fusion proteins are illustrated in Figures 33A-33C. In some embodiments, fusion proteins comprising an anti-PD-1 antibody, or fragment or variant thereof, fused to an adenosine deaminase (e.g., ADA2) can elicit synergistic anti-tumor effects due to the simultaneous blockade of the interaction between PD-L1 on tumor cells and PD-1 on immune cells, and targeted reduction of extracellular adenosine within the tumor microenvironment.

[0202] In some embodiments, the adenosine deaminase (e.g., ADA2) is activated by the variable region (V) of the heavy chain of a PD-1 antibody, or fragment / variant thereof. H ) of the heavy chain of a PD-1 antibody, or fragment / variant thereof (e.g., Figure 4a). In other embodiments, adenosine deaminase is fused to the IgG of a PD-1 antibody (e.g., Figure 4a). In certain aspects, the IgG is IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the IgG is IgG4. In another embodiment, the IgG4 is SEQ ID NO: 146 (wild type), SEQ ID NO: 291, SEQ ID NO: 292, or SEQ ID NO: 147 (S108P). In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the variable region (V) of the heavy chain of a PD-1 antibody, or fragment / variant thereof, via a linker. H In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the V H In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the variable region of the light chain (V) of a PD-1 antibody, or fragment / variant thereof. L In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to the variable region (V) of the light chain of the PD-1 antibody, or fragment / variant thereof, via a linker. L In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the V LIn one embodiment, the adenosine deaminase is fused to the V constant region of the PD-1 antibody, or fragment / variant thereof, via a linker. H or V L It is fused to the N-terminus or C-terminus of

[0203] In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the Fab of a PD-1 antibody, or fragment / variant thereof. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the Fab of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the (Fab)2 of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the (Fab')2 of a PD-1 antibody, or fragment / variant thereof. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the (Fab')2 of a PD-1 antibody, or fragment / variant thereof, via a linker. In one embodiment, the adenosine deaminase is fused via a linker to the N-terminus or C-terminus of the Fab or (Fab)2 of the PD-1 antibody, or fragment / variant thereof.

[0204] In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the Fv of a PD-1 antibody, or fragment / variant thereof. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the Fv of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the (Fv)2 of a PD-1 antibody, or fragment / variant thereof, via a linker. In one aspect, an adenosine deaminase is fused to the N-terminus or C-terminus of the Fv or (Fv)2 of a PD-1 antibody, or fragment / variant thereof, via a linker.

[0205] In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the scFv of a PD-1 antibody, or fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the scFv of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the scFv-Fc of a PD-1 antibody, or fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the scFv-Fc of a PD-1 antibody, or fragment / variant thereof, via a linker. In one aspect, adenosine deaminase is fused to the N-terminus or C-terminus of the scFv or scFv-Fc of a PD-1 antibody, or fragment / variant thereof, via a linker.

[0206] In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the Fc of a PD-1 antibody, or fragment / variant thereof. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the Fc of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, an adenosine deaminase (e.g., ADA2) is linked to the C-terminal Fc of a PD-1 antibody, or fragment / variant thereof, via a linker. In some embodiments, an adenosine deaminase (e.g., ADA2) is linked to the N-terminal Fc of a PD-1 antibody, or fragment / variant thereof. In some embodiments, an adenosine deaminase (e.g., ADA2) is linked to the N-terminal Fc of a PD-1 antibody, or fragment / variant thereof, via a linker.

[0207] In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to a diabody of a PD-1 antibody, or fragment / variant thereof. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to a diabody of a PD-1 antibody, or fragment / variant thereof via a linker. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to a triabody of a PD-1 antibody, or fragment / variant thereof via a linker. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to a minibody of a PD-1 antibody, or fragment / variant thereof. In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to a minibody of a PD-1 antibody, or fragment / variant thereof via a linker.

[0208] In some embodiments, adenosine deaminase (e.g., ADA2) is expressed by the VNAR In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. NAR In one embodiment, the adenosine deaminase is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. NAR It is fused to the N-terminus or C-terminus of

[0209] In some embodiments, adenosine deaminase (e.g., ADA2) is expressed by the V H In some embodiments, an adenosine deaminase (e.g., ADA2) is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. H In one embodiment, the adenosine deaminase is fused to the V of the PD-1 antibody, or fragment / variant thereof, via a linker. H It is fused to the N-terminus or C-terminus of H.

[0210] In some embodiments, the variable region of the heavy chain (V H ) comprises one or more polypeptide sequences set forth in any one of SEQ ID NOs: 1-7. In some embodiments, the variable region (V) of the heavy chain of the PD-1 antibody, or fragment / variant thereof, H ) comprises one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to any one of the polypeptide sequences set forth in SEQ ID NOs: 1-7 and 149-164.

[0211] In some embodiments, the variable region of the heavy chain (V L) comprises one or more polypeptide sequences set forth in any one of SEQ ID NOs: 8-13 and 148. In some embodiments, the variable region (V) of the heavy chain of the PD-1 antibody, or fragment / variant thereof, L ) comprises one or more polypeptide sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identity to any one of the sequences set forth in polypeptide SEQ ID NOs: 8-13, and 148.

[0212] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:6. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 12. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:6. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12. L ) and

[0213] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 280.

[0214] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:12 and the sequence set forth in SEQ ID NO:280.

[0215] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 281.

[0216] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:12 and the sequence set forth in SEQ ID NO:281.

[0217] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:7. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 13. LIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:7. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 13. L ) and

[0218] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 13 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 282.

[0219] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:13 and the sequence set forth in SEQ ID NO:282.

[0220] In some embodiments, the fusion protein comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 13 and a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 283.

[0221] In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO:13 and the sequence set forth in SEQ ID NO:283.

[0222] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 1. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:1. H ) is included.

[0223] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:2. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:2. H ) is included.

[0224] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:3. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:3. H ) is included.

[0225] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:4. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:4. H ) is included.

[0226] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:5. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:5. H ) is included.

[0227] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:6. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:6. H ) is included.

[0228] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:7. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:7. H ) is included.

[0229] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 149. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149. H ) is included.

[0230] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 150. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 150. H ) is included.

[0231] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 151. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 151. H ) is included.

[0232] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 152. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 152. H ) is included.

[0233] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 153. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 153. H ) is included.

[0234] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 154. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 154. H ) is included.

[0235] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 155. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 155. H ) is included.

[0236] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 156. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 156. H ) is included.

[0237] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 157. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157. H ) is included.

[0238] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) is included.

[0239] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 159. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 159. H ) is included.

[0240] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 160. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 160. H ) is included.

[0241] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 161. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 161. H ) is included.

[0242] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 162. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 162. H ) is included.

[0243] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 163. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 163. H ) is included.

[0244] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 164. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 164. H ) is included.

[0245] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:8. LIn some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) is included.

[0246] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:9. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:9. L ) is included.

[0247] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 10. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 10. L ) is included.

[0248] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:11. LIn some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:11. L ) is included.

[0249] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 12. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 12. L ) is included.

[0250] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 13. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 13. L ) is included.

[0251] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 148. LIn some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 148. L ) is included.

[0252] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 15. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 15.

[0253] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 280. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:280.

[0254] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 281. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:281.

[0255] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 282. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:282.

[0256] In some embodiments, the fusion protein comprises a polypeptide sequence set forth in SEQ ID NO: 283. In some embodiments, the fusion protein comprises a polypeptide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:283.

[0257] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:5. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:5. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0258] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 149. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0259] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 157. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0260] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. H ) and a light chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO: 8. L In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) and a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) and

[0261] In some embodiments, the fusion protein comprises a heavy chain variable region (V) comprising the polypeptide sequence set forth in SEQ ID NO:5. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:5. H ) is included.

[0262] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 149. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 149. H ) is included.

[0263] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 157. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 157. H ) is included.

[0264] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. H In some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) is included.

[0265] In some embodiments, the fusion protein comprises a variable region of the heavy chain (V) comprising the polypeptide sequence set forth in SEQ ID NO: 158. HIn some embodiments, the fusion protein comprises a heavy chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO: 158. H ) is included.

[0266] In some embodiments, the fusion protein comprises a variable region of the light chain (V) comprising the polypeptide sequence set forth in SEQ ID NO:8. L In some embodiments, the fusion protein comprises a light chain variable region (V) that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical to the sequence set forth in SEQ ID NO:8. L ) is included.

[0267] Linker In some embodiments, the linker comprises one or more polypeptide sequences set forth in any one of SEQ ID NOS: 17-34. In some embodiments, the linker may be a flexible linker. Flexible linkers may be applied when the domains to be joined require some degree of movement or interaction. Flexible linkers may be composed of small, non-polar (e.g., Gly), or polar (e.g., Ser or Thr) amino acids. Flexible linkers may have a sequence consisting primarily of a string of Gly and Ser residues (a "GS" linker). A non-limiting example of a flexible linker may have the sequence (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. By adjusting the copy number "n," the length of this exemplary GS linker may be optimized to achieve appropriate separation of functional domains or to maintain necessary inter-domain interactions. In addition to the GS linker, other flexible linkers can be used in recombinant fusion proteins. In some embodiments, the flexible linker can have a sequence of (Gly)n, where n can be 6, 7, or 8. In some cases, the flexible linker can also be rich in small or polar amino acids, such as Gly and Ser, but can contain additional amino acids, such as Thr and Ala, to maintain flexibility. In some cases, the linkers described herein can be rigid linkers. Rigid linkers can be used to maintain a constant distance between the domains of the fusion proteins described herein, or fragments or variants thereof. Examples of rigid linkers can be alpha-helix-forming linkers, Pro-rich sequences, (XP)n, X-Pro backbones, and (EAAAK)n (n = 1-6), to name a few. An inflexible linker may optionally exhibit a relatively rigid structure by adopting an α-helical structure or by containing multiple Pro residues.In some embodiments, the immune checkpoint inhibitor, such as a PD-1 inhibitor, and a cytokine trap (e.g., a TGF-β trap) capable of neutralizing a cytokine (e.g., TGF-β) in a fusion protein, or fragment or variant thereof, described herein, can be separated by an intervening sequence encoding an intervening linker polypeptide. In some embodiments, the immune checkpoint inhibitor, such as a PD-1 inhibitor, and ADA2 (or mutant thereof) in a fusion protein, or fragment or variant thereof, described herein, can be separated by an intervening sequence encoding an intervening linker polypeptide. In certain embodiments, the linker polypeptide is selected from the group consisting of:

[0268] [Table 1] The present invention includes a linker polypeptide as disclosed in

[0269] In some embodiments, the linker can be a flexible linker, a non-flexible linker, an in vivo cleavable linker, or any combination thereof. In some cases, the linker can link functional domains together (as in the case of flexible and non-flexible linkers) or release the functional domains in vivo, as in the case of in vivo cleavable linkers. In some embodiments, the linker can improve biological activity, increase expression yield, and achieve desired pharmacokinetics. In some embodiments, the linker can also include a hydrazone, peptide, disulfide, or thioether.

[0270] In some cases, the linker sequences described herein may include flexible linkers. Flexible linkers may be applied when the domains to be joined require some degree of movement or interaction. Flexible linkers may be composed of small, non-polar (e.g., Gly), or polar (e.g., Ser or Thr) amino acids. Flexible linkers may have a sequence consisting primarily of a string of Gly and Ser residues (a "GS" linker). An example of a flexible linker may have the sequence (GS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a flexible linker may have the sequence (Gly)n, where n can be 6, 7, or 8. In some cases, flexible linkers may also be rich in small or polar amino acids, such as Gly and Ser, but may contain additional amino acids, such as Thr and Ala, to maintain flexibility. In other cases, polar amino acids such as Lys and Glu can be used to improve solubility. The length of these non-limiting exemplary linkers can be optimized by adjusting the copy number "n" to achieve appropriate separation of functional domains, and the length of these non-limiting exemplary linkers can be optimized to maintain necessary inter-domain interactions. In addition to the GS linker, other flexible linkers can also be utilized in the fusion proteins, or fragments or variants thereof, described herein. In some cases, flexible linkers can also be rich in small or polar amino acids, such as Gly and Ser, but can contain additional amino acids, such as Thr and Ala, to maintain flexibility. In other cases, polar amino acids such as Lys and Glu can be used to improve solubility.

[0271] The flexible linker incorporated into the linker sequence described herein may be rich in small or polar amino acids, such as Gly and Ser, to provide good flexibility and solubility. A flexible linker may be an appropriate choice when a specific movement or interaction is desired for the domain of the fusion protein, or a fragment or variant thereof. In addition, a flexible linker may be used as a passive linker that does not have an inflexible structure but maintains the distance between functional domains. The length of the flexible linker may be adjusted to allow the fusion protein, or a fragment or variant thereof, to fold properly and to allow optimal biological activity.

[0272] The linkers described herein may optionally further comprise a rigid linker. Rigid linkers can be used to maintain a constant distance between domains of the fusion proteins described herein, or fragments or variants thereof. Examples of rigid linkers include alpha-helix-forming linkers, Pro-rich sequences, (XP)n, X-Pro backbones, and (EAAAK)n (n=1-6), to name a few. Rigid linkers may optionally exhibit a relatively rigid structure by adopting an alpha-helical structure or by containing multiple Pro residues.

[0273] In some embodiments, the linkers described herein may be cleavable linkers. In other cases, the linkers are not cleavable. A non-cleavable linker may covalently join the functional domains of a fusion protein, or a fragment or variant thereof, together to act as a single molecule through in vivo or ex vivo processes. The linker may also be cleavable in vivo. A cleavable linker can be introduced to release a free functional domain in vivo. A cleavable linker can be cleaved in the presence of a reducing agent, a protease, to name a few. For example, reduction of a disulfide bond can be used to create a cleavable linker. In the case of a disulfide linker, a cleavage event via disulfide exchange with a thiol, such as glutathione, could result in cleavage. In other cases, cleavage of a linker within a recombinant fusion protein in vivo can also be carried out by proteases that may be expressed in specific cells or tissues in vivo under pathological conditions (e.g., cancer or inflammation) or that may be restricted to certain cellular compartments. In some cases, a cleavable linker can enable targeted cleavage. For example, the specificity of many proteases can result in slow cleavage of the linker within a restricted compartment. A cleavable linker can also include a hydrazone, peptide, disulfide, or thioether. For example, a hydrazone can confer stability in serum. In other cases, a hydrazone can enable cleavage within an acidic compartment. The acidic compartment can have a pH of up to 7. A linker can also include a thioether. The thioether can be non-reducing. The thioether can be designed for intracellular proteolysis.

[0274] In some cases, the linker may be an engineered linker. For example, the linker may be designed to include chemical features such as hydrophobicity. In some cases, at least two different linker polypeptide sequences may encode the same polypeptide linker sequence. The method for designing the linker may be a computational method. In some cases, the computational method may include a graphical method. Computational methods may be used to search for suitable peptides from a library of three-dimensional peptide structures derived from a database. For example, the Brookhaven Protein Data Bank (PDB) may be used to measure the spacing distance between selected amino acids of the linker. In some cases, the polypeptide linker may also include one or more GS linker sequences, such as (GS), (GS), (SG), (GSG), and (SGSG), where n may be any number from 0 to 15.

[0275] Methods for Treating Cancer Also provided herein are methods for treating cancer with a fusion protein comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor, and a cytokine trap (e.g., a TGF-β trap) capable of neutralizing a cytokine (e.g., TGF-β). Also provided herein are methods for treating cancer with a fusion protein comprising an immune checkpoint inhibitor, such as a PD-1 inhibitor, and an adenosine deaminase protein. The development of monoclonal antibodies targeting blockade of immune checkpoint pathways, such as the PD1 and PD-L1 / 2 signaling pathways and the CTLA-4 and CD80 / 86 signaling pathways, has revolutionized cancer treatment and demonstrated durable clinical activity in several cancer indications, including, but not limited to, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, MSI-high colorectal cancer, Merkel cell carcinoma, and Hodgkin lymphoma. Despite durable responses, response rates remain extremely low, and some patients develop resistance leading to disease progression. Furthermore, in several indications, such as ovarian, gastroesophageal, prostate, pancreatic, and many other cancers, checkpoint inhibitors have failed to demonstrate any substantial clinical response.

[0276] The failure of checkpoint blockade may be attributed to the complexity of immunosuppressive factors present in the tumor microenvironment. These factors may include, but are not limited to, suppressor cells such as myeloid-derived suppressor cells and tumor-associated macrophages (TAMs); inhibitory cytokines and growth factors such as TGF-β and interleukin-10 (IL-10); and metabolic derivatives such as adenosine and by-products of indoleamine 2,3-dioxygenase (IDO). The anti-PD1-TGFβRII fusion protein presented herein is an example of a therapeutic that can target two negative inhibitory pathways within the tumor microenvironment. These pathways may include cell-intrinsic interactions with the immune system mediated by tumor cells, in which PD-1 / PD-L1 interactions may play a key role, and cell-extrinsic interactions mediated by immunosuppressive cytokines, in which TGF-β may be a prominent member.

[0277] In some embodiments, the cancer is, but is not limited to, glioblastoma, colorectal cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, and renal cancer. In addition, the fusion proteins described herein may be suitable for application in cancers such as non-small cell lung cancer (NSCL) and melanoma, which have low response rates to checkpoint blockade and where TGF-β is highly expressed.

[0278] Cancers include, but are not limited to, B-cell cancers, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as, e.g., alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic prostate cancer, hormone refractory prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematopoietic tissue cancer, and the like. Other non-limiting examples of cancer types applicable to the methods encompassed by the present disclosure include human sarcomas and human carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, triple-negative breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, liver cancer, hepatocellular carcinoma (HCC), choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer. , bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.In some embodiments, the cancer whose phenotype is determined by the methods of the present disclosure is an epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancer may also be characterized in various other ways, including, but not limited to, serous carcinoma, endometrial carcinoma, mucinous carcinoma, clear cell carcinoma, Brenner carcinoma, or carcinoma of unknown nature. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma.

[0279] In certain embodiments, the anti-PD1-TGFβRII fusion proteins provided herein are examples of therapies that may be used to treat cancers that have an average response rate of about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% to standard therapies (including, but not limited to, chemotherapy and current clinical trial treatments). In certain embodiments, the anti-PD1-ADA2 fusion proteins provided herein are examples of therapies that may be used to treat cancers that have an average response rate of about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% to standard therapies (including, but not limited to, chemotherapy and current clinical trial treatments). Such cancers include, but are not limited to, Hodgkin's lymphoma, melanoma, non-small cell lung cancer (NSCLC), microsatellite instability-high (MSI) or mismatch repair (MMR) deficient solid tumors, CSCC, RCC, CRC, melanoma, Merkel cell carcinoma, bladder cancer, RCC, hepatocellular carcinoma (HCC), head and neck cancer (H&N), cervical cancer, gastric cancer, small cell lung cancer (SCLC), endometrial cancer, mesothelioma, ovarian cancer, triple-negative breast cancer (TNBC), breast cancer, colorectal cancer (CRC), pancreatic cancer, and prostate cancer.

[0280] Combination Therapy In some embodiments, the fusion protein is administered as a combination therapy with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a biological molecule such as an antibody. For example, treatment may involve administering the fusion protein in combination with an antibody against a tumor-associated antigen, including, but not limited to, an antibody that binds to EGFR, HER2 / ErbB2, and / or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF antibody or a VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX). In some embodiments, the additional therapeutic agent comprises an agent such as a small molecule. For example, treatment may involve the combined administration of the fusion proteins provided herein, or fragments or variants thereof, with small molecules that act as inhibitors of tumor-associated antigens, including, but not limited to, EGFR, HER2 (ErbB2), and / or VEGF. In some embodiments, the fusion proteins, or fragments or variants thereof, are administered in combination with a protein kinase inhibitor selected from the group consisting of gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatinib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor. In another embodiment, the additional therapeutic agent is chemotherapy, or T REGIn certain embodiments, the therapeutic agent is cyclophosphamide or an anti-CTLA4 antibody. In another embodiment, the additional therapeutic agent reduces the presence of myeloid-derived suppressor cells. In a further embodiment, the additional therapeutic agent is carbotaxol. In a further embodiment, the additional therapeutic agent is ibrutinib.

[0281] In some embodiments, the method may further comprise one or more checkpoint inhibitors in combination with the fusion protein, or fragment or variant thereof, described herein. In some embodiments, the additional checkpoint inhibitor may be an anti-CTLA-4 antibody. Anti-CTLA-4 antibodies (e.g., ipilimumab) have demonstrated durable anti-tumor activity and prolonged survival in participants with advanced melanoma, resulting in its approval by the Food and Drug Administration (FDA) in 2011. See Hodi et al., Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. (2010) Aug 19; 363(8):711-23. In some embodiments, the one or more checkpoint inhibitors may be an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody may be full-length atezolizumab (anti-PD-L1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1), or a fragment or variant thereof. In some embodiments, the one or more checkpoint inhibitors may be any one or more of a CD27 inhibitor, a CD28 inhibitor, a CD40 inhibitor, a CD122 inhibitor, a CD137 inhibitor, an OX40 (also known as CD134) inhibitor, a GITR inhibitor, an ICOS inhibitor, or any combination thereof. In some embodiments, the one or more checkpoint inhibitors may be any one or more of an A2AR inhibitor, a B7-H3 (also known as CD276) inhibitor, a B7-H4 (also known as VTCN1) inhibitor, a BTLA inhibitor, an IDO inhibitor, a KIR inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, or any combination thereof.

[0282] In certain embodiments, the additional therapeutic agent comprises a second immunotherapeutic agent. In some embodiments, the additional immunotherapeutic agent includes, but is not limited to, a colony-stimulating factor, an interleukin, an antibody that blocks immunosuppressive function (e.g., an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-CD3 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody), an antibody that enhances immune cell function (e.g., an anti-GITR antibody, an anti-OX-40 antibody, an anti-CD40 antibody, or an anti-4-1BB antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), a soluble ligand (e.g., GITRL, GITRL-Fc, OX-40L, OX-40L-Fc, CD40L, CD40L-Fc, 4-1BB ligand, or 4-1BB ligand-Fc), or a member of the B7 family (e.g., CD80, CD86). In some embodiments, the additional immunotherapeutic agent targets CTLA-4, CD28, CD3, PD-L1, TIGIT, GITR, OX-40, CD-40, or 4-1BB.

[0283] In some embodiments, the additional therapeutic agent is an additional immune checkpoint inhibitor. In some embodiments, the additional immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-4-1BB antibody, or an anti-OX-40 antibody. In some embodiments, the additional therapeutic agent is an anti-TIGIT antibody. In some embodiments, the additional therapeutic agent is an anti-PD-L1 antibody selected from the group consisting of BMS935559 (MDX-1105), atexolizumab (MPDL3280A), durvalumab (MEDI4736), and avelumab (MSB0010718C). In some embodiments, the additional therapeutic agent is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab (YERVOY) and tremelimumab. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from the group consisting of BMS-986016 and LAG525. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody selected from the group consisting of MEDI6469, MEDI0562, and MOXR0916. In some embodiments, the additional therapeutic agent is an anti-4-1BB antibody selected from the group consisting of PF-05082566. In some embodiments, the fusion protein, or fragment or variant thereof, may be administered in combination with a biomolecule selected from the group consisting of cytokines, adrenomedullin (AM), angiopoietin (Ang), BMP, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), GDF9, HGF, HDGF, IGF, migration stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, PlGF, gamma-IFN, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

[0284] cytokines In some embodiments, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variant, or a combination thereof. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is at least one of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and functional variants and fragments. In some embodiments, the cytokine may be membrane-bound or secreted. In some embodiments, the cytokine is soluble IL-15, a soluble IL-15 / IL-15Rα complex (e.g., ALT-803). In certain cases, the interleukin may comprise membrane-bound IL-15 (mbIL-15), or a fusion of IL-15 with IL-15Rα. In some embodiments, the mbIL-15 is a membrane-bound chimeric IL-15 that can be co-expressed with engineered immune effector cells described herein. In some embodiments, the mbIL-15 comprises full-length IL-15 (e.g., a native IL-15 polypeptide), or a fragment or variant thereof, fused in-frame with full-length IL-15Rα, or a fragment or variant thereof. Optionally, the IL-15 is indirectly linked to the IL-15Rα via a linker. In some cases, mbIL-15 is as described in Hurton et al., "Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells," PNAS 2016. In some cases, the cytokine is expressed in the same immune effector cell as the CAR.

[0285] In some embodiments, mbIL-15 is expressed with a cell tag, such as HER1t, HER-1t-1, CD20t-1, or CD20, as described herein. mbIL-15 can be expressed in frame with HER1t, HER-1t-1, CD20t-1, or CD20.

[0286] In some embodiments, mbIL-15 can be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a ligand-inducible promoter of a gene switch. In some cases, the inducible promoter can be a small molecule ligand-inducible, two-polypeptide, ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch.

[0287] In another aspect, the interleukin can include IL-12. In some embodiments, the IL-12 is single-chain IL-12 (scIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, or intercalated IL-12. In some cases, the IL-12 variant is as described in International Publication Nos. WO 2015 / 095249, WO 2016 / 048903, and WO 2017 / 062953, all of which are incorporated by reference in their entireties. In some embodiments, the cytokine described above can be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a ligand-inducible promoter of a gene switch. In some cases, the inducible promoter can be a small molecule ligand-inducible, two-polypeptide, ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch.

[0288] In some embodiments, the fusion proteins described herein can be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a ligand-inducible promoter of a gene switch. In some cases, the inducible promoter can be a small molecule ligand-inducible, two-polypeptide ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch.

[0289] Genetic Switch Provided herein are gene switch polypeptides, polynucleotides encoding ligand-inducible gene switch polypeptides, and methods and systems incorporating these polypeptides and / or polynucleotides. The term "gene switch" refers to the combination of a response element associated with a promoter, e.g., an ecdysone receptor (EcR)-based system in which the response element and promoter modulate the expression of a gene in which they are incorporated in the presence of one or more ligands. Tightly regulated inducible gene expression systems or gene switches are useful for a variety of applications, such as gene therapy, intracellular, large-scale protein production, cell-based high-throughput screening assays, functional genomics, and regulating traits in transgenic plants and animals. Such inducible gene expression systems can include ligand-inducible heterologous gene expression systems.

[0290] Early forms of EcR-based gene switches used Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) polypeptides and demonstrated that these receptors transactivate reporter genes in mammalian cell lines and transgenic mice in the presence of the steroid ponasterone A (Christopherson et al., 1992; No et al., 1996). Subsequently, Suhr et al., 1998, showed that the nonsteroidal ecdysone agonist tebufenozide induces high-level transactivation of reporter genes in mammalian cells via the silkworm EcR (BmEcR) in the absence of an exogenous heterodimer partner.

[0291] PCT International Patent Application / US Application No. 97 / 05330 (WO 97 / 38117) and PCT International Patent Application / US Application No. 99 / 08381 (WO 99 / 58155) disclose a method for modulating the expression of an exogenous gene, in which a DNA construct containing an exogenous gene and an ecdysone response element is activated by a second DNA construct containing an ecdysone receptor, which binds to the ecdysone response element and induces gene expression in the presence of its ligand and, optionally, in the presence of a receptor capable of acting as a silent partner. In this example, the ecdysone receptor was isolated from Drosophila melanogaster. Typically, such systems require the presence of a silent partner, preferably a retinoid X receptor (RXR), to achieve optimal activation. In mammalian cells, the insect ecdysone receptor (EcR) heterodimerizes with the mammalian retinoid X receptor (RXR), which can be used to regulate the expression of target or heterologous genes in a ligand-dependent manner. PCT International Patent Application / U.S. Application No. 98 / 14215 (WO 99 / 02683) discloses that an ecdysone receptor isolated from the silkworm, Bombyx mori, is functional in mammalian systems without the need for an exogenous dimer partner.

[0292] U.S. Patent No. 6,265,173 discloses that various members of the steroid / thyroid superfamily of receptors can be combined with the Drosophila melanogaster ultraspiracle receptor (USP) or a fragment thereof, containing at least the dimerization domain of USP, for use in gene expression systems. U.S. Patent No. 5,880,333 discloses a heterodimer system of Drosophila melanogaster EcR and ultraspiracle (USP) for use in plants, in which the transactivation and DNA-binding domains are located on two different hybrid proteins. In each of these cases, the transactivation domain and DNA binding domain (as native EcR, as in PCT Int. Pat. Appl. / US Ser. No. 98 / 14215, or as modified EcR, as in PCT Int. Pat. Appl. / US Ser. No. 97 / 05330) were combined into a single molecule, and the other heterodimeric partner, USP or RXR, was used in their native state.

[0293] PCT International Patent Application / US Application No. 01 / 0905 discloses an ecdysone receptor-based inducible gene expression system in which the transactivation domain and the DNA-binding domain are separated from each other by being placed on two different proteins, resulting in a significant reduction in background activity in the absence of a ligand and a significant increase in activity above background in the presence of a ligand. This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the two systems disclosed in PCT / US Application Nos. 97 / 05330 and 98 / 14215. The two-hybrid system is thought to take advantage of the ability of the interacting protein pair to better position the transcription activation domain relative to the DNA-binding domain so that, when the DNA-binding domain binds to the DNA-binding site on the gene, the transactivation domain more effectively activates the promoter (see, e.g., U.S. Patent No. 5,283,173). The two-hybrid gene expression system contains two gene expression cassettes: a first gene expression cassette encoding a DNA-binding domain fused to a nuclear receptor polypeptide, and a second gene expression cassette encoding a transactivation domain fused to a different nuclear receptor polypeptide. In the presence of a ligand, a conformational change is induced in the antibody that promotes interaction with the TGF-β cytokine trap, which is thought to result in dimerization of the DNA-binding domain and the transactivation domain. Because the DNA-binding domain and the transactivation domain are present on two different molecules, background activity is greatly reduced in the absence of a ligand.

[0294] Certain modifications of the two-hybrid system can also result in improved sensitivity to nonsteroidal ligands, such as diacylhydrazines, compared with steroidal ligands, such as ponasterone A ("PonA") or muristerone A ("MurA"). That is, compared with steroids, nonsteroidal ligands resulted in greater gene transcription activity at lower ligand concentrations. Furthermore, the two-hybrid system avoids some of the side effects resulting from overexpression of RXR, which can occur when unmodified RXR is used as the switching partner. In preferred two-hybrid systems, the native DNA-binding and transactivation domains of EcR or RXR are removed, resulting in a reduced likelihood of these hybrid molecules interacting with other steroid hormone receptors present in the cell, thereby resulting in reduced side effects.

[0295] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily, classified into subfamily 1, group H (herein referred to as "group H nuclear receptors"). Members of each group share 40-60% amino acid identity within the E (ligand-binding) domain (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97: 161-163). In addition to the ecdysone receptor, other members of this nuclear receptor subfamily 1, group H, include ubiquitous receptor (UR), orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXR-interacting protein 15 (RIP-15), liver x receptor beta (LXRβ), steroid hormone receptor-like protein (RLD-1), liver x receptor (LXR), liver x receptor alpha (LXRα), farnesoid x receptor (FXR), receptor-interacting protein 14 (RIP-14), and farnesol receptor (HRR-1).

[0296] In some cases, the inducible promoter ("IP") can be a small molecule ligand-inducible, two-polypeptide, ecdysone receptor-based gene switch, such as Intrexon Corporation's RHEOSWITCH® gene switch. In some cases, the gene switch can be a gene switch as described in PCT / US Appl. No. 2001 / 009050 (WO 2001 / 070816); U.S. Patent Nos. 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT / US Appl. No. 2001 / 030608 (WO 2002 / 029075); U.S. Patent Nos. 5,811,512; ... US Patent Nos. 8,105,825; 8,168,426; PCT / US Application No. 2002 / 005235 (WO 2002 / 066613); US Patent Application No. 10 / 468,200 (US Publication No. 20120167239); PCT / US Application No. 2002 / 005706 (WO 2002 / 066614); US Patent Nos. 7,531,326; and 8,236,556. No. 8,598,409; PCT / US Application No. 52002 / 005090 (WO 2002 / 066612); U.S. Patent No. 8,715,959 (U.S. Publication No. 20060100416); PCT / US Application No. 2002 / 005234 (WO 2003 / 027266); U.S. Patent Nos. 7,601,508; 7,829,676; 7,919,269; No. 8,030,067; PCT / US Application No. 52002 / 005708 (WO 2002 / 066615); U.S. Patent Application No. 10 / 468,192 (U.S. Application No. 20110212528); PCT / US Application No. 2002 / 005026 (WO 2003 / 027289); U.S. Patent Nos. 7,563,879; 8,021,878; 8,497,093;PCT / US Application No. 2005 / 015089 (WO 2005 / 108617); U.S. Patent Nos. 7,935,510; 8,076,454; PCT / US Application No. 52008 / 011270 (WO 2009 / 045370); U.S. Patent Application No. 12 / 241,018 (U.S. Publication No. 20090136465); PCT / US Application No. 2008 / 011563 (WO 2009 / 048560); U.S. Patent Application No. 12 / 247,738 (U.S. Publication No. 20090123441); PCT / US Application No. 2009 / 005510 (WO 2010 / 042189 ); may be selected from, but is not limited to, ecdysone-based receptor components of any of the systems described in U.S. Patent Application Publication No. 13 / 123,129 (U.S. Publication No. 20110268766); PCT / U.S. Application No. 2011 / 029682 (WO 2011 / 119773); U.S. Patent Application Publication No. 13 / 636,473 (U.S. Publication No. 20130195800); PCT / U.S. Application No. 2012 / 027515 (WO 2012 / 122025); WO 2018 / 132494 (PCT / U.S. Application No. 2018 / 013196) and U.S. Patent No. 9,402,919.

