Compositions and Methods Comprising Protease-Activating Therapeutics

Abstract

The present disclosure relates to the manipulation of collagen-binding modifications of masked therapeutic agents containing one or more tumor-associated protease cleavage sites. When exposed to tumor-associated proteases in the tumor microenvironment, the polypeptide is cleaved, thereby unmasking the therapeutic agent and reducing off-target side effects and toxicity associated with systemic administration. Thus, an aspect of the present disclosure relates to a polypeptide comprising a therapeutic agent linked to a masking agent via a linker, wherein the linker contains one or more tumor-associated protease cleavage sites, the masking agent blocks the therapeutic agent from binding to its therapeutic target, and the polypeptide is operably linked to a collagen-binding domain or a tumor-targeting agent.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 878,574, filed July 25, 2019, which is incorporated by reference in its entirety. [Background technology]

[0002] background Cytokine cancer immunotherapy using interleukin (IL)-12 has shown strong antitumor efficacy in both mice and humans. However, some IL12 clinical trials have been terminated or failed due to its severe toxicity. IL12 has not been approved for clinical use so far. Immunotherapy serves to activate immune responses, so side effects are usually due to drug action on healthy organs. There is a need in the art for strategies to reduce the toxicity of therapeutic treatments. Summary of the Invention

[0003] The present disclosure relates to the manipulation of collagen-binding modification of masked therapeutic agents that contain one or more tumor-associated protease cleavage sites.When exposed to tumor-associated proteases in tumor microenvironment, the polypeptide is cleaved, thereby unmasking the therapeutic agent and reducing the off-target side effects and toxicity associated with systemic administration.Therefore, an aspect of the present disclosure relates to a polypeptide that comprises a therapeutic agent linked to a masking agent through a linker, the linker comprises one or more tumor-associated protease cleavage sites, the masking agent blocks the association of the therapeutic agent with its therapeutic target, and the polypeptide is functionally linked to a collagen-binding domain or a tumor targeting agent.

[0004] A further aspect of the present disclosure relates to a polypeptide comprising a cytokine linked to a masking agent through a linker, the linker comprising one or more tumor-associated protease cleavage sites, and the masking agent comprising a cytokine receptor polypeptide or a fragment thereof that specifically binds to the cytokine. A masking agent refers to a molecule that blocks the association of a therapeutic agent with at least one binding partner. In some embodiments, the therapeutic agent comprises an antibody, and the binding partner comprises an antigen. In some embodiments, the therapeutic agent comprises a cytokine, and the binding partner comprises a receptor polypeptide.

[0005] Further aspects relate to compositions comprising the polypeptides of the present disclosure. Further aspects relate to nucleic acids encoding the polypeptides of the present disclosure, and host cells comprising the nucleic acids and / or polypeptides of the present disclosure. Also provided are methods for producing the polypeptides, comprising expressing the nucleic acids of the present disclosure in host cells and isolating the expressed polypeptides. Further aspects relate to methods for treating cancer, comprising administering the polypeptides or compositions of the present disclosure to a subject in need thereof, such as a subject with cancer.

[0006] In some embodiments, the cytokine comprises interleukin-12 (IL12) and the masking agent comprises an interleukin 12 receptor (IL12R) polypeptide or an IL12-binding fragment thereof. In some embodiments, IL12 comprises one or both of a p35 subunit and a p40 subunit. In some embodiments, IL12 comprises a p35 subunit and a p40 subunit linked through a disulfide bond. In some embodiments, IL12 comprises a p35 subunit and a p40 subunit linked through a peptide linker. In some embodiments, the IL12R polypeptide or fragment comprises an interleukin 12 receptor beta 1 (IL12Rβ1), or a fragment thereof. In some embodiments, the IL12R polypeptide or fragment comprises an interleukin 12 receptor beta 2 (IL12Rβ2), or a fragment thereof. In some embodiments, the IL12Rβ1 polypeptide comprises one or both of fibronectin domains D1 and D2.

[0007] In some embodiments, the masking agent is fused to the N-terminus of the p35 subunit of IL12, and a linker comprising a tumor-associated protease cleavage site is between the masking agent and the p35 subunit of IL12. In some embodiments, the masking agent is fused to the C-terminus of the p35 subunit of IL12, and a linker comprising a tumor-associated protease cleavage site is between the masking agent and the p35 subunit of IL12. In some embodiments, the masking agent is fused to the C-terminus of the p40 subunit of IL12, and a linker is between the masking agent and the p40 subunit of IL12. In some embodiments, the masking agent is fused to the N-terminus of the p40 subunit of IL12, and a linker is between the masking agent and the p40 subunit of IL12. In some embodiments, the cytokine comprises interleukin-2 (IL-2) and the masking agent comprises interleukin 2 receptor alpha subunit (IL-2Rα), interleukin 2 receptor beta subunit (IL-2Rβ), interleukin 2 receptor gamma subunit (IL-2Rγ), a fragment, or a combination of fragments thereof. In some embodiments, the cytokine comprises interferon gamma (IFNγ) and the masking agent comprises interferon gamma receptor 1 (IFNγR1), interferon gamma receptor 2 (IFNγR2), a fragment, or a combination of fragments thereof.

[0008] In some embodiments, the polypeptide comprises at least two tumor-associated protease cleavage sites. In some embodiments, the polypeptide comprises at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, or 8 tumor-associated protease cleavage sites, or any range derivable therein. In some embodiments, the tumor-associated protease cleavage site comprises a uPA, matrix metalloproteinase, or thrombin cleavage site. In some embodiments, the tumor-associated protease cleavage site comprises at least one tumor-associated protease cleavage site described herein. In some embodiments, the tumor-associated cleavage site comprises a cleavage site having one of the amino acids of SEQ ID NO: 13, 14, 49, 51, 55, 109-190. In some embodiments, the polypeptide comprises at least two different tumor-associated protease cleavage sites. In some embodiments, the polypeptide comprises at least 2, 3, or 4 different tumor-associated protease cleavage sites, or any range derivable therein. In some embodiments, the polypeptide comprises at least two of the same tumor-associated protease cleavage site. In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, or 8 of the same protease cleavage sites, or any range derivable therein. In embodiments comprising more than one protease cleavage site, the protease cleavage sites may be adjacent or may have intervening amino acids. In some embodiments, at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, or any range derivable therein, separate one tumor-associated protease cleavage site from another tumor-associated protease cleavage site.

[0009] In some embodiments, the cytokine comprises an anti-inflammatory cytokine. In some embodiments, the cytokine comprises a pro-inflammatory cytokine.

[0010] In some embodiments, the polypeptide is conjugated to a tumor targeting agent. In some embodiments, the tumor targeting agent comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment comprises a stroma targeting antibody or a stroma binding fragment thereof. In some embodiments, the antibody or binding fragment specifically binds to fibronectin, an alternative splice domain of fibronectin, collagen, tenascin, periostin, syndecan, proteoglycan, or a tumor stromal cell specific antigen. In some embodiments, the antibody or binding fragment specifically binds to extra domain A (EDA) or extra domain B (EDB) of fibronectin. In some embodiments, the tumor targeting agent comprises a Fab that specifically binds to an alternative splice domain of fibronectin that includes extra domain A (EDA). In some embodiments, the tumor targeting agent comprises an antibody or antigen-binding fragment thereof that specifically binds to a tumor associated antigen. Other tumor targeting agents include those listed in US20140294723A1, WO2001062298A2, WO1997045544A1, WO2006119897A2, WO2006050834A2, WO2008120101A2, WO2010078916A1, which are incorporated herein by reference.

[0011] In some embodiments, the tumor targeting agent comprises a collagen binding domain. In some embodiments, the polypeptide comprises at least two collagen binding domains. In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, or 6 collagen binding domains. In some embodiments, the polypeptide comprises a collagen binding domain derived from decorin or von Willebrand factor (VWF).

[0012] In some embodiments, the polypeptide further comprises a serum protein conjugated to the polypeptide. In some embodiments, the serum protein is conjugated to the polypeptide through a peptide bond. In some embodiments, the serum protein comprises albumin or a fragment thereof. In some embodiments, the serum protein is at least 40, 45, 50, 55, 60, 65, 70, or 75 kDa (or any range derivable therein).

[0013] In some embodiments, the polypeptide comprises a second linker. In some embodiments, the second linker comprises glycine and serine amino acid residues. In some embodiments, the polypeptide comprises a third, fourth, or fifth linker. In some embodiments, the third, fourth, or fifth linker comprises glycine and serine amino acid residues. In some embodiments, the linker is n = 1, 2, 3, 4, 5, 6, 7, or 8, or any range derivable therein (GGGS). n , (SEQ ID NO:48), or GGGSGGGS (SEQ ID NO:47). In some embodiments, the second linker comprises n=6 (GGGS) n (SEQ ID NO:48). In some embodiments, the polypeptide comprises a protein tag. In some embodiments, the protein tag comprises a 6H tag. In some embodiments, the protein tag comprises a protein tag described herein. In some embodiments, the polypeptide is not operably linked to a particle, nanovesicle, or liposome. In some embodiments, the composition does not comprise a liposome, particle, or nanovesicle.

[0014] In some embodiments, the method or disclosure relates to the treatment of skin cancer, such as for the treatment of melanoma. In some embodiments, the method of the disclosure further comprises administering one or more additional cancer therapies. In some embodiments, the additional therapy is as described herein. In some embodiments, the subject is undergoing or will undergo immunotherapy. In some embodiments, the method further comprises administering immunotherapy. In certain embodiments, the immunotherapy comprises an immune checkpoint inhibitor. The immune checkpoint inhibitor can be an anti-PD-1 monoclonal antibody or an anti-CTLA-4 monoclonal antibody. Additional exemplary immune checkpoint proteins that can be inhibited in embodiments of the disclosure are described herein. In some embodiments, the immune checkpoint inhibitor comprises one or more of nivolumab, pembrolizumab, pidilizumab, ipilimumab, or tremelimumab. In some embodiments, the immune checkpoint therapy is a monotherapy. The term monotherapy, in the context of immune checkpoint therapy, refers to the administration of one immune checkpoint inhibitor during the course of treatment. A monotherapy can be a therapy that includes only one of a PD-1, PDL1, PDL2, CTLA-4, B7-1, or B7-2 inhibitor. In some embodiments, the immune checkpoint inhibitor therapy includes a combination therapy. For example, the combination therapy can be a combination of (i) a PD-1, PDL1, or PDL2 inhibitor and (ii) a CTLA-4, B7-1, or B7-2 inhibitor. Particular combination therapies include those that include an anti-PD-1 antibody and an anti-CTLA-4 antibody. Additional immunotherapies useful in the methods and compositions of the disclosure are described herein. In some embodiments, the immunotherapy or additional therapy is administered before, after, or simultaneously with the polypeptide. In some embodiments, the polypeptide or composition is administered systemically. In some embodiments, the polypeptide or composition is administered by a route of administration described herein. In some embodiments, the polypeptide or composition is administered by intravenous injection. In some embodiments, the subject has previously been treated with a cancer therapy.In some embodiments, the subject is determined to be non-responsive to a previous treatment or the subject has experienced non-specific toxicity to a previous treatment.

[0015] As used herein, the term "cytokine polypeptide" refers to a polypeptide that is a cytokine or its receptor-binding domain and retains a portion of the cytokine activity.

[0016] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to gene products that include polymers of amino acids.

[0017] The terms "subject", "mammal" and "patient" are used interchangeably. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a clinical trial animal such as a mouse, rat, rabbit, dog, horse, or fly, zebrafish, etc.

[0018] The methods and compositions are intended to include any exclusion of any of the embodiments described herein.

[0019] As used herein, the terms "or" and "and / or" are used to combine or describe components mutually exclusively. For example, "x, y, and / or z" can mean "x" only, "y" only, "z" only, "x, y, and z", "(x and y) or z", "x or (y and z)" or "x or y or z". It is specifically contemplated that x, y, or z can be specifically excluded from an embodiment.

[0020] Throughout this application, the term "about" is used in its plain and ordinary sense in the field of cell biology to indicate that a value includes the standard deviation of error for the apparatus or method used to determine that value.

