Methods and compositions for treating cancer using collagen-bound drug carriers

Abstract

The present disclosure relates to tumor-targeted drug carriers that efficiently deliver cytotoxic agents to the tumor microenvironment, resulting in improved antitumor efficacy. An aspect of the present disclosure relates to a polypeptide comprising an albumin or IgG Fc domain polypeptide operably linked to a collagen-binding domain. A further aspect relates to a composition comprising the polypeptide, nucleic acid, or cell of the present disclosure.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 856,468, filed June 3, 2019, which is incorporated by reference in its entirety.

[0002] II. FIELD OF THEINVENTION The present invention relates generally to the field of medicine. More specifically, the present invention relates to compositions and methods involving nucleotide constructs, proteins, and drug carriers for treating cancer. [Background technology]

[0003] III. Background Serum albumin (SA) is the most abundant protein in blood. Several compounds, including small molecules, peptides, and cytokines, have been fused, conjugated, or co-formulated with SA for improved drug delivery to disease lesions. The exceptionally long plasma half-life and / or hydrophilicity of SA contribute to improved drug pharmacokinetics, safety, and efficacy.

[0004] Doxorubicin (Dox) is a small molecule anticancer drug approved by the US Food and Drug Administration (FDA) to treat a wide range of cancers. Dox is internalized into cells via passive transmembrane diffusion and disrupts DNA function, causing the death of proliferating cells. Although Dox treatment prolongs survival in some patient populations, antitumor efficacy is not dramatic, in part due to acquired drug resistance. The poor Dox therapeutic index also limits its therapeutic use. Indeed, significant toxicity of Dox has been reported in outpatient clinics, including myelosuppression, excessive inflammation, and cardiac toxicity. Dox is often used in combination with other chemotherapeutic agents. Other approaches to improve the efficacy and maximum tolerated dose of Dox are liposomal formulations (Doxil) and the use of a maleimide derivative of Dox (aldoxorubicin) with a pH-sensitive cleavable linker, developed to achieve in situ conjugation with cysteine-34 (in the human sequence) of circulating SA.

[0005] Although strategies for passive targeting of cytotoxic agents to tumors have been developed, there is a need in the art for more targeted therapies that can increase the efficacy of cytotoxic agents while reducing nonspecific toxicity and decreasing the minimum effective dose required to achieve a therapeutic effect. Summary of the Invention

[0006] Herein, we describe a tumor-targeted drug carrier that provides improved antitumor efficacy by efficient delivery of cytotoxic agents to tumor microenvironment. An aspect of the present disclosure relates to a polypeptide comprising an albumin polypeptide or an IgG Fc domain polypeptide operably linked to a collagen binding domain. A further aspect relates to a composition comprising the polypeptide, nucleic acid, or cell of the present disclosure. A further aspect relates to a nucleic acid encoding the polypeptide of the present disclosure. A further aspect relates to a cell comprising the nucleic acid or polypeptide of the present disclosure.

[0007] A further aspect of the present disclosure relates to a method for making a polypeptide comprising expressing a nucleic acid of the present disclosure in a cell and isolating the expressed polypeptide.

[0008] A further aspect relates to a method for treating cancer comprising administering a polypeptide, nucleic acid or composition of the present disclosure. A further aspect relates to a method for reducing non-specific toxicity of a treatment comprising a cytotoxic agent in a subject, comprising administering a polypeptide or composition of the present disclosure to the subject. The term "non-specific toxicity" refers to toxicity or cell death of non-cancerous cells.

[0009] A further aspect relates to a method for increasing accumulation of a cytotoxic agent in a tumor in a subject, comprising administering to the subject a polypeptide, nucleic acid, or composition of the disclosure.

[0010] A further aspect relates to a method for targeted delivery of a cytotoxic agent to tumor vasculature, comprising administering a polypeptide or composition of the present disclosure to a subject. A further aspect relates to a method for treating a tumor in a subject, comprising administering a polypeptide or composition of the present disclosure to the tumor or to the subject. In some aspects, the method is for inhibiting tumor growth or tumor progression. The inhibition can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any range derivable therein), at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any range derivable therein), or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any range derivable therein).

[0011] In some embodiments, the polypeptide is operably linked to the cytotoxic agent. The term "operably linked" refers to covalent or non-covalent attachment. In some embodiments, the attachment is covalent. In some embodiments, the attachment is non-covalent. In some embodiments, the polypeptide is covalently linked to the cytotoxic agent. In some embodiments, the peptide is non-covalently linked to the cytotoxic agent. In some embodiments, the polypeptide is linked to the cytotoxic agent through a cleavable linker. In some embodiments, the cleavable linker comprises a pH cleavable linker. In some embodiments, the linker comprises a hydrazone linker. In some embodiments, the linker is cleaved at a pH less than 7.4. In some embodiments, the linker is cleaved at an acidic pH. In some embodiments, optimal cleavage of the linker is at a pH of 4.5, 5, 5.5, 6, or 6.5 (or any range therein). Optimal cleavage refers to a pH at which at least 75, 80, 85, 90, 95, or 99% of cleavage occurs in solution or in vitro for a period of less than 6, 5, 4, 3, 2, 1, 0.5, or 0.25 hours (or any range derivable therein). In some embodiments, the polypeptide is linked to the cytotoxic agent and / or collagen-binding polypeptide through a bifunctional linker.

[0012] In some embodiments, the cytotoxic agent comprises doxorubicin. In some embodiments, the cytotoxic agent comprises a derivative of doxorubicin. In some embodiments, the cytotoxic agent comprises aldoxorubicin. In some embodiments, the cytotoxic agent is a cytotoxic agent described herein. In some embodiments, the cytotoxic agent is conjugated to the polypeptide prior to administration of the polypeptide. In some embodiments, in situ conjugation of the cytotoxic agent is excluded.

[0013] In some embodiments, the polypeptide is covalently linked to the collagen binding domain through a peptide bond. In some embodiments, the polypeptide comprises a collagen binding domain from decorin or von Willebrand factor (vWF). In some embodiments, the collagen binding domain comprises a polypeptide having at least 80% identity to SEQ ID NO:1, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO:1-4 or 11-14, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO: 1, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO: 2, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO:3, or a fragment thereof.In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO: 4, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO: 11, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO: 12, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO: 13, or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide having at least 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% identity to SEQ ID NO:14, or a fragment thereof.

[0014] In some embodiments, the polypeptide is covalently linked to an albumin polypeptide. In some embodiments, the albumin polypeptide comprises a polypeptide having 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% (or any range derivable therein) identity to one of SEQ ID NOs:7-10.

[0015] In some embodiments, the polypeptide is covalently linked to an IgG Fc domain polypeptide. In some embodiments, the IgG Fc domain polypeptide comprises a polypeptide having 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% (or any range derivable therein) identity to one of SEQ ID NOs:15-18.

[0016] In some embodiments, the collagen binding domain is at the amino terminus of the albumin polypeptide. In some embodiments, the collagen binding domain is at the carboxy terminus of the albumin polypeptide. The phrases "amino-terminal" or "carboxy-terminal" refer to the relative position of one polypeptide to another polypeptide. For example, when a polypeptide is "amino-terminal", it is linked to the N-terminal amine group of the other polypeptide. However, there may be an intervening sequence between the two polypeptides or domains. Similarly, a polypeptide "carboxy-terminal" refers to a polypeptide linked to the carboxy terminus of another polypeptide or domain. In some embodiments, a cytotoxic agent is linked to the amino terminus of the collagen binding domain. In some embodiments, a cytotoxic agent is linked to the carboxy terminus of the collagen binding domain. In some embodiments, a cytotoxic agent is linked to the amino terminus of the albumin polypeptide. In some embodiments, a cytotoxic agent is linked to the carboxy terminus of the albumin polypeptide. In some embodiments, a collagen binding domain is at the amino terminus of an IgG Fc domain polypeptide.

[0017] In some embodiments, the collagen binding domain is at the carboxy terminus of the IgG Fc domain polypeptide. In some embodiments, the cytotoxic agent is linked to the amino terminus of the collagen binding domain. In some embodiments, the cytotoxic agent is linked to the carboxy terminus of the collagen binding domain. In some embodiments, the cytotoxic agent is linked to the amino terminus of the IgG Fc domain polypeptide. In some embodiments, the cytotoxic agent is linked to the carboxy terminus of the IgG Fc domain polypeptide.

[0018] In some embodiments, the polypeptide comprises a linker between the IgG Fc domain polypeptide and the collagen binding domain. In some embodiments, the linker comprises glycine and serine amino acid residues. In some embodiments, the linker comprises GGGS (SEQ ID NO: 19), (GGGS) n (SEQ ID NO:20) , or (GGGS) 2 (SEQ ID NO:5) Includes.

[0019] In some embodiments, the polypeptide comprises a linker between the albumin polypeptide and the collagen binding domain. In some embodiments, the linker comprises glycine and serine amino acid residues. In some embodiments, the linker comprises the amino acid residues GGGS (SEQ ID NO: 19) , (GGGS) n (SEQ ID NO:20) , or (GGGS) 2 (SEQ ID NO:5) In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more (or any range derivable therein).

[0020] In some embodiments, the polypeptide is not operably linked to a particle, nanovesicle, or liposome. In some embodiments, the polypeptide is not operably linked to a nanoparticle or a solid support such as a microplate or bead. In some embodiments, the composition does not include a liposome, particle, or nanovesicle. In some embodiments, the composition does not include a nanoparticle or a solid support such as a microplate or bead.

[0021] In some embodiments, the polypeptide comprises at least two collagen binding domains. In some embodiments, the polypeptide comprises at least 2, 3, 4, 5, 6, 7, or 8 collagen binding domains. The collagen binding domains may be in tandem or at both the amino and carboxy termini of the albumin or IgG Fc domain polypeptide.

[0022] In some embodiments, the ratio of cytotoxic agent to albumin is 3: 1. In some embodiments, the ratio of cytotoxic agent to albumin is at least 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 7:1, 8:1, 9:1, or 10:1 (or any range derivable therein), and at most 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. (or any range derivable therein), or exactly 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 7:1, 8:1, 9:1, or 10:1 (or any range derivable therein). In some embodiments, the ratio of albumin polypeptide to collagen binding domain is 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, or 2:1 (or any range derivable therein).

[0023] In some embodiments, the ratio of cytotoxic agent to IgG Fc domain is 3: 1. In some embodiments, the ratio of cytotoxic agent to IgG Fc domain is at least 0.5: 1, 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1 (or any range derivable therein), and at most 0.5: 1, 1: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1. (or any range derivable therein), or exactly 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 7:1, 8:1, 9:1, or 10:1 (or any range derivable therein). In some embodiments, the ratio of IgG Fc domain polypeptide to collagen binding domain is 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, or 2:1 (or any range derivable therein).

[0024] In some embodiments, the subject has cancer. In some embodiments, the subject has breast cancer or colon cancer, or the tumor is a breast tumor or colon tumor. In some embodiments, the subject has a cancer described herein, or a tumor derived from a cancer described herein. In some embodiments, the cancer or tumor comprises a solid tumor. In some embodiments, hematological tumors or cancers are excluded.

[0025] In some embodiments, non-specific toxicity is reduced compared to the toxicity of the same cytotoxic agent linked to albumin and not linked to a collagen binding domain. For example, non-specific toxicity can be reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% in a polypeptide that includes a collagen binding domain compared to the same polypeptide that does not have a collagen binding domain. In some embodiments, the accumulation of the cytotoxic agent in the tumor is increased compared to the dose of the same cytotoxic agent linked to albumin and not linked to a collagen binding domain. In some embodiments, the increase is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%.

[0026] In some embodiments, non-specific toxicity is reduced compared to the toxicity of the same cytotoxic agent linked to an IgG Fc domain and not linked to a collagen binding domain. For example, non-specific toxicity can be reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% in a polypeptide that includes a collagen binding domain compared to the same polypeptide that does not have a collagen binding domain. In some embodiments, the accumulation of the cytotoxic agent in the tumor is increased compared to the dose of the same cytotoxic agent linked to an IgG Fc domain and not linked to a collagen binding domain. In some embodiments, the increase is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%.

[0027] In some embodiments, the method further comprises administration of an additional cancer therapy. In some embodiments, the subject is undergoing or will undergo immunotherapy. In some embodiments, the subject is determined to be non-responsive to immunotherapy. In some embodiments, the subject has refractory cancer. In some embodiments, the subject is a subject who has experienced toxicity associated with a previous therapy or previous immunotherapy. In some embodiments, the method further comprises administration of immunotherapy. In some embodiments, the immunotherapy is administered before, after, or simultaneously with the polypeptide. In some embodiments, the immunotherapy comprises checkpoint inhibitor therapy. In some embodiments, the checkpoint inhibitor therapy comprises single checkpoint inhibitor therapy, which indicates that only one checkpoint inhibitor is administered. In some embodiments, the checkpoint inhibitor therapy comprises combination checkpoint inhibitor therapy, which indicates that at least two checkpoint inhibitors are administered, such as an inhibitor against PD-1 and an inhibitor against CTLA-4. In some embodiments, the checkpoint inhibitor therapy comprises a PD-1 antibody. In some embodiments, the checkpoint inhibitor therapy comprises one or more checkpoint inhibitors described herein.

[0028] In some embodiments, the polypeptide and the additional therapy are administered in the same composition. In some embodiments, the polypeptide and the additional therapy are administered in separate compositions. In some embodiments, the composition of the present disclosure further comprises one or more immune checkpoint inhibitors. In some embodiments, the composition of the present disclosure comprises a PD-1 antibody. In some embodiments, the composition of the present disclosure comprises a CTLA-4 antibody. In some embodiments, the composition of the present disclosure comprises a PD-1 and a CTLA-4 antibody.

[0029] In some embodiments, the polypeptide or composition is administered systemically. In some embodiments, the polypeptide or composition is administered by intravenous injection. In some embodiments, the polypeptide or composition is administered intratumorally or peritumorally. In some embodiments, the polypeptide or composition is administered via a route of administration described herein.

[0030] In some embodiments, the administered dose of the cytotoxic agent is less than the minimum effective amount of the cytotoxic agent not conjugated to a collagen binding domain. In some embodiments, the administered dose of the cytotoxic agent is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (or any range derivable therein) less than the minimum effective amount of the cytotoxic agent not conjugated to a collagen binding domain. In some embodiments, the administered dose of the cytotoxic agent is less than the minimum effective amount of the cytotoxic agent conjugated to an albumin polypeptide and not conjugated to a collagen binding domain. In some embodiments, the administered dose of the cytotoxic agent is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (or any range derivable therein) less than the minimum effective amount of the cytotoxic agent conjugated to an albumin polypeptide and not conjugated to a collagen binding domain. In some embodiments, the administered dose of the cytotoxic agent is less than the minimum effective amount of the cytotoxic agent conjugated to the IgG Fc domain polypeptide and not conjugated to a collagen binding domain. In some embodiments, the administered dose of the cytotoxic agent is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (or any range derivable therein) less than the minimum effective amount of the cytotoxic agent conjugated to the IgG Fc domain polypeptide and not conjugated to a collagen binding domain. In some embodiments, the subject has been previously treated with a cytotoxic agent. In some embodiments, the subject has been determined to be non-responsive to a previous treatment or the subject has experienced non-specific toxicity to a previous treatment. In some embodiments, the subject experiences more than two, three, four, or five immune-related adverse events in response to a previous therapy.

[0031] In some embodiments, the compositions and polypeptides of the present disclosure provide targeted delivery of cytotoxic agents. Such targeted delivery can result in reduced cardiac damage, prolonged survival, reduced effective dose concentration, increased tumor infiltrating lymphocytes, increased CD8 expression, and decreased cytotoxicity, as compared to compositions comprising the same polypeptides that do not have a collagen binding domain. + It may provide an increase in cytotoxic T cells, an increase in natural killer cells, a reduction in inflammatory cytokines such as IFN-g, TNF-a, IL-5 and IL-6, or a harmless reduction in red blood cell, white blood cell, hematocrit and / or hemoglobin concentrations.

[0032] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to gene products.

[0033] 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 mouse, rat, rabbit, dog, donkey, or laboratory animal, such as fruit fly, zebrafish, etc.

[0034] In some embodiments, the patient has previously been treated for cancer. In some embodiments, the subject was resistant to the previous cancer treatment. In some embodiments, the subject was determined to be a poor responder to the previous cancer treatment.

[0035] It is contemplated that the methods and compositions include the exclusion of any of the aspects described herein.

[0036] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "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.

[0037] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the art of cell biology to indicate that a value includes the standard deviation of error for the device or method being utilized to determine the value.

[0038] 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."

[0039] 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. [The present invention 1001] A polypeptide comprising an albumin polypeptide or an IgG Fc domain polypeptide operably linked to a collagen binding domain. [The present invention 1002] The polypeptide of the present invention 1001 operably linked to a cytotoxic agent. [The present invention 1003] The polypeptide of the present invention 1002 which is covalently linked to a cytotoxic agent. [The present invention 1004] The polypeptide of the invention 1002 or 1003, linked to a cytotoxic agent via a cleavable linker. [The present invention 1005] The polypeptide of the invention 1004, wherein the cleavable linker comprises a pH cleavable linker. [The present invention 1006] The polypeptide of the present invention 1005, wherein the linker comprises a hydrazone linker. [The present invention 1007] The polypeptide of claim 1005 or 1006, wherein the linker is cleaved at a pH below 7.4. [The present invention 1008] The polypeptide of any of claims 1001 to 1007, linked to a cytotoxic agent and / or a collagen-binding polypeptide via a bifunctional linker. [The present invention 1009] The polypeptide of any one of claims 1002 to 1008, wherein the cytotoxic agent comprises doxorubicin. [The present invention 1010] The polypeptide of any of claims 1001 to 1009, which is covalently linked to a collagen-binding domain via a peptide bond. [The present invention 1011] The polypeptide of any one of claims 1001 to 1010, comprising a collagen-binding domain derived from decorin or von Willebrand factor (VWF). [The present invention 1012] The polypeptide of any of claims 1001 to 1011, wherein the collagen binding domain is at the amino terminus of the albumin polypeptide or the IgG Fc domain polypeptide. [The present invention 1013] The polypeptide of any of claims 1001 to 1012, comprising a linker between the albumin polypeptide or the IgG Fc domain polypeptide and the collagen-binding domain. [The present invention 1014] The polypeptide of the invention 1013, wherein the linker comprises glycine and serine amino acid residues. [The present invention 1015] The linker is GGGS, (GGGS) n , or (GGGS) 2 The polypeptide of the present invention comprising: [The present invention 1016] The polypeptide of any one of claims 1001 to 1015, which is not operably linked to a particle, nanovesicle, or liposome. [The present invention 1017] The polypeptide of any one of claims 1001 to 1016, comprising at least two collagen-binding domains. [The present invention 1018] The polypeptide of any of claims 1002 to 1017, wherein the ratio of the cytotoxic agent to albumin is 3:1. [The present invention 1019] A composition comprising any one of the polypeptides of the present invention 1001 to 1018. [The present invention 1020] The composition of the present invention 1019 does not include a liposome, particle, or nanovesicle. [The present invention 1021] A nucleic acid encoding any one of the polypeptides of the present invention 1001 to 1018. [The present invention 1022] A cell comprising a nucleic acid of the present invention. [The present invention 1023] A method for making a polypeptide comprising expressing a nucleic acid of the invention 1021 in a cell and isolating the expressed polypeptide. [The present invention 1024] A method for treating cancer, comprising the step of administering any one of the polypeptides of the present inventions 1001 to 1018 or the composition of the present inventions 1019 or 1020. [The present invention 1025] A method for reducing non-specific toxicity of a treatment involving a cytotoxic agent in a subject, the method comprising administering to the subject a polypeptide of any one of 1002 to 1018 of the present invention or a composition of 1019 or 1020 of the present invention. [The present invention 1026] The method of claim 1025, wherein the subject has cancer. [The present invention 1027] 1027. The method of claim 1025 or 1026, wherein the non-specific toxicity is reduced compared to the toxicity of the same cytotoxic agent linked to an albumin or IgG Fc domain polypeptide and not linked to a collagen binding domain. [The present invention 1028] A method for increasing accumulation of a cytotoxic agent in a tumor of a subject, the method comprising administering to the subject any one of the polypeptides of the present invention 1002 to 1018 or the composition of the present invention 1019 or 1020. [The present invention 1029] The method of claim 1028, wherein accumulation of the cytotoxic agent in the tumor is increased compared to a dose of the same cytotoxic agent linked to an albumin or IgG Fc domain polypeptide and not linked to a collagen binding domain. [The present invention 1030] A method for targeted delivery of a cytotoxic agent to tumor vasculature, comprising administering to a subject a polypeptide of any one of claims 1002 to 1018 or a composition of claim 1019 or 1020. [The present invention 1031] The method of any one of 1024, or 1026 to 1030, wherein the cancer or tumor comprises a solid tumor. [The present invention 1032] The method of any of claims 1024, or 1026-1030, wherein the cancer comprises breast or colon cancer, or the tumor comprises a tumor in the breast or colon. [The present invention 1033] The method of any of claims 1024 to 1032, further comprising administration of one or more additional cancer therapies. [The present invention 1034] Any of the methods of claims 1024 to 1033, wherein the subject is undergoing or intends to undergo immunotherapy. [The present invention 1035] The method of any one of claims 1024 to 1034, further comprising administration of an immunotherapy. [The present invention 1036] The method of claim 1035, wherein the immunotherapy is administered before, after, or simultaneously with the polypeptide. [The present invention 1037] The method according to any one of claims 1034 to 1036, wherein the immunotherapy comprises checkpoint inhibitor therapy. [The present invention 1038] The method of claim 1037, wherein the checkpoint inhibitor therapy comprises a PD-1 antibody. [The present invention 1039] The method of any of claims 1024 to 1038, wherein the polypeptide or composition is administered systemically. [The present invention 1040] The method of claim 1039, wherein the polypeptide or composition is administered by intravenous injection. [The present invention 1041] The method of any of claims 1024 to 1040, wherein the administered dose of the cytotoxic agent is less than the minimum effective dose of the cytotoxic agent that is not linked to a collagen binding domain. [The present invention 1042] Any of the methods of claims 1024 to 1041, wherein the administered dose of the cytotoxic agent is less than the minimum effective amount of the cytotoxic agent conjugated to an albumin polypeptide or an IgG Fc domain polypeptide and not linked to a collagen binding domain. [The present invention 1043] The method of any of claims 1024 to 1042, wherein the subject has previously been treated with a cytotoxic agent. [The present invention 1044] The method of claim 1043, 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. [Brief description of the drawings]

[0040] 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.

[0041] [Figure 1-1]Figure 1A-H Synthesis and characterization of Dox-CBD-SA. (A) Schematic of drug delivery via CBD-SA. (B) Synthesis scheme of Dox-CBD-SA. (C) Affinity of CBD-SA and SA for collagen type I and collagen type III (Kd values ​​shown) was measured by ELISA. ND = not determined due to low signal. [Concentration] vs [Signal] graph is shown in Figure 7. Two experimental replicates. (D) Dox conjugation ratio per protein is presented. Values ​​were calculated based on the results of BCA protein quantification assay (protein) and absorbance at 495 nm (Dox) (mean ± SD of three experimental replicates). (E) Dox release kinetics from Dox-CBD-SA under three different pH conditions was evaluated by fluorescence (excitation at 495 nm, emission at 590 nm) (n = 3, mean ± SD. Two experimental replicates). (F) MMTV-PyMT cells were seeded and incubated overnight. Dox, Dox-SA or Dox-CBD-SA were added (red). Cells were also stained with lysotracker (green). Scale bar = 20 μm. Representative pictures are presented. Two experimental replicates. (G, H) Cytotoxicity of Dox variants against MMTV-PyMT or MC38 cells in vitro (n = 6, mean ± SEM). Two experimental replicates. [Figure 1-2] Please refer to the description of Figure 1-1. [Diagram 2]Figure 2A-D Dox-CBD-SA shows comparable plasma pharmacokinetics to Dox-SA and higher tumor accumulation than aldoxorubicin and Dox-SA. (A) Aldoxorubicin, Dox-SA or Dox-CBD-SA (5 mg / kg Dox basis) were administered by tail vein injection (i.v.) to tumor-free FVB mice. Plasma was collected at the indicated time points. Dox plasma concentrations were measured by fluorescence (mean ± SEM, n = 4 for aldoxorubicin, n = 5 for Dox-SA and Dox-CBD-SA). (B) Plasma half-life of Dox was calculated using the exponential biphasic decay: MFI (t) = Ae-αt + Be-βt. t1 / 2, α, fast clearance half-life; t1 / 2, β, slow clearance half-life. (Mean ± SEM, n = 4 for aldoxorubicin, n = 5 for Dox-SA and Dox-CBD-SA). (C) MMTV-PyMT tumor-bearing mice were treated with aldoxorubicin, Dox-SA, or Dox-CBD-SA (4.16 mg / kg Dox basis). At the indicated time points, tumors were harvested and the amount of Dox in the tumors was quantified (mean ± SEM, n = 5 for 2 h and n = 7 for 24 h per group). (D) 100 μg of DyLight 488-labeled SA or an equimolar amount of DyLight 488-labeled CBD-SA was injected iv into MMTV-PyMT tumor-bearing mice. One hour after injection, tumors were harvested and analyzed for fluorescence by confocal microscopy. Tissues were also stained with DAPI and anti-CD31 antibodies. Scale bar = 100 μm. Representative images of three tumors each. Two experimental replicates. Statistical analysis was performed using ANOVA with Tukey's test. *p < 0.05; **p < 0.01; NS = not significant. [Figure 3-1]Figure 3A-L Dox-CBD-SA enhanced antitumor efficacy and lymphocyte infiltration into tumors in the MMTV-PyMT breast cancer model. (A) 5 × 105 MMTV-PyMT cells were inoculated into FVB mice on day 0. Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg / kg Dox base) were injected iv on day 7. The graph depicts the tumor volume until the first mouse died (mean ± SEM). (B) Survival rate. (C-F) Individual tumor growth curves. CR indicates complete response frequency. Three experimental replicates. (G-L) 5 × 105 MMTV-PyMT cells were inoculated on day 0. Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg / kg Dox base) were injected iv on day 7. Lymphocytes in the tumor were extracted on day 14, followed by flow cytometry analysis. (G-I) Graphs depict the number of (G) CD45+CD8+CD3+ T cells, (H) CD45+CD4+CD3+ T cells, and (I) CD45+NK1.1+CD3- NK cells per mg of tumor weight. Bars represent the mean ± SEM. (J-L) Graphs show [CD45+CD8+CD3+ T cells per mg of tumor weight] (J), [CD45+CD4+CD3+ T cells per mg of tumor weight] (K) or [CD45+NK1.1+CD3- NK cells per mg of tumor weight] (L) vs [tumor weight] for two experimental replicates. Statistical analysis was performed by (A, H, I) ANOVA with Tukey's test or (G) Kruskal-Wallis test followed by Dunn's test, or (B) log-rank (Mantel-Cox) test. *p < 0.05; **p < 0.01; NS = not significant. [Figure 3-2] Please refer to the description of Figure 3-1. [Figure 4]Figure 4A-G Dox-CBD-SA treatment shows reduced toxicity. 20 mg / kg aldoxorubicin or Dox-CBD-SA (Dox-based) was administered to tumor-free FVB mice by tail vein injection on day 0. (A-D) Plasma cytokine concentrations on day 3. (E) Red blood cell counts on day 6. (F) White blood cell counts on day 3. (G) Spleen weights on day 16. Data represent mean ± SEM. Two experimental replicates. Statistical analysis was performed using ANOVA with Tukey's test. *p < 0.05; **p < 0.01; NS = not significant. [Diagram 5] Figure 5A-H Dox-CBD-SA treatment completely eradicates established MC38 tumors in combination with anti-PD-1 checkpoint inhibitors. 5 × 105 MC38 cells were inoculated on day 0. Mice were injected iv with 5 mg / kg (Dox basis) aldoxorubicin or Dox-CBD-SA on days 6, 9, and 12. They were also injected ip with αPD-1 on days 10 and 13. (A) Experimental schedule. (B) Graph depicts tumor volume until the first mouse died (mean ± SEM), CR = complete response. (C) Survival rate. (D-G) Individual tumor growth curves. CR indicates complete response frequency. (H) On day 60, survivors treated with Dox-CBD-SA + αPD-1 were challenged again by subcutaneous injection of 5 × 105 MC38 cells. Naive mice were also challenged with the same amount of cells as the control group. The number of mice that developed palpable tumors is shown. Two experimental replicates. Statistical analysis was performed using the log-rank (Mantel-Cox) test for survival curves. *p < 0.05; **p < 0.01; NS = not significant. [Figure 6] Confirmation of CBD fusion to SA by MALDI-TOF MS analysis. CBD-SA was analyzed by MALDI-TOF MS. The abscissa is the mass-to-charge ratio (m / z) and the ordinate is the intensity of the charged ion. Two experimental replicates. [Figure 7]Figure 7A-B. Binding affinity of CBD-SA for collagen type I and type III. The affinity of CBD-SA for (A) collagen type I and (B) collagen type III was determined by ELISA (n = 4, mean ± SD). Graphs of [concentration] vs [signal] are shown. Two experimental replicates. [Figure 8] SDS-PAGE analysis of mouse SA and CBD-SA conjugated with Dox. Dox-SA and Dox-CBD-SA were analyzed by SDS-PAGE with Coomassie blue staining. R reduced; NR non-reduced. Representative images are presented. Two experimental replicates. [Figure 9] Figure 9A-B Hydrodynamic size. (A) The size of unconjugated CBD-SA, Dox-CBD-SA and Dox-CBD-SA reconstituted after lyophilization was measured by DLS. (B) The size of unconjugated SA and Dox-SA was also measured. Two experimental replicates. [Figure 10] Binding interface between collagen type III and the A3 domain of von Willebrand factor. Crystal structure of the A3 domain (CBD) of von Willebrand factor in complex with collagen type III (PDB 4DMU). Images were processed using UCSF Chimera. Lysines are shown as blue. [Figure 11] In vitro release kinetics of Dox from Dox-SA. The release kinetics of Dox from Dox-SA under three different pH conditions was assessed by fluorescence (excitation at 495 nm, emission at 590 nm, n = 3, mean ± SD). Two experimental replicates. [Figure 12]FIG. 12A-B Plasma pharmacokinetics of DyLight 800-labeled SA and CBD-SA. Tumor-free FVB mice were administered 200 μg of DyLight 800-labeled SA or CBD-SA via tail vein injection (i.v.). Plasma was collected at the indicated time points. (A) Signal intensity of each sample was normalized to the mean signal intensity of samples collected 1 min post-injection (mean ± SEM, n = 4). (B) Plasma half-life of labeled SA and CBD-SA was calculated using the exponential biphasic decay: MFI(t) = Ae-t + Be-βt. t1 / 2, β, slow clearance half-life. (mean ± SEM, n = 4). One experimental replicate. [Figure 13] FIG. 13A-B. Changes in hematological values ​​in mice administered 20 mg / kg aldoxorubicin or Dox-CBD-SA. (A) Hematocrit and (B) hemoglobin concentration 6 days after injection. Two experimental replicates. Statistical analysis was performed using ANOVA with Tukey's test. **p < 0.01; NS = not significant. [Figure 14] Histological analysis of major organs after Dox-CBD-SA treatment. Tumor-free FVB mice were administered Dox-CBD-SA (20 mg / kg) on ​​day 0. On day 16, heart, liver, kidneys, and lungs were harvested and processed to obtain tissue sections (n ​​= 7 for untreated, n = 5 for Dox-CBD-SA). Scale bar = 200 μm. H&E stained histological images were blindly evaluated and no significant abnormalities were observed after Dox-CBD-SA treatment. Representative images are shown. Two experimental replicates. [Figure 15] Figure 15A-B MC38 tumor rechallenge and weight change of MC38 tumor-bearing mice during treatment. (A) The graph depicts tumor size (mean ± SEM) of Dox-CBD-SA + αPD-1 treated survivors rechallenged with MC38 cells. Naive mice were also challenged with the same amount of cells as the control group. The number of mice that developed palpable tumors is shown. (B) Weight change (mean ± SEM) of mice during treatment in Figure 5. The line represents 85% of the initial weight. Two experimental replicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description Small molecule anticancer drugs distribute widely in tissues and induce systemic side effects, so drug modification to improve their pharmacokinetics and biodistribution has been attempted. Active targeting of tumor-specific or tumor-associated antigens for drug delivery is a therapeutic strategy. However, this essentially limits the scope of applicable cancers and may also lead to drug resistance due to antigen-selective cell targeting and killing, and the antigen may be lost by mutation. Herein, we engineered CBD-SA (collagen binding domain-serum albumin) to overcome these problems. Unlike other active activation strategies, CBD-SA does not require prior investigation of tumor-associated antigen expression, since collagen is nearly ubiquitously expressed in tumors and CBD accesses the tumor stroma via abnormal vasculature in the tumor microenvironment. CBD-SA then binds to exposed collagen, transforming the tumor stroma into a reservoir for chemotherapeutic agents.

[0043] Cardiac toxicity is the main drawback of Dox, which limits its lifetime accumulation. Surprisingly, we found that even 20 mg / kg of Dox-CBD-SA did not show any signs of cardiac damage. This suggests that Dox pre-conjugated with CBD-SA is less cardiac toxic than free Dox, which irreversibly damages cardiac tissue at an accumulation of 15 mg / kg in mouse models. Importantly, the accumulation of 15 mg / kg is almost equivalent to the maximum accumulation in humans. The potency and non-specific toxicity of the conjugate are somewhat surprising and unexpected, since it can be hypothesized that CBD-SA may accumulate in undesirable sites in the body, such as the liver, kidneys, and wounds, where collagen may be exposed through fenestrated or leaky endothelium. However, we did not observe pathological damage in the liver and kidneys after 20 mg / kg of Dox-CBD-SA. Thus, the present disclosure describes a novel strategy for targeting cytotoxic agents to the tumor environment.

[0044] I. Targeting Polypeptides A. Collagen-binding domain Von Willebrand factor (vWF) is a blood coagulation factor that binds to both type I and type III collagen, as well as to the adhesion receptor GPIb on platelets. Upon injury, collagen beneath the endothelium is exposed to plasma, and vWF-collagen binding initiates the thrombosis cascade. The vWF A domain has the highest affinity for collagen among non-bacterial derived proteins / peptides reported.

[0045] In some embodiments, the polypeptide comprises a collagen binding domain derived from decorin, hi some embodiments, the collagen binding domain comprises a decorin peptide such as LRELHLNNNC (SEQ ID NO:11) derived from bovine or LRELHLDNNC (SEQ ID NO:12) derived from human.

[0046] In some embodiments, the collagen binding domain has the following amino acid sequence: It includes a peptide fragment derived from human decorin represented by TIFF0007680375000001.tif48160.

[0047] In some embodiments, the collagen-binding peptide is a peptide derived from von Willebrand factor (vWF). The sequence of human vWF is as follows: Includes TIFF0007680375000002.tif194160TIFF0007680375000003.tif165159.

[0048] In some embodiments, the peptide is derived from the vWF A3 domain, which is derived from the human sequence, residues 1670-1874 (residues 907-1111 of mature vWF) and has the following sequence: TIFF0007680375000004.tif26160.

[0049] In some embodiments, the ECM peptide has the following amino acid sequence: Contains all or a fragment of vWF A3 represented by TIFF0007680375000005.tif27160.

[0050] In some embodiments, the collagen binding domain has the following sequence: The polypeptide having the sequence TIFF0007680375000006.tif26160.

[0051] In some embodiments, the polypeptide has the following sequence: TIFF0007680375000007.tif107160.

[0052] Exemplary peptides include all or a portion of any one of SEQ ID NOs: 1-4 or 11-14. A collagen binding domain can be 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 any range derivable therein) identity to a polypeptide of the disclosure, such as SEQ ID NOs: 1-4 or 11-14.

[0053] B. Linker A linker sequence may be included in the polypeptide.For example, at least 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, 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, 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more (or any range derivable therein), 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 or more (or any range derivable therein), 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, 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 any range derivable therein) may separate the antibody and peptide.

[0054] In some embodiments, the albumin polypeptide, the IgG Fc domain polypeptide, the collagen binding domain and / or the cytotoxic agent are covalently linked. For example, the cytotoxic agent can be covalently linked to the collagen binding domain. In some embodiments, the cytotoxic agent is covalently linked to the albumin polypeptide. In some embodiments, the cytotoxic agent is covalently linked to the IgG Fc domain polypeptide. In some embodiments, a linker is between the cytotoxic agent and the collagen binding domain or the albumin polypeptide. In some embodiments, a linker is between the cytotoxic agent and the collagen binding domain or the IgG Fc domain polypeptide. In some embodiments, the albumin polypeptide is covalently linked to the collagen domain. In some embodiments, the IgG Fc domain polypeptide is covalently linked to the collagen domain. In some embodiments, the linker is between the albumin polypeptide and the collagen binding domain. In some embodiments, the linker is between the IgG Fc domain polypeptide and the collagen binding domain. In some embodiments, the linker comprises a bifunctional linker. A linker, such as an amino acid or peptidomimetic sequence, can be inserted between the peptide and / or antibody sequence. The linker may have one or more properties, including flexible conformation, inability to form regular secondary structures, or hydrophobicity or charge that may facilitate or interact with either domain. Examples of amino acids commonly found in flexible protein regions may include Gly, Asn, and Ser. For example, a suitable peptide linker may be GGGSGGGS (SEQ ID NO:5) or (GGGS)n (SEQ ID NO:6), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein). Other near-neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence may vary without significantly affecting the function or activity of the fusion protein (see, for example, U.S. Patent No. 6,087,329).In certain aspects, the linker is at least 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, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 11 0, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 (or any range derivable therein), at most 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 (or any range derivable therein), or exactly 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 (or any range derivable therein), The peptides and antibody heavy or light chains can be linked by peptide sequences having from about 1 to 25 amino acid residues. Exemplary linkers may 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). Exemplary linkers include C. N(where N=1-100 carbon atoms). In some embodiments, the linker can be a dipeptide linker, such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or maleimidocaproic-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 (as found in lysines and the N-terminus of proteins or peptides). Additionally, the linker can be maleimidocaproyl (mc). In some embodiments, covalent attachment can be achieved by use of Traut's reagent.

[0055] C. Albumin In some embodiments, the albumin polypeptide is of murine origin. In some embodiments, the albumin polypeptide is of human origin.

[0056] In some embodiments, the albumin polypeptide has the following sequence: TIFF0007680375000008.tif77159.

[0057] In some embodiments, the albumin polypeptide has the following sequence: TIFF0007680375000009.tif78160.

[0058] In some embodiments, the albumin polypeptide has the following sequence: TIFF0007680375000010.tif77159.

[0059] In some embodiments, the albumin polypeptide has the following sequence: TIFF0007680375000011.tif78159.

[0060] D. Fc domain from human IgG Similar to albumin, the Fc domain from human IgG is used to enhance drug half-life because the Fc domain also has a cellular recycling system similar to albumin. In some embodiments, the albumin polypeptide is of human origin.

[0061] In some embodiments, the hIgG1 Fc polypeptide has the following sequence (IGHG1, 99-330): TIFF0007680375000012.tif34159.

[0062] In some embodiments, the hIgG2 Fc polypeptide has the following sequence (IGHG2, 99-326): TIFF0007680375000013.tif34160.

[0063] In some embodiments, the hIgG3 Fc polypeptide has the following sequence (IGHG3, 99-376): TIFF0007680375000014.tif41159.

[0064] In some embodiments, the hIgG4 Fc polypeptide has the following sequence (IGHG4, 99-327): TIFF0007680375000015.tif34159.

[0065] II. Cytotoxic Agents An embodiment of the present disclosure relates to an albumin-collagen binding domain conjugate linked to a cytotoxic agent. An embodiment of the present disclosure relates to an IgG Fc domain-collagen binding domain conjugate linked to a cytotoxic agent. Cytotoxic agents include enzyme inhibitors, such as dihydrofolate reductase inhibitors, thymidylate synthase inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, anthracycline family drugs, vinca drugs, mitomycins, bleomycins, cytotoxic nucleosides, pteridine family drugs, diynenes, podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxol.

[0066] Members of these classes include, for example, taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives, such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere, retinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamicin, enediynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, maytansinoids (including DM1), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF) and maytansinoids (DM4) and their analogs.

[0067] Cytotoxic agents also include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin, maytansinoids, and calicheamicin, hemiasterin. Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0068] Cytotoxic agents such as maytansinoids, dolastatins, auristatins, trichothecenes, calicheamicins, and CC1065, as well as derivatives of these toxins that have toxin activity, may also be used. Other cytotoxic agents include BCNU, streptozocin, vincristine, and 5-fluorouracil, the family of agents known collectively as LL-E33288 complexes described in U.S. Patents 5,053,394, 5,770,710, and esperamicin (U.S. Patent 5,877,296). Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitgellin, restrictocin, phenomycin, enomycin, and trichothecenes. See, e.g., WO 93 / 21232 published Oct. 28, 1993. In some embodiments, the cytotoxic agent comprises a chemotherapeutic agent described herein.

[0069] III. Further Treatments A. Immunotherapy In some embodiments, the method includes the administration of 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 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.

[0078] 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.

[0079] Anti-human-CTLA-4 antibodies (or VH domains 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; formerly known 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.

[0080] 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).

[0081] 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.

[0082] 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.

[0083] 3. Dendritic cell therapy Dendritic cell therapy induces an anti-tumor response 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 target cancer antigens. One example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0084] 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).

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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 to 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.

[0089] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding and T-cell activation functions. The general premise of CAR-T cells is to artificially create T cells targeted against 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. Once a T cell is engineered to become a CAR-T cell, it acts as a "living drug". 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 usually a single-chain variable fragment (scFv). A key aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of the molecule that is targeted.

[0090] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19.

[0091] 5. Cytokine therapy Cytokines are proteins produced by many types of cells present in tumors. They can modulate the immune response. Tumors often use cytokines to promote tumor growth and reduce immune responses. These immunomodulatory effects allow them to be used as drugs to elicit an immune response. Two commonly used cytokines are interferons and interleukins.

[0092] 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).

[0093] Interleukins have a number of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0094] 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.

[0095] 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.

[0096] It is contemplated that the cancer treatment may exclude any of the cancer treatments described herein.Furthermore, aspects of the present disclosure include patients who have previously undergone treatment with the therapies described herein, patients who are currently undergoing treatment with the therapies described herein, or patients who have not undergone treatment with the therapies described herein.In some embodiments, the patient is a patient who is determined to be resistant to the therapies described herein.In some embodiments, the patient is a patient who is determined to be sensitive to the therapies described herein.

[0097] B. Oncolytic Viruses In some embodiments, the additional therapeutic agent comprises oncolytic virus. Oncolytic virus is a virus that selectively infects and kills cancer cells. When infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy remaining tumors. Oncolytic virus is not only believed to cause direct destruction of tumor cells, but also to stimulate host anti-tumor immune response for long-term immunotherapy.

[0098] C. polysaccharides In some embodiments, the additional treatment comprises polysaccharides.Certain compounds found in mushrooms, mainly polysaccharides, can upregulate immune system and 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.

[0099] D. Neoantigens In some embodiments, the additional therapy includes administration of neoantigens. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. CD8 in cancer lesions identified using RNA sequencing data +The presence of T cells is elevated in tumors with high mutational burden. Levels of transcripts associated with natural killer cell and T cell cytolytic activity positively correlate with mutational burden in many human tumors.

[0100] 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.

[0101] 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 is administered at about 15 mg / m for 5 days every 3 weeks for a total of 3 courses. 2 to about 20 mg / m 2 Effective doses for use in clinical applications are contemplated, including: In some embodiments, the amount of cisplatin delivered to a cell and / or subject 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 were used alone.

[0102] 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.

[0103] Doxorubicin is poorly absorbed and is preferably administered intravenously. In certain embodiments, a suitable intravenous dose for adults is about 60 mg / m2 at approximately 21 day intervals. 2 to about 75 mg / m 2 or about 25 mg / m on each of 2 or 3 consecutive days repeated at intervals of about 3 to about 4 weeks. 2 to about 30 mg / m 2 or once weekly at approximately 20 mg / m 2The lowest dose should be used in elderly patients if there is prior myelosuppression caused by previous chemotherapy or neoplastic bone marrow infiltration, or if the drug is combined with other myelopoietin-suppressing drugs.

[0104] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards include mechlorethamine (HN 2 ), 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 initially for about 2 to about 5 days, 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 a body cavity.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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 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), at most 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), 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, ionizing radiation is administered in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any range derivable therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any range derivable therein), 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 spaced apart 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.

[0110] 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 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), at most 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), 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 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), at most 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), or exactly 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, at least 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, or 100 times, at most 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, 15 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 times, 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, or 100 fractionated doses are administered (or any range derivable therein). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therein) fractionated doses are administered per day. In some embodiments, the IL-10 receptor is activated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times (or any range derivable therein), and at most 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 times (or any range derivable therein), 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, or 30 (or any range derivable therein) fractionated doses are administered per week.

[0111] 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).

[0112] Removal of part or all of the 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 at various dosages.

[0113] 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.

[0114] IV. Nucleic acids In certain embodiments, there is a recombinant nucleic acid that encodes a polypeptide described herein.

[0115] 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.

[0116] 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 skilled 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 can include a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide can be encoded by a nucleic acid that includes variants that have slightly different nucleic acid sequences but still encode the same or substantially similar proteins (see above).

[0117] Certain embodiments include isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding a polypeptide (e.g., an interacting component that is a polymerase, an RNA polymerase, one or more truncated polymerase domains or polypeptides) that drives gene transcription dependent on polymerase activity from the polymerase domain when the interacting components interact. The term "recombinant" can be used in connection with the name of a polypeptide or specific polypeptide, and generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is the product of replication of such a molecule.

[0118] Regardless of the length of the coding sequence itself, the nucleic acid segment 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 its 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.

[0119] In some embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; the variants include 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 provided herein using the methods described herein (e.g., BLAST analysis with standard parameters). In some aspects, the isolated polynucleotide comprises a nucleotide sequence that encodes a polypeptide having at least 90%, preferably 95% and more identity over the entire length of the sequence to the amino acid sequences described herein; or a nucleotide sequence complementary to the isolated polynucleotide.

[0120] A. Vector The polypeptide may be encoded by a nucleic acid molecule. The nucleic acid molecule may be in the form of a nucleic acid 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. The 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 that are homologous to the sequence in the cell or nucleic acid, but at a location within the host cell or nucleic acid where they are not normally found. 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). The vector may be used in a host cell to produce a polymerase, an RNA polymerase, one or more truncated polymerase domains, or an interacting component that is fused, attached or linked to one or more truncated RNA polymerase domains.

[0121] 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.

[0122] B. Cell The present disclosure provides a method for modifying a target RNA of interest, particularly in a prokaryotic cell, a eukaryotic cell, tissue, organ, or organism, more particularly in a mammalian cell, tissue, organ, or organism. The target RNA can be contained in a nucleic acid molecule in a cell. In some embodiments, the target RNA is in a eukaryotic cell, such as a mammalian cell or a plant cell. The mammalian cell can be a human, non-human primate, bovine, porcine, rodent, or murine cell. The cell can be a non-mammalian eukaryotic cell, such as poultry, fish, or shrimp. The plant cell can be of a crop plant, such as cassava, corn, sorghum, wheat, or rice. The plant cell can also be of an algae, tree, or vegetable. The RNA regulation induced in a cell by the methods, systems, and compositions of the present disclosure can be such that the cell and the progeny of the cell are modified for improved production of a biological product, such as an antibody, starch, alcohol, or other desired cell output. Regulation of the RNA induced in a cell can be such that the cell and its progeny contain a modification that alters the biological product that is produced.

[0123] The mammalian cell may be a human or non-human mammalian cell, such as a primate, bovine, ovine, porcine, canine, rodent, or Leporidae cell, such as a monkey, bovine, ovine, porcine, canine, rabbit, rat, or mouse cell. The cell may be a non-mammalian eukaryotic cell, such as a poultry bird (e.g., chicken), vertebrate fish (e.g., salmon), or crustacean (e.g., oyster, clam, lobster, shrimp) cell. The cell may also be a plant cell. The plant cell may be of a monocotyledonous or dicotyledonous plant, or may be of a crop or cereal plant, such as cassava, com, sorghum, soybean, wheat, oat, or rice. The plant cell may also be from an algae, a tree or productive plant, a fruit or vegetable (e.g., a tree such as a citrus tree, e.g., an orange, grapefruit or lemon tree; a peach or nectarine tree; an apple or pear tree; a nut tree such as an almond or walnut or pistachio tree; a Solanaceae plant; a plant of the genus Brassica; a plant of the genus Lactuca; a plant of the genus Spinacia; a plant of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).

[0124] As used herein, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All of these terms include any and all subsequent progeny thereof. 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 capable of replicating a vector or expressing 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.

[0125] Some vectors may use control sequences that allow replication and / or expression in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above host cells to maintain them and allow replication of the vector. Similarly, techniques and conditions that would allow large-scale production of the vector, as well as production of the nucleic acid encoded by the vector and its cognate polypeptide, protein, or peptide, are understood and known.

[0126] C. Expression Systems There are many expression systems that contain at least some or all of the compositions discussed above.Prokaryotic and / or eukaryotic based systems can be used in the embodiments to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides.For example, the vectors, fusion proteins, RNA hairpin binding proteins, RNA targeting molecules, RNA regulatory domains and accessory proteins of the present disclosure can utilize expression systems, such as inducible or constitutive expression systems.Many such systems are commercially available and widely available.

[0127] 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.

[0128] In addition to the disclosed expression systems, 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 would know how to express vectors, such as expression constructs, and how to produce nucleic acid sequences or their cognate polypeptides, proteins, or peptides.

[0129] V. Proteinaceous Compositions Polypeptides or polynucleotides of the disclosure, such as those comprising or encoding an albumin polypeptide linked to a collagen binding domain, 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: At least 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, 1, 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, 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, 1500, 2000 or more, or any range derivable therein, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 212, 213, 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, 1500, 2000 or more, or any range derivable therein, 5, 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, 1 30, 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, 1, 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, 300, 400, 500, 550, 1000, 1500, 2000 or more consecutive amino acids or nucleic acids, or any range derivable therein, It may be 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.

[0130] Polypeptides or polynucleotides of the disclosure, such as those comprising or encoding an albumin polypeptide linked to a collagen binding domain, are described in SEQ ID NO:NO: 1-18, 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, 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, 1500, 2000 or more contiguous amino acids, or any range derivable therein.

[0131] In some embodiments, the polypeptide has the sequence SEQ ID NO:1-18 amino acids 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, 14 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, 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, 2 07, 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, 5 72, 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, 615, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 (or any range derivable therein).

[0132] In some embodiments, the polypeptide has the sequence SEQ ID NO: 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, 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, 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, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 contiguous amino acids (or any range derivable therein).

[0133] In some embodiments, the polypeptide is selected from the group consisting of any of SEQ ID NOs: 1-18. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 12 0, 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, 210, 211, 212, 213, 2 14, 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, 5, 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, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 (or any range derivable therein), At most 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, 4 4, 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, 1 83, 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, 21 4, 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, 5 79, 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, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 (or any range derivable therein), 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, 4, 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, 1 83, 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, 21 4, 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, 5 80, 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, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 contiguous amino acids (or any range derivable therein); SEQ ID NO: 1 to 18 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, 8 1, 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, 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, 5 Starting with 74, 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, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000.

[0134] In some embodiments, the polypeptide has a sequence similar to any one of SEQ ID NOs: 1-18, but differs from any one of SEQ ID NOs: 1-18 by 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%, 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 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% similar, identical or homologous, or at most 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, or exactly 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, SEQ ID NO: 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 of SEQ ID NO: 1 to 18 , 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, 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, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 contiguous amino acids (or any range derivable therein).

[0135] The polypeptides of the present disclosure include At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 12 0, 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, 210, 211, 212, 213, 2 14, 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, 5, 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, 599, 598, 599, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 599, 590, 591, 594, 595, 596, 597, 598, 599, 599, 590, 599, 590, 591, 592, 593, 4, 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), At most 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, 4 4, 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, 1 83, 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, 21 4, 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), 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, 4, 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, 1 83, 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, 21 4, 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) of may include substitutions.

[0136] The substitutions may be made at amino acid or nucleic 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, 120, 121, , 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, 1 14, 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, 1 45, 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, 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, 5 72, 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, 650, 700, 750, 800, 850, 900, 1000, 1500, or 2000 (or any range derivable therein).

[0137] The polypeptides described herein are At least 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, 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 range derivable therefrom), At most 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, 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, 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, 2 05, 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 range derivable therein), or Exactly 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, 9 ...7, 98, 99, 99, 90, 91, 92, 93, 94, 95, 98, 99, 99, 90, 91, 3, 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, 20 4, 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 range derivable therein) The sequence may be of fixed length of 100 amino acids.

[0138] 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 nonpolar or uncharged amino acid, and vice versa.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] VI. Combination Therapy The compositions and related methods of the present disclosure, particularly the administration of a polypeptide comprising an albumin polypeptide or an IgG Fc domain polypeptide linked to a collagen binding domain, may also be used in combination with the administration of an additional therapy, such as an additional therapy described herein or in combination with other conventional therapies known in the art.

[0150] 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.

[0151] Various combination regimens of therapeutic agents and treatments can be utilized.Non-limiting examples of such combinations are shown below, where therapeutic agent, for example, the composition disclosed herein, is "A", and second agent, for example, additional agent, chemotherapeutic agent, or checkpoint inhibitor, described herein or known in the art, is "B". TIFF0007680375000016.tif18128

[0152] In some embodiments, more than one course of treatment may be utilized. It is contemplated that multiple courses may be performed.

[0153] VII. 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.

[0154] 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.

[0155] "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.

[0156] 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).

[0157] VIII. 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.

[0158] 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.

[0159] Other routes of administration are also contemplated. For example, the construct and the agent may be administered in association with a carrier. In some embodiments, the carrier is a nanoparticle or microparticle. In some embodiments, the nanoparticle or microparticle is a tumor-targeting nanoparticle or microparticle. For example, the carrier may further comprise a targeting moiety that targets the carrier to a tumor. The targeting moiety may be a binding agent (e.g., an antibody, such as an scFv, or other antigen binding agent) that specifically recognizes tumor cells. In some embodiments, the construct is encapsulated within the carrier. In some embodiments, the construct is covalently or non-covalently bound to the carrier surface. In some embodiments, the carrier is a liposome. In further embodiments, the carrier molecules described herein are excluded.

[0160] Particles can have variable dimensional structures and are variously known as microspheres, microparticles, nanoparticles, nanospheres, or liposomes. Such particulate formulations can be formed by covalent or non-covalent binding of constructs to particles. In some embodiments, the particles described herein are excluded.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] Typically, for an adult human (body weight of approximately 70 kilograms), about 0.1 mg to about 3000 mg (including all values ​​and ranges therebetween), or about 5 mg to about 1000 mg (including all values ​​and ranges therebetween), or about 10 mg to about 100 mg (including all values ​​and ranges therebetween) of the compound is administered. It will be understood that such dosage ranges are exemplary only, and that administration may be adjusted depending on factors known to those skilled in the art.

[0169] In certain embodiments, the subject Approximately 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, 10.0, 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, 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, 300305, 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, 510, 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, 9 70, 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, 3900, 4000, 4100, 42 00, 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, At least 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, 10.0, 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, 2 8, 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, 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, 510, 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, 97 0, 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, 3900, 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, 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, 10.0, 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, 2 8, 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, 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, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 5 90, 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, 3900, 4000, 4100, 4 200, 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, is administered.

[0170] 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 state. 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 state. 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

[0171] IX. Working Example 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.

[0172] Example 1 - Engineered collagen-bound serum albumin as a drug-conjugate carrier for cancer therapy Serum albumin (SA) is used as a carrier to deliver cytotoxic agents to tumors via passive targeting. To further improve the tumor targeting ability of SA, we sought to develop an approach to retain SA-drug conjugates within tumors through a combination of passive and active targeting. SA was recombinantly fused with the collagen-binding domain (CBD) of von Willebrand factor and bound within the tumor interstitium after extravasation due to tumor vascular permeability. Doxorubicin (Dox) was conjugated to CBD-SA via a pH-sensitive linker. Dox-CBD-SA treatment significantly suppressed tumor growth compared to both Dox-SA and aldoxorubicin treatment in a mouse model of breast cancer. Dox-CBD-SA efficiently stimulated host antitumor immunity and caused complete eradication of MC38 colon carcinoma when used in combination with an anti-PD-1 checkpoint inhibitor. Dox-CBD-SA reduced adverse events compared to aldoxorubicin. Thus, the engineered CBD-SA may be a versatile and clinically relevant drug conjugate carrier protein for the treatment of solid tumors.

[0173] A. Introduction Doxorubicin (Dox) is a small molecule anticancer drug approved by the US Food and Drug Administration (FDA) to treat a wide range of cancers. Dox is internalized into cells via passive transmembrane diffusion and disrupts DNA function, causing the death of proliferating cells. Although Dox treatment prolongs survival in some patient populations, antitumor efficacy is not dramatic, in part due to acquired drug resistance. The poor Dox therapeutic index also limits its therapeutic use. Indeed, significant toxicity of Dox has been reported in outpatient clinics, including bone marrow suppression, excessive inflammation, and cardiac toxicity (13, 14). To improve efficacy, Dox is often used in combination with other chemotherapeutic agents. Herein, we designed a recombinant mouse SA (CBD-SA) whose N-terminus was fused to the C-terminus of the VWF A3 domain, and aldoxorubicin was conjugated to the CBD-SA via a pH-dependent cleavable hydrazone bond prior to injection (i.e., Dox-CBD-SA).(21) We evaluated the engineered CBD-SA as a tumor-targeted drug carrier, thereby obtaining improved antitumor efficacy through efficient Dox delivery to the tumor microenvironment.

[0174] B. Results 1. CBD-SA binds to collagen and can be conjugated to Dox We synthesized Dox-CBD-SA conjugates to target the tumor microenvironment (Figure 1A, B). We first investigated the binding ability of CBD-SA to recombinant collagen protein in vitro. (GGGS) 2 Linker (SEQ ID NO: 5) was used to attach CBD to the N-terminus of mouse SA, and SA was recombinantly expressed. The molecular weight of CBD-SA was analyzed by MALDI-TOF MS (Figure 6). Strong binding affinity of CBD-SA to collagen types I and III (dissociation constant (Kd) values ​​in the nM range) was observed (Figure 1C, Figure 7). For Dox conjugation, we first thiolated the lysine residues of CBD-SA using 2-iminothiolane (also known as Traut's reagent). Aldoxorubicin was then covalently conjugated to CBD-SA. Unmodified SA was also conjugated with aldoxorubicin in the same manner (Dox-SA). SDS-polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions showed that purified Dox-SA and Dox-CBD-SA were monomeric (Figure 8). The hydrodynamic size of CBD-SA was measured before and after Dox conjugation (Figure 9). The results also showed that CBD-SA exists in a monomeric form and that Dox conjugation did not change this property even after lyophilization / reconstitution cycles. Approximately three Dox molecules were conjugated per SA and CBD-SA molecule (Figure 1D). Of note, our conjugation method would not affect the binding ability of CBD-SA to collagen, since there are no cysteine ​​or lysine residues in the binding interface between the VWF A3 domain and human collagen III (Figure 10, PDB: 4DMU (22)). This interface is also far from the C-terminal fusion site for the SA domain.

[0175] 2. Dox is released under acidic pH conditions Since Dox is linked to SA with a pH-sensitive cleavable linker, we investigated the release kinetics of Dox from the conjugate under different pH conditions (Figure 1E). After 48 h of incubation, Dox release from Dox-CBD-SA reached a maximum at pH 5.0 and pH 6.5 (reported tumor microenvironment conditions). In contrast, only about 20% of Dox was released at pH 7.4 after 48 h. Dox-SA showed a similar release profile (Figure 11). These data indicate a pH-dependent release of Dox from the conjugate, consistent with previously reported release kinetics of small molecule chemicals linked via hydrazone bonds (21).

[0176] 3. Dox conjugates are taken up by cancer cells and retain their cytotoxicity We compared the intracellular localization of Dox conjugates with free drug using confocal laser scanning microscopy by detecting the fluorescence of Dox. Since Dox is the primary drug for breast cancer (23), here we chose mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse breast cancer as an experimental model. MMTV-PyMT cells were cultured in the presence of Dox or Dox conjugates, and then their intracellular uptake was evaluated (Figure 1F). After 1 h of incubation, free Dox was detected in the cytoplasm, intracellular acidic organelles, and selectively in the nucleus, indicating that its delivery is mediated by passive transmembrane diffusion. In contrast, 1 h after the addition of either Dox-SA or Dox-CBD-SA, the cytoplasm did not show strong fluorescence compared to unconjugated Dox. Instead, scattered fluorescence was observed, with some dots colocalizing with lysosomes, suggesting that both Dox-SA and Dox-CBD-SA were internalized via endocytosis. 24 hours after addition of the Dox conjugates, Dox-derived fluorescence was also observed in the nucleus, suggesting that the acidic pH of intracellular organelles induced drug release from the conjugates. Next, we investigated the cytotoxicity of the different Dox forms in vitro. MMTV-PyMT or MC38 colon carcinoma cells were seeded and incubated in the presence of Dox forms for 3 days. Viability assays showed that all three Dox forms had comparable cytotoxicity in vitro (Figure 1G, H).

[0177] 4. Dox-CBD-SA demonstrates plasma pharmacokinetics comparable to aldoxorubicin and accumulates in tumors Aldoxorubicin has a significantly longer plasma half-life compared to Dox because it reacts with endogenous SA quickly after intravenous (iv) administration (18). We tested the plasma pharmacokinetics of aldoxorubicin with or without preconjugation of SA and CBD-SA in tumor-free FVB mice. Similar plasma half-lives of aldoxorubicin, Dox-SA, and Dox-CBD-SA were observed after iv injection (Figure 2A, B). We also investigated the plasma pharmacokinetics of SA and CBD-SA fluorescently labeled with a pH-insensitive linker (Figure 12). The results showed that the half-lives of each protein conjugated with either Dox or a dye were similar, suggesting that release of Dox from the conjugate does not occur in the blood circulation.

[0178] Next, we hypothesized that CBD fusion to SA would increase the amount of Dox in tumors through active targeting to collagen in the tumor microenvironment. To test this hypothesis, we measured the amount of Dox in tumor tissues after a single iv administration. Dox-CBD-SA showed significantly higher tumor accumulation of Dox compared to aldoxorubicin and Dox-SA at 2 hours after administration (Figure 2C). Conjugation with CBD-SA similarly achieved the highest tumor accumulation of Dox 24 hours after injection, showing a significant increase compared to aldoxorubicin. Histological analysis revealed that fluorescently labeled CBD-SA colocalized with CD31 staining in tumor tissues, demonstrating that CBD-SA targets tumor vasculature (Figure 2D). These data demonstrate that CBD fusion to SA to which Dox is conjugated allows Dox to target tumors, resulting in enhanced tumor accumulation of Dox.

[0179] 5. Dox-CBD-SA demonstrates superior efficacy in the MMTV-PyMT mouse breast cancer model Motivated by the plasma pharmacokinetics and tumor accumulation studies, we evaluated the antitumor efficacy of Dox-CBD-SA in vivo. MMTV-PyMT orthotopic tumor-bearing mice received a single iv injection of the Dox form (5 mg / kg Dox basis) via the tail vein. Dox-SA and Dox-CBD-SA significantly inhibited tumor growth, whereas aldoxorubicin did not (Figure 3A, C-F). This suggests that prior conjugation of Dox with SA provides a higher therapeutic effect than in situ conjugation of aldoxorubicin with endogenous SA. Notably, Dox-CBD-SA showed a greater therapeutic effect compared to Dox-SA. Dox-CBD-SA treatment significantly prolonged survival compared to all other groups (Figure 3B) and induced complete tumor remission in 2 out of 12 mice. These data demonstrate that the CBD-fused SA functions as a superior Dox carrier compared with unmodified SA in terms of antitumor efficacy.

[0180] 6. Dox-CBD-SA enhances lymphocyte infiltration into tumors Dox has been reported to induce ICD, which stimulates immune responses against antigens from necrotic cells (15). Indeed, ICD increases the number of tumor-infiltrating lymphocytes (TILs), which are a useful prognostic marker for multiple types of cancer, such as colorectal and breast cancer (24, 25). We analyzed TILs, particularly T cells and natural killer (NK) cells, after Dox-CBD-SA treatment. Lymphocytes were extracted from tumors and analyzed by flow cytometry 7 days after treatment with different Dox forms. Dox-CBD-SA, but not aldoxorubicin or Dox-SA, significantly increased the number of intratumoral CD8 cells per unit tumor burden. + T cells, CD4 + Dox-CBD-SA treatment significantly increased the number of CD8 T cells and NK cells (Figure 3G-I). + Dox-CBD-SA indeed increased the number of tumor-infiltrating CD8 T cells by more than 2-fold compared to the other treatment groups (Figure 3G). Plots of individual tumor size and TIL cell counts show that Dox-CBD-SA indeed increased tumor size and tumor-infiltrating CD8 T cells by more than 2-fold compared to the other treatment groups (Figure 3G). + T cells, CD4+ These data revealed that the number of lymphocytes, especially CD8 + These results suggest that enhanced infiltration of cytotoxic T cells may contribute to the superior antitumor efficacy of Dox-CBD-SA.

[0181] 7. Dox-CBD-SA Shows Reduced Toxicity Because conjugated aldoxorubicin is only released very slowly from SA under physiological pH (Figure 1E), we hypothesized that Dox-CBD-SA would show reduced toxicity compared to aldoxorubicin. We used tumor-free FVB mice to evaluate adverse events after a single injection of aldoxorubicin or Dox-CBD-SA (20 mg / kg Dox basis). Administration of aldoxorubicin increased plasma concentrations of inflammatory cytokines such as IFN-γ, TNF-α, IL-5, and IL-6, but Dox-CBD-SA did not (Figure 4A-D). Aldoxorubicin treatment also induced significant decreases in red blood cell (RBC) count, white blood cell (WBC) count, hematocrit, and hemoglobin concentration (Figure 4E, F, Figure 13). In contrast, the adverse effects of Dox-CBD-SA on hematological values ​​were mild. Only a significant decrease in WBC count was observed compared to the untreated group. Aldoxorubicin administration induced splenomegaly, whereas Dox-CBD-SA treatment did not (Figure 4G). Histological analysis revealed that Dox-CBD-SA treatment did not result in observable damage in the heart, liver, kidney, or lung (Figure 14). These data suggest that pre-conjugation of Dox with CBD-SA reduced toxicity in various aspects.

[0182] 8. Dox-CBD-SA in combination with anti-PD-1 antibody (αPD-1) eradicates MC38 tumors Based on the observation of an increase in TILs induced by Dox-CBD-SA treatment (Figures 3G-L), we hypothesized that Dox-CBD-SA combination therapy with CPIs would show greater therapeutic efficacy compared to aldoxorubicin combination therapy with CPIs. To test this hypothesis, we selected αPD-1 as the most widely used CPI in outpatient clinics (26). Importantly, αPD-1 is used in combination with Dox in clinical trials (e.g., NCT02648477). We investigated the antitumor efficacy of aldoxorubicin and Dox-CBD-SA in combination with αPD-1 using the MC38 colon carcinoma model, which is immunogenic (27) but not curable by Dox monotherapy (28). C57BL / 6 mice were treated with 5 × 10 5 MC38 cells were inoculated subcutaneously. The treatment schedule is shown in Figure 5A. Aldoxorubicin or Dox-CBD-SA was administered to mice 6, 9, and 12 days after tumor inoculation. Because Dox-CBD-SA increases the number of TILs, we injected 100 μg of αPD-1 twice (days 10 and 13) one day after Dox treatment. Dox-CBD-SA + αPD-1 therapy completely eradicated established MC38 tumors (mean tumor volume was approximately 100 mm on day 6). 3 5B,G) and significantly prolonged the survival of mice compared to all other groups (Fig. 5C). In other treatment groups, some mice failed to show a complete response, and the average tumor size gradually increased (Fig. 5B,D-F). In survivors treated with Dox-CBD-SA + αPD-1, mice rechallenged with MC38 cells without additional therapy did not develop palpable tumors, demonstrating that they had acquired a strong immunological antitumor memory (Fig. 5H, Fig. 15A). No mice showed more than 15% weight loss during treatment (Fig. 15B). These data indicate that Dox-CBD-SA synergizes with αPD-1 through the induction of ICD, providing additional antitumor effects that could not be achieved with a comparable dose of aldoxorubicin + αPD-1.

[0183] C. Discussion Small molecule anticancer drugs distribute widely in tissues and induce systemic side effects, so modifications of drugs to improve their pharmacokinetics and biodistribution have been attempted. Nanoparticle formulations (17) doxorubicin or SA-reactive (18, 19) doxorubicin show improved pharmacokinetics and accumulation in tumors, based in part on pathologically abnormal vasculature (5). However, this effect may not always be effective in human cancers due to their heterogeneity (29). Thus, drugs that rely only on passive targeting may have room for improvement. Active targeting of tumor-specific or tumor-associated antigens for drug delivery is another therapeutic strategy. However, this essentially limits the range of applicable cancers and may also lead to drug resistance due to antigen-selective cell targeting and killing, and the antigen may be lost by mutation (30). Herein, we engineered CBD-SA to overcome these problems. Unlike other active activation strategies, CBD-SA does not require prior investigation of tumor-associated antigen expression. This is because collagen is nearly ubiquitously expressed in tumors, and CBD has access to the tumor stroma via abnormal vasculature in the tumor microenvironment (6). CBD-SA then binds to the exposed collagen (Fig. 1C, Fig. 7), transforming the tumor stroma into a reservoir for chemotherapy. Dox conjugation to CBD-SA showed significantly higher accumulation of Dox in tumor tissues compared with aldoxorubicin and Dox-SA (Fig. 2C). After accumulation of Dox-CBD-SA in tumor tissues, the hydrazone bond (Fig. 1E) (21), which can be cleaved under the mildly acidic conditions of the tumor microenvironment, allows sustained release of Dox from CBD-SA. At the same time, tumor cells are known to internalize SA (1). Notably, CBD fusion did not alter the cellular uptake of SA (Fig. 1F), indicating that Dox-CBD-SA can also be delivered into cells as efficiently as Dox-SA. Thus, part of the Dox release may occur in the tumor interstitium while Dox-CBD-SA is still bound to the matrix, and part may occur in the endolysosomal compartment after endocytosis.The relatively low molecular weight of CBD-SA (88 kDa, Figure 6) may be beneficial in terms of diffusion into tumor tissue (32).

[0184] In terms of antitumor efficacy, Dox-CBD-SA significantly inhibited the growth of MMTV-PyMT breast cancer and prolonged the survival of mice compared to aldoxorubicin and Dox-SA (Figure 3A-F). Since Dox-CBD-SA showed the highest accumulation in tumor tissues in vivo, Dox-CBD-SA should more efficiently induce tumor cell death via inhibition of tumor cell proliferation. In addition to this effect, a single injection of Dox-CBD-SA resulted in a long-term therapeutic effect despite its faster plasma clearance half-life (Figure 2A, B). This could be explained by our observation that Dox-CBD-SA treatment induced a higher number and density of TILs compared to Dox-SA and aldoxorubicin treatment (Figure 3G-L). Therefore, the antitumor mechanism of action of Dox-CBD-SA may not only be direct cell killing but also the stimulation of the host's antitumor immunity. Dox-CBD-SA efficiently accumulates in tumors and thus may induce ICD and tumor antigen exposure to the immune system more efficiently than aldoxorubicin and Dox-SA. As a result, Dox-CBD-SA synergistically eradicated MC38 colon carcinoma when administered in combination with αPD-1 (Figure 5B, G). The improved therapeutic efficacy of Dox-SA and Dox-CBD-SA compared to aldoxorubicin (Figure 3A-F) also indicates that pre-conjugation of Dox before injection provides higher antitumor efficacy. In addition to rapid clearance from blood circulation, in situ conjugation of aldoxorubicin with other sulfhydryl compounds such as cysteine, glutathione, fibronectin, or α1-antitrypsin in plasma (18) is also a possible cause of the inefficient therapeutic efficacy of aldoxorubicin.

[0185] Cardiac toxicity is the main drawback of Dox, which limits its lifetime accumulation (13). Histological analysis revealed that even a dose of 20 mg / kg of Dox-CBD-SA did not show any signs of cardiac damage (Figure 14). This suggests that Dox pre-conjugated with CBD-SA is less cardiotoxic than free Dox, which irreversibly damages cardiac tissue at an accumulation of 15 mg / kg in a mouse model (34). Importantly, the accumulation of 15 mg / kg is similar to the maximum accumulation in humans (35).

[0186] Regarding the manufacturing process, we conjugated Dox using Traut's reagent, which allows for precise control of the drug conjugation ratio (36). This method has little risk of abolishing the binding between CBD and collagen, because there are no lysine residues at the binding interface between the VWF A3 domain and collagen (Figure 10) (22). In addition, SA contains approximately seven times as many lysine residues as the CBD sequence, which also suggests a low risk of unfavorable conformational changes of CBD upon conjugation. Traut's reagent has also been used in a CD70-targeting ADC (MDX-1203, Bristol-Myers Squibb) (37), demonstrating its applicability for technology transfer. Because CBD-SA is produced in high yields (approximately 70-100 mg / L HEK293 cell culture), we propose that pre-conjugation of Dox to CBD-SA would generate high antitumor efficacy in a simple and technology transferable production method.

[0187] The reduction in nonspecific toxicity is unexpected, since one would expect CBD-SA to accumulate in undesirable sites in the body, such as the liver, kidneys, and wounds, where collagen may be exposed through fenestrated or leaky endothelium. However, we did not observe pathological damage in the liver and kidneys after administration of 20 mg / kg Dox-CBD-SA (Figure 14). Furthermore, the increased efficacy of the polypeptide is somewhat unexpected, since previous work in this field has shown that chemical conjugation can generally decrease the half-life of SA. It has been reported that methotrexate conjugation accelerated the clearance of methotrexate-SA conjugates from the circulation in a manner that was dependent on the drug:protein ratio (38). Thus, it is possible that the half-lives of Dox-SA and Dox-CBD-SA were significantly shorter than the reported half-life (t 1 / 2 It is surprising that the improvement in treatment efficiency was achieved despite the shorter treatment time (β = 35 (h)) than that used in the previous study (39).

[0188] In conclusion, Dox-CBD-SA accumulated in tumors and activated host antitumor immunity. As a result, Dox-CBD-SA monotherapy suppressed the growth and prolonged survival of orthotopic MMTV-PyMT breast tumors. More importantly, combined therapy of αPD-1-mediated immune checkpoint inhibition and Dox-CBD-SA completely eradicated tumors in the immunogenic MC38 model. CBD fusion provided active targeting capability to SA, which is classically used as a passively targeted drug carrier, enabling effective drug delivery from the systemic circulation to tumors. CBD-SA is expected to be non-immunogenic and biologically tolerated because it is composed of two proteins (VWF A3 domain and SA) that are naturally present in blood. Furthermore, CBD-SA acts independently on tumor type-specific antigens, providing a wide applicability to various types of solid tumors as a drug carrier. Therefore, CBD-SA may have potential for clinical technology transfer to cancer therapy as an antitumor drug carrier.

[0189] D. Materials and Methods 1. Study design This study was designed to validate the strategy of anticancer drug delivery to tumors by engineered collagen-bound SA as a drug conjugation carrier. Specifically, we tested whether the antitumor efficacy of Dox-CBD-SA against mouse models of breast and colon carcinoma is improved compared to its unmodified form. The side effects of Dox-CBD-SA were also tested using tumor-free mice. We measured the combined aspects of tumor growth, anticancer immune response, and toxicity after treatment. No statistical methods were used to predetermine the required sample size, but the sample size was determined based on estimates from pilot experiments and published results to ensure significant results in the appropriate statistical tests. CBD-SA was produced by multiple individuals to ensure reproducibility. All experiments were repeated at least twice, except for Figure 12 (once). In the animal experiments, mice were randomly divided into treatment groups in cages and treated in the same way immediately before the first Dox-CBD-SA injection. Samples were excluded from analysis only if animals developed health problems for reasons unrelated to treatment, in violation of animal welfare guidelines. 3 Exceeds 600 mm for MC38 models 3 Survival endpoints were reached when > 0.05%. The n values ​​used to calculate statistics are indicated in the figures or in the figure legends. Drug administration and pathological analyses were performed in a blinded manner. Statistical methods are described in the "Statistical Analysis" section.

[0190] 2. Cell culture Mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) cells were derived from spontaneously arising mammary tumors in FVB-Tg (MMTV-PyMT) transgenic mice as previously described (9). The MC38 colon carcinoma cell line was kindly provided by the R. Weichselbaum laboratory (University of Chicago). DMEM (Gibco) supplemented with 110 mg / L sodium pyruvate, 10% heat-inactivated FBS, and 1% penicillin / streptomycin was used for both cell lines. Cell lines were checked for mycoplasma contamination by the IMPACT I pathogen test (IDEXX BioResearch).

[0191] 3. Mice Female FVB mice, 8-12 weeks old, were obtained from Charles River and Jackson Laboratory. Female C57BL / 6 mice, 8-12 weeks old, were obtained from Jackson Laboratory. All animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee at the University of Chicago.

[0192] 4. Production and purification of CBD-SA The CBD-SA protein was designed, produced, and purified in the same manner as the previously reported CBD protein (9). A sequence encoding a fusion of human VWF A3 domain residues Cys1670–Gly1874 (907–1111 of mature VWF) with mouse SA (amino acids 25–608 of the entire SA) without the propeptide was synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. For further purification of the recombinant protein, a His-tag (6 His SEQ ID NO:21 ) was inserted at the C-terminus. 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 6The cells were diluted in fresh medium at a density of 1000 cells / mL. 2 μg / mL of plasmid DNA, 2 μg / mL of linear 25 kDa polyethyleneimine (Polysciences), and OptiPRO SFM media (final concentration 4%, Thermo Fisher Scientific) were added. The culture flasks were incubated at 37 °C for 2 h in 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). The 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 1000 mM 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. The protein was verified to be >90% pure by SDS-PAGE.

[0193] 5. MALDI-TOF MS Purified CBD-SA was analyzed by MALDI-TOF MS (Bruker Ultraflextreme MALDI TOF / TOF) as previously described (9). Data acquisition was performed using Bruker flexControl™ and data processing was performed using Bruker flexAnalysis™. First, a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) was prepared in 50:50 acetonitrile:1% TFA in water as the solvent. CBD-SA (5 μL, 0.1 mg / mL) and matrix solution (25 μL) in PBS were mixed and 1 μL of the mixture was dropped onto an MTP 384 ground steel target plate. The droplet was dried in a stream of nitrogen gas. All samples were analyzed using the high-mass linear positive mode method with 2500 laser shots at 75% laser intensity. Measurements were externally calibrated in three points using a mixture of carbonic anhydrase, phosphorylase B, and bovine SA.

[0194] 6. Binding affinity assay The binding affinity of CBD-SA to collagen was tested as previously described (9). 96-well ELISA plates (Greiner Bio-One) were coated overnight at 37°C with collagen I or collagen III (10 μg / mL each in PBS) and then blocked with 2% BSA in PBS containing 0.05% Tween 20 (PBS-T) for 1 h at room temperature. The wells were then washed with PBS-T and further incubated with increasing concentrations of CBD-SA for 2 h at room temperature. After three washes with PBS-T, the wells were incubated with biotin-conjugated Ab against mouse SA for 1 h at room temperature. After washing, bound CBD-SA was detected by measuring the absorbance at 450 nm using tetramethylbenzidine substrate and subtracting the absorbance at 570 nm. Apparent Kd values ​​were obtained by nonlinear regression analysis in Prism software (version 7, GraphPad) assuming one-site specific binding.

[0195] 7. Synthesis of Dox conjugates Mouse SA or CBD-SA was dissolved in PBS containing 2 mM EDTA. Four molar equivalents of Traut's reagent dissolved in PBS containing 2 mM EDTA were added and incubated at room temperature in the dark for 1 hour. Excess Traut's reagent was removed by Zeba spin desalting column (Thermo fisher scientific). 15 molar equivalents of aldoxorubicin (MedChemExpress) dissolved in 10 mM sodium phosphate buffer (pH 5.9) were added and incubated at room temperature for 1 hour and at 4°C overnight in the dark. To quench the reaction, 20 molar equivalents of L-cysteine ​​(Sigma-Aldrich (pharmaceutical grade) dissolved in PBS containing 2 mM EDTA) was added relative to aldoxorubicin. Unreacted Dox precipitate was removed by centrifugation (10000×g, 5 min). The supernatant was further purified by Zeba spin desalting column followed by ultrafiltration using Amicon-Ultra (Merck, 10K MWCO). The concentration of Dox in the final product was determined using a molar extinction coefficient of 10650 (L mol-1 cm -1 ) was used to quantify the absorbance at 495 nm. The protein content was measured by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0196] 8. Dynamic Light Scattering (DLS) The hydrodynamic size of the Dox conjugates in PBS was measured using a Zetasizer Nano ZS (Malvern). Conjugates were analyzed immediately after synthesis or lyophilized and stored at -20°C until use.

[0197] 9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed as described in our previous study (9). 4–20% gradient gels (Bio-Rad) were used, and 0.5 μg of each Dox conjugate was loaded with or without reduction with 10 mM DTT. After electrophoresis, SimplyBlue SafeStain (Thermo Fisher Scientific) was used for gel staining according to the manufacturer's instructions. Images were acquired using a ChemiDoc XRS+ system (Bio-Rad).

[0198] 10. pH-dependent release of Dox from the conjugate The release profile of Dox from the conjugates was determined using a Slide-A-Lyzer MINI dialysis device (Thermo fisher scientific, 10K MWCO). The Dox conjugates were diluted in PBS (pH 6.5 or 7.4) or 0.1 M acetate buffer (pH 5.0) to a final concentration of 100 μM (Dox basis). 150 μL of each sample was loaded into the device and dialyzed against 50 mL of buffer. Dialysis was performed using a magnetic stirrer. The stage temperature was set at 37°C and the samples were protected from light during dialysis. The dialysates were collected at various time points and stored at 4°C until the end of sample collection. The dialysates were loaded in duplicate (90 μL / well) into a 96-well black plate. Fluorescence was determined using excitation at 495 nm and emission at 590 nm. Serial dilutions of doxorubicin hydrochloride were prepared in the same buffer to generate a standard curve.

[0199] 11. Cellular uptake of Dox conjugates To evaluate the cellular uptake of Dox conjugates, MMTV-PyMT cells were seeded at 5000 cells / well in 96-well high content imaging plates (Corning) and incubated overnight. Cells were washed with PBS and treated with free Dox, Dox-SA, or Dox-CBD-SA dissolved in DMEM (110 mg / L sodium pyruvate, 10% heat-inactivated FBS, 1% penicillin / streptomycin, phenol red (-)) at a concentration equivalent to 50 μM of Dox. After incubation, cells were washed twice and treated with 75 nM Lysotracker Deep Red and further incubated for 30 min at 37 °C. Cells were washed twice and observed under an IX83 microscope (Olympus) at 60x magnification. Images were processed using ImageJ software (NIH). Scale bar; 20 μm.

[0200] 12. In vitro cytotoxicity MMTV-PyMT or MC38 cells were seeded at 3000 cells / well in 96-well tissue culture plates (BD Falcon) and incubated overnight. Cells were washed with DMEM (110 mg / L sodium pyruvate, 10% heat-inactivated FBS, 1% penicillin / streptomycin, phenol red (-)) and 80 μL / well of DMEM was added. Serial dilutions of aldoxorubicin, Dox-SA, or Dox-CBD-SA in PBS were then added (20 μL / well). Cells were incubated at 37°C for 3 days and viability was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega) according to the manufacturer's instructions. Cells treated with 80 μL / well of DMEM + 20 μL / well of PBS were defined as 100% viability, and cell-free wells containing the same mixture were defined as 0% viability. Half-maximal inhibitory concentration (IC 50 ) values ​​were obtained by nonlinear regression analysis in Prism software ([inhibitor] vs normalized response).

[0201] 13. Plasma Pharmacokinetics of Dox Conjugates We referred to a previous report on polypeptide-Dox nanoparticles (40). To measure the pharmacokinetics of Dox, aldoxorubicin, Dox-SA, or Dox-CBD-SA were injected intravenously into female FVB mice at 5 min, 30 min, 1 h, 4 h, 12 h, 25 h, 50 h, and 75 h after injection. Blood samples were collected in EDTA-coated tubes at 5 min, 30 min, 1 h, 4 h, 12 h, 25 h, 50 h, and 75 h after injection. Blood samples were stored at 4 °C until the end of sample collection. Samples were centrifuged (2000 × g, 5 min) and plasma was collