Fusion constructs and methods of use thereof

Abstract

The present disclosure provides compositions comprising a fusion protein or a fragment or variant thereof, which fusion protein or fragment or variant comprises an anti-PD1 antibody or a fragment / variant thereof and a TGF-β trap. The present disclosure provides compositions comprising a fusion protein or a fragment or variant thereof, which fusion protein or fragment or variant comprises an anti-PD1 antibody or a fragment / variant thereof and an ADA2 polypeptide. The present disclosure also provides methods of treating cancer using the compositions.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 695,623, filed Jul. 9, 2018; 62 / 695,627, filed Jul. 9, 2018; 62 / 863,710, filed Jun. 19, 2019; 62 / 864,367, filed Jun. 20, 2019; and 62 / 866,420, filed Jun. 25, 2019, which are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION

[0003] Recently, monoclonal antibody-based cancer immunotherapies that interrupt inhibitory signals delivered to the adaptive immune system have shown promise clinically. With the FDA approval of CTLA-4 antibody inhibitors (such as ipilimumab) and PD-1 inhibitors (such as pembrolizumab, nivolumab), there are now more treatment options available for treating solid tumors including lung cancer, renal cell carcinoma, and ovarian cancer. However, in most indications co-expressing PD-1 / PD-L1 and TGF-β (e.g., ovarian cancer, gastric cancer, and colorectal cancer), little or no response to immune checkpoint inhibitors has been observed. Thus, there is a continuing need in the art to obtain safer and more effective cancer treatment methods.

[0004] Incorporation by reference

[0005] All publications, patents, and patent applications mentioned in this specification are hereby 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

[0006] Provided herein is a fusion protein comprising: (a) an antibody or a fragment or variant of the antibody that binds to programmed cell death protein-1 (PD-1); and (b) a transforming growth factor beta (TGF-β) cytokine trap; wherein one or more polypeptides of the fusion protein are linked by a linker.

[0007] 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 shown in any 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.

[0008] In one embodiment, the TGFβR is transforming growth factor β receptor II (TGFβRII) or a functional fragment thereof. In one embodiment, the functional fragment of TGFβRII is the extracellular domain (ECD) of TGFβRII. In one embodiment, the ECD binds TGF-β1. In one embodiment, the ECD binds TGF-β3. In one embodiment, the ECD binds both TGF-β1 and TGF-β3. In one embodiment, the ECD binds TGF-β1 and TGF-β3, but does not bind TGF-β2. In one embodiment, the TGF-β cytokine trap comprises a sequence that is at least 80% identical to the sequence shown 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 shown in SEQ ID NO: 14, SEQ ID NO. 141, or SEQ ID NO: 142.

[0009] In one embodiment, the TGF-β cytokine trap comprises the sequence shown 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 the S108P mutation. In one embodiment, the IgG4 is linked to the TGF-β cytokine trap via the linker. In one embodiment, the fragment of the antibody is Fab, (Fab)2, (Fab’)2, Fv, (Fv)2, or scFv.

[0010] In one embodiment, the antibody comprises the variable region of the heavy chain of the antibody (VH ) and the variable region of the light chain (V L ). In one embodiment, the linker connects the variable region of the heavy chain (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, in the fusion protein, the variable region of the heavy chain (V H ) is connected to the variable region of the light chain (V L ) by a second linker. In one embodiment, the second linker comprises a sequence shown in any one of SEQ ID NOs: 17-34. In one embodiment, the variable region of the heavy chain (V H ) is at least 80% identical to a sequence shown in any one of SEQ ID NOs: 1-7 and 149-164.

[0011] In one embodiment, the variable region of the light chain (V L ) is at least 80% identical to a sequence shown in any one of SEQ ID NOs: 8-13 and 148. In one embodiment, the variable region of the heavy chain (V H ) comprises a sequence shown in any one of SEQ ID NOs: 1-7 and 149-164. In one embodiment, the variable region of the light chain (V L ) comprises a sequence shown in any one of SEQ ID NOs: 8-13 and 148. In one embodiment, the variable region of the heavy chain (V H ) is at least 90% identical to the sequence shown in SEQ ID NO: 6, and the variable region of the light chain (V L ) is at least 90% identical to the sequence shown in SEQ ID NO: 12. 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.

[0012] In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO: 15 and the sequence shown in SEQ ID NO: 16. In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO: 15 and the sequence shown in SEQ ID NO: 143. In one embodiment, the variable region of the heavy chain (VH) is at least 90% identical to the sequence shown in SEQ ID NO: 7, and the variable region of the light chain is at least 90% identical to the sequence shown in SEQ ID NO: 13. In one embodiment, the variable region of the heavy chain (V H)comprises the sequence shown in SEQ ID NO:7, and the light chain variable region (V L ) comprises the sequence shown in SEQ ID NO:13.

[0013] In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO:296 and the sequence shown in SEQ ID NO:145. In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO:296 and the sequence shown in SEQ ID NO:144. In one embodiment, the antibody further comprises a fragment crystallizable region (F C ). In one embodiment, the F C is a human F C 1, F C 2, F C 3, F C 4 or a fragment thereof. In one embodiment, the F C further comprises one or more mutations. In one embodiment, the antibody comprises the scFv and the F C fragment. In one embodiment, the TGFβ cytokine trap comprises the anti-TGFβ antibody or its antigen-binding fragment. 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.

[0014] In one embodiment, the TGFβ cytokine trap comprises the TGFβ inhibitory peptide or its variant. In one embodiment, the TGFβ inhibitory peptide comprises the sequence shown in any one of SEQ ID NO.193 - 227.

[0015] Provided herein is a polynucleotide encoding the fusion protein disclosed herein.

[0016] Provided herein is an expression vector comprising a polynucleotide encoding the fusion protein disclosed herein, wherein the polynucleotide is 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 gene switch based on two polypeptide ecdysone receptors. In one embodiment, the vector is an adenovirus vector.

[0017] The present disclosure provides a pharmaceutical composition 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.

[0018] The present disclosure provides a method for treating cancer, comprising contacting a cell with (a) a fusion protein disclosed herein, (b) a polynucleotide encoding a 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.

[0019] The present disclosure provides a method for treating a subject having cancer, the method comprising administering a composition comprising a fusion protein comprising (a) an antibody or a fragment or variant of the antibody that binds to programmed cell death protein-1 (PD-1), and (b) 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 linked by a linker.

[0020] 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 shown 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 that is at least 80% identical to the sequence shown 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 shown in SEQ ID NO: 14, SEQ ID NO. 141, or SEQ ID NO: 142.

[0021] 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 contains a mutation at position 108 of SEQ ID NO: 146 or SEQ ID NO: 292. In one embodiment, the mutation is the S108P mutation. In one embodiment, the fragment of the antibody is the Fab, (Fab)2, (Fab’)2, Fv, (Fv)2, or scFv of the antibody. In one embodiment, the antibody or the fragment of the antibody or the variant of the antibody comprises a heavy chain variable region (V H ) and a light chain variable region (V L ). In one embodiment, the linker connects the heavy chain variable region (V H ) to the TGF-β cytokine trap. In one embodiment, the linker connects the light chain variable region (V L ) to the TGF-β cytokine trap.

[0022] In one embodiment, the heavy chain variable region (V H ) is connected to the light chain variable region (V L ) by a second linker. In one embodiment, the heavy chain variable region (V H ) is at least 80% identical to the sequence shown in any one of SEQ ID NOs: 1-7 and 149-164. In one embodiment, the light chain variable region (V L ) is at least 80% identical to the sequence shown in any one of SEQ ID NOs: 8-13 and 148. In one embodiment, the heavy chain variable region (V H ) comprises the sequence shown in any one of SEQ ID NOs: 1-7 and 149-164.

[0023] In one embodiment, the light chain variable region (V L ) comprises the sequence shown in any one of SEQ ID NOs: 8-13 and 148. In one embodiment, the heavy chain variable region (V H ) is at least 90% identical to the sequence shown in SEQ ID NO: 6, and the light chain variable region (V L ) is at least 90% identical to the sequence shown in SEQ ID NO: 12. In one embodiment, the heavy chain variable region (V H ) comprises the sequence shown in SEQ ID NO: 6, and the light chain variable region (V L)Comprises the sequence shown in SEQ ID NO:12. In one embodiment, the fusion protein comprises the sequences shown in SEQ ID NO:15 and SEQ ID NO:16.

[0024] In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO:15 (VL5 Igg4) and the sequence shown in SEQ ID NO:143. In one embodiment, the variable heavy chain (VH) is at least 90% identical to the sequence shown in SEQ ID NO:7, and the variable light chain (V L ) is at least 90% identical to the sequence shown in SEQ ID NO:13. In one embodiment, the variable heavy chain comprises the sequence shown in SEQ ID NO:7, and the variable light chain comprises the sequence shown in SEQ ID NO:13. In one embodiment, the fusion protein comprises the sequences shown in SEQ ID NO:296 and SEQ ID NO:145. In one embodiment, the fusion protein comprises the sequences shown in SEQ ID NO:296 and SEQ ID NO:144.

[0025] In one embodiment, the antibody further comprises a fragment crystallizable region (F C ). In one embodiment, the F C is human F C 1, F C 2, F C 3, F C 4 or a fragment thereof. In one embodiment, the F C further comprises one or more mutations. In one embodiment, the antibody comprises the scFv and the F C fragment. In one embodiment, the cancer is a refractory cancer. In one embodiment, the subject is unresponsive 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.

[0026] 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 a 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, gastric cancer, bladder cancer, liver cancer, Hodgkin lymphoma, lung cancer, skin cancer, kidney cancer or head and neck cancer.

[0027] 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.

[0028] In one embodiment, the chimeric antigen receptor comprises an antigen-binding domain capable of binding to an epitope on CD19, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, 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.

[0029] In one embodiment, the effective amount of the engineered T cells is at least 10 2 cells / kg. In one embodiment, the effective amount of the engineered T cells is at least 10 4 cells / kg. In one embodiment, the effective amount of the engineered T cells is at least 10 5 cells / kg. 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.

[0030] The present disclosure provides a method for treating cancer in a subject in need thereof, comprising (a) administering a composition comprising a fusion protein comprising an antibody or a fragment or variant of the antibody that binds to programmed cell death protein-1 (PD-1); and a transforming growth factor receptor (TGFβR) protein or a functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked 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.

[0031] In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO:15 and the sequence shown in SEQ ID NO:294. In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO:296 and the sequence shown in SEQ ID NO:295. In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO:15 and the sequence shown in SEQ ID NO:294. In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO:13 and the sequence shown in SEQ ID NO:295.

[0032] The present disclosure particularly discloses a fusion protein, comprising: (a) an antibody or a fragment or variant of the antibody that binds to programmed cell death protein-1 (PD-1); and (b) an adenosine deaminase (ADA) protein or a functional fragment thereof;

[0033] wherein one or more polypeptides of the fusion protein are linked by a linker. In one embodiment, the linker comprises (G4S)n, where n is 2, 3, 4, 5 or 6. In some embodiments,

[0034] the linker comprises (Gly)n, where n is 6, 7 or 8. In another embodiment, 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 any of the sequences shown in SEQ ID NOs:17-34.

[0035] 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 IDNO: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 that is at least 80% identical to the sequence shown in SEQ ID NO:284 or any one of 273-279. In certain embodiments, the adenosine deaminase (ADA) protein comprises the sequence shown in SEQ ID NO:284 or any one of 273-279.

[0036] 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 the S108P mutation. In some instances, the fragment of the antibody is the Fab, (Fab)2, (Fab’)2, Fv, (Fv)2, or scFv of the antibody. In one embodiment, the antibody or the fragment of the antibody or the variant of the antibody comprises a heavy chain variable region (V H ) and a light chain variable region (V L ). In one embodiment, the linker connects the heavy chain variable region (V H ) to adenosine deaminase 2 (ADA2) or a mutant or variant thereof. In another embodiment, the linker connects the light chain variable region (V L ) to adenosine deaminase 2 (ADA2) or a mutant or variant thereof.

[0037] In one instance, the heavy chain variable region (V H ) is connected to the light chain variable region (V L ) by a second linker. In one embodiment, the heavy chain variable region (V H ) is at least 80% identical to the sequence shown in any one of SEQ ID NO:1-7 and 149-164. In another instance, the light chain variable region (V L) is at least 80% identical to the sequence shown in any of SEQ ID NOs: 8-13 and 148. In a further embodiment, the heavy chain variable region (V H ) comprises the sequence shown in any of SEQ ID NOs: 1-7 and 149-164. In a further embodiment, the light chain variable region (V L ) comprises the sequence shown in any of SEQ ID NOs: 8-13 and 148. In one embodiment, the heavy chain variable region (VH) is at least 90% identical to the sequence shown in SEQ ID NO: 6 (VH6), and the light chain variable region (VL) is at least 90% identical to the sequence shown in SEQ ID NO: 12 (VL5). In an embodiment, the heavy chain variable region (V H ) comprises the sequence shown in SEQ ID NO: 6, and the light chain variable region (V L ) comprises the sequence shown in SEQ ID NO: 12.

[0038] In one instance, the fusion protein comprises the sequence shown in SEQ ID NO: 12 (VL5) and the sequence shown in SEQ ID NO: 280 (VH6 IgG4(mut)-ADA2 wt). In another instance, the fusion protein comprises the sequence shown in SEQ ID NO: 12 (VL5) and the sequence shown in SEQ ID NO: 281 (VH6 igG4(mut)ADA2 mut 7). In one instance, the heavy chain variable region (V H ) is at least 90% identical to the sequence shown in SEQ ID NO: 7 (VH7), and the light chain variable region (V L ) is at least 90% identical to the sequence shown in SEQ ID NO: 13 (VL6). In one instance, the heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO: 7 (VH7), and the light chain variable region (V L ) comprises the sequence shown in SEQ ID NO: 13 (VL6). In another instance, the fusion protein comprises the sequence shown in SEQ ID NO: 13 (VL6) and the sequence shown in SEQ ID NO: 282 (VH7 igG4(mut)-ADA2wt). In one instance, the fusion protein comprises the sequence shown in SEQ ID NO: 13 (VL6) and the sequence shown in SEQ ID NO: 283 (VH7 igG4(mut)ADA2 mut 7).

[0039] The present disclosure provides a polynucleotide encoding a fusion protein. The present disclosure further provides an expression vector comprising the polynucleotide encoding the fusion protein of any 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-inducible gene switch based on two polypeptides, the ecdysone receptor. In some embodiments, the vector is an adenovirus vector.

[0040] The present disclosure provides a method for treating cancer, comprising contacting a cell with the fusion protein, the polynucleotide encoding the fusion protein, or the expression vector. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a mammalian cell.

[0041] The present disclosure further provides a pharmaceutical composition comprising the fusion protein; or the polynucleotide encoding the fusion protein; or the expression vector, and a pharmaceutically acceptable excipient.

[0042] The present disclosure provides a method for treating a subject having cancer, the method comprising administering a composition comprising a fusion protein comprising an antibody or a fragment or a variant of the antibody that binds to programmed cell death protein-1 (PD-1); and an adenosine deaminase protein or a functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked 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), or ADA2 mutant 6 (SEQ ID NO:278), ADA2 mutant 7 (SEQ ID NO:279), or wild-type ADA2 (SEQ ID NO:284).

[0043] In certain cases, 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 cases, the linker comprises any of the sequences shown in SEQ ID NOs: 17-34.

[0044] The present invention provides a method for treating a subject suffering from cancer. In certain cases, the 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 lymphoma, lung cancer, skin cancer, kidney cancer or head and neck cancer. In certain cases, the skin cancer is cutaneous squamous cell carcinoma, melanoma or basal cell carcinoma. In other cases, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). In some cases, the breast cancer is triple negative breast cancer (TNBC).

[0045] In another embodiment, there is provided a method for treating a subject suffering from cancer, the method comprising administering a composition comprising a fusion protein comprising an antibody or a fragment or a variant of the antibody that binds to programmed cell death protein-1 (PD-1); and an adenosine deaminase protein or a functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked by a linker. In a further embodiment, the method for treating a subject suffering from cancer further comprises administering an effective amount of T cells engineered to express a foreign receptor.

[0046] In some cases, the foreign 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 on CD19, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor alpha, 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.

[0047] In one embodiment, the effective amount of the engineered T cells is at least 10 2 cells / kg. In another embodiment, the effective amount of the engineered T cells is at least 10 4 cells / kg. In a further embodiment, the effective amount of the engineered T cells is at least 10 5 cells / kg.

[0048] In a further embodiment, the engineered T cells further express a cytokine. In another embodiment, the cytokine is a fusion protein comprising IL-15 and IL-15Rα.

[0049] Provided herein is a method of treating cancer in a subject in need thereof, comprising administering a composition comprising a fusion protein comprising an antibody or a fragment or a variant of the antibody that binds to programmed cell death protein-1 (PD-1); and an adenosine deaminase protein or a functional fragment thereof; wherein one or more polypeptides of the fusion protein are linked 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The features of the invention are set forth with particularity in the appended claims. The features and advantages of the disclosure will be better understood by reference to the following detailed description of illustrative embodiments that utilize the principles of the disclosure and the accompanying drawings, in which:

[0051] Figure 1 is a schematic diagram of PD-1 / PD-L1 in immunosuppression.

[0052] Figure 2 is a schematic diagram of TGF-β in immunosuppression.

[0053] Figure 3 shows a TGF-β-related gene cluster (enriched in stages III / IV) associated with metastatic disease and poor prognosis in a subset of ovarian cancer patients.

[0054] Figure 4A 、 Figure 4B and Figure 4C show the design of the anti-PD1-TGFRII fusion protein design. In other exemplary embodiments, ADA2 can be fused with anti-PD1.

[0055] Figure 5 is a graph showing the blockade of PD-1 / PD-L1 interaction by anti-PD1 (VH6-VL5) IgG1-TGFβRII and anti-PD1 (VH6-VL5) IgG4-TGFβRII.

[0056] Figure 6 is a graph showing the neutralization of TGF-β1 isotype signaling by anti-PD1 (VH6-VL5) IgG1-TGFβRII and anti-PD1 (VH6-VL5) IgG4-TGFβRII.

[0057] Figure 7 Graph showing the neutralization of TGF-β2 isotypes by anti-PD1 (VH6-VL5) IgG1-TGFβRII and anti-PD1 (VH6-VL5) IgG4-TGFβRII.

[0058] Figure 8 Graph showing the neutralization of TGF-β3 isotype signaling by anti-PD1 (VH6-VL5) IgG1-TGFβRII and anti-PD1 (VH6-VL5) IgG4-TGFβRII.

[0059] Figure 9A 、 Figure 9B and Figure 9C The graphs in

[0060] Figure 9D and Figure 9E show that, compared to anti-PD1 or control antibodies, the proliferation and IFN-γ production of stimulated PBMCs are enhanced in a dose-dependent manner in the presence of anti-PD1-TGFRII fusion proteins. + Occupancy of the PD1 receptor on CD8

[0061] Figure 9F and Figure 9G The graphs in

[0062] Figure 9H and Figure 9I show the occupancy of the PD1 receptor on CD8+ T cells and IFN-γ production when anti-PD1-TGFRII fusion proteins are added to co-cultures of PBMCs and colorectal cancer (colorectal adenocarcinoma) cell lines.

[0063] Figure 9J and Figure 9K The graphs in

[0064] Figure 9L and Figure 9M show the concentrations of TGF-β1 and TGF-β2 in the supernatants of co-cultures of PBMCs and colorectal cancer (colorectal adenocarcinoma) cells in the presence of anti-PD1-TGFRII fusion proteins, respectively, compared to anti-PD1 alone.

[0065] Figures 10A - 10D Shows the effect of anti-PD1-TGFRII fusion protein treatment on T cell proliferation and activation in the presence of recombinant TGF-β1.

[0066] Figures 11A - 11F Shows 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.

[0067] Figure 12A Shows the effect of anti-PD1-TGFRII fusion protein on tumor growth in a humanized mouse model of colorectal cancer compared to anti-PD1 alone.

[0068] Figure 12B Shows that treatment with anti-PD1-TGFRII fusion protein significantly increased the CD8 + T cells to T reg ratio in tumors in a humanized mouse model of colorectal cancer.

[0069] Figure 12C Shows the effect of anti-PD1-TGFRII fusion protein treatment on the expression level of perforin in a humanized mouse model of colorectal cancer compared to anti-PD1 treatment.

[0070] Figure 12D and Figure 12E Shows the effect of anti-PD1-TGFRII fusion protein treatment on the concentrations of TGF-β1 and TGF-β2 in a humanized mouse model of colorectal cancer compared to anti-PD1 treatment, respectively.

[0071] Figure 13A Shows that treatment with anti-PD1-TGFRII fusion protein significantly improved the production of IFNγ in an in vitro model of head and neck cancer compared to anti-PD1 treatment. Figures 13B - 13G Shows that treatment with anti-PD1-TGFRII fusion protein significantly increased T cell function, as confirmed by gene expression analysis of various pathways.

[0072] Figure 14A Shows the effect of anti-PD1-TGFRII fusion protein on tumor growth in a humanized mouse model of head and neck cancer compared to anti-PD1 alone.

[0073] Figure 14B Shows the survival rate of tumor-bearing mice in a humanized mouse model of head and neck cancer when treated with anti-PD1-TGFRII fusion protein compared to treatment with anti-PD1 alone or isotype control.

[0074] Figure 14C Shows the ratio of CD8 + T cells to regulatory T cells in the tumors of mice treated with anti-PD1-TGFRII fusion protein in a humanized mouse model of head and neck cancer.

[0075] Figure 14D and Figure 14E Shows the effect of anti-PD1-TGFRII fusion protein on the concentrations of TGF-β1 and TGF-β2 compared to anti-PD1 alone in a humanized mouse model of head and neck cancer.

[0076] Figure 14F Shows the effect of anti-PD1-TGFRII fusion protein on the production of IFN-γ in a humanized mouse model of head and neck cancer.

[0077] Figures 15A - 15B Shows the effect of anti-PD1(VH7 / VL6)--TGFRII fusion protein on IFN-γ production and TGF-β1 concentration in samples from patients with primary colorectal cancer.

[0078] Figure 15C Shows the gene expression analysis of samples from patients with primary colorectal cancer co-cultured with anti-PD1(VH7 / VL6)--TGFRII fusion protein.

[0079] Figure 16 The graphs shown depict the results of the cytotoxicity assay of anti-PD1(VH7 / VL6)-TGFRII fusion protein compared to anti-PD-L1--TGFRII fusion protein.

[0080] Figure 17 The graphs in

[0081] Figure 18A and Figure 18B depict the results of the cytotoxicity assay of anti-PD1(VH6 / VL5)-TGFRII fusion protein combined with CD33 CAR-T and anti-PD1(VH7 / VL6)-TGFRII fusion protein combined with CD33 CAR-T, respectively, compared to anti-PD-1 combined with CAR T cells and compared to CAR T cells alone.

[0082] Figure 19A and Figure 19B The graphs in depict the lysis of tumor cells using anti-PD1(VH6 / VL5)-TGFRII fusion protein and anti-PD1(VH7 / VL6)-TGFRII fusion protein, respectively, when co-cultured with NK cells.

[0083] Figure 20 The figures therein depict Biacore assays in which TGF-b1 and PD1 are simultaneously bound by the anti-PD1 (VH6 / VL5)-TGFRII fusion protein using different linkers.

[0084] Figure 21 It is a figure showing the blockade of PD-1 / PD-L1 interaction by anti-PD1 IgG4-ADA2.

[0085] Figure 22 It is a figure showing the ADA2 enzyme activity measured for anti-PD1 hIgG1-ADA2 and anti-PD1 hIgG4-ADA2.

[0086] Figures 23A - 23C It is a figure showing the effect of various variants of anti-PD1 and anti-PD1-ADA2 fusion proteins on PD-L1 / PD-1 interaction.

[0087] Figures 24A - 24F It is a figure showing the enzyme activity of various variants of anti-PD1-ADA2 fusion proteins measured by ADA enzyme activity.

[0088] Figure 25 It is a figure showing the enzyme activity of anti-PD1-mutADA2 compared to anti-PD1-wtADA2 measured by ADA enzyme activity.

[0089] Figures 26A - 26D It is a figure depicting the effect of variants of anti-PD1-wtADA2 on T cell proliferation.

[0090] Figure 26E It is a figure depicting the production of IFNγ induced by anti-PD1-wtADA2 compared to anti-PD1 or isotype control.

[0091] Figures 27A - 27B It is a figure depicting the effectiveness of wtADA2 and mutADA2 in reversing adenosine-mediated inhibition of T cell proliferation.

[0092] Figure 28 It is a figure depicting the effect of variants of anti-PD1-ADA2 fusion proteins on the blockade of PD1-PDL1 interaction.

[0093] Figure 29 It is a figure showing the enzyme activity of anti-PD1-ADA2-scFv-Fc measured by ADA enzyme activity.

[0094] Figure 30 It is a bar graph showing the enzyme activity of variants of anti-PD1-ADA2 measured by ADA enzyme activity.

[0095] Figures 31A - 31B It is a graph depicting the effects of anti-PD1-wtADA2 on IFN-γ production and tumor-infiltrating lymphocyte (TIL) proliferation in tumors of primary CRC patients.

[0096] Figure 32 It is a graph depicting the effects of anti-PD1 on tumor volume in a humanized mouse model of lung cancer compared to anti-PD1-wtADA2.

[0097] Figures 33A - 33C Shows the design of the anti-PD1-adenosine deaminase 2 (ADA2) design - anti-PD1-ADA2. Detailed Description of the Invention

[0098] The following description and examples detail embodiments of the present disclosure.

[0099] It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are variations and modifications to the present disclosure, which are all encompassed within the scope of the present disclosure.

[0100] All terms should be understood as they would be understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0101] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0102] Although the various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

[0103] The following definitions supplement those in the art and are specific to this application and should not be attributed to any related or unrelated circumstances, such as any co-owned patents or applications. Preferred materials and methods are described herein, but any methods and materials similar or equivalent to those described herein may be used in testing the practice of the present disclosure. Thus, the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0104] Definitions

[0105] In this application, unless otherwise specifically stated, the use of the singular form includes the plural form. It must be noted that, unless the context clearly indicates otherwise, as used in this specification, the singular forms "a", "an", and "the" include plural referents.

[0106] In this application, unless otherwise specified, the use of "or" means "and / or". As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents are used interchangeably. These terms can express, specifically referring to any combination. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" can mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C". The term "or" can be used conjunctively or disjunctively, unless the context specifically indicates disjunctive use.

[0107] Furthermore, the use of the term "comprising" and other forms such as "including", "containing", and "having" is not restrictive.

[0108] The mention of "some embodiments", "embodiments", "an embodiment", or "other embodiments" in this specification means that the specific features, structures, or characteristics described in connection with that embodiment are included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0109] As used in this specification and the claims, the words "comprising" (and any form of comprising), "having" (and any form of having), "including" (and any form of including), or "containing" (and any form of containing) are inclusive or open-ended and do not exclude other unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented using any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0110] As used herein, the term "about" and its grammatical equivalents with respect to a reference numerical value can include the numerical value itself and a range of values that are plus or minus 10% of that numerical value.

[0111] The terms “about” or “approximately” mean within an acceptable error range of a particular value as determined by a person of ordinary skill in the art, which acceptable error range will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with practice in the art, “about” can mean within one or more standard deviations. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, an amount of “about 10” includes 10 and any amount from 9 to 11. In yet another example, the term “about” with respect to a reference numerical value can also include a numerical range that is 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 particular value, preferably within 5-fold, more preferably within 2-fold. In cases where a particular value is described in the present application and claims, unless otherwise indicated, the term “about” is to be presumed to mean within the acceptable error range of that particular value.

[0112] 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, triple-stranded DNA, as well as double-stranded and single-stranded RNA. It also includes modified forms (e.g., by methylation and / or by capping) and unmodified forms of polynucleotides. The term also is intended to include molecules containing non-naturally occurring nucleotides or synthetic nucleotides, as well as nucleotide analogs.

[0113] 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 cells by, for example, transfection, transformation, or transduction. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic lipid-mediated transfection; tungsten particle-facilitated particle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). After growing infectious particles in suitable packaging cells, phage or viral vectors can be introduced into host cells, many of which packaging cells are commercially available.

[0114] As used herein, "polypeptide", "peptide" and their grammatical equivalents refer to polymers of amino acid residues. The polypeptides may optionally include proteins that are glycosylated or otherwise modified that are typical for a given protein in a given cellular environment. The polypeptides and proteins (including functional portions and functional variants thereof) disclosed herein may include 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-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxy phenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 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 the expression of the polypeptides described herein in engineered cells 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 ethylization), ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bonds, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0115] In the context of two nucleic acid sequences or the amino acid sequences of polypeptides, the term "identical" and its grammatical equivalents or "sequence identity" as used herein refer to residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, typically about 50 to about 200, more typically about 100 to about 150 contiguous positions, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences have been optimally aligned. Methods for sequence alignment for comparison are well known in the art. The optimal alignment of sequences for comparison can be conducted 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. U.S.A., 85:2444 (1988); computerized implementations of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program of Intelligentics, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is described in detail 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). Alignments are usually also performed by inspection 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 a fragment thereof, e.g., as determined by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.Similarly, nucleic acids can also be described with reference to a starting nucleic acid. For example, they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to the reference nucleic acid or a fragment thereof, as determined, for example, using default parameters by BLASTN (or CLUSTAL, or any other available alignment software). When describing that a molecule has a certain percentage of sequence identity with a larger molecule, this means that when the two molecules are optimally aligned, according to the order of the optimal alignment of the two molecules, the residues of the said percentage in the smaller molecule find matching residues in the larger molecule.

[0116] The term "substantially identical" and its grammatical equivalents as applied to nucleic acid or amino acid sequences refer to nucleic acid or amino acid sequences that contain sequences having at least 90% or higher, at least 95%, at least 98% and at least 99% sequence identity compared to a reference sequence using standard procedures such as those described above (e.g., BLAST). For example, the BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program defaults to 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)). The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence used for the optimal alignment of the two sequences (which does not include additions or deletions). The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the resulting value by 100 to obtain the percentage of sequence identity. In some embodiments, there is substantial identity over a region of a sequence of at least about 50 residues, 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 region.

[0117] "Homology" is generally inferred from sequence identity between two or more nucleic acids or proteins (or their sequences). The exact percentage of sequence identity between sequences that can be used to establish homology varies with the nucleic acids and proteins under discussion, but sequence identity as low as 25% is routinely used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or higher, can also be used to establish homology. Methods for determining the percentage of sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. Nucleic acids and / or nucleic acid sequences are "homologous" when they are naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are "homologous" when the encoding DNA of the protein and / or protein sequence is naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules may be referred to as "homologs". For example, any naturally occurring protein can be modified by any available mutagenesis method. When expressed, the polypeptide encoded by the mutagenized nucleic acid is homologous to the protein encoded by the original nucleic acid.

[0118] As used herein, the term "isolated" and its grammatical equivalents refer to removing a nucleic acid from its natural environment. As used herein, the term "purified" and its grammatical equivalents refer to a molecule or composition of increased purity, whether taken from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, where "purity" is a relative term and not "absolute purity". However, it should be understood that nucleic acids and proteins can be formulated with diluents or adjuvants and still be considered isolated for practical purposes. For example, when used for introduction into cells, 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 substantially free, i.e., more than about 50% free, more than about 70% free, more than about 90% free of polynucleotides, proteins, polypeptides and other molecules that are naturally associated with the nucleic acid, polypeptide, protein or other compound.

[0119] "Expression vector" or "vector" refers to any genetic element, such as a plasmid, chromosome, virus, transposon, which acts as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replicating under its own control) or capable of replicating by insertion into the host cell chromosome, to which another polynucleotide segment has been attached in order to effect replication and / or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, phages, and cosmids. The vector may contain polynucleotide sequences necessary to effect ligation or insertion of the vector into the desired host cell and to effect expression of the attached segment. Such sequences vary according to the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, the expression vector may be capable of directly expressing the nucleic acid sequence product encoded therein without vector ligation or integration into the host cell DNA sequence. In some embodiments, the vector is an "episomal expression vector" or "episome", which is capable of replicating in the host cell and exists within the host cell as an extrachromosomal segment of DNA in the presence of an appropriately selected pressure (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 utilizing Epstein-Barr nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). Vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of episomal vectors that use the T-antigen and the SV40 origin of replication in place of EBNA1 and oriP. The vector may also contain a selectable marker gene.

[0120] As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that permits cells expressing the nucleic acid sequence to be specifically selected or eliminated in the presence of the corresponding selective agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publications 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. Patents 5,122,464 and 5,770,359.

[0121] As used herein, the term "coding sequence" refers to a polynucleotide segment that encodes a protein or polypeptide. This region or sequence is bounded by a start codon near the 5'-end and a stop codon near the 3'-end. The coding sequence may also be referred to as an open reading frame.

[0122] As used herein, the term "operably linked" refers to the physical and / or functional linkage of a DNA segment to another DNA segment such that the segments function in their intended manner. A DNA sequence is operably linked to a regulatory sequence, such as a promoter, enhancer, and / or silencer, when the DNA sequence encoding a gene product is linked to the regulatory sequence in such a way as to permit direct or indirect regulation of the transcription of the DNA sequence. For example, a DNA sequence is operably linked to a promoter when the DNA sequence is linked downstream of the transcription start site of the promoter, in the correct reading frame with the transcription start site, and permits transcription to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when the enhancer or silencer is linked to the DNA sequence in such a way as to increase or decrease, respectively, the transcription of the DNA sequence. Enhancers and silencers can be located upstream of, downstream of, or embedded within the coding region of a DNA sequence. If a signal sequence is expressed as a preprotein involved in polypeptide secretion, the DNA of the signal sequence is operably linked to the DNA encoding the polypeptide. The linkage of a DNA sequence to a regulatory sequence is typically accomplished by ligating at appropriate restriction sites or by inserting an adaptor or linker into the sequence using restriction endonucleases known to those of skill in the art.

[0123] As used herein, the term "induce" and its grammatical equivalents refer to an increase in the transcription, promoter activity, and / or expression of a nucleic acid sequence caused by a transcriptional regulator relative to a certain basal transcription level.

[0124] The term "transcriptional regulator" refers to a biochemical element that is used under certain environmental conditions to block or inhibit the transcription of a promoter-driven DNA sequence (e.g., a repressor or a nuclear corepressor), or to permit or stimulate the transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or an enhancer).

[0125] As used herein, the term "enhancer" refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence operably linked thereto. Enhancers can be located thousands of bases away from the coding region of the nucleic acid sequence and can mediate binding of regulatory factors, patterns of DNA methylation, or alterations in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as cloned polynucleotides (from depository centers such as ATCC and other commercial or individual sources) or can be obtained within cloned polynucleotides. Many polynucleotides that contain a promoter (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, within, or downstream of the coding sequence. The term "Ig enhancer" refers to enhancer elements derived from enhancer regions located within the immunoglobulin (Ig) locus (such enhancers include, for example, the heavy chain (μ) 5' enhancer, the light chain (κ) 5' enhancer, the κ and μ intronic enhancers, and the 3' enhancer) (see generally Paul W.E. (ed.), Fundamental Immunology, 3rd ed., Raven Press, New York (1993), pp. 353-363; and U.S. Patent 5,885,827).

[0126] The term "promoter" refers to a polynucleotide region that initiates transcription of a coding sequence. A promoter is located near the transcriptional start site of a gene, on the same strand and upstream (towards the 5' region of the sense strand) of the DNA. Some promoters are constitutive, in that they are active in a cell under all circumstances, while other promoters are regulated to become active in response to a specific stimulus, such as an inducible promoter. The term "promoter activity" and its grammatical equivalents refer to the degree of expression of a nucleotide sequence operably linked to the promoter whose activity is being measured. Promoter activity can be directly measured by determining the amount of RNA transcript produced (e.g., by Northern blot analysis) or indirectly measured by determining the amount of product encoded by the linked nucleic acid sequence (such as a reporter nucleic acid sequence linked to the promoter).

[0127] As used herein, an "inducible promoter" refers to a promoter that is induced to be active by the presence or absence of a transcriptional regulator (e.g., a biological or abiotic factor). Inducible promoters are useful because the expression of genes operably linked to them can be turned on or off at certain developmental stages of an organism or in specific tissues. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters, and light-regulated promoters. Inducible promoters can be part of a gene switch or genetic switch.

[0128] As used herein, "T cell" or "T lymphocyte" is a type of lymphocyte that plays an important role in cell-mediated immunity. They can be distinguished from other lymphocytes such as B cells and natural killer (NK) cells by the presence of a T cell receptor (TCR) on the cell surface.

[0129] 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. Antibodies can be from any animal source. Antibodies 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 full antibody, including a single-chain full antibody. In some embodiments, the antibody can be a fragment of an antibody, which can include but is not limited to Fab, Fab', F(ab’)2, Fd (composed of V H and CH1), Fv fragment (composed of V H and V L ), single-chain variable fragment (scFv), single-chain antibody, disulfide-linked variable fragment (dsFv), and a fragment containing V L or V H domain. A full antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains an N-terminal variable (V H ) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (V L ) region and a C-terminal constant (C L ) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. V H region and V LThe regions have a similar general structure, each containing four framework regions that are relatively conserved in sequence. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, designated CDR1, CDR2, and CDR3, form the "hypervariable regions" of the antibody, which are responsible for antigen binding. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entirety, where the definitions include overlapping or subsets of amino acid residues when compared to each other. Preferably, the term "CDR" is the CDR defined by Kabat based on sequence comparison. CDRH1, CDRH2, and CDRH3 denote the heavy chain CDRs, while CDRL1, CDRL2, and CDRL3 denote the light chain CDRs.

[0130] The terms "fragment of an antibody", "antibody fragment", "fragment of the antibody", "antigen-binding portion", or grammatical equivalents thereof are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). For example, an antibody fragment desirably comprises one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Non-limiting examples of antibody fragments include (1) Fab fragments, which are monovalent fragments consisting of V L 、V H 、C L and CH1 domains; (2) F(ab’)2 fragments, which are bivalent fragments comprising two Fab fragments that are linked by a disulfide bond at the hinge region; (3) Fv fragments, which consist of the V L and V H domains of a single arm of an antibody; (4) single-chain Fv (scFv), which consists of two domains of an Fv fragment (i.e., V L and V HA single-chain molecule composed of the linker 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, wherein each polypeptide chain comprises a V L linked to a V H by a peptide linker that is too short to allow pairing between the V H and V L on the same polypeptide chain, thereby driving pairing between complementary domains on different V H -V L polypeptide chains to generate 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 8,603,950.

[0131] "Antigen recognition portion", "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 portion is an antibody, an antibody-like molecule, or a fragment thereof.

[0132] The term "conservative amino acid substitution" or "conservative mutation" refers to the substitution of one amino acid with another amino acid having common properties. A functional way to define the common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, G.E. and Schirmer, R.H., Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analysis, groups of amino acids can be defined, wherein amino acids within a group preferentially exchange with each other and thus are most similar to each other in their effect on the overall protein structure (Schulz, G.E. and Schirmer, R.H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, e.g., lysine substitution for arginine and vice versa, such that the positive charge can be maintained; glutamate substitution for aspartate and vice versa, such that the negative charge can be maintained; serine substitution for threonine, such that the free -OH can be maintained; glutamine substitution for asparagine, such that the free -NH2 can be maintained. Alternatively or additionally, a functional variant may comprise the amino acid sequence of a reference protein that has at least one non-conservative amino acid substitution.

[0133] The term "non-conservative mutation" refers to amino acid substitutions between different groups, for example, lysine substituting for tryptophan, or phenylalanine substituting for serine, and so on. In such cases, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the homologous parental protein.

[0134] As used herein, the term "proliferative disease" refers to a unified concept in which the excessive proliferation of cells and / or the renewal of the cell matrix significantly contributes to the pathogenesis of diseases including cancer. In some embodiments, the proliferative disease is cancer.

[0135] As used herein, "patient" or "subject" refers to a mammalian subject diagnosed with, suspected of having, or developing a proliferative disorder such as cancer. In some embodiments, the term "patient" refers to a mammalian subject having 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 can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female. "A patient in need" or "a subject in need" as used herein refers to a patient diagnosed with, or suspected of having, a disease or disorder, such as but not limited to cancer.

[0136] "Administering" as used herein refers to providing a patient or subject with one or more of the compositions described herein. By way of example and not limitation, administration of the composition (e.g., injection) can be by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by infusion over time. Alternatively or concurrently, administration can be by the oral route. In addition, administration can also be by surgical deposition of cell aggregates or pellets or placement of a medical device. In embodiments, the compositions of the present disclosure can comprise engineered cells or host cells expressing the nucleic acid sequences described herein, or a vector comprising at least one of the nucleic acid sequences described herein, in an amount effective to treat or prevent a proliferative disorder. The pharmaceutical composition can comprise the target cell population described herein, as well as one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0137] As used herein, the term "treatment", "treating" or grammatical equivalents thereof refer to obtaining a desired pharmacological and / or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect cures the disease and / or adverse symptoms caused by the disease, in whole or in part. In some embodiments, the term "treatment" may include "preventing" a disease or condition.

[0138] As used herein, "treatment period" refers to a treatment cycle, e.g., the administration of a therapeutic agent that can be repeated (e.g., on a regular schedule). In embodiments, the dosing regimen may have one or more periods during which the therapeutic agent is not administered between treatment periods. For example, a treatment period may include a fusion protein administered (before, concurrently, or after) in combination with a second therapeutic agent, e.g., CAR-T cells.

[0139] As used herein, the term "co-administer", "co-administration", "co-dosing", or "co-providing" refers to delivering two (or more) different treatments to a subject during the course of the subject's disease, e.g., after the subject has been diagnosed with the condition and before the condition has been cured or eliminated or treatment has stopped for other reasons, delivering two or more treatments. In some embodiments, the delivery of one treatment is still ongoing at the start of the delivery of the second treatment, so there is an overlap in dosing. This is sometimes referred to herein as "simultaneous" or "parallel delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective due to co-administration. For example, the second treatment is more effective, e.g., the same effect is observed with less of the second treatment compared to when the second treatment is administered in the absence of the first treatment, or the second treatment alleviates symptoms to a greater extent, or a similar situation is observed for the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters related to the condition is greater than that observed when delivering one treatment in the absence of the other treatment. The effects of the two treatments can be partially additive, fully additive, or more than additive. The delivery can be such that the effect of the first treatment delivered can still be detected when the second treatment is delivered.

[0140] In some embodiments, the first and second treatments can be administered simultaneously (e.g., at the same time) in the same or separate compositions, or sequentially. Sequential administration refers to administering one treatment before (e.g., immediately before, less than 5, 10, 15, 30, 45, 60 minutes before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 or more hours before; 4, 5, 6, 7, 8, 9 or more days before; 1, 2, 3, 4, 5, 6, 7, 8 or more weeks before) the administration of an additional (e.g., second) treatment. The order of administration of the first and second treatments can also be reversed.

[0141] The terms "therapeutically effective amount", "therapeutic amount", "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or grammatical equivalents thereof refer to an amount that is effective, at the required dosage and for a period of time, to achieve the desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the compositions described herein to elicit the desired response in one or more subjects. The precise amount of the compositions of the present disclosure to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0142] Alternatively, the pharmacological and / or physiological effect of administering one or more of the compositions described herein to a patient or subject can be "prophylactic", i.e., the effect completely or partially prevents a disease or its symptoms. A "prophylactically effective amount" refers to an amount that is effective, at the required dosage and for a period of time, to achieve the desired prophylactic result (e.g., prevention of the onset of a disease).

[0143] Programmed cell death protein and other checkpoint inhibitors

[0144] 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 escape. In some cases, tumor cells, accessory cells such as myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), and antigen-presenting cells (APC) in the tumor microenvironment may overexpress PDL-1. In some cases, PD-1 can be upregulated in "exhausted" T cells and can signal to inhibit effector T cell function when bound to its ligands (PDL-1, PDL2, and CD80). Blockade of the PD-1 / PD-L1 pathway with anti-PD-1 or anti-PD-L1 can restore the function of exhausted T cells and promote the killing of tumor cells ( Figure 1 ).

[0145] In some embodiments, the fusion protein comprising a PD-1 inhibitor and a TGF-β trap may 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 functional fragments or variants thereof. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab.

[0146] Fusion protein

[0147] In some embodiments, the fusion proteins or fragments or variants thereof provided herein comprise a PD-1 inhibitor or antibody fused to a cytokine trap via a linker.

[0148] In some embodiments, the PD-1 inhibitor can be an antibody targeting PD-1 or a fragment or variant of the antibody. In some embodiments, the fusion protein comprising pembrolizumab can be fused to a cytokine trap (e.g., a TGF-β trap). In some embodiments, the fusion protein comprising nivolumab can be fused to a cytokine trap (e.g., a TGF-β trap).

[0149] In some embodiments, the fusion proteins or fragments or variants thereof described herein comprise a cytokine trap as described above and an antibody targeting an immune checkpoint gene, a fragment or variant of the antibody. In some embodiments, an antibody targeting an immune checkpoint such as cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and programmed death ligand-1 (PDL1), or a fragment or variant of the antibody, can be fused to a TGF-β trap via a linker. In some embodiments, the PD-L1 inhibitor is atezolizumab.

[0150] In some embodiments, the fusion proteins or fragments or variants thereof provided herein comprise a PD1 inhibitor or antibody fused to adenosine deaminase (e.g., ADA2) or a functional variant or derivative thereof as described herein.

[0151] Cytokine trap

[0152] Cytokines have an impact on many biological processes. Inhibiting cytokines can have clinical benefits, for example, in cancer. Several cytokines have been shown to be causative agents of various diseases. Such cytokines include, but are not limited to, IL-1, IL-4, Il-6, TNF-α, TGF-β, and their various isotypes. As used herein, the term "cytokine trap" refers to an agent or neutralizing agent that blocks the action of cytokines. Examples of such cytokine traps can include, but are not limited to, the extracellular domain of a cytokine receptor, an antibody that binds to a cytokine, and a peptide that binds to a cytokine (e.g., an inhibitory peptide). 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 a further embodiment, a cytokine trap targeting TGF-β (e.g., a TGF-β trap) can 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 inhibitory peptides of TGF-β1, TGF-β2, and / or TGF-β3.

[0153] Transforming growth factor

[0154] Transforming growth factor-β (TGF-β) is a group of multifunctional peptides that can control proliferation, differentiation, and other functions in many cell types. TGF-β can act synergistically with TGF-α in inducing transformation. It can also act as a negative autocrine growth factor. Dysregulation of TGF-β activation and signaling can lead to apoptosis. Many cells can synthesize TGF-β, and almost all cells have specific receptors for this peptide. TGF-β1, TGF-β2, and TGF-β3 all act through the same receptor signaling system. TGF-β1 can play an important role in controlling the immune system and can display different activities on different types of cells or cells at different developmental stages. Most immune cells (or white blood cells) can secrete TGF-β1. TGF-β1 is a peptide of 112 amino acid residues derived from the C-terminus of a precursor protein by proteolytic cleavage. TGF-β is a secreted small polypeptide that can signal through a type II serine / threonine kinase dimer receptor (TGFβRII), which can recruit and phosphorylate a type I dimer receptor (TGFβRI). TGFβRI can phosphorylate and activate SMAD, which may be a transcription factor that regulates genes involved in cell proliferation, differentiation, apoptosis, and growth. Many advanced cancers are known to overexpress TGF-β and TGFβR, thus promoting the formation of invasive tumors. Inhibiting the TGFB signaling pathway may be a key therapeutic strategy for treating cancer.

[0155] Some T cells (such as regulatory T cells) can release TGF-β1 to inhibit the action of other T cells. The activity of TGF-β1 can prevent the IL-1- and IL-2-dependent proliferation of activated T cells, as well as the activation of resting helper T cells and cytotoxic T cells. Similarly, TGF-β1 can inhibit the secretion and activity of many other cytokines including, but not limited to, interferon-γ, tumor necrosis factor α (TNF-α), and various interleukins. It can also reduce the expression level of cytokine receptors such as the IL-2 receptor to downregulate the activity of immune cells. However, TGF-β1 can also increase the expression of certain cytokines in T cells and can promote their proliferation, especially in the case of immature cells ( Figure 2 ).

[0156] TGF-β1 can have similar effects on B cells, which vary according to the differentiation state of the cells. It can inhibit the proliferation of B cells and can stimulate the apoptosis of B cells, and can play a role in controlling the expression of antibodies, transferrin, and MHC class II proteins on immature and mature B cells.

[0157] The effects of TGF-β1 on macrophages and monocytes are likely to be mainly inhibitory; this cytokine can inhibit the proliferation of these cells and can prevent them from generating reactive oxygen species (such as superoxide (O2 - )) and nitrogen (such as nitric oxide (NO)) intermediates. However, as with other cell types, TGF-β1 can also have the opposite effect on myeloid-derived cells. For example, TGF-β1 can act as a chemotactic agent, guiding the immune response to certain pathogens; macrophages and monocytes can respond chemotactically to low levels of TGF-β1. In addition, the effects of TGF-β1 can increase the expression of monocyte cytokines (including IL-1α, IL-1β, and TNF-α) and the phagocytic killing of macrophages ( Figure 2 ).

[0158] Transforming growth factor-βIII (TGF-β3) is a subset of the cytokine family and is responsible for a variety of functions, including cell proliferation, embryogenesis, immune system regulation, and differentiation.

[0159] Transforming growth factor-β receptor II (TGFβRII)

[0160] TGF-β receptors (TGFβRs) are single-pass serine / threonine kinase receptors. They can exist in several different isoforms, which can be homodimers or heterodimers. The number of characterized ligands in the TGF-β superfamily can far exceed the number of known receptors, suggesting promiscuity between ligand and receptor interactions. Three TGF-β superfamily receptors (TGFβRs) that are specific for TGF-β can be distinguished by their structural and functional properties. TGFβRI (ALK5) and TGFβRII may have similar ligand-binding affinities and can only be distinguished from each other by peptide mapping. Both TGFβRI and TGFβRII have high affinity for TGF-β1 and low affinity for TGF-β2. TGFβRIII (β-glycan) may have high affinity for both homodimeric TGF-β1 and TGF-β2 and additionally high affinity for the heterodimeric TGF-β1,2. TGFβ receptors can also bind to TGF-β3. The so-called "TGFβRII" or "TGF-β receptor II" refers to a polypeptide having the amino acid sequence of wild-type human TGF-β receptor type 2 isoform A (e.g., the amino acid sequence (SEQ ID NO:289) of NCBI Reference Sequence (RefSeq) accession number NP_001020018), or having the amino acid sequence of wild-type human TGF-β receptor type 2 isoform B (e.g., the amino acid sequence (SEQ ID NO:290) of NCBI RefSeq accession number NP_003233) or a polypeptide having a sequence substantially identical 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 TGF-β binding activity of the wild-type sequence. The polypeptide of expressed TGFβRII lacks a signal sequence.

[0161] TGF-β1 can reduce the efficacy of MHC II in astrocytes and dendritic cells, which in turn reduces the activation of an appropriate helper T cell population. TGF-β1 can promote tumor growth with cancer progression and, in some embodiments, does not inhibit the inflammatory cell response but can promote regulatory T cell function. TGF-β1 can be produced by tumor cells, tumor-associated fibroblasts, regulatory T cells, and immature myeloid cells. TGF-β1 can inhibit T cell priming and promote an exhausted phenotype. TGF-β1 can inhibit the anti-tumor activity of innate immune cell populations including natural killer cells, macrophages, and dendritic cells. TGF-β receptor II can be upregulated by tumor-associated myeloid cells and can promote metastasis.

[0162] TGF-β trap fusion protein or a fragment or variant thereof

[0163] The present disclosure provides a fusion protein or a fragment or variant thereof, which comprises an immune checkpoint inhibitor, such as a PD-1 inhibitor or antibody, and a cytokine trap that can neutralize a cytokine (e.g., TGF-β). In some cases, the cytokine trap can be a TGF-β trap comprising SEQ ID NO. 142 (also referred to as TGF-βRII or a fragment or variant thereof). Examples of the TGF-β trap can include, but are not limited to, the extracellular domain (ECD) of a receptor (e.g., TGFβRII) 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 comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) 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 NO: 166, 168, 169, 171, 173, 175 or 177. In some embodiments, the anti-TGF-β antibody comprises a light chain variable region (V L ), and the light chain variable region (V L ) 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 NO: 165, 167, 170, 172, 174, 176 or 178. In certain embodiments, the TGF-β trap can specifically bind to or have a high affinity for TGF-β1 or TGF-β2 or TGF-β3. In other embodiments, the TGF-β trap can specifically bind to or have a high affinity for TGF-β1, TGF-β2 and TGF-β3. In other embodiments, the TGF-β trap can specifically bind to or have a high affinity for TGF-β1 and TGF-β3. In further embodiments, the TGF-β trap can have a low affinity for or not bind to TGF-β2.

[0164] The fusion proteins or fragments or variants thereof provided herein (e.g., a PD-1 inhibitor or antibody fused to a cytokine trap such as a TGF-β trap) can elicit a synergistic anti-tumor effect due to simultaneously blocking, for example, the interaction between PD-L1 on tumor cells and PD-1 on immune cells, and neutralizing, for example, TGF-β in the tumor microenvironment. Without being bound by theory, this effect is obtained from simultaneously blocking two major immune escape mechanisms and the targeted depletion of TGF-β in the tumor microenvironment by a single molecular entity. This depletion can be achieved by one or more of the following: (1) anti-PD-1 targeting of tumor cells; (2) binding of TGF-β in the tumor microenvironment by a TGF-β trap (e.g., TGFbRII); and / or (3) disruption of bound TGF-β by PD-L1 receptor-mediated endocytosis. The fusion proteins or fragments or variants thereof provided herein (e.g., a PD-1 inhibitor or antibody fused to a cytokine trap such as a TGF-β trap) can also promote natural killer cell-mediated tumor cell killing.

[0165] (i) A TGFβRII trap fusion protein or a fragment or variant thereof

[0166] In some embodiments, the fusion protein or a fragment or variant thereof further comprises a cytokine trap and a PD-1 inhibitor or an anti-PD-1 antibody or a fragment or variant thereof. In some embodiments, it is a fusion protein or a fragment or variant thereof that comprises a cytokine trap (e.g., a TGF-β trap) 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 protein 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 shown in SEQ ID NO:14. In some embodiments, the cytokine receptor sequence in the fusion protein or a 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:14. In some embodiments, the cytokine receptor sequence in the fusion protein 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 shown in SEQ ID NO:141. In some embodiments, the cytokine receptor sequence in the fusion protein or a 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 protein 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 shown in SEQ ID NO:142. In some embodiments, the cytokine receptor sequence in the fusion protein or a 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:142. In some embodiments, the cytokine receptor sequence in the fusion protein or a fragment or variant thereof described herein binds TGF-β1 and / or TGF-β3, but does not bind TGF-β2.In certain embodiments, the cytokine receptor sequence in the fusion protein described herein, or a fragment or variant thereof, binds only to TGF-β1. In certain embodiments, the cytokine receptor sequence in the fusion protein described herein, or a fragment or variant thereof, binds only to TGF-β3. In certain embodiments, the cytokine receptor sequence in the fusion protein described herein, or a fragment or variant thereof, binds only to TGF-β1 and / or TGF-β3, but has low affinity or no affinity for TGF-β2.

[0167] 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 portion is fused to TGFβRII or a fragment thereof (e.g., the ECD of TGFβRII) via a connector. 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 connector.

[0168] In some embodiments, the PD-1 antibody fragment or variant is Fab, Fab2, (Fab')2, Fv, (Fv)2, scFv, scFv-Fv of the PD-1 antibody. C 、F C In some embodiments, the PD-1 antibody fragment is a single domain antibody of the PD-1 antibody. In some embodiments, the single domain antibody is a V NAR or V H H fragment.

[0169] Non-limiting exemplary fusion proteins are Figures 4A - 4C In some embodiments, a fusion protein comprising an anti-PD-1 antibody or a fragment or variant thereof fused to a TGF-β trap can induce a synergistic anti-tumor effect due to simultaneous blocking of the interaction between PD-L1 on tumor cells and PD-1 on immune cells and neutralizing TGF-β in the tumor microenvironment. Without being bound by theory, this effect is obtained from the simultaneous blocking of two major immune escape mechanisms and targeted depletion of TGF-β in the tumor microenvironment by a single molecule entity. The depletion is achieved as follows: (1) PD-1 targeting of tumor cells; (2) binding of TGF-β in the tumor microenvironment by the TGF-β trap (e.g., TGFβRII); and (3) destruction of bound TGF-β by PD-L1 receptor-mediated endocytosis.

[0170] In some embodiments, the TGF-β trap (e.g., TGFβRII) is coupled to the heavy chain variable region (V H) Fusion. In other embodiments, the TGF-β trap is fused to the IgG of the PD-1 antibody (e.g., Figure 4A ). In certain aspects, the IgG is IgG1, IgG2, IgG3 or IgG4. In an embodiment, 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, the TGF-β trap (e.g., TGFβRII) is fused via a linker to the heavy chain variable region (V H ) of the PD-1 antibody or its fragment / variant. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused via a linker to the V H constant region of the PD-1 antibody or its fragment / variant. Examples of the V H sequence of the PD-1 antibody or its fragment or variant include but are not limited to SEQ ID NO:1-7 and 149-164. Examples of the V L sequence of the PD-1 antibody or its fragment or variant include but are not limited to SEQ ID NO:8-13 and 148. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused to the light chain variable region (V L ) of the PD-1 antibody or its fragment / variant. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused via a linker to the V L constant region of the PD-1 antibody or its fragment / variant. In some embodiments, the TGF-β trap (e.g., TGFβRII) is fused via a linker to the light chain variable region (V L ) of the PD-1 antibody or its fragment / variant. In one aspect, the TGF-β trap (e.g., TGFβRII) is fused via a linker to the N-terminus or C-terminus of the VL or VH chain or its fragment / variant.

[0171] The terms "anti-PD1 (VL / VH)-TGFβRII" or "anti-PD1 (VH / VL)-TGFβRII" are used interchangeably and denote a specific VL or VH used in the fusion protein. In one embodiment, the term "anti-PD1 (VL / VH)-TGFβRII" or "anti-PD1 (VH / VL)-TGFβRII" refers to a TGF-β trap (e.g., TGFβRII) fused to the heavy chain constant region of anti-PD1, or refers to a TGF-β trap (e.g., TGFβRII) fused to the light chain constant region of anti-PD1.

[0172] In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a Fab of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to a Fab of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a Fab2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to a Fab2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to a (Fab’)2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to a (Fab’)2 of a PD-1 antibody or a fragment / variant thereof. In one aspect, a TGF-β trap (e.g., TGFβRII) is fused via a linker to the N-terminus or C-terminus of a Fab or (Fab’)2 of a PD-1 antibody or a fragment / variant thereof.

[0173] In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to an Fv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to an Fv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to an (Fv)2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to an (Fv)2 of a PD-1 antibody or a fragment / variant thereof. In one aspect, a TGF-β trap (e.g., TGFβRII) is fused via a linker to the N-terminus or C-terminus of an Fv or (Fv)2 of a PD-1 antibody or a fragment / variant thereof.

[0174] In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to an scFv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to an scFv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to an scFv-F C fusion. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to an scFv-F C fusion. In one aspect, a TGF-β trap (e.g., TGFβRII) is fused via a linker to the N-terminus or C-terminus of an scFv or scFv-Fc of a PD-1 antibody or a fragment / variant thereof.

[0175] In some embodiments, a TGF-β trap (such as TGFβRII) is fused to an anti-PD-1 antibody or a fragment / variant thereof F C In some embodiments, a TGF-β trap (such as TGFβRII) is fused via a linker to an anti-PD-1 antibody or a fragment / variant thereof F C In some embodiments, a TGF-β trap (such as TGFβRII) is linked to the C-terminal F of an anti-PD-1 antibody or a fragment / variant thereof C In some embodiments, a TGF-β trap (such as TGFβRII) is linked via a linker to the C-terminal F of an anti-PD-1 antibody or a fragment / variant thereof C In some embodiments, a TGF-β trap (such as TGFβRII) is linked to the N-terminal F of an anti-PD-1 antibody or a fragment / variant thereof C In some embodiments, a TGF-β trap (such as TGFβRII) is linked via a linker to the N-terminal F of an anti-PD-1 antibody or a fragment / variant thereof C linked

[0176] In some embodiments, a TGF-β trap (such as TGFβRII) is fused to a diabody of an anti-PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (such as TGFβRII) is fused via a linker to a diabody of an anti-PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (such as TGFβRII) is fused to a triabody of an anti-PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (such as TGFβRII) is fused via a linker to a triabody of an anti-PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (such as TGFβRII) is fused to a minibody of an anti-PD-1 antibody or a fragment / variant thereof. In some embodiments, a TGF-β trap (such as TGFβRII) is fused via a linker to a minibody of an anti-PD-1 antibody or a fragment / variant thereof. In one aspect, a TGF-β trap (such as TGFβRII) is fused via a linker to the N-terminus or C-terminus of a minibody of an anti-PD-1 antibody or a fragment / variant thereof

[0177] In some embodiments, a TGF-β trap (such as TGFβRII) is fused to the V of an anti-PD-1 antibody or a fragment / variant thereof NAR In some embodiments, a TGF-β trap (such as TGFβRII) is fused via a linker to the V of an anti-PD-1 antibody or a fragment / variant thereof NAR In all of the embodiments described, a TGF-β trap (such as TGFβRII) is fused via a linker to the N-terminus or C-terminus of the V of an anti-PD-1 antibody or a fragment / variant thereof NAR fused

[0178] In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused to the VH of a PD-1 antibody or a fragment / variant thereof. H In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to the VH of a PD-1 antibody or a fragment / variant thereof. H In all of the foregoing embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to the N-terminus or C-terminus of the VH of a PD-1 antibody or a fragment / variant thereof. H

[0179] 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 via a linker 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 at least one extracellular domain of TGFβRII. In some embodiments, the PD-1 antibody portion is fused via a linker to at least one extracellular domain of TGFβRII. Non-limiting exemplary fusion proteins are shown in Figures 4A - 4C

[0180] In some embodiments, the PD-1 antibody fragment is a Fab, Fab2, (Fab’)2, Fv, (Fv)2, scFv, scFv-F C , F C , diabody, triabody, or minibody of the PD-1 antibody. In some embodiments, the PD-1 antibody fragment is a single-domain antibody of the PD-1 antibody. In some embodiments, the single-domain antibody is a VH or VL fragment of the PD-1 antibody. NAR H

[0181] In some embodiments, the heavy-chain variable region (VH) of the PD-1 antibody or a fragment / variant thereof H comprises one or more polypeptide sequences as shown in any one of SEQ ID NOs: 1-7. In some embodiments, the heavy-chain variable region (VH) of the PD-1 antibody or a 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 shown in SEQ ID NOs: 1-7 and 149-164.

[0182] In some embodiments, the heavy chain variable region (V L ) of the PD-1 antibody or its fragment / variant comprises one or more polypeptide sequences as shown in any one of SEQ ID NOs: 8-13 and 148. In some embodiments, the heavy chain variable region (V L ) of the PD-1 antibody or its fragment / variant comprises one or more polypeptide sequences that have 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 with any one of the polypeptide sequences shown in SEQ ID NOs: 8-13 and 148.

[0183] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 6, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO: 12. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) 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 shown in SEQ ID NO: 6, and the light chain variable region (V L ) 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 shown in SEQ ID NO: 12.

[0184] 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 shown 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 shown in SEQ ID NO: 16.

[0185] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:15 and the sequence shown in SEQ ID NO:16.

[0186] 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 shown 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 shown in SEQ ID NO:143.

[0187] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:15 and the sequence shown in SEQ ID NO:143.

[0188] 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 shown 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 shown in SEQ ID NO:294.

[0189] In embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:15 and the sequence shown in SEQ ID NO:294.

[0190] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:7, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:13. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H) 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 shown in SEQ ID NO:7, and the variable region of the light chain (V L ) 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 shown in SEQ ID NO:13.

[0191] 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 shown 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 shown in SEQ ID NO:145.

[0192] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:296 and the sequence shown in SEQ ID NO:145.

[0193] 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 shown 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 shown in SEQ ID NO:144.

[0194] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:296 and the sequence shown in SEQ ID NO:144.

[0195] 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 shown 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 shown in SEQ ID NO: 295.

[0196] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 296 and the sequence shown in SEQ ID NO: 295.

[0197] 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 shown 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 shown in SEQ ID NO: 16.

[0198] 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 shown 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 shown in SEQ ID NO: 143.

[0199] 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 shown 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 shown in SEQ ID NO:145. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:145.

[0200] In some embodiments, the fusion protein comprises a linker and a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:16, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:15. In some embodiments, the fusion protein comprises a linker and a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) 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 shown in SEQ ID NO:16, and the light chain variable region (V L ) 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 shown in SEQ ID NO:15.

[0201] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), wherein the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:1. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), wherein the heavy chain variable region (V His 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 shown in SEQ ID NO:1.

[0202] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:2. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:2.

[0203] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:3. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:3.

[0204] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:4. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:4.

[0205] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H)comprises a polypeptide sequence as shown in SEQ ID NO:5. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:5.

[0206] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:6. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:6.

[0207] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:7. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:7.

[0208] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:5, and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), and this heavy chain variable region (V H) 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 shown in SEQ ID NO:5, and the variable light chain region (V L ) 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 shown in SEQ ID NO:8.

[0209] In some embodiments, the fusion protein comprises a variable heavy chain region (V H ) and a variable light chain region (V L ), the variable heavy chain region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:149, and the variable light chain region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a variable heavy chain region (V H ) and a variable light chain region (V L ), the variable heavy chain region (V H ) 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 shown in SEQ ID NO:149, and the variable light chain region (V L ) 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 shown in SEQ ID NO:8.

[0210] In some embodiments, the fusion protein comprises a variable heavy chain region (V H ) and a variable light chain region (V L ), the variable heavy chain region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:157, and the variable light chain region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a variable heavy chain region (V H ) and a variable light chain region (V L ), the variable heavy chain region (V H) 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 shown in SEQ ID NO:157, and the variable light chain region (V L ) 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 shown in SEQ ID NO:8.

[0211] In some embodiments, the fusion protein comprises a variable heavy chain region (V H ) and a variable light chain region (V L ), the variable heavy chain region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:158, and the variable light chain region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a variable heavy chain region (V H ) and a variable light chain region (V L ), the variable heavy chain region (V H ) 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 shown in SEQ ID NO:158, and the variable light chain region (V L ) 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 shown in SEQ ID NO:8.

[0212] In some embodiments, the fusion protein comprises a variable heavy chain region (V H ), the variable heavy chain region (V H ) comprises the polypeptide sequence as shown in SEQ ID NO:5. In some embodiments, the fusion protein comprises a variable heavy chain region (V H ), the variable heavy chain region (V H ) 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 shown in SEQ ID NO:5.

[0213] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 149. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 149.

[0214] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 157. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 157.

[0215] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 158.

[0216] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V His 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 shown in SEQ ID NO: 158.

[0217] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO: 8. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO: 8.

[0218] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 149. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 149.

[0219] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 150. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 150.

[0220] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H)Comprises a polypeptide sequence as shown in SEQ ID NO: 151. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 151.

[0221] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO: 152. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 152.

[0222] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO: 153. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 153.

[0223] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO: 154. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V His 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 shown in SEQ ID NO: 154.

[0224] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 155. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) 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 shown in SEQ ID NO: 155.

[0225] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 156. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) 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 shown in SEQ ID NO: 156.

[0226] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 157. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) 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 shown in SEQ ID NO: 157.

[0227] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H)comprises a polypeptide sequence as shown in SEQ ID NO:158. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:158.

[0228] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:159. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:159.

[0229] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:160. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:160.

[0230] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:161. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H) 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 shown in SEQ ID NO:161.

[0231] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:162. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:162.

[0232] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:163. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:163.

[0233] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:164. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:164.

[0234] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L) comprises a polypeptide sequence as shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:8.

[0235] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises a polypeptide sequence as shown in SEQ ID NO:9. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:9.

[0236] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises a polypeptide sequence as shown in SEQ ID NO:10. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:10.

[0237] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises a polypeptide sequence as shown in SEQ ID NO:11. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:11.

[0238] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:12. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:12.

[0239] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:13. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:13.

[0240] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:148. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:148.

[0241] In some embodiments, the fusion protein comprises the polypeptide sequence shown 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 shown in SEQ ID NO:15.

[0242] In some embodiments, the fusion protein comprises the polypeptide sequence as 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.

[0243] In some embodiments, the fusion protein comprises the polypeptide sequence as 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.

[0244] In some embodiments, the fusion protein comprises the polypeptide sequence as 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.

[0245] In some embodiments, the fusion protein comprises the polypeptide sequence as 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.

[0246] (ii) anti-TGFβ antibody-fusion protein or a fragment or variant thereof

[0247] In other embodiments, the cytokine trap is an antibody, antibody fragment or antibody variant against TGF-β. In such embodiments, such antibodies can include pan-neutralizing anti-TGFβ antibodies or anti-receptor antibodies that block the binding of the receptor to TGFβ1, 2 and / or 3. In certain embodiments, the antibody fragment or variant is a Fab, Fab2, (Fab’)2, Fv, (Fv)2, scFv, scFv-F C 、F C, diabody, triabody or minibody. 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. Examples of VH sequences of the anti-TGF-β antibody or fragment or variant thereof include but are not limited to SEQ ID NO.166, 168, 169, 171, 173, 175 and 177. Examples of VL sequences of the anti-TGF-β antibody or fragment or variant thereof include but are not limited to SEQ ID NO:165, 167, 170, 172, 174, 176 and 178. In some embodiments, the anti-TGF-β antibody comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) 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 NO:166, 168, 169, 171, 173, 175 or 177. In some embodiments, the anti-TGF-β antibody comprises a light chain variable region (V L ), and the light chain variable region (V L ) 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 NO:165, 167, 170, 172, 174, 176 or 178.

[0248] (iii) TGF-β antagonist peptide-fusion protein or fragment or variant thereof

[0249] In certain embodiments, the cytokine trap may include a TGF-β antagonist peptide or a TGF-β inhibitory peptide. Such peptides can be generated de novo using phage display. In one embodiment, such peptides may be derived from a segment of a TGF-β isoform or a TGF-β receptor. Examples of TGF-β antagonist 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 may be fused via a linker to the V H and / or V L of a PD-1 antibody or a fragment / variant thereof. In another embodiment, one peptide may be fused to a PD-1 antibody or a fragment / variant thereof. In a further embodiment, more than one peptide may be fused to a PD-1 antibody or a fragment / variant thereof. When more than one peptide is used, the peptides may be fused with or without a linker to form a multimer. When more than one peptide is used, the same peptide may be used to form a multimer. Alternatively, a combination of two or more different peptides may be used to form a multimer.

[0250] Adenosine:

[0251] Adenosine is a key immunomodulator in the tumor microenvironment. Extracellular adenosine inhibits inflammatory responses after binding to the A2A adenosine receptor (A2AR), which is the major subtype expressed in most immune cells. Several tumors express high levels of CD39 and CD73, which are extracellular nucleotidases responsible for converting ATP and ADP to AMP and AMP to adenosine, respectively. Thus, adenosine promotes the inhibitory activity of regulatory T cells by inducing the expression of Foxp3, CD39, and CD73. Additionally, hypoxia induces the accumulation of extracellular adenosine in the tumor microenvironment by inducing CD39 and CD73. Additionally, the high levels of extracellular adenosine in the tumor microenvironment are maintained by hypoxia-inducible factor (HIF)-dependent inhibition of the nucleoside transporter ENT-1 and by preventing the repositioning of adenosine into the intracellular space and the inhibition of adenosine kinase, which forms AMP. Thus, targeting the reduction of extracellular adenosine in the tumor microenvironment is understood to enhance immune cell function and promote tumor cell killing.

[0252] In the embodiments provided herein, a fusion protein comprising adenosine deaminase (such as ADA2) is used to target the reduction of extracellular adenosine in the tumor microenvironment.

[0253] Adenosine deaminase

[0254] Adenosine deaminase (also known as adenosine aminohydrolase, or ADA) irreversibly deaminates adenosine, converting it to the related nucleoside inosine by substituting an amino group with a keto group. Inosine can then be de-ribosylated (removed from ribose) by another enzyme called purine nucleoside phosphorylase (PNP), converting it to hypoxanthine. The breakdown of adenosine from food and the turnover of nucleic acids in tissues require ADA. Its main function in humans is the development and maintenance of the immune system. However, ADA has also been found to be associated with epithelial cell differentiation, neurotransmission, and pregnancy maintenance. There are 2 isoforms of ADA: ADA1 and ADA2.

[0255] ADA1 is found in most body cells, especially lymphocytes and macrophages, where it exists not only in the cytosol and nucleus but also in an extracellular form attached to dipeptidyl peptidase-4 (also known as CD26) on the cell membrane. ADA1 is mainly involved in intracellular activities and exists in two forms: a small form (monomer) and a large form (dimer). The conversion from the small to the large form is regulated by a "conversion factor" in the lung.

[0256] ADA2 was first identified in the human spleen. It was subsequently found in other tissues, including macrophages, where it coexists with ADA1. These two isoforms regulate the ratio of adenosine to deoxyadenosine. ADA2 is mainly present in human plasma and serum and exists mainly in the form of a homodimer. ADA2 is the main form present in human plasma and is increased in many diseases, especially those related to the immune system: for example, rheumatoid arthritis, psoriasis, and sarcoidosis. In most cancers, the plasma ADA2 isoform is also increased. ADA2 is not ubiquitous but coexists with ADA1 in monocytes-macrophages.

[0257] ADA2 fusion protein or its fragments or variants

[0258] The present disclosure provides a fusion protein or a fragment or variant thereof, which comprises an immune checkpoint inhibitor, such as a PD-1 inhibitor or an antibody, and an adenosine deaminase (such as ADA2) that can neutralize adenosine. The fusion protein or a fragment or variant thereof provided herein (e.g., a PD-1 inhibitor or an antibody fused with an adenosine deaminase (such as ADA2)) can elicit a synergistic anti-tumor effect due to simultaneously blocking the interaction between, for example, PD-L1 on tumor cells and PD-1 on immune cells, and neutralizing adenosine in, for example, the tumor microenvironment. Without being bound by theory, this effect is obtained from simultaneously blocking two major immune escape mechanisms and the targeted depletion of adenosine in the tumor microenvironment by a single molecular entity. The depletion can be achieved by one or more of the following: (1) anti-PD-1 targeting of tumor cells; (2) binding of adenosine deaminase (such as ADA2) to adenosine in the tumor microenvironment; and (3) disruption of the bound adenosine by PD-L1 receptor-mediated endocytosis.

[0259] In some embodiments, the adenosine deaminase (such as ADA2) is part of a fusion protein or a fragment or variant thereof that further comprises a PD-1 inhibitor or an antibody or a fragment or variant thereof. In some embodiments, it is a fusion protein or a fragment or variant thereof that comprises an adenosine deaminase (such as ADA2) optionally fused to a 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 protein described herein comprises the adenosine deaminase (such as ADA2) described herein or a functional variant or derivative thereof. Examples of ADA2 and variants are described in WO 2016061286, 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 protein or a fragment or variant thereof provided herein comprises a PD-1 inhibitor or an antibody fused to an ADA protein or a functional fragment thereof via a linker. In some embodiments, the PD-1 inhibitor can be an antibody targeting PD-1 or a fragment or variant of the antibody.

[0260] 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 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: 279.

[0261] In some embodiments, the fusion protein or fragment or variant thereof described herein comprises an adenosine deaminase (such as ADA2) as described above and an antibody targeting an immune checkpoint gene, a fragment or variant of the antibody. In some embodiments, an antibody targeting an immune checkpoint such as cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and programmed cell death-1 (PD-1), or a fragment or variant of the antibody, can be fused to an adenosine deaminase (such as ADA2) via a linker.

[0262] In some embodiments, the PD-1 antibody portion is fused to an adenosine deaminase (such as ADA2) or a fragment thereof. In some embodiments, the PD-1 antibody portion is fused to an adenosine deaminase (such as 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.

[0263] In some embodiments, the PD-1 antibody fragment or variant is a Fab, Fab2, (Fab’)2, Fv, (Fv)2, scFv, scFv-F C 、F C 、diabody, triabody or minibody of the PD-1 antibody. In some embodiments, the PD-1 antibody fragment is a single-domain antibody of the PD-1 antibody. In some embodiments, the single-domain antibody is a VH or VL fragment of the PD-1 antibody. NAR or V H H fragment.

[0264] Non-limiting exemplary fusion proteins are shown in Figures 33A - 33C In some embodiments, a fusion protein comprising an anti-PD-1 antibody or a fragment or variant thereof fused to an adenosine deaminase (such as ADA2) can elicit a synergistic anti-tumor effect by simultaneously blocking the interaction between PD-L1 on tumor cells and PD-1 on immune cells and targeting the reduction of extracellular adenosine in the tumor microenvironment.

[0265] In some embodiments, the adenosine deaminase (such as ADA2) is fused to the variable heavy chain (VH) of the PD-1 antibody or its fragment / variant H) Fusion. 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 an embodiment, 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 via a linker to the variable region of the heavy chain (V H ) Fusion. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the V H of a PD-1 antibody or a fragment / variant thereof. L ) Fusion. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the variable region of the light chain (V L ) of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the variable region of the light chain (V L ) of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the V H or V L of a PD-1 antibody or a fragment / variant thereof at the N-terminus or C-terminus.

[0266] In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the Fab of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the Fab of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the Fab2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the Fab2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the (Fab’)2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the (Fab’)2 of a PD-1 antibody or a fragment / variant thereof. In one aspect, adenosine deaminase is fused via a linker to the N-terminus or C-terminus of the Fab or Fab2 of a PD-1 antibody or a fragment / variant thereof.

[0267] In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the Fv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the Fv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the (Fv)2 of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the (Fv)2 of a PD-1 antibody or a fragment / variant thereof. In one aspect, adenosine deaminase is fused via a linker to the N-terminus or C-terminus of the Fv or (Fv)2 of a PD-1 antibody or a fragment / variant thereof.

[0268] In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the scFv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the scFv of a PD-1 antibody or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the scFv-F of a PD-1 antibody or a fragment / variant thereof. C fusion. In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the scFv-F of a PD-1 antibody or a fragment / variant thereof. C fusion. In one aspect, adenosine deaminase is fused via a linker to the N-terminus or C-terminus of the scFv or scFv-F of a PD-1 antibody or a fragment / variant thereof. C

[0269] In some embodiments, adenosine deaminase (e.g., ADA2) is fused to the F of a PD-1 antibody or a fragment / variant thereof. C In some embodiments, adenosine deaminase (e.g., ADA2) is fused via a linker to the F of a PD-1 antibody or a fragment / variant thereof. C In some embodiments, adenosine deaminase (e.g., ADA2) is linked to the C-terminal F of a PD-1 antibody or a fragment / variant thereof. C In some embodiments, adenosine deaminase (e.g., ADA2) is linked via a linker to the C-terminal F of a PD-1 antibody or a fragment / variant thereof. C In some embodiments, adenosine deaminase (e.g., ADA2) is linked to the N-terminal F of a PD-1 antibody or a fragment / variant thereof. C In some embodiments, adenosine deaminase (e.g., ADA2) is linked via a linker to the N-terminal F of a PD-1 antibody or a fragment / variant thereof. C connection.

[0270] In some embodiments, adenosine deaminase (such as ADA2) is fused to a bispecific antibody of a PD-1 antibody or its fragment / variant. In some embodiments, adenosine deaminase (such as ADA2) is fused to a bispecific antibody of a PD-1 antibody or its fragment / variant via a linker. In some embodiments, adenosine deaminase (such as ADA2) is fused to a trispecific antibody of a PD-1 antibody or its fragment / variant. In some embodiments, adenosine deaminase (such as ADA2) is fused to a trispecific antibody of a PD-1 antibody or its fragment / variant via a linker. In some embodiments, adenosine deaminase (such as ADA2) is fused to a minibody of a PD-1 antibody or its fragment / variant. In some embodiments, adenosine deaminase (such as ADA2) is fused to a minibody of a PD-1 antibody or its fragment / variant via a linker.

[0271] In some embodiments, adenosine deaminase (such as ADA2) is fused to the V NAR of a PD-1 antibody or its fragment / variant. In some embodiments, adenosine deaminase (such as ADA2) is fused to the V NAR of a PD-1 antibody or its fragment / variant via a linker. In one aspect, adenosine deaminase is fused to the N-terminus or C-terminus of the V NAR of a PD-1 antibody or its fragment / variant via a linker.

[0272] In some embodiments, adenosine deaminase (such as ADA2) is fused to the V H H of a PD-1 antibody or its fragment / variant. In some embodiments, adenosine deaminase (such as ADA2) is fused to the V H H of a PD-1 antibody or its fragment / variant via a linker. In one aspect, adenosine deaminase is fused to the N-terminus or C-terminus of the V H H of a PD-1 antibody or its fragment / variant via a linker.

[0273] In some embodiments, the heavy chain variable region (V H ) of a PD-1 antibody or its fragment / variant comprises one or more polypeptide sequences as shown in any one of SEQ ID NO: 1-7. In some embodiments, the heavy chain variable region (V H ) of a PD-1 antibody or its fragment / variant comprises one or more polypeptide sequences that have 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 shown in SEQ ID NO: 1-7 and 149-164.

[0274] In some embodiments, the heavy chain variable region (V L ) of the PD-1 antibody or its fragment / variant comprises one or more polypeptide sequences as shown in any one of SEQ ID NOs: 8-13 and 148. In some embodiments, the heavy chain variable region (V L ) of the PD-1 antibody or its fragment / variant comprises one or more polypeptide sequences that have 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 with any one of the polypeptide sequences shown in SEQ ID NOs: 8-13 and 148.

[0275] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 6, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO: 12. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) 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 shown in SEQ ID NO: 6, and the light chain variable region (V L ) 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 shown in SEQ ID NO: 12.

[0276] 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 shown 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 shown in SEQ ID NO: 280.

[0277] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:12 and the sequence shown in SEQ ID NO:280.

[0278] 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 shown 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 shown in SEQ ID NO:281.

[0279] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:12 and the sequence shown in SEQ ID NO:281.

[0280] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:7, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:13. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) 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 shown in SEQ ID NO:7, and the light chain variable region (V L ) 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 shown in SEQ ID NO:13.

[0281] 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 shown 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 shown in SEQ ID NO:282.

[0282] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:13 and the sequence shown in SEQ ID NO:282.

[0283] 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 shown 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 shown in SEQ ID NO:283.

[0284] In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:13 and the sequence shown in SEQ ID NO:283.

[0285] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) comprises the polypeptide sequence as shown in SEQ ID NO: 1. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) 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.

[0286] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H) comprises a polypeptide sequence as shown in SEQ ID NO:2. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:2.

[0287] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:3. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:3.

[0288] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:4. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:4.

[0289] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:5. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:5.

[0290] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:6. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:6.

[0291] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:7. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:7.

[0292] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:149. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:149.

[0293] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:150. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H) 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 shown in SEQ ID NO:150.

[0294] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:151. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:151.

[0295] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:152. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:152.

[0296] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:153. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:153.

[0297] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H)comprises a polypeptide sequence as shown in SEQ ID NO: 154. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 154.

[0298] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO: 155. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 155.

[0299] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO: 156. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 156.

[0300] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO: 157. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H) 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 shown in SEQ ID NO: 157.

[0301] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 158. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 158.

[0302] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 159. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 159.

[0303] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 160. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO: 160.

[0304] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H)comprises a polypeptide sequence as shown in SEQ ID NO:161. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:161.

[0305] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:162. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:162.

[0306] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:163. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) 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 shown in SEQ ID NO:163.

[0307] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V H ) comprises a polypeptide sequence as shown in SEQ ID NO:164. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and this heavy chain variable region (V His 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 shown in SEQ ID NO: 164.

[0308] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO: 8. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO: 8.

[0309] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO: 9. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO: 9.

[0310] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO: 10. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO: 10.

[0311] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L)comprises a polypeptide sequence as shown in SEQ ID NO:11. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:11.

[0312] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises a polypeptide sequence as shown in SEQ ID NO:12. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:12.

[0313] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises a polypeptide sequence as shown in SEQ ID NO:13. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:13.

[0314] In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) comprises a polypeptide sequence as shown in SEQ ID NO:148. In some embodiments, the fusion protein comprises a light chain variable region (V L ), and this light chain variable region (V L ) 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 shown in SEQ ID NO:148.

[0315] In some embodiments, the fusion protein comprises the polypeptide sequence shown 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 shown in SEQ ID NO:15.

[0316] In some embodiments, the fusion protein comprises the polypeptide sequence shown 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 shown in SEQ ID NO:280.

[0317] In some embodiments, the fusion protein comprises the polypeptide sequence shown 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 shown in SEQ ID NO:281.

[0318] In some embodiments, the fusion protein comprises the polypeptide sequence shown 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 shown in SEQ ID NO:282.

[0319] In some embodiments, the fusion protein comprises the polypeptide sequence shown 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 shown in SEQ ID NO:283.

[0320] In some embodiments, the fusion protein comprises a heavy chain variable region (V H) and a light chain variable region (V L ), the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:5, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), the heavy chain variable region (V H ) 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 shown in SEQ ID NO:5, and the light chain variable region (V L ) 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 shown in SEQ ID NO:8.

[0321] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:149, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), the heavy chain variable region (V H ) 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 shown in SEQ ID NO:149, and the light chain variable region (V L ) 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 shown in SEQ ID NO:8.

[0322] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), the heavy chain variable region (VH ) comprises the polypeptide sequence shown in SEQ ID NO:157, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), and the heavy chain variable region (V H ) 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 shown in SEQ ID NO:157, and the light chain variable region (V L ) 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 shown in SEQ ID NO:8.

[0323] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), and the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:158, and the light chain variable region (V L ) comprises the polypeptide sequence shown in SEQ ID NO:8. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), and the heavy chain variable region (V H ) 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 shown in SEQ ID NO:158, and the light chain variable region (V L ) 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 shown in SEQ ID NO:8.

[0324] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), and the heavy chain variable region (V H ) comprises the polypeptide sequence as shown in SEQ ID NO:5. In some embodiments, the fusion protein comprises a heavy chain variable region (VH )), the heavy chain variable region (V H ) 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 shown in SEQ ID NO:5.

[0325] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:149. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) 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 shown in SEQ ID NO:149.

[0326] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:157. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) 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 shown in SEQ ID NO:157.

[0327] In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) comprises the polypeptide sequence shown in SEQ ID NO:158. In some embodiments, the fusion protein comprises a heavy chain variable region (V H ), the heavy chain variable region (V H ) 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 shown in SEQ ID NO:158.

[0328] In some embodiments, the fusion protein comprises a heavy chain variable region (VH ), the variable region of the heavy chain (V H ) comprises the polypeptide sequence shown in SEQ ID NO: 158. In some embodiments, the fusion protein comprises a variable region of the heavy chain (V H ), the variable region of the heavy chain (V H ) 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 shown in SEQ ID NO: 158.

[0329] In some embodiments, the fusion protein comprises a variable region of the light chain (V L ), the variable region of the light chain (V L ) comprises the polypeptide sequence shown in SEQ ID NO: 8. In some embodiments, the fusion protein comprises a variable region of the light chain (V L ), the variable region of the light chain (V L ) 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 shown in SEQ ID NO: 8.

[0330] Linker

[0331] In some embodiments, the linker comprises one or more polypeptide sequences shown in any of SEQ ID NOs: 17 - 34. In some embodiments, the linker can be a flexible linker. A flexible linker can be applied when the domains being linked require a degree of movement or interaction. Flexible linkers can be composed of small non - polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. A flexible linker can have a sequence consisting mainly of segments of Gly and Ser residues ("GS" linker). Non - limiting examples of flexible linkers can 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 the exemplary GS linker can be optimized to achieve proper isolation of functional domains or to maintain essential inter - domain interactions. In addition to GS linkers, other flexible linkers can also be used for recombinant fusion proteins. In some embodiments, the flexible linker can have the sequence (Gly)n, where n can be 6, 7, or 8. In some cases, the flexible linker can also be rich in small amino acids 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 linker described herein can be a rigid linker. Rigid linkers can be used to maintain a fixed distance between domains of the fusion proteins or fragments or variants thereof described herein. Non - limiting examples of rigid linkers can be: α - helix - forming linkers, Pro - rich sequences, (XP)n, X - Pro scaffolds, (EAAAK)n (n = 1 - 6). In some cases, rigid linkers can exhibit a relatively rigid structure by adopting an α - helix structure or by containing multiple Pro residues. In some embodiments, an immune checkpoint inhibitor, such as a PD - 1 inhibitor, and a cytokine trap (e.g., a TGF - β trap) that can neutralize a cytokine (e.g., TGF - β) in the fusion protein or fragment or variant thereof described herein can be separated by an intervening sequence encoding an intervening linker polypeptide. In some embodiments, an immune checkpoint inhibitor, such as a PD - 1 inhibitor, and ADA2 (or a mutant thereof) in the 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 comprises the sequences disclosed in the following table:

[0332] Table 1. Linker Amino Acid Sequences and Polynucleotide Sequences

[0333]

[0334]

[0335] In some embodiments, the linker can be a flexible linker, a rigid linker, an in vivo cleavable linker, or any combination thereof. In some cases, the linker can connect functional domains together (as in flexible and rigid linkers) or release free functional domains in vivo, as in an in vivo cleavable linker. In some embodiments, the linker can improve bioactivity, increase expression yield, and achieve an ideal pharmacokinetic profile. In some embodiments, the linker can also comprise a hydrazone, a peptide, a disulfide bond, or a thioester.

[0336] In some cases, the linker sequences described herein can include flexible linkers. Flexible linkers can be applied when the domains being linked require a degree of movement or interaction. Flexible linkers can be composed of small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Flexible linkers can have sequences consisting primarily of segments of Gly and Ser residues (“GS” linkers). Examples of flexible linkers can have the sequence (G4S)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the flexible linker can have the sequence (Gly)n, where n can be 6, 7, or 8. 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 increase solubility. By adjusting the copy number “n”, the length of these non-limiting exemplary linkers can be optimized to achieve proper separation of functional domains or to maintain necessary inter-domain interactions. In addition to GS linkers, other flexible linkers can also be used for 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 increase solubility.

[0337] The flexible linkers included in the linker sequences described herein can be rich in small or polar amino acids, such as Gly and Ser, to provide good flexibility and solubility. Flexible linkers can be a suitable choice when the fusion protein domains or fragments or variants thereof require specific movement or interaction. Additionally, although flexible linkers do not have a rigid structure, they can be used as inert linkers to maintain the distance between functional domains. The length of the flexible linker can be adjusted to allow proper folding or to achieve optimal bioactivity of the fusion protein or fragments or variants thereof.

[0338] In some cases, the linker described herein may further comprise a rigid linker. The rigid linker can be used to maintain a fixed distance between domains of the fusion protein or its fragments or variants described herein. Examples of rigid linkers can be, by way of example, α-helix forming linkers, Pro-rich sequences, (XP)n, X-Pro scaffolds, (EAAAK)n (n = 1-6). In some cases, a rigid linker can exhibit a relatively rigid structure by adopting an α-helical structure or by containing multiple Pro residues.

[0339] In some embodiments, the linker described herein can be a cleavable linker. In other cases, the linker is non-cleavable. A non-cleavable linker can covalently join the functional domains of a fusion protein or its fragments or variants to act as one molecule during an in vivo or in vitro process. The linker can also be in vivo cleavable. A cleavable linker can be introduced to release free functional domains in vivo. By way of example, a cleavable linker can be cleaved by a reducing reagent, the presence of a protease. For example, cleavage can be generated by the reduction of a disulfide bond. In the case of a disulfide bond linker, cleavage can be generated by a cleavage event of disulfide exchange with a thiol such as glutathione. In other cases, in vivo cleavage of the linker in a recombinant fusion protein can also be carried out by a protease that is expressed in vivo or confined to certain cellular compartments in a particular cell or tissue under pathological conditions such as cancer or inflammation. In some cases, a cleavable linker can allow targeted cleavage. For example, the specificity of many proteases can provide slower linker cleavage in a confined compartment. A cleavable linker can also contain a hydrazone, a peptide, a disulfide bond or a thioester. For example, a hydrazone can confer serum stability. In other cases, a hydrazone can allow cleavage in an acidic compartment. The acidic compartment can have a pH of up to 7. The linker can also include a thioether. The thioether can be non-reducible. The thioether can be designed for intracellular proteolytic degradation.

[0340] In some cases, the linker can be an engineered linker. For example, the linker can be designed to incorporate chemical properties such as hydrophobicity. In some cases, at least two different linker polypeptide sequences can encode the same polypeptide linker sequence. The method of designing the linker can be computational. In some cases, the computational method can include graphical techniques. Computational methods can be used to search a library of three-dimensional peptide structures derived from a database for suitable peptides. For example, the Brookhaven Protein Data Bank (PDB) can be used to span the spatial distance between selected amino acids across the linker. In some cases, the polypeptide linker can also include one or more GS linker sequences, such as (G4S)n, (GS)n, (SG)n, (GSG)n, and (SGSG)n, where n can be any number from zero to fifteen.

[0341] Method for treating cancer

[0342] The present disclosure also provides a method 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) that can neutralize a cytokine (e.g., TGF-β). The present disclosure also provides a method 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 that target and block 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 they have shown durable clinical activity in a variety of 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, the response rates remain extremely low, and a number of patients have developed resistance, leading to disease progression. In addition, checkpoint inhibitors have failed to show any substantial clinical response in a number of indications such as ovarian cancer, gastroesophageal cancer, prostate cancer, pancreatic cancer, and many other cancers.

[0343] Failure of checkpoint blockade can be attributed to the complexity of immunosuppressive factors present in the tumor microenvironment. These factors can include, but are not limited to, inhibitory cells such as myeloid-derived suppressor cells, tumor-associated macrophages (TAM); inhibitory cytokines and growth factors such as TGF-β and interleukin 10 (IL-10); and metabolic derivatives such as adenosine and indoleamine 2,3-dioxygenase (IDO) by-products. The anti-PD1-TGFβRII fusion proteins provided herein are examples of therapies that can target two negative inhibitory pathways in the tumor microenvironment. These pathways can include tumor cell-mediated cell-intrinsic interactions, in which the PD-1 / PD-L1 interaction can play a major role, and immunosuppressive cytokine-mediated cell-extrinsic interactions, of which TGF-β may be a major member.

[0344] 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. Additionally, the fusion proteins described herein can be applicable to indications such as in non-small cell carcinoma (NSCL) and melanoma, in which the response rate to checkpoint blockade is low and TGF-β is highly expressed.

[0345] Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases (such as alpha-chain disease, gamma-chain disease, and mu-chain disease), benign monoclonal gammopathy, and immunocyte amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (such as metastatic, 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, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancers of the hematopoietic tissues, etc. Other non-limiting examples of cancer types applicable to the methods covered by the present disclosure include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, 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, bronchial cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, liver cancer, hepatocellular carcinoma (HCC), choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain diseases. 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, kidney 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 (such as serous ovarian cancer), or breast cancer.Epithelial cancers can be characterized in various other ways, including but not limited to serous, endometrioid, mucinous, clear cell, Brenner type, or undifferentiated. In some embodiments, the present disclosure is for the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including but not limited to mantle cell lymphoma.

[0346] In certain embodiments, the anti-PD1-TGFβRII fusion proteins provided herein are examples of therapies useful for treating cancers having 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, chemoimmunotherapy, and current clinical trial therapies. In certain embodiments, the anti-PD1-ADA2 fusion proteins provided herein are examples of therapies useful for treating cancers having 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, chemoimmunotherapy, and current clinical trial therapies. Such cancers include but are not limited to Hodgkin lymphoma, melanoma, non-small cell lung cancer (NSCLC), microsatellite instability (MSI) high 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, prostate cancer.

[0347] Combination therapy

[0348] In some embodiments, the fusion protein is administered as part of a combination therapy with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a biomolecule, such as an antibody. For example, the treatment may involve co-administering the fusion protein with an antibody against a tumor-associated antigen (including but not limited to antibodies that bind 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 acts as an angiogenesis inhibitor (e.g., an anti-VEGF or 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, the treatment may involve co-administering the fusion protein or a fragment or variant thereof provided herein with a small molecule that acts as an inhibitor against a tumor-associated antigen (including but not limited to EGFR, HER2 (ErbB2), and / or VEGF). In some embodiments, the fusion protein or a fragment or variant thereof is co-administered with a protein kinase inhibitor selected from the group consisting of gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, 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 a chemotherapy or other inhibitor that reduces the number of T REG cells. In 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.

[0349] 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 shown durable anti-tumor activity and prolonged survival in participants with advanced melanoma and thus received approval from the US 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.

[0350] 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, colony stimulating factors, interleukins, antibodies that block immunosuppressive functions (e.g., anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-PD-L1 antibody, anti-TIGIT antibody), antibodies that enhance immune cell function (e.g., anti-GITR antibody, anti-OX-40 antibody, anti-CD40 antibody or anti-4-1BB antibody), toll-like receptors (e.g., TLR4, TLR7, TLR9), soluble ligands (e.g., GITRL, GITRL-Fc, OX-40L, OX-40L-Fc, CD40L, CD40L-Fc, 4-1BB ligand or 4-1BB ligand-Fc) or B7 family members (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.

[0351] 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 BMS935559 (MDX-1105), atezolizumab (MPDL3280A), durvalumab (MEDI4736), and avelumab (MSB0010718C). In some embodiments, the additional therapeutic agent is an anti-CTLA-4 antibody selected from ipilimumab (YERVOY) and tremelimumab. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from BMS-986016 and LAG525. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody selected from MEDI6469, MEDI0562, and MOXR0916. In some embodiments, the additional therapeutic agent is an anti-4-1BB antibody selected from PF-05082566. In some embodiments, the fusion protein or fragment or variant thereof can be co-administered 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, neurotrophin, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, PlGF, γ-IFN, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

[0352] Cytokine

[0353] In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variants or combinations thereof. In some cases, the cytokine is an interleukin. In some cases, 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 thereof. In some embodiments, the cytokine can be membrane-bound or secreted. In an embodiment, the cytokine is soluble IL-15, soluble IL-15 / IL-15Rα complex (e.g., ALT-803). In certain cases, the interleukin can comprise membrane-bound IL-15 (mbIL-15) or a fusion of IL-15 with IL-15Rα. In some embodiments, mbIL-15 is a membrane-bound chimeric IL-15, which can be co-expressed with the modified immune effector cells described herein. In some embodiments, the mbIL-15 comprises full-length IL-15 (e.g., native IL-15 polypeptide) or a fragment or variant thereof fused in-frame with full-length IL-15Rα, a fragment, or a variant thereof. In some cases, the IL-15 is indirectly linked to IL-15Rα via a linker. In some cases, the mbIL-15 is as described by 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.

[0354] In some embodiments, mbIL-15 is expressed together 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.

[0355] 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 gene-switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible bipartite polypeptide ecdysone receptor-based gene switch, such as gene switch.

[0356] 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, embedded IL-12. In some cases, IL-12 variants are described in WO2015 / 095249, WO2016 / 048903, WO2017 / 062953, which are hereby incorporated by reference in their entirety. In some embodiments, the cytokines described above can be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene-switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible bipartite polypeptide ecdysone receptor-based gene switch, such as gene switch.

[0357] In some embodiments, the fusion proteins as described herein can be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene-switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible bipartite polypeptide ecdysone receptor-based gene switch, such as gene switch.

[0358] gene switch

[0359] The present disclosure provides gene-switch polypeptides, polynucleotides encoding ligand-inducible gene-switch polypeptides, and methods and systems comprising such polypeptides and / or polynucleotides. The term "gene switch" refers to a response element associated with a promoter, such as a combination with an ecdysone receptor (EcR)-based system, which regulates the expression of a gene incorporating the response element and the promoter in the presence of one or more ligands. Tightly regulated inducible gene expression systems or gene switches can be used in a variety of applications, such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, functional genomics, and the regulation of traits in transgenic plants and animals. Such inducible gene expression systems can include ligand-inducible heterologous gene expression systems.

[0360] Early forms of EcR-based gene switches used Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) polypeptides and showed that these receptors transactivated 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 non-steroidal ecdysone agonist tebufenozide induced high-level transactivation of reporter genes in mammalian cells through Bombyx mori EcR (BmEcR) in the absence of exogenous heterodimeric partners.

[0361] International Patent Applications PCT / US97 / 05330 (WO 97 / 38117) and PCT / US99 / 08381 (WO99 / 58155) disclose methods for regulating the expression of exogenous genes 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, in the presence of its ligand, optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression. In this instance, the ecdysone receptor was isolated from Drosophila melanogaster. Typically, such systems require the presence of a silent partner, preferably the retinoid X receptor (RXR), to provide optimal activation. In mammalian cells, the insect ecdysone receptor (EcR) is capable of heterodimerizing with the mammalian retinoid X receptor (RXR) and thus used to regulate the expression of target or heterologous genes in a ligand-dependent manner. International Patent Application PCT / US98 / 14215 (WO 99 / 02683) discloses that an ecdysone receptor isolated from the silkworm moth Bombyx mori is functional in mammalian systems and does not require an exogenous dimeric partner.

[0362] U.S. Patent 6,265,173 discloses that individual 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 USP dimerization domain for use in gene expression systems. U.S. Patent 5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system for use in plants in which the transactivation domain and the DNA binding domain are located on two different hybrid proteins. In each of these cases, the transactivation domain and the DNA binding domain (such as the native EcR in International Patent Application PCT / US98 / 14215 or the modified EcR in International Patent Application PCT / US97 / 05330) are incorporated into a single molecule and the other heterodimeric partner (USP or RXR) is used in its native state.

[0363] International Patent Application PCT / US01 / 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 placing them on two different proteins, resulting in a greatly reduced background activity in the absence of ligand, while in the presence of ligand, the activity is significantly increased compared to the background. Compared with the two systems disclosed in Applications PCT / US97 / 05330 and PCT / US98 / 14215, this two-hybrid system is a significantly improved inducible gene expression regulatory system. It is believed that this two-hybrid system utilizes the following ability of a pair of interacting proteins: they place the transcriptional activation domain in a more favorable position relative to the DNA-binding domain, such that when the DNA-binding domain binds to the DNA-binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Patent 5,283,173). This two-hybrid gene expression system comprises two gene expression cassettes; the first encodes a DNA-binding domain fused to a nuclear receptor polypeptide, and the second encodes a transactivation domain fused to a different nuclear receptor polypeptide. In the presence of ligand, it is believed that a conformational change is induced, which promotes the interaction of the antibody with the TGF-β cytokine trap, resulting in the dimerization of the DNA-binding domain and the transactivation domain. Since the DNA-binding domain and the transactivation domain are located on two different molecules, the background activity is greatly reduced in the absence of ligand.

[0364] Certain modifications of the two-hybrid system can also provide improved sensitivity to non-steroid ligands (e.g., diacylhydrazine) compared to steroid ligands (e.g., ponasterone A ("PonA") or muristerone A ("MurA")). That is, non-steroid ligands provide higher gene transcription activity at lower ligand concentrations compared to steroids. In addition, the two-hybrid system avoids some side effects due to overexpression of RXR that may occur when using unmodified RXR as a switch partner. In a preferred two-hybrid system, the native DNA-binding domain and transactivation domain of EcR or RXR are eliminated, and thus, these hybrid molecules have less opportunity to interact with other steroid hormone receptors present in the cell, resulting in reduced side effects.

[0365] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily and is classified into subfamily 1, group H (referred to herein as "group H nuclear receptors"). Members of each group have 40-60% amino acid identity in 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 β (LXRβ), steroid hormone receptor-like protein (RLD-1), liver X receptor (LXR), liver X receptor (LXRα), farnesoid X receptor (FXR), receptor interacting protein 14 (RIP-14), and heme receptor (HRR-1).

[0366] In some cases, an inducible promoter ("IP") can be a small molecule ligand-inducible gene switch based on a dual polypeptide ecdysone receptor, such as that of Intrexon Corporation Gene switches. In some cases, the gene switch can be selected from ecdysone-based receptor components, such as those described in any of the systems described in but not limited to the following: PCT / US2001 / 009050 (WO 2001 / 070816); U.S. Patent 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT / US2001 / 030608 (WO 2002 / 029075); U.S. Patent 8,105,825; 8,168,426; PCT / US52002 / 005235 (WO 2002 / 066613); U.S. Application 10 / 468,200 (U.S. Publication 20120167239); PCT / US2002 / 005706 (WO 2002 / 066614); U.S. Patent 7,531,326; 8,236,556; 8,598,409; PCT / U52002 / 005090 (WO 2002 / 066612); U.S. Patent 8,715,959 (U.S. Publication 20060100416); PCT / US2002 / 005234 (WO 2003 / 027266); U.S. Patent 7,601,508; 7,829,676; 7,919,269; 8,030,067; PCT / U52002 / 005708 (WO 2002 / 066615); U.S. Application 10 / 468,192 (U.S. Publication 20110212528); PCT / US2002 / 005026 (WO 2003 / 027289); U.S. Patent 7,563,879; 8,021,878; 8,497,093; PCT / US2005 / 015089 (WO 2005 / 108617); U.S. Patent 7,935,510; 8,076,454; PCT / U52008 / 011270 (WO 2009 / 045370); U.S. Application 12 / 241,018 (U.S. Publication 20090136465); PCT / US2008 / 011563 (WO 2009 / 048560); U.S. Application 12 / 247,738 (U.S. Publication 20090123441); PCT / US2009 / 005510 (WO 2010 / 042189); U.S. Application 13 / 123,129 (U.S. Publication 20110268766); PCT / US2011 / 029682 (WO 2011 / 119773); U.S. Application 13 / 636,473 (U.S. Publication 20130195800); PCT / US2012 / 027515 (WO 2012 / 122025); WO 2018 / 132494 (PCT / US2018 / 013196);and U.S. Patent No. 9,402,919; each of which is incorporated herein by reference in its entirety.;

[0367] As used herein, the term "ligand" applied to a ligand-activated ecdysone receptor-based gene switch is a small molecule (such as a diacylhydrazine compound) with different solubilities, which has the ability to activate the gene switch to stimulate gene expression (i.e., where ligand-induced expression of a polynucleotide (e.g., mRNA, miRNA, etc.) and / or polypeptide is provided). Examples of such ligands include, but are not limited to, those described in: WO 2004 / 072254 (PCT / US2004 / 003775); WO 2004 / 005478 (PCT / US2003 / 021149); WO 2005 / 017126 (PCT / US2004 / 005149); WO 2004 / 078924 (PCT / US2004 / 005912); WO 2008 / 153801 (PCT / US2008 / 006757); WO 2009 / 114201 (PCT / US2009 / 001639); WO 2013 / 036758 (PCT / US2012 / 054141); WO 2014 / 144380 (PCT / US2014 / 028768); and WO 2016 / 044390 (PCT / US2015 / 050375); each of which is incorporated herein by reference in its entirety.

[0368] Examples of ligands also include, but are not limited to: ecdysteroids such as ecdysone, 20-hydroxyecdysone, pterosterone A, muristerone A, etc., 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazines such as those disclosed in U.S. Pat. Nos. 6,258,603, 6,013,836, 5,117,057, 5,530,028, 5,378,726, 7,304,161, 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; oxadiazolines such as those described in U.S. Pat. Nos. 8,669,072 and 8,895,306; dibenzoylalkylcyanohydrazines such as those disclosed in European Application 2,461,809; N-alkyl-N,N'-diaroylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application 234,994; N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S. Pat. No. 4,985,461; aminoketones such as those described in U.S. Pat. Nos. 7,375,093, 8,129,355, and 9,802,936, each of which is incorporated herein by reference, and other similar substances including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterols, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-α-6-α-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, farnesol (framesol), bile acids, 1,1-diphosphonates, juvenile hormone III, etc. 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, for example, WO 2008 / 153801 (PCT / US2008 / 006757) and WO 2013 / 036758 (PCT / US2012 / 054141), both of which are incorporated herein by reference in their entirety.

[0369] For example, ligands for the ecdysone receptor-based genetic switch can be selected from any suitable ligand. Naturally occurring ecdysone or ecdysone analogs (such as 20-hydroxyecdysone, muristerone A, cyasterone A, cyasterone B, cyasterone C, 26-iodocyasterone A, inokosterone, or 26-methylsulfonyl inokosterone) and non-steroidal inducers can be used as ligands for the genetic switch of the present invention. U.S. Patent No. 6,379,945 describes an insect steroid receptor (“HEcR”) isolated from Heliothis virescens that can act as a genetic switch responsive to both steroids and certain non-steroidal inducers. In this system and many other systems responsive to both steroids and non-steroidal inducers, non-steroidal inducers have distinct advantages over steroids for several reasons, including, for example: lower production costs, metabolic stability, absence in insects, plants, or mammals, and environmental acceptability. U.S. Patent No. 6,379,945 describes the use of two dibenzoylhydrazines - 1,2-dibenzoyl-1-tert-butylhydrazine and tebufenozide (N-(4-ethylbenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine) as ligands for the ecdysone-based genetic switch. Other dibenzoylhydrazines are also included as ligands in the present invention, such as those 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 U.S. Patent No. 6,147,282. Other non-limiting examples of ecdysone ligands are 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, 1,2-diacylhydrazines, N'-substituted-N,N′-disubstituted hydrazines, dibenzoylalkylcyanohydrazines, N-substituted-N-alkyl-N,N-diaroylhydrazines, N-substituted-N-acyl-N-alkyl, carbonylhydrazines, or N-aroyl-N'-alkyl N'-aroylhydrazines. (See U.S. Patent No. 6,723,531).

[0370] In one embodiment, the ligand for the ecdysone-based genetic switch system is a diacylhydrazine ligand or a chiral diacylhydrazine ligand. The ligand used in the genetic switch system can be a compound of formula I

[0371]

[0372] Wherein A is an alkoxy group, an arylalkoxy group or an aryloxy group; B is an optionally substituted aryl group or an optionally substituted heteroaryl group; and R1 and R2 are independently an optionally substituted alkyl group, an arylalkyl group, a hydroxyalkyl group, a haloalkyl group, an optionally substituted cycloalkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted heterocycle, an optionally substituted aryl group or an optionally substituted heteroaryl group; or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

[0373] In another embodiment, the ligand can be an enantiomerically enriched compound of formula II

[0374]

[0375] Wherein A is an alkoxy group, an arylalkoxy group, an aryloxy group, an arylalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group; B is an optionally substituted aryl group or an optionally substituted heteroaryl group; and R1 and R2 are independently an optionally substituted alkyl group, an arylalkyl group, a hydroxyalkyl group, a haloalkyl group, an optionally substituted cycloalkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted heterocycle, an optionally substituted aryl group or an optionally substituted heteroaryl group; provided that R1 is not equal to R2; wherein the absolute configuration at the asymmetric carbon atom bearing R1 and R2 is predominantly S; or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

[0376] In certain embodiments, the ligand can be an enantiomerically enriched compound of formula III

[0377]

[0378] Wherein A is an alkoxy group, an arylalkoxy group, an aryloxy group, an arylalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group; B is an optionally substituted aryl group or an optionally substituted heteroaryl group; and R1 and R2 are independently an optionally substituted alkyl group, an arylalkyl group, a hydroxyalkyl group, a haloalkyl group, an optionally substituted cycloalkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted heterocycle, an optionally substituted aryl group or an optionally substituted heteroaryl group; provided that R1 is not equal to R2; wherein the absolute configuration at the asymmetric carbon atom bearing R1 and R2 is predominantly R; or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

[0379] 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 with at least 95% enantiomeric excess, or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.

[0380] When used in conjunction with an ecdysone-based gene switch system, the diacylhydrazine ligands of Formula I and the chiral diacylhydrazine ligands of Formula II or III provide a means for externally temporally regulating the expression of the therapeutic polypeptides or therapeutic polynucleotides of the present invention. See U.S. Patents: 8,076,517; 8,884,060; and 9,598,355; each of which is hereby incorporated by reference in its entirety.

[0381] The ligands used in the present invention can form salts. As used herein, the term "salt" refers to acid and / or base salts formed with inorganic and / or organic acids and bases. Additionally, when a compound of Formula I, II, or III contains both a basic moiety and an acidic moiety, zwitterions ("inner salts") can form and are included in the term "salt" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are used, but other salts are also useful, e.g., in separation or purification steps that can be used during preparation. Salts of the compounds of Formula I, II, or III can be formed, for example, by reacting the compound with an amount (e.g., an equivalent) of an acid or base in a medium (such as a medium in which the salt precipitates) or an aqueous medium, followed by lyophilization.

[0382] Ligands containing a basic moiety can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (e.g., salts formed with acetic acid or trihaloacetic acids such as trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptonates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 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 (e.g., salts formed with sulfuric acid), sulfonates (such as those salts mentioned herein), tartrates, thiocyanates, toluenesulfonates, undecanoates, etc.

[0383] Ligands containing an acidic moiety can form salts with a variety of organic and inorganic bases. Exemplary base salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts formed with organic bases (e.g., organic amines) such as benzathine, dicyclohexylamine, hydrabamine (formed from N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, tert-butylamine, and salts formed with amino acids such as arginine, lysine, etc.

[0384] Non-limiting examples of ligands for inducible gene expression systems also include those utilizing the FK506 binding domain and are FK506, cyclosporin A, or rapamycin. FK506, rapamycin, and their analogs are disclosed in U.S. Patents 6,649,595, 6,187,757, 7,276,498, and 7,273,874.

[0385] In some embodiments, the diacylhydrazine ligand for inducible gene expression is administered in a unit daily dose of about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or 120 mg. In some embodiments, the diacylhydrazine ligand is administered in a unit daily dose of about 5 mg. In some embodiments, the diacylhydrazine ligand is administered in a unit daily dose of about 10 mg. In some embodiments, the diacylhydrazine ligand is administered in a unit daily dose of about 15 mg. In some embodiments, the diacylhydrazine ligand is administered daily in a unit daily dose of about 20 mg.

[0386] In some embodiments, combination therapies with two or more therapeutic agents can use agents that act via different mechanisms of action, although this is not required. Combining agents with different mechanisms of action can result in additive or synergistic effects. Combination therapy can allow for lower doses of each agent than those used in monotherapy, thus reducing toxic side effects and / or increasing the therapeutic index of the agent. Combination therapy can reduce the likelihood of the development of drug-resistant cancer cells. In some embodiments, the combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) tumors / cancer cells.