[0021] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes any unspecified elements, steps, or ingredients. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of "comprising" can also be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0022] [The present invention 1001] A polypeptide comprising a cytokine linked to a masking agent via a linker, the linker comprising one or more tumor-associated protease cleavage sites, and the masking agent comprising a cytokine receptor polypeptide or a fragment thereof that specifically binds to the cytokine. [The present invention 1002] The polypeptide of the present invention 1001, wherein the cytokine comprises IL12 and the masking agent comprises an IL12R polypeptide or an IL12-binding fragment thereof. [The present invention 1003] The polypeptide of the present invention 1002, wherein IL12 comprises one or both of the p35 and p40 subunits. [The present invention 1004] The polypeptide of the present invention 1003, wherein IL12 comprises a p35 subunit and a p40 subunit linked through a disulfide bond. [The present invention 1005] The polypeptide of the present invention 1003, wherein IL12 comprises a p35 subunit and a p40 subunit linked through a peptide linker. [The present invention 1006] The polypeptide of any one of claims 1002 to 1005, wherein the IL12R polypeptide or fragment thereof comprises IL12Rβ1 or a fragment thereof. [The present invention 1007] The polypeptide of the present invention, wherein the IL12Rβ1 polypeptide comprises one or both of the fibronectin domains D1 and D2. [The present invention 1008] The polypeptide of any of claims 1002 to 1007, wherein a masking agent is fused to the N-terminus of the p35 subunit of IL12, and a linker is between the masking agent and the p35 subunit of IL12. [The present invention 1009] The polypeptide of any of claims 1002 to 1007, wherein a masking agent is fused to the C-terminus of the p40 subunit of IL12, and a linker is located between the masking agent and the p40 subunit of IL12. [The present invention 1010] The polypeptide of the present invention 1001, wherein the cytokine comprises IL-2 and the masking agent comprises IL-2Rα, IL-2Rβ, a fragment thereof, or a combination of fragments. [The present invention 1011] The polypeptide of the present invention 1001, wherein the cytokine comprises IFNγ and the masking agent comprises IFNγR1, IFNγR2, a fragment thereof, or a combination of fragments. [The present invention 1012] The polypeptide of any of claims 1001 to 1011, comprising at least two tumor-associated protease cleavage sites. [The present invention 1013] The polypeptide of any of claims 1001 to 1012, wherein the tumor-associated protease cleavage site comprises a uPA, a matrix metalloproteinase, or a thrombin cleavage site. [The present invention 1014] The polypeptide of any one of claims 1001 to 1013, wherein the cytokine comprises a proinflammatory cytokine. [The present invention 1015] The polypeptide of any of claims 1001 to 1014, conjugated to a tumor targeting agent. [The present invention 1016] The polypeptide of the present invention, wherein the tumor targeting agent comprises an antibody or an antigen-binding fragment thereof. [The present invention 1017] The polypeptide of the invention 1016, wherein the antibody or antigen-binding fragment comprises a stroma targeting antibody or stroma binding fragment. [The present invention 1018] The polypeptide of the invention, wherein the antibody or binding fragment specifically binds to fibronectin, an alternative splice domain of fibronectin, collagen, tenascin, periostin, syndecan, proteoglycan, or a tumor stromal cell-specific antigen. [The present invention 1019] The polypeptide of the present invention, wherein the antibody or binding fragment specifically binds to the extra domain A (EDA) or extra domain B (EDB) of fibronectin. [The present invention 1020] The polypeptide of the present invention, wherein the tumor targeting agent comprises a Fab that specifically binds to an alternative splice domain of fibronectin that contains extra domain A (EDA). [The present invention 1021] The polypeptide of the present invention, wherein the tumor targeting agent comprises an antibody or an antigen-binding fragment thereof that specifically binds to a tumor-associated antigen. [The present invention 1022] The polypeptide of the present invention, wherein the tumor targeting agent comprises a collagen binding domain. [The present invention 1023] A polypeptide of the invention 1022 comprising at least two collagen binding domains. [The present invention 1024] The polypeptide of the present invention 1022 or 1023, comprising a collagen-binding domain derived from decorin or von Willebrand factor (VWF). [The present invention 1025] The polypeptide of any one of claims 1001 to 1024, further comprising a serum protein conjugated to the polypeptide. [The present invention 1026] The polypeptide of the present invention 1025, wherein the serum protein is conjugated to the polypeptide through a peptide bond. [The present invention 1027] The polypeptide of the present invention 1025 or 1026, wherein the serum protein contains albumin. [The present invention 1028] The polypeptide of any one of the present inventions 1001 to 1027, including a second linker. [The present invention 1029] The polypeptide of the present invention 1028, wherein the second linker contains glycine and serine amino acid residues. [The present invention 1030] The polypeptide of the present invention 1029, wherein the linker contains SEQ ID NO: 47 or SEQ ID NO: 48. [The present invention 1031] The polypeptide of any one of the present inventions 1001 to 1030, including a protein tag. [The present invention 1032] The polypeptide of any one of the present inventions 1001 to 1031, which is not functionally linked to particles, nanovesicles, or liposomes. [The present invention 1033] A composition comprising the polypeptide of any one of the present inventions 1001 to 1032. [The present invention 1034] The composition of the present invention 1033, which does not contain liposomes, particles, or nanovesicles. [The present invention 1035] A nucleic acid encoding the polypeptide of any one of the present inventions 1001 to 1032. [The present invention 1036] A host cell comprising the nucleic acid of the present invention 1035. [The present invention 1037] A method for producing a polypeptide, comprising the steps of expressing the nucleic acid of the present invention 1035 in a cell and isolating the expressed polypeptide. [The present invention 1038] A method for treating cancer, comprising the step of administering the polypeptide of any one of the present inventions 1001 to 1032 or the composition of the present invention 1033 or 1034. [The present invention 1039] The method of the present invention 1038, further comprising the step of administering one or more additional cancer therapies. [The present invention 1040] The method of the present invention 1038 or 1039, wherein the subject is receiving or will receive immunotherapy. [The present invention 1041] The method of any one of the present inventions 1038 to 1040, further comprising the step of administering immunotherapy. [The present invention 1042] The method of the present invention 1040 or 1041, wherein the immunotherapy comprises an immune checkpoint inhibitor. [The present invention 1043] The method of the present invention 1042, wherein the immune checkpoint inhibitor comprises an anti-PD-1 monoclonal antibody or an anti-CTLA-4 monoclonal antibody. [The present invention 1044] The method of claim 1043, wherein the immune checkpoint inhibitor comprises one or more of nivolumab, pembrolizumab, pidilizumab, ipilimumab, or tremelimumab. [The present invention 1045] The method of any of claims 1041 to 1044, wherein the immunotherapy is administered before, after or simultaneously with the polypeptide. [The present invention 1046] The method of any of claims 1038 to 1045, wherein the polypeptide or composition is administered systemically. [The present invention 1047] The method of claim 1046, wherein the polypeptide or composition is administered by intravenous injection. [The present invention 1048] The method of any of claims 1038 to 1047, wherein the subject has previously been treated with a cancer therapy. [The present invention 1049] The method of claim 1048, wherein the subject has been determined to be non-responsive to a previous treatment or the subject is experiencing non-specific toxicity to a previous treatment. It is specifically contemplated that any limitation discussed with respect to one embodiment of the present invention may be applied to any other embodiment of the present invention.Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention.Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, for example in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legend Descriptions. [Brief description of the drawings]

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1A] Figure 1A-C. Schematic of fusion of IL12Rβ1 recombinant protein to IL12. (A) Schematic of fusion of IL12Rβ1 recombinant protein to IL12. The IL12Rβ1-IL12 receptor binding site is not exposed in peripheral tissues, but tumor-specific proteases expose the IL12 binding site within the tumor. (B-C) Structure of IL12Rβ1 fibronectin I and II domain fusion to IL12. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Diagram 2] Figure 2A-B. Fusion of IL12Rβ1 fibronectin I and II domains to IL12 inactivates IL12 activity in vitro and in vivo. (A) IFNγ concentration after in vitro splenocyte culture. After 2 days of culture in the presence of IL12, IFNγ concentration in the supernatant was measured by ELISA. (B) 5x105 B16F10 cells were inoculated on day 0. IL12 variants (25μg, IL12 base) were injected iv on day 7. On day 9, blood was collected and IFNγ concentration in serum was determined by ELISA. [Figure 3-1]FIG. 3A-P. IL12Rβ1 fusions reduce potential treatment-related toxicity of IL12. (A) In vitro cleavage of IL12Rβ1-VP-IL12 and IL12Rβ1-LS-IL12 by MMP and uPA. IL-12, IL12Rβ1-VP-IL12 and IL12Rβ1-LS-IL12 were treated with either assay buffer only (blank), MMP2, MMP9 or uPA for 30 min at 37° C. The decrease in molecular weight from approximately 105 kDa to approximately 60 kDa indicates proteolytic cleavage of IL12Rβ1-VP-IL12 and IL12Rβ1-LS-IL12. (B) In vitro activity of IL12Rβ1-VP-IL12, IL12Rβ1-LS-IL12 and uncleavable IL12Rβ1-(G3S)11-IL12. IL-12, IL12Rβ1-VP-IL12, IL12Rβ1-LS-IL12 and IL12Rβ1-(G3S)11-IL12 were applied to preactivated murine CD8+ T cells at the indicated concentrations (n ​​= 2 per condition) and STAT4 phosphorylation was assessed by flow cytometry. Dose-response correlations and half-maximal activation values ​​are shown. (C) In vitro activity of IL12Rβ1-VP-IL12, IL12Rβ1-LS-IL12 after treatment with proteases. IL12Rβ1-VP-IL12 or IL12Rβ1-LS-IL12 were first treated with MMP2 or uPA, respectively. Cleaved constructs or IL12 were applied to preactivated murine CD8+ T cells at the indicated concentrations (n ​​= 2 per condition) and STAT4 phosphorylation was assessed by flow cytometry. Dose-response relationships and half-maximal activation values ​​are shown. (D) In ​​vivo toxicity of IL12Rβ1-VPLS-IL12-CBD in healthy mice. C57BL / 6 mice were treated iv with either PBS, IL12 or IL12Rβ1-VPLS-IL12-CBD (doses shown on an IL12 molar basis) on days 0, 3 and 6. On days 2, 5 and 8, mice were bled and serum was analyzed for the presence of proinflammatory cytokines using the LEGENDplex Cytokine Release Syndrome Panel. (E) White blood cell counts and (F) platelet counts were measured by hematology analyzer.(G-N) Blood toxicity markers were analyzed after IL12Rβ1-IL12 injection into non-tumor-bearing mice. The graphs depict the analysis of liver injury markers (blood albumin concentration, total protein, alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, and alkaline phosphatase activity), kidney injury markers (total bilirubin), pancreatic injury markers (amylase), and lung injury markers (CO2 concentration). (O-P) Blood toxicity markers (ALT activity and amylase) were analyzed after IL12Rβ1-IL12 and anti-PD-1 antibody injection into B16F10 tumor-bearing mice. Statistical analysis was performed using ANOVA with Tukey's test. *p < 0.05 **p < 0.01; NS = not significant. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Diagram 3-4] See description of Figure 3-1. [Figure 3-5] See description of Figure 3-1. [Diagram 3-6] See description of Figure 3-1. [Diagram 3-7] See description of Figure 3-1. [Figure 4A]Figures 4A-D. IL12Rβ1-IL12 with a cleavable linker treatment reduces the growth rate of B16F10 melanoma. 5×105 B16F10 cells were inoculated on day 0. IL12 (25 μg), equimolar IL12 variants, or PBS were administered i.v. on (A) day 8 or (B) day 7. (A-B) IL12Rβ1-IL12 with a uPA protease-cleavable linker (LS) was used. (C) IL12 Rβ1-IL12 (50 μg) and IL12 (5 μg) with a uPA and MMP protease-cleavable linker (VP-LS) were injected i.v. every 3 days starting on day 7. (D) 100 μg of IL12Rβ1-IL12 with cleavable linkers (HP, VP, and LS) was injected i.v. on days 7 and 10. An anti-PD-1 antibody was injected i.p. on days 7, 10, and / or 13. The graph depicts the tumor volume until the first mouse died. Tumor volume is presented as mean ± SEM. n = 3-4 [Figure 4B] See the description of Figure 4A. [Figure 4C] See the description of Figure 4A. [Figure 4D] See the description of Figure 4A.

Mode for Carrying Out the Invention

[0024] Detailed Description Cytokines are important factors of antitumor activity, but to date many of them have not been transferred to the clinic. IL12 is one of the most potent antitumor cytokines, but due to its high toxicity, clinical trials have been terminated or failed. Therefore, reducing its toxicity is an important strategy to transfer it to the clinic. To improve CBD-IL12 therapy, a domain of the IL12 receptor IL12Rβ1 was fused to IL12 to form IL12Rβ1-IL12. This fusion is inactive, but the inclusion of an MMP or thrombin cleavage site between the receptor masking agent and the cytokine generates a procytokine that can be activated within the tumor microenvironment. We have demonstrated that the immunotoxicity of IL12 is thus reduced and that fusion of IL12Rβ1-IL12 with a protease-sensitive linker retains therapeutic utility. The inventors have also found that introducing multiple cleavage sites into the linker (e.g., tandem MMP, tandem thrombin, and MMP-thrombin dyads and repeats) may increase protease sensitivity and enhance the antitumor efficacy of IL12Rβ1-IL12 therapy. Furthermore, the use of collagen binding domains fused to the masked therapeutic molecules of the present disclosure is particularly useful because the CBD increases the retention of the masked therapeutic in the tumor microenvironment, which prolongs the exposure of the masked therapeutic to proteases and increases the local concentration of the unmasked therapeutic. In conclusion, the inventors have developed a technology to reduce the toxicity of therapeutic agents by fusing cytokine receptors to cytokines. Tumor-specific proteases cleave the linker to activate cytokines within the tumor.

[0025] I. Polypeptide Aspects A. Therapeutic and Masking Agents Aspects of the present disclosure relate to a therapeutic agent and a masking agent that binds to the therapeutic agent and inhibits the association of the therapeutic agent with its target, thereby reducing toxicity associated with the therapeutic agent. The polypeptide of the present disclosure contains a tumor-associated protease cleavage site that unmasks the therapeutic agent when it encounters the associated protease. The protease is a protease that is enriched in the tumor microenvironment, so that when administered systemically, a reduction in active therapeutic agent is observed in normal tissues compared to systemic administration of an unmasked therapeutic agent.

[0026] 1. Cytokines In some embodiments, the therapeutic agent comprises a cytokine or a therapeutic polypeptide derived from a cytokine. In certain embodiments, the cytokine comprises a functionally active fragment of the cytokine. In some embodiments, the functionally active cytokine fragment binds to and activates a corresponding receptor. In some embodiments, the cytokine comprises IL12. IL12 is a heterodimeric glycosylated cytokine composed of disulfide-linked p35 (approximately 35 kDa) and p40 (approximately 40 kDa) subunits. The human IL12 p35 sequence is as follows: It is represented by TIFF0007682150000001.tif26159. The human IL12 p40 sequence is: Represented by TIFF0007682150000002.tif41159. The mouse IL12 p35 sequence is: It is represented by TIFF0007682150000003.tif26159. The mouse IL12 p40 sequence is: Represented by TIFF0007682150000004.tif41159.

[0027] Suitable IL12 masking agents include polypeptides that bind to IL12 and inhibit the binding of IL12 to other molecules, such as IL12R. Exemplary polypeptides include polypeptides derived from IL12R, such as IL12Rβ1 and IL12Rβ2.

[0028] Mouse IL12Rβ1 has the following amino acid sequence: TIFF0007682150000005.tif129160.

[0029] Human IL12Rβ1 has the following amino acid sequence: It is represented by a polypeptide having TIFF0007682150000006.tif85159.

[0030] Human IL12Rβ2 has the following amino acid sequence: Mouse IL12Rβ2 is represented by a polypeptide having the following amino acid sequence: It is represented by a polypeptide having TIFF0007682150000008.tif107160.

[0031] In some embodiments, the cytokine comprises a polypeptide comprising an amino acid sequence of SEQ ID NO:3-6, or a polypeptide comprising the amino acid sequence of a fragment of a polypeptide represented by SEQ ID NO:3-6, or a polypeptide having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity to a polypeptide of SEQ ID NO:3-6 or a fragment thereof.

[0032] In some embodiments, the cytokine comprises an IL12 polypeptide and the masking agent comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or 19-22, or a fragment of a polypeptide represented by SEQ ID NO:2, or 19-22, or a polypeptide having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity to a polypeptide of SEQ ID NO:2, or 19-22 or a fragment thereof.

[0033] In some embodiments, the cytokine comprises IL-2. The human IL-2 sequence is TIFF0007682150000009.tif19159. The mouse IL-2 sequence is Contains TIFF0007682150000010.tif19159.

[0034] In some embodiments, the masking agent for IL-2 comprises an IL-2R polypeptide. In some embodiments, the IL-2R polypeptide comprises a polypeptide derived from an IL-2R beta, IL-2R alpha, or IL-2R gamma subunit. The human interleukin-2 receptor subunit beta has the following amino acid sequence: Contains TIFF0007682150000011.tif26159. Mouse interleukin-2 receptor subunit beta has the following amino acid sequence: The human interleukin-2 receptor subunit alpha has the following amino acid sequence: The mouse interleukin-2 receptor subunit alpha has the following amino acid sequence: The human interleukin-2 receptor subunit gamma has the following amino acid sequence: The mouse interleukin-2 receptor subunit gamma has the following amino acid sequence: TIFF0007682150000016.tif34160.

[0035] In some embodiments, the cytokine comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:23 or 24, or a polypeptide comprising the amino acid sequence of a fragment of the polypeptide represented by SEQ ID NO:23 and 24, or a polypeptide having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity to a polypeptide of SEQ ID NO:23 or 24 or a fragment thereof.

[0036] In some embodiments, the cytokine comprises an IL-2 polypeptide and the masking agent comprises a polypeptide comprising the amino acid sequence of SEQ ID NOs:27-32, or the amino acid sequence of a fragment of a polypeptide represented by SEQ ID NOs:27-32, or a polypeptide having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity to a polypeptide of SEQ ID NOs:27-32 or a fragment thereof.

[0037] In some embodiments, the cytokine comprises IFNγ. Murine IFNγ has the following sequence: Human IFNγ contains the following sequence: IFNγ can be a functional fragment, such as a C-terminal truncation. For example, the IFNγ polypeptide can be at least 30, 31, 32, 33, 34, 35, 36, 37 of SEQ ID NO:25 or 26. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132 contiguous amino acids of SEQ ID NO:25 or 26. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9 1, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132 (or any range derivable therein).

[0038] In some embodiments, the masking agent for the IFNγ polypeptide comprises a polypeptide from IFNγ receptor 1 or IFNγ receptor 2. Human IFNγ receptor 1 has the following sequence: TIFF0007682150000019.tif34159 contains the following sequence: TIFF0007682150000020.tif34159. Human IFNγ receptor 2 has the following sequence: Contains TIFF0007682150000021.tif34160. Mouse IFNγ receptor 2 has the following sequence: Contains TIFF0007682150000022.tif43162.

[0039] In some embodiments, the cytokine comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:25 or 26, or a polypeptide comprising the amino acid sequence of a fragment of the polypeptide represented by SEQ ID NO:25 and 26, or a polypeptide having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity to the polypeptide of SEQ ID NO:25 or 26 or a fragment thereof.

[0040] In some embodiments, the cytokine comprises an IFNγ polypeptide and the masking agent comprises a polypeptide comprising the amino acid sequence of SEQ ID NOs:33-36, or the amino acid sequence of a fragment of a polypeptide represented by SEQ ID NOs:33-36, or a polypeptide having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity to a polypeptide of SEQ ID NOs:33-36 or a fragment thereof.

[0041] In embodiments of the present disclosure, the masking agent can be a polypeptide or a functional fragment of a polypeptide described herein. In some embodiments, the masking agent comprises a receptor polypeptide or a fragment thereof that binds to a cytokine.

[0042] 2. Antibodies In some embodiments, the therapeutic agent comprises an antibody, such as a therapeutic antibody. In some embodiments, the therapeutic antibody is modified by site-specific substitution of an amino acid in either the heavy or light chain variable region with a cysteine ​​(Cys). The sulfhydryl (SH) group in the side chain of the substituted-in Cys serves as a chemical handle for attaching a masking agent that interferes with the ability of the antibody to bind to its antigen. The masking agent can be a group that sterically blocks antibody-antigen binding, but does not otherwise specifically interact with either the antibody or the antigen.

[0043] Alternatively, the masking agent may interact with the antibody, for example, by electrostatic or van der Waals forces. The tumor-associated protease cleavage site may be between or link the masking agent and the antibody.

[0044] In some embodiments, masking agent can have its own pharmacological activity after its release by cleavage of tumor-associated protease cleavage site.Cys substitution site is selected so that the replacement of original amino acid with Cys does not adversely affect the ability of antibody to specifically and strongly bind to its antigen.Furthermore, removal of masking agent can leave residual chemical group still covalently bonded to Cys.

[0045] In one embodiment, there is provided a prodrug antibody according to formula (I): (ML) m -Ab I wherein Ab is an antibody in which at least one amino acid in its heavy or light chain variable region has been replaced by Cys, where the replaced amino acid (a) is in a framework region; (b) has at least 30% side chain exposure and (c) is within 10 A, preferably 5 A, of a CDR amino acid; M is a masking agent that inhibits binding of the Ab to its antigen; each L is independently a linker moiety attached to M and to the Ab, where L comprises a tumor-associated cleavage site and is attached to the Ab at said Cys; and m is 1, 2, 3, or 4.

[0046] In some embodiments, at least one substituted amino acid in the antibody Ab is at Kabat position number 1, 3, 5, 19, 23, 25, 43, 46, 68, 72, 74, 75, 76, 82a, 82b, 83, 84, 85, or 105 of the heavy chain variable region or at Kabat position number 1, 3, 5, 7, 8, 18, 20, 45, 57, 60, 63, 65, 66, 67, 69, 77, or 100 of the light chain variable region. In some embodiments, at least one substituted amino acid in the antibody Ab is at Kabat position number 23 of the heavy chain or Kabat position number 67 of the light chain. In some embodiments, an antibody is provided that has a Cys at Kabat position number 67 of the light chain. The antibody can be an anti-CTLA4 antibody or an anti-CD137 antibody. In some embodiments, an antibody is provided that has a Cys at Kabat position number 23 of the heavy chain. The antibody can be an anti-CTLA4 antibody or an anti-CD137 antibody.

[0047] The masked therapeutic antibody of the present disclosure can be polyclonal, monoclonal, murine, human, humanized, or chimeric. Suitable amino acids in the heavy and light chain variable regions for substitution with Cys are framework amino acids whose side chains are solvent exposed, preferably at least 30% exposed, so that the substituted inserted Cys is accessible for attachment of a masking agent. It is also important that the substituted-out amino acid is close to the CDR amino acid so that the masking agent can effectively interfere with antibody-antigen binding. A distance of 10 A or less, more preferably 5 A or less, is preferred. Preferred positions for Cys substitution include position number 23 in the heavy chain variable region and position number 67 in the light chain variable region, as numbered by Kabat. Both positions are in the framework region of each variable region. Cys can be inserted and substituted at these positions by site-directed substitution techniques well known in the art. Substitution at the first site is represented by the abbreviation V, ... L X67C, where X represents the amino acid being substituted out. In natural antibodies, this site is highly conserved and is often Ser. Substitutions at the second site can also be made to V H It can be called X23C.

[0048] The masked antibody of the present disclosure can have substitutions in the VH region or the VL region, or both.If the antibody has only one of these substitutions, the theoretical maximum number of blocking moiety-linker compounds that can be attached is 2, but the masked antibody preparation may be statistically assayed for a smaller number, reflecting the chemical inefficiency of the attachment process.If the antibody has both substitutions, the theoretical maximum number is 4.

[0049] In some embodiments, the antibody is a bispecific antibody with two different pairs of heavy and light chains.Thus, the masked antibody of the present disclosure can be a bispecific antibody with only one heavy / light chain pair masked, or a bispecific antibody with both heavy / light chain pairs masked.It is also known to replace amino acids in VH or VL regions with Cys in order to introduce sulfhydryl side chains suitable for conjugation by maleimide addition chemistry to generate antibody-drug conjugates.See, for example, Eigenbrot et al. 2007 and Bhakta et al. 2016).

[0050] Masking agents that can be used to impede or block the activity of a masked antibody with its antigen include polyethylene glycol (PEG), albumin binding polypeptides, adnectins, peptides, and soluble globular proteins such as albumin or fibrinogen. In some embodiments, the blocking agent contains a 2 kDa -(CH 2 CH 2 PEG having a molecular weight of at least about 2 kDa, and preferably 5 kDa, corresponding to PEG having about 115 -(CH 2 CH 2 O)-repeating units, corresponding to PEG having a molecular weight of at least about 5 kDa.

[0051] The antibody having Cys described herein can be conjugated to a masking agent having a maleimide end group by Michael addition of Cys sulfhydryl (SH), as known in the art.The procedure of such conjugation is well known in the art; for example, see Shepard et al, WO 2017 / 112624 A 1 (2017), which is incorporated herein by reference.Further examples of specific masking agents for therapeutic antibodies are disclosed in WO2019036433, which is incorporated herein by reference.

[0052] In further embodiments, the therapeutic agent may be an antibody whose variable region is masked by linking the N-terminus of the variable region chain to a coiled-coil-forming peptide. The coiled-coil-forming peptides associate with each other to form a coiled coil (i.e., each peptide forms a coil, and these coils are coiled around each other). The coiled coil may sterically inhibit the binding of the antibody binding site to its target. In some embodiments, the antibody comprises a bivalent antibody. The non-covalent bond between the coiled-coil-forming peptides is sufficient to form a stable coiled coil that inhibits the binding of the antibody variable region; for example, it is not necessary for the coiled-coil-forming peptides to be further linked by a disulfide bridge between the terminal cysteines of each peptide. The presence of non-naturally occurring cysteines is potentially disadvantageous, as it may cause misfolding or misbinding problems. Masking of the antibody in this manner can reduce the binding affinity (and cytotoxic activity in the case of ADC) by more than 100-fold. Antibodies can be masked in this manner without significantly compromising expression, purification, conjugation, pharmacokinetics, or binding or other activity upon unmasking.

[0053] In some embodiments, the masking agent includes a coiled coil. The coiled coil-forming peptides are peptide pairs that can associate with each other to form a coiled coil. "Coiled coil" is a technical term referring to a bundle of alpha helices wound into a superhelical structure. Leucine zipper-forming peptides are an example of peptides that associate to form a coiled coil. The coiled coils formed in the present disclosure are typically formed from two coiled coil-forming peptides. The coiled coil can be formed by alpha helices on the peptides in a parallel or antiparallel orientation. The coiled coil is further characterized by the packing of amino acid side chains in the core of the bundle, called knob-into-hole, where a residue (knob) from one helix packs into a space (hole) surrounded by four side chains of the opposing helix. The residues involved in the knob-into-hole interaction are usually hydrophobic, while the outer residues are hydrophilic, so the sequence of the coiled coil exhibits a "heptad" repeat in the chemical nature of the side chains. Examples of consensus formulas for heptad repeats in coiled coil-forming peptides are provided by WO2011034605, which is incorporated herein by reference.

[0054] In some embodiments, the coiled coil includes Formula II: (XI, X2, X3, X4, X5, X6, X7)n II Wherein XI is a hydrophobic amino acid or asparagine; X2, X3 and X6 are any amino acids; X4 is a hydrophobic amino acid; and X5 and X7 are each charged amino acid residues.

[0055] Examples of coiled coils are as follows: including TIFF0007682150000023.tif172160.

[0056] The coiled-coil-forming peptide is linked to the N-terminus of the antibody variable region via a linker that contains a tumor-associated protease cleavage site. A typical antibody comprises heavy and light chain variable regions, in which the coiled-coil-forming peptide is linked to the N-terminus of each. A bivalent antibody has two binding sites, which may or may not be the same. In a normal monospecific antibody, the binding sites are the same, and the antibody has two identical pairs of light and heavy chains. In this case, each heavy chain is linked to the same coiled-coil-forming peptide, and each light chain is linked to the same coiled-coil-forming peptide (which may or may not be the same as the peptide linked to the heavy chain).

[0057] In bispecific antibodies, the binding sites are different and are formed from two different pairs of heavy and light chains. The binding sites can have specificity for different targets or different epitopes on the same target. When the binding sites have specificity for different targets, the targets can be on the same cell (e.g., two different surface antigens on cancer cells) or on two different cells (e.g., one surface antigen on cancer cells and one on immune cells such as T cells). For example, one binding site of bispecific antibodies can be directed to CD3 or 4-1BB.

[0058] In a bispecific antibody, the heavy and light chain variable regions of one binding site can each be linked to a coiled-coil forming peptide. The heavy and light chain variable regions of the other binding site may or may not also be linked to a coiled-coil peptide. When both heavy and light pairs of both binding sites are both linked to coiled-coil peptides, usually both heavy chain variable regions are linked to the same type of coiled-coil forming peptide, as are both light chain variable regions. Masking of both binding sites can be useful, for example, when both binding sites have specificity for the same surface antigen on tumors. Masking of one binding site but not both can be useful, for example, when one binding site is specific for a tumor surface antigen and the other is specific for a surface antigen on immune cells. The binding site with specificity for tumor surface antigens or immune cell antigens can be masked. Some bispecific antibodies with specificity for both tumor surface antigens and immune cells have masking of both sites.

[0059] Coiled coils can be formed from the same peptide forming homodimers or two different peptides forming heterodimers. In the case of homodimer formation, the antibody light chain and the heavy chain are linked to the same coiled coil-forming peptide. In the case of heterodimer formation, the antibody light chain and the heavy chain are linked to different coiled coil peptides. For some pairs of coiled coil-forming peptides, it is preferable that one of the pair is linked to the antibody heavy chain and the other is linked to the antibody light chain, although the reverse orientation is also possible.

[0060] Each antibody chain can be linked to a single coiled-coil forming peptide or multiple such peptides in tandem (e.g., 2, 3, 4 or 5 copies of the peptide). In the latter case, the peptides in the tandem linkage are usually the same. Also, when tandem linkage is utilized, the light and heavy chains are usually linked to the same number of peptides.

[0061] Linking an antibody chain to a coiled-coil forming peptide can reduce the binding affinity of the antibody by, for example, at least 10, 50, 100, 200, 500, 1000, 1500, 2000, 4000, 5000 or 10,000 fold compared to the same antibody without such linkage or after cleavage of such linkage. For some such antibodies, the binding affinity is reduced by 50-10,000, 50-5000, 50-4000, 50-1000, 100-10,000, 100-5000, 100-4000, 200-10,000, 200-5000, 50-1500, 100-1500, 200-1500, 200-1000, 500-1500, 50-1000, 100-1000, 200-1000, 500-1000, 50-500, 100-500 fold.

[0062] Antibodies include non-human antibodies, humanized antibodies, human antibodies, chimeric antibodies, and veneered antibodies, nanobodies, dAbs, scFV's, Fabs, etc. Some such antibodies include antibodies specific for cancer cell antigens, preferably those on the cell surface that can be internalized into the cell upon antibody binding. Targets to which antibodies can be directed include receptors and their ligands or counterreceptors on cancer cells (e.g., CD3, CD19, CD20, CD22, CD30, CD33, CD34, CD40, CD44, CD52, CD70, CD79a, CD123, Her-2, EphA2, lymphocyte-associated antigen 1, VEGF or VEGFR, CTLA-4, LIV-1, Nectin-4, CD74, and SLTRK-6).

[0063] In some embodiments, the antibody is brentuximab or brentuximab vedotin, anti-CD30, alemtuzumab, anti-CD52, rituximab, anti-CD20, trastuzumab Her / neu, nimotuzumab, cetuximab, anti-EGFR, bevacizumab, anti-VEGF, palivizumab, anti-RSV, abciximab, GpIIb / IIIa, infliximab, adalimumab, certolizumab, golimumab TNF-α, basiliximab, daclizumab, anti-IL-2, omalizumab, anti-IgE, gemtuzumab or vadastuximab, anti These include CD33, natalizumab, anti-VLA-4, vedolizumab α4β7, belimumab, anti-BAFF, otelixizumab, teplizumab, anti-CD3, ofatumumab, ocrelizumab, epratuzumab, anti-CD22, alemtuzumab, eculizumab, canakinumab, mepolizumab, reslizumab, tocilizumab, ustekinumab, and briakinumab.

[0064] Further aspects are described in WO2018107125, which is incorporated herein by reference.

[0065] B. Collagen-binding domain Collagen is an extracellular matrix (ECM) protein that regulates various cell biological functions, such as proliferation, differentiation, and adhesion in both normal and tumor tissues (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). Collagen is the most abundant protein in the mammalian body, occurring as one or more of 28 isoforms in almost all tissues (Ricard-Blum, Cold Spring Harb Perspect Biol 3:a004978, 2011). The subendothelial space of blood vessels is rich in collagen. Collagen is almost absent in blood due to its insolubility under physiological conditions (Dubois et al., Blood 107:3902-06, 2006; Bergmeier and Hynes, Cold Spring Harb Perspect Biol 4:a005132, 2012). Tumor vasculature has been reported to be permeable due to abnormal structures (Nagy et al., British journal of cancer 100:865, 2009). Thus, collagen is exposed in tumors due to their leaky vasculature (Liang et al., Journal of controlled release 209:101-109, 2015; Liang et al., Sci Rep 6:18205, 2016; Yasunaga et al., Bioconjugate Chemistry 22:1776-83, 2011; Xu et al. The Journal of cell biology 154:1069-80, 2001; Swartz and Lund, Nat Rev Cancer 12:210-19). Tumor tissue also contains increased amounts of collagen compared to normal tissue (Zhou et al. J Cancer 8:1466-76, 2017; Provenzano et al. BMC Med 6:11, 2008).

[0066] Von Willebrand factor (vWF) is a blood coagulation factor that binds to both collagen types I and III, and to the adhesion receptor GPIb on platelets (Lenting et al., Journal of thrombosis and haemostasis:JTH 10:2428-37, 2012; Shahidi Advances in experimental medicine and biology 906:285-306, 2017). Upon injury, collagen beneath the endothelium is exposed to plasma, and vWF-collagen binding initiates the thrombogenic cascade (Shahidi Advances in experimental medicine and biology 906:285-306, 2017; Wu et al. Blood 99:3623-28, 2002). Among the reported proteins / peptides of nonbacterial origin, the vWF A domain has the highest affinity for collagen (Addi et al., Tissue Engineering Part B: Reviews, 2016). In particular, within the A domain, the A3 domain of vWF has been reported to be a collagen binding domain (CBD) (Ribba et al. Thrombosis and Haemostasis 86:848-54, 2001). As mentioned above, the present inventors considered that fusion protein with vWF A3 CBD could realize targeted cytokine immunotherapy due to the exposure of collagen by the leaky vasculature of tumors even in the case of systemic injection.

[0067] In some embodiments, the collagen binding domain comprises a polypeptide derived from decorin. Exemplary decorin polypeptides include those having the following sequences: Examples include human decorin, represented by TIFF0007682150000024.tif48159, or fragments thereof, a peptide derived from human decorin: LRELHLDNNC (SEQ ID NO:41), and a peptide derived from bovine decorin: LRELHLNNNC (SEQ ID NO:44).

[0068] In some embodiments, the CBD comprises a polypeptide fragment derived from vWF. In some embodiments, the CBD comprises the amino acid sequence: vWF A1, derived from the human sequence, residue numbers 1237-1458 (474-695 of mature VWF) or a fragment thereof, represented by TIFF0007682150000025.tif34160.

[0069] In some embodiments, the CBP has the following amino acid sequence: Contains all or a fragment of vWF A3, represented by TIFF0007682150000026.tif56160.

[0070] In some embodiments, the CBP has the following amino acid sequence: It contains a vWF A3 domain polypeptide with a 6H tag having TIFF0007682150000027.tif26160.

[0071] In some embodiments, the CBP comprises a peptide or polypeptide derived from von Willebrand factor (vWF), such as a collagen-binding peptide derived from vWF. The sequence of human vWF is as follows: Includes TIFF0007682150000028.tif85159TIFF0007682150000029.tif245160TIFF0007682150000030.tif26158.

[0072] In some embodiments, the peptide is derived from the vWF A3 domain and has the following amino acid sequence (or a fragment thereof): TIFF0007682150000031.tif26160.

[0073] A CBP peptide or polypeptide can be a peptide having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity to the CBD peptides or peptide fragments listed above, such as SEQ ID NOs:1, 37-41, and 44-46.

[0074] C. Linker In some embodiments, the polypeptide comprises or further comprises a linker. The linker can be between any two domains of the polypeptide. In some embodiments, the polypeptide comprises a linker between the CBP and the cytokine. In some embodiments, the polypeptide comprises a linker between the CBP and the serum polypeptide. In some embodiments, the polypeptide comprises a linker between the masking agent and the cytokine. In some embodiments, the polypeptide comprises a linker between the albumin and the cytokine. In some embodiments, the polypeptide comprises a linker between the therapeutic agent and the masking agent. In some embodiments, the polypeptide comprises a linker between the therapeutic agent and the CBP. In some embodiments, the linker comprises one or more tumor-associated protease cleavage sites. A tumor-associated protease cleavage site refers to a cleavage site recognized by a protease that is highly upregulated or enriched in the tumor microenvironment. A tumor-associated protease cleavage site may not be tumor-specific (meaning that the protease is expressed only in the tumor), but is tumor-enriched, meaning that the protease is expressed at a higher level in the tumor microenvironment than in normal tissues or most normal tissues. In some embodiments, the tumor-associated protease cleavage site comprises an amino acid sequence recognized and cleaved by matrix metalloproteinase. For example, the tumor-associated protease cleavage site can be a site cleaved by MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, or a combination thereof. In some embodiments, the tumor-associated protease cleavage site comprises an MMP response sequence of SEQ ID NO:13: GLLSGRSDNH. In some embodiments, the tumor-associated protease cleavage site can be a site cleaved by thrombin. In some embodiments, the thrombin responsive sequence comprises SEQ ID NO:14: LVPRGS.

[0075] In some embodiments, two polypeptides, such as two of CBP, serum protein, therapeutic agent, masking agent, and cytokine, can be linked through a bifunctional linker. A linker, such as an amino acid or peptidomimetic sequence, can be inserted between the peptide and / or antibody sequences. In one embodiment, a fynomer domain is linked to the heavy (H) or light (L) chain immediately following the last amino acid at the amino (NH2) or carboxy (C) terminus of the heavy (H) or light (L) chain. The linker can have one or more properties including a flexible three-dimensional structure, inability to form a regular secondary structure, or hydrophobicity or charge that promotes or interacts with any domain. Examples of amino acids commonly found in flexible protein regions can include Gly, Asn, and Ser. For example, suitable peptide linkers can be GGGSGGGS (SEQ ID NO:47) or (GGGS)n (SEQ ID NO:48), where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein). Other substantially neutral amino acids such as Thr and Ala can also be used in the linker sequence. The length of the linker sequence can vary without significantly affecting the function or activity of the fusion protein (see, e.g., U.S. Patent No. 6,087,329). Examples of linkers can also include chemical moieties and conjugating agents such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), and disuccinimidyl tartrate (DST). Further examples of linkers can include linear carbon chains such as CN (where N = 1 to 100 carbon atoms, e.g., N = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more).In some embodiments, the linker can be a dipeptide linker, such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. In some embodiments, the linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation is performed via a maleimide group that reacts with sulfhydryls (thiols, --SH), while its sulfo-NHS ester is reactive towards primary amines (found in lysine and the N-terminus of proteins or peptides). Additionally, the linker can be maleimidocaproyl (mc).

[0076] In some embodiments, the linker comprises one or more polypeptides that are cleavable by, i.e., are substrates for, enzymes (proteases) that are uniquely expressed or overexpressed in cancer or tumor microenvironments compared to healthy tissues or organs. Preferably, the enzymes are found in the extracellular environment of tumors. Examples of such proteases include aspartic proteases (e.g., renin), fibroblast activation protein (FAP), aspartic cathepsins (e.g., cathepsin D, caspase 1, caspase 2, etc.), cysteine ​​cathepsins (e.g., cathepsin B), cysteine ​​proteases (e.g., legumain), a disintegrin / metalloproteinase (ADAM, e.g., ADAM8, ADAM9), a disintegrin / metalloproteinase with thrombospondin motifs (ADAMTS, e.g., For example, ADAMTSl), integral membrane serine proteases (e.g., matriptase 2, MT-SPl / matriptase, TMPRSS2, TMPRSS3, TMPRSS4), kallikrein-related peptidases (KLKs, e.g., KLK4, KLK5), matrix metalloproteases (e.g., MMP-1, MMP-2, MMP-9), and serine proteases (e.g., cathepsin A, coagulation factor proteases, e.g., elastase, plasmin, thrombin, PSA, uPA, factor Vila, factor Xa, and HCV NS3 / 4). Preferably, the protease is fibroblast activation protein (FAP), urokinase-type plasminogen activator (uPA, urokinase), MT-SP1 / matriptase, legumain, or matrix metalloproteases (particularly MMP-1, MMP-2, and MMP-9). Those skilled in the art will understand that the choice of enzyme and corresponding cleavable peptide will depend on the disease to be treated and the proteases expressed by the affected tissue or organ.

[0077] Further examples of tumor-associated protease sites include LSGRSDNH (SEQ ID NO:49), which is cleaved by urokinase, matriptase, or legumain; VPLSLYS (SEQ ID NO:50), which is cleaved by MMP2 or MMP9; PLGLAG (SEQ ID NO:51), which is cleaved by MMP2; VLVPMAMMAS (SEQ ID NO:52), which is cleaved by MMP1; XXQAR(A / V)X (SEQ ID NO:53), where X is any amino acid cleaved by matriptase; AGPR (SEQ ID NO:54), which is cleaved by matriptase; AANL (SEQ ID NO:55) and PTNL (SEQ ID NO:56), which are cleaved by legumain; and TSGRSANP (SEQ ID NO:57).

[0078] Linker sequences can be included in the polypeptides of the present disclosure, for example, at least, at most, or exactly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, A linker having 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (or a range derivable therein) can separate or be between any two of the CBP, serum protein, therapeutic agent, masking agent, and cytokine.

[0079] The linker can comprise the sequence of SEQ ID NO:13, 14, or 47-57, or a peptide having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or a range derivable therein) identity to SEQ ID NO:13, 14, or 47-57.

[0080] Further examples of tumor-associated protease sites include those in the table below. TIFF0007682150000032.tif242160TIFF0007682150000033.tif58160

[0081] D. Serum Proteins In some embodiments, the polypeptide of the present disclosure is further linked to serum protein. Serum proteins include, for example, albumin, globulin, and fibrinogen. Globulins include alpha 1 globulin, alpha 2 globulin, beta globulin, and gamma globulin. Albumin can be mouse, human, bovine, or any other homologous albumin protein. In some embodiments, albumin includes human serum albumin, which is encoded by the ALB gene and is exemplified by the following amino acid sequence: TIFF0007682150000034.tif77160.

[0082] In some embodiments, the albumin comprises mouse albumin having the following sequence: TIFF0007682150000035.tif78160.

[0083] In some embodiments, the serum protein comprises a polypeptide of SEQ ID NO:42 or 43, or a fragment thereof, or a polypeptide having 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or a range derivable therein) identity to SEQ ID NO:42, 43, or a fragment thereof.

[0084] E. Polypeptide Aspects Specific polypeptide embodiments are exemplified below. TIFF0007682150000036.tif142150TIFF0007682150000037.tif243150TIFF0007682150000038.tif228150TIFF0007682150000039.tif126150

[0085] In some embodiments, the polypeptide has at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or a range derivable therein) identity to a polypeptide of the disclosure, such as SEQ ID NOs:7-12, and 15-18, or a fragment thereof.

[0086] F. Protein Tags In some embodiments, the polypeptide further comprises a protein tag. The protein tag can be used, for example, in protein purification and / or immunoassays. Exemplary protein tags include AviTag (GLNDIFEAQKIEWHE (SEQ ID NO:82)), a peptide that allows biotinylation by the enzyme BirA, such that the protein can be isolated by streptavidin, calmodulin-tag, a peptide that is bound by the protein calmodulin, TIFF0007682150000040.tif5128, polyglutamic acid tag (EEEEEE (SEQ ID NO:84)), a peptide that efficiently binds to anion exchange resins such as Mono-Q, E-tag (GAPVPYPDPLEPR (SEQ ID NO:102)), a peptide recognized by an antibody, FLAG-tag (DYKDDDDK (SEQ ID NO:85)), a peptide recognized by an antibody, HA-tag (YPYDVPDYA (SEQ ID NO:86)), a peptide derived from hemagglutinin that is recognized by an antibody, His-tag (HHHHHH (SEQ ID NO:87)), a sequence of 5-10 histidines bound by a nickel or cobalt chelate, Myc-tag (EQKLISEEDL (SEQ ID NO:88)), a peptide derived from c-myc that is recognized by an antibody, the NE-tag (TKENPRSNQEESYDDNES (SEQ ID NO:89)), a novel 18 amino acid synthetic peptide recognized by a monoclonal IgG1 antibody, useful in a wide range of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; the S-tag (KETAAAKFERQHMDS (SEQ ID NO:90)), a peptide derived from ribonuclease A; and the SBP-tag (SBP-tag), a peptide that binds to streptavidin. TIFF0007682150000041.tif4142, Softag 1 (SLAELLNAGLGGS (SEQ ID NO:92)) for mammalian expression, Softag 3 (TQDPSRVG (SEQ ID NO:93)) for prokaryotic expression, Strep-tag (Strep-tag II: WSHPQFEK (SEQ ID NO:94)), a peptide that binds to streptavidin or a modified streptavidin called streptactin, TC-tag (CCPGCC (SEQ ID NO:95)), a tetracysteine ​​tag recognized by FlAsH and ReAsH biarsenicals, V5-tag (GKPIPNPLLGLDST (SEQ ID NO:96)), a peptide recognized by an antibody, VSV-tag (YTDIEMNRLGK (SEQ ID NO:97)), a peptide recognized by an antibody, Xpress-tag (DLYDDDDK (SEQ ID NO:98)), a peptide recognized by an antibody, covalent peptide tags, Isopeptag (TDKDMTITFTNKKDAE (SEQ ID NO:99)), a peptide that is covalently attached to the pyrin-C protein, SpyTag (AHIVMVDAYKPTK (SEQ ID NO:100)), a peptide that is covalently attached to the SpyCatcher protein, SnoopTag (KLGDIEFIKVNK (SEQ ID NO:101)), a peptide that is covalently attached to the SnoopCatcher protein, and BCCP (Biotin Carboxyl Carrier Protein), a protein domain that is biotinylated by BirA to enable recognition by streptavidin.These include the Glutathione-S-Transferase-Tag, a protein that binds immobilized glutathione, the Green Fluorescent Protein-Tag, a protein that spontaneously fluoresces and can be bound by nanobodies, the HaloTag, a mutant bacterial haloalkane dehalogenase that covalently binds reactive haloalkane substrates, allowing attachment to a wide variety of substrates, the Maltose Binding Protein-Tag, a protein that binds amylose agarose, the Nus-Tag, the Thioredoxin-Tag, the Fc-Tag, which is derived from the immunoglobulin Fc domain and allows dimerization and solubilization and can be used for purification on Protein-A Sepharose, the Designed Intrinsically Disordered Tag, which contains disorder-promoting amino acids (P, E, S, T, A, Q, G,), and the Ty-Tag. In some embodiments, the polypeptide comprises the 6H tag of SEQ ID NO:87.

[0087] II. Proteinaceous Compositions A polypeptide or polynucleotide of the disclosure, such as a CBD, serum protein, therapeutic agent, masking agent, linker, or cytokine polypeptide, may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acid or nucleic acid substitutions, or may be selected from the group consisting of SEQ ID NO: NO: 1 to 190, at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 11 6, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 1 63, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more or any derivable therein The sequence may be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous to a sequence of consecutive amino acids or nucleic acids in the range of

[0088] A polypeptide of the disclosure, such as a CBD, serum protein, therapeutic agent, masking agent, linker, or cytokine polypeptide, may be selected from the group consisting of SEQ ID NO:NO: 1-190, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 1 46, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 2 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or any range derivable therein.

[0089] In some embodiments, the polypeptide of the disclosure has SEQ ID NO:1-190 amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 , 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 2 06, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any range derivable therein).

[0090] In some embodiments, a polypeptide of the disclosure, such as a CBD, serum protein, therapeutic agent, masking agent, linker, or cytokine polypeptide, is selected from the group consisting of SEQ ID NO: NO: At least, at most, about, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 , 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 1 94, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532 , 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563 , 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 contiguous amino acids (or any range derivable therein).

[0091] In some embodiments, a polypeptide, such as a CBD, serum protein, therapeutic agent, masking agent, linker, or cytokine polypeptide, is a polypeptide that is at least, at most, or about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous to one of SEQ ID NOs: 1-190. NO: At least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 20A, 201, 4, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 37, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 598, 599, 590, 591, 592, 593, 594, 595, 596, 597, 598, 59 ...8, 599, 590, 591, 591, 592, 593, 594, 595, 596, 597, 598, 599, 599, 590, 5 81, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 contiguous amino acids (or any range derivable therein).

[0092] A polypeptide of the disclosure, such as a CBD, serum protein, therapeutic agent, masking agent, linker, or cytokine polypeptide, can be at least, at most, or about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) similar, identical, or homologous to one of SEQ ID NOs:1-190.

[0093] The polypeptides and nucleic acids of the disclosure may be at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 11 5, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 , 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 1 75, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 20 6, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 , 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 57 1, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 substitutions (or any range derivable therein).

[0094] Substitution is by SEQ ID NO: 1 amino acid position number from 1 to 190 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 , 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 13 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 1 6, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 1 77, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 2 08, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615. One or more of these substitutions may be specifically excluded from the embodiments.

[0095] or a CBD, serum protein, therapeutic agent, masking agent, linker, or cytokine polypeptide having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% identity, or having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% identity, or having 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% identity, The peptides, polypeptides, and proteins of the disclosure may be selected from the group consisting of amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200(or any range derivable therein) and starting at amino acid position number 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1081, 1092, 1093, 1094, 1 9, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 15, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or 205 (or any range derivable therein).

[0096] Substitutional variants typically involve the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modify one or more properties of the polypeptide, with or without losing other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, changing alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine. Alternatively, the substitution may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve the substitution of one residue with a chemically different residue, such as a polar or charged amino acid in place of a non-polar or uncharged amino acid and vice versa. One or more of these substitutions may be specifically excluded from the embodiment.

[0097] The proteins may be recombinant or in vitro synthesized. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. It is contemplated that bacteria containing such variants may be practiced in the compositions and methods. As a result, the proteins may not be isolated.

[0098] The term "functionally equivalent codon" is used herein to refer to codons that code for the same amino acid, such as the six codons for arginine or serine, and also to codons that code for biologically equivalent amino acids.

[0099] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially as set forth in one of the sequences disclosed herein, so long as they meet the above criteria, including the maintenance of the biological protein activity to which expression of the protein is associated. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0100] The following is a discussion based on changing the amino acids of a protein to create an equivalent or possibly improved second generation molecule. For example, certain amino acids can be substituted for other amino acids in a protein structure without significant loss of interactive binding ability. For example, structures such as enzyme catalytic domains or interacting components can have substituted amino acids to maintain such functions. Since it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in the protein sequence and in the underlying DNA coding sequence, and still produce a protein with similar properties. Thus, the inventors contemplate that various changes can be made in the DNA sequence of a gene without significant loss of its biological usefulness or activity.

[0101] In other embodiments, it is intended to modify the function of a polypeptide by introducing one or more substitutions. For example, certain amino acids can be substituted for other amino acids in a protein structure, with the intention of modifying the interactive binding ability of interacting components. For example, structures such as protein interaction domains, nucleic acid interaction domains, and catalytic sites can have amino acids substituted to modify such functions. Since it is the interaction ability and properties of a protein that define the functional activity of a protein, certain amino acid substitutions can be made in a protein sequence and in the underlying DNA coding sequence, and still produce a protein with different properties. Thus, it is contemplated by the inventors that various changes can be made in the DNA sequence of a gene to significantly change its biological usefulness or activity.

[0102] When making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological functions on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic index characteristics of amino acids contribute to the secondary structure of the resulting protein and further dictate the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0103] Similarly, it is understood in the art that the substitution of similar amino acids can be made effectively based on hydrophilicity. US Patent No. 4,554,101, which is incorporated herein by reference, states that the maximum local average hydrophilicity of a protein, as governed by the hydrophilicity of its neighboring amino acids, correlates with the biological properties of the protein. It is understood that one amino acid can be substituted with another amino acid that has a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0104] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account various characteristics such as those mentioned above are well known and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0105] In certain embodiments, all or part of the proteins described herein can be synthesized in solution or on solid support according to conventional techniques.Various automated synthesizers are commercially available and can be used according to known protocols.See, for example, Stewart and Young, (1984); Tarn et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference.Alternatively, recombinant DNA technology can be used, in which the nucleotide sequence encoding the peptide or polypeptide is inserted into an expression vector, which is transformed or transfected into a suitable host cell, and cultured under suitable conditions for expression.

[0106] In one embodiment, it includes the use of gene transfer into cells, including microorganisms, for the production and / or display of proteins. The gene for the protein of interest can be transferred into a suitable host cell, followed by culturing the cell under suitable conditions. Nucleic acids encoding virtually any polypeptide can be used. The construction of recombinant expression vectors and the elements contained therein are discussed herein. Alternatively, the protein produced can be an endogenous protein that is normally synthesized by the cell used to produce the protein.

[0107] III. Nucleic acids In certain embodiments, the present disclosure relates to recombinant polynucleotides that encode the proteins, polypeptides, and peptides of the invention, such as CBD, serum proteins, therapeutic agents, masking agents, linkers, or cytokine polypeptides and / or other molecules. Thus, certain embodiments relate to nucleotides that encode the CBD, serum proteins, therapeutic agents, masking agents, linkers, or cytokine polypeptides, and fragments thereof.

[0108] As used in this application, the term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated free of total genomic nucleic acid. Included within the scope of the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. A polynucleotide, in some aspects, includes a regulatory sequence that is substantially isolated from a natural gene or protein coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present in a polynucleotide.

[0109] In this regard, the terms "gene", "polynucleotide", or "nucleic acid" are used to refer to a nucleic acid (including any sequences required for proper transcription, post-translational modification, or localization) that encodes a protein, polypeptide, or peptide. As will be understood by those of skill in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide includes any of the following polynucleotides that encode one or more amino acid sequences described or referenced herein, including all values ​​and ranges between: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, , 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 65 0, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 106 It can include a contiguous nucleic acid sequence of 0, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more (or any range derivable therein) nucleotides, nucleosides, or base pairs.It is also contemplated that a particular polypeptide may be encoded by a nucleic acid including variants that have slightly different nucleic acid sequences but encode the same or substantially similar proteins.

[0110] In certain embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding the disclosed polypeptides or peptides. The term "recombinant" can be used in conjunction with a polynucleotide or polypeptide and generally refers to a polypeptide or polynucleotide that has been produced and / or manipulated in vitro, or that is the product of replication of such a molecule.

[0111] In other aspects, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding the polypeptides or peptides of the present disclosure.

[0112] The nucleic acid segments used in this disclosure may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and therefore their total length may vary considerably. Therefore, it is considered that nucleic acid fragments of almost any length can be used, with the total length being preferably limited by the ease of purification and the intended use in the recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can code for a polypeptide sequence with additional heterologous coding sequences, for example, to allow purification, transport, secretion, post-translational modification of the polypeptide, or to allow therapeutic utility, such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the sequence encoding the modified polypeptide, with "heterologous" referring to a polypeptide that is not the same as the modified polypeptide.

[0113] In certain embodiments, the disclosure provides polynucleotide variants having substantial identity to the sequences disclosed herein; the variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, including all values ​​and ranges between, compared to the polynucleotide sequences of the disclosure using the methods described herein (e.g., BLAST analysis with standard parameters).

[0114] The present disclosure also contemplates the use of polynucleotides that are complementary to all of the above polynucleotides.

[0115] A. Vector The polypeptides of the present disclosure may be encoded by a nucleic acid molecule contained in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted to introduce it into a cell where it can be replicated and expressed. A nucleic acid sequence may be "heterologous", which in context means that the nucleic acid sequence is foreign to the cell into which the vector is introduced or to the nucleic acid into which it is incorporated, including sequences at a location within the host cell or nucleic acid that is homologous to the sequence in the cell or nucleic acid, but is not normally found therein. Vectors include DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well equipped to construct vectors through standard recombinant techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both of which are incorporated herein by reference). In addition to encoding the polypeptides of the present disclosure, vectors can encode other polypeptide sequences, such as one or more other bacterial peptides, tags, or immunogenicity enhancing peptides. Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al., 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage.

[0116] The term "expression vector" refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that perform other functions and are described herein.

[0117] B. Promoters and Enhancers A "promoter" is a regulatory sequence. A promoter is typically a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. The phrases "operably arranged," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to a nucleic acid sequence to control the transcription initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0118] Of course, it may be important to use a promoter and / or enhancer that efficiently directs the expression of the DNA segment in the cell type or organism selected for expression. Those skilled in the art of molecular biology are generally aware that a combination of promoter, enhancer, and cell type is used to express proteins (see Sambrook et al., 2001, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, or inducible, and in certain embodiments, may direct high-level expression of the introduced DNA segment under specific conditions, such as large-scale production of recombinant protein or peptide.

[0119] The particular promoter used to control the expression of the polynucleotide encoding the peptide or protein of the present invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a target cell, preferably a bacterial cell. When a human cell is targeted, it is preferred to position the polynucleotide coding region adjacent to and under the control of a promoter capable of being expressed in a human cell. Generally speaking, such promoters may include either bacterial promoters, human promoters, or viral promoters.

[0120] C. Initiation signals and internal ribosome binding sites (IRES) Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be readily able to determine this and provide the necessary signals.

[0121] In certain embodiments of the present invention, internal ribosome entry site (IRES) elements are used to create multigenic or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can link heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to create polycistronic messages. Multiple genes can be efficiently expressed using a single promoter / enhancer and a single message can be transcribed (see U.S. Patent Nos. 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0122] D. Selectable and Screenable Markers In certain embodiments of the present invention, cells containing the nucleic acid construct of the present disclosure can be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, the marker confers an identifiable change to the cell, allowing easy identification of cells containing the expression vector. Generally, a selectable marker confers a property that allows for selection. A positive selectable marker is one whose presence allows for its selection, whereas a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0123] E. host cell As used herein, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All of these terms include any and all subsequent generations of their progeny. It will be understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism that can replicate a vector or express a heterologous gene encoded by the vector. Host cells can and have been used as recipients of vectors or viruses. Host cells may be "transfected" or "transformed", which refer to the process by which exogenous nucleic acid, such as a sequence encoding a recombinant protein, is transferred or introduced into a host cell. Transformed cells include the primary subject cell and its progeny.

[0124] Host cells can be of prokaryotic or eukaryotic origin, including bacteria, yeast cells, insect cells, and mammalian cells, for replicating the vector or expressing part or all of the nucleic acid sequence. Numerous cell lines and cultures are available as host cells and are available from the American Type Culture Collection (ATCC), an organization that serves as an archive of living cultures and genetic material (www.atcc.org).

[0125] F. Expression Systems There are numerous expression systems that contain at least some or all of the compositions discussed above. Prokaryotic and / or eukaryotic based systems can be used for use in the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially available and widely available.

[0126] The insect cell / baculovirus system can provide high levels of protein expression of heterologous nucleic acid segments, as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and can be purchased, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® under the name BACPACK™ BACULOVIRUS EXPRESSION SYSTEM.

[0127] In addition to the disclosed expression systems of the present invention, other examples of expression systems include the COMPLETE CONTROL Inducible Mammalian Expression System from STRATAGENE®, which includes a synthetic ecdysone-inducible receptor or its pET expression system, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which has the T-REX™ (Tetracycline Regulated Expression) System, an inducible mammalian expression system using a full-length CMV promoter. INVITROGEN® also offers a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. Those skilled in the art will know how to express vectors, such as expression constructs, nucleic acid sequences, or methods to produce their cognate polypeptides, proteins, or peptides.

[0128] IV. Further Treatments A. Immunotherapy In some embodiments, the method includes administering cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapies can be classified as active, passive or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAA); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAA. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapies useful in the methods of the present disclosure are described below.

[0129] 1. Checkpoint Inhibitors and Combination Treatments Aspects of the present disclosure can include administration of immune checkpoint inhibitors (also referred to as checkpoint inhibitor therapy), which are further described below.

[0130] a. PD-1, PD-L1, and PD-L2 inhibitors PD-1 can act in the tumor microenvironment where T cells encounter infection or tumors. Activated T cells upregulate PD-1 and continue to express PD-1 in peripheral tissues. Cytokines such as IFN-gamma induce the expression of PD-L1 on epithelial and tumor cells. PD-L2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to tissues during immune responses. The inhibitors of the present disclosure may block one or more functions of PD-1 and / or PD-L1 activity.

[0131] Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0132] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1 inhibitor is a molecule that inhibits the binding of PD-L1 to its ligand binding partner. In certain aspects, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 inhibitor is a molecule that inhibits the binding of PD-L2 to its ligand binding partner. In certain aspects, the PD-L2 binding partner is PD-1. The inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art, as described in U.S. Patent Application Publication Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0133] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion or a PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-L1 inhibitor comprises AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, Lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0134] In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PD-L2 inhibitor, such as rHIgM12B7.

[0135] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding to the same epitope on PD-1, PD-L1, or PD-L2 as the aforementioned antibodies, and / or binds to the same epitope on PD-1, PD-L1, or PD-L2 as the aforementioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) of variable region amino acid sequence identity with the aforementioned antibodies.

[0136] b. CTLA-4, B7-1, and B7-2 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA-4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA-4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. Activation of T cells through T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitor of the present disclosure can block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks CTLA-4 and B7-2 interaction.

[0137] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0138] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; also known formerly as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998 can be used in the methods disclosed herein. The disclosure of each of the aforementioned publications is incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0139] An additional anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424).

[0140] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) of variable region amino acid sequence identity with the aforementioned antibodies.

[0141] 2. Inhibition of costimulatory molecules In some embodiments, the immunotherapy comprises inhibitors of costimulatory molecules. In some embodiments, the inhibitors comprise inhibitors of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0142] 3. Dendritic cell therapy Dendritic cell therapy induces anti-tumor responses by having dendritic cells present tumor antigens to lymphocytes, thereby activating the lymphocytes and stimulating them to kill other cells that present the antigens. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells help in cancer antigen targeting. One example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0143] One way to induce dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small portions of proteins that correspond to protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to boost immune and antitumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0144] Dendritic cells can also be activated in vivo by expressing GM-CSF in tumor cells, which can be accomplished by genetically engineering the tumor cells to produce GM-CSF or by infecting the tumor cells with an oncolytic virus that expresses GM-CSF.

[0145] Another strategy is to remove dendritic cells from the patient's blood and activate them ex vivo. The dendritic cells are activated in the presence of tumor antigens, which can be single tumor-specific peptides / proteins or tumor cell lysates (a solution of destroyed tumor cells). These cells (with optional adjuvants) are injected to elicit an immune response.

[0146] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, inducing dendritic cells to mature and provide immunity against tumors.

[0147] 4. CAR-T cell therapy Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine new specificities with immune cells to target cancer cells. Usually, these receptors transfer the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because parts from different sources are fused together. CAR-T cell therapy refers to the treatment using such transformed cells for cancer therapy.

[0148] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding function and T cell activation function. A general premise of CAR-T cells is to artificially create T cells that are targeted to markers found on cancer cells. Scientists can remove T cells from a person, genetically modify them, and return them to the patient to attack cancer cells. When T cells are engineered to become CAR-T cells, they act as "living drugs." CAR-T cells create a link between an extracellular ligand recognition domain and an intracellular signaling molecule, which activates the T cell. The extracellular ligand recognition domain is typically a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is a way to ensure that only cancerous tumor cells, not normal cells, are targeted. The specificity of CAR-T cells is determined by the selection of the molecule to be targeted.

[0149] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta). In some embodiments, CAR-T therapy targets CD19.

[0150] 5. Cytokine Therapy Cytokines are proteins produced by many types of cells present within tumors. They can regulate the immune response. Tumors often utilize cytokines to grow the tumor and reduce the immune response. Due to these immunomodulatory effects, it becomes possible to use them as drugs to elicit an immune response. Two commonly used cytokines are interferon and interleukin.

[0151] Interferons are produced by the immune system. They are usually involved in antiviral responses, but are also used in cancer. They are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ).

[0152] Interleukins have a number of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0153] 6. Adoptive T cell therapy Adoptive T cell therapy is a form of passive immunization by transfusion of T cells (adoptive cell transfer). T cells are found in blood and tissues and are usually activated when they find a foreign pathogen. Specifically, T cells become activated when their surface receptors encounter cells that present a portion of a foreign protein on their surface antigen. These can be either infected cells or antigen-presenting cells (APCs). They are found in normal tissues and in tumor tissues, in which case they are known as tumor-infiltrating lymphocytes (TILs). They are activated by the presence of APCs, such as dendritic cells, that present tumor antigens. These cells can attack tumors, but the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0154] Several methods have been developed to produce and obtain tumor-targeted T cells. T cells specific for tumor antigens can be removed from tumor samples (TIL) or filtered from the blood. Subsequent activation and culture is performed ex vivo, followed by reinfusion. Activation can be achieved through gene therapy or by exposing the T cells to tumor antigens.

[0155] Cancer treatment is contemplated to be able to exclude any of the cancer treatments described herein. Further, aspects of the present disclosure include patients who have previously received treatment with the therapies described herein, patients who are currently receiving treatment with the therapies described herein, or patients who have never received treatment with the therapies described herein. In some aspects, the patient is a patient determined to be resistant to the therapies described herein. In some aspects, the patient is a patient determined to be sensitive to the therapies described herein.

[0156] B. Oncolytic virus In some aspects, the additional therapy includes an oncolytic virus. An oncolytic virus is a virus that selectively infects cancer cells and kills them. When infected cancer cells are destroyed by tumor lysis, they release new infectious virus particles or virions that help destroy the remaining tumor. Oncolytic viruses are thought to not only cause direct destruction of tumor cells, but also stimulate the host's anti-tumor immune response for long-term immunotherapy.

[0157] C. Polysaccharide In some aspects, the additional therapy includes a polysaccharide. Certain compounds found in mushrooms, mainly polysaccharides, can upregulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophages, NK cells, T cells, and immune system cytokines and are being investigated in clinical trials as immunological adjuvants.

[0158] D. Neoantigen In some embodiments, the additional therapy comprises administering neoantigens. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, identified using RNA sequencing data, is higher in tumors with high mutation load. The levels of transcripts associated with natural killer cell and T cell cytolytic activity are positively correlated with mutation load in many human tumors.

[0159] E. Chemotherapy In some embodiments, the additional therapy comprises chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related substances (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) Natural products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adrenal cortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0160] Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes, such as intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and in certain embodiments, effective doses used in clinical applications include about 15 mg / m2 to about 20 mg / m2 for 5 days every 3 weeks for a total of 3 courses. In some embodiments, the amount of cisplatin delivered to cells and / or subjects in conjunction with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount that would be delivered if cisplatin was used alone.

[0161] Other suitable chemotherapeutic agents include anti-microtubule agents, such as paclitaxel ("taxol") and doxorubicin hydrochloride ("doxorubicin"). The combination of an Egr-1 promoter / TNFα construct delivered via an adenoviral vector and doxorubicin has been found to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combined treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0162] Doxorubicin is poorly absorbed and is preferably administered intravenously.In certain embodiments, suitable intravenous doses for adults include about 60 mg / m2 to about 75 mg / m2 at intervals of about 21 days, or about 25 mg / m2 to about 30 mg / m2 on each of two or three consecutive days repeated at intervals of about 3 weeks to about 4 weeks, or about 20 mg / m2 once a week.The lowest dose should be used in elderly patients when there is previous myelosuppression or neoplastic bone marrow infiltration caused by previous chemotherapy, or when the drug is combined with other myelopoiesis suppressing drugs.

[0163] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards may include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN® available from Mead Johnson, NEOSTAR® available from Adria) is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous doses include, for example, about 40 mg / kg to about 50 mg / kg in divided doses for about 2 to about 5 days initially, or about 10 mg / kg to about 15 mg / kg every about 7 to about 10 days, or about 3 mg / kg to about 5 mg / kg twice weekly, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse gastrointestinal effects, the intravenous route is preferred. Drugs may also be administered intramuscularly, by infiltration, or into body cavities.

[0164] Further suitable chemotherapeutic agents include pyrimidine analogs such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU) and floxuridine (fluorodeoxyuridine; FudR). 5-FU can be administered to a subject at a dosage anywhere from about 7.5 to about 1000 mg / m2. Furthermore, 5-FU dosing schedules can be for various periods, for example, up to 6 weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0165] Another suitable chemotherapeutic agent, gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), is recommended for the treatment of advanced and metastatic pancreatic cancer and therefore would be useful in the present disclosure for these cancers as well.

[0166] The amount of chemotherapeutic agent delivered to the patient can be variable. In one suitable embodiment, the chemotherapeutic agent can be administered in an amount effective to cause the arrest or regression of cancer in the host when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent can be administered in an amount anywhere from 2 to 10,000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. For example, the chemotherapeutic agent can be administered in an amount about 20 times less, about 500 times less, or even about 5000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct and for determining effective dosages. For example, such compounds can be tested in suitable animal model systems, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, etc., prior to testing in humans. As described in the examples, in vitro testing can also be used to determine suitable combinations and dosages.

[0167] F. Radiation Therapy In some embodiments, the additional or previous therapy comprises radiation, such as ionizing radiation.As used herein, "ionizing radiation" refers to radiation that comprises particles or photons that have sufficient energy or can produce sufficient energy through nuclear interaction to produce ionization (gain or loss of electrons).An exemplary and preferred ionizing radiation is x-rays.Means for delivering x-rays to target tissue or cells are well known in the art.

[0168] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any range derivable therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any range derivable therein). If more than one dose is administered, the doses may be separated by about 1, 4, 8, 12, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks, or any range derivable therein.

[0169] In some embodiments, the amount of IR may be presented as a total dose of IR, which is administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 (or any range derivable therein). In some embodiments, the total dose is administered in fractions of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any range derivable therein).In some embodiments, the IL-10 antibody is at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 1 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractional doses are administered (or any range derivable therein). In some embodiments, at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fractional doses (or any range derivable therein) are administered per day. In some embodiments, at least, but not more than about 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, or 30 (or any range derivable therein) fractionated doses are administered per week.

[0170] G. Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive, diagnostic, or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies, such as treatments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs surgery).

[0171] Removal of part or all of cancer cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment may be performed by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be of various dosages.

[0172] H. Other Agents It is contemplated that other agents may be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. It is contemplated that cell adhesion inhibitors improve the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the embodiments of the present invention to improve the efficacy of the treatment.

[0173] V. Combination Therapy The compositions and related methods of the present disclosure, particularly the administration of the masked therapeutic agents of the present disclosure, may also be used in combination with administering additional therapies, such as those described herein, or in combination with other conventional therapies known in the art for the treatment of cancer.

[0174] The therapeutic compositions and treatments disclosed herein may precede, co-occur, and / or follow another treatment or drug by intervals ranging from minutes to weeks.In the embodiment in which drugs are applied separately to cells, tissues or organisms, it will generally be ensured that no significant period between each delivery time point passes so that the therapeutic agents can still exert beneficial combined effects on cells, tissues or organisms.For example, in such a case, it is contemplated that cells, tissues or organisms may be contacted with two, three, four or more drugs or treatments at substantially the same time (i.e., within less than about one minute). In other aspects, one or more therapeutic agents or treatments are administered 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours before and / or after administration of another therapeutic agent or treatment. The present invention can be administered or provided within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or more, and any range derivable therein.

[0175] Various combination regimens of therapeutic agents and treatments can be utilized. Non-limiting examples of such combinations are shown below, where a therapeutic agent, e.g., a composition disclosed herein, is "A", and a second agent, e.g., an additional agent or treatment, described herein or known in the art, is "B". TIFF0007682150000042.tif13128

[0176] In some embodiments, more than one course of treatment may be utilized. It is contemplated that multiple courses may be performed.

[0177] VI. Treatment method The present methods and compositions relate to methods for treating cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is non-lymphatic. In some embodiments, the cancer is breast cancer or colon cancer.

[0178] The composition of the present disclosure can be used for in vivo, in vitro or ex vivo administration. The route of administration of the composition can be, for example, intratumoral, intradermal, subcutaneous, intravenous, intralymphatic and intraperitoneal administration. In some embodiments, administration is intratumoral or intralymphatic or peritumoral. In some embodiments, the composition is directly administered into cancer tissue or lymph node.

[0179] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.

[0180] Cancers suitable for treatment include, but are not limited to, tumors of all types, locations, sizes and characteristics. The disclosed methods and compositions are useful for treating, for example, pancreatic cancer, colon cancer, acute myeloid leukemia, adrenal cortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, pediatric cerebellar or cerebral basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumor brain tumor, visual pathway and hypothalamic glioma, breast cancer, certain breast cancers, e.g., non-invasive breast tumors Ductal carcinoma, Invasive ductal carcinoma, Tubular adenocarcinoma of the breast, Medullary carcinoma of the breast, Mucinous carcinoma of the breast, Papillary carcinoma of the breast, Cribriform carcinoma of the breast, Invasive lobular carcinoma, Inflammatory breast cancer, Lobular carcinoma in situ, Male breast cancer, Paget's disease of the nipple, Phyllodes tumor of the breast, Recurrent and / or metastatic breast cancer, Luminal A or B breast cancer, Triple negative / basaloid breast cancer, and HER2-overexpressing breast cancer, Cancer of the lymphatic system, Bronchial adenoma / carcinoid, Tracheal carcinoma, Burkitt's lymphoma, Carcinoid tumor, Carcinoid tumor of childhood, Gastrointestinal cancer of unknown primary, Central nervous system Lymphoma of the system, Primary cerebellar astrocytoma, Cerebral astrocytoma / malignant glioma in children, Cervical cancer in children, Cancer in children, Chronic lymphocytic leukemia, Chronic myeloid leukemia, Chronic myeloproliferative disorder, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Ewing's, Extragonadal germ cell tumors of children, Extrahepatic bile duct cancer, Eye cancer, Retinoblastoma, Gallbladder cancer, Gastric / stomach cancer, Gastrointestinal carcinoid tumors, Gastrointestinal stromal tumors (GIST), Germ cell tumors: extracranial, extragonadal or ovarian, Gestational trophoblastic Tumors, brain stem gliomas, gliomas, pediatric cerebral astrocytoma, pediatric visual pathway and hypothalamic gliomas, gastric carcinoid, hairy cell leukemia, head and neck cancer, cancer of the heart, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas, pediatric intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphoblastic (also called acute lymphocytic) leukemia, acute myeloid (also called acute myelogenous) leukemia,Chronic lymphocytic (also called chronic lymphocytic leukemia), chronic myelogenous (also called chronic myeloid leukemia), hairy cell cancer of the lip and oral cavity, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin (old classification of all lymphomas other than Hodgkin) lymphoma, primary central nervous system lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma of childhood, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma of adults, mesothelioma of children, metastatic cervical squamous Neck cancer, oral cavity (mouth) cancer, multiple endocrine neoplasia, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia of adults, acute myeloid leukemia of children, multiple myeloma, chronic myeloproliferative disorders, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (superficial epithelial and stromal tumors), ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, islet cell sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pediatric pituitary adenoma, plasma cell neoplasms / multiple myeloma, pleuropulmonary It is suitable for treating blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, pediatric salivary gland carcinoma sarcoma, Ewing's sarcoma family of tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine Sezary syndrome sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer, Merkel cell small cell lung cancer, cancer of the small intestine, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma of unknown primary, metastatic gastric cancer, supratentorial primitive neuroectodermal tumor, pediatric T-cell lymphoma, testicular cancer, throat cancer, thymoma, pediatric thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma of the endometrium, vaginal cancer, visual pathway and hypothalamic glioma, pediatric vulvar cancer, and Wilms' tumor (kidney cancer).

[0181] VII. Pharmaceutical Compositions and Methods In some embodiments, the pharmaceutical composition is administered to a subject. Different aspects involve administering an effective amount of the composition to a subject. In some embodiments, the composition comprising the inhibitor can be administered to a subject or patient to treat cancer or reduce the size of a tumor. Furthermore, such compounds can be administered in combination with additional cancer therapy.

[0182] The composition can be formulated for parenteral administration, for example, for injection via intravenous, catheter injection, intraarterial injection, intramuscular, subcutaneous, or intraperitoneal routes.Typically, such compositions can be prepared as injections, either as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions by adding liquids prior to injection can also be prepared; and the preparations can be emulsified.The preparation of such formulations will be known to those skilled in the art in light of this disclosure.Other routes of administration include intratumoral, peritumoral, intralymphatic, injection into cancer tissue, and injection into lymph nodes.In some embodiments, administration is systemic.

[0183] The pharmaceutical forms suitable for injection include sterile aqueous solution or dispersion; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powder for extemporaneous preparation of sterile injectable solution or dispersion.In all cases, the form must be sterile and fluid enough to be easily squirted.It should also be stable under the conditions of manufacture and storage, and must be protected from the contaminating action of microorganisms, such as bacteria and fungi.

[0184] The carrier may also be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, such as sugars and sodium chloride. Prolonged absorption of the injectable composition can be achieved by the use of an absorption-delaying agent in the composition, for example, aluminum monostearate and gelatin.

[0185] Sterile injectable solution is prepared by incorporating the required amount of active compound into suitable solvent with various other components as listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile medium that contains the basic dispersion medium and the other components as listed above.For the sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technique, which allows the powder of active component plus other desired components to be obtained from the solution that has been previously sterilized and filtered.

[0186] As used herein, the term "pharmaceutical acceptable" refers to a compound, substance, composition and / or dosage form that is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals, without undue toxicity, irritation, allergic response or other significant complications, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutical acceptable carrier" refers to a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulant, involved in carrying or transporting a chemical agent.

[0187] As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound that is modified by converting an acidic or basic moiety present in the parent compound into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example, formed with non-toxic inorganic or organic acids. Pharmaceutically acceptable salts may be synthesized from parent compounds that contain a basic or acidic moiety by conventional chemical methods.

[0188] Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will determine the appropriate dose for each individual subject in any event. The effective amount of therapeutic or prophylactic composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to a physically separate unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response discussed above with its administration, i.e., appropriate route and regimen. The amount administered will depend on the desired effect, both according to the number of treatments and the unit dose. The exact amount of the composition will also depend on the judgment of the practitioner and will be specific to each individual. Factors that affect the dosage include the subject's physical and clinical condition, the route of administration, the intended purpose of treatment (whether symptom relief or cure), and the efficacy, stability and toxicity of the specific composition.

[0189] Once formulated, solutions will be administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0190] Typically, for a human adult (weighing approximately 70 kilograms), a compound of about 0.1 mg to about 3000 mg (including any value and range therebetween), or about 5 mg to about 1000 mg (including any value and range therebetween), or about 10 mg to about 100 mg (including any value and range therebetween) is administered. It will be understood that such dosage ranges are merely exemplary and that the administration can be adjusted according to factors known to those of ordinary skill in the art.

[0191] In certain embodiments, a subject is administered an amount that is about, at least about, or at most about: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7 ,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 1 0.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 4, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 51 0, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3 900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms (mcg) or μg / kg or micrograms / kg / min or mg / kg / min or micrograms / kg / hour or mg / kg / hour, or any range derivable therein.

[0192] Dosing can be as needed, or can be every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 hours (or any range derivable therein) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times per day (or any range derivable therein). Dosing can be first administered before or after symptoms of a disease condition. In some embodiments, a patient is first administered a dose of the regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range derivable therein) or 1, 2, 3, 4 or 5 days (or any range derivable therein) after the patient develops or shows signs or symptoms of a disease condition. The patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more (or any range derivable therein), or until symptoms of the disease state have disappeared or been reduced, or until 6, 12, 18 or 24 hours or 1, 2, 3, 4 or 5 days after symptoms of the infection have disappeared or been reduced. EXAMPLES

[0193] VIII. Working Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art will recognize that the procedures disclosed in the following examples are procedures that the inventors have found to work well in the practice of the present disclosure and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art will recognize in light of the present disclosure that many changes may be made in the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0194] Example 1: Masked cytokines reduce toxicity and increase tumor-specific activation Cytokine cancer immunotherapy using interleukin (IL)-12 has shown potent antitumor efficacy in both mice and humans. However, some IL12 clinical trials have been terminated or failed due to its severe toxicity. IL12 has not been approved for clinical use to date (1).

[0195] Immunotherapy serves to activate immune responses, so side effects are usually due to drug action in healthy organs. One solution to overcome the toxicity problem is to suppress cytokine action in healthy tissues. One solution involves the conversion of cytokines into prodrugs that are inactive in healthy tissues and in the systemic circulation, but are activated locally at disease sites. In antibody format, this concept has been developed as probodies (2). IL12 fusions to anti-IL12 antibodies that cover the IL12 receptor binding site have been developed very recently, but in vivo, the success of this approach involving antibody domains has not been shown (3). Described herein are procytokines (prodrugs in cytokine format) that include a masking agent and a cytokine linked via a substrate peptide (tumor-associated protease cleavage site) that is cleaved by enzymes present in the tumor microenvironment, such as matrix metalloproteinases (MMPs). MMPs are a family of proteases that are specifically activated in the tumor microenvironment to cleave their recognition site. The resulting procytokines were shown to be locally activated within the tumor microenvironment, providing an opportunity to act as tumor-targeted immunotherapies.

[0196] IL12 is a heterodimeric glycosylated cytokine composed of disulfide-linked p35 (approximately 35 kDa) and p40 (approximately 40 kDa) subunits. IL12 is secreted as an early proinflammatory cytokine by activated antigen-presenting cells (APCs) in response to infection. Upon binding to the IL12 receptor on CD4+ T cells, IL12 promotes Th1 polarization and triggers IFNγ production (4). IL12 is considered to be an ideal antitumor therapeutic cytokine because it activates both the innate and adaptive immune arms of the immune system. Despite this, systemic administration of recombinant human IL12 (rh-IL12) has shown unsatisfactory results in clinical trials due to intolerable toxicity, leading to the cessation of trials with systemic IL12 (5). The main barrier to recombinant IL12 therapy stems from the inability to reach sufficiently high local concentrations within tumors, thus motivating the development of tumor-targeted recombinant IL12 therapy to trigger the full therapeutic potential of this cytokine (1). Herein, we attempted to develop a methodology for masking the binding site for IL12 to improve its toxicity. We used the cytokine-binding domain of this receptor protein, IL12 receptor beta 1 (IL12Rβ1) fibronectin I and II domains, as a mask for the IL12 binding site.

[0197] The inventors have shown that chemical conjugation of CBD protein to CPI and recombinant fusion to IL-2 results in enhanced antitumor efficacy compared to its unmodified form. The inventors have combined this CBD technology with a procytokine format; this combination results in the prolonged presence of masked procytokines in the enzymatic microenvironment, enhancing the enzymatic unmasking of the procytokine. This approach is also applicable to probodies.

[0198] A. Results The inventors designed an IL12Rβ1 fusion to IL12 protein using the first two fibronectin domains D1 and D2 of the receptor (Figure 1). After protein expression by HEK293 cells, it was purified by histidine tag affinity purification and size exclusion column purification.

[0199] To test the activity of IL12Rβ1-IL12, splenocytes were cultured in vitro for 2 days in the presence of IL12Rβ1-IL12. IL12Rβ1 fusion to IL12 (in both cases of IL12Rβ1 fused to p35 and p40) significantly decreased the concentration of IFNγ, a major downstream molecule of IL12, in the medium compared to IL12, suggesting that IL12Rβ1 fusion to IL12 decreased IL12 activity (Figure 2A). Since IFNγ is a major downstream molecule of IL12 immunological action and systemic IFNγ release causes systemic toxicity, the inventors measured plasma IFNγ concentration (Figure 2B). IL12 and its variants were injected into B16F10 tumor-bearing mice, and plasma was collected 2 days after injection, which is the time when IFNγ levels are highest based on previous experiments. In the absence of a protease cleavage sequence linking the IL12Rβ1 domains, IL12Rβ1-IL12 (in both cases of IL12Rβ1 fused to p35 and p40) showed undetectable levels of IFNγ in plasma, while unmodified IL12 showed high IFNγ concentrations.

[0200] Next, we expressed IL12Rβ1-IL12, which contains a protease cleavage site inserted between IL12Rβ1 and IL12, to achieve tumor-specific IL12 activation. We tested whether IL12Rβ1-VP-IL12, which contains VPLSLYS (SEQ ID NO:50), could be proteolytically cleaved by the VPLSLYS (SEQ ID NO:50)-recognizing enzymes MMP2 and MMP9 (FIG. 3A). SDS-PAGE analysis revealed that MMP2 and MMP9 completely cleaved the IL12Rβ1 portion, resulting in the IL12 heterodimer (approximately 60 kDa). Similarly, IL12Rβ1-LS-IL12, which contains the LSGRSDNH (SEQ ID NO:49) substrate, was cleaved by uPA. As shown in FIG. 3A, these enzymes did not affect IL12 itself.

[0201] Next, we tested whether fusion of IL12Rβ1 with the p35 chain could reduce the biological activity of IL12 (Figure 3B). As a proxy for IL12 biological activity, we measured STAT4 phosphorylation in response to various amounts of cytokines. The EC 50 The EC value for the pro-IL12 construct was 16.3 pM. 50 The values ​​were approximately 80-fold lower. To determine whether treatment of IL12Rβ1-VP-IL12 with MMP2 and IL12Rβ1-LS-IL12 with uPA could result in a functional response in T cells, we compared the bioactivity of the cleaved and uncleaved constructs with that of IL12 (Figure 3C). In accordance with previous results, the uncleaved construct showed an approximately 80-fold reduction in STAT4 phosphorylation. Nevertheless, treatment of the pro-IL12 construct with each protease fully restored IL12 bioactivity, showing a dose-response correlation similar to that of unmodified IL12.

[0202] To determine whether IL12Rβ1-VPLS-IL12-CBD also reduces toxicity in vivo, the inventors dosed healthy mice three times every three days with unmodified IL12 or escalating doses of IL12Rβ1-VPLS-IL12-CBD (Figure 3D). Two days after each injection, blood was collected from the mice and serum was analyzed for the presence of inflammatory biomarkers using the LEGENDplex technology. The data suggest that IL12Rβ1-VPLS-IL12-CBD exhibited an enhanced safety profile when compared to IL12. Importantly, IL12Rβ1-VPLS-IL12-CBD 5 μg did not upregulate any of the toxicity markers to a detectable level when compared to PBS-treated mice, and IL12Rβ1-VPLS-IL12-CBD 100 μg was generally tolerated to a greater extent when compared to IL12-treated mice. Next, the blood cell counts two days after IL12 injection in B16F10 tumor-bearing mice were tested. IL12 and CBD-IL12 induced a decrease in the number of white blood cells in the blood, while none of the variants of IL12Rβ1-IL12 did (Figure 3E). Platelet counts were maintained in all cases. These data suggest that the fusion of IL12Rβ1 to IL12 reduces systemic toxicity (Figure 3F). Blood toxicity markers were analyzed by a biochemical analyzer three days after IL12Rβ1-IL12 injection into non-tumor-bearing mice. When injecting 25 μg or 100 μg of IL12Rβ1-IL12, liver injury markers (serum albumin concentration, total protein, alanine aminotransferase (ALT) activity, aspartic acid aminotransferase (AST) activity, and alkaline phosphatase activity), kidney injury markers (total bilirubin), pancreas injury markers (amylase), lung injury markers (CO 2The concentrations of IL12Rβ1-IL12 in the 25- or 50-μg groups were comparable to those in the PBS-injected group (Figure 3G-N). Mice injected with 25 μg or 50 μg of IL12 showed a marked decrease in blood albumin concentration, as well as increases in ALT activity, AST activity, total bilirubin, and amylase, suggesting toxicity to several organs. When injected with 200 μg of IL12Rβ1-IL12, one out of three mice responded with elevated AST activity, total bilirubin, and amylase. These data suggest that the maximum tolerated dose of IL12Rβ1-IL12 is at least >100 μg. When we combined IL12Rβ1-IL12 and anti-PD-1 blocking antibodies, ALT activity and amylase levels were not dramatically increased compared to wild-type IL12 therapy (Figure 3OP).

[0203] Next, we used the B16F10 model to test the antitumor efficacy of IL12Rβ1-IL12 fused with IL12Rβ1 (Figure 4). IL12Rβ1-IL12 with uPA and thrombin cleavage sites showed antitumor effects. In contrast, IL12Rβ1-IL12 without a cleavable sequence showed no antitumor efficacy (Figure 4B). These data demonstrate that IL12Rβ1-IL12 is activated in tumors by proteases and shows antitumor efficacy. Furthermore, the antitumor efficacy of IL12Rβ1-IL12 with CBD demonstrated higher antitumor efficacy than the variant without CBD, demonstrating that the effect of CBD in prolonging the presence of the procytokine in the enzymatic environment of the tumor enhances its activation. We also tested lower doses of IL12Rβ1-VPLS-IL12 and found that both IL12Rβ1-VPLS-IL12 and IL12Rβ1-VPLS-IL12-CBD were as effective as IL12 (Figure 4C). Next, we tested whether IL12Rβ1-IL12, which has uPA and MMP cleavage sites, synergized with anti-PD-1 blockade therapy (Figure 4D). Anti-PD-1 antibody therapy did not cure any of the B16F10 mice, but combination therapy of IL12Rβ1-LSHPVP-IL12 and anti-PD-1 antibody cured all five B16F10 tumors. As a result, we found that IL12Rβ1-IL12 enhanced the therapeutic effect of anti-PD-1 therapy.

[0204] B. Conclusion Cytokines are important factors of antitumor activity, but to date many of them have not been transferred to the clinic. IL12 is one of the most potent antitumor cytokines, but due to its high toxicity, clinical trials have been terminated or failed. Therefore, reducing its toxicity is an important strategy to transfer it to the clinic. To improve CBD-IL12 therapy, we fused a domain of the IL12 receptor IL12Rβ1 to IL12 to form IL12Rβ1-IL12. This fusion is inactive, but the inclusion of an MMP or thrombin cleavage site between the receptor mask and the cytokine generates a procytokine that can be activated within the tumor microenvironment. We demonstrated that the immunotoxicity of IL12 is thus reduced and that fusion of IL12Rβ1-IL12 with a protease-sensitive linker retains therapeutic utility.

[0205] Because we observed that a single repeat of an MMP or thrombin cleavage site between the receptor mask and the cytokine confers antitumor efficacy, we hypothesized that introducing multiple cleavage sites into the linker (e.g., tandem MMP, tandem thrombin, and MMP-thrombin dyads and repeats) might increase protease sensitivity and enhance the antitumor efficacy of IL12Rβ1-IL12 therapy.

[0206] In conclusion, the development of a technology to reduce cytokine toxicity by fusing a cytokine receptor to the cytokine is described herein. Tumor-specific proteases cleave the linker and activate the cytokine within the tumor.

[0207] C. Materials and Methods 1. Production and purification of recombinant fusion proteins of VWF A3 domain, IL12Rβ1 and IL12 Protein production and purification were performed as previously described (7). Sequences encoding human VWF A3 domain residues Cys1670–Gly1874 (907–1111 in mature VWF), mouse IL12, and IL12Rβ1 fusion proteins were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. A sequence encoding 6 His was added to the N-terminus for further purification of the recombinant protein. Suspension-adapted HEK-293F cells were routinely maintained in serum-free FreeStyle 293 Expression Medium (Gibco). On the day of transfection, cells were cultured at 1 × 10 6 The cells were seeded in fresh medium at a density of 1000 cells / ml. Plasmid DNA (2 μg / ml), linear 25 kDa polyethyleneimine (Polysciences) (2 μg / ml), and OptiPRO SFM medium (final concentration 4%, Thermo Fisher) were added sequentially. The culture flasks were incubated at 37 °C for 2 h at 4 °C for 1 h at 5% CO. 2 The cells were agitated by orbital shaking at 135 rpm at 37°C in the presence of 0.1% ethanol. Seven days after transfection, the cell culture medium was harvested by centrifugation and filtered through a 0.22 μm filter. The medium was loaded onto a HisTrap HP 5 ml column (GE Healthcare) using AKTA pure 25 (GE Healthcare). Wash buffer (20 mM imidazole, 20 mM NaH 2 PO 4 , 0.5 M NaCl, pH 7.4), followed by column washing with 500 mM imidazole (20 mM NaH 2 PO 4 The protein was eluted with a gradient of 0.1 M NaCl (pH 7.4) at 20 °C. The eluate was further purified by size-exclusion chromatography using a HiLoad Superdex 200PG column (GE healthcare). All purification steps were performed at 4 °C. Expression of laminin LG domain was determined by Western blotting with an anti-His tag antibody (BioLegend), and the protein was confirmed to be >90% pure by SDS-PAGE. TIFF0007682150000043.tif133150TIFF0007682150000044.tif231150TIFF0007682150000045.tif255150

[0208] 2. Pro-IL12 cleavage assay Recombinant mouse matrix metalloproteinase-2 (MMP2), MMP9 and recombinant human urokinase plasminogen activator (uPA) were purchased from R&D. As MMP2 and MMP9 were supplied in their proenzyme forms, MMPs were first activated with 1 mM p-aminophenylmercuric acetate (APMA, Sigma) for 2 h at 37°C. After activation, MMPs were incubated at 37°C for 2 h in 150 mM NaCl, 50 mM Tris, 10 mM CaCl at pH = 7.5. 2 MMPs and cytokines were diluted in assay buffer containing 0.05% Brij-35. The final concentrations of MMP2, MMP9 and cytokines were 2 μg / mL, 5 μg / mL and 50 μg / mL, respectively. Cleavage was performed for 30 min at 37°C. Samples were then analyzed by gel electrophoresis. Cleavage with uPA was performed according to the manufacturer's protocol. The concentration of uPA was 10 μg / mL.

[0209] 3. In vitro stimulation assay EasySep Mouse CD8 + Mouse CD8 T cells were isolated from the spleens of C57BL / 6 mice using a Stem Cell T cell isolation kit. + T cells were purified. Purified CD8 + T cells (10 6Cells (cells / mL) were pre-coated with 2 μg / mL α-CD3 (clone 17A2, Bioxcell) and activated for 3 days in 6-well plates supplemented with soluble 5 μg / mL α-CD28 (clone 37.51, BioLegend) and 30 ng / mL mouse IL-2 (Peprotech). The medium was IMDM (Gibco) containing 10% heat-inactivated FBS, 1% penicillin / streptomycin and 50 μM 2-mercaptoethanol (Sigma Aldrich). After 3 days in culture, activated CD8 + T cells were rested in fresh medium for 6 h and transferred to 96-well plates (50,000 cells / well). The indicated amounts of IL-12 or pro-IL12 variants were administered to CD8 + T cells were applied for 20 min at 37°C to induce STAT4 phosphorylation. Cells were immediately fixed with BD Phosflow Lyse / Fix buffer for 10 min at 37°C and then permeabilized with BD Phosflow Perm Buffer III for 30 min on ice. Cells were stained with an Alexa Fluor (AF) 647-conjugated antibody against pSTAT4 (clone 38, BD), which recognizes phosphorylation at Tyr693. Staining was performed for 1 h at room temperature (RT) in the dark. Cells were acquired on a BD Fortessa X-20 and data were analyzed using FlowJo (Treestar). pSTAT4 + The mean fluorescence intensity (MFI) of the population was plotted against cytokine concentration. Dose-response curves were fitted using Prism (v8, GraphPad).

[0210] 4. Mice and cell lines Mice and cell lines were prepared as previously described (8). Eight to 12 week old C57BL / 6 mice were obtained from Charles River laboratories. Experiments were performed with approval from the Institutional Animal Care and Use Committee of the University of Chicago. B16F10 cells were obtained from the American Type Culture Collection and cultured according to the manufacturer's instructions. All cell lines were checked for mycoplasma contamination by pathogen testing IMPACT I (IDEXX BioResearch).

[0211] 5. Plasma cytokine concentration analysis Measurements are performed as previously described (8). 5 × 10 5 B16F10 melanoma cells were injected intradermally into the left side of the back of each 9-week-old C57BL / 6 mouse. Seven or eight days later, mice were administered 25 μg IL12 and equimolar IL12 variants. 100 μg anti-PD-1 antibody (clone: ​​29F.1A12, BioXCell) was injected ip. Two days after IL12 injection, blood samples were collected in heparinized tubes containing EDTA, followed by centrifugation. Plasma cytokine concentrations were measured with Ready-SET-Go! ELISA kits (eBioscience) or LEGENDplex kits (BioLegend) according to the manufacturer's protocols. LEGENDplex results were analyzed using a BD Fortessa X-20 flow cytometry system and FlowJo.

[0212] 6. Analysis of toxicity markers in serum Nine-week-old C57BL / 6 mice were used. Mice were administered IL12 and IL12 variants iv. Three days after IL12 injection, blood samples were collected in tubes followed by clotting and centrifugation. Serum injury markers were measured by Vet Axcel (Alfa Wassermann) according to the manufacturer's protocol.

[0213] 7. Antitumor efficacy of IL12Rβ1-IL12 against B16F10 tumors Assays are performed as previously described (8). A total of 5 × 10 5 B16F10 cells were inoculated intradermally into the left side of the back of each C57BL / 6 mouse. Seven, eight, and / or ten days later, mice were injected iv with IL12 (25 μg), IL12Rβ1-IL12 (equimolar or 100 μg IL12 base), or CBD-IL12 (equimolar). 100 μg of anti-PD-1 antibody (clone: ​​29F.1A12, BioXCell) was injected ip on days 7, 10, and / or 13. Tumors were measured with digital calipers beginning 8 days after tumor inoculation, and volumes were calculated as ellipsoids, where V = 4 / 3 × 3.14 × depth / 2 × width / 2 × height / 2. If either tumor volume was 500 mm 3 Mice were sacrificed when > 0.05 mg / kg were reached.

[0214] IX. Example 2 - Cytokine Aspects The technique of masking the receptor binding site by fusing the cytokine receptor domain can be applied to other anti-tumor cytokines. In this example, we teach the receptor fusion to IL-2 and IFNγ to create other procytokines that are pro-IL-2 and pro-IFNγ. In all versions, MMP and / or thrombin responsive cleavage sites are inserted between the receptor and the cytokine. Exemplary embodiments of cytokines and masking agents are provided below. TIFF0007682150000046.tif56160

[0215] Example 3 - Addition of serum proteins for long-term circulation Procytokines can be improved by CBD fusion to provide long-term retention in tumors and / or albumin fusion to provide long-term circulation. Cytokines generally have a very short half-life in the blood (9). Because procytokine technology relies on proteases in the body (i.e., tumors), it is important to increase the retention time of the procytokine injected in the tumor. We have taken two approaches to improve the CBD-cytokine platform. The first step is to fuse a collagen-binding domain to the procytokine. As described in Example 1, CBD can target and retain the fusion protein in the tumor due to the nature of the tumor vasculature. Thus, the activity of the CBD-procytokine is more specific in the tumor, resulting in enhanced efficacy and safety. This is a kind of dual tumor targeting system.

[0216] Another step is to extend the blood half-life of the procytokine. It is hypothesized that extending the blood half-life of the injected cytokine will further enhance the efficacy of the CBD-procytokine, since it will have more opportunities to contact tumor tissue. This can be achieved by fusing albumin to the CBD-procytokine or the procytokine. Thus, these further aspects of tumor-targeted cytokines with extended blood half-life that are only active within the tumor microenvironment are further contemplated.

[0217] Although certain embodiments have been described above with some degree of specificity or with reference to one or more individual embodiments, those skilled in the art may make numerous modifications to the embodiments of the present disclosure without departing from the scope of the present invention. Moreover, aspects of any embodiment described above may be combined with aspects of any other embodiment described, where appropriate, to form further embodiments having equivalent or different characteristics and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or to several embodiments. Any reference to a published patent application or other publication is specifically incorporated herein by reference to the disclosure content of the publication / publication. The claims should not be construed as including means-plus-function or step-plus-function limitations unless such limitations are expressly stated in a given claim using the phrase(s) "means for" or "step for," respectively.

[0218] References The following references and publications, referenced throughout the specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007682150000047.tif158161

Claims

1. A polypeptide comprising IL12 linked to IL12R through a linker, the linker comprising one or more of an MMP, thrombin, and uPa protease cleavage site. (a) IL12 contains both or one of the p35 and p40 subunits; or (b) IL12 comprises a p35 subunit and a p40 subunit linked through a disulfide bond; or (c) IL12 comprises a p35 subunit and a p40 subunit linked through a peptide linker; The polypeptide of claim 1.

3. (a) the IL12R comprises IL12Rβ1, optionally wherein the IL12Rβ1 comprises one or both of the fibronectin domains D1 and D2; and / or (b) the IL12R is fused to the N-terminus of the p35 subunit of IL12, and a linker is between the IL12R and the p35 subunit of IL12; or (c) IL12R is fused to the C-terminus of the p40 subunit of IL12, and a linker is between the IL12R and the p40 subunit of IL12; The polypeptide of claim 2.

4. The polypeptide described in claim 1, wherein the polypeptide comprises IL12Rβ1 including fibronectin domains D1 and D2 linked to IL12 via a linker, and the linker comprises SEQ ID NO:138, 50 and 49.

5. (a) the polypeptide comprises at least two protease cleavage sites; and / or (b) the protease cleavage site comprises SEQ ID NO: 138, 49 and / or 50; and / or (c) the IL12 comprises proinflammatory IL12; and / or (d) a polypeptide comprising: (i) an antibody or antigen-binding fragment thereof, comprising a stroma-targeting antibody or stroma-binding fragment thereof, wherein, optionally, the antibody or binding fragment specifically binds to fibronectin, an alternative splice domain of fibronectin, a collagen, a tenascin, a periostin, a syndecan, a proteoglycan, or a tumor stromal cell-specific antigen, and optionally, (I) the antibody or binding fragment specifically binds to the extra domain A (EDA) or extra domain B (EDB) of fibronectin, or (II) the antibody or binding fragment comprises a Fab that specifically binds to an alternative splice domain of fibronectin that contains extra domain A (EDA); the antibody or antigen-binding fragment thereof; or (ii) an antibody or antigen-binding fragment thereof that specifically binds to a tumor-associated antigen; or (iii) a collagen-binding domain, optionally (I) the polypeptide comprises at least two collagen-binding domains; and / or (II) the polypeptide comprises a collagen-binding domain derived from decorin or von Willebrand factor (VWF); The collagen-binding domain Conjugated to A polypeptide according to any one of claims 1 to 4.

6. (a) the polypeptide further comprises a serum protein conjugated to the polypeptide, and optionally (i) the serum protein is conjugated to the polypeptide through a peptide bond; and / or (ii) the serum protein comprises albumin; and / or (b) the polypeptide comprises a second linker, optionally comprising glycine and serine amino acid residues, and further optionally, wherein the linker comprises SEQ ID NO:47 or SEQ ID NO:48; and / or (c) the polypeptide comprises a protein tag; and / or (d) the polypeptide is not operably linked to a particle, nanovesicle, or liposome; A polypeptide according to any one of claims 1 to 5.

7. A composition comprising a polypeptide according to any one of claims 1 to 6.

8. 8. The composition of claim 7, which does not contain liposomes, particles or nanovesicles.

9. A nucleic acid encoding the polypeptide of any one of claims 1 to 6.

10. A host cell comprising the nucleic acid of claim 9.

11. 10. A method for making a polypeptide comprising expressing the nucleic acid of claim 9 in a cell and isolating the expressed polypeptide.

12. A polypeptide according to any one of claims 1 to 6 or a composition according to claim 7 or 8 for use in a method for treating cancer.

13. (a) the method further comprises administering one or more additional cancer therapies; and / or (b) the subject is undergoing or will undergo immunotherapy; and / or (c) the method further comprises administering immunotherapy, optionally administered before, after, or simultaneously with the polypeptide; 13. A polypeptide or composition for use according to claim 12.

14. 13. The polypeptide or composition for use of claim 13(b) or 13(c), wherein the immunotherapy comprises an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor comprises an anti-PD-1 monoclonal antibody or an anti-CTLA-4 monoclonal antibody, and further optionally wherein the immune checkpoint inhibitor comprises one or more of nivolumab, pembrolizumab, pidilizumab, ipilimumab, or tremelimumab.

15. (a) the polypeptide or composition is administered systemically, optionally wherein the polypeptide or composition is administered by intravenous injection; and / or (b) the subject has been previously treated with a cancer therapy, and optionally, the subject has been determined to be non-responsive to the previous treatment or the subject has experienced non-specific toxicity to the previous treatment; A polypeptide or composition for use according to any one of claims 12 to 14.

16. A polypeptide or composition for use according to any one of claims 12 to 14, wherein the polypeptide comprises an IL12Rβ1 polypeptide comprising fibronectin domains D1 and D2 linked to IL12 via a linker, the linker comprising MMP and uPa protease cleavage sites, the cancer comprises melanoma, and the subject is undergoing or will undergo anti-PD-1 monotherapy or anti-PDL1 monotherapy.

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