Fusion constructs and methods of using the same

Abstract

To provide a fusion protein, a pharmaceutical composition comprising the fusion protein, and a method of treating a cancer.SOLUTION: A fusion protein comprises: a. an antibody, a fragment of the antibody or a variant of the antibody that binds to programmed cell death protein-1; and b. a transforming growth factor beta (TGF-β) cytokine trap; where one or more polypeptides of the fusion protein are connected by a linker. In one aspect of the fusion protein, the cytokine trap comprises a transforming growth factor receptor 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.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Recently, monoclonal antibody-based immunotherapy based on the interruption of inhibitory signals transmitted to the adaptive immune system has been proposed. Cancer immunotherapy using CTLA-4 antibodies has shown promise in clinical trials. ipilimumab), and PD-1 inhibitors (e.g., pembrolizumab, nivolumab With FDA approval of EGFR-1, today there are a wide range of treatments available, including for lung cancer, renal cell carcinoma, and ovarian cancer. Many treatment options are available for treating advanced tumors. PD-L1 and TGF-β are co-expressed in most indications (e.g., ovarian, gastric, and rectal cancer). In the colon and rectum, little to no response to immune checkpoint inhibitors has been observed. Therefore, there is a need in the art for safer and more effective treatments for cancer. There continues to be a need to obtain it.

[0003] Incorporation by Reference All publications, patents, and patent applications mentioned herein are to be construed as separate entities each individually identified as such. Where any patent or patent application is specifically and individually indicated to be incorporated by reference, to the same extent as if each of the preceding claims were incorporated by reference herein. Summary of the Invention

[0004] As used herein, (a) an antibody that binds to programmed cell death protein 1 (PD-1), or or a fragment of said antibody, or a variant of said antibody; and (b) a transforming enzyme. Transforming Growth Factor Beta (TGF-β) cytokine trap, A fusion protein is provided in which multiple polypeptides are connected by a linker. is shown.

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

[0006] In one embodiment, the TGFβR is transforming growth factor beta receptor II. (TGFβRII), or said functional fragment. The functional fragment of TGFβRII is the extracellular domain (ECD) of TGFβRII. In one embodiment, the ECD binds to TGF-β1. In one embodiment, the ECD binds to TGF-β1 and TGF-β3. In one embodiment, the ECD binds to TGF-β1 and TGF-β3. In one embodiment, the TGF-β cytokine tracker The fragment contains at least one 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 a sequence that is 80% identical to the TGF-β cytokine , SEQ ID NO: 14, SEQ ID NO: 141, or SEQ ID NO: 142.

[0007] In one embodiment, the TGF-β cytokine trap is 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 IgG In one embodiment, the IgG4 is one of SEQ ID NO: 146 or SEQ ID NO: 292. In one embodiment, the mutation comprises a S108P mutation at position 08. In one embodiment, the IgG4 is linked to the TGF-β receptor via the linker. In one embodiment, the fragment of the antibody is Fab , (Fab)2, (Fab')2, Fv, (Fv)2, or scFv.

[0008] In one embodiment, the antibody comprises a heavy chain variable region (V H ) and the light chain variable region (V L In one embodiment, the linker comprises the variable region (V H )of, to the TGF-β cytokine trap. In one embodiment, the linker The variable region of the light chain (V L ) to the TGF-β cytokine trap. In embodiments, the variable region of the heavy chain (V H ) is a second linker in the fusion protein The variable region (V L ) is connected to the second The linker comprises a sequence shown in any one of SEQ ID NOs: 17 to 34. In this form, the variable region (V H ) is SEQ ID NO: 1 to 7 and 149 to 164 is at least 80% identical to the sequence shown in any one of

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

[0010] In one embodiment, the fusion protein comprises a sequence shown in SEQ ID NO: 15 and a sequence shown in SEQ ID NO: 1 In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO: 15. and the sequence shown in SEQ ID NO: 143. Variable domain (V H ) is at least 90% identical to the sequence shown in SEQ ID NO: 7, and The variable region is at least 90% identical to the sequence shown in SEQ ID NO: 13. In this form, the variable region (V H ) comprises the sequence shown in SEQ ID NO: 7, The variable region (V L ) comprises the sequence shown in SEQ ID NO:13.

[0011] In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 145. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 29 6 and the sequence set forth in SEQ ID NO: 144. The body further comprises a fragment crystallizable region (Fc). In some embodiments, the Fc is human Fc1, human Fc2, human Fc3, human Fc4, or any of these. In one embodiment, the Fc further comprises one or more mutations. In one embodiment, the antibody comprises the scFv and the Fc fragment. The TGFβ cytokine trap is an anti-TGFβ antibody or an antigen-binding In one embodiment, the anti-TGFβ antibody comprises a polypeptide fragment selected from SEQ ID NOs: 166, 168, 169, 171, 173, 175, or 177 and a VH encoded by SEQ ID NO: 165, 1 and a VL encoded by 67, 170, 172, 174, 176, or 178. .

[0012] In one embodiment, the TGFβ cytokine trap is or a variant thereof. In one embodiment, the TGFβ inhibitory peptide comprises SEQ ID NO: 193 to 227.

[0013] As used herein, a polynucleotide encoding the fusion protein disclosed herein is It is presented.

[0014] As used herein, a polynucleotide encoding the fusion protein disclosed herein is an expression vector comprising the polynucleotide operably linked to a promoter; In one embodiment, the promoter is a constitutive promoter, a tissue-specific promoter, or a In one embodiment, the inducible promoter is - a small-molecule ligand-induced, two-polypeptide, ecdysone receptor-based In one embodiment, the vector is an adenoviral vector. do.

[0015] As used herein, there are provided (a) fusion proteins disclosed herein; (b) fusion proteins disclosed herein; or (c) a polynucleotide encoding a fusion protein as disclosed herein. A pharmaceutical composition is provided that includes the expression vector, and (d) a pharmaceutically acceptable excipient.

[0016] Provided herein are methods of treating cancer, comprising: (a) treating cells with a fusion protein comprising a fusion protein described herein; (b) a polynucleotide encoding the fusion protein disclosed herein; or (c) contacting the cell culture medium with an expression vector disclosed herein. In one embodiment, the cell is a cancer cell. In one embodiment, the cell is a mammalian cell.

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

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

[0019] In one embodiment, the antibody is an immunoglobulin G (IgG) antibody. In one embodiment, the IgG is IgG1, IgG2, IgG3, or IgG4. In this case, the IgG4 suddenly has a sequence at position 108 of SEQ ID NO: 146 or SEQ ID NO: 292. In one embodiment, the mutation is an S108P mutation. The fragments of the antibody include Fab, (Fab)2, (Fab')2, F of the antibody. In one embodiment, the antibody or the fragment of the antibody The fragments or variants of the antibodies may comprise the variable region of the heavy chain (V H ) and the variable region of the light chain (V L In one embodiment, the linker comprises the variable region (V H ) to the T In one embodiment, the linker connects the light chain to a GF-β cytokine trap. The variable region (V L ) to the TGF-β cytokine trap.

[0020] In one embodiment, the variable region of the heavy chain (V H ) is connected to said light chain by a second linker Variable region (V L In one embodiment, the variable region (VH )teeth a sequence shown in any one of SEQ ID NOs: 1 to 7 and 149 to 164, and at least In one embodiment, the variable region (V L ) is SEQ ID NO: 8 13 and 148. In one embodiment, the variable region (V H ) are SEQ ID NOs: 1 to 7 and 149 to 16 Contains one of the four.

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

[0022] In one embodiment, the fusion protein comprises the sequence shown in SEQ ID NO: 15 (VL5 Ig g4) and the sequence set forth in SEQ ID NO: 143. In one embodiment, the variable region of the heavy chain The VH region is at least 90% identical to the sequence shown in SEQ ID NO: 7, and Variable region (V L ) is at least 90% identical to the sequence shown in SEQ ID NO: 13. In embodiments, the variable region of the heavy chain comprises the sequence set forth in SEQ ID NO: 7, and the variable region of the light chain comprises the sequence set forth in SEQ ID NO: 8. The variable region comprises the sequence set forth in SEQ ID NO: 13. In one embodiment, the fusion protein comprises , the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 145. In one embodiment, the fusion protein comprises a sequence as set forth in SEQ ID NO: 296 and a sequence as set forth in SEQ ID NO: 144. and an array to be

[0023] In one embodiment, the antibody further comprises a fragment crystallizable region (Fc). In the above, the Fc is human Fc1, human Fc2, human Fc3, human Fc4, or any of these. In one embodiment, the Fc further comprises one or more mutations. In one embodiment, the antibody comprises the scFv and the Fc fragment. In one embodiment, the subject is a patient receiving a PD-1 antibody or CTL therapy. In one embodiment, the method comprises one or more of the following steps: In one embodiment, the method further comprises administering a further anti-cancer agent. The anti-cancer drugs used are PD-1 inhibitors, PD-L1 inhibitors, or CTLA-4 inhibitors. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody, or a fragment or variant thereof. It is the body.

[0024] In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody, or a fragment thereof. In one embodiment, the method 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, ovarian cancer, or ovarian cancer. Endometrial cancer, colorectal cancer, stomach cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer cancer, stomach cancer, bladder cancer, liver cancer, Hodgkin's lymphoma, lung cancer, skin cancer, kidney cancer, or It is head and neck cancer.

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

[0026] In one embodiment, the chimeric antigen receptor is CD19, BCMA, CD44, alpha-folate receptor. body, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, H ER3, folate binding protein, GD2, GD3, IL-13R-α2, KDR, EDB -F, mesothelin, CD22, EGFR, folate receptor α, MUC-1, MUC-4, MU C-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, Antibodies that bind to epitopes on EGFRvIII, CD123, or VEGF-R2 In one embodiment, the antigen-binding domain comprises an antigen-binding domain selected from the group consisting of SEQ ID NOs: 37-56. In one embodiment, the antigen-binding domain comprises a sequence selected from SEQ ID NOs: 35-3. 6.

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

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

[0029] In one embodiment, the fusion protein comprises a sequence as set forth in SEQ ID NO: 15 and a sequence as set forth in SEQ ID NO: 2. 94. In one embodiment, the fusion protein comprises the sequence set forth in SEQ ID NO: 296 and the sequence set forth in SEQ ID NO: 295. In one embodiment, the fusion The 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: 2. and the sequence shown in SEQ ID NO:95.

[0030] As used herein, inter alia, (a) an antibody that binds to programmed cell death protein 1 (PD-1) (b) an antibody, a fragment of said antibody, or a variant of said antibody; and (b) an adenosine dehydrogenase (ADD) antibody. a fusion protein comprising the aminase (ADA) protein, or a functional fragment thereof, A fusion protein in which one or more polypeptides are connected by a linker is displayed. In one embodiment, the linker is (G4S)n, where n is 2, 3, 4, 5, or In some embodiments, the linker comprises (Gly)n, where n is 6, 7, or 8. In another embodiment, the linker comprises (EAAAK)n, wherein , n is 1, 2, 3, 4, 5, or 6. In certain embodiments, phosphorus The car includes A(EAAAK)ALEA(EAAAK)A. In one particular embodiment The linker comprises a sequence shown in any one of SEQ ID NOs: 17 to 34.

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

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

[0033] In one example, the heavy chain variable region (V H ) is connected by a second linker to the light chain variable region (V L )fart In one embodiment, the variable region of the heavy chain (V H ) are SEQ ID NOs: 1 to 7 and 149 to 164. In another example, the variable region of the light chain (V L ) is any one of SEQ ID NOs: 8 to 13 and 148 In a further embodiment, the heavy chain is at least 80% identical to the sequence shown in Variable region (V H ) is shown in any one of SEQ ID NOs: 1 to 7 and 149 to 164 In a further embodiment, the variable region of the light chain (V L ) are SEQ ID NOs: 8 to 13 and 148. In one embodiment, the heavy chain variable The region (VH) is at least 90% identical to the sequence (VH6) shown in SEQ ID NO: 6. The variable region of the light chain (VL) has the sequence shown in SEQ ID NO: 12 (VL5) and at least 9 In one embodiment, the heavy chain variable region (V H ) is shown in SEQ ID NO: 6 the variable region (V L ) comprises the sequence shown in SEQ ID NO:12.

[0034] In one example, 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 the fusion protein, the sequence shown in SEQ ID NO: 12 (VL5) and the sequence shown in SEQ ID NO: 28 1 (VH6 igG4(mut)ADA2 mut 7). is the variable region of the heavy chain (V H ) is a sequence shown in SEQ ID NO: 7 (VH7) and at least 9 0% identical to the variable region of the light chain (V L ) is the sequence shown in SEQ ID NO: 13 (VL6 In one example, the variable region of the heavy chain (VH) is at least 90% identical to SEQ ID NO: 7. and a light chain variable region (VH7) comprising the sequence shown inL ) is shown in SEQ ID NO: 13 In another example, the fusion protein comprises the sequence (VL6) shown in SEQ ID NO: 13. VL6), and the sequence shown in SEQ ID NO: 282 (VH7 igG4(mut)ADA2 In one example, 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) is included.

[0035] Provided herein are polynucleotides encoding the fusion proteins. Further provided herein is a polynucleotide encoding a fusion protein according to any one of the above aspects. an expression vector comprising a nucleotide, the polynucleotide being operably linked to a promoter; In some embodiments, the promoter is a constitutive promoter, a tissue promoter, or a In some embodiments, the promoter is a specific promoter or an inducible promoter. The promoter is a small-molecule ligand-inducible, two-polypeptide ecdysone receptor In some embodiments, the vector is an adenoviral vector. It is Tar.

[0036] Provided herein are methods of treating cancer, comprising administering to cells a fusion protein, a fusion protein-containing a polynucleotide encoding the protein, or an expression vector In some embodiments, the cells are cancer cells. The cells are mammalian cells.

[0037] Further provided herein are fusion proteins; or polynucleotides encoding fusion proteins. or an expression vector and a pharmaceutically acceptable excipient. can be.

[0038] Provided herein is a method of treating a subject with cancer, comprising administering to a subject a therapeutically effective amount of a programmed cell death protein. an antibody that binds to PD-1, or a fragment of said antibody, or a variant of said antibody; and and adenosine deaminase protein, or a fusion protein containing this functional fragment. and administering to the subject a composition comprising one or more polypeptides of the fusion protein. In one particular case, the adenovirus is connected by a linker. The adenosine deaminase protein is adenosine deaminase 2 (ADA2). In embodiments, the adenosine deaminase (ADA) protein is 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 ( ADA2 mutant 6 (SEQ ID NO: 278), or ADA2 mutant 7 (SEQ ID NO: 279), Variant 7 (SEQ ID NO: 279), or wild-type ADA2 (SEQ ID NO: 284) Includes one.

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

[0040] Provided herein are methods of treating a subject with cancer. Cancers include mesothelioma, glioblastoma, endometrial cancer, colorectal cancer, gastric cancer, cervical cancer, and ovarian cancer. pancreatic cancer, prostate cancer, breast cancer, stomach cancer, bladder cancer, liver cancer, Hodgkin's lymphoma, lung cancer Cancer of the skin, kidney, or head and neck. In certain cases, skin cancer is In other cases, lung cancer is non-small cell lung cancer. Lung cancer (NSLC) or small cell lung cancer (SCLC). In some cases, breast cancer is It is triple-negative breast cancer (TNBC).

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

[0042] In some cases, the exogenous receptor is a chimeric antigen receptor. The receptor is an engineered T cell receptor. In one case, the chimeric antigen receptor is CD19, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP -2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3, I L-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, folate receptor α, MUC-1, MUC-4, MUC-16, MAGE-A1, h5T4, PSMA, T AG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF-R In some embodiments, the antigen-binding domain binds to an epitope in The binding domain comprises a sequence selected from SEQ ID NOs: 37-56, and in another embodiment, The antigen-binding domain comprises a sequence selected from SEQ ID NOs: 35-36.

[0043] In one embodiment, an effective amount of engineered T cells is at least 10 cells per kg. 2 Individual In another embodiment, an effective amount of engineered T cells is at least 10 cells per kg. 4 pieces In a further embodiment, the effective amount of engineered T cells is at least 100 mg of cells per kg. 10 5 There are individuals.

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

[0045] Provided herein is a method of treating cancer in a subject in need thereof, comprising administering a protease inhibitor to a subject. an antibody that binds to ram cell death protein 1 (PD-1), or a fragment of said antibody; or a variant of said antibody; and an adenosine deaminase protein, or a functional fragment thereof. administering a composition comprising a fusion protein comprising: or a plurality of polypeptides are connected by a linker; and and administering one or more doses of an effective amount of engineered T cells, The method includes the steps of: .

[0046] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the present invention and its advantages may be obtained by reference to exemplary embodiments in which the principles of the present disclosure are employed. This can be had by reference to the following detailed description and accompanying drawings, which set forth: [Brief explanation of the drawings]

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

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

[0049] The present disclosure is not limited to the particular embodiments described herein, and as such It should be understood that variations and modifications may occur to those skilled in the art. It will be recognized that these are encompassed within its scope.

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

[0051] The section headings used herein are for organizational purposes only and do not limit the scope of the subject matter described. should not be considered as limiting the subject matter.

[0052] While various features of the disclosure may be described in the context of a single embodiment, the features may also be provided separately. Conversely, the terms "common" and "commonly used" may be used interchangeably herein, and may be presented in any suitable combination. Although, for clarity, the present disclosure may be described in the context of individual embodiments, the present disclosure also may be described in the context of individual embodiments. It can be implemented in one embodiment.

[0053] The following definitions supplement those in the art and are intended to orient the present application and any Related or unrelated matters, e.g., commonly owned, belonging to any patent or application Any methods and materials similar or equivalent to those described herein are not intended to be limiting. Although various materials and methods may be used in carrying out the tests of this disclosure, preferred materials and methods are described herein. Accordingly, the terminology used herein refers to specific embodiments and methods. are for descriptive purposes only and are not intended to be limiting. There is no.

[0054] definition In this application, unless otherwise stated, the use of the singular refers to the plural. As used herein, unless the context clearly dictates otherwise, Unless otherwise specified, the singular forms "a", "an" and "the" are used in conjunction with the plural It must be noted that the referent is included.

[0055] In this application, unless stated otherwise, the use of "or" means "and" As used herein, "and / or" and "these" mean The terms "any combination of" and "any combination of" and their grammatical equivalents may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following terms "A, B, and / or C" or "A, B, The phrase "A; B; C; or any combination thereof" is used to mean "individual A; individual B; individual C; A and B; B and C; A and C; and A, B, and C. Therefore, unless disjunctive use is mentioned, the term "or" is used conjunctively. It can be used disjunctively or disjunctively.

[0056] Furthermore, in addition to the term "including," "including," and " The use of other forms, such as "included," is also non-limiting.

[0057] As used herein, "some embodiments," "an embodiment," "one embodiment," or References to "other embodiments" may include modifications to the particular features, structures, or aspects described in connection with the embodiments. The features are included in at least some embodiments of the present disclosure, but all embodiments include: It means that it is not necessarily included.

[0058] As used in this specification and claims, "comprising" means "comprise" and "comprises" are examples of "comprising" g)), "having" ("have" and "having any form of "has," "including," ("include" and "includes" are examples of "including" any form of "containing") or "containing" ("contain " and "contains," or any form of "containing" The term "list" is inclusive or open-ended and may include additional, unlisted elements or methods. Any embodiment discussed herein does not exclude any method steps of the present disclosure. Alternatively, it is contemplated that the present invention may be practiced with respect to compositions, and vice versa. Additionally, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

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

[0060] The terms "about" or "approximately" refer to an approximate value for a particular value, as determined by one of ordinary skill in the art. , within an acceptable error range, which in part depends on how the value is measured or determined. That is, within the error range that depends on the limits of the measurement system. For example, "about" means that According to practice in the art, this can mean within one standard deviation or more than one standard deviation. Alternatively, "about" can mean up to 20%, e.g., up to 10%, or up to 5% of a given value. In another example, an amount of "about 10" can mean between 10 and 9. In yet another example, the use of "about" in relation to a reference numerical value includes any amount between 1 and 11. The word also means a value ± 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or Alternatively, particularly with respect to a biological system or process, The term "about" also means within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold. Where specific values ​​are recited in this application and claims, they are not necessarily the same. Unless otherwise stated, "within acceptable error range" is used for a particular value. The term "about" should be assumed.

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

[0062] As used herein, "transfection," "transformation," or "transduction" " refers to the addition of one or more exogenous polynucleotides to a cell by using physical or chemical methods. The term "polynucleotide" refers to the introduction of a polynucleotide into a host cell. The nucleic acid sequences and vectors can be used for, e.g., transfection, transformation, or transformation. A number of transfection methods are known in the art. It is well known, for example, calcium phosphate DNA co-precipitation method (e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Hu See Mana Press (1991); DEAE-dextran method; electroporation; cation Liposome-mediated transfection; tungsten particle-promoted gene gun method (Jo hnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation ( Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage vectors or Viral vectors can be expressed in suitable packaging cells, many of which are commercially available. After the infectious particles have been propagated, they can be introduced into host cells.

[0063] As used herein, "polypeptide," "peptide," and their grammatical equivalents A polypeptide refers to a polymer of amino acid residues. A polypeptide may be optionally glycosylated or or other modifications typical for a given protein within a given intracellular environment. The polypeptides and proteins disclosed in the document (including functional portions and functional variants thereof) (including) contains synthetic amino acids in place of one or more naturally occurring amino acids Such synthetic amino acids are known in the art and can be prepared, for example, by the synthesis of aminocyclohexane. Xanthate, norleucine, α-amino-n-decanoic acid, homoserine, S-acetyl trans-3-hydroxyproline and trans-4 -hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4 -chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β -hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexane Silalanin, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4 -Tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoaromatic acid amide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxy Roxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexyl α-aminocycloheptanecarboxylic acid, α-(2-amino-2-nor Bornane)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homofuran The present disclosure provides methods for the preparation of recombinant proteins in engineered cells. The expression of the polypeptides or proteins described herein may be It is further contemplated that the modification of one or more amino acids of a protein may be associated with post-translational modification of the protein. Non-limiting examples of post-translational modifications include phosphorylation, acylation, including acetylation and formylation, glycosylation, and glycosylation. Cosylation (including N-linked and O-linked), amidation, hydroxylation, methylation and alkylation, including ethylation, ubiquitination, pyrrolidone carboxylic acid addition, disulfide Formation of amide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation These include ionization, geranylation, glypionation, lipoylation, and iodination.

[0064] As used herein in the context of two nucleic acid or amino acid sequences of a polypeptide The terms "identical" or "sequence identity" and their grammatical equivalents are used interchangeably. Two words that are the same when aligned with respect to the maximal anaphoric relation across the comparison range As used herein, a "comparison window" refers to a range of at least about 20, typically , about 50 to about 200, more usually about 100 to about 150 consecutive positions. After optimally aligning the two sequences, the sequences are divided into the same number of It refers to a segment that can be compared to a reference sequence by consecutive positions. The optimal sequence alignment for comparison is based on the Smith method. and Waterman, Adv. Appl. Math., 2:482 (1981) local homology algorithm; Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); Pearson and Lipman, Proc. Nat. Acad. Sci USA, 85:2444 (1988); Computerized implementation of algorithms (Intelligentics, Mountai n View Calif.'s PC / Gene program, CLUSTAL, Wi sconsin Genetics Software Package, Geneti cs Computer Group (GCG), 575 Science Dr., M GAP, BESTFIT, BLAST in Washington, Wis., USA This can be done by a variety of methods, including but not limited to FASTA, FASTA, and TFASTA. For more information on the CLUSTAL program, see Higgins and Sharp, Gene, 73:237-244 (198 8) and Higgins and Sharp, CABIOS, 5:151-153 (1989), Corpet et al., Nucleic Acid s Res., 16:10881-10890 (1988), Huang et al., Computer Applications in the Biosci. ences, 8:155-165 (1992), and Pearson et al., Methods in Molecular Biology, 24: 307-331 (1994). Alignment can also be performed by visual and manual inspection. In one class of embodiments, the present invention provides a method for detecting a position of a target object. The polypeptides can be searched using, for example, BLASTP (or CL) with default parameters. USTAL, or any other available alignment software) and a sequence similar to the reference polypeptide or fragment thereof, but at least 80%, 85%, 90%, 98% identical. 99%, or 100% identical. Similarly, nucleic acids are also described in reference to a starting nucleic acid. They can also be used, for example, as in BLAST, using default parameters. N (or CLUSTAL, or any other available alignment software) 50%, 60%, 70%, 75%, 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%, can be 0%, 85%, 90%, 98%, 99%, or 100% identical. , when we say that a molecule has a certain percentage of sequence identity to a larger molecule, this means that If two molecules are optimally aligned, the percentage of residues in the small molecule is This means finding matching residues within a larger molecule according to the optimally aligned order. do.