[0387] In certain embodiments, in addition to administering the fusion protein or fragment or variant thereof described herein, the method or treatment further comprises administering at least one additional therapeutic agent. The at least one additional therapeutic agent can be administered before, simultaneously with, and / or after the administration of the agent. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

[0388] Therapeutic agents that can be co-administered with the fusion proteins, fragments, or variants described herein include chemotherapeutic agents. Thus, in some embodiments, the methods or treatments involve co-administering the agents described herein with a chemotherapeutic agent or a mixture of chemotherapeutic agents. In some embodiments, the methods may further include one or more anti-tumor agents, such as cisplatin, capecitabine, or 5-fluorouracil, in combination with the fusion proteins, fragments, or variants described herein. Treatment with the agents can be performed before, during, or after administration of chemotherapy. Co-administration can include co-administering in a single pharmaceutical formulation or using separate formulations, or sequential administration in either order, but generally over a period of time such that all active agents can exert their biological activity simultaneously. The preparation and dosing regimens of such chemotherapeutic agents can be used according to the manufacturer's instructions or determined empirically by a person skilled in the art. The preparation and dosing regimens of such chemotherapy are also described in The Chemotherapy SourceBook, 4th Edition, 2008, edited by M.C. Perry, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0389] Useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, duocarmycins, etoposide, fluoropyrimidines, ionophores, lexitropsin, nitrosoureas, platinol, purine antimetabolites, puromycin, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an anti-mitotic agent, a topoisomerase inhibitor, or an angiogenesis inhibitor.

[0390] The fusion proteins, fragments, or variants provided herein can 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). For example, a panel of tumor antigens can be used, for example, in the majority of cancer patients.

[0391] Fusion proteins associated with chimeric receptors

[0392] In some embodiments, the fusion protein is administered as part of a combination therapy together 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 include co-administering the fusion protein and the chimeric receptor. In one embodiment, the treatment can include co-administering the fusion protein with a modified effector cell comprising a chimeric receptor. In one embodiment, the treatment can include sequential administration of a modified effector cell comprising a chimeric receptor, followed by the fusion protein. In another embodiment, the treatment can include sequential administration of the fusion protein, followed by a modified effector cell comprising a chimeric receptor. In one aspect, there can be a lag of at least 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, or 24 hours between administrations. In another aspect, there can be a lag of 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 days or more between administrations.

[0393] In some embodiments, the modified immune effector cells are modified immune cells comprising T cells and / or natural killer cells. T cells, or T lymphocytes, are a subtype of white blood cells involved in cell-mediated immunity. Exemplary T cells include T helper cells, cytotoxic T cells, TH17 cells, memory stem cell-like T cells (TSCM), naive T cells, memory T cells, effector T cells, regulatory T cells, or natural killer T cells. In certain aspects, the embodiments described herein include preparing and / or expanding modified immune effector cells (e.g., T cells, Tregs, NK cells, or NK T cells), which includes transfecting cells with an expression vector containing a DNA (or RNA) construct encoding a chimeric receptor.

[0394] In some embodiments, the present disclosure includes modified effector cells that comprise a chimeric receptor expressed on the cell surface. In some cases, the chimeric receptor comprises an antigen-binding domain capable of recognizing and binding to a tumor antigen (e.g., a tumor-associated antigen or a tumor-specific antigen). In some cases, the antigen-binding domain comprises an antibody or a binding fragment, such as Fab, Fab’, F(ab’)2, F(ab’)3, scFv, sc(Fv)2, dsFv, diabody, microbody, and nanobody or a binding fragment thereof. In some cases, the antigen-binding domain comprises an scFv. In some cases, the chimeric receptor comprises an scFv (e.g., a chimeric antigen receptor (CAR)). In some cases, the chimeric antigen receptor comprises a pattern recognition receptor. In other cases, the chimeric receptor comprises an engineered T cell receptor (TCR).

[0395] The present disclosure further provides an immune effector cell that comprises a cell tag used 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, and the HER1t comprises the polypeptide sequence of SEQ ID NO:68. In some cases, the cell tag comprises HER1t-1, and the HER1t-1 comprises the polypeptide sequence of SEQ ID NO:69.

[0396] Chimeric antigen receptor (CAR)

[0397] A chimeric antigen receptor (CAR) is an engineered receptor that transplants exogenous specificity onto an immune effector cell. In some cases, the CAR comprises an extracellular domain and an intracellular domain, and the extracellular domain comprises an antigen-binding domain, a stalk region, and a transmembrane domain. In some cases, the intracellular domain further comprises one or more intracellular signaling domains. In some cases, the CAR described herein comprises an antigen-binding domain, a stalk region, a transmembrane domain, one or more co-stimulatory domains, and a signaling domain for T cell activation.

[0398] The antigen-binding domain can comprise the complementarity-determining regions of a monoclonal antibody, the variable region of a monoclonal antibody, and / or antigen-binding fragments thereof. Complementarity-determining regions (CDRs) are short amino acid sequences found in the variable domains of antigen receptor (e.g., immunoglobulin and T cell receptor) proteins that are complementary to the antigen and thus provide the receptor with specificity for that particular antigen. Each polypeptide chain of an antigen receptor can 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 scFv. In some cases, the antigen-binding domain is Fab. In some cases, the antigen-binding domain is Fab. In some cases, the antigen-binding domain is F(ab’)2. In some cases, the antigen-binding domain is Fv.

[0399] In some embodiments, the CARs described herein comprise an antigen-binding domain capable of binding to an epitope on CD19, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-a2, 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 capable of binding to an epitope on MUC16. In some embodiments, the CARs described herein comprise an antigen-binding domain capable of binding to an epitope on CD19 or CD33. In some cases, the CARs described herein comprise an antigen-binding domain capable of binding to an epitope on CD19. In some cases, the CARs described herein comprise an antigen-binding domain capable of binding to an epitope on CD33. In further embodiments, the CARs described herein comprise an autoantigen or an antigen-binding region capable of binding to an epitope on HLA-A2, myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1, or type II collagen.

[0400] In some embodiments, the CARs and methods described herein can be used to treat hyperproliferative diseases such as cancer, autoimmune diseases, or to treat infections such as viral, bacterial, or parasitic infections. In some aspects, the CARs target antigens that are elevated in cancer cells, autoimmune cells, or cells infected with a virus, bacterium, or parasite. Pathogens that can be targeted include, but are not limited to, Plasmodium, Trypanosoma, Aspergillus, Candida, hepatitis A, hepatitis B, hepatitis C, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogen. Autoimmune diseases can include graft-versus-host disease, rheumatoid arthritis, lupus, 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.

[0401] 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 viral pathogens of the following families: Adenoviridae, Epstein-Barr virus (EBV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), JC virus, BK virus, Papillomaviridae, Herpes simplex virus family (HSV), Herpesviridae, Hepadnaviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses cause smallpox, influenza, mumps, 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, Escherichia coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Treponema, and Salmonella. In some embodiments, the pathogen receptor Dectin-1 can be used to generate CARs that recognize carbohydrate structures on the cell wall of fungi such as Aspergillus. In another embodiment, the CAR can be prepared based on an antibody that recognizes viral determinants (e.g., glycoproteins from CMV and Ebola) to interrupt viral infection and pathology.

[0402] In some embodiments, an "stalk region" or "spacer" or "hinge" region is used to link the antigen-binding domain to the transmembrane domain. In some cases, the "stalk domain" or "stalk region" comprises any oligonucleotide or polypeptide that serves to link the transmembrane domain to the extracellular domain or the cytoplasmic domain in a polypeptide chain. In some embodiments, it is flexible enough to allow the antigen-binding domain to be oriented in different directions, thus facilitating antigen recognition. In some cases, the stalk region comprises the hinge region from IgG1. In alternative cases, the stalk region comprises the CH2CH3 region of an immunoglobulin and optionally a portion of CD3. In some cases, the stalk region comprises the CD8α hinge region, the IgG4-Fc 12-amino acid hinge region (ESKYGPPCPPCP) or the IgG4 hinge region, as described in WO / 2016 / 073755.

[0403] The transmembrane domain can be from a natural or synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Suitable transmembrane domains can include the transmembrane regions of the α, β or ζ chains of the T cell receptor; or the transmembrane regions from CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. Alternatively, the transmembrane domain can be synthetic and can comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine is found at one or both ends of the synthetic transmembrane domain. Optionally, in some embodiments a short oligonucleotide or polypeptide linker of 2 to 10 amino acids in length can form a connection between the transmembrane domain of the CAR and the cytoplasmic signaling domain. In some embodiments, the linker is a glycine-serine linker.

[0404] In some embodiments, the transmembrane domain comprises the CD8α transmembrane domain or the CD3ζ transmembrane domain. In some embodiments, the transmembrane domain comprises the CD8α transmembrane domain. In other embodiments, the transmembrane domain comprises the CD3ζ transmembrane domain.

[0405] The intracellular domain may include one or more co-stimulatory domains. Exemplary co-stimulatory 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 include one or more or two or more of the co-stimulatory 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 include one or more or two or more of the co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, OX40 (CD134), or fragments or combinations thereof. In some cases, the CARs described herein include one or more or two or more of the co-stimulatory domains selected from CD8, CD28, 4-1BB (CD137), or fragments or combinations thereof. In some cases, the CARs described herein include one or more or two or more of the co-stimulatory domains selected from CD28, 4-1BB (CD137), or fragments or combinations thereof. In some cases, the CARs described herein include the co-stimulatory domains CD28 and 4-1BB (CD137), or respective fragments thereof. In some cases, the CARs described herein include the co-stimulatory domains CD28 and OX40 (CD134), or respective fragments thereof. In some cases, the CARs described herein include the co-stimulatory domains CD8 and CD28, or respective fragments thereof. In some cases, the CARs described herein include the co-stimulatory domain CD28, or a fragment thereof. In some cases, the CARs described herein include the co-stimulatory domain 4-1BB (CD137), or a fragment thereof. In some cases, the CARs described herein include the co-stimulatory domain OX40 (CD134), or a fragment thereof. In some cases, the CARs described herein include the co-stimulatory domain CD8, or a fragment thereof.

[0406] In some embodiments, the intracellular domain further includes a signaling domain for T cell activation. In some cases, the signaling domain for T cell activation includes a domain derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some cases, the signaling domain for T cell activation includes a domain derived from CD3ζ.

[0407] In some embodiments, the CARs described herein are administered to a subject together with one or more additional therapeutic agents, the additional therapeutic agents including but not limited to cytokines described herein. In further embodiments, the immune effector cells expressing the CARs described herein express membrane-bound IL-15 (“mIL-15 or mbIL-15”). In aspects of the invention, the mbIL-15 comprises a fusion protein between IL-15 and IL-15Rα. In further embodiments, the 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 some cases, the CAR and the cytokine are expressed in separate vectors. In certain cases, the vector can be a lentiviral vector, a retroviral vector or a Sleeping Beauty transposon.

[0408] CD19-specific CAR

[0409] CD19 is a cell surface glycoprotein of the immunoglobulin superfamily and is mainly found in malignant B lineage cells. In some cases, CD19 has also been detected in solid tumors such as pancreatic cancer, liver cancer and prostate cancer.

[0410] In some embodiments, the CD19-specific CARs described herein are provided, 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 embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by FMC63. In some embodiments, the scFv and / or VH / VL domains are derived from FMC63. FMC63 generally refers to a murine monoclonal IgG1 antibody against Nalm-1 and -16 cells expressing human CD19 (Ling, N.R. et al. (1987). Leucocyte typing III. 302). In some cases, the CD19-specific CAR-T cells further comprise a transmembrane domain selected from the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0411] 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, the CD19-specific CAR-T cells described herein include those 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[0412] In some embodiments, the CD19-specific CAR-T cells described herein include those comprising an scFv antigen-binding domain, and 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134), or a fragment or combination thereof; and a signaling domain from CD3ζ.

[0413] In some embodiments, the CD19-specific CAR-T cells described herein comprise the anti-CD19 antibody described in US20160152723. In some embodiments, the CD19-specific CAR-T cells described herein comprise the anti-CD19 antibody described in WO2015 / 123642. In some embodiments, the CD19-specific CAR-T cells described herein comprise an anti-CD19 scFv derived from clone FMC63 (Nicholson et al. Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma. Mol. Immunol., 34:1157-1165, 1997).

[0414] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharma, Inc.). In some embodiments, the CD19-specific CAR-T cells described herein include those 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0415] In some embodiments, the CD19-specific CAR-T cells described herein include those comprising an scFv antigen-binding domain, and 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0416] In some embodiments, the CD19-specific CAR-T cells described herein comprise the anti-CD19 antibody or a fragment or derivative thereof described in WO2015187528.

[0417] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis). In some embodiments, the CD19-specific CAR-T cells described herein include those 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0418] In some embodiments, described herein are CD19 - specific CAR - T cells comprising a scFv antigen - binding domain, and 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co - stimulatory domains selected from CD27, CD28, 4 - 1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0419] In some embodiments, the antigen - binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (Cellectis). In some embodiments, described herein are CD19 - specific CAR - T cells, wherein 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co - stimulatory domains selected from CD27, CD28, 4 - 1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0420] In some embodiments, described herein are CD19 - specific CAR - T cells comprising a scFv antigen - binding domain, and 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co - stimulatory domains selected from CD27, CD28, 4 - 1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0421] In some embodiments, the antigen - binding domain recognizes an epitope on CD19 that is also recognized by BPX - 401 (Bellicum). In some embodiments, described herein are CD19 - specific CAR - T cells, wherein 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co - stimulatory domains selected from CD27, CD28, 4 - 1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0422] In some embodiments, the CD19-specific CAR-T cells described herein include those comprising a scFv antigen-binding domain, and 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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0423] In some cases, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by lamtomumab (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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0424] In some embodiments, the CD19-specific CAR-T cells described herein, 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 lamtuzumab (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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0425] In some cases, the CD19-specific CAR-T cells described herein comprise a scFv antigen-binding domain, and the antigen-binding domain recognizes an epitope on CD19 that is also recognized by FMC63, lintuzumab (Amgen), cötuximab (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), dinutuximab (Seattle Genetics), B4 (or DI-B4) (Merck Serono), teprotumumab (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 the CD8α transmembrane domain or the CD3ζ transmembrane domain; one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof; and a signaling domain from CD3ζ.

[0426] CD33-specific CAR

[0427] "CD33" is a 67 kDa single-pass transmembrane glycoprotein and is a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) superfamily. CD33 is characterized by a V-set Ig-like domain responsible for sialic acid binding and a C2-set Ig-like domain in its extracellular domain. Alternative splicing of CD33 mRNA results in a shorter isoform (CD33m) lacking the V-set Ig-like domain and the disulfide bond connecting the V-set and C2-set Ig-like domains. In healthy subjects, CD33 is mainly expressed as a myeloid differentiation antigen found on normal multipotent bone marrow precursors, unipotent colony-forming cells, monocytes, and mature granulocytes. CD33 is expressed on more than 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 portion of the blasts in the vast 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 blasts, CD33 can also be expressed on the stem cells that give rise to AML.

[0428] In embodiments, the antigen-binding portion of the CARs described herein are specific for CD33 (CD33 CARs). When expressed on the cell surface, the CD33-specific CAR redirects the specificity of T cells to human CD33. In embodiments, the antigen-binding domain comprises a single-chain antibody fragment (scFv) comprising the variable light chain domain (VL) and the variable heavy chain domain (VH) of a monoclonal anti-CD33 antibody specific for the target antigen, linked by a flexible linker such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is M195, m2H12, DRB2, and / or My9-6. In embodiments, the scFv is humanized, such as hM195. In some embodiments, the antigen-binding portion may comprise a VH and a VL linked in a defined orientation, e.g., from the N-terminus to the C-terminus, VH-linker-VL or VL-linker-VH.

[0429] In embodiments, the CARs described herein comprise 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).

[0430] In embodiments, the CARs described herein comprise 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).

[0431] In embodiments, the CARs described herein comprise 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).

[0432] In embodiments, the CARs described herein comprise 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).

[0433] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0434] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0435] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0436] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0437] MUC16-specific CAR

[0438] MUC16 is a large carbohydrate antigen, also known as CA-125. MUC16 is encoded by the MUC16 gene located on human chromosome 19. MUC16 is a highly glycosylated multi-domain type I transmembrane protein containing three domains. The C-terminal domain contains multiple extracellular SEA (sea urchin sperm protein, enterokinase, and agrin) modules with autocatalytic activity. SEA has two proteolytic sites near the transmembrane (TM) domain. The cleaved large domain, known as 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-bound protein domain is called the MUC16ecto domain. A MUC16 antibody has been identified that specifically binds to the extracellular domain of MUC16 retained on the surface of tumor cells. "MUC16 overexpression" in a target cell (such as a cancer cell) refers to a higher level of MUC16 protein and / or mRNA expressed by the target cell compared to a control cell (such as a non-cancer cell, normal cell, etc.).

[0439] In embodiments, the antigen-binding portion of the CARs 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 embodiments, the antigen-binding domain comprises a single-chain antibody fragment (scFv) that comprises the variable light chain domain (VL) and variable heavy chain domain (VH) of a monoclonal anti-MUC16 antibody specific for the target antigen, linked by a flexible linker such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is MUC16-1scFv (SEQ ID NO:43-44), MUC16-2 scFv (SEQ ID NO:45-46), MUC16-3 scFv (SEQ ID NO:47-48), MUC16-4 scFv (SEQ ID NO:49-50), MUC16-5 scFv (SEQ ID NO:51-52), MUC16-6 scFv (SEQ ID NO:53-54), or MUC16-7 scFv (SEQ ID NO:55-56). In embodiments, the scFv is humanized.

[0440] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0441] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0442] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0443] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0444] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0445] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0446] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0447] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0448] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0449] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0450] In embodiments, the CARs described herein comprise an antigen-binding portion that comprises 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).

[0451] In an embodiment, the CAR described herein comprises an antigen-binding portion that comprises 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).

[0452] In an embodiment, the CAR described herein comprises an antigen-binding portion that comprises 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).

[0453] In an embodiment, the CAR described herein comprises an antigen-binding portion that comprises 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).

[0454] In some embodiments, the antigen-binding portion may comprise a VH and a VL joined in a defined orientation, e.g., from the N-terminus to the C-terminus, VH-linker-VL or VL-linker-VH.

[0455] Engineered T cell receptor (TCR)

[0456] In some embodiments, the chimeric receptor comprises an engineered T cell receptor. The T cell receptor (TCR) consists of two chains (αβ or γδ), which pair on the T cell surface to form a heterodimeric receptor. In some cases, the αβ TCR is expressed on the majority of T cells in vivo and is known to be involved in the recognition of specific MHC-restricted antigens. Each α and β chain consists of two domains: a constant domain (C), which anchors the protein to the cell membrane and associates with invariant subunits of the CD3 signal transducer; and a variable domain (V), which confers antigen recognition through six loops called complementarity-determining regions (CDRs). In some cases, each V domain contains three CDRs; for example, CDR1, CDR2, and CDR3, where CDR3 is the hypervariable region. These CDRs interact with the complex formed by binding of the antigen peptide to a protein encoded by the major histocompatibility complex (pepMHC) (e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, or HLA-DRB1 complex). In some cases, the constant domain further contains a joining region that links the constant domain to the variable domain. In some cases, the β chain further contains a shorter diversity region that forms part of the joining region.

[0457] In some cases, such TCRs are reactive to specific tumor antigens (e.g., NY-ESO, Mage A3, Titin). In other cases, such TCRs are reactive to specific neoantigens expressed within the patient's tumor (i.e., patient-specific mutations, somatic mutations, nonsynonymous mutations expressed by the tumor). In some cases, the engineered TCR can be affinity-enhanced.

[0458] In some embodiments, the TCR is described using the International ImMunoGeneTics (IMGT) TCR nomenclature, and the TCR is linked to the IMGT public database of TCR sequences. For example, there can be several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions, distinguished by their framework, CDR1, CDR2, and CDR3 sequences. Thus, a Vα type can be designated in the IMGT nomenclature by a unique TRAV number. For example, "TRAV21" defines a TCR Vα region with a unique framework and CDR1 and CDR2 sequences, as well as a CDR3 sequence that is defined in part by an amino acid sequence conserved between TCRs but also includes an amino acid sequence that varies between TCRs. Similarly, "TRBV5-1" defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences, as well as a CDR3 sequence that is only partially defined.

[0459] In some cases, the β-chain diversity region is denoted in the IMGT nomenclature by the abbreviation TRBD.

[0460] In some cases, the unique sequences defined by the IMGT nomenclature are well known and accessible to workers in the TCR field. For example, they are visible in the IMGT public database and in "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

[0461] In some embodiments, for example, the αβ heterodimeric TCR is transfected as full-length chains with cytoplasmic and transmembrane domains. In some cases, the TCR contains introduced disulfide bonds between residues of the corresponding constant domains, as described, for example, in WO 2006 / 000830.

[0462] In some cases, the TCRs described herein are in single-chain form, see, for example, WO 2004 / 033685. Single-chain forms include αβ TCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L-Vβ-Cβ types, where Vα and Vβ are the TCRα and β variable regions, respectively, Cα and Cβ are the TCRα and β constant regions, respectively, and L is a linker sequence. In certain embodiments, the single-chain TCRs of the invention can have introduced disulfide bonds between residues of the corresponding constant domains, as described in WO 2004 / 033685.

[0463] The TCRs described herein can be associated with a detectable label, a therapeutic agent, or a PK-modifying moiety.

[0464] Exemplary detectable labels for diagnostic purposes include, but are not limited to, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0465] Therapeutic agents that can be associated with the TCRs described herein include immunomodulators, radiolabeled compounds, enzymes (such as perforin), or chemotherapeutic agents. To ensure toxic effects at the desired location, the toxin can be within a liposome linked to the TCR, such that the compound is released in a controlled manner. In some cases, the controlled release minimizes disruptive effects during in vivo transport and ensures maximum effect of the toxin after the TCR binds to the relevant antigen-presenting cell.

[0466] In some embodiments, other suitable therapeutic agents include, for example:

[0467] a. Small molecule cytotoxic agents, such as compounds with the ability to kill mammalian cells and a molecular weight less than 700 daltons. Such compounds may also contain toxic metals capable of having a cytotoxic effect. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreateglucuronate, auristatin E, vincristine, and doxorubicin;

[0468] b. Peptide cytotoxins, i.e., proteins or fragments thereof with the ability to kill mammalian cells. For example, ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNases, and RNases;

[0469] c. Radionuclides, i.e., unstable elemental isotopes that decay with the emission of one or more of α or β particles or γ rays. For example, 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 TCR or its multimer;

[0470] d. Immunostimulants, i.e., immune effector molecules that stimulate an immune response. For example, cytokines such as IL-2 and IFN-γ,

[0471] e. Superantigens and their mutants;

[0472] f. TCR-HLA fusions;

[0473] g. Chemokines such as IL-8, platelet factor 4, melanoma growth stimulating activity, etc.;

[0474] h. Antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28, or anti-CD16);

[0475] i. Alternative protein scaffolds with antibody-like binding properties

[0476] j. Complement activators; and

[0477] k. Xenoprotein domains, alloprotein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0478] Dose

[0479] The appropriate dosages of the fusion proteins and modified immune effector cells used will depend on the age and weight of the subject and the specific drugs used. The dosages and treatment regimens of the fusion proteins and modified immune effector cells can be determined by a person skilled in the art.

[0480] In certain embodiments, the fusion protein is administered by injection (e.g., subcutaneously or intravenously) at a dose of 1 to 30 mg / kg, such as about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. In some embodiments, the fusion protein is administered at a dose of about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, or about 40 mg / kg. In some embodiments, the fusion protein is administered at a dose of about 1-3 mg / kg or about 3-10 mg / 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 / kg. The dosing schedule can vary from, for example, once a week to once every 2, 3, or 4 weeks. In one embodiment, the fusion protein is administered every other week at a dose of about 10 to 20 mg / kg. In another embodiment, the fusion protein is administered at a dose of about 1 mg / kg every two weeks, about 3 mg / kg every two weeks, 10 mg / kg every two weeks, 3 mg / kg every four weeks, or 5 mg / kg every four weeks.

[0481] In other embodiments, the fusion protein is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 200 mg to 500 mg, such as 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 (e.g., flat dose). 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 - 450 mg or about 300 - 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 dosing schedule can vary from, for example, once a week 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 to 400 mg once every three weeks or once every four weeks. In one embodiment, the fusion protein is administered at a dose of about 300 mg once every three weeks. In one embodiment, the fusion protein is administered at a dose of about 400 mg once every four weeks. In one embodiment, the fusion protein is administered at a dose of about 300 mg once every four weeks. In one embodiment, the fusion protein is administered at a dose of about 400 mg once every three weeks. The fusion protein can be administered one or more times, e.g., once, twice, three times, four times, five times, six times, seven times, or more. In one embodiment, the fusion protein is administered six times. The fusion protein can be administered at least 5 days, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 25, 30, 35, or 40 days, after administration of cells expressing a CAR, such as 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 cells expressing a CAR, such as MUC16 - specific CAR - expressing cells or CD33 - specific CAR - expressing cells.

[0482] The fusion protein 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 intravenous infusion at a rate greater than 20 mg / min, such as 20 - 40 mg / min, and generally greater than or equal to 40 mg / min, to achieve a dose of about 35 to 440 mg / m2, generally about 70 to 310 mg / m2, and more generally about 110 to 130 mg / m2. In an embodiment, the fusion protein can be administered by intravenous infusion at a rate less than 10 mg / min, preferably less than or equal to 5 mg / min, to achieve a dose of about 1 to 100 mg / m2, preferably about 5 to 50 mg / m2, about 7 to 25 mg / m2, and more preferably about 10 mg / m2.

[0483] The fusion protein can be administered by intravenous infusion at a rate greater than 20 mg / min, such as 20 - 40 mg / min, and generally greater than or equal to 40 mg / min, to achieve a dose of about 35 to 440 mg / m2, generally about 70 to 310 mg / m2, and more generally about 110 to 130 mg / m2. In an embodiment, an infusion rate of about 110 to 130 mg / m2 reaches a level of about 3 mg / kg. In other embodiments, the fusion protein can be administered by intravenous infusion at a rate less than 10 mg / min, such as less than or equal to 5 mg / min, to achieve a dose of about 1 to 100 mg / m2, such as about 5 to 50 mg / m2, about 7 to 25 mg / m2, or about 10 mg / m2. In some embodiments, the antibody is infused over a period of about 30 minutes.

[0484] Dose of Modified Effector Cells

[0485] In some embodiments, an amount of modified effector cells is administered to a subject in need thereof, and this amount is determined based on efficacy and the potential to induce cytokine-related toxicity. In some cases, the amount of modified effector cells comprises about 10 5 to about 10 9 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 5 to about 10 8 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 5 to about 10 7 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 6 to about 10 9 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 6from about 0 to about 10 8 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 7 to about 10 9 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 5 to about 10 6 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 6 to about 10 7 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 7 to about 10 8 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 8 to about 10 9 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 9 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 8 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 7 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 6 modified effector cells / kg. In some cases, the amount of modified effector cells comprises about 10 5 modified effector cells / kg.

[0486] In some embodiments, the modified effector cells are modified T cells. In some cases, the modified T cells are CAR-T cells. In some cases, the amount of CAR-T cells comprises about 10 5 to about 10 9 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 5 to about 10 8 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 5 to about 10 7 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 6 to about 10 9 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 6 to about 10 8CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 7 to about 10 9 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 5 to about 10 6 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 6 to about 10 7 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 7 to about 10 8 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 8 to about 10 9 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 9 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 8 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 7 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 6 CAR-T cells / kg. In some cases, the amount of CAR-T cells comprises about 10 5 CAR-T cells / kg.

[0487] In some embodiments, the CAR-T cells are CD19-specific CAR-T cells. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 5 to about 10 9 CD19-specific CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 5 to about 10 8 CD19-specific CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 5 to about 10 7 CD19-specific CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 6 to about 10 9 CD19-specific CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 6 to about 10 8 CD19-specific CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 107 to about 10 9 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 5 to about 10 6 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 6 to about 10 7 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 7 to about 10 8 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 8 to about 10 9 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 9 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 8 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 7 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 6 CAR-T cells / kg. In some cases, the amount of CD19-specific CAR-T cells comprises about 10 5 CAR-T cells / kg.

[0488] In some embodiments, the modified T cells are engineered TCR T cells. In some cases, the amount of engineered TCR T cells comprises about 10 5 to about 10 9 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises about 10 5 to about 10 8 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises about 10 5 to about 10 7 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises about 10 6 to about 10 9 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises about 10 6 to about 10 8 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises about 10 7 to about 10 9TCR cells / kg. In some cases, the amount of engineered TCR cells comprises from about 10 5 to about 10 6 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises from about 10 6 to about 10 7 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises from about 10 7 to about 10 8 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises from about 10 8 to about 10 9 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises from about 10 9 to about 10 8 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises from about 10 7 to about 10 6 TCR cells / kg. In some cases, the amount of engineered TCR cells comprises from about 10 5 TCR cells / kg.

[0489] It should be noted that the 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, the specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges set forth herein are only exemplary and are not intended to limit the scope or practice of the claimed composition.

[0490] Expression and purification system of the fusion protein

[0491] The fusion proteins or fragments or variants thereof described herein can be produced from cells as follows: Culturing a host cell transformed with an expression vector comprising one or more polynucleotides encoding the fusion protein or fragment or variant thereof under conditions and for a time sufficient to permit expression of the fusion protein or fragment or variant 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 of using them are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning – A Laboratory Manual – Third Edition, Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors, and suitable host cells can vary according to a number of factors and can be readily 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).

[0492] Following expression, the fusion protein or fragment or variant thereof can be purified or isolated. The terms “purified” or “isolated” as applied to any fusion protein or fragment or variant thereof described herein can refer to a polypeptide or protein that has been separated or purified from the 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 in which the protein is expressed). Generally, a polypeptide is purified when it comprises 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%, at least 99.9% by weight of the total protein in the sample.

[0493] The fusion proteins or fragments or variants thereof described herein can be isolated or purified in a variety of ways depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse phase HPLC chromatography. For example, a fusion protein or fragment or variant thereof can be purified using a standard anti-fusion protein antibody affinity column. Ultrafiltration and diafiltration techniques, as well as protein concentration, are also useful. See, e.g., Scopes (1994) “Protein Purification, 3rd ed.,” Springer-Verlag, New York City, N.Y. The degree of purification necessary can vary depending on the intended use. In some cases, the expressed fusion protein or fragment or variant thereof does not need to be purified.

[0494] Methods for determining the yield or purity of a purified fusion protein or fragment or variant thereof can include, for example, Bradford assay, ultraviolet spectrophotometry, 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 stains such as Coomassie blue or colloidal silver stain). Once expressed, purified, or purified after expression, in vitro or in vivo assays, such as any of those described herein, can be used to assay for any of the various desired properties of the fusion proteins or fragments or variants thereof described herein. For example, the ability of the fusion proteins or fragments or variants thereof described herein to, e.g., inhibit PD-1 and capture TGF-β can be assayed.

[0495] A variety of techniques can be used to generate the fusion proteins or fragments or variants thereof described herein. For example, a polynucleotide encoding a fusion protein or fragment or variant thereof described herein can be inserted into an expression vector containing transcriptional and translational regulatory sequences, which can include, for example, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptional terminator signals, polyadenylation signals, and enhancer or activator sequences. In some embodiments, the regulatory sequences can include a promoter and transcriptional start and stop sequences. Additionally, the expression vector can include more than one replication system, enabling it to be maintained in two different organisms, e.g., for expression in mammalian or insect cells and for cloning and amplification in a prokaryotic host.

[0496] Several possible vector systems can be used to express a fusion protein or a fragment or variant thereof from a nucleic acid in mammalian cells (e.g., host cells). For example, one class of vectors relies on the integration of the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be selected by co-introducing a drug resistance gene such as Escherichia coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can be ligated 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 the ability to replicate autonomously as 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). The expression vector can be introduced into cells (e.g., host cells) in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is mainly determined by the targeted cell type, as described below. Non-limiting exemplary methods can include CaPO4 precipitation, liposome fusion, lipofection, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0497] Suitable host cells for expressing a fusion protein or a fragment or variant thereof can include, but are not limited to, yeast, bacteria, insects, plants, and mammalian cells as described above. Of interest are bacteria such as Escherichia coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), and primary cell lines (e.g., primary mammalian cells). In some embodiments, the fusion protein or a fragment or variant thereof can be expressed in Chinese hamster ovary (CHO) cells or a suitable myeloma cell line such as (NS0). Suitable cell lines also include, for example, BHK-21 (baby hamster kidney) cells; 293 (human embryonic kidney) cells; HMEpC (human mammary epithelial cells); 3T3 (mouse embryonic fibroblast) cells.

[0498] The methods described herei...

Claims

1. A fusion protein comprising: (a) An IgG4 antibody or an antigen-binding fragment thereof, comprising: (i) a first polypeptide comprising a heavy chain variable region (V H region) consisting of the amino acid sequence of SEQ ID NO: 6; and (ii) a second polypeptide comprising a light chain variable region (V L region) consisting of the amino acid sequence of SEQ ID NO: 12; and (b) A TGF-β trap consisting of the amino acid sequence of SEQ ID NO:14; wherein the IgG4 antibody or its antigen-binding fragment is linked to the TGF-β trap via a peptide linker.

2. The fusion protein according to claim 1, wherein the peptide linker consists of the amino acid sequence (Gly Gly Gly Gly Ser) n wherein n is 2, 3, 4, 5 or 6.

3. The fusion protein according to claim 1, wherein the peptide linker comprises the amino acid sequence (Gly Gly Gly Gly Ser) n , where n is 2, 3, 4, 5 or 6.

4. The fusion protein according to claim 1, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:

33.

5. The fusion protein according to claim 1, wherein the peptide linker comprises the amino acid sequence (Glu Ala Ala Ala Lys) n , where n is 1, 2, 3, 4, 5 or 6.

6. The fusion protein according to claim 1, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:

31.

7. The fusion protein according to claim 1, wherein the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs:17-34.

8. The fusion protein according to claim 1, wherein the IgG4 antibody or its antigen-binding fragment further comprises the amino acid sequence of SEQ ID NO:146 or 292, but has a mutation at position 108 thereof.

9. The fusion protein according to claim 8, wherein the mutation is S108P.

10. The fusion protein according to claim 1, wherein the antigen-binding fragment of the IgG4 antibody is Fab, (Fab)2, (Fab’)2, Fv, (Fv)2 or scFv.

11. The fusion protein according to claim 1, wherein the peptide linker links the first polypeptide to the TGF-β trap.

12. The fusion protein according to claim 1, wherein the peptide linker links the second polypeptide to the TGF-β trap.

13. The fusion protein according to claim 1, wherein a second peptide linker links the first polypeptide and the second polypeptide.

14. The fusion protein according to claim 13, wherein the second peptide linker comprises the amino acid sequence of any one of SEQ ID NOs:17-34.

15. The fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:

16.

16. The fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:

143.

17. The fusion protein according to claim 1, wherein the IgG4 antibody or its antigen-binding fragment further comprises a fragment crystallizable region (F C ).

18. The fusion protein according to claim 17, wherein the F C is human F C 1, F C 2, F C 3, F C 4 or a fragment thereof.

19. The fusion protein according to claim 17, wherein said F C further comprises one or more mutations.

20. The fusion protein according to claim 1, wherein the IgG4 antibody or its antigen-binding fragment comprises scFv and F C fragment.

21. The fusion protein according to claim 1, which comprises the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:

294.

22. A pharmaceutical composition comprising: (a) The fusion protein according to any one of claims 1-21; and (b) A pharmaceutically acceptable excipient.

23. Use of the fusion protein according to any one of claims 1-21 in the preparation of a medicament for treating cancer in a subject in need of such treatment.

24. The use according to claim 23, wherein the cancer is a refractory cancer.

25. The use according to claim 23, wherein the subject is non-responsive to treatment with a PD-1 antibody or a CTLA-4 antibody.

26. The use according to claim 23, wherein the medicament further comprises one or more additional anti-cancer agents.

27. The use according to claim 26, wherein the additional anti-cancer agent is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, 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.

28. The use according to claim 27, wherein the PD-1 inhibitor is an anti-PD-1 antibody or a functional fragment or variant thereof.

29. The use according to claim 27, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody or a functional fragment or variant thereof.

30. The use according to claim 23, wherein the drug further comprises a cytokine.

31. The use according to claim 23, wherein the drug comprises a fusion protein, the fusion protein comprising: (a) IL-15 or a functional variant thereof; and (b) IL-15Rα or a functional variant thereof.

32. The use according to claim 23, wherein the subject is a mammal.

33. The use according to claim 23, wherein the subject is a human.

34. The use according to claim 23, wherein the 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 lymphoma, lung cancer, skin cancer, kidney cancer or head and neck cancer.

35. The use according to claim 34, wherein the cancer is colorectal cancer, ovarian cancer, head and neck cancer or AML.

36. The use according to claim 34, wherein the cancer is cutaneous squamous cell carcinoma, melanoma or basal cell carcinoma.

37. The use according to claim 34, wherein the cancer is non-small cell lung cancer (NSLC) or small cell lung cancer (SCLC).

38. The use according to claim 34, wherein the cancer is triple negative breast cancer (TNBC).

39. The use according to claim 23, wherein the drug further comprises an effective amount of T cells engineered to express a foreign receptor.

40. The use according to claim 39, wherein the foreign receptor is a chimeric antigen receptor.

41. The use according to claim 40, wherein the chimeric antigen receptor is an engineered T cell receptor.

42. The use according to claim 40, wherein the chimeric antigen receptor comprises an antigen-binding domain that binds to an epitope on CD19, BCMA, CD44, α-folate receptor, CAIX, CD30, CD33, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-a2, 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.

43. The use according to claim 42, wherein the antigen-binding domain comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 37-56.

44. The use according to claim 42, wherein the antigen-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 35 or 36.

45. The use according to claim 41, wherein the engineered T cell further expresses a polypeptide comprising a cytokine.

46. The use according to claim 45, wherein the polypeptide comprising a cytokine is a fusion protein, and the fusion protein comprises: (a) IL-15 or a functional variant thereof; and (b) IL-15Rα or a functional variant thereof.

47. The use according to claim 46, wherein the polypeptide comprising a cytokine comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

69.

48. The use according to claim 46, wherein the polypeptide comprising a cytokine comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

69.

49. The use according to claim 46, wherein the polypeptide comprising a cytokine comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

69.

50. The use according to claim 46, wherein the polypeptide comprising a cytokine comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

69.

51. The use according to claim 46, wherein the polypeptide comprising a cytokine comprises the amino acid sequence of SEQ ID NO: 69, or a sequence that differs from SEQ ID NO: 69 by no more than conservative amino acid substitutions.

52. The use according to claim 46, wherein the polypeptide comprising a cytokine comprises the amino acid sequence of SEQ ID NO: 69.

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