[0297] As used herein, the term "ligand," as applied to a ligand-activated ecdysone receptor-based gene switch, refers to various soluble small molecules (such as diacylhydrazine compounds) that have the ability to activate a gene switch and stimulate gene expression (i.e., upon activation, result in ligand-induced expression of polynucleotides (e.g., mRNA, miRNA, etc.) and / or polypeptides). Examples of such ligands are disclosed in WO 2004 / 072254 (PCT / US Appl. No. 2004 / 003775); WO 2004 / 005478 (PCT / US Appl. No. 2003 / 021149); WO 2005 / 017126 (PCT / US Appl. No. 2004 / 005149); WO 2004 / 078924 (PCT / US Appl. No. 2004 / 005912); WO 2008 / 153801, each of which is incorporated herein by reference in its entirety. (PCT / US Appl. No. 2008 / 006757); WO 2009 / 114201 (PCT / US Appl. No. 2009 / 001639); WO 2013 / 036758 (PCT / US Appl. No. 2012 / 054141); WO 2014 / 144380 (PCT / US Appl. No. 2014 / 028768); and WO 2016 / 044390 (PCT / US Appl. No. 2015 / 050375).

[0298] Examples of ligands also include, but are not limited to, ecdy steroids such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, 9-cis-retinoic acid, synthetic analogs of retinoic acid, each of which is incorporated herein by reference, including U.S. Pat. Nos. 6,258,603; 6,013,836; 5,117,057; 5,530,028; and 5,378,726; 7,304,16 N,N'-diacylhydrazines, such as those disclosed in U.S. Pat. Nos. 7,851,220; 8,748,125; 9,272,986; 7,456,315; 7,563,928; 8,524,948; 9,102,648; 9,169,210; 9,255,273; and 9,359,289; U.S. Pat. dibenzoylalkylcyanohydrazines such as those disclosed in European Application No. 2,461,809; N-alkyl-N,N'-diaroylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; and oxazolines as described in European Application No. 234,994. N-acyl-N-alkylcarbonylhydrazines such as the N-acyl-N-alkylcarbonylhydrazines described in U.S. Pat. No. 4,985,461; N-aroyl-N-alkyl-N'-aroylhydrazines such as the N-aroyl-N-alkyl-N'-aroylhydrazines described in U.S. Pat. No. 4,985,461; amidoketones such as the amidoketones described in U.S. Pat. Nos. 7,375,093; 8,129,355; and 9,802,936;and 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterols, 22(R)hydroxycholesterol, 24(S)hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, farnesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the present invention include RG-115819 (3,5-dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-methyl-3-methoxy-benzoyl)-hydrazide), RG-115932 ((R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115830 (3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide). See, e.g., WO 2008 / 153801 (PCT / US Appl. No. 2008 / 006757); and WO 2013 / 036758 (PCT / US Appl. No. 2012 / 054141), both of which are incorporated herein by reference in their entireties.

[0299] For example, the ligand for an ecdysone receptor-based gene switch can be selected from any suitable ligand. Both naturally occurring ecdysone or ecdysone analogs (e.g., 20-hydroxyecdysone, muristerone A, ponasterone A, ponasterone B, ponasterone C, 26-iodoponasterone A, inokosterone, or 26-mesyl inokosterone) and non-steroidal inducers can be used as ligands for the gene switch of the present invention. U.S. Patent No. 6,379,945 describes an insect steroid receptor ("HEcR") isolated from Heliothis virescens, which can act as a gene switch responsive to both steroids and certain non-steroidal inducers. Nonsteroidal inducers have distinct advantages over steroids in this system, as well as in many other systems that respond to both steroids and nonsteroidal inducers, for several reasons, including, for example, low production costs, metabolic stability, absence in insects, plants, or mammals, and environmental acceptability. U.S. Patent No. 6,379,945 describes the usefulness of two dibenzoylhydrazines, 1,2-dibenzoyl-1-tert-butylhydrazine and tebufenozide (N-(4-ethylbenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine), as ligands for ecdysone-based gene switches. The present invention also includes other dibenzoylhydrazines as ligands, such as the dibenzoylhydrazines disclosed in U.S. Patent No. 5,117,057. The use of tebufenozide as a chemical ligand for the ecdysone receptor from Drosophila melanogaster is also disclosed in US Pat. No. 6,147,282.Further non-limiting examples of ecdysone ligands are 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, 1,2-diacylhydrazine, N'-substituted-N,N'-disubstituted hydrazine, dibenzoylalkylcyanohydrazine, N-substituted-N-alkyl-N,N-diaroylhydrazine, N-substituted-N-acyl-N-alkyl, carbonylhydrazine, or N-aroyl-N'-alkyl-N'-aroylhydrazine (see U.S. Pat. No. 6,723,531).

[0300] In one embodiment, the ligand for the ecdysone-based gene switch system is a diacylhydrazine ligand or a chiral diacylhydrazine ligand. The ligand used in the gene switch system has Formula I:

[0301] [ka] wherein A is alkoxy, arylalkyloxy, or aryloxy; B is optionally substituted aryl or optionally substituted heteroaryl; R and R are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclo, optionally substituted aryl, or optionally substituted heteroaryl; or a pharmaceutically acceptable salt, hydrate, crystalline, or amorphous form thereof. The compound may be:

[0302] In another embodiment, the ligand has Formula II:

[0303] [ka] wherein A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl; B is optionally substituted aryl or optionally substituted heteroaryl; R and R are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclo, optionally substituted aryl, or optionally substituted heteroaryl, provided that R is not identical to R; and the absolute configuration at R and R, which possess the asymmetric carbon atom, is predominantly S; or a pharmaceutically acceptable salt, hydrate, crystalline, or amorphous form thereof. The compound may be an enantiomerically enriched compound of the formula:

[0304] In certain embodiments, the ligand has formula III:

[0305] [ka] wherein A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl; B is optionally substituted aryl or optionally substituted heteroaryl; R and R are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclo, optionally substituted aryl, or optionally substituted heteroaryl, provided that R is not identical to R; and the absolute configuration at R and R, which possess the asymmetric carbon atom, is predominantly R; or a pharmaceutically acceptable salt, hydrate, crystalline, or amorphous form thereof. The compound may be an enantiomerically enriched compound of the formula:

[0306] In one embodiment, the ligand can be (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide, or a pharmaceutically acceptable salt, hydrate, crystalline form, or amorphous form thereof, having an enantiomeric excess of at least 95%.

[0307] Diacylhydrazine ligands of Formula I and chiral diacylhydrazine ligands of Formula II or III, when used with ecdysone-based gene switch systems, provide a means for exogenous transient regulation of expression of therapeutic polypeptides or therapeutic polynucleotides of the invention. See U.S. Patent Nos. 8,076,517; 8,884,060; and 9,598,355, each of which is incorporated herein by reference in its entirety.

[0308] The ligands used in the present invention may form salts. As used herein, the term "salt(s)" refers to acidic and / or basic salts formed with inorganic acids and inorganic bases and / or organic acids and organic bases. In addition, when a compound of Formula I, II, or III contains both a basic and an acidic moiety, zwitterions ("internal salts") may be formed and are included in the term "salt(s)" as used herein. For example, in isolation or purification steps that may be employed during preparation, pharmaceutically acceptable salts (i.e., non-toxic salts, physiologically acceptable salts) are used, although other salts are also useful. Salts of compounds of Formula I, II, or III can be formed, for example, by reacting the compound with an amount of acid or base, such as an equivalent amount, in a medium, such as a medium in which the salt precipitates, or in an aqueous medium, followed by lyophilization.

[0309] Ligands containing a basic moiety can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetate (such as acetate formed with acetic acid or trihaloacetic acid, e.g., trifluoroacetic acid), adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, disulfate, borate, butyrate, citrate, camphor, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride (formed with hydrochloric acid), hydrobromide (formed with hydrogen bromide), iodide, and the like. salts, such as nitrates, 2-hydroxyethanesulfonates, lactates, maleates (formed with maleic acid), methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as sulfates formed with sulfuric acid), sulfonates (such as the sulfonates referred to herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.

[0310] Ligands containing an acidic moiety can form salts with a variety of organic and inorganic bases. Exemplary base salts include alkali metal salts such as ammonium salts, sodium salts, lithium salts, and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts with organic bases (e.g., organic amines), such as benzathine, dicyclohexylamine, hydrabamine (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, and the like, and salts with amino acids such as arginine and lysine.

[0311] Non-limiting examples of ligands for inducible gene expression systems also include ligands that utilize the FK506 binding domain, such as FK506, cyclosporin A, or rapamycin. FK506, rapamycin, and analogs thereof are disclosed in U.S. Patent Nos. 6,649,595; 6,187,757; 7,276,498; and 7,273,874.

[0312] In some embodiments, the diacylhydrazine ligand for inducible gene expression is administered at a unit dose of about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or 120 mg per day. In some embodiments, the diacylhydrazine ligand is administered at a unit dose of about 5 mg per day. In some embodiments, the diacylhydrazine ligand is administered at a unit dose of about 10 mg per day. In some embodiments, the diacylhydrazine ligand is administered at a unit dose of about 15 mg per day. In some embodiments, the diacylhydrazine ligand is administered daily at a unit dose of about 20 mg per day.

[0313] In some embodiments, combination therapy with two or more therapeutic agents may, but is not required to, use agents that act by different mechanisms of action. Combination therapy using agents with different mechanisms of action may result in additive or synergistic effects. Combination therapy may allow for lower doses of each agent than would be used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index(ies) of the agents. Combination therapy may reduce the likelihood of resistant cancer cells developing. In some embodiments, combination therapy includes a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects tumor / cancer cells (e.g., inhibits or kills tumor / cancer cells).

[0314] In certain embodiments, in addition to administering a fusion protein, or fragment or variant thereof, described herein, the method or treatment further comprises administering at least one additional therapeutic agent. The additional therapeutic agent may be administered prior to, contemporaneously with, and / or subsequent to administration of the agent. In some embodiments, the at least one additional therapeutic agent comprises one, two, three, or more additional therapeutic agents.

[0315] Therapeutic agents that can be administered in combination with the fusion proteins, or fragments or variants thereof, described herein include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the administration of the agents described herein in combination with a chemotherapeutic agent or a cocktail of chemotherapeutic agents. In some embodiments, the method may further include one or more anti-neoplastic agents, such as cisplatin, capecitabine, or 5-fluorouracil, in combination with the fusion proteins, or fragments or variants thereof, described herein. The drug treatment can occur prior to, contemporaneously with, or subsequent to the administration of chemotherapy. The combined administration can include coadministration in a single pharmaceutical formulation, coadministration using separate formulations, or generally sequential administration within a time period such that all active agents can exert their biological activities simultaneously. The preparations and administration schedules for such chemotherapy can be used according to the manufacturer's instructions or as empirically determined by those skilled in the art. Preparations and administration schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, MC Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0316] Useful classes of chemotherapeutic agents include, for example, antitubulin agents, auristin, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., cisplatin, platinum complexes such as mono(platinum), bis(platinum), and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosoureas, platinol, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc. In certain embodiments, the second therapeutic agent is an alkylating agent, antimetabolite, antimitotic agent, topoisomerase inhibitor, or angiogenesis inhibitor.

[0317] The fusion proteins provided herein, or fragments or variants thereof, may be used alone or in combination with conventional treatment regimens, such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated). A set of tumor antigens may be useful, for example, in most cancer patients.

[0318] Fusion proteins in combination with chimeric receptors In some embodiments, the fusion protein is administered as a combination therapy with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chimeric receptor, such as a chimeric antigen receptor or an engineered T cell receptor. For example, the treatment can involve simultaneous administration of the fusion protein with the chimeric receptor. In one embodiment, the treatment can involve co-administration of the fusion protein with engineered effector cells comprising the chimeric receptor. In one embodiment, the treatment can involve sequential administration of the fusion protein followed by the engineered effector cells comprising the chimeric receptor. In another embodiment, the treatment can involve sequential administration of the fusion protein followed by the engineered effector cells comprising the chimeric receptor. In one aspect, there can be a time difference of at least 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, or 24 hours between the administrations. In another embodiment, there can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 75, 90 or more days between administrations.

[0319] In some embodiments, the engineered effector cells are engineered immune cells, including T cells and / or natural killer cells. T cells or T lymphocytes are a subtype of white blood cell involved in cell-mediated immunity. Exemplary T cells include helper T cells, cytotoxic T cells, TH17 cells, stem cell memory T cells (TSCM), naive T cells, memory T cells, effector T cells, regulatory T cells, or natural killer T cells. In certain aspects, embodiments described herein include the generation and / or expansion of engineered immune effector cells (e.g., T cells, Tregs, NK cells, or NK T cells), comprising transfecting the cells with an expression vector containing a DNA (or RNA) construct encoding a chimeric receptor.

[0320] Some embodiments of the present specification describe engineered effector cells that include a chimeric receptor expressed on the cell surface. In some cases, the chimeric receptor includes an antigen-binding region that can recognize and bind to a tumor antigen, such as a tumor-associated antigen or a tumor-specific antigen. In some cases, the antigen-binding region includes an antibody or a binding fragment, such as Fab, Fab', F(ab')2, F(ab')3, scFv, sc(Fv)2, dsFv, diabody, minibody, and nanobody, or a binding fragment thereof. In some cases, the antigen-binding region includes an scFv. In some cases, the chimeric receptor includes an scFv (e.g., a chimeric antigen receptor (CAR)). In some cases, the chimeric antigen receptor includes a pattern recognition receptor. In other cases, the chimeric receptor includes an engineered T cell receptor (TCR).

[0321] Further provided herein are immune effector cells comprising a cell tag for use as a kill switch, a selection marker, a biomarker, or a combination thereof. In some embodiments, the cell tag comprises HER1t, HER1t-1, CD20t-1, or CD20. In some cases, the cell tag comprises HER1t, wherein the HER1t comprises the polypeptide sequence of SEQ ID NO: 68. In some cases, the cell tag comprises HER1t-1, wherein the HER1t-1 comprises the polypeptide sequence of SEQ ID NO: 69.

[0322] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) are engineered receptors that transfer exogenous specificity to immune effector cells. In some cases, CARs comprise an extracellular domain (ectodomain) containing an antigen-binding domain, a stalk region, a transmembrane domain, and an intracellular domain (endodomain). In some cases, the intracellular domain further comprises one or more intracellular signaling domains. In some cases, the CARs described herein comprise an antigen-binding domain, a stalk region, a transmembrane domain, one or more costimulatory domains, and a signaling domain for T cell activation.

[0323] The antigen-binding domain may comprise a complementarity-determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. A complementarity-determining region (CDR) is a short amino acid sequence found in the variable domain of an antigen receptor (e.g., immunoglobulin and T-cell receptor) protein that is complementary to an antigen and thus confers its specificity to the receptor for this particular antigen. Each polypeptide chain of an antigen receptor may contain three CDRs (CDR1, CDR2, and CDR3). In some cases, the antigen-binding domain comprises F(ab')2, Fab', Fab, Fv, or scFv. In some cases, the antigen-binding domain is an scFv. In some cases, the antigen-binding domain is a Fab. In some cases, the antigen-binding domain is a Fab'. In some cases, the antigen-binding domain is a F(ab')2. In some cases, the antigen-binding domain is an Fv.

[0324] In some embodiments, the CARs described herein comprise an antigen-binding domain that binds to an epitope on CD19, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor α, mucins such as MUC-1, MUC-4, or MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R2. In one embodiment, the CARs described herein comprise an antigen-binding domain that binds to an epitope on MUC16. In some embodiments, the CARs described herein comprise an antigen-binding domain that binds to an epitope on CD19 or CD33. In some instances, the CARs described herein comprise an antigen-binding domain that binds to an epitope on CD19. In some instances, the CARs described herein comprise an antigen-binding domain that binds to an epitope on CD33. In further embodiments, the CARs described herein comprise an autoantigen-binding region or an antigen-binding region that binds to an epitope on HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1, or type II collagen.

[0325] In some embodiments, the CARs and methods described herein can be used to treat hyperproliferative diseases such as cancer, autoimmune diseases, or infections such as viral, bacterial, or parasitic infections. In some aspects, the CAR targets an antigen that grows in cancer cells, autoimmune cells, or cells infected with viruses, bacteria, or parasites. Pathogens that can be targeted include, but are not limited to, Plasmodium, trypanosomes, Aspergillus, Candida, hepatitis A virus, hepatitis B virus, hepatitis C virus, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or Ebola. The autoimmune disease can include graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, celiac disease, Crohn's disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, or autoimmune uveitis.

[0326] The pathogen recognized by the CAR can be essentially any type of pathogen, but in some embodiments, the pathogen is a fungus, bacterium, or virus. Exemplary viral pathogens include Adenoviridae, Epstein-Barr virus (EBV), cytomegalovirus (CMV), respiratory syncytial (RS) virus, JC virus, BK virus, viruses of the HPV, HSV, HHV family, viruses of the Hepatitis family, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae pathogens. Exemplary pathogenic viruses cause smallpox, influenza, parotitis, measles, chickenpox, Ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella.In some embodiments, the pathogen receptor Dectin-1 can be used to create CARs that recognize carbohydrate structures on the cell wall of fungi, such as Aspergillus. In another embodiment, CARs can be made that block viral infection and pathology based on antibodies that recognize viral determinants (e.g., glycoproteins from CMV and Ebola).

[0327] In some embodiments, a "stalk region," "spacer," or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, a "stalk domain" or "stalk region" comprises any oligonucleotide or polypeptide that functions within a polypeptide chain to link the transmembrane domain to the extracellular or cytoplasmic domain. In some embodiments, the "stalk domain" or "stalk region" is sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some cases, the stalk region comprises a hinge region derived from IgG1. In alternative cases, the stalk region comprises an immunoglobulin CH2CH3 region and, optionally, a portion of CD3. In some cases, the stalk region comprises the CD8α hinge region, the 12-amino acid hinge region of IgG4-Fc (ESKYGPPCPPCP), or an IgG4 hinge region, as described in WO 2016 / 073755.

[0328] The transmembrane domain may be derived from a natural or synthetic source. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Suitable transmembrane domains may include the transmembrane regions of the alpha, beta, or zeta chains of the T cell receptor, or may include transmembrane regions from CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic and may contain hydrophobic residues such as leucine and valine. In some embodiments, a phenylalanine, tryptophan, and valine triplet is found at one or both ends of a synthetic transmembrane domain. Optionally, in some embodiments, a short oligonucleotide or polypeptide linker, between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In some embodiments, the linker is a glycine-serine linker.

[0329] In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain or a CD3ζ transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In other embodiments, the transmembrane domain comprises a CD3ζ transmembrane domain.

[0330] The intracellular domain may comprise one or more costimulatory domains. Exemplary costimulatory domains include, but are not limited to, CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof. In some cases, the CARs described herein comprise one or more, or two or more, costimulatory domains selected from CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof. In some cases, the CARs described herein comprise one or more, or two or more, costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, OX40 (CD134), or fragments or combinations thereof. In some cases, the CARs described herein comprise one or more, or two or more, costimulatory domains selected from CD8, CD28, 4-1BB (CD137), or fragments or combinations thereof. In some cases, the CARs described herein comprise one or more, or two or more, costimulatory domains selected from CD28, 4-1BB (CD137), or fragments or combinations thereof. In some cases, the CARs described herein comprise costimulatory domains CD28 and 4-1BB (CD137), or their respective fragments. In some cases, the CARs described herein comprise costimulatory domains CD28 and OX40 (CD134), or their respective fragments. In some cases, the CARs described herein comprise costimulatory domains CD8 and CD28, or their respective fragments. In some cases, the CARs described herein comprise costimulatory domains CD28, or a fragment thereof. In some cases, the CARs described herein comprise a costimulatory domain, 4-1BB (CD137), or a fragment thereof. In some cases, the CARs described herein comprise a costimulatory domain, OX40 (CD134), or a fragment thereof.In some cases, the CARs described herein comprise a costimulatory domain, CD8, or a fragment thereof.

[0331] In some embodiments, the intracellular domain further comprises a signaling domain for T cell activation. In some cases, the signaling domain for T cell activation comprises a domain derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In some cases, the signaling domain for T cell activation comprises a domain derived from CD3 zeta.

[0332] In some embodiments, a CAR described herein is administered to a subject along with one or more additional therapeutic agents, including, but not limited to, a cytokine described herein. In a further embodiment, immune effector cells expressing a CAR described herein express membrane-bound IL-15 ("mIL-15 or mbIL-15"). In aspects of the invention, mbIL-15 comprises a fusion protein of IL-15 and IL-15Rα. In further embodiments, mbIL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 69. In certain cases, the CAR and cytokine are expressed in separate vectors. In specific cases, the vectors can be lentiviral vectors, retroviral vectors, or Sleeping Beauty transposons.

[0333] CD19-specific CAR CD19 is a cell surface glycoprotein of the immunoglobulin superfamily that is found primarily in malignant B-cell lines. In some cases, CD19 has also been detected in solid tumors, such as pancreatic cancer, liver cancer, and prostate cancer.

[0334] In some embodiments, CD19-specific CARs are described herein, in which the antigen-binding domain comprises F(ab')2, Fab', Fab, Fv, or scFv. In some cases, the antigen-binding domain recognizes an epitope on CD19. In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by FMC63. In some embodiments, the scFv domain and / or VH / VL domain are derived from FMC63. FMC63 generally refers to a mouse IgG1 monoclonal antibody (Ling, NR, el al. (1987). Leucocyte typing III. 302) raised against Nalm-1 and Nalm-16 cells expressing human CD19. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134), or a fragment or combination thereof; and a signaling domain derived from CD3 zeta.

[0335] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some embodiments herein, CD19-specific CAR-T cells are described in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta.

[0336] Some embodiments herein describe CD19-specific CAR-T cells that comprise an antigen-binding domain of an scFv, wherein the antigen-binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta.

[0337] In some embodiments, the CD19-specific CAR-T cells described herein comprise an anti-CD19 antibody described in U.S. Patent Application Publication No. 20160152723. In some embodiments, the CD19-specific CAR-T cells described herein comprise an anti-CD19 antibody described in WO 2015 / 123642. In some embodiments, the CD19-specific CAR-T cells described herein comprise an anti-CD19scFv (Nicholson et al. Construction and characterization of a functional CD19-specific single chain Fv fragment for immunotherapy of B lineage leukemia and lymphoma. Mol. Immunol., 34:1157-1165, 1997) derived from clone FMC63.

[0338] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharma, Inc.). Some embodiments herein describe CD19-specific CAR-T cells, in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3ζ.

[0339] Some embodiments herein describe CD19-specific CAR-T cells that include an antigen-binding domain of an scFv, where the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19. In some cases, the CD19-specific CAR-T cells further include a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3ζ.

[0340] In some embodiments, the CD19-specific CAR-T cells described herein comprise an anti-CD19 antibody described in WO2015187528, or a fragment or derivative thereof.

[0341] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis). Some embodiments described herein are CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta.

[0342] Some embodiments herein describe CD19-specific CAR-T cells that comprise an antigen-binding domain of an scFv, where the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta.

[0343] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (Cellectis). Some embodiments described herein are CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3ζ.

[0344] Some embodiments herein describe CD19-specific CAR-T cells that comprise an antigen-binding domain of an scFv, where the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3ζ.

[0345] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401 (Bellicum). In some embodiments herein, CD19-specific CAR-T cells are described, in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta.

[0346] Some embodiments herein describe CD19-specific CAR-T cells that comprise an antigen-binding domain of an scFv, where the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401. In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3ζ transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3ζ.

[0347] In some cases, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximab ravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed). In some cases, the CD19-specific CAR further comprises a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134), or fragments or combinations thereof; and a signaling domain derived from CD3 zeta.

[0348] In some embodiments, the present specification describes CD19-specific CAR-T cells, wherein the antigen-binding domain comprises F(ab')2, Fab', Fab, Fv, or scFv. In some cases, the antigen-binding domain recognizes an epitope on CD19. In some cases, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximab ravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed). In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain derived from CD3 zeta.

[0349] In some cases, the CD19-specific CAR-T cells described herein comprise an antigen-binding domain of an scFv that recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximab ravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomab paptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed). In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from a CD8 alpha transmembrane domain or a CD3 zeta transmembrane domain; one or more costimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof; and a signaling domain derived from CD3 zeta.

[0350] CD33-specific CAR "CD33" is a 67-kDa single-pass transmembrane glycoprotein and a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) superfamily. CD33 is characterized by a V-set Ig-like domain and a C2-set Ig-like domain within its extracellular domain, which are responsible for sialic acid binding. Alternative splicing of CD33m RNA results in a shorter isoform (CD33m) that lacks the V-set Ig-like domain and the disulfide bond linking the V-set Ig-like domain and the C2-set Ig-like domain. In healthy subjects, CD33 is primarily expressed as a myeloid differentiation antigen found on normal multipotent myeloid precursors, unipotent colony-forming cells, monocytes, and mature granulocytes. CD33 is expressed on over 80% of myeloid leukemia cells but not on normal hematopoietic stem cells or mature granulocytes (Andrews, R. et al., The L4F3 antigen is expressed by unipotent and multipotent colony-forming cells but not by their precursors, Blood, 68(5):1030-5 (1986)). CD33 has been reported to be expressed on malignant myeloid cells, activated T cells, and activated NK cells, and is found on at least a subset of blast cells in the majority of AML patients (Pollard, J. et al., Correlation of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin-containing chemotherapy in childhood AML, Blood, 119(16):3705-11 (2012)). In addition to its widespread expression on AML blast cells, CD33 can also be expressed on stem cells underlying AML.

[0351] In some embodiments, the antigen-binding portion of the CAR described herein is specific for CD33 (CD33 CAR). When expressed on the cell surface, the CD33-specific CAR redirects T cell specificity to human CD33. In some embodiments, the antigen-binding domain comprises a single-chain antibody fragment (scFv) comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) of a target antigen-specific anti-CD33 monoclonal antibody, the VL and VH being connected by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In some embodiments, the scFv is M195, m2H12, DRB2, and / or My9-6. In some embodiments, the scFv is a humanized scFv, e.g., hM195. In some embodiments, the antigen-binding portion can comprise a VH and VL directionally linked, for example, from N-terminus to C-terminus, with a VH-linker-VL or VL-linker-VH.

[0352] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 35 (hM195 VL).

[0353] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 36 (hM195 VH).

[0354] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 37 (M2H12 VH).

[0355] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 38 (M2H12 VL).

[0356] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 39 (DRB2 VH).

[0357] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 40 (DRB2 VL).

[0358] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 41 (My9-6 VH).

[0359] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 42 (My9-6 VL).

[0360] MUC16-specific CAR MUC16, also known as CA-125, is a large carbohydrate antigen. It is encoded by the MUC16 gene located on human chromosome 19. MUC16 is a three-domain, highly glycosylated, multidomain type I transmembrane protein. The C-terminal domain contains multiple extracellular SEA (sea urchin sperm protein, enterokinase, and agrin) modules with autoproteolytic activity. SEA possesses two proteolytic sites proximal to the transmembrane (TM) domain. The large, cleaved domain, designated CA-125, is released into the circulation at acidic pH. CA-125 is commonly used as a disease biomarker for ovarian cancer. A highly conserved, truncated, extracellular, membrane-tethered protein domain is referred to as the MUC16 extracellular domain. A MUC16 antibody was identified that specifically bound to the extracellular domain of MUC16 retained on the tumor cell surface. "Overexpression of MUC16" by a cell of interest (e.g., a cancer cell) refers to MUC16 protein and / or mRNA being expressed at elevated levels by the cell of interest compared to a control cell (e.g., a non-cancerous cell, a normal cell).

[0361] In some embodiments, the antigen-binding portion of the CAR described herein is specific for MUC16 (MUC16 CAR). When expressed on the cell surface, the MUC16-specific CAR redirects the specificity of T cells to human MUC16. In some embodiments, the antigen-binding domain comprises a single-chain antibody fragment (scFv) comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) of a target antigen-specific anti-MUC16 monoclonal antibody, the VL and VH being connected by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In some embodiments, the scFv is MUC16-scFv (SEQ ID NOs: 43 to 44), MUC16-scFv (SEQ ID NOs: 45 to 46), MUC16-scFv (SEQ ID NOs: 47 to 48), MUC16-scFv (SEQ ID NOs: 49 to 50), MUC16-scFv (SEQ ID NOs: 51 to 52), MUC16-scFv (SEQ ID NOs: 53 to 54), or MUC16-scFv (SEQ ID NOs: 55 to 56). In some embodiments, the scFv is humanized scFv.

[0362] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 43 (MUC16-1).

[0363] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44 (MUC16-1).

[0364] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 45 (MUC16-2).

[0365] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46 (MUC16-2).

[0366] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 47 (MUC16-3).

[0367] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 48 (MUC16-3).

[0368] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 49 (MUC16-4).

[0369] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 50 (MUC16-4).

[0370] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 51 (MUC16-5).

[0371] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 52 (MUC16-5).

[0372] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 53 (MUC16-5).

[0373] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 54 (MUC16-6).

[0374] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 55 (MUC16-7).

[0375] In embodiments, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 56 (MUC16-7).

[0376] In some embodiments, the antigen-binding portion can comprise a VH and VL directionally linked, for example, from N- to C-terminus, with VH-linker-VL or VL-linker-VH.

[0377] Engineered T cell receptor (TCR) In some embodiments, the chimeric receptor comprises an engineered T cell receptor. T cell receptors (TCRs) are composed of two chains (αβ or γδ) that pair on the surface of T cells to form a heterodimeric receptor. In some cases, αβ TCRs are expressed on the majority of T cells in the body and are known to be involved in recognizing specific MHC-restricted antigens. Each of the α and β chains is composed of two domains: a constant domain (C) that anchors the protein to the cell membrane and associates with the invariant subunit of the CD3 signaling apparatus; and a variable domain (V) that confers antigen recognition via a six-loop loop, termed the complementarity-determining region (CDR). In some cases, each V domain comprises three CDRs, e.g., CDR1, CDR2, and CDR3, with CDR3 being the hypervariable region. These CDRs interact with a complex formed between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pepMHC) (e.g., HLA-A complex, HLA-B complex, HLA-C complex, HLA-DPA1 complex, HLA-DPB1 complex, HLA-DQA1 complex, HLA-DQB1 complex, HLA-DRA complex, or HLA-DRB1 complex). In some cases, the constant domain further comprises a junction region connecting the constant domain to the variable domain. In some cases, the beta chain further comprises a short diversity region that constitutes part of the junction region.

[0378] In some cases, such TCRs are reactive with specific tumor antigens, e.g., NY-ESO, Mage A3, Titin. In other cases, such TCRs are reactive with specific neoantigens expressed within the patient's tumor (i.e., patient-specific, somatic, non-synonymous mutations expressed by the tumor). In some cases, engineered TCRs can be affinity-enhanced.

[0379] In some embodiments, TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and linked to IMGT's public database of TCR sequences. For example, there may be several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions, identified by their framework, CDR1, CDR2, and CDR3 sequences. Thus, each Vα type may be referred to by a unique TRAV number in the IMGT nomenclature. For example, "TRAV21" defines a TCR Vα region with unique framework, CDR1, and CDR2 sequences, and a CDR3 sequence that is partially defined by amino acid sequences that are conserved from TCR to TCR, but also includes amino acid sequences that vary from TCR to TCR. Similarly, "TRBV5-1" defines a TCR Vβ region with unique framework, CDR1, and CDR2 sequences, but whose CDR3 sequence is only partially defined.

[0380] The beta chain diversity region is sometimes referred to by the abbreviation TRBD in the IMGT nomenclature.

[0381] In some cases, the unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the field of TCRs, for example, they can be found in the public databases by IMGT and in "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

[0382] In some embodiments, the αβ heterodimer TCR is transfected as a full-length chain, e.g., having both a cytoplasmic domain and a transmembrane domain. Optionally, the TCR contains an introduced disulfide bond between residues of each constant domain, e.g., as described in WO 2006 / 000830.

[0383] In some instances, the TCRs described herein are in a single-chain format (see, e.g., WO 2004 / 033685). Single-chain formats include αβ TCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L-Vβ-Cβ formats, where Vα and Vβ are the TCR α and TCR β variable regions, respectively, Cα and Cβ are the TCR α and TCR β constant regions, respectively, and L is a linker sequence. In certain embodiments, the single-chain TCRs of the invention may have disulfide bonds introduced between residues of the respective constant domains, as described in WO 2004 / 033685.

[0384] The TCRs described herein may be associated with a detectable label, a therapeutic agent, or a PK-modifying moiety.

[0385] For diagnostic purposes, exemplary detectable labels include, but are not limited to, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and imaging reagents.

[0386] Therapeutic agents that can be associated with the TCRs described herein include immunomodulators, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents. Toxins could be present inside liposomes linked to the TCR so that the compound is released in a controlled manner to ensure that the toxic effect is exerted at the desired location. In some cases, sustained release minimizes damaging effects during transport within the body and ensures that the toxin exerts its maximum effect after binding of the TCR to the target antigen-presenting cell.

[0387] In some embodiments, the additional, suitable therapeutic agent is, for example, a. Small molecule cytotoxic agents, e.g., compounds with the ability to kill mammalian cells and having a molecular weight of less than 700 daltons. Such compounds may also contain toxic metals capable of cytotoxicity. Furthermore, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release a cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmicin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, solfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, auristin E, vincristine, and doxorubicin; b. Peptide cytotoxins, i.e., proteins or fragments thereof with the ability to kill mammalian cells, such as ricin, diphtheria toxin, pseudomonas exotoxin A, DNase and RNase; c. Radionuclides, i.e., unstable isotopes of elements that decay with the synchronic emission of one or more of alpha or beta particles, or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents can be used to facilitate the association of these radionuclides with high-affinity TCRs or multimers thereof; d. Immunostimulatory agents, i.e., immune effector molecules that stimulate the immune response, such as cytokines such as IL-2 and IFN-γ; e. Superantigens and their mutants; f.TCR-HLA fusion; g. Chemokines such as IL-8, crystalloid factor 4, and melanoma growth stimulatory protein; h. Antibodies or fragments thereof, including anti-T cell determinant antibodies or NK cell determinant antibodies (e.g., anti-CD3 antibodies, anti-CD28 antibodies, or anti-CD16 antibodies); i. Alternative protein scaffolds with antibody-like binding characteristics; j. complement activators; and k. Includes heterologous protein domains, homologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0388] dose The appropriate dose of the fusion protein and modified immune effector cells used will depend on the age and weight of the subject and the particular drug used. The dose and treatment regimen of the fusion protein and modified immune effector cells can be determined by one skilled in the art.

[0389] In certain embodiments, the fusion protein is administered by injection (e.g., subcutaneous or intravenous injection) at a dose of about 1-30 mg per kg, e.g., about 5-25 mg per kg, about 10-20 mg per kg, about 1-5 mg per kg, or about 3 mg per kg. In some embodiments, the fusion protein is administered at a dose of about 1 mg per kg, about 3 mg per kg, about 5 mg per kg, about 10 mg per kg, about 20 mg per kg, about 30 mg per kg, or about 40 mg per kg. In some embodiments, the fusion protein is administered at a dose of about 1-3 mg per kg, or about 3-10 mg per kg. In some embodiments, the fusion protein is administered at a dose of about 0.5-2, 2-4, 2-5, 5-15, or 5-20 mg per kg. The administration schedule can vary, e.g., from once weekly to once every 2, 3, or 4 weeks. In one embodiment, the fusion protein is administered at a dose of about 10-20 mg per kg every two weeks, or at a dose of about 1 mg per kg once every two weeks, about 3 mg per kg once every two weeks, 10 mg per kg once every two weeks, 3 mg per kg once every four weeks, or 5 mg per kg once every four weeks.

[0390] In other embodiments, the fusion protein is administered by injection (e.g., subcutaneous or intravenous injection) at a dose (e.g., a flat dose) of about 200 mg to 500 mg, e.g., about 250 mg to 450 mg, about 300 mg to 400 mg, about 250 mg to 350 mg, about 350 mg to 450 mg, or about 300 mg, or about 400 mg. In some embodiments, the fusion protein is administered at a dose of about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg. In some embodiments, the fusion protein is administered at a dose of 200 or 300 mg. In some embodiments, the fusion protein is administered at a dose of about 250 to 450 mg, or about 300 to 400 mg. In some embodiments, the fusion protein is administered at a dose of about 200-300 mg, 250-350 mg, 300-400 mg, 350-450 mg, or 400-500 mg. The administration schedule can vary, for example, from once weekly to once every 2, 3, 4, 5, or 6 weeks. In one embodiment, the fusion protein is administered at a dose of about 300 mg-400 mg once every 3 or 4 weeks. In one embodiment, the fusion protein is administered at a dose of about 300 mg once every 3 weeks. In one embodiment, the fusion protein is administered at a dose of about 400 mg once every 4 weeks. In one embodiment, the fusion protein is administered at a dose of about 300 mg once every 4 weeks. In one embodiment, the fusion protein is administered at a dose of about 400 mg once every 3 weeks. The fusion protein can be administered one or more times, for example, 1, 2, 3, 4, 5, 6, 7 or more times. In one embodiment, the fusion protein is administered six times. The fusion protein can be administered at least 5 days, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 25, 30, 35, or 40 days after administration of the CAR-expressing cells, for example, MUC16-, CD33-, CD19-, or BCMA-specific CAR-expressing cells. In some embodiments, the fusion protein can be administered about 8 days or about 15 days after administration of the CAR-expressing cells, for example, MUC16-, CD33-, CD19-, or BCMA-specific CAR-expressing cells.

[0391] Fusion proteins can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is intravenous injection or infusion. For example, the fusion protein can be administered by 1 ml 2 Approximately 35-440 mg per 1 m 2 Approximately 70-310 mg per 1 m 2 The fusion protein may be administered by intravenous infusion at a rate greater than 20 mg / min, e.g., 20-40 mg / min, typically 40 mg / min or greater, to reach a dose of about 110-130 mg per minute. In some embodiments, the fusion protein is administered at a rate greater than 1 mg / min. 2 Approximately 1 to 100 mg per 1 ml, preferably 1 ml 2 Approximately 5 to 50 mg per 1 m 2 More preferably, it is about 7 to 25 mg per m 2 The active ingredient may be administered by intravenous infusion at a rate of less than 10 mg / minute, preferably 5 mg / minute or less, to reach a dose of about 10 mg per minute.

[0392] The fusion protein is 2 Approximately 35-440 mg per 1 m 2 Approximately 70-310 mg per 1 m 2 The drug may be administered by intravenous infusion at a rate greater than 20 mg / min, e.g., 20-40 mg / min, typically 40 mg / min or greater, to reach a dose of about 110-130 mg per minute. 2 Injection rates of about 110-130 mg per kg achieve levels of about 3 mg per kg. 2 Approximately 1 to 100 mg per unit, e.g., 1 m 2 Approximately 5 to 50 mg per 1 m 2 Approximately 7 to 25 mg per 1 m 2 The antibody may be administered by intravenous infusion at a rate of less than 10 mg / minute, e.g., 5 mg / minute or less, to reach a dose of about 10 mg per minute. In some embodiments, the antibody is infused over a period of about 30 minutes.

[0393] Dose of engineered effector cells In some embodiments, an amount of modified effector cells is administered to a subject in need thereof, the amount being determined based on efficacy and potential to induce cytokine-associated toxicity. In some cases, the amount of modified effector cells is about 10 modified effector cells per kg. 5 ~about 10 9 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 5 ~about 10 8 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 5 ~about 10 7 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 6 ~about 10 9 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 6 ~about 10 8 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 7 ~about 10 9 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 5 ~about 10 6 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 6 ~about 10 7 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 7 ~about 10 8 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 8 ~about 10 9 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 9 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 8In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 7 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 6 In some cases, the amount of engineered effector cells is about 10 engineered effector cells per kg. 5 Includes pieces.

[0394] In some embodiments, the engineered effector cells are engineered T cells. In some cases, the engineered T cells are CAR-T cells. In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 9 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 8 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 7 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 6 ~about 10 9 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 6 ~about 10 8 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 7 ~about 10 9 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 6 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 6 ~about 10 7 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 7 ~about 10 8 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 8 ~about 10 9In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 9 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 8 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 7 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 6 In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 5 Includes pieces.

[0395] In some embodiments, the CAR-T cells are CD19-specific CAR-T cells. Optionally, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 9 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 8 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 7 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 6 ~about 10 9 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 6 ~about 10 8 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 7 ~about 10 9 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 5 ~about 10 6 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 6 ~about 10 7In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 7 ~about 10 8 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 8 ~about 10 9 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 9 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 8 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 7 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 6 In some cases, the amount of CD19-specific CAR-T cells is about 10 CAR-T cells per kg. 5 Includes pieces.

[0396] In some embodiments, the modified T cells are engineered TCR T-cells. Optionally, the amount of engineered TCR T-cells is about 10 TCR cells per kg. 5 ~about 10 9 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 5 ~about 10 8 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 5 ~about 10 7 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 6 ~about 10 9 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 6 ~about 10 8 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 7 ~about 10 9 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 5 ~about 10 6In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 6 ~about 10 7 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 7 ~about 10 8 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 8 ~about 10 9 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 9 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 8 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 7 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 6 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 5 Includes pieces.

[0397] It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or application of the claimed compositions.

[0398] Fusion protein expression and purification system The fusion proteins, or fragments or variants thereof, described herein can be produced from host cells transformed with an expression vector containing a polynucleotide encoding one or more fusion proteins, or fragments or variants thereof, by culturing the cells under conditions and for a sufficient period of time to allow expression of the fusion proteins, or fragments or variants thereof. For example, polypeptides expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see, Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning - A Laboratory Manual - 3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The selection of codons, appropriate expression vectors, and appropriate host cells can vary depending on numerous factors and can be easily optimized as needed. The fusion proteins, or fragments or variants thereof, described herein can be expressed in mammalian cells or other expression systems, including, but not limited to, yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0399] After expression, the fusion protein, or fragment or variant thereof, can be purified or isolated. The terms "purified" or "isolated," as applied to any of the fusion proteins, or fragments or variants thereof described herein, can refer to a polypeptide or protein that has been separated or purified from components that naturally accompany it (e.g., proteins or other naturally occurring biological or organic molecules), e.g., other proteins, lipids, and nucleic acids in the prokaryote expressing the protein. Typically, a polypeptide is purified if it constitutes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% by weight of the total protein in a sample.

[0400] The fusion proteins described herein, or fragments or variants thereof, can be isolated or purified in various ways, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic methods, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, fusion proteins, or fragments or variants thereof, can be purified using a standard anti-fusion protein antibody affinity column. Ultrafiltration and dialysis, followed by protein concentration, are also useful. See, for example, Scopes (1994) "Protein Purification, 3rd edition," Springer-Verlag, New York City, NY. The degree of purification required can vary depending on the desired use. In some cases, purification of the expressed fusion protein, or fragment or variant thereof, is not necessary.

[0401] Methods for determining the yield or purity of purified fusion proteins, or fragments or variants thereof, can include, for example, Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, Amido Black protein assay, high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (e.g., using protein dyes such as Coomassie Blue or colloidal silver dyes). Once expressed, purified, or after post-expression purification, the fusion proteins described herein, or fragments or variants thereof, can be assayed for any one of the listed desired properties using an in vitro or in vivo assay, such as any of the in vitro or in vivo assays described herein. For example, the fusion proteins described herein, or fragments or variants thereof, can be assayed for their ability to inhibit PD-1 and capture TGF-β, for example.

[0402] The fusion proteins described herein, or fragments or variants thereof, can be produced using various techniques.For example, the polynucleotide encoding the fusion proteins described herein, or fragments or variants thereof, can be inserted into an expression vector containing transcriptional and translational regulatory sequences, which can include, for example, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, transcriptional terminator signals, polyadenylation signals, and enhancer or activator sequences.In some embodiments, the regulatory sequences can include promoter sequences, and transcriptional initiation and termination sequences.In addition, the expression vector can contain more than one replication system so that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression, and in prokaryotic hosts for cloning and amplification.

[0403] Several possible vector systems are available for expressing fusion proteins, or fragments or variants thereof, from nucleic acids in mammalian cells (e.g., host cells). For example, one class of vectors relies on integration of the desired gene sequence into the host cell genome. Cells that have stably integrated DNA can be selected by co-introducing a drug resistance gene such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene may be linked to the DNA gene sequence to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). Another class of vectors utilizes DNA elements that confer autonomous replication capability to extrachromosomal plasmids. These vectors can be derived from animal viruses such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), polyomavirus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79). Expression vectors can be introduced into cells (e.g., host cells) in a form suitable for subsequent expression of the nucleic acid. The method of introduction will, in large part, be dictated by the target cell type, as discussed below. Non-limiting exemplary methods can include CaPO4 precipitation, liposome fusion, lipofectin, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0404] Suitable host cells for expression of the fusion protein, or fragment or variant thereof, can include, but are not limited to, yeast cells, bacterial cells, insect cells, plant cells, and, as described above, mammalian cells. Cells of interest include bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines (e.g., primary mammalian cells). In some embodiments, the fusion protein, or fragment or variant thereof, is expressed in Chinese hamster ovary (CHO) cells or a suitable myeloma cell line, such as NSO. Suitable cell lines also include, for example, BHK-21 (baby hamster kidney) cells; 293 (human embryonic kidney) cells; HMEpC (human mammary epithelial cells); and 3T3 (mouse embryonic fibroblast) cells.

[0405] The methods described herein result in the expression and purification of fusion proteins, or fragments or variants thereof, in a variety of cell-based expression systems, including protein production in bacterial cells, mammalian cells, insect cells, yeast cells, and Chlamydomonas cells. Protein expression can be constitutive or inducible with inducers such as copper sulfate, galactose, sugars such as methanol, methylamine, thiamine, tetracycline, or IPTG. After the fusion protein, or fragments or variants thereof, are expressed in the host cells, the host cells are lysed to release the fusion protein, or fragments or variants thereof, for purification. Methods for lysing various host cells are featured in "Sample Preparation—Tools for Protein Research" EMD Bioscience and in the Current Protocols in Protein Sciences (CPPS). A non-limiting exemplary purification method is affinity chromatography, such as ion metal affinity chromatography, using nickel, cobalt, or zinc affinity resins for histidine-tagged fusion proteins, or fragments or variants thereof. A method for purifying histidine-tagged proteins is described by Clontech, using their own Talonx cobalt resin, and by Novagen in the 10th edition of their pET system manual. Another non-limiting exemplary purification strategy is immunoaffinity chromatography, for example, a resin conjugated with anti-myc antibodies can be used to affinity purify, for example, myc-tagged fusion proteins, or fragments or variants thereof.Enzymatic digestion with serine proteases, such as thrombin and enterokinase, cleaves and releases the fusion protein, or fragments or variants thereof, from the histidine or myc tag, leaving the histidine and myc tags attached to the affinity resin while releasing the fusion protein, or fragments or variants thereof, from the affinity resin.

[0406] Methods for introducing polynucleotides encoding fusion proteins, or fragments or variants thereof, into cells (e.g., host cells) and expressing them are known in the art. In the context of expression vectors, vectors containing polynucleotides encoding fusion proteins, or fragments or variants thereof, can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0407] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, gene gun, microinjection, electroporation, etc. Methods for producing cells containing...

Claims

1. (a) an immune checkpoint inhibitor comprising an antibody or a functional fragment thereof that binds to PD-1, the immune checkpoint inhibitor comprising either (i) or (ii): (i) a first polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 6, the first polypeptide comprising the variable region (V Ｈ and a second polypeptide comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12, the first polypeptide comprising each of the three CDRs of SEQ ID NO: 12, the second polypeptide comprising the variable region (V Ｌ a second polypeptide comprising each of the three CDRs of (ii) a first polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 7, provided that the first polypeptide comprises each of the three CDRs of the variable region of the heavy chain (VH) of pembrolizumab, and a second polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13, provided that the second polypeptide comprises each of the three CDRs of the variable region of the light chain (VL) of pembrolizumab; and (b) a transforming growth factor beta (TGF-β) cytokine trap comprising an amino acid sequence having at least 90% identity to any of SEQ ID NO: 14, SEQ ID NO: 141, and SEQ ID NO:

142. A fusion protein comprising:

2. The fusion protein of claim 1, wherein the immune checkpoint inhibitor is connected to the TGF-β cytokine trap by a linker comprising any of SEQ ID NOs: 17-34.

3. The fusion protein of claim 1, wherein the TGF-β cytokine trap comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

14.

4. The fusion protein of claim 3, wherein the TGF-β cytokine trap binds to TGF-β1 or TGF-β3.

5. The fusion protein of claim 3, wherein the TGF-β cytokine trap binds to TGF-β1 and TGF-β3.

6. The fusion protein of claim 5, wherein the TGF-β cytokine trap binds to TGF-β1 and TGF-β3, but does not bind to TGF-β2.

7. The fusion protein of claim 1, wherein the TGF-β cytokine trap comprises an amino acid sequence having at least 95% identity to any of SEQ ID NO:14, SEQ ID NO:141 and SEQ ID NO:

142.

8. The fusion protein of claim 1, wherein the TGF-β cytokine trap comprises SEQ ID NO:

14.

9. The fusion protein of claim 1 , wherein the antibody is an immunoglobulin G (IgG) antibody.

10. The fusion protein of claim 9, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

11. The fusion protein of claim 1, wherein the antibody is an IgG4 antibody and comprises SEQ ID NO:146 or SEQ ID NO:292, but with a mutation at position 108 of SEQ ID NO:146 or SEQ ID NO:

292.

12. The fusion protein of claim 11, wherein the mutation is S108P.

13. The fusion protein of claim 10, wherein the TGF-β cytokine trap is linked to the IgG4 antibody by a linker.

14. The immune checkpoint inhibitor is a Fab, (Fab) ２ , (Fab′) ２ , Fv, (Fv) ２ , or scFv.

15. The fusion protein of claim 1, wherein a linker connects the first polypeptide of the immune checkpoint inhibitor to the TGF-β cytokine trap.

16. 2. The fusion protein of claim 1, wherein a linker connects the second polypeptide of the immune checkpoint inhibitor to the TGF-β cytokine trap.

17. The fusion protein of claim 1, wherein (a) a first linker links either the first or second polypeptide of the immune checkpoint inhibitor to the TGF-β cytokine trap, and (b) a second linker links the first polypeptide of the immune checkpoint inhibitor to the second polypeptide.

18. The fusion protein of claim 17, wherein the second linker comprises any of SEQ ID NOs: 17 to 34.

19. An immune checkpoint inhibitor comprising: (a) (i) a first polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 6, provided that the first polypeptide comprises each of the three CDRs of the variable region of the heavy chain (VH) of nivolumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 12, provided that the second polypeptide comprises each of the three CDRs of the variable region of the light chain (VL) of nivolumab; or (b) (i) a first polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 7, provided that the first polypeptide comprises each of the three CDRs of the heavy chain variable region (VH) of pembrolizumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 13, provided that the second polypeptide comprises each of the three CDRs of the light chain variable region (VL) of pembrolizumab; The fusion protein of claim 1 , comprising any one of:

20. The fusion protein of claim 1, wherein the fusion protein comprises SEQ ID NO:15 and SEQ ID NO:16 or SEQ ID NO:

143.

21. An immune checkpoint inhibitor, (a) (i) a first polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:6, provided that the first polypeptide comprises each of the three CDRs of the variable region of the heavy chain (VH) of nivolumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:12, provided that the second polypeptide comprises each of the three CDRs of the variable region of the light chain (VL) of nivolumab; or (b) (i) a first polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 7, provided that the first polypeptide comprises each of the three CDRs of the heavy chain variable region (VH) of pembrolizumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 13, provided that the second polypeptide comprises each of the three CDRs of the light chain variable region (VL) of pembrolizumab; The fusion protein of claim 1 , comprising any one of:

22. An immune checkpoint inhibitor, (a) (i) a first polypeptide comprising an amino acid sequence having at least 99.5% identity to SEQ ID NO:6, provided that the first polypeptide comprises each of the three CDRs of the variable region of the heavy chain (VH) of nivolumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 99.5% identity to SEQ ID NO:12, provided that the second polypeptide comprises each of the three CDRs of the variable region of the light chain (VL) of nivolumab; or (b) (i) a first polypeptide comprising an amino acid sequence having at least 99.5% identity to SEQ ID NO: 7, provided that the first polypeptide comprises each of the three CDRs of the heavy chain variable region (VH) of pembrolizumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 99.5% identity to SEQ ID NO: 13, provided that the second polypeptide comprises each of the three CDRs of the light chain variable region (VL) of pembrolizumab; The fusion protein of claim 1 , comprising any one of:

23. An immune checkpoint inhibitor, (a) (i) a first polypeptide comprising SEQ ID NO:6; and (ii) a second polypeptide comprising SEQ ID NO:12; or (b) (i) a first polypeptide comprising SEQ ID NO:7; and (ii) a second polypeptide comprising SEQ ID NO:13; The fusion protein of claim 1 , comprising any one of:

24. The fusion protein of claim 1, wherein the fusion protein comprises (a) the amino acid sequence of SEQ ID NO:296 and (b) the amino acid sequence of SEQ ID NO:144 or SEQ ID NO:

145.

25. The fusion protein of claim 1 , wherein the immune checkpoint inhibitor comprises a fragment crystallizable region (Fc).

26. The fusion protein of claim 25, wherein the Fc is a human Fc selected from Fc1, Fc2, Fc3, Fc4, or a functional fragment thereof.

27. The fusion protein of claim 25, wherein the Fc further comprises one or more mutations.

28. The fusion protein of claim 1 , wherein the immune checkpoint inhibitor comprises an scFv and an Fc.

29. The fusion protein of claim 1 comprising SEQ ID NO:15 and SEQ ID NO:

294.

30. The fusion protein of claim 1 comprising SEQ ID NO:296 and SEQ ID NO:

295.

31. A polynucleotide encoding a fusion protein according to any one of claims 1 to 30.

32. 31. An expression vector comprising a polynucleotide encoding the fusion protein of any one of claims 1 to 30, said polynucleotide operably linked to a promoter.

33. (a)(i) a fusion protein according to any one of claims 1 to 30; (ii) a polynucleotide encoding the fusion protein according to any one of claims 1 to 30; or (iii) an expression vector comprising the polynucleotide according to (a)(ii) linked to a promoter; and (b) a pharma- ceutically acceptable excipient 23. A pharmaceutical composition comprising:

34. 31. A pharmaceutical composition for treating cancer in a subject, comprising a fusion protein according to any one of claims 1 to 30.

35. A pharmaceutical composition for treating cancer in a subject, comprising the polynucleotide of claim 31.

36. A pharmaceutical composition for treating cancer in a subject, comprising the vector described in claim 32.

37. 34. The pharmaceutical composition of claim 33, further comprising a chimeric antigen receptor.

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