[0065] The term "substantially identical" as applied to nucleic acid or amino acid sequences and The grammatical equivalent thereof is that a nucleic acid sequence or an amino acid sequence is encoded by a program described above, e.g. For example, at least 100% of the total number of matches was obtained by comparing the matched sequences with a reference sequence using BLAST with standard parameters. 90% or more sequence identity, at least 95%, at least 98%, and at least 99% sequence identity. For example, the BLASTN program (for nucleotide sequences) uses 11 as the default. Using a word length (W) of 10, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses a default search of 3 Code length (W), expectation value (E) of 10, and BLOSUM62 scoring matrix (See Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992) Percent sequence identity is calculated by dividing two optimally aligned sequences by the number of sequences in the comparison window. The portion of the polynucleotide sequence within the comparison window is determined by comparing the two sequences. For optimal alignment of the sequences, compare with the reference sequence (without additions or deletions) It may contain additions or deletions (i.e., gaps). The percentage is the number of identical nucleobases or amino acids. Determine the number of positions where the amino acid residue occurs in both sequences to determine the number of matched positions. The number of matched positions is divided by the total number of positions in the comparison region, and the result is multiplied by 100 to obtain the matched position. In some embodiments, substantial identity is calculated by determining the percentage of sequence identity. over a region of the sequence at least about 50 residues in length, and over a region of at least about 100 residues In some embodiments, the sequence spans at least about 150 residues and is substantially In some embodiments, the sequences are substantially identical over the entire length of the coding region. be.

[0066] "Homology" generally refers to the degree of similarity between two or more nucleic acids or proteins (or The identity between sequences is useful for establishing homology. The exact percentage varies with the nucleic acid and protein in question, but is at least 25% A sequence identity of 100% or less is used to establish homology. High levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or Sequence identities greater than this can also be used to establish homology. is a method for determining percent sequence identity (e.g., using default parameters) , BLASTP, and BLASTN) are described, but these are publicly available. Nucleic acids and / or nucleic acid sequences may be derived from a common ancestral nucleic acid or ancestral nucleic acid, whether natural or artificial. Proteins and / or protein sequences are "homologous" if they are derived from the amino acid sequence. Their coding DNA, natural or artificial, is derived from a common ancestral nucleic acid or ancestral nucleic acid sequence. Homologous molecules can be referred to as "homologs." For example, any Naturally occurring proteins can be modified by any available mutagenesis method. Upon expression, this mutant nucleic acid produces a polypeptide homologous to the protein encoded by the original nucleic acid. Encodes a polypeptide.

[0067] As used herein, the term "isolated" and its grammatical equivalents means to isolate an organism from its natural environment. As used herein, the term "purified" and its grammatical meanings are Equivalents are those in which the molecule or composition is isolated from nature (e.g., genomic DNA and mRNA). whether derived from DNA (including DNA fragments), synthesized (including cDNA), and / or grown under laboratory conditions Whether the product is amplified by a specific factor, the purity is increased, and in this case "purity" is a relative term. On the other hand, nucleic acids and proteins are free from diluents or Although it may be formulated with an adjuvant, for practical purposes it is best to use it in isolation. For example, when used for introduction into a cell, the nucleic acid may be Typically, it is mixed with an acceptable carrier or diluent. The term "qualitatively purified" and its grammatical equivalents refer to nucleic acids, polypeptides, proteins, Polynucleotides, proteins, polypeptides, or other compounds with which they are naturally associated. essentially free, i.e., greater than about 50%, of peptides and other molecules, More than 70% free, more than about 90% free, nucleic acid sequences, polypeptides, proteins It refers to a substance or other compound.

[0068] An "expression vector" or "vector" is an autonomous vector that mediates replication of a polynucleotide within a cell. behaves as a unit (i.e., is capable of replicating under its own control), or is any genetic element that can replicate upon insertion into a host cell chromosome, e.g. , plasmids, chromosomes, viruses, transposons, and the like, which are complexes of joined segments. and / or by incorporating another polynucleotide segment into it to effect its production and / or expression. Suitable vectors include plasmids, transposons, Vectors include, but are not limited to, bacteriophages and cosmids. Ligation or insertion of the vector into a host cell of the joined segments is carried out. The host may contain polynucleotide sequences necessary to effect expression of the host. Varies depending on the host organism: promoter sequences that effect transcription, enhancers that increase transcription nucleotide sequence, a ribosome binding site sequence, and a transcription / translation termination sequence. The vector involves ligation or integration of the vector into the DNA sequence of the host cell. It may be possible to directly express the product of a nucleic acid sequence encoded therein without transfection. In some embodiments, the vector is capable of replicating in the host cell and is capable of being cloned into a suitable cell. In the presence of chemical pressure, "episodes" persist as extrachromosomal segments of DNA within the host cell. These vectors are "gene expression vectors" or "episomes" (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004). Representative, commercially available episomal expression vectors include: Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV ) origin of replication (oriP), including, but not limited to, episomal plasmids. The vectors pREP4, pCEP4, and pREP7, as well as Invitrogen pcDNA3.1 and Stratagene from (Carlsbad, Calif.) pBK-CMV (LaJolla, Calif.) contains EBNA1 and oriP. Alternatively, episomal vectors using the T antigen and SV40 origin of replication may be used, but are not limited to: The vector may also contain a selectable marker gene.

[0069] As used herein, the term "selectable marker gene" refers to a gene that directs the expression of a nucleic acid sequence. Cells are specifically selected in the presence of the corresponding selection agent, either forward or backward. Suitable selectable marker genes are publicly known in the art. For example, International Patent Application Publication No. 1992 / 08796 and 1994 / 28143 Brochure, Wigler et al., Proc. Natl. Acad. Sci. USA, 7 7: 3567 (1980), O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527 (1981), Mul Ligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981), Colberre-Garapin et a l., J. Mol. Biol., 150:1 (1981), Santerre et al., Gene, 30: 147 (1984), Kent et al. al., Science, 237: 901-903 (1987), Wigler et al., Cell, 11: 223 (1977), Szybalsk a & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026 (1962), Lowy et al., Cell, 2 2: 817 (1980), and U.S. Pat. Nos. 5,122,464 and 5,770 ,359.

[0070] As used herein, a "coding sequence" refers to a sequence that encodes a protein or polypeptide. A region or sequence may have an opening near the 5' end. A start codon is bounded by the 3' end, and a stop codon is bounded near the 3' end. It may also be referred to as an open reading frame.

[0071] As used herein, "operably linked" refers to the ability of a DNA segment to interact with another DNA segment. a physical and / or functional linkage to the A segment, wherein the segment is It refers to a linkage that allows the gene products to function in their intended manner. The DNA sequence to be loaded may be, for example, a promoter, enhancer, and / or Regulatory sequences, such as silencers, can modulate the transcription of DNA sequences either directly or operably linked to a regulatory sequence when linked in an indirectly enabling manner For example, a DNA sequence may be selected if it is located downstream of the transcription start site of a promoter. ligated to the promoter in the correct reading frame relative to the transcription start site. When a DNA sequence is inserted into a target region, allowing transcription elongation to proceed through the DNA sequence, An enhancer or silencer is operably linked to a promoter. are ligated to a DNA sequence to increase or decrease transcription of the DNA sequence, respectively. When inserted, it is operably linked to a DNA sequence encoding a gene product. Enhancers and silencers are often located upstream of the coding region of a DNA sequence. It may be located downstream of this or embedded within it. Null sequences are signal transduction pathways when expressed as a preprotein that participates in the secretion of the polypeptide. The DNA for the polypeptide sequence is operably linked to the DNA encoding the polypeptide. Linkage of the A sequence to the regulatory sequence is typically by ligation at appropriate restriction sites. or by using restriction endonucleases known to those skilled in the art to insert This is achieved via an adapter or linker.

[0072] As used herein, the terms "induce" and "induce" and their grammatical equivalents The substance is a compound that controls the transcription of a nucleic acid sequence, the activity of a promoter, and and / or refers to an increase in the expression of a promoter relative to some basal transcription level.

[0073] The term "transcriptional regulator" refers to a promoter-driven DNA Biochemical elements that act to prevent or inhibit transcription of a sequence (e.g., repressors) promoter proteins or nuclear inhibitory proteins), or under certain environmental conditions, A biosynthetic molecule that acts to enable or stimulate the transcription of a motor-driven DNA sequence. "Enhanced expression" refers to a genetic element (e.g., inducer or enhancer) that mediates a specific target gene expression.

[0074] As used herein, the term "enhancer" refers to, for example, a gene that is operably linked to An enhancer refers to a DNA sequence that increases the transcription of a nucleic acid sequence. They can be located many kilobases away from the target region, allowing for regulatory factor binding, DNA These may mediate changes in the methylation patterns of the target gene or in the DNA structure. Numerous enhancers are known and have been cloned from various sources. available as a peptide or within a cloned polynucleotide (e.g., (e.g., from depositories such as ATCC, as well as other commercial or private sources). Many polynucleotides, including promoters (such as the commonly used CMV promoter), It also includes enhancer sequences. Enhancers may be located upstream of a coding sequence. It may be located within this or downstream of it. The term "enhancer" refers to an enhancer region that maps within the immunoglobulin (Ig) locus. Enhancer elements derived from (such enhancers include, for example, heavy chain (mu) 5' enhancer, light chain (kappa) 5' enhancer, kappa intron enhancer This refers to enhancers that are intronic and mu intronic enhancers, as well as 3' enhancers. In general, see Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New Y See Work (1993), pages 353-363, and U.S. Pat. No. 5,885,827. sea ​​bream).

[0075] "Promoter" refers to a region of a polynucleotide that induces transcription of a coding sequence A promoter is a gene that is located near the transcription start site of a gene, on the same strand of DNA, and A promoter is located upstream (towards the 5' region of the sense strand). Constitutive promoters are active under all conditions, whereas other promoters, e.g. An inducible promoter is one that is regulated and active in response to a specific stimulus. The term "promoter activity" and its grammatical equivalents refer to the activity of a gene or molecule whose activity is measured. It refers to the degree of expression of a nucleotide sequence operably linked to a promoter. The activity can be determined by determining the amount of RNA transcripts produced, for example, by Northern blot analysis. It can also be measured directly by using a reporter nucleic acid linked to a promoter. Indirectly, by determining the amount of product encoded by linked nucleic acid sequences, such as sequences. It can also be measured quantitatively.

[0076] As used herein, an "inducible promoter" refers to a promoter that is regulated by a transcriptional regulator, e.g., a biological factor. Or refers to a promoter that is induced to activity by the presence or absence of an abiotic factor. Inducible promoters are promoters that allow expression of genes operably linked to them to be expressed in an organism or in a particular They are useful because they can be turned on or off at certain stages of tissue development. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, and Nodal promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis These promoters include regulated promoters, temperature-regulated promoters, and light-regulated promoters. The inducible promoter can be part of or be a gene switch.

[0077] As used herein, "T cell" or "T lymphocyte" refers to a cell-mediated immune response. T cells or T lymphocytes are a type of lymphocyte that play a central role in B cells and natural killer cells due to the presence of T cell receptors (TCR) on their cell surface They can be distinguished from other lymphocytes, such as NK cells.

[0078] As used herein, the term "antibody," also known as immunoglobulin (Ig), refers to a substance that binds to a specific antigen. The term may refer to a monoclonal or polyclonal antibody. The term "monoclonal antibody" as used in refers to a antibody produced by a single clone of B cells. In contrast, a "polyclonal antibody" refers to an antibody that binds to the same epitope. A population of antibodies produced by different B cells that bind to different epitopes of the same antigen. The antibody may be of any animal origin. The antibody may be of any type, including IgG (IgG1, IgG2, Ig IgA (including IgA1 and IgA2), IgD, IgE, IgF, IgG, IgH, IgIg ... In some embodiments, the antibody may be a single chain whole antibody. In some embodiments, the antibody may be a whole antibody, including Fab, Fab', F(a) b')2, Fd(V H and CH1), Fv fragment (V H and V L consisting of Single chain variable fragments (scFv), single chain antibodies, disulfide-linked variable fragments (dsFv), and V L Domain or V H These may include, but are not limited to, fragments containing the domain, A whole antibody typically consists of four polypeptides: a heavy (H) chain polypeptide; It consists of two identical copies of the ribosomal peptide and two identical copies of the light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, Each light chain contains an N-terminal variable (VL) region and a C-terminal variable (CH2, CH3) region. , and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains are The VH region and VL region form the antigen-binding site of an antibody. They share a common general structure, including four framework regions that are relatively conserved. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, known as DR1, CDR2, and CDR3, are responsible for binding to the antigen. These specific regions form the "hypervariable regions" of the antibody. Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of interest munological interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987 ), as well as MacCallum et al., J. Mol. Biol. 262:732-745 (1996) In this case, the definition refers to overlapping amino acid residues, or amino acids, when compared against each other. Preferably, the term "CDR" is used to refer to a subset of amino acid residues of a CDR that are identical to those of a CDR based on sequence comparison. The CDRs are those defined by Kabat, based on the CDRs CDRH1, CDRH2, and CDRH 3 denotes the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 denote the light chain CDRs. Display.

[0079] As used herein, the terms "antibody fragment," "antibody fragment," "antibody fragment," "antigen-binding portion" and "antibody fragment" are used interchangeably. The term "antigen" or its grammatical equivalents refers to an antibody that retains the ability to specifically bind to an antigen. Used interchangeably to mean one or more pieces or parts of a body (generally Ho (See, e.g., Illiger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Antibody fragments can be For example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Non-limiting examples of antibody fragments include (1 ) a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain (2) a Fab fragment linked by a disulfide bridge in the stalk region; (3) a single arm of an antibody; (4) an Fv fragment consisting of the VL and VH domains of a single Fv fragments joined by a linker that allows them to be synthesized as a polypeptide chain of Single-chain Fv (s), which are monovalent molecules consisting of two domains (i.e., VL and VH) cFv) (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. Biotechno I., 16: 778 (1998); and (5) each polypeptide chain is a member of the same polypeptide. The peptide linker is too short to allow pairing between VH and VL on the peptide chain. , comprising a VH connected to a VL, thereby having two functional antigen-binding sites. between complementary domains on different VH-VL polypeptide chains to create a dimeric molecule. The present invention also includes diabodies, which are dimers of polypeptide chains that drive the pairing of Antibody fragments are known in the art, and are described, for example, in U.S. Pat. No. 8,603,950. , which is described in more detail.

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

[0081] The term "conservative amino acid substitution" or "conservative mutation" refers to a substitution of one amino acid. Refers to the substitution of another amino acid with a common characteristic. The functional scheme that defines the amino acid changes between corresponding proteins of homologous organisms is The aim is to analyze the standardized frequency of of Protein Structure, Springer-Verlag, New York (1979). Amino acids within a group are preferentially exchanged with each other, thus affecting the overall protein structure. It is possible to define a group of amino acids that are most similar to each other in their effect on (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations are Amino acid substitutions, e.g., lysine for arginine, which may maintain a positive charge. replacement of ribonucleotides with glutamic acid, and vice versa, so as to maintain a negative charge; Substitution of aspartic acid and vice versa; substitution of serine with a free -OH group and replacement of threonine with glutamine, which may maintain free -NH2. Alternatively, or in addition, the functional variant may comprise at least one substitution of α, β ... The amino acid sequence of the reference protein may include one or more non-conservative amino acid substitutions.

[0082] The term "non-conservative mutation" refers to an amino acid substitution between different groups, e.g., a lysine These include the substitution of tryptophan with phenylalanine, or the substitution of serine with phenylalanine. In this case, the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions preferably do not impair or disrupt the biological activity of functional variants. may enhance the biological activity of the functional variant such that the .

[0083] As referred to herein, a "proliferative disorder" refers to a disorder characterized by excessive proliferation of cells and / or cell maturation. The unified theory is that excessive turnover of ribosomal proteins significantly contributes to the pathogenesis of diseases, including cancer. In some embodiments, the proliferative disease is cancer.

[0084] As used herein, a "patient" or "subject" refers to a person diagnosed with a proliferative disorder, such as cancer. refers to a mammalian subject diagnosed with or suspected of having or developing In some embodiments, the term "patient" refers to a person at risk of developing a proliferative disorder, such as cancer. Exemplary patients include humans, apes, dogs, pigs, and cattle. animals, cats, horses, goats, sheep, rodents, and other animals that may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female. As used herein, a "patient in need thereof" or a "subject in need thereof" is used to refer to, for example, Have been diagnosed with a disease or disorder, such as, but not limited to, cancer; or patients suspected of having it.

[0085] As used herein, "administering" refers to administering one or more of the compositions described herein. By way of example and not limitation, a composition may be considered to be administered to a patient or subject. Administration of the substance, e.g., injection, can be by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (i d.) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more of these routes can be used. Parenteral administration can be, for example, For example, it can be by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by oral route. It may also be by bolus or cell pellet deposition surgery, and the design of medical devices. In some embodiments, the compositions of the present disclosure comprise a nucleic acid as described herein. An engineered or host cell that expresses a sequence or sequences as described herein. a vector comprising one or more nucleic acid sequences in an amount effective to treat or prevent a proliferative disorder; Pharmaceutical compositions can be used to target cell populations described herein by administering one or more pharmaceutical or may be included in combination with a physiologically acceptable carrier, diluent, or excipient. Suitable compositions include buffer solutions such as neutral buffered saline and phosphate buffered saline; glucose, malate, and the like. carbohydrates such as ammonium, sucrose or dextran, mannitol; proteins; Polypeptides or amino acids such as glycine; antioxidants; EDTA or glutathione adjuvants (e.g., aluminum hydroxide); and preservatives. do.

[0086] As used herein, the terms "treatment," "treating," or the phrases thereof Legal equivalents refer to obtaining the desired pharmacological and / or physiological effect. In this embodiment, the effect is therapeutic, i.e., the effect is effective against the disease and / or disorder. In some embodiments, the term "to partially or completely cure adverse symptoms that may be attributed to The term "treating" can include "preventing" a disease or condition.

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

[0088] As used herein, "administered in combination" or "co-administration" or "co-administration" refers to The terms "administering" or "co-administering" refer to two (or more) ), different treatments may be delivered to a subject during the course of the subject's illness, e.g., After an elephant is diagnosed with a disorder, the disorder is cured or disappears, or treatment is not effective against other This means that two or more treatments are delivered before being discontinued for any reason. In some embodiments, the administration of the second treatment is initiated after the second treatment has begun, so that overlap occurs. However, delivery of one treatment is still achieved. In some cases, this is referred to herein as "simultaneous delivery." In other embodiments, the delivery of another treatment begins with the delivery of the other treatment. In either case, in some embodiments, the delivery of one treatment is completed before the other. Due to the combined administration, the treatments are more effective. For example, the second treatment is more effective. For example, a lower dose of the second treatment may be given with comparable efficacy, or the second treatment may be given at a dose higher than the first. Symptoms that occur to a greater extent in the absence of one treatment than when the second treatment is administered A similar situation exists for the first or second treatment. In this case, delivery is achieved by reducing symptoms or other parameters associated with the disorder, which may be achieved by other treatments. The reduction observed with one treatment delivered in the absence of The effects of the two treatments may be partially additive, fully additive, or The delivery may be more than additive. The delivery may be more than additive. The effect of the device can be delivered so that it is still detectable.

[0089] In some embodiments, the first treatment and the second treatment are administered by the same or separate compositions. , may be administered simultaneously (e.g., at the same time point), or sequentially. Sequential administration refers to administration of a compound prior to the administration of a further treatment, e.g., a secondary treatment (e.g., 5, 1 0, 15, 30, 45, 60 minutes or less, immediately before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 hours or longer; 4, 5, 6 , 7, 8, 9 days or more; 1, 2, 3, 4, 5, 6, 7, 8 weeks or refers to the administration of one treatment at a time (or more periods prior to the administration of the first treatment) The order of administration of the primary and secondary treatments may also be reversed.

[0090] "Therapeutically effective amount," "therapeutic amount," "immunologically effective amount," "antitumor effective amount," "tumor-inhibiting amount" The term "effective amount," or grammatical equivalents thereof, refers to the amount of a compound needed to achieve a desired therapeutic result. A therapeutically effective amount refers to an amount that is effective for a necessary period of time at a necessary dosage. The subject's condition, age, sex, and weight, and the ability to elicit a desired response in one or more subjects. The amount of the administered dose may vary depending on factors such as the ability of the compositions described herein to The exact amount of the disclosed compositions will depend on the patient's (subject's) age, weight, tumor size, infection or metastasis. It can be determined by a doctor, taking into consideration the degree and individual differences in the condition.

[0091] Alternatively, administering one or more compositions described herein to a patient or subject The pharmacological and / or physiological effects of may also be "prophylactic," i.e., The effect may be complete or partial prevention of the disease or its symptoms. "Effective amount" refers to the dosage required to achieve the desired prophylactic result (e.g., prevention of disease onset). In this context, it refers to an amount that is effective for the necessary period of time. Programmed cell death proteins and other checkpoint inhibitors

[0092] PD-1 or CD279 (cluster of differentiation Programmed cell death protein 1, also known as 279, is an immune checkpoint inhibitor. The PD-1 / PD-L1 signaling axis promotes tumor-mediated immune evasion. In some cases, PDL-1 acts on tumor cells, myeloid-derived suppressor cells in the tumor microenvironment, and cells (MDSCs), tumor-associated macrophages (TAMs), and antigen-presenting cells (APCs). In some cases, PD-1 can be overexpressed by "exhausted" T cells. It can be upregulated by its ligands (PDL-1, PDL2, and CD8 0), it can signal to suppress effector T cell function. Blockade of the D-1 / PD-L1 pathway with anti-PD-1 or anti-PD-L1 enhances the proliferation of exhausted T cells. This may restore function and promote tumor cell killing (Figure 1).

[0093] In some embodiments, a fusion protein comprising a PD-1 inhibitor and a TGF-β trap. The full-length, nivolumab (anti-PD-1), MK-3945 (anti-PD-1), and pembrolizumab Mab (anti-PD-1), pidilizumab (anti-PD-1), REGN2810 (anti-PD-1), AMP-224 (anti-PD-1), MEDI0680 (anti-PD-1), PDR001 (anti-P D-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.

[0094] Fusion proteins In some embodiments, the fusion proteins presented herein, or fragments or The mutants are PD-1 inhibitors or cytokine traps fused via a linker. contains PD-1 antibodies.

[0095] In some embodiments, the PD-1 inhibitor is an antibody that targets PD-1, or It may be an antibody fragment or an antibody variant. In some embodiments, pembrolizumab fusion proteins containing fused to a cytokine trap (e.g., TGF-β trap) In some embodiments, the fusion protein comprising nivolumab can be used to inhibit cytokine trajectories. The protein may be fused to a trap (eg, a TGF-β trap).

[0096] In some embodiments, the fusion proteins, or fragments or The mutants express the cytokine trap and immune checkpoint genes described above. In some embodiments, the antibody comprises a targeting antibody, antibody fragment, or variant. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death ligands Antibodies targeting immune checkpoints, such as protease inhibitor 1 (PDL1), or antibodies The fragment or antibody variant is fused to the TGF-β trap molecule via a linker. In some embodiments, the PD-L1 inhibitor is atezolizumab.

[0097] In some embodiments, the fusion proteins presented herein, or fragments or The mutants may be adenosine deaminase (e.g., ADA2) as described herein, or including a PD1 inhibitor or PD1 antibody fused to this functional variant or derivative. .

[0098] Cytokine Trap Cytokines affect many biological processes. Several cytokines have been shown to be effective in a variety of diseases, including cancer. These cytokines have been shown to be causative agents in the disease. -1, IL-4, IL-6, TNF-α, TGF-β, and various other As used herein, "cytokine tracker" refers to a cytokine that is a cytokine that is a protein that is involved in the production of cytokines. The term "blocker" refers to a blocker or neutralizer of cytokine action. An example of a kine trap is the extracellular domain of a cytokine receptor, which binds to the cytokine. These may include antibodies and peptides that bind to cytokines (e.g., inhibitory peptides). In one embodiment, the cytokine is TGF-β. In one embodiment, the cytokine is TGF-β1. In one embodiment, the cytokines are TGF-β1 and TGF-β3. In a further embodiment, a cytokine trap ( For example, TGF-β traps are proteins that bind to the extracellular domain of TGF-βRII or to mutations in this domain. antibodies (e.g., SEQ ID NOs: 141 and 142), anti-TGF-β antibodies, and TGF-β1 , TGF-β2, and / or TGF-β3 inhibitory peptides.

[0099] Transforming Growth Factor Transforming growth factor β (TGF-β) promotes proliferation, differentiation, and proliferation in many cell types. TGF-β is a multifunctional set of peptides that can regulate the phenotype, proliferation, and other functions of the cells. It may act synergistically with TGF-α in inducing transformation. TGF-β also It can also act as a negative autocrine growth factor. Dysregulation can result in apoptosis. Many cells synthesize TGF-β. Almost all of these have specific receptors for this peptide. TGF-β1, TGF-β2, and TGF-β3 all have the same receptor. TGF-β1 plays an important role in regulating the immune system. They may play different roles on different cell types or at different stages of development. Most immune cells (or white blood cells) can secrete TGF-β1. TGF-β1 is derived from the C-terminus of the precursor protein by proteolytic cleavage. It is a 112 amino acid peptide that binds to TGF-β, a small secreted polypeptide. It is a type II serine receptor that can recruit and phosphorylate the type I dimeric receptor (TGFβRI). It can signal via a dimeric TGF / threonine kinase receptor (TGFβRII). TGFβRI mediates cell proliferation, differentiation, apoptosis, and We have identified SMADs, which are potential transcription factors that regulate genes involved in cell proliferation and growth. Many advanced cancers express both TGF-β and TGFβR, It is known that overexpression of TGFB signaling pathway promotes invasive tumorigenesis. Inhibition of this pathway may be a key therapeutic strategy in the treatment of cancer.

[0100] Some T cells (e.g., regulatory T cells) release TGF-β1 to stimulate the production of other T cells. It can inhibit the IL-1 / IL-2-dependent proliferation of activated T cells and the use of resting helpers. The activation of T cells and cytotoxic T cells can be prevented by the activity of TGF-β1. Similarly, TGF-β1 inhibits the production of interferon-γ, tumor necrosis factor alpha (TNF-α), and many other cytokines, including but not limited to various interleukins TGF-β1 also acts on cytokines such as the IL-2 receptor, inhibiting their secretion and activity. However, TGF-α can downregulate the activity of immune cells by reducing the expression levels of TGF-α receptors. -β1 can also increase the expression of certain cytokines in T cells, particularly Furthermore, when cells are immature, their proliferation can be promoted (Figure 2).

[0101] TGF-β1 may have a similar effect on B cells, which may be due to the It can vary depending on the differentiation state. TGF-β1 can inhibit B cell proliferation and apoptosis. These antibodies, transactivators, and receptors on immature and mature B cells can stimulate B cell proliferation and differentiation. May play a role in regulating the expression of ferrin and MHC class II proteins .

[0102] The effects of TGF-β1 on macrophages and monocytes are primarily inhibitory This cytokine inhibits the proliferation of these cells and their reactive oxygen species. (e.g., superoxide (O2 - )) intermediates, and nitrogen (e.g., nitric oxide (N However, as in other cell types, TGF-β1 may prevent the production of intermediates. It can also have the opposite effect on bone marrow-derived cells. For example, TGF-β1 inhibits the chemical They can act as attractants and direct the immune response towards some pathogens; Macrophages and monocytes can respond chemotactically to low levels of TGF-β1. In addition, expression of monocytic cytokines (including IL-1α, IL-1β, and TNF-α) , and phagocytic killing by macrophages can also be increased by the action of TGF-β1 ( Figure 2).

[0103] Transforming growth factor-beta, a subset of the cytokine family III (TGF-β3) mediates many functions, including cell proliferation, embryogenesis, immune system regulation, and differentiation. It contributes to a wide range of functions.

[0104] Transforming growth factor β receptor II (TGFβRII) TGF-β receptor (TGFβR) is a single-transmembrane serine / threonine kinase receptor TGFβR is a complex of several molecules, which may be homodimers or heterodimers. They can exist in several different isoforms. The number of ligands far exceeds the number of known receptors. This suggests that the interaction is promiscuous. Three typical TGF-β superfamily receptors (TGFβRs) are known for their structure and TGFβRI (ALK5) and TGFβRII can be distinguished by their specific functions and functional properties. They have similar ligand binding affinities and are indistinguishable from each other by peptide mapping alone. Both TGFβRI and TGFβRII act in response to TGF-β1. It may have high affinity for TGF-β2 and low affinity for TGF-β2. βRIII (β-glycan) is a β-glycan that binds both homodimers, TGF-β1 and TGF-β2. It has high affinity for TGF-β1,2, which is a heterodimer. TGF-β receptors also have high affinity for TGF-β3. "TGFβRII" or "TGFβ receptor II" refers to the wild-type human TGFβ receptor II. A polypeptide having a TGFβ receptor isoform A sequence (see, for example, NCBI Reference Sequence Amino acid sequence (RefSeq) accession number: NP_001020018 (SEQ ID NO: 289) a polypeptide having the sequence, or the wild-type human type 2 TGFβ receptor isoform B sequence (e.g., NCBI RefSeq accession number: NP_003233 (SEQ ID NO: 290) or the amino acid sequence of SEQ ID NO: 289 or SEQ ID NO: 290 and substantially TGFβRII refers to a sequence that has substantially the same sequence as the wild-type sequence of TGFβ binding. At least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35% of the activity The expressed TGFβRI may be retained at 50%, 75%, 90%, 95%, or 99%. The polypeptide of I lacks a signal sequence.

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

[0106] TGF-β trap fusion protein, or a fragment or variant thereof As used herein, immune checkpoint inhibitors, such as PD-1 inhibitors or PD-1 antibodies and a cytokine trap capable of neutralizing a cytokine (e.g., TGF-β). The polypeptide, or a fragment or variant thereof, is displayed. TGF-β traps include SEQ ID NO: 142, TGF-β traps (also known as TGF-βR II, or a fragment or variant thereof). Examples include the extracellular domain (ECD) of a receptor (e.g., TGFβRII), or this functional domain. and TGF-β inhibitory peptides (e.g., SEQ ID NOs: 193-22). 7), or anti-TGF-β antibodies. The anti-TGF-β antibodies are those represented by SEQ ID NOs: 166, 168, 169, 171, 173, 175, or or 177, and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, at least heavy chain variable regions (V) that are at least 99%, at least 99.5%, or 100% identical H ) In some embodiments, the anti-TGF-β antibody comprises SEQ ID NOs: 165, 167, 170, 172, 174, 176, or 178, and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L In certain embodiments, the TGF-β trap comprises: specifically bind to TGF-β1 or TGF-β2 or TGF-β3, or In another embodiment, the TGF-β trap may have high affinity for TG Specifically binds to or reacts with TGF-β1, TGF-β2, and TGF-β3 In another embodiment, the TGF-β trap may have high affinity for TGF-β. 1 and TGF-β3, or has high affinity for them. In a further embodiment, the TGF-β trap has an affinity for TGF-β2. The tea may be low or may not bond to it.

[0107] The fusion proteins provided herein, or fragments or variants thereof (e.g., TG PD-1 inhibitors or PD-β fused to cytokine traps such as F-β traps 1 antibody) inhibits the interaction between, for example, PD-L1 on tumor cells and PD-1 on immune cells. The relative effects of TGF-β on the immune system are due to the simultaneous blockade of TGF-β and the neutralization of TGF-β, for example, within the tumor microenvironment. Without being bound by theory, this effect may be due to the fact that single molecular entities The simultaneous blockade of two major immune evasion mechanisms by the body and the TGF-β receptor in the tumor microenvironment This depletion is achieved by targeted depletion of anti-PD-β. (1) targeting tumor cells; (2) TGF-β traps (e.g., TGFbR II) binding to TGF-β within the tumor microenvironment; and / or (3) PD-L1 disruption of TGF-β binding via receptor-mediated endocytosis; This can be achieved by a number of methods. The fusion proteins presented herein, or fragments or fragments thereof, Mutants (e.g., PD-α fused to a cytokine trap such as TGF-β trap) PD-1 inhibitors or PD-1 antibodies) also inhibit natural killer cell-mediated killing of tumor cells. It can also promote

[0108] (i) a TGFβRII trap fusion protein, or a fragment or variant thereof In some embodiments, the fusion protein, or a fragment or variant thereof, also comprises a site Kine traps, and PD-1 inhibitors or anti-PD-1 antibodies, or fragments thereof In some embodiments, the present invention also includes a cytokine trap (e.g., TGF-β The fusion protein comprising the cleaved fragment or variant thereof may optionally be The PD-1 inhibitor is fused to the PD-1 inhibitor via a cleavable or non-cleavable linker. In some embodiments, the cytokine trap (e.g., TGF-β trap) In some embodiments, the IL-1 receptor is a TGFβRII receptor (e.g., TGFβRII). The cytokine receptor sequence in the fusion protein to be produced may be a receptor (e.g., TGFβR II) the extracellular domain (ECD) of the polypeptide or a functional variant or derivative thereof. In some embodiments, the extracellular domain (ECD) of TGFβRII is set forth in SEQ ID NO:14. In some embodiments, the fusion protein comprises a polypeptide sequence that is The cytokine receptor sequence in the protein, or a fragment or variant thereof, is set forth in SEQ ID NO: At least 50%, at least 55%, at least 60% with 14 polypeptide sequences , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% In some embodiments, the polypeptide sequence has 0.5%, or 100% identity to the polypeptide sequence. In the fusion proteins described herein, the sequence of the cytokine receptor may be a receptor (e.g., For example, the extracellular domain (ECD) of TGFβRII, or a functional variant or In some embodiments, the extracellular domain (ECD) of TGFβRII is In some embodiments, the polypeptide sequence described herein comprises the polypeptide sequence set forth in SEQ ID NO: 141. The fusion protein, or a fragment or variant thereof, as described above, is a cytokine receptor. The homologous sequence has at least 50%, at least 55%, or both of the amino acid sequence of SEQ ID NO: 141. %, at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 95%, at least 9 9%, at least 99.5%, or 100% identity to a polypeptide sequence In some embodiments, the cytokine receptor in the fusion proteins described herein The sequence of the present invention is derived from the extracellular domain (ECD) of a receptor (e.g., TGFβRII) or from the ECD of this receptor. In some embodiments, the extracellular domain of TGFβRII is a functional variant or derivative thereof. The ECD comprises the polypeptide sequence set forth in SEQ ID NO: 142. In some embodiments, the fusion protein, or fragment or variant thereof, as described herein, The cytokine receptor sequence has at least 5′ homology with the polypeptide sequence of SEQ ID NO: 142. 0%, at least 55%, at least 60%, at least 65%, at least 70%, 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. In some embodiments, the fusion protein described herein comprises a polypeptide sequence. Alternatively, the cytokine receptor sequence in this fragment or variant may be TGF-β1 or and / or TGF-β3, but not TGF-β2. In the fusion protein, or a fragment or variant thereof, as described herein, In certain embodiments, the cytokine receptor sequence binds only to TGF-β1. The cytochrome P450 in the fusion protein, or fragment or variant thereof, as described herein In certain embodiments, the tokine receptor sequence binds only to TGF-β3. The cytokinin in the fusion protein, or a fragment or variant thereof, as described in the document The receptor sequence binds only TGF-β1 and / or TGF-β3, but not TGF-β2. has low or no affinity for .

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

[0110] In some embodiments, the PD-1 antibody fragment or variant is a PD-1 antibody fragment, such as a Fab fragment, F fragment, or F fragment. ab2, (Fab')2, Fv, (Fv)2, scFv, scFv-Fc, Fc, Dia In some embodiments, the PD-1 antibody is a PD-1 antibody, a PD-1 triabody, or a PD-1 minibody. The antibody fragment is a single domain antibody of an antibody against PD-1. In some embodiments, the single domain The antibody is a PD-1 antibody, NAR Fragment or V H It is the H fragment.

[0111] Non-limiting, exemplary fusion proteins are illustrated in Figures 4A-4C. an anti-PD-1 antibody, or a fragment or variant thereof, fused to a TGF-β trap Fusion proteins containing PD-L1 bind to PD-1 on tumor cells and PD-L1 on immune cells. Synergistic effects due to simultaneous blockade of TGF-β interactions and neutralization of TGF-β within the tumor microenvironment Without being bound by theory, this effect may be due to the binding of a single molecular entity to the This results in the simultaneous blockade of two major immune evasion mechanisms and the upregulation of TGF-β in the tumor microenvironment. This depletion is achieved by (1) targeting PD-1-mediated tumor suppression. (2) targeting tumor cells; (3) by TGF-β traps (e.g., TGFβRII) , binding to TGF-β within the tumor microenvironment; and (3) PD-L1 receptor-mediated endoscopy. This is achieved by disrupting the binding of TGF-β via mitosis.

[0112] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. The variable region (V) of the heavy chain of the antibody or a fragment / variant thereof H ) have been merged into other implementations. In this form, the TGF-β trap is fused to the IgG of the PD-1 antibody (e.g., Figure 4A). In certain embodiments, the IgG is an IgG1, IgG2, IgG3, or IgG In one embodiment, the IgG is IgG4. In another embodiment, the IgG is IgG5. 4 is SEQ ID NO: 146 (wild type), SEQ ID NO: 291, SEQ ID NO: 292, or SEQ ID NO: 1 47(S108P). In some embodiments, a TGF-β trap (e.g., TGF βRII) is a potential heavy chain of a PD-1 antibody, or a fragment / variant thereof, via a linker. Variable domain (V H In some embodiments, the TGF-β trap (e.g., , TGFβRII) is linked via a linker to a PD-1 antibody, or a fragment / variant thereof, V H PD-1 antibody, or a fragment or variant thereof, V H Examples of sequences include, but are not limited to, SEQ ID NOs: 1-7 and 149-164. PD-1 antibody, or a fragment or variant thereof, L Examples of sequences are SEQ ID NOs: 8 to 13 and and 148. In some embodiments, TGF-β trap ( For example, TGFβRII) is a PD-1 antibody or a fragment / variant thereof, which binds to the variable region of the light chain. Area(V L In some embodiments, the TGF-β trap (e.g., T GFβRII) via a linker to the V of a PD-1 antibody, or a fragment / variant thereof. L In some embodiments, the TGF-β trap (e.g., T GFβRII) via a linker to the light chain of a PD-1 antibody, or a fragment / variant thereof. The variable region (V L In one embodiment, the TGF-β trap (e.g., T GFβRII) is a VL chain or VH chain, or a fragment / variant thereof, via a linker. It is fused to the N- or C-terminus of the variant.

[0113] "Anti-PD1 (VL / VH)-TGFβRII" or "Anti-PD1 (VH / VL)-TG The terms "FβRII" and "FβRII" are used interchangeably and refer to the specific In one embodiment, the VL or VH is an anti-PD1 (VL / VH)-TGFβR The terms "anti-PD1 (VH / VL)-TGFβRII" and "anti-PD1 (VH / VL)-TGFβRII" refer to the heavy chain of anti-PD1. refers to a TGF-β trap (e.g., TGFβRII) fused to the constant region of the chain, or Or, alternatively, a TGF-β trap (e.g., For example, TGFβRII.

[0114] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. In some embodiments, the TGF-β is fused to a Fab of the TGF-β nucleotide or a fragment / variant thereof. -β trap (e.g., TGFβRII) is linked via a linker to a PD-1 antibody, or In some embodiments, the TGF-β tracking antibody is fused to the Fab of this fragment / variant. The fragment (e.g., TGFβRII) binds to the Fab2 of the PD-1 antibody or its fragment / variant. In some embodiments, a TGF-β trap (e.g., a TGFβRII) is fused to ) is fused via a linker to the Fab2 of a PD-1 antibody or its fragment / variant. In some embodiments, the TGF-β trap (e.g., TGFβRII) is D-1 antibody, or a fragment / variant thereof, fused to (Fab')2. In this form, the TGF-β trap (e.g., TGFβRII) is attached to the PD via a linker. In one embodiment, the antibody is fused to (Fab')2 of the Fab'-1 antibody, or a fragment / variant thereof. TGF-β trap (e.g., TGFβRII) is linked to a PD-1 antibody via a linker. or this fragment / variant is fused to the N-terminus or C-terminus of Fab or (Fab')2. are combined.

[0115] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. In some embodiments, the TGF- The β-trap (e.g., TGFβRII) can be linked to a PD-1 antibody or In some embodiments, the TGF-β trap ( For example, TGFβRII) binds to PD-1 antibodies or their fragments / variants (Fv)2. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to the (Fv)2 of a PD-1 antibody, or a fragment / variant thereof. In one embodiment, the TGF-β trap (e.g., TGFβRII) comprises a linker. The N-terminus or Fv or (Fv)2 of the PD-1 antibody, or fragment / variant thereof, is or fused to the C-terminus.

[0116] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. In some embodiments, the TG is fused to an scFv of the TG or a fragment / variant thereof. F-β traps (e.g., TGFβRII) can be linked to PD-1 antibodies or In some embodiments, the TGF-β receptor is fused to the scFv of this fragment / variant. RAP (e.g., TGFβRII) is a PD-1 antibody or its fragment / variant scF In some embodiments, the TGF-β trap (e.g., TG FβRII) is a scFv of a PD-1 antibody or its fragment / variant via a linker. -Fc. In one embodiment, a TGF-β trap (e.g., TGFβRII) ) is a fragment or variant of a PD-1 antibody or fragment / variant thereof, linked via a linker. It is fused to the N-terminus or C-terminus of cFv-Fc.

[0117] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. In some embodiments, the TGF- The β-trap (e.g., TGFβRII) can be linked to a PD-1 antibody or In some embodiments, the TGF-β trap ( For example, TGFβRII) is a PD-1 antibody or a fragment / variant thereof, which binds to the C-terminal Fc In some embodiments, a TGF-β trap (e.g., a TGFβRI) is linked to I) is linked to the C-terminal Fc of a PD-1 antibody or a fragment / variant thereof via a linker. In some embodiments, a TGF-β trap (e.g., TGFβRII) is linked to ) is linked to the N-terminal Fc of the PD-1 antibody, or fragment / variant thereof. In some embodiments, the TGF-β trap (e.g., TGFβRII) is linked via a linker. and linked to the N-terminal Fc of the PD-1 antibody, or fragment / variant thereof.

[0118] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. In some embodiments, the diabody is fused to a diabody of the present invention, or a fragment / variant thereof. The TGF-β trap (e.g., TGFβRII) is linked to a PD-1 antibody via a linker. or a fragment / variant thereof. TGF-β traps (e.g., TGFβRII) are targeted to PD-1 antibodies, or fragments / variants thereof. In some embodiments, the TGF-β trap ( For example, TGFβRII) is linked to a PD-1 antibody, or a fragment / mutant thereof, via a linker. In some embodiments, the TGF-β trap (e.g., a TGF-β trap) is fused to a triabody of the TGF-β receptor. For example, TGFβRII) binds to PD-1 antibodies or minibodies of its fragments / mutants. In some embodiments, a TGF-β trap (e.g., TGFβRII) is fused via a linker to a PD-1 antibody or a minibody of this fragment / variant. In one embodiment, the TGF-β trap (e.g., TGFβRII) is via the N-terminus or C-terminus of the PD-1 antibody, or fragment / variant thereof, or minibody. are fused to the end.

[0119] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. V of this body, or fragments / variants NAR In some embodiments, the TG F-β traps (e.g., TGFβRII) can be linked to PD-1 antibodies or is the V of this fragment / variant NAR In all embodiments described, TGF-β trap (e.g., TGFβRII) is linked to a PD-1 antibody via a linker. or this fragment / variant, V NAR It is fused to the N-terminus or C-terminus of

[0120] In some embodiments, the TGF-β trap (e.g., TGFβRII) is a PD-1 antagonist. V of this body, or fragments / variants H In some embodiments, the TGF -β trap (e.g., TGFβRII) is linked via a linker to a PD-1 antibody, or V of this fragment / variant HIn all embodiments described, the TG F-β traps (e.g., TGFβRII) can be linked to PD-1 antibodies or is the fragment / variant of V H It is fused to the N-terminus or C-terminus of H.

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

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

[0123] In some embodiments, the variable region of the heavy chain (V H) is one or more polypeptides shown in any one of SEQ ID NOs: 1 to 7 In some embodiments, the heavy chain of a PD-1 antibody, or fragment / variant thereof, comprises a sequence Variable domain (V H ) is shown in any one of SEQ ID NOs: 1 to 7 and 149 to 164 at least 50%, at least 55%, or At least 60%, at least 65%, at least 70%, at least 75%, at least At least 80%, at least 85%, at least 90%, at least 95%, at least 99% , one or more polypeptides having at least 99.5%, or 100% identity Contains code arrays.

[0124] In some embodiments, the variable region of the heavy chain (V L ) is one or more of the sequences shown in any one of SEQ ID NOs: 8 to 13 and 148 In some embodiments, the PD-1 antibody, or fragment / mutation thereof, comprises the polypeptide sequence of The variable region of the heavy chain (V L ) are polypeptides shown in SEQ ID NOs: 8 to 13 and 148. At least 50%, at least 55%, at least 6 0%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least Contains one or more polypeptide sequences with 99.5% or 100% identity .

[0125] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO:6. The variable region of the heavy chain (V H) and a light chain comprising the polypeptide sequence set forth in SEQ ID NO: 12. The variable region (V L In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:6. and at least 50%, at least 55%, at least 60%, at least 6 5%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or The variable regions of the heavy chains (V H ) and the sequence shown in SEQ ID NO: 12 and at least at least 50%, at least 55%, at least 60%, at least 65%, at least 7 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical , the light chain variable region (V L ) and

[0126] In some embodiments, the fusion protein comprises a sequence as set forth in SEQ ID NO: 15 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least and sequences that are 95%, at least 99%, at least 99.5%, or 100% identical. , the sequence shown in SEQ ID NO: 16 and at least 50%, at least 55%, at least 6 0%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical sequences.

[0127] In some embodiments, the fusion protein comprises a sequence shown in SEQ ID NO: 15 and a sequence shown in SEQ ID NO: 1 6.

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

[0129] In some embodiments, the fusion protein comprises a sequence shown in SEQ ID NO: 15 and a sequence shown in SEQ ID NO: 1 43.

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

[0131] In one embodiment, the fusion protein comprises a sequence as set forth in SEQ ID NO: 15 and a sequence as set forth in SEQ ID NO: 29. 4.

[0132] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO:7. The variable region of the heavy chain (V H ) and a light chain comprising the polypeptide sequence set forth in SEQ ID NO: 13. The variable region (V L In some embodiments, the fusion protein comprises the nucleotide sequence shown in SEQ ID NO:7. and at least 50%, at least 55%, at least 60%, at least 6 5%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or The variable regions of the heavy chains (V H ) and the sequence shown in SEQ ID NO: 13 and at least at least 50%, at least 55%, at least 60%, at least 65%, at least 7 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical , the light chain variable region (V L ) and

[0133] In some embodiments, the fusion protein comprises at least one sequence selected from the group consisting of the sequence set forth in SEQ ID NO:296. At least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, less sequences that are 95%, at least 99%, at least 99.5%, or 100% identical to and the sequence set forth in SEQ ID NO: 145, and at least 50%, at least 55%, at least At least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 99%, less It also includes sequences that are 99.5% or 100% identical.

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

[0135] In some embodiments, the fusion protein comprises at least one sequence selected from the group consisting of the sequence set forth in SEQ ID NO:296. At least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, less sequences that are 95%, at least 99%, at least 99.5%, or 100% identical to and the sequence set forth in SEQ ID NO: 144, and At least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 99%, less It also includes sequences that are 99.5% or 100% identical.

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

[0137] In some embodiments, the fusion protein comprises at least one sequence selected from the group consisting of the sequence set forth in SEQ ID NO:296. At least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, less sequences that are 95%, at least 99%, at least 99.5%, or 100% identical to and the sequence set forth in SEQ ID NO: 295, and at least 50%, at least 55%, at least At least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 99%, less It also includes sequences that are 99.5% or 100% identical.

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

[0139] In some embodiments, the fusion protein comprises a sequence as set forth in SEQ ID NO: 12 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least and sequences that are 95%, at least 99%, at least 99.5%, or 100% identical. , the sequence shown in SEQ ID NO: 16 and at least 50%, at least 55%, at least 6 0%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5% or 100% identical sequences.

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

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

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

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

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

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

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

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

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

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

[0150] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO:5. The variable region of the heavy chain (V H ) and a light chain comprising the polypeptide sequence set forth in SEQ ID NO:8. Variable region (V L In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:5. and at least 50%, at least 55%, at least 60%, at least 65%. %, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, at least 99%, at least 99.5%, or 1 The variable regions of the heavy chains (V H ) and the sequence shown in SEQ ID NO: 8 and at least At least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, less 95%, at least 99%, at least 99.5%, or 100% identical to the original The variable region of the chain (V L ) and

[0151] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 149. The variable region of the heavy chain (V H ) and a polypeptide sequence as set forth in SEQ ID NO:8. The variable region of the chain (V L In some embodiments, the fusion protein comprises SEQ ID NO: 14 9 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical heavy chain variable regions (V H ) and the sequence shown in SEQ ID NO: 8; At least 50%, at least 55%, at least 60%, at least 65%, at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L ) and

[0152] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 157. The variable region of the heavy chain (V H ) and a polypeptide sequence as set forth in SEQ ID NO:8. The variable region of the chain (V L In some embodiments, the fusion protein comprises SEQ ID NO: 15 7 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical heavy chain variable regions (V H ) and the sequence shown in SEQ ID NO: 8; At least 50%, at least 55%, at least 60%, at least 65%, at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L ) and

[0153] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (VH ) and a polypeptide sequence as set forth in SEQ ID NO:8. The variable region of the chain (V L In some embodiments, the fusion protein comprises SEQ ID NO: 15 8 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical heavy chain variable regions (V H ) and the sequence shown in SEQ ID NO: 8; At least 50%, at least 55%, at least 60%, at least 65%, at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L ) and

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

[0155] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 149. The variable region of the heavy chain (V HIn some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0156] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 157. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 157 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0157] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0158] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

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

[0160] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 149. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0161] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 150. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0162] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 151. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 151 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0163] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 152. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 152 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0164] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 153. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 153 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0165] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 154. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0166] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 155. The variable region of the heavy chain (V HIn some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0167] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 156. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0168] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 157. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 157 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0169] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0170] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 159. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0171] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 160. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0172] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 161. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 161 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0173] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 162. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 162 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0174] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 163. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 163 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0175] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 164. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

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

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

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

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

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

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

[0182] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 148. The variable region of the light chain (V L In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical light chain variable regions (V L ) is included.

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

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

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

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

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

[0188] (ii) an anti-TGFβ antibody fusion protein, or a fragment or variant thereof; In other embodiments, the cytokine trap is an antibody, antibody fragment, or antibody against TGF-β. is an antibody variant. In such embodiments, such antibodies are anti-TGFβ pan-neutralizing antibodies Anti-TGFβ receptors that block the binding of TGFβ1, 2, and / or 3 to the body or receptors In certain embodiments, the antibody fragment or variant is a TGFβ antibody. Fab, (Fab)2, (Fab')2, Fv, (Fv)2, scFv, scFv- In one embodiment, the antibody is an Fc, an Fc, a diabody, a triabody, or a minibody. In the present invention, an anti-TGF-β antibody, or a fragment or variant thereof, is capable of inhibiting TGF-β1, TGF-β In certain embodiments, an anti-TGF-β antibody or In certain embodiments, the fragment or variant binds to TGF-β1. The TGF-β antibody, or fragment or variant thereof, binds to TGF-β3. In embodiments, the anti-TGF-β antibody, or fragment or variant thereof, is and TGF-β2. In certain embodiments, an anti-TGF-β antibody, or fragment thereof, The fragments or variants bind to TGF-β1 and TGF-β3. Examples of VH sequences of the ... 1, 173, 175, and 177. TGF-β antibodies, Examples of VL sequences of the ... 2, 174, 176, and 178. In some embodiments, The anti-TGF-β antibodies are those represented by SEQ ID NOs: 166, 168, 169, 171, 173, 175, or or 177, and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, at least heavy chain variable regions (V) that are at least 99%, at least 99.5%, or 100% identical H ) In some embodiments, the anti-TGF-β antibody comprises SEQ ID NOs: 165, 167, 170, 172, 174, 176, or 178, and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L ) is included.

[0189] (iii) a TGF-β antagonistic peptide fusion protein, or a fragment thereof, or is a mutant In certain embodiments, the cytokine trap is a TGF-β antagonist peptide. Such peptides may include phage derivatizations, phage derivatizations, or TGF-β inhibitory peptides. In one embodiment, such peptides can be generated de novo using a , a segment of TGF-β isoform(s) or TGF-β receptor(s) An example of a TGF-β antagonist peptide is SEQ ID NO: 193 Examples of TGF-β inhibitory peptides include, but are not limited to, those having SEQ ID NOs. In one embodiment, the peptide may be a peptide of the present invention, including, but not limited to, peptides 193-227. PD-1 antibodies or their fragments / variants via a linker. H and / or V L To In another embodiment, one peptide may be fused to a PD-1 antibody, or a fragment / antibody thereof. In a further embodiment, more than one peptide may be fused to a PD-1 antibody. The peptide may be fused to the polypeptide or a fragment / variant thereof. If the peptides are fused with a linker to form concatamers, In some cases, the peptides are fused together, and in other cases, they are fused together without a linker. If the peptides are identical, the same peptides can be used to form the concatamers. Two or more different peptide combinations are used to form the catamers. Ugh.

[0190] Adenosine: Adenosine is a key immunomodulator within the tumor microenvironment. Extracellular adenosine is A2A adenosine receptors (A2AR), a subtype that is predominantly expressed in half of immune cells, Upon binding, they suppress the inflammatory response. Some tumors also bind to ATP and ADP, respectively. to AMP, and AMP to adenosine. They express high levels of the enzymes CD39 and CD73. Syn inhibits regulatory T cell proliferation by inducing the expression of Foxp3, CD39, and CD73. In addition, hypoxia also promotes the suppressive activity of IL-1 cells through the induction of CD39 and CD73. In addition, the tumor microenvironment induces the accumulation of extracellular adenosine. High levels of extracellular adenosine are responsible for the upregulation of the nucleotide transporter ENT-1. , hypoxia-inducible factor (HIF)-dependent inhibition, and adenosine redistribution within the intracellular lumen. This is maintained by the inhibition of adenosine kinase, which prevents the formation of AMP. Therefore, targeting the reduction of extracellular adenosine within the tumor microenvironment may be beneficial for immune response. It is understood that the purpose of this is to enhance immune cell function and promote the killing of tumor cells.

[0191] In some embodiments herein, the antibody comprises an adenosine deaminase (e.g., ADA2). Fusion proteins targeting reduction of extracellular adenosine in the tumor microenvironment The fusion protein is displayed.

[0192] Adenosine deaminase Adenosine deaminase (also known as adenosine aminohydrolase or ADA) ADA, which is known to be adenosine-containing ATP, irreversibly deaminates adenosine, converting the amino group to a ketone group. This is converted to the related nucleoside, inosine, via substitution with Inosine is converted to hypoxanthine by the purine nucleoside phosphorylation reaction. It is deribosylated (removed from the ribose) by another enzyme called protease (PNP) ADA is required for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues. In humans, its primary function is the development and maintenance of the immune system. The association of α with epithelial cell differentiation, neurotransmission, and maintenance of pregnancy has also been observed. There are two isoforms of DA: ADA1 and ADA2.

[0193] ADA1 is found in most somatic cells, especially in lymphocytes and macrophages. Here, ADA1 is not only present in the cytosol and nucleus, but also dipeptidyl Extracellular form on the cell membrane, conjugated to peptidase 4 (also known as CD26) ADA1 is mostly responsible for intracellular activity and exists as a small form (monomer) and It exists in both small and large forms (dimers). Interconversion from the small to the large form occurs in the lungs. It is regulated by a "transformation factor".

[0194] ADA2 was first identified in human spleen. Later, ADA2 was also expressed in macrophages. ADA2 is also found in other tissues, including the brain, where it coexists with ADA1. The isoforms regulate the ratio of adenosine to deoxyadenosine. ADA2 It is found primarily in human plasma and serum and exists primarily as a homodimer. A2 is the major form present in human plasma and is associated with many diseases, particularly those of the immune system. Diseases: Increased in, for example, rheumatoid arthritis, psoriasis, and sarcoidosis. The ADA2 isoform is also elevated in most cancers. However, it co-localizes with ADA1 in monocytes-macrophages.

[0195] ADA2 fusion protein, or a fragment or variant thereof As used herein, immune checkpoint inhibitors, such as PD-1 inhibitors or PD-1 antibodies and an adenosine deaminase (e.g., ADA2) that can neutralize adenosine. The fusion proteins, or fragments or variants thereof, are provided herein. Proteins, or fragments or variants thereof (e.g., adenosine deaminase (e.g., PD-1 inhibitors or PD-1 antibodies fused to PD-1 receptors (ADA2) can be used to target, for example, PD-1 receptors on tumor cells. and simultaneous blockade of the interaction between PD-L1 on the immune cells and PD-1 on the immune cells, e.g. This may induce a synergistic antitumor effect due to the neutralization of adenosine in the tumor microenvironment. Without being bound by theory, this effect is due to the fact that two major immune escape routes are mediated by a single molecular entity. Simultaneous blockade of immune defense mechanisms and targeted adenosine release within the tumor microenvironment This depletion is achieved by: (1) targeting tumor cells with anti-PD-1; (2) adenosine deaminase (e.g., ADA2) in the tumor microenvironment binding to adenosine; and (3) via PD-L1 receptor-mediated endocytosis. This can be achieved by one or more of the following: disruption of the bond of adenosine.

[0196] In some embodiments, the adenosine deaminase (e.g., ADA2) also inhibits PD- fusion proteins comprising PD-1 inhibitors or PD-1 antibodies, or fragments or variants thereof, or or a portion of a fragment or variant thereof. Fusion proteins containing an enzyme (e.g., ADA2), or a fragment or variant thereof, can be used in any Optionally, fused to a PD-1 inhibitor via a cleavable or non-cleavable linker In some embodiments, the adenosine deaminase is 2 (ADA2). In some embodiments, the fusion proteins described herein are Adenosine deaminase (e.g., ADA2), or a protein capable of carrying out this function, as described herein. Examples of ADA2 and variants thereof are provided by reference. The present invention is described in WO2016061286, the entire contents of which are incorporated herein by reference. In some embodiments, the TGF-β cytokine trap is ADA2 mutant 1. , ADA2 mutant 2, ADA2 mutant 3, ADA2 mutant 4, ADA2 mutant Mutant 5, ADA2 mutant 6, or ADA2 mutant 7 In some embodiments, the fusion proteins presented herein, or fragments thereof, The ADA protein or its functional fragment is fused to the ADA protein or its functional fragment via a linker. In some embodiments, the PD-1 inhibitor or PD-1 antibody is can be an antibody, or a fragment or variant of an antibody, that targets PD-1.

[0197] In some embodiments, the fusion proteins, or fragments or The variant has at least 50%, at least 50%, or at least 50% identity with the polypeptide sequence of SEQ ID NO: 284. 5%, at least 60%, at least 65%, at least 70%, 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 a polypeptide sequence In some embodiments, the fusion proteins, or fragments thereof, described herein The variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, at least polypeptide sequences having at least 99%, at least 99.5%, or 100% identity to the In some embodiments, the fusion proteins, or fragments thereof, described herein also include or variants that are at least 50%, at least partially identical to the polypeptide sequence of SEQ ID NO: 274. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, less polypeptide sequences that have 99%, at least 99.5%, or 100% identity with In some embodiments, the fusion protein, or fragment thereof, described herein. Alternatively, the variant may have at least 50%, at least 50% similarity to the polypeptide sequence of SEQ ID NO: 37275. at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, Polypeptides with at least 99%, at least 99.5%, or 100% identity In some embodiments, the fusion proteins described herein or A fragment or variant of the polypeptide sequence of SEQ ID NO: 276 has at least 50% similarity to the polypeptide sequence of SEQ ID NO: 276. At least 55%, at least 60%, at least 65%, at least 70%, at least At least 75%, at least 80%, at least 85%, at least 90%, at least 95% , polypeptides having at least 99%, at least 99.5%, or 100% identity In some embodiments, the fusion proteins described herein include a nucleotide sequence. The fragment or variant has at least 50% similarity to the polypeptide sequence of SEQ ID NO:277. , at least 55%, at least 60%, at least 65%, at least 70%, less 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 a polypeptide In some embodiments, the fusion proteins described herein also comprise a peptide sequence. The fragment or variant has at least 50% homology with the polypeptide sequence of SEQ ID NO: 278. %, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 9 5%, at least 99%, at least 99.5%, or 100% identity In some embodiments, the fusion proteins or or a fragment or variant thereof, having at least 5'-fold identical sequences with the polypeptide sequence of SEQ ID NO: 279. 0%, at least 55%, at least 60%, at least 65%, at least 70%, 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. Contains the polypeptide sequence.

[0198] In some embodiments, the fusion proteins, or fragments or The mutants include those described above that encode adenosine deaminase (e.g., ADA2) and immunoglobulins. Includes antibodies, antibody fragments or variants that target checkpoint genes. In some embodiments, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and Antibodies targeting immune checkpoints such as programmed cell death 1 (PD-1) The antibody, or a fragment or variant of the antibody, is linked to adenosine deaminase ( For example, it can be fused to an ADA2 molecule.

[0199] In some embodiments, the PD-1 antibody portion inhibits adenosine deaminase (e.g., ADA 2), or a fragment thereof. In some embodiments, the PD-1 antibody portion is fused to: It is fused to an adenosine deaminase (eg, ADA2) via a linker. In some embodiments, the PD-1 antibody portion binds to at least one domain of ADA2. In some embodiments, the PD-1 antibody portion is fused to the ADA via a linker. 2, fused to at least one domain.

[0200] In some embodiments, the PD-1 antibody fragment or variant comprises a PD-1 antibody fragment, such as a Fab fragment ( Fab)2, (Fab')2, Fv, (Fv)2, scFv, scFv-Fc, Fc, Da In some embodiments, the PD A PD-1 antibody fragment is a single domain antibody of a PD-1 antibody. Domain antibodies are PD-1 antibodies with V NAR Fragment or V H It is the H fragment.

[0201] Non-limiting, exemplary fusion proteins are illustrated in Figures 33A-33C. In some embodiments, an anti-PD-1 antibody fused to an adenosine deaminase (e.g., ADA2), or a fusion protein containing this fragment or variant, which binds to PD-L1 on tumor cells and to immune cells. Simultaneous blockade of the interaction between PD-1 on immune cells and extracellular antigens within the tumor microenvironment This may induce synergistic antitumor effects due to targeting of adenosine reduction.

[0202] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. or a fragment / variant thereof, H ) Other implementations In this embodiment, adenosine deaminase is fused to the IgG of the PD-1 antibody (e.g., Figure 4a). In certain embodiments, the IgG is an IgG1, IgG2, IgG3, or IgG1. In one embodiment, the IgG is IgG4. In another embodiment, the IgG is IgG4. G4 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 a potential linker between the heavy chain of a PD-1 antibody, or a fragment / variant thereof, and Variable domain (V H In some embodiments, the enzyme is fused to an adenosine deaminase (e.g., For example, ADA2) is a PD-1 antibody, or a fragment / variant thereof, linked via a linker to the V H In some embodiments, the fusion protein is fused to the constant region of an adenosine deaminase (e.g., For example, ADA2) is a PD-1 antibody or its fragment / variant, which is a light chain variable region (V L ) In some embodiments, the enzyme is fused to an adenosine deaminase (e.g., ADA2 ) is a light chain variable region ( V L In some embodiments, the enzyme is fused to an adenosine deaminase (e.g., A DA2) is a PD-1 antibody or its fragment / variant, linked via a linker to a V L Stationary In one embodiment, the adenosine deaminase is fused to a region via a linker. PD-1 antibodies, or fragments / variants thereof, V H or V L to the N-terminus or C-terminus of It is fused together.

[0203] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. or a fragment / variant thereof. The ATP deaminase (e.g., ADA2) is linked to the PD-1 antibody, or In some embodiments, the adenosine deaminase is fused to the Fab fragment / variant. The enzyme (e.g., ADA2) binds to the (Fab)2 of the PD-1 antibody or its fragment / variant. In some embodiments, the adenosine deaminase (e.g., ADA2) is fused to , fused via a linker to (Fab)2 of a PD-1 antibody, or a fragment / variant thereof. In some embodiments, adenosine deaminase (e.g., ADA2) is involved in the PD fused to (Fab')2 of the Fab'-1 antibody, or a fragment / variant thereof. In this state, adenosine deaminase (e.g., ADA2) binds to PD-1 via a linker. In one embodiment, the adenovirus is fused to the (Fab')2 of an antibody, or fragment / variant thereof. The PD-1 antibody, or a fragment / mutant thereof, is linked to the PD-1 antibody via a linker. It is fused to the N-terminus or C-terminus of Fab or (Fab)2.

[0204] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. or a fragment / variant thereof. The deaminase (e.g., ADA2) is attached to the PD-1 antibody, or fragment thereof, via a linker. In some embodiments, the Fv of the adenosine deaminase ( For example, ADA2) is fused to a PD-1 antibody, or a fragment / variant thereof (Fv)2. In some embodiments, the adenosine deaminase (e.g., ADA2) fused to a PD-1 antibody or its fragment / variant (Fv)2 via a CAR In one embodiment, the adenosine deaminase is linked to the PD-1 antibody or the The fragment / variant of (Fv) is fused to the N-terminus or C-terminus of Fv or (Fv)2.

[0205] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. or a fragment / variant thereof. The syndeaminase (e.g., ADA2) is linked to the PD-1 antibody or In some embodiments, the adenosine deaminase (ADDM) fragment / variant is fused to an scFv of the adenosine deaminase (ADDM) fragment / variant. Enzymes (e.g., ADA2) bind to PD-1 antibodies or scFv-F of fragments / variants thereof. In some embodiments, the enzyme is fused to adenosine deaminase (e.g., ADA). 2) is linked to the scFv-Fc of a PD-1 antibody or its fragment / variant via a linker. In one embodiment, the adenosine deaminase is fused to PD- 1. The N-terminus or C-terminus of the scFv or scFv-Fc of an antibody or fragment / variant thereof are fused to the end.

[0206] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. or a fragment / variant thereof. The deaminase (e.g., ADA2) is attached to the PD-1 antibody, or fragment thereof, via a linker. In some embodiments, the Fc fragment / variant is fused to adenosine deaminase ( For example, ADA2) is linked to the C-terminal Fc of PD-1 antibody or its fragment / variant. In some embodiments, the adenosine deaminase (e.g., ADA2) is It is linked to the C-terminal Fc of the PD-1 antibody or its fragment / variant via a linker. In some embodiments, adenosine deaminase (e.g., ADA2) is involved in the PD In some embodiments, the Fc domain is linked to the N-terminus of the Fc domain of the .DELTA.-1 antibody or fragment / variant thereof. In this state, adenosine deaminase (e.g., ADA2) binds to PD-1 via a linker. It is linked to the N-terminal Fc of the antibody, or fragment / variant thereof.

[0207] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. or a fragment / variant thereof. Adenosine deaminase (e.g., ADA2) is linked to a PD-1 antibody or or a fragment / variant thereof is fused to a diabody. The PD-1 antibody or its fragment / variant is a target of ATP. In some embodiments, the antibody is fused to an adenosine deaminase (e.g., adenosine deaminase). For example, ADA2) is a PD-1 antibody or its fragment / variant via a linker. In some embodiments, the antibody is fused to an adenosine deaminase (e.g., , ADA2) are fused to a PD-1 antibody or its fragment / mutant minibody. In some embodiments, the adenosine deaminase (e.g., ADA2) is via fusion to a minibody of a PD-1 antibody or its fragment / variant.

[0208] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. , or V of this fragment / variant NAR In some embodiments, the adenovirus is fused to The syndeaminase (e.g., ADA2) is linked to the PD-1 antibody or Fragments / variants of V NAR In one embodiment, the adenosine deaminase is fused to , a PD-1 antibody, or a fragment / variant thereof, via a linker, NAR The N-terminus or fused to the C-terminus.

[0209] In some embodiments, an adenosine deaminase (e.g., ADA2) is activated by a PD-1 antibody. , or V of this fragment / variant H In some embodiments, the adenosine is fused to H. The ATP deaminase (e.g., ADA2) is linked to the PD-1 antibody, or Fragment / Variant V H In one embodiment, the adenosine deaminase is fused to PD-1 antibodies, or fragments / variants thereof, via a linker. H N-terminus or C of H are fused to the end.

[0210] In some embodiments, the variable region of the heavy chain (V H ) is one or more polypeptides shown in any one of SEQ ID NOs: 1 to 7 In some embodiments, the heavy chain of a PD-1 antibody, or fragment / variant thereof, comprises a sequence Variable domain (V H ) are the polypeptide sequences shown in SEQ ID NOs: 1 to 7 and 149 to 164 At least 50%, at least 55%, at least 60%, or at least at least 65%, at least 70%, 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 one or more polypeptide sequences.

[0211] In some embodiments, the variable region of the heavy chain (V L ) is one or more of the sequences shown in any one of SEQ ID NOs: 8 to 13 and 148 In some embodiments, the PD-1 antibody, or fragment / mutation thereof, comprises the polypeptide sequence of The variable region of the heavy chain (V L ) are shown in polypeptide SEQ ID NOs: 8-13, and 148 At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, at least 99%, at least and one or more polypeptide sequences with at least 99.5% or 100% identity. nothing.

[0212] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO:6. The variable region of the heavy chain (V H ) and a light chain comprising the polypeptide sequence set forth in SEQ ID NO: 12. The variable region (V L In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:6. and at least 50%, at least 55%, at least 60%, at least 6 5%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or The variable regions of the heavy chains (V H ) and the sequence shown in SEQ ID NO: 12 and at least at least 50%, at least 55%, at least 60%, at least 65%, at least 7 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical , the light chain variable region (V L ) and

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

[0214] In some embodiments, the fusion protein comprises a sequence shown in SEQ ID NO: 12 and a sequence shown in SEQ ID NO: 2. and the sequence shown in SEQ ID NO:80.

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

[0216] In some embodiments, the fusion protein comprises a sequence shown in SEQ ID NO: 12 and a sequence shown in SEQ ID NO: 2. and the sequence shown in SEQ ID NO:81.

[0217] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO:7. The variable region of the heavy chain (V H ) and a light chain comprising the polypeptide sequence set forth in SEQ ID NO: 13. The variable region (V L In some embodiments, the fusion protein comprises the nucleotide sequence shown in SEQ ID NO:7. and at least 50%, at least 55%, at least 60%, at least 6 5%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or The variable regions of the heavy chains (V H ) and the sequence shown in SEQ ID NO: 13 and at least at least 50%, at least 55%, at least 60%, at least 65%, at least 7 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical , the light chain variable region (V L ) and

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

[0219] In some embodiments, the fusion protein comprises a sequence shown in SEQ ID NO: 13 and a sequence shown in SEQ ID NO: 2. and the sequence shown in SEQ ID NO:82.

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

[0221] In some embodiments, the fusion protein comprises a sequence shown in SEQ ID NO: 13 and a sequence shown in SEQ ID NO: 2. and the sequence shown in SEQ ID NO:83.

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

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

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

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

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

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

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

[0229] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 149. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0230] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 150. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0231] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 151. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 151 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0232] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 152. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 152 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0233] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 153. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 153 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0234] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 154. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0235] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 155. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0236] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 156. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0237] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 157. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 157 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0238] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0239] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 159. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0240] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 160. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0241] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 161. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 161 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0242] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 162. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 162 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0243] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 163. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 163 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0244] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 164. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

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

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

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

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

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

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

[0251] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 148. The variable region of the light chain (V L In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical light chain variable regions (V L ) is included.

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

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

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

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

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

[0257] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO:5. The variable region of the heavy chain (V H ) and a light chain comprising the polypeptide sequence set forth in SEQ ID NO:8. Variable region (V L In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO:5. and at least 50%, at least 55%, at least 60%, at least 65%. %, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, at least 99%, at least 99.5%, or 1 The variable regions of the heavy chains (V H ) and the sequence shown in SEQ ID NO: 8 and at least At least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, less 95%, at least 99%, at least 99.5%, or 100% identical to the original The variable region of the chain (V L ) and

[0258] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 149. The variable region of the heavy chain (V H ) and a polypeptide sequence as set forth in SEQ ID NO:8. The variable region of the chain (V L In some embodiments, the fusion protein comprises SEQ ID NO: 14 9 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical heavy chain variable regions (V H ) and the sequence shown in SEQ ID NO: 8; At least 50%, at least 55%, at least 60%, at least 65%, at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L ) and

[0259] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 157. The variable region of the heavy chain (V H ) and a polypeptide sequence as set forth in SEQ ID NO:8. The variable region of the chain (V L In some embodiments, the fusion protein comprises SEQ ID NO: 15 7 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical heavy chain variable regions (V H ) and the sequence shown in SEQ ID NO: 8; At least 50%, at least 55%, at least 60%, at least 65%, at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L ) and

[0260] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (V H ) and a polypeptide sequence as set forth in SEQ ID NO:8. The variable region of the chain (V L In some embodiments, the fusion protein comprises SEQ ID NO: 15 8 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, at least 99%, at least 99.5%, or 100% identical heavy chain variable regions (V H ) and the sequence shown in SEQ ID NO: 8; At least 50%, at least 55%, at least 60%, at least 65%, at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95%, at least 99%, at least 99.5%, or 100% identical The variable region of the light chain (V L ) and

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

[0262] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 149. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0263] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 157. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. 157 , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0264] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (V HIn some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

[0265] In some embodiments, the fusion protein comprises the polypeptide sequence set forth in SEQ ID NO: 158. The variable region of the heavy chain (V H In some embodiments, the fusion protein comprises the sequence At least 50%, at least 55%, at least 60% of the sequence shown in FIG. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 99%, at least 99% 0.5%, or 100% identical heavy chain variable regions (V H ) is included.

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

[0267] Linker In some embodiments, the linker is selected from the group consisting of: In some embodiments, the linker comprises one or more polypeptide sequences that are A flexible linker can be a linker that allows the joined domains to move or The flexible linker can be applied when interaction is required. composed of amino acids that are either polar (e.g., Gly) or polar (e.g., Ser or Thr) The flexible linker may be a sequence consisting primarily of a string of Gly and Ser residues ("G A non-limiting example of a flexible linker is (Gly-Gly-Gly -Gly-Ser)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 By adjusting the copy number "n", functional domains can be The length of this exemplary GS linker may be optimized to achieve adequate separation of Therefore, the length of this exemplary GS linker is optimized to maintain the necessary interdomain interactions. In addition to the GS linker, other flexible linkers can also be used to construct recombinant fusion proteins. In some embodiments, the flexible linker can be (Gly)n, where n is 6. , 7, or 8]. Optionally, the flexible linker may also have a sequence of May be rich in small or polar amino acids such as Gly and Ser, but flexible It may contain additional amino acids such as Thr and Ala to maintain the sequence. In some cases, the linkers described herein can be non-flexible linkers. The functional linker may be a fusion protein, or a fragment or variant thereof, as described herein. Examples of rigid linkers include those that maintain a constant distance between the domains. Alpha helix-forming linkers, Pro-rich sequences, (XP)n, X-, to name a few. The rigid linker may be a Pro backbone, (EAAAK)n (n=1 to 6). Therefore, by adopting an α-helical structure or by containing multiple Pro residues, In some embodiments, the fusion Immune checkpoint inhibitors, such as PD-1 inhibitors, in proteins, or fragments or variants thereof and cytokines that can neutralize cytokines (e.g., TGF-β). A trap (e.g., a TGF-β trap) is a protein encoding an intervening linker polypeptide. In some embodiments, the fusion sequences described herein may be separated by intervening sequences. Immune checkpoint inhibitors, such as PD-1 inhibitors, in proteins, or fragments or variants thereof The cross-linking inhibitor and ADA2 (or a mutant thereof) are connected via an intervening linker polypeptide. In certain embodiments, the peptides may be separated by an intervening sequence encoding the linker. The polypeptides are listed in the table below:

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

[0269] In some embodiments, the linker may be a flexible linker, a non-flexible linker, an in vivo or a cleavable linker, or any combination thereof. links the functional domains together (in the case of flexible and non-flexible linkers) In some cases, such as in the case of in vivo cleavable linkers, the functional domain In some embodiments, the linker may be: It is possible to improve biological activity and increase expression yield, achieving desired pharmacokinetics In some embodiments, the linker may also be a hydrazone, peptide, disulfide, Alternatively, it may comprise a thioether.

[0270] In some cases, the linker sequences described herein may include flexible linkers. Flexible linkers are useful when the domains being joined require some degree of movement or interaction. Flexible linkers can be small, non-polar (e.g., Gly), or polar (e.g., The flexible linker may be composed of amino acids (e.g., Ser or Thr). It may have a sequence consisting primarily of a group and a string of Ser residues ("GS" linker). Examples of linkers include (G4S)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or or 10]. In some embodiments, the flexible linker may have a sequence of (Gly )n, where n can be 6, 7, or 8. Anchoring may also be rich in small or polar amino acids, such as Gly and Ser. but contain additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu improve solubility. By adjusting the copy number "n", the number of functional domains can be The lengths of these non-limiting exemplary linkers are optimized to achieve adequate separation. These non-limiting examples and examples may be used to maintain the necessary inter-domain interactions. In some cases, the length of the flexible linker is optimized. In addition to the GS linker, other flexible linkers are also suitable. may also be utilized in the fusion proteins, or fragments or variants thereof, described herein. Optionally, the flexible linker may also contain small amino acids such as Gly and Ser. Alternatively, it may be rich in polar amino acids, but may contain Thr and Ala to maintain flexibility. In other cases, they may contain additional amino acids such as Lys and Glu. Polar amino acids can be used to improve solubility.

[0271] The flexible linkers incorporated into the linker sequences described herein provide good flexibility. Small or polar amino acids, such as Gly and Ser, are used to confer stability and solubility. The flexible linker may be rich in amino acids. If desired, the domain of the protein, or a fragment or variant thereof, may be selected appropriately. In addition, the flexible linker does not have a rigid structure, but is a linker between functional domains. It may also be used as a passive linker to maintain distance. The length of the flexible linker is , fusion protein, or fragments or variants thereof, In some cases, the compound may be adjusted to allow optimal biological activity. There are also cases where this is the case.

[0272] The linkers described herein can optionally further comprise a non-flexible linker. The rigid linker may be a fusion protein, or a fragment or fragments thereof, as described herein. can be used to maintain a constant distance between the domains of the mutant. Examples include alpha helix-forming linkers, Pro-rich sequences, (XP), to name a few. n, X-Pro backbone, (EAAAK)n (n=1 to 6). In some cases, it may be due to its α-helical structure or the presence of multiple Pro residues. This allows the structure to be relatively rigid.

[0273] In some embodiments, the linkers described herein can be cleavable linkers. In other cases, the linker is not cleavable. A non-cleavable linker is one that is The fusion proteins are then combined to act as one molecule through in vivo or ex vivo processes. The functional domains of the protein, or fragments or variants thereof, are covalently joined together. The linker may also be cleavable in vivo. The anchor is introduced in a way that releases free functional domains in vivo. Cleavable linkers can be cleaved in the presence of reducing agents, proteases, to name a few. For example, reduction of a disulfide bond can be used to cleave the cleavable linker. In the case of disulfide linkers, thiols such as glutathione can be used. A cleavage event via disulfide exchange with could result in cleavage. In vivo cleavage of the linker in the recombinant fusion protein also In vivo, under pathological conditions (e.g., cancer or inflammation), within specific cells or tissues proteases, which may be expressed within the cell or may be restricted to specific cellular compartments. In some cases, the cleavable linker may be used to target the cleavage. For example, the specificity of many proteases is due to the slow binding of linkers within the constraint compartment. Cleavable linkers also include hydrazones, peptides, disulfides, For example, hydrazones may also contain thioethers, which improve serum stability. In other cases, the hydrazone may allow cleavage within the acidic compartment. The solution may have a pH of up to 7. The linker may also include a thioether. Thioethers are designed for intracellular proteolysis. It is possible.

[0274] In some cases, the linker can be an engineered linker. For example, the linker can be a hydrophobic linker. In some cases, at least two different The linker polypeptide sequences may encode the same polypeptide linker sequence. The method of designing the car can be a computational method. Computational methods may include graphical methods. A suitable peptide is selected from a library of three-dimensional peptide structures derived from the database. For example, the Brookhaven Protein Data Base nk (PDB) to measure the spacing distance between selected amino acids in the linker. Optionally, the polypeptide linker may also comprise one or more GS linker sequences. Columns, e.g., (G4S)n, (GS)n, (SG)n, (GSG)n, and (SGSG )n, where n can be any number from 0 to 15.

[0275] Methods for Treating Cancer Also referred to herein are immune checkpoint inhibitors, such as PD-1 inhibitors, and cytokines. cytokine traps (e.g., TGF-β) that can neutralize cytokines (e.g., TGF-β); Also provided herein are methods of treating cancer with fusion proteins comprising a fusion protein comprising a fusion protein (RAP) and a fusion protein (RAP). In addition, immune checkpoint inhibitors such as PD-1 inhibitors and adenosine deaminase Also provided are methods of treating cancer with fusion proteins comprising PD1 and and PD-L1 / 2 signaling pathways, as well as CTLA-4 and CD80 / 8 Targeting blockade of immune checkpoint pathways, such as the 6-mediated signaling pathway The development of monoclonal antibodies has revolutionized cancer treatment, and has led to the development of new treatments for melanoma, non-small cell lung cancer, renal cell carcinoma, Bladder cancer, head and neck squamous cell carcinoma, MSI-rich colorectal cancer, Merkel cell carcinoma, and Durable efficacy in several cancer indications, including but not limited to, Johnson & Johnson lymphoma Despite the durability of responses, the response rate remains extremely low. Some patients develop resistance leading to disease progression. In some indications, such as thyroid, prostate, pancreatic, and many other cancers, checkpoint Inhibitors have failed to show any substantial clinical response.

[0276] Failure of checkpoint blockade is due to the complexity of immunosuppressive factors present in the tumor microenvironment. These factors may be attributed to myeloid-derived suppressor cells, tumor-associated macrophages (TAMs), inhibitory cytokines, and growth factors such as TGF-β, and Interleukin-10 (IL-10); and adenosine and indoleamine 2,3-diamine may include, but are not limited to, metabolic derivatives such as by-products of oxygenase (IDO) As presented herein, the anti-PD1-TGFβRII fusion protein inhibits TGFβRII activity within the tumor microenvironment. These are examples of therapeutics that can target two negative inhibitory pathways: PD-1 / PD-L1 interactions may play a key role in tumor cell-mediated The immune system is a key player in cell-specific interactions, of which TGF-β may be a prominent member. and extracellular interactions mediated by immunosuppressive cytokines.

[0277] In some embodiments, the cancer is glioblastoma, colorectal cancer, gastric cancer, cervical cancer, ovarian cancer, or Cancers that are of concern include, but are not limited to, pancreatic cancer, prostate cancer, breast cancer, and renal cancer. Additionally, the fusion proteins described herein have low response rates to checkpoint blockade. TGF-β is highly expressed in non-small cell lung cancer (NSCL) and melanoma. It may be suitable for application in

[0278] The cancer is a B-cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia Hemoglobinemia, e.g., heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal antibodies, gammopathy, as well as immune cell amyloidosis, melanoma, breast cancer, lung cancer Cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic prostate cancer, hormone deficiency) refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, Peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or throat cancer Head cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, Including, but not limited to, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and hematopoietic tissue cancer Other non-limiting examples of cancer types applicable to the methods encompassed by the present disclosure include: , human sarcomas and human carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic Sarcoma, chondroma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, 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, skin cancer Sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, Liver cancer, hepatocellular carcinoma (HCC), choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer , bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocyte tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrocyte tumor tumors, meningiomas, melanomas, neuroblastomas, retinoblastomas; leukemias, e.g., acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, leukemia, and erythroleukemia); chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), Includes multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In embodiments, the cancer whose phenotype is determined by the methods of the present disclosure is bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer cancer, such as, but not limited to, ovarian, pancreatic, prostate, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In yet another embodiment, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, or intestinal cancer. Cancer of the cervix, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. cancer, endometrial cancer, mucinous cancer, clear cell carcinoma, Brenner carcinoma, or cancer of unknown nature In some embodiments, the molecule may be characterized in a variety of other ways, including, but not limited to: The present disclosure relates to lymphomas or subtypes thereof, including but not limited to mantle cell lymphoma. The present invention is useful in the treatment, diagnosis, and / or prognosis of various types of cancer.

[0279] In certain embodiments, the anti-PD1-TGFβRII fusion proteins presented herein are Proteins are used in standard treatments (including but not limited to chemotherapy and current clinical trials). (I) for approximately 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 5 have an average response rate of 5, 60, 65, 70, 75, 80, 85, 90, or 95% In certain embodiments, the therapeutics provided herein are examples of therapeutics that may be used to treat cancer. As shown, the anti-PD1-ADA2 fusion protein is a promising treatment option for patients with PD-1 / PD-2 / ADA2-associated leukemia. (including but not limited to) approximately 0, 5, 10, 15, 20, 2 5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or are examples of therapies that can be used to treat cancer with an average response rate of 95%. These cancers include Hodgkin's lymphoma, melanoma, non-small cell lung cancer (NSCLC), and Microsatellite instability (MSI) or mismatch repair (MMR) deficient solid tumors , CSCC, RCC, CRC, melanoma, Merkel cell carcinoma, bladder cancer, RCC, hepatocellular carcinoma (HCC), head and neck cancer (H&N), cervical cancer, gastric cancer, small cell lung cancer (SCLC), Endometrial cancer, mesothelioma, ovarian cancer, triple-negative breast cancer (TNBC), breast cancer, including, but not limited to, colorectal cancer (CRC), pancreatic cancer, and prostate cancer.

[0280] Combination Therapy In some embodiments, the fusion protein is administered as a combination therapy with an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a biological molecule such as an antibody. The treatment involves the binding of fusion proteins to EGFR, HER2 / ErbB2, and / or VEG Combination with antibodies against tumor-associated antigens, including but not limited to antibodies that bind to F. In certain embodiments, the additional therapeutic agent specifically targets a cancer stem cell marker. In certain embodiments, the additional therapeutic agent is an angiogenesis inhibitor (e.g., an anti-angiogenic antibody). For example, an antibody that is an anti-VEGF antibody or a VEGF receptor antibody. Further treatments include bevacizumab (AVASTIN), ramucirumab, and trastuzumab. Mab (HERCEPTIN), Pertuzumab (OMNITARG), Panitumumab (VE CTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX) In some embodiments, the additional therapeutic agent comprises a drug such as a small molecule. The position of the fusion protein presented herein, or a fragment or variant thereof, is R, HER2 (ErbB2), and / or VEGF, This may involve the combined administration of small molecules that act as inhibitors against tumor-associated antigens. In embodiments, the fusion protein, or a fragment or variant thereof, is gefitinib (IRE) or SSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatinib (lapatanib), vandetanib (ZACTIMA), AEE788, CI-1033, Ziranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib ( GW786034B) in combination with a protein kinase inhibitor In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor. In embodiments, the additional therapeutic agent is chemotherapy, or T REG Reducing the number of cells, other In certain embodiments, the therapeutic agent is cyclophosphamide or an anti-CTL inhibitor. In another embodiment, the additional therapeutic agent is an antibody that inhibits the presence or absence of myeloid-derived suppressor cells. In a further embodiment, the additional therapeutic agent is carbotaxol. In a further embodiment, the additional therapeutic agent is ibrutinib.

[0281] In some embodiments, the methods involve using a fusion protein, or a fragment thereof, as described herein. and further comprising one or more checkpoint inhibitors in combination with the fragment or variant. In some embodiments, the additional checkpoint inhibitor is an anti-CTLA-4 antibody. Anti-CTLA-4 antibodies (e.g., ipilimumab) have been shown to be effective in treating patients with advanced melanoma. demonstrated durable antitumor activity and prolonged survival in patients with rheumatoid arthritis. This led to its approval by the Food and Drug Administration (FDA). d survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. ( 2010) Aug 19; 363(8):711-23. In some embodiments, one or more The checkpoint inhibitor may be an anti-PD-L1 antibody. L1 antibodies include full-length atezolizumab (anti-PD-L1), avelumab (anti-PD-L1), and duplex PD-L1. It may be ruvalumab (anti-PD-L1), or a fragment or variant thereof. In embodiments, the one or more checkpoint inhibitors include a CD27 inhibitor, a CD28 inhibitor, agents, CD40 inhibitors, CD122 inhibitors, CD137 inhibitors, OX40 (as CD134 inhibitors (also known as GITR inhibitors), ICOS inhibitors, or any combination thereof In some embodiments, one or more chains may be any one or more of the chains. Checkpoint inhibitors include the A2AR inhibitor, B7-H3 (also known as CD276) inhibitors, B7-H4 (also known as VTCN1) inhibitors, BTLA inhibitors, IDO inhibitor, KIR inhibitor, LAG3 inhibitor, TIM-3 inhibitor, VISTA inhibitor, or It can be any one or more of any combination of these.

[0282] In certain embodiments, the additional therapeutic agent comprises a second immunotherapeutic agent. In this condition, further immunotherapeutic agents are used, such as colony-stimulating factors, interleukins, and immunosuppressive agents. Blocking antibodies (e.g., anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD3 antibodies, 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 receptor ( For example, TLR4, TLR7, and TLR9), soluble ligands (for example, GITRL, GI TRL-Fc, OX-40L, OX-40L-Fc, CD40L, CD40L-Fc, 4 -1BB ligand, or 4-1BB ligand-Fc), or a member of the B7 family In some embodiments, the present invention provides a method for the preparation of immunoglobulins, including, but not limited to, CD80, CD86, and CD87. , Further immunotherapeutic agents include CTLA-4, CD28, CD3, PD-L1, TIGIT, Targeting GITR, OX-40, CD-40, or 4-1BB.

[0283] In some embodiments, the additional therapeutic agent is an additional immune checkpoint inhibitor In some embodiments, the additional immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti- CTLA-4 antibody, anti-CD28 antibody, anti-TIGIT antibody, anti-LAG3 antibody, anti-TIM3 antibody The antibody is 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. The therapeutic agents are BMS935559 (MDX-1105), atexolizumab ( MPDL3280A), durvalumab (MEDI4736), and avelumab (MS B0010718C). In some embodiments, the additional therapeutic agent is selected from ipilimumab (YERVOY) and tremelimumab. In some embodiments, the additional treatment is an anti-CTLA-4 antibody selected from the group consisting of: The agent is an anti-LAG- In some embodiments, the additional therapeutic agent is MEDI6469, MEDI0 562, and MOXR0916. In some embodiments, the additional therapeutic agent is selected from the group consisting of PF-05082566. In some embodiments, the fusion protein, or a fragment thereof, is an anti-4-1BB antibody. Alternatively, the mutants may inhibit cytokines such as adrenomedullin (AM) and angiopoietin (A ng), 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), N GF, neurotrophin, PDGF, thrombopoietin, TGF-α, TGF-β, T NF-α, VEGF, PlGF, gamma-IFN, IL-1, IL-2, IL-3, IL -4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18 The compound may be administered in combination with a biomolecule selected from the group consisting of:

[0284] cytokines In some cases, the cytokine is at least one of a chemokine, an interferon, an interferon-like compound, and a phospholipid. Interleukins, lymphokines, tumor necrosis factors, or variants thereof, or combinations thereof Optionally, the cytokine is an interleukin. Optionally, the interleukin Tarleukin is 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, I L-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 In some embodiments, the polypeptide is at least one of a fully functional variant and a fragment. The cytokine may be membrane-bound or secreted. The kinases are soluble IL-15, soluble IL-15 / IL-15Rα complexes (e.g., AL In certain cases, the interleukin is membrane-bound IL-15 ( mbIL-15), or a fusion of IL-15 with IL-15Rα. In embodiments, mbIL-15 is used in combination with engineered immune effector cells, as described herein. In some embodiments, mbIL-1 is a membrane-bound chimeric IL-15 that can be co-expressed with mbIL-1. 5 is a full-length IL-15Rα fragment or a mutant thereof fused in-frame with the full-length IL-15Rα fragment or mutant thereof. IL-15 (e.g., a native IL-15 polypeptide), or a fragment or variant thereof In some cases, IL-15 is indirectly linked to IL-15Rα via a linker. In some cases, mbIL-15 is linked to Hurton et al., "Tethered IL-1 5 augments antitumor activity and promotes a stem-cell memory subset in tumor-sp As described in “Primitive T cells,” PNAS 2016. The tokine is expressed in the same immune effector cells as the CAR.

[0285] In some embodiments, mbIL-15 is a HER1t, HER2, or HER3 antibody, as described herein. It is expressed with a cell tag such as CD20t-1, CD20t-1, or CD20. L-15 binds HER1t, HER-1t-1, CD20t-1, or CD20 to It may be expressed in frame.

[0286] In some embodiments, mbIL-15 is an inducible promoter for gene transcription. In one aspect, the inducible promoter is a promoter that is under the control of a ligand-induced gene switch. Optionally, the inducible promoter is RHEOSWIT CH® gene switches, small molecule ligand-induced, two-polypeptide Therefore, it may be an ecdysone receptor-based gene switch.

[0287] In another aspect, the interleukin can include IL-12. L-12 is a synthetic IL-12 derived from single-chain IL-12 (scIL-12), protease-sensitive IL-12, and anxiety-inducing IL-12. IL-12 is a type of IL-12 that is expressed in the body as a single molecule. -12 variants, all of which are incorporated herein by reference in their entirety. International Publication No. 2015 / 095249, International Publication No. 2016 / 048 Described in Pamphlet No. 903 and Pamphlet No. WO 2017 / 062953 In some embodiments, the cytokines described above are expressed by the gene For transcription, the gene may be under the control of an inducible promoter. In one embodiment, the inducible promoter The promoter may be a ligand-inducible promoter of a gene switch. The promoter can be coupled to a small molecule ligand, such as the RHEOSWITCH® gene switch. An ecdysone receptor-based gene switch with two polypeptides induced by cyclin. It's possible.

[0288] In some embodiments, the fusion proteins described herein are used to induce transcription of a gene. The gene may be under the control of an inducible promoter. In one embodiment, the inducible promoter is a gene sequence. Optionally, the inducible promoter may be a ligand-inducible promoter of the gene. provides small molecule ligand-inducible gene switches, such as the RHEOSWITCH® gene switch. It may be an ecdysone receptor-based gene switch with two polypeptides.

[0289] Genetic Switch As used herein, gene switch polypeptides, ligand-inducible gene switch polypeptides, Polynucleotides encoding these polypeptides and / or polypeptides Methods and systems for incorporating nucleotides are presented. The term "gene switch" refers to It refers to the combination of a promoter and its associated response elements, e.g., one or more ligands. In the presence of a nucleotide, the expression of a gene containing a response element and promoter is modulated. This refers to the ecdysone receptor (EcR)-based system that regulates the expression of a tightly regulated inducible gene. Gene expression systems or gene switches are useful for gene therapy, intracellular, large-scale protein expression. Creation, cell-based high-throughput screening assays, functional genomics, etc. and the regulation of traits in transgenic plants and animals. Such an inducible gene expression system is useful for a variety of applications. It may comprise an expression system.

[0290] An early form of EcR-based gene switch was discovered in Drosophila melanogaster ( anogaster EcR (DmEcR) polypeptide and mouse (Mus musculus) RXR (MmRXR) polypeptides, and these receptors bind to the steroid ponasteride. In mammalian cell lines and transgenic mice in the presence of phenobarbital A, reporter have been shown to transactivate genes (Christopherson et al., 1992;No et al. , 1996). Subsequently, Suhr et al., 1998, reported that a nonsteroidal ecdysone agonist, Tebufenozide is a cytosolic compound that inhibits the growth of silkworms (Bombyx mori) in the absence of exogenous heterodimer partners. ori)EcR(BmEcR) via high levels of reporter genes in mammalian cells It was shown that it induces transactivation of

[0291] PCT International Patent Application / US Application No. 97 / 05330 (International Publication No. 97 / 381 17) and PCT International Patent Application / U.S. Application No. 99 / 08381 (WO 99 / 58155) modulates the expression of exogenous genes a DNA construct comprising an exogenous gene and an ecdysone response element; The construct reacts with the ligand for the construct and, optionally, with a silent partner. In the presence of a receptor that can act as a Methods of activation by a second DNA construct containing an ecdysone receptor that induces gene expression In this example, the ecdysone receptor was expressed in Drosophila melanogaster (Dr. Typically, such systems have been isolated from Escherichia coli (Bombyx mori) for optimal activation. To achieve this, the presence of a silent partner, preferably the retinoid X receptor (RXR), is required. In mammalian cells, the insect ecdysone receptor (EcR) is required for the mammalian ecdysone receptor. Heterodimerizes with rhodopsin X receptor (RXR), thereby targeting target genes or heterologous genes. It can be used to regulate gene expression in a ligand-dependent manner. Patent application / US application No. 98 / 14215 specification (International Publication No. 99 / 02683 pamphlet The ecdysone receptor isolated from the silkworm moth, Bombyx mori, It has been shown to be functional in mammalian systems without the need for an exogenous dimer partner. It shows.

[0292] U.S. Patent No. 6,265,173 describes a steroid / thyroid superfamily of receptors. Various members of the Millipore Standards are incorporated herein by reference in their entirety, at least in accordance with the U.S.P. Drosophila melanogaster ultraconjugates containing dimerization domains It is disclosed that the spirulina receptor (USP) or a fragment thereof can be combined with the spirulina receptor (USP) or a fragment thereof. U.S. Patent No. 5,880,333 describes the use of cinnamon in plants. Drosophila melanogaster EcR and Ultraspiracle (USP) A heterodimer system according to the present invention, wherein the transactivation domain and the DNA binding domain are The present invention discloses a heterodimeric system, which is located on two different hybrid proteins. In each of these cases, a transactivation domain and a DNA-binding domain (P Native EcR as described in CT International Patent Application / US Application No. 98 / 14215. or as in PCT International Patent Application / US Application No. 97 / 05330 and (as modified EcR) into a single molecule, and other The heterodimer partners were used in their native state.

[0293] PCT International Patent Application / US Application No. 01 / 0905 discloses a transactivation domain The DNA-binding domain and the ATP-binding domain are separated from each other by placing them on two different proteins. In the absence of ligand, an ecdysone receptor-based inducible gene expression system was shown to produce background This results in a significant reduction in the activity of the ligand, which is above background This results in a significant increase in activity over that of the two high The hybrid system is described in PCT / US Application No. 97 / 05330 and PCT / US Application No. A significant improvement over the two systems disclosed in 98 / 14215. The two-hybrid system is an inducible gene expression modulation system. When the domain binds to the DNA binding site on the gene, the transactivation domain activates the promoter. Pairs of interacting proteins interact with transcriptional activation proteins to activate the motor more efficiently. This is thought to take advantage of the ability to place the DNA domain in a more favorable position relative to the DNA binding domain. (See, for example, U.S. Patent No. 5,283,173.) The Lid gene expression system expresses a DNA-binding domain fused to a nuclear receptor polypeptide. a first gene expression cassette encoding a first nuclear receptor polypeptide fused to a second nuclear receptor polypeptide; A second gene expression cassette encoding a transactivation domain, In the presence of a ligand, the antibody expresses a TGF-β cytokine. This induces a conformational change that promotes interaction with the DNA This is thought to result from dimerization of the binding domain with the transactivation domain. The DNA-binding domain and the transactivation domain are located on two different molecules. In the absence of ligand, background activity is greatly reduced because .

[0294] Certain modifications of the two-hybrid system also involve the use of steroidal ligands, e.g., ponasteroids. Non-steroidal anti-inflammatory drugs (NSAIDs) when compared with PonA or Muristerone A (MurA) It may also result in improved sensitivity to steroid ligands, e.g., diacylhydrazines. That is, compared to steroids, nonsteroidal ligands are more potent at low ligand concentrations. Furthermore, the two-hybrid system demonstrated that unmodified RXR Some of the effects are due to overexpression of RXR, which may occur when RXR is used as a switching partner. In a preferred two-hybrid system, the natural D of EcR or RXR is The NA-binding and transactivation domains were removed, resulting in the The hybrid molecule has the potential to interact with other steroid hormone receptors present in the cell. This reduces the likelihood of side effects.

[0295] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily. Classified into subfamily 1, group H (referred to herein as "group H nuclear receptors") Members of each group share 40–60% amino acid identity within the E (ligand-binding) domain. (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor r Subfamily, 1999; Cell 97: 161-163). In addition to the ecdysone receptor, this nuclear receptor Other members of subfamily 1, group H, are ubiquitous receptors (URs), orphan receptors, and steroid hormone nuclear receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXR interacting Liver protein 15 (RIP-15), liver x receptor beta (LXRβ), steroid hormone receptor receptor-like protein (RLD-1), liver x receptor (LXR), liver x receptor α (LXRα ), farnesoid x receptor (FXR), receptor-interacting protein 14 (RIP-1 4), and the farnesol receptor (HRR-1).

[0296] In some cases, the inducible promoter ("IP") is a promoter available from Intrexon Corp. Small molecule ligand-induced A potential, two-polypeptide, ecdysone receptor-based gene switch In some cases, the gene switches are incorporated herein by reference in their entirety. PCT / US Application No. 2001 / 009050 (International Publication No. 2001 / 070816 pamphlet); U.S. Patent No. 7,091,038; U.S. Patent No. 7,7 Specification No. 76,587; Specification No. 7,807,417; Specification No. 8,202,718 Details: PCT / US Application No. 2001 / 030608 (International Publication No. 2002 / 02 9075; U.S. Patent Nos. 8,105,825; 8,168, 426; PCT / US Application No. 2002 / 005235 (International Publication No. WO 2002 / 005235); 02 / 066613 Brochure); U.S. Patent Application Publication No. 10 / 468,200 Specification Publication No. 20120167239; PCT / US Application No. 2002 / 00 5706 (International Publication No. WO 2002 / 066614); U.S. Pat. , 531,326; 8,236,556; 8,598,409 No. PCT / US Application No. 52002 / 005090 (International Publication No. 2002 No. 8,715,959 (U.S. Patent Publication No. PCT / US Application No. 2002 / 005234 (International Publication No. WO 2003 / 027266); U.S. Patent No. 7,601,500 Specification No. 8; Specification No. 7,829,676; Specification No. 7,919,269; Specification No. 7,919,269; No. 8,030,067; PCT / US Application No. 52002 / 005708 ( WO 2002 / 066615; U.S. Patent Application Publication No. 10 / 468 ,192 (U.S. Publication No. 20110212528); PCT / U.S. Application No. 2002 / 005026 specification (International Publication No. 2003 / 027289 pamphlet) ;U.S. Patent Nos. 7,563,879; 8,021,878; 8, No. 497,093; PCT / US Application No. 2005 / 015089 (International Publication Patent Publication No. 2005 / 108617; U.S. Patent No. 7,935,510 ; PCT / US Application No. 52008 / 011270 Detailed description (International Publication No. 2009 / 045370); U.S. Patent Application Publication No. 12 / 241,018 (U.S. Publication No. 20090136465); PCT / U.S. Application No. 2008 / 011563 specification (International Publication No. 2009 / 048560 pamphlet U.S. Patent Application Publication No. 12 / 247,738 (U.S. Publication No. 2009012 3441); PCT / US Application No. 2009 / 005510 (International Publication No. 2010 / 042189); U.S. Patent Application Publication No. 13 / 123,129 Specification (U.S. Publication No. 20110268766); PCT / U.S. Application No. 2011 / No. 029682 (International Publication No. WO 2011 / 119773); U.S. Pat. Application Publication No. 13 / 636,473 (U.S. Publication No. 20130195800 PCT / US Application No. 2012 / 027515 (International Publication No. 2012 / 122 No. 025 Brochure); International Publication No. 2018 / 132494 Brochure (PCT / No. 2018 / 013196 and U.S. Pat. No. 9,402,919 Ecdysone-based receptor-based constructs of any of the systems described in the specification. The components may be selected from, but are not limited to:

[0297] When applied to a ligand-activated ecdysone receptor-based gene switch, this As used herein, the term "ligand" refers to a molecule that activates a gene switch and induces a genetic Various soluble small molecules (e.g., diacylhydrazine compounds) have the ability to stimulate gene expression. ) (i.e., in this activation, polynucleotides (e.g., mRNA, miRNA and / or result in ligand-induced expression of the polypeptide. Examples of such ligands are listed in the accompanying patents, each of which is incorporated herein by reference in its entirety. WO 2004 / 072254 (PCT / US Application No. 2004 / 003775 specification); International Publication No. 2004 / 005478 pamphlet (PCT / U.S. Application No. 2003 / 021149; International Publication No. 2005 / 017126 Pamphlet (PCT / US Application No. 2004 / 005149); International Publication No. 04 / 078924 Brochure (PCT / US Application No. 2004 / 005912 Details International Publication No. 2008 / 153801 (PCT / US Application No. 2008 / 006757 specification); International Publication No. 2009 / 114201 pamphlet (PCT / U.S. Application No. 2009 / 001639; International Publication No. 2013 / 036758 Pamphlet (PCT / US Application No. 2012 / 054141); International Publication No. 14 / 144380 Brochure (PCT / US Application No. 2014 / 028768 Details and WO 2016 / 044390 (PCT / US Application No. 2016 / 044390). 015 / 050375) It will not be done.

[0298] Examples of ligands also include, but are not limited to, ecdysone, 20-hydroxyecdysone ecdysteroids such as ponasterone A and muristerone A, and 9-cis-retinoin and synthetic analogs of retinoic acid, each of which is incorporated herein by reference. US Patent No. 6,258,603; US Patent No. 6,013,836; US Patent No. 5 ,117,057; 5,530,028; and 5,378, Specification No. 726; Specification No. 7,304,161; Specification No. 7,851,220; Specification No. 8,748,125; Specification No. 9,272,986; Specification No. 7,456, Specification No. 315; Specification No. 7,563,928; Specification No. 8,524,948; Specification No. 9,102,648; Specification No. 9,169,210; Specification No. 9,255, 273; and N,N disclosed in US Pat. No. 9,359,289. N,N'-diacylhydrazines such as '-diacylhydrazine; U.S. Patent No. 8,669, 072; and oxalates as described in US Pat. No. 8,895,306. Zolin; a dibenzoyl azoline disclosed in European Application No. 2,461,809 dibenzoylalkylcyanohydrazines such as dibenzoylalkylcyanohydrazine; U.S. Patent No. 5, The N-alkyl-N,N'-diaroyl groups disclosed in 225,443 N-Alkyl-N,N'-diaroylhydrazines such as hydrazine; European Application No. 234,9 N-acyl-N-alkylcarbonylhydrazines, such as those disclosed in the specification of No. 94 Which N-acyl-N-alkylcarbonylhydrazine; U.S. Patent No. 4,985,461 N-aroyl-N-alkyl-N'-aroylhydrazines described in the specification N-aroyl-N-alkyl-N'-aroylhydrazines such as U.S. Pat. No. 7,375 ,093; 8,129,355; and 9,802,936 Amidoketones such as those described in the specification; and 3,5-di- tert-Butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetyl Harpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxy 25-epoxycholesterol, 25-epoxycholesterol, T0901317, 5-alf α-6-alpha-epoxycholesterol-3-sulfate (ECHS ), 7-ketocholesterol-3-sulfate, farnesol ), bile acids, 1,1-biphosphonate esters, juvenile hormone II Examples of diacylhydrazine ligands useful in the present invention include RG-115 819 (3,5-dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl) -N'-(2-methyl-3-methoxy-benzoyl)-hydrazide), RG-11593 2((R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N' -(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-11583 0(3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2- ethyl-3-methoxy-benzoyl)-hydrazide). For example, any of them WO 2008 / 15, which are incorporated herein by reference in their entirety. Pamphlet No. 3801 (PCT / US Application No. 2008 / 006757); and and International Publication No. 2013 / 036758 (PCT / US Application No. 2012 / 0 See US Pat. No. 5,414,141.

[0299] For example, the ligand for the ecdysone receptor-based gene switch can be any suitable Ligands can be selected from naturally occurring ecdysone or ecdysone analogs (e.g., , 20-hydroxyecdysone, muristerone A, ponasterone A, ponasterone B, ponasterone Ponasterone C, 26-iodoponasterone A, inokosterone or 26-mesyl inokosterone Both steroids and nonsteroidal inducers are useful as ligands for the gene switches of the present invention. U.S. Patent No. 6,379,945 discloses a method for producing a fake American tar. Insect steroid receptor ("HEcR") isolated from the box moth (Heliothis virescens) and which is responsive to both steroids and certain non-steroidal inducers. We describe HEcR, which can act as a gene switch. The amide-based inducers have, for example, low production costs, metabolic stability, and the ability to be used in insects, plants, or mammals. For several reasons, including the absence of microbial communities and environmental acceptability, this system and In many other systems, which are responsive to both steroid and non-steroid inducers, , which have clear advantages over steroids. is a compound of two dibenzoylhydrazines, 1,2-dibenzoyl-1-tert-butyl hydrazine, and tebufenozide (N-(4-ethylbenzoyl)-N'-(3, 5-dimethylbenzoyl)-N'-tert-butyl-hydrazine) ecdysone b The present invention describes the utility of ribosomal kinases as ligands for gene switches. Also, as a ligand, the compound disclosed in U.S. Pat. No. 5,117,057 Also included are other dibenzoylhydrazines such as dibenzoylhydrazine. ecdysone receptor from Drosophila melanogaster The use of hydroxybenzoates as chemical ligands for hydroxybenzoates is also described in U.S. Pat. No. 6,147,282. Further non-limiting examples of ecdysone ligands include 3,5-di-t ert-Butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetyl Rupazide, 1,2-diacylhydrazine, N'-substituted-N,N'-disubstituted hydrazine, di Benzoylalkylcyanohydrazine, N-substituted-N-alkyl-N,N-diaroylhydrazine hydrazine, N-substituted-N-acyl-N-alkyl, carbonylhydrazine, or N-acyl and N'-aroyl-N'-alkyl N'-aroylhydrazines (U.S. Pat. No. 6,723,533). (See specification No. 1).

[0300] In one embodiment, the ligand for the ecdysone-based gene switch system is a diacyl hydrazine ligands or chiral diacylhydrazine ligands. The ligands used in the thiol system are represented by Formula I:

[0301] [ka] wherein A is alkoxy, arylalkyloxy, or aryloxy; is optionally substituted aryl or optionally substituted heteroaryl; R and R2 independently optionally represents a substituted alkyl, arylalkyl, hydroxyalkyl, alkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclo, optionally substituted aryl or optionally substituted heteroaryl; or pharmaceutically acceptable salts thereof salts, hydrates, crystalline forms, or amorphous forms] The compound may be:

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

[0303] [ka] [wherein A is alkoxy, arylalkyloxy, aryloxy, arylalkyl B is aryl, optionally substituted aryl, or optionally substituted heteroaryl; , optionally substituted aryl, or optionally substituted heteroaryl; R and and R2, R1 is not identical to R2; R1 and R2 have an asymmetric carbon atom; Independently, optionally, substituted alkyl groups, provided that the absolute configuration in alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally Substituted heterocyclo, optionally substituted aryl, or optionally substituted heteroaryl or a pharmaceutically acceptable salt, hydrate, crystalline form, or non-crystalline form thereof. in crystalline form] The compound may be an enantiomerically enriched compound of the formula:

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

[0305] [ka] [wherein A is alkoxy, arylalkyloxy, aryloxy, arylalkyl B is aryl, optionally substituted aryl, or optionally substituted heteroaryl; , optionally substituted aryl, or optionally substituted heteroaryl; R and and R2, R1 is not identical to R2; R1 and R2 have an asymmetric carbon atom; independently and optionally, substituted alkyl groups, provided that the absolute configuration in alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally Substituted heterocyclo, optionally substituted aryl, or optionally substituted heteroaryl or a pharmaceutically acceptable salt, hydrate, crystalline form, or non-crystalline form thereof. in crystalline form] The compound may be an enantiomerically enriched compound of the formula:

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

[0307] A diacylhydrazine ligand of formula I and a chiral diacylhydrazine ligand of formula II or III The ecdysone-based gene switch system of the present invention can be used in conjunction with the ecdysone-based gene switch system. of exogenous transient regulation of the expression of a therapeutic polypeptide or therapeutic polynucleotide. Each of which is incorporated herein by reference in its entirety. U.S. Patent Nos. 8,076,517 and 8,884,060 and US Pat. Nos. 9,598,355.

[0308] The ligands used in the present invention may form salts. The term "several" refers to inorganic acids and bases and / or organic acids and bases. In addition, the compounds of formula I, II, or When a compound of III contains both a basic and an acidic moiety, it is called a zwitterion ("internal"). As used herein, the term "salt(s)" refers to the salts of the compounds of the present invention. For example, isolation or purification steps that may be employed during preparation include: Pharmaceutically acceptable salts (i.e., non-toxic, physiologically acceptable salts), although other salts are also useful, are preferred. A salt of a compound of Formula I, II, or III can be used, for example, by reacting the compound The substance is added to a medium, such as a medium in which salts precipitate, or to an aqueous medium, with an amount of acid or can be formed by reaction with a base followed by lyophilization.

[0309] Ligands containing a basic moiety can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts are acetates (with acetic acid or trihaloacetic acid, e.g., trifluoroacetic acid). acetates, adipates, alginates, ascorbates, asparagus, Glycates, benzoates, benzenesulfonates, disulfates, borates, butyrates, and citrates Salt, camphor, camphorsulfonate, cyclopentanepropionate, digluconate, Dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, hydrochloride (formed with hydrochloric acid), Hydrobromide (formed with hydrogen bromide), hydroiodide, 2-hydroxyethane sulfonate, lactate, maleate (formed with maleic acid), methanesulfonate Salt (formed with methanesulfonic acid), 2-naphthalenesulfonate, nicotinate , nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate Salt, picrate, pivalate, propionate, salicylate, succinate, sulfate ( sulfonates (such as sulfates formed with sulfuric acid), sulfonates (the sulfonates referred to herein etc.), toluenesulfonates such as tartrates, thiocyanates, and tosylates, undeca Contains phosphates, etc.

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

[0311] Non-limiting examples of ligands for inducible gene expression systems also include FK506, cyclosporin, Ligands that utilize the FK506 binding domain, such as porin A or rapamycin, are also available. FK506, rapamycin, and their analogs are described in U.S. Pat. Specification No. 49,595; Specification No. 6,187,757; Specification No. 7,276,498 and US Pat. No. 7,273,874.

[0312] In some embodiments, the diacylhydrazine ligand for inducible gene expression is 1 Daily unit doses are approximately 5, 10, 15, 20, 25, 30, 35, 40, 50, 60 In some embodiments, the dose is 70, 80, 90, 100, or 120 mg. The diacylhydrazine ligand is administered as a unit dose of approximately 5 mg per day. In some embodiments, the diacylhydrazine ligand is administered at a daily unit dose of about 1 In some embodiments, the diacylhydrazine ligand is administered as 0 mg per day. In some embodiments, the diacylhydramine is administered as a unit dose of about 15 mg per dose. The Jin Ligand is administered daily at a unit dose of about 20 mg per day.

[0313] In some embodiments, combination therapy with two or more therapeutic agents may have different mechanisms of action. Drugs that act by different mechanisms may be used, but this is not required. Combination therapy using different drugs may result in additive or synergistic effects. Combination therapy allows for lower doses of each drug than would be used in monotherapy, thereby , reducing toxic side effects and / or increasing the therapeutic index(ies) of the drug. In some embodiments, the combination therapy may reduce the likelihood of resistant cancer cells developing. Therapy may be a treatment that affects the immune response (e.g., enhances or activates the response). therapeutic agents, and agents that affect tumor / cancer cells (e.g., inhibit or Contains therapeutic agents that kill or destroy bacteria.

[0314] In certain embodiments, the fusion proteins, or fragments thereof, described herein In addition to administering the antibody or variant, the method or treatment may also include administering at least one additional antibody or variant. The method further comprises administering a therapeutic agent to the subject, the additional therapeutic agent being administered prior to administration of the agent. In some embodiments, at least one of the following may be administered simultaneously and / or sequentially: One additional therapeutic agent includes one, two, three, or more additional therapeutic agents.

[0315] In combination with a fusion protein, or a fragment or variant thereof, as described herein Therapeutic agents that may be administered include chemotherapeutic agents. Thus, in some embodiments, the method or treatment is combined with a chemotherapy agent or a cocktail of chemotherapy agents. In some embodiments, the method involves administering an agent described herein. cisplatin in combination with the fusion protein, or a fragment or variant thereof, as described above. and one or more antineoplastic agents, such as cyclosporine, capecitabine, or 5-fluorouracil. Treatment with the agent may be administered prior to, concurrently with, or in addition to the administration of chemotherapy. The combined administration can occur either by co-administration in a single pharmaceutical formulation or by using separate formulations. Co-administration, or generally, co-administration, where all active agents exert their biological activity simultaneously. Such a preparation for chemotherapy may include sequential administration within a time period such that The dosage and administration schedule may be determined according to the manufacturer's instructions or empirically by one skilled in the art. The preparation and administration schedule for such chemotherapy may be See also The Chemotherapy Source Book, 4th Edition, 2008, MC Perry, Also listed by Editor, Lippincott, Williams & Wilkins, Philadelphia, PA .

[0316] Useful classes of chemotherapeutic agents include, for example, antitubulin agents, auristin, DNA minor groove inhibitors, binding agents, DNA replication inhibitors, alkylating agents (e.g., cisplatin, mono(platinum), bis (platinum), and platinum complexes such as trinuclear platinum complexes, as well as carboplatin), anthraquinone Icrin, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycin, eto Posides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, pla Thiaminol, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes In certain embodiments, the compounds include benzodiazepines, topoisomerase inhibitors, vinca alkaloids, and the like. The second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor , or an angiogenesis inhibitor.

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

[0318] Fusion proteins in combination with chimeric receptors In some embodiments, the fusion protein is administered as a combination therapy with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chimeric antigen receptor or an engineered T cell receptor. For example, treatment involves the synthesis of a fusion protein with a chimeric receptor. In one embodiment, the treatment involves simultaneous administration of the fusion protein and the chimeric receptor. In one embodiment, the treatment involves co-administration of the fusion protein with engineered effector cells. The method involves the sequential administration of engineered effector cells containing chimeric receptors followed by the administration of a target gene. In another embodiment, the treatment involves the administration of engineered effector cells comprising a chimeric receptor. In one embodiment, the administration may involve sequential administration of the fusion protein, with at least one subsequent administration. All 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, or 24 In another embodiment, there is 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, 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, 4 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 75, 90 days or more.

[0319] In some embodiments, the engineered effector cells are T cells and / or natural killer cells. T cells or T lymphocytes are modified immune cells, including T cells. T cells or T lymphocytes are involved in cell-mediated immunity. Exemplary T cells include helper T cells, cytotoxic T cells, and T cells. 17 cells, stem cell memory T cells (TSCM), naive T cells, memory T cells, These include effector T cells, regulatory T cells, or natural killer T cells. In aspects, embodiments described herein involve the production of engineered immune effector cells (e.g., T cells). generation and / or expansion of T cells (Tregs, NK cells or NK T cells), The cells are infected with an expression vector containing a DNA (or RNA) construct encoding the chimeric receptor. The method includes producing and / or expanding the cell line, including transfecting the cell line with the desired cell line.

[0320] In some embodiments herein, modified enzymes are provided, including chimeric receptors expressed on the cell surface. In some cases, the chimeric receptor is a tumor antigen, e.g., a For example, antigen binding molecules capable of recognizing and binding to tumor-associated or tumor-specific antigens. In some cases, the antigen-binding region comprises an antibody or binding fragment, e.g., Fab, Fab', F(ab')2, F(ab')3, scFv, sc(Fv)2, ds Fv, diabodies, minibodies, and nanobodies, or binding fragments thereof Optionally, the antigen-binding region comprises an scFv. Optionally, the chimeric receptor The antibody comprises an scFv (e.g., a chimeric antigen receptor (CAR)). Antigen receptors include pattern recognition receptors. In other cases, chimeric receptors are used to engineer T cells. Contains the TCR.

[0321] Further provided herein are kill switches, selectable markers, biomarkers, or any of these. Immune effector cells containing cell tags for use in combination are presented. In this embodiment, the cell tag is HER1t, HER1t-1, CD20t-1, or C D20. Optionally, the cell tag comprises HER1t, wherein said HER1t has the sequence In some instances, the cell tag comprises a polypeptide sequence of HER1t-1. The HER1t-1 comprises the polypeptide sequence of SEQ ID NO:69.

[0322] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) are molecules that transfer exogenous specificity to immune effector cells. In some cases, CARs are engineered receptors that contain an antigen-binding domain. The extracellular domain (ectodomain), stalk region, transmembrane domain, and intracellular domain In some cases, the intracellular domain comprises one or more cytoplasmic domains (endodomains). In some instances, the CARs described herein further comprise an intracellular signaling domain. , antigen-binding domain, stalk region, transmembrane domain, 1 or 2 domains for T cell activation contains multiple costimulatory domains and a signaling domain.

[0323] The antigen-binding domain is the complementarity-determining region of a monoclonal antibody. The complementarity determining regions (CDRs) may comprise a variable region, a complementarity determining region (CDR), ... ) is complementary to the antigen and therefore transmits to the receptor its specificity for this particular antigen. of antigen receptor (e.g., immunoglobulin and T cell receptor) proteins that mediate A short sequence of amino acids found within the variable domain of each polypeptide chain of an antigen receptor. may contain three CDRs (CDR1, CDR2, and CDR3). The antigen-binding domain is F(ab')2, Fab', Fab, Fv, or scFv. Optionally, the antigen-binding domain is an scFv. Optionally, the antigen-binding domain is an scFv. Optionally, the antigen-binding domain is a Fab. Optionally, the antigen-binding domain is a Fab'. Optionally, the antigen-binding domain is F(ab')2. The synthetic domain is Fv.

[0324] In some embodiments, the CARs described herein are capable of expressing 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, M Mucins such as UC-4 or MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, or VEGF- In one embodiment, the antibody comprises an antigen-binding domain that binds to an epitope in R2. The CAR described in the document contains an antigen-binding domain that binds to an epitope on MUC16. In some embodiments, the CARs described herein are expressed on CD19 or CD33. In some cases, the antibody may comprise an antigen-binding domain that binds to an epitope of a target polypeptide. The CAR comprises an antigen-binding domain that binds to an epitope on CD19. Therefore, the CARs described herein contain an antigen-binding domain that binds to an epitope on CD33. In a further embodiment, the CAR described herein comprises a target polypeptide comprising HLA-A2, Myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAd Autoantigens that bind to epitopes on CAM1, SDF1, or type II collagen It comprises a binding domain or an antigen-binding domain.

[0325] In some embodiments, the CARs and methods described herein are used to treat hyperproliferative disorders such as cancer. Treatment of sexual disorders, autoimmune diseases, or viral, bacterial, or parasitic infections, In some embodiments, CARs can be used to treat infections, such as cancer cells, autoimmune cells, and the like. Targets antigens that grow within cells, or cells infected with viruses, bacteria, or parasites Pathogens that can be targeted include, but are not limited to, Plasmodium genus (Plasmodium), Trypanosomes, Aspergillus, Candida dida), Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, HSV, HPV, R SV, EBV, CMV, JC virus, BK virus, or Ebola pathogens. Immunologic diseases include graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, celiac disease, Crohn's disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis, neuromyelitis optica , ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis , pemphigus vulgaris, or autoimmune uveitis.

[0326] The pathogen recognized by CAR can be essentially any type of pathogen. In some embodiments, the pathogen is a fungus, bacterium, or virus. The infectious pathogens are the Adenoviridae, Epstein-Barr virus (EBV), V), cytomegalovirus (CMV), RS (Respiratory Syncyt ial) virus, JC virus, BK virus, HPV, HSV, HHV family Viruses, Hepatitis family viruses, Picornaviridae, Herpes Herpesviridae, Hepadnaviridae, Flaviviridae Flaviviridae, Retroviridae, Orthomyxoviridae (Orthomyxoviridae), Paramyxoviridae, Papovaviridae (Papovaviridae), genus Polyomavirus, family Rhabdoviridae Exemplary pathogenic viruses include pathogens of the families Pseudoviridae, Pseudoviridae, and Togaviridae. Viruses include smallpox, influenza, mumps, measles, chickenpox, Ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, us), Cryptococcus, Histoplasma, Includes the genera Pneumocystis and Stachybotrys. Typical pathogenic bacteria include Streptococcus, Pseudomonas, udomonas, Shigella, Campylobacter, Staphylococcus aureus Staphylococcus, Helicobacter, E. coli i), Rickettsia, Bacillus, Bordetella a), Chlamydia, Spirochetes, and Salmonella In some embodiments, the pathogen receptor Dectin 1 is a member of the Aspergillus oryzae family. CAR, which recognizes carbohydrate structures on the cell walls of fungi such as Aspergillus In another embodiment, viral determinants (e.g., CMV and Based on antibodies that recognize the glycoprotein derived from Ebola, CARs can be made that block the pathology.

[0327] In some embodiments, the "stalk region," "spacer," or "hinge" region is It is used to link the antigen-binding domain to the transmembrane domain. The "stalk domain" or "stalk region" is the region within a polypeptide chain that separates the transmembrane domain. Any oligonucleotide that functions to link to the extracellular or cytoplasmic domain. In some embodiments, the "stalk domain" or The "stalk region" is a region in which the antigen-binding domains are oriented in different directions to facilitate antigen recognition. In some cases, the stalk region is flexible enough to allow the Alternatively, the stalk region may be derived from an immunoglobulin G or IgG1. The CH2CH3 region of the serotonin and optionally a portion of CD3. The region is described in WO 2016 / 073755. CD8α hinge region, 12 amino acid hinge region of IgG4-Fc (ESKYGPPCP PCP), or IgG4 hinge region.

[0328] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be any membrane-bound or transmembrane protein. Suitable transmembrane domains include the alpha and beta chains of the T cell receptor. , or may contain the transmembrane domain of the zeta chain, and may be involved in CD28, CD3 epsilon, CD3 Zeta, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD3 3, CD37, CD64, CD80, CD86, CD134, CD137, or CD1 Alternatively, the transmembrane domain may be synthetic. and may contain hydrophobic residues such as leucine and valine. At one or both ends of the transmembrane domain, phenylalanine, tryptophan, and and valine triplets are found. Optionally, in some embodiments, 2 to 10 amino acids Short oligonucleotide or polypeptide linkers, the length of which is between amino acids may form a link between the transmembrane domain and the cytoplasmic signaling domain of CAR. In some embodiments, the linker is a glycine-serine linker.

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

[0330] The intracellular domain may include one or more costimulatory domains. Exemplary costimulatory domains include: The markers are CD8, CD27, CD28, 4-1BB (CD137), ICOS, and DAP10. , DAP12, OX40 (CD134), or fragments or combinations thereof. In some cases, the CARs described herein are not limited to CD8, CD2 7, CD28, 4-1BB(CD137), ICOS, DAP10, DAP12, OX4 0 (CD134), or a fragment or combination thereof. It may include one or more of, or two or more of, the following: The CARs described herein are CD27, CD28, 4-1BB (CD137), IC Co-stimulatory proteins selected from OS, OX40 (CD134), or fragments or combinations thereof. Contains one or more, or two or more, of the following domains: In the case of , the CAR described herein is a CD8, CD28, 4-1BB (CD13 7), or a fragment or combination thereof, In some cases, the term "a" includes "one or more," "a plurality," or "two or more." The CAR to be carried may be CD28, 4-1BB (CD137), or a fragment thereof or one or more, or two or more, of the costimulatory domains selected from the combination In some cases, the CARs described herein include a co-stimulatory domain. CD28 and 4-1BB (CD137), or their respective fragments. In some instances, the CARs described herein contain a co-stimulatory domain, CD28 and OX40 (CD134), or their respective fragments. The CARs described herein contain co-stimulatory domains, CD8 and CD28, as well as In some cases, the CARs described herein contain fragments of each of these. In some cases, the stimulatory domain of CD28, or a fragment thereof, may be included. The CAR carried is a costimulatory domain, 4-1BB (CD137), or a fragment thereof. In some instances, the CARs described herein comprise a costimulatory domain, OX 40 (CD134), or a fragment thereof. The AR contains the costimulatory domain, CD8, or a fragment thereof.

[0331] In some embodiments, the intracellular domain comprises a signaling domain for T cell activation. In some instances, the signaling domain for T cell activation further comprises a TCR Zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon , CD5, CD22, CD79a, CD79b, or CD66d-derived domains In some cases, the signaling domain for T cell activation is derived from CD3ζ Contains the domain.

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

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

[0334] In some embodiments herein, the antigen-binding domain is selected from the group consisting of F(ab')2, Fab', CD19-specific CARs are described, including Fab, Fv, or scFv. In some embodiments, the antigen-binding domain recognizes an epitope on CD19. In this state, the antigen-binding domain is an endogenous domain on CD19 that is also recognized by FMC63. In some embodiments, the scFv domain and / or VH / VL recognize a target antigen. The domain is derived from FMC63. FMC63 is generally a domain that expresses human-derived CD19. Mouse IgG1 model elicited against Nalm-1 and Nalm-16 cells expressing IgG1 This refers to monoclonal antibodies (Ling, NR, el al. (1987). Leucocyte typing III. 302). In some cases, CD19-specific CAR-T cells express the CD8 alpha transmembrane domain or or the CD3ζ transmembrane domain; CD27, CD28, 4- 1BB (CD137), ICOS, DAP10, OX40 (CD134), or any of these and one or more costimulatory domains selected from fragments or combinations of: It further comprises a signaling domain derived from

[0335] In some embodiments, the antigen-binding domain is selected from the group consisting of JCAR014, JCAR015, JC AR017, or 19-28z CAR (Juno Therapeutics) Some embodiments herein recognize an epitope on CD19 that is also recognized by In this study, the antigen-binding domains of JCAR014, JCAR015, JCAR017, and is also recognized by CAR (Juno Therapeutics) Described is a CD19-specific CAR-T cell that recognizes an epitope on CD19. In some cases, CD19-specific CAR-T cells express the CD8 alpha transmembrane domain. a transmembrane domain selected from the group consisting of CD1, CD27, CD28, CD3ζ transmembrane domain, and CD3ζ transmembrane domain; , 4-1BB(CD137), ICOS, DAP10, DAP12, OX40(CD13 4), or a fragment or combination thereof, and a signaling domain derived from CD3ζ.

[0336] In some embodiments herein, the antigen-binding domain comprises an scFv antigen-binding domain. Main is JCAR014, JCAR015, JCAR017, or 19-28z C on CD19, which is also recognized by AR (Juno Therapeutics) CD19-specific CAR-T cells that recognize the epitope have been described. In this study, CD19-specific CAR-T cells were engineered to express either the CD8 alpha transmembrane domain or the CD3ζ A transmembrane domain selected from the group consisting of CD27, CD28, 4-1BB (CD 137), ICOS, DAP10, OX40 (CD134), or fragments thereof one or more costimulatory domains selected from the combination; and a synergistic effect of a co-stimulatory domain derived from CD3ζ. It further comprises a signal transduction domain.

[0337] In some embodiments, the CD19-specific CAR-T cells described herein are The anti-CD19 antibody described in Patent Application Publication No. 20160152723 In some embodiments, the CD19-specific CAR-T cells described herein are Anti-CD19 antibodies described in International Patent Publication No. 2015 / 123642 In some embodiments, the CD19-specific CAR-T cells described herein include antibodies. is an anti-CD19 scFv derived from clone FMC63 (Nicholson et al. construction and characterization of a functional CD19 specific single chain Fv f ragment for immunotherapy of B lineage leukaemia and lymphoma. Mol. Immunol., 34 :1157-1165, 1997).

[0338] In some embodiments, the antigen-binding domain is KTE-C19 (Kite Pharm This antibody recognizes an epitope on CD19 that is also recognized by Sigma-Aldrich Co., Inc. In some embodiments of the present invention, the antigen-binding domain is also recognized by KTE-C19. Described is a CD19-specific CAR-T cell that recognizes an epitope on CD19. In some cases, CD19-specific CAR-T cells express the CD8 alpha transmembrane domain. a transmembrane domain selected from the group consisting of CD1, CD27, CD28, CD3ζ transmembrane domain, and CD3ζ transmembrane domain; , 4-1BB(CD137), ICOS, DAP10, DAP12, OX40(CD13 4), or a fragment or combination thereof, and a signaling domain derived from CD3ζ.

[0339] In some embodiments herein, the antigen-binding domain comprises an scFv antigen-binding domain. The main antibody recognizes an epitope on CD19 that is also recognized by KTE-C19. CD19-specific CAR-T cells have been described. Targeted CAR-T cells are derived from either the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain. Selected transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS , DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof and a signal derived from CD3ζ; It further comprises a transduction domain.

[0340] In some embodiments, the CD19-specific CAR-T cells described herein are available from international publications. The anti-CD19 antibody described in the pamphlet of Patent Publication No. 2015187528, includes fragments or derivatives thereof.

[0341] In some embodiments, the antigen-binding domain is a polypeptide of the type designated CTL019 (Novartis). Some embodiments herein recognize an epitope on CD19 that is also recognized by In this study, the antigen-binding domain of CTL019 is located on the CD19 epitope, which is also recognized by CTL019. CD19-specific CAR-T cells that recognize a target antigen have been described. CD19-specific CAR-T cells express either the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain. A transmembrane domain selected from transmembrane domains; CD27, CD28, 4-1BB (CD1 37), ICOS, DAP10, DAP12, OX40 (CD134), or any of these one or more costimulatory domains selected from the group consisting of fragments or combinations thereof; and It further comprises a signaling domain from which it is derived.

[0342] In some embodiments herein, the antigen-binding domain comprises an scFv antigen-binding domain. The main antibody recognizes an epitope on CD19 that is also recognized by CTL019. , CD19-specific CAR-T cells are described. In some cases, CD19-specific CAR-T cells are selected from CD8 alpha transmembrane domain or CD3 zeta transmembrane domain. Selected transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof and one or more costimulatory domains selected from the group consisting of: It further includes a reach domain.

[0343] In some embodiments, the antigen-binding domain is UCART19 (Cellectis) It recognizes an epitope on CD19 that is also recognized by In this embodiment, the antigen-binding domain is CD19, which is also recognized by UCART19. CD19-specific CAR-T cells that recognize the above epitopes have been described. In this case, CD19-specific CAR-T cells are expressed using the CD8 alpha transmembrane domain or CD A transmembrane domain selected from the 3ζ transmembrane domains; CD27, CD28, 4-1BB ( CD137), ICOS, DAP10, DAP12, OX40 (CD134), or one or more costimulatory domains selected from fragments or combinations thereof; and It further contains a signaling domain derived from 3ζ.

[0344] In some embodiments herein, the antigen-binding domain comprises an scFv antigen-binding domain. The main antibody recognizes an epitope on CD19 that is also recognized by UCART19. CD19-specific CAR-T cells have been described. Targeted CAR-T cells are derived from either the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain. Selected transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS , DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof and a signal derived from CD3ζ; It further comprises a transduction domain.

[0345] In some embodiments, the antigen-binding domain is selected from the group consisting of BPX-401 (Bellicum). The antibody recognizes an epitope on CD19 that is also recognized by the antibody. In this embodiment, the antigen-binding domain is located on CD19, which is also recognized by BPX-401. CD19-specific CAR-T cells that recognize the epitope In this case, CD19-specific CAR-T cells are expressed as CD8 alpha transmembrane domain or CD3 A transmembrane domain selected from the ζ transmembrane domain; CD27, CD28, 4-1BB (C D137), ICOS, DAP10, DAP12, OX40 (CD134), or one or more costimulatory domains selected from fragments or combinations thereof; and CD3 It further contains a signaling domain derived from ζ.

[0346] In some embodiments herein, the antigen-binding domain comprises an scFv antigen-binding domain. The main antibody recognizes an epitope on CD19 that is also recognized by BPX-401. CD19-specific CAR-T cells have been described. Targeted CAR-T cells are derived from either the CD8 alpha transmembrane domain or the CD3 zeta transmembrane domain. Selected transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS , DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof and a signal derived from CD3ζ; It further comprises a transduction domain.

[0347] In some cases, the antigen-binding domain is selected from the group consisting of blinatumomab (Amgen), coltuximab (Campaigner), and gliomas with ... Bu-Rabtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-55 1 (Medimmune), denintuzumab-mafodotin (Seattle Genet ics), B4 (or DI-B4) (Merck Serono), taplitumomab Paptox (National Cancer Institute), XmAb 5 871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex) or an epitope on CD19 that is also recognized by AFM11 (Affimed) In some cases, CD19-specific CARs recognize the CD8 alpha transmembrane domain. a transmembrane domain selected from the group consisting of CD1, CD27, CD28, CD3ζ transmembrane domain, and CD3ζ transmembrane domain; , 4-1BB (CD137), ICOS, DAP10, OX40 (CD134), or one or more costimulatory domains selected from fragments or combinations thereof; and C It further contains a signaling domain derived from D3ζ.

[0348] In some embodiments herein, the antigen-binding domain is selected from the group consisting of F(ab')2, Fab', CD19-specific CAR-T cells, including Fab, Fv, or scFv, have been described. In some cases, the antigen-binding domain recognizes an epitope on CD19. The antigen-binding domain is a marker for blinatumomab (Amgen), coltuximab laravel (Amgen), and Butansine (ImmunoGen Inc. / Sanofi-aventis), MOR 208 (Morphosys AG / Xencor Inc.), MEDI-551 (M edimmune), denintuzumab-mafodotin (Seattle Genetics ), B4 (or DI-B4) (Merck Serono), taplitumomab papt XmAb 5871 (National Cancer Institute) (Amgen / Xencor, Inc.), MDX-1342 (Medarex), and identifies an epitope on CD19 that is also recognized by AFM11 (Affimed). In some cases, CD19-specific CAR-T cells recognize the CD8 alpha transmembrane domain. A transmembrane domain selected from the main or CD3ζ transmembrane domain; CD27, CD2 8, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD1 34), or a fragment or combination thereof, and further comprising a signaling domain derived from CD3ζ.

[0349] In some cases, the CD19-specific CAR-T cells described herein contain scFv antibodies. The antigen-binding domain is a cytochrome P450 antibody, which is a cytochrome P450 antibody. Tuximab-ravtansine (ImmunoGen Inc. / Sanofi-Avent is), MOR208 (Morphosys AG / Xencor Inc.), MED I-551 (Medimmune), denintuzumab-mafodotin (Seattle G enetics), B4 (or DI-B4) (Merck Serono), Taplitz Momab-Paptox (National Cancer Institute), Xm Ab 5871 (Amgen / Xencor, Inc.), MDX-1342 (Meda rex), or AFM11 (Affimed), on CD19 In some cases, CD19-specific CAR-T cells recognize epitopes of CD8 antigens. a transmembrane domain selected from the group consisting of the C transmembrane domain, the C transmembrane domain, and the CD3ζ transmembrane domain; D27, CD28, 4-1BB(CD137), ICOS, DAP10, DAP12, O X40 (CD134), or a fragment or combination thereof. and a signaling domain derived from CD3ζ.

[0350] CD33-specific CAR "CD33" is a 67kDa single-pass transmembrane glycoprotein that binds sialic acid. It is a member of the immunoglobulin-like lectin (Siglec) superfamily. 33 contains a V-set Ig-like domain within its extracellular domain that is responsible for binding sialic acid. CD33 mRNA is characterized by a C2 set Ig-like domain. Alternative splicing results in the V-set Ig-like domains, as well as the V-set Ig-like domains. A short Ig-like domain lacking the disulfide bond connecting the Ig-like domain to the C2-set Ig domain. In healthy subjects, CD33 is expressed on normal multipotent bone marrow progenitors. Found on progenitors, unipotent colony-forming cells, monocytes, and mature granulocytes, bone marrow compartments CD33 is expressed primarily as a cytotoxic antigen on over 80% of myeloid leukemia cells. but not expressed on normal hematopoietic stem cells or mature granulocytes (Andrews, R. et al. , The L4F3 antigen is expressed by unipotent and multipotent colony-forming cells s but not by their precursors, Blood, 68(5):1030-5 (1986)). CD33 is a It has been reported to be expressed on myeloid cells, activated T cells, and activated NK cells. , found on at least a subset of blast cells in the majority of AML patients ( Pollard, J. et al., Correlation of CD33 expression level with disease characteristics stics and response to gemtuzumab ozogamicin containing chemotherapy in childhood AML, Blood, 119(16):3705-11 (2012)). In addition to widespread expression on AML blast cells, CD33 can also be expressed on stem cells that underlie AML.

[0351] In embodiments, the antigen-binding portion of the CAR described herein is specific for CD33. CD33-specific CARs are expressed on the cell surface and In some embodiments, the specificity of T cells is redirected to human CD33. The synthetic domain is a light chain variable domain of an anti-CD33 monoclonal antibody specific for the target antigen. a heavy chain variable domain (VL) and a heavy chain variable domain (VH) comprising a glycine-serine linker or A single chain comprising a VL and a VH connected by a flexible linker such as a Whitlow linker In some embodiments, the scFv includes M195, m2H12, , DRB2, and / or My9-6. In some embodiments, the scFv is human In some embodiments, the antigen-binding portion is a modified scFv, e.g., hM195. For example, from the N-terminus to the C-terminus, VH-linker-VL or VL-linker-VH. The VH and VL may comprise a symmetrically linked VH and VL.

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

[0353] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 36 ( hM195 VH) and at least 90%, 91%, 92%, 93%, 94%, 95%, VH polypeptides with 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises:

[0354] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 37 ( M2H12 VH) and at least 90%, 91%, 92%, 93%, 94%, 95%, VH polypeptides with 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises:

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

[0356] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 39 ( DRB2 VH) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 VH polypeptides having 6%, 97%, 98%, 99%, or 100% identity to the VH polypeptides. The antigen-binding portion comprises:

[0357] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 40 ( DRB2 VL) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0358] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 41 ( My9-6 VH) and at least 90%, 91%, 92%, 93%, 94%, 95%, VH polypeptides with 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises:

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

[0360] MUC16-specific CAR MUC16 is a large carbohydrate antigen, also known as CA-125. UC16 is encoded by the MUC16 gene located on human chromosome 19. C16 is a three-domain, highly glycosylated, multidomain type I membrane It is a transmembrane protein. The C-terminal domain contains multiple nucleotides with autoproteolytic activity. Extracellular SEA (sea urchin sperm protein, enterokinase, and agrin) module SEA contains two proteins proximal to the transmembrane (TM) domain. The large truncated domain, designated CA-125, is degradable at acidic pH. CA-125 is generally used as a disease biomarker for ovarian cancer. Used as a carrier. Highly conserved, truncated, extracellular, membrane-tethered The protein domain is called the MUC16 extracellular domain. A MUC16 antibody that specifically bound to the retained extracellular domain of MUC16 was identified. "Overexpression of MUC16" by target cells (such as cancer cells) means that are expressed at higher levels compared to control cells (non-cancerous cells, normal cells, etc.), Refers to MUC16 protein and / or mRNA.

[0361] In embodiments, the antigen-binding portion of the CAR described herein binds to MUC16. MUC16-specific CAR is expressed on the cell surface. When administered, it redirects the specificity of T cells to human MUC16. The antigen-binding domain is a light chain of an anti-MUC16 monoclonal antibody specific for the target antigen. A variable domain (VL) and a heavy chain variable domain (VH) comprising glycine-serine VL and VH connected by a flexible linker such as a Carr or Whitlow linker In some embodiments, the scFv comprises a single chain antibody fragment (scFv) comprising MUC16 -scFv (SEQ ID NOs: 43 to 44), MUC16-scFv (SEQ ID NOs: 45 to 46), M UC16-scFv (SEQ ID NOs: 47 to 48), MUC16-scFv (SEQ ID NOs: 49 to 5 0), MUC16-scFv (SEQ ID NOs: 51-52), MUC16-scFv (SEQ ID NO: 53-54), or MUC16-scFv (SEQ ID NOs: 55-56). In some embodiments, the scFv is a humanized scFv.

[0362] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 43 ( MUC16-1) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0363] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 44 ( MUC16-1) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 VH polypeptides having 6%, 97%, 98%, 99%, or 100% identity to the VH polypeptides. The antigen-binding portion comprises:

[0364] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 45 ( MUC16-2) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0365] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 46 ( MUC16-2) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 VH polypeptides having 6%, 97%, 98%, 99%, or 100% identity to the VH polypeptides. The antigen-binding portion comprises:

[0366] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 47 ( MUC16-3) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0367] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 48 ( MUC16-3) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 VH polypeptides having 6%, 97%, 98%, 99%, or 100% identity to the VH polypeptides. The antigen-binding portion comprises:

[0368] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 49 ( MUC16-4) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0369] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 50 ( MUC16-4) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 VH polypeptides having 6%, 97%, 98%, 99%, or 100% identity to the VH polypeptides. The antigen-binding portion comprises:

[0370] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 51 ( MUC16-5) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0371] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 52 ( MUC16-5) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0372] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 53 ( MUC16-5) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 VH polypeptides having 6%, 97%, 98%, 99%, or 100% identity to the VH polypeptides. The antigen-binding portion comprises:

[0373] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 54 ( MUC16-6) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0374] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 55 ( MUC16-7) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity to a VL polypeptide. The antigen-binding portion comprises:

[0375] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 56 ( MUC16-7) and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 VH polypeptides having 6%, 97%, 98%, 99%, or 100% identity to the VH polypeptides. The antigen-binding portion comprises:

[0376] In some embodiments, the antigen-binding portion comprises, e.g., from N-terminus to C-terminus, a VH- VH and VL linked in a direction with VL-linker-VH or VL-linker-VH It may include.

[0377] Engineered T cell receptor (TCR) In some embodiments, the chimeric receptor comprises an engineered T cell receptor. R) consists of two chains (αβ) that pair on the surface of T cells to form a heterodimeric receptor. In some cases, the αβ TCR is present on most T cells in the body. The α and β chains are expressed in the IL-1 receptor and are known to be involved in the recognition of specific MHC-restricted antigens. Each of these domains contains two domains: one that anchors the protein to the cell membrane and one that mediates the CD3 signal. A constant domain (C) associated with the invariant subunit of the transmitter; and a complementarity-determining region (CDR). ) that confers antigen recognition via a six-loop domain called the variable domain (V) In some cases, each V domain comprises three CDRs, e.g., CDR3, which are hypervariable regions. These CDRs comprise CDR1, CDR2, and CDR3, which are the major tissue-specific CDRs. formed between antigenic peptides bound to proteins encoded by the synthetic complex complex (pepMHC) (e.g., HLA-A complex, HLA-B complex, HLA-C complex) coalescence, HLA-DPA1 complex, HLA-DPB1 complex, HLA-DQA1 complex, H HLA-DQB1 complex, HLA-DRA complex, or HLA-DRB1 complex) In some cases, the constant domains act as a linker connecting the constant domains to the variable domains. Optionally, the beta strand further comprises a short junction region that forms part of the junction region. It further comprises a diversity region.

[0378] In some cases, such TCRs target specific tumor antigens, e.g., NY-ESO, Mag e A3, reactive with Titin. In other cases, such TCRs are expressed in tumors in patients. specific neoantigens (i.e., tumor-expressed, patient-specific, In some cases, engineered TCRs are reactive with affinity The effect can be enhanced.

[0379] In some embodiments, the TCR is from International Immunogene The TCRs are described using the TCR nomenclature by tics (IMGT), and Link to public databases for sequences, e.g., their framework sequences , several types identified by CDR1 sequences, CDR2 sequences, and CDR3 sequences There are 10 types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions. Thus, the types of Vα are identified by unique TRAV numbers in the IMGT nomenclature. For example, "TRAV21" may be referred to as a unique framework sequence, as well as a C DR1 and CDR2 sequences and amino acids conserved from TCR to TCR The amino acid sequence is partially defined by the TCR but also varies from TCR to TCR. The "TRBV5-1" region defines the TCR Vα region having the CDR3 sequence, including the CDR3 sequence. " has a unique framework sequence, and CDR1 and CDR2 sequences, The CDR3 sequences define a region of the TCR Vβ that is only partially defined.

[0380] The beta chain diversity region is sometimes abbreviated as TRBD in the IMGT nomenclature. It will be imposed.

[0381] In some cases, the unique sequences defined by the IMGT nomenclature are widely known and These are available to those working in the field of CR. For example, these are public data from IMGT. Bass and “T cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

[0382] In some embodiments, a TCR that is an αβ heterodimer comprises, for example, a cytoplasmic domain and The polypeptide is transfected as a full-length chain having both the transmembrane domain and the transmembrane domain. TCRs are described, for example, in WO 2006 / 000830. The constant domains contain disulfide bonds introduced between the residues of each of the constant domains.

[0383] In some cases, the TCRs described herein are in a single chain format (e.g., (See WO 2004 / 033685). Single-stranded format are Vα-L-Vβ type, Vβ-L-Vα type, Vα-Cα-L-Vβ type, Vα-L-Vβ- αβ TCR polypeptides of the Cβ type, Vα-Cα-L-Vβ-Cβ type [in format, Vα and Vβ are the TCRα and TCRβ variable regions, respectively; Cβ and Cβ are the TCR α and TCR β constant regions, respectively, and L is a linker. In one particular embodiment, the single chain TCR of the invention comprises the sequence Each constant domain described in brochure 004 / 033685 The amino acid sequence may have a disulfide bond introduced between the residues of

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

[0385] For diagnostic purposes, exemplary detectable labels include fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and the like. Examples of suitable immunosuppressants include, but are not limited to, immunoglobulins, antibodies, and imaging agents.

[0386] Therapeutic agents that may be associated with the TCRs described herein include immunomodulators, radioactive Toxic effects include, but are not limited to, compounds, enzymes (e.g., perforin), or chemotherapeutic agents. To ensure that the compound is released in a controlled manner, The enzyme could be present inside the liposome linked to the TCR. This release minimizes the damaging effects of transport within the body and allows the toxin to reach the target antigen-presenting cells. Ensures maximum effect after TCR engagement.

[0387] In some embodiments, the additional, suitable therapeutic agent is, for example, a. Small molecule cytotoxic agents, e.g., compounds with the ability to kill mammalian cells , compounds having a molecular weight of less than 700 daltons. Such compounds also have cytotoxic effects. Furthermore, these small molecules may also contain toxic metals that can have a negative effect on cells. The damaging agent may also be a prodrug, i.e., a drug that breaks down or is converted under physiological conditions. It is understood that the term also includes compounds that release cytotoxic agents upon contact with the target molecule. Examples of cisplatin, maytansine derivatives, raccelericin, calicheamicin, docetaxel Cel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitochondrial Xantrone, solfimer sodium, photofrin II, temozolomide, topotecan , trimetrexate glucuronate, auristin E, bile including cristine, and doxorubicin; b. Peptide cytotoxins, i.e., proteins with the ability to kill mammalian cells proteins or fragments thereof, e.g., ricin, diphtheria toxin, Pseudomonas nas) fungal exotoxin A, DNase, and RNase; c. Radionuclides, i.e., unstable isotopes of elements that emit alpha or beta particles Isotopes that decay with the synchronic emission of one or more of the following: Iodine-131, Rhenium-186, Indium-111, Yttrium-90, Bismuth-210 and 213, actinium 225, and astatine 213; and the chelating agent , which facilitates the association of these radionuclides with high affinity TCRs or multimers thereof. Can be used to facilitate; d. Immunostimulatory agents, i.e., immune effector molecules that stimulate the immune response. For example, I cytokines such as L-2 and IFN-γ; e. Superantigens and their mutants; f.TCR-HLA fusion; g. Chemokines such as IL-8, crystalloid factor 4, and melanoma growth stimulatory protein; h. Anti-T cell determinant antibodies or NK cell determinant antibodies (e.g., anti-CD3 antibodies, anti-CD2 8 antibody, or anti-CD16 antibody); i. Alternative protein scaffolds with antibody-like binding characteristics; j. complement activators; and k. Heterologous protein domains, homologous protein domains, viral / bacterial proteins Contains protein domains, viral / bacterial peptides.

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

[0389] In certain embodiments, the fusion protein is present in an amount of about 1-30 mg per kg, e.g., Approximately 5 to 25 mg per kg, approximately 10 to 20 mg per kg, approximately 1 to 5 mg per kg injection (e.g., subcutaneous or intravenous) at a dose of about 3 mg per g, or per kg. In some embodiments, the fusion protein is administered at a dose of about 1 ml per kg. g, approximately 3 mg per kg, approximately 5 mg per kg, approximately 10 mg per kg, approximately Administered at a dose of approximately 20 mg per kg, approximately 30 mg per kg, or approximately 40 mg per kg In some embodiments, the fusion protein is administered at a concentration of about 1-3 mg per kg, or In some embodiments, the fusion protein is administered at a dose of about 3-10 mg per kg. , at doses of approximately 0.5 to 2, 2 to 4, 2 to 5, 5 to 15, or 5 to 20 mg per kg The administration schedule can vary, for example, from once weekly to once every 2, 3, or 4 weeks. In one embodiment, the fusion protein is administered at a dose of about 10-20 mg per kg every two weeks. In another embodiment, the fusion protein is administered in an amount of 100 mg / kg once every two weeks. Approximately 1 mg per kg, once every 2 weeks. Approximately 3 mg per kg, once every 2 weeks. 10 mg per kg once every 4 weeks, or 3 mg per kg once every 4 weeks It is administered at a dose of 5 mg per day.

[0390] In other embodiments, the fusion protein is about 200 mg to 500 mg, for example, about 250 mg. mg~450mg, approx. 300mg~400mg, approx. 250mg~350mg, approx. 350m doses of 100 mg to 450 mg, or about 300 mg, or about 400 mg (e.g., for flat The drug is administered by injection (e.g., subcutaneous or intravenous injection) in a small amount. In embodiments, the fusion protein is administered in an amount of about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg. It is administered in doses of about 400 mg, about 450 mg, or about 500 mg. In embodiments, the fusion protein is administered in a dose of 200 or 300 mg. In embodiments, the fusion protein is about 250-450 mg, or about 300-400 mg. In some embodiments, the fusion protein is administered in a dose of about 200-300 mg, 2 50-350mg, 300-400mg, 350-450mg, or 400-500mg The administration schedule may be, for example, once every week to 2, 3, 4, 5, or In one embodiment, the fusion protein is administered at a dose of 3 or 4 weeks. In one embodiment, the fusion protein is administered at a dose of about 300 mg to 400 mg once per day. The protein is administered at a dose of about 300 mg once every three weeks. The protein is administered at a dose of about 400 mg once every four weeks. The fusion protein is administered at a dose of about 300 mg once every four weeks. The fusion protein is administered at a dose of approximately 400 mg once every three weeks. The protein may be administered one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, or More than this number of times may be administered. In one embodiment, the fusion protein is administered over six times. The fusion protein is administered to a CAR-expressing cell, e.g., MUC16, CD33, At least 5 days after administration of the CD19- or BCMA-specific CAR-expressing cells, for example, about 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 20, 25, 30, 35, or 4 In some embodiments, the fusion protein may be administered to a CAR-expressing cell, e.g., on day 0. For example, after administration of MUC16-specific CAR-expressing cells or CD33-specific CAR-expressing cells Administration may occur about 8 days or about 15 days later.

[0391] The fusion protein may be administered by a variety of methods known in the art, many of which are therapeutic. For therapeutic applications, the preferred route / mode of administration is intravenous injection or infusion. The fusion protein is 2 Approximately 35-440 mg per 1 m 2 Approximately 70 per 310 mg, more typically 1 m 2 Achieve a dose of approximately 110-130 mg per day Thus, the dose is greater than 20 mg / min, e.g., 20-40 mg / min, typically 40 mg In some embodiments, the fusion tank may be administered by intravenous infusion at a rate of 100 mg / min or more. The protein is 1m 2 Approximately 1 to 100 mg per 1 ml, preferably 1 ml 2 Approximately 5 to 50 mg per 1m 2 More preferably, it is about 7 to 25 mg per m 2 At a dose of approximately 10 mg per Intravenous at a rate of less than 10 mg / min, preferably 5 mg / min or less, to achieve It may be administered by injection.

[0392] The fusion protein is 2 Approximately 35-440 mg per 1 m 2 Approximately 70 per...

Claims

1. (a) an immune checkpoint inhibitor; and (b) adenosine deaminase (ADA) or a functional fragment or variant thereof A fusion protein comprising:

2. A fusion protein as described in claim 1, wherein the immune checkpoint inhibitor binds to an immune checkpoint molecule.

3. The fusion protein described in claim 2, wherein the immune checkpoint molecule is programmed cell death protein-1 (PD-1) or cytotoxic T-lymphocyte-associated protein-4 (CTLA-4).

4. The fusion protein described in claim 2, wherein the immune checkpoint molecule is PD-1.

5. An immune checkpoint inhibitor, (a) a first polypeptide having at least 80% identity to any one of SEQ ID NOs: 6, 7, 1-5, or 149-164; and (b) a second polypeptide having at least 80% identity to any one of SEQ ID NOs: 12, 13, 8-11, or 148. The fusion protein of claim 4, comprising:

6. An immune checkpoint inhibitor, (a) a first polypeptide having at least 90% identity to any one of SEQ ID NOs: 6, 7, 1-5, or 149-164; and (b) a second polypeptide having at least 90% identity to any one of SEQ ID NOs: 12, 13, 8-11, or 148. The fusion protein of claim 4, comprising:

7. An immune checkpoint inhibitor, (a) a first polypeptide having at least 95% identity to any one of SEQ ID NOs: 6, 7, 1-5, or 149-164; and (b) a second polypeptide having at least 95% identity to any one of SEQ ID NOs: 12, 13, 8-11, or 148. The fusion protein of claim 4, comprising:

8. An immune checkpoint inhibitor, （a） (i) a first polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 6, wherein the first polypeptide comprises three complementarity-determining regions (CDRs) of the variable region (VH) of the heavy chain of nivolumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 12, wherein the second polypeptide comprises the three CDRs of the variable region (VL) of the light chain of nivolumab; or （b） (i) a first polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 7, wherein the first polypeptide comprises three CDRs of the variable region (VH) of the heavy chain of pembrolizumab; and (ii) a second polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13, wherein the second polypeptide comprises the three CDRs of the variable region (VL) of the light chain of pembrolizumab; or The fusion protein of claim 4, comprising:

9. An immune checkpoint inhibitor, (a) a first polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 6, wherein the first polypeptide comprises three CDRs of the variable region (VH) of the heavy chain of nivolumab; and (b) a second polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 12, wherein the second polypeptide comprises three CDRs of the variable region (VL) of the light chain of nivolumab. The fusion protein of claim 4, comprising:

10. An immune checkpoint inhibitor, (a) a first polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 7, wherein the first polypeptide comprises three CDRs of the variable region (VH) of the heavy chain of pembrolizumab; and (b) a second polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13, wherein the second polypeptide comprises three CDRs of the variable region (VL) of the light chain of pembrolizumab. The fusion protein of claim 4, comprising:

11. The fusion protein described in claim 5, wherein the immune checkpoint inhibitor further comprises a third polypeptide having an amino acid sequence of any one of SEQ ID NOs: 291, 147, 292, or 146.

12. The fusion protein described in claim 5, wherein the immune checkpoint inhibitor further comprises a third polypeptide having an amino acid sequence of SEQ ID NO: 291 or 147.

13. The fusion protein described in claim 1, wherein the immune checkpoint inhibitor comprises an antibody or a functional fragment or variant thereof.

14. The fusion protein described in claim 13, wherein the antibody is an IgG antibody.

15. The fusion protein described in claim 13, wherein the antibody is an IgG4 antibody.

16. The fusion protein of claim 13, wherein the immune checkpoint antibody is Fab, (Fab) 2 , (Fab') 2 , Fv, (Fv) 2 , or scFv.

17. The fusion protein described in claim 1, wherein ADA or a functional fragment or variant thereof is adenosine deaminase 2 (ADA2) or a functional fragment or variant thereof.

18. The fusion protein described in claim 13, comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 279, 284, or 273-278.

19. The fusion protein described in claim 13, comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 279, 284, or 273-278.

20. A fusion protein described in claim 13, comprising an amino acid sequence having at least 95% identity with any one of SEQ ID NOs: 279, 284, or 273-278.

21. A fusion protein described in claim 13, comprising an amino acid sequence having at least 90% identity with either one of SEQ ID NOs: 279 or 284.

22. The fusion protein of claim 1, wherein the immune checkpoint inhibitor is connected to ADA or a functional fragment or variant thereof by a linker comprising the amino acid sequence of any one of SEQ ID NOs: 32, 17-31, 33, or 34.

23. The fusion protein of claim 1, wherein the immune checkpoint inhibitor is connected to ADA or a functional fragment or variant thereof by a linker comprising the amino acid sequence of SEQ ID NO: 32 (n is 2).

24. The fusion protein described in claim 5, wherein the linker links the first polypeptide of the immune checkpoint inhibitor to ADA or a functional fragment or variant thereof.

25. (a) (i) a polypeptide having at least 80% identity to SEQ ID NO: 12; and (ii) a polypeptide having at least 80% identity to SEQ ID NO: 281 or 280; or （b） (i) a polypeptide having at least 80% identity to SEQ ID NO: 13; and (ii) a polypeptide having at least 80% identity to SEQ ID NO: 283 or 282 The fusion protein of claim 1 , comprising:

26. (a) (i) a polypeptide having at least 90% identity to SEQ ID NO: 12; and (ii) a polypeptide having at least 90% identity to SEQ ID NO: 281 or 280; or （b） (i) a polypeptide having at least 90% identity to SEQ ID NO: 13; and (ii) a polypeptide having at least 90% identity to SEQ ID NO: 283 or 282 The fusion protein of claim 1 , comprising:

27. ​​(a) (i) a polypeptide having at least 95% identity to SEQ ID NO: 12; and (ii) a polypeptide having at least 95% identity to SEQ ID NO: 281 or 280; or （b） (i) a polypeptide having at least 95% identity to SEQ ID NO: 13; and (ii) a polypeptide having at least 95% identity to SEQ ID NO: 283 or 282 The fusion protein of claim 1 , comprising:

28. (a) (i) a polypeptide having the sequence of SEQ ID NO: 12; and (ii) a polypeptide having the sequence of SEQ ID NO: 281 or 280; or （b） (i) a polypeptide having the sequence of SEQ ID NO: 13; and (ii) a polypeptide having the sequence of SEQ ID NO: 283 or 282 The fusion protein of claim 1 , comprising:

29. A polynucleotide encoding a fusion protein described in any one of claims 1 to 28.

30. An expression vector comprising a polynucleotide encoding the fusion protein described in any one of claims 1 to 28.

31. (a) (i) a fusion protein according to any one of claims 1 to 28; (ii) a polynucleotide encoding the fusion protein; or (ii) an expression vector comprising the polynucleotide linked to a promoter; and (b) pharmaceutically acceptable excipients 10. A pharmaceutical composition comprising:

32. The pharmaceutical composition of claim 31, further comprising a modified effector cell comprising a chimeric antigen receptor (CAR).

33. The pharmaceutical composition of claim 32, wherein the CAR binds to an epitope in CD19, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-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.

34. Use of any of the following in the manufacture of a medicament for treating cancer in a subject in need thereof: (a) a fusion protein according to any one of claims 1 to 28; (b) a polynucleotide encoding the fusion protein of (a); or (c) An expression vector comprising the polynucleotide of (b) linked to a promoter.

35. A pharmaceutical composition described in any one of claims 31 to 33, which is a pharmaceutical composition for treating cancer.

36. The pharmaceutical composition described in claim 35, wherein the cancer is mesothelioma, glioblastoma, endometrial cancer, colorectal cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, stomach cancer, bladder cancer, liver cancer, Hodgkin's lymphoma, lung cancer, skin cancer, renal cancer, or head and neck cancer.

37. The pharmaceutical composition described in claim 35, wherein the cancer is colorectal cancer or pancreatic cancer.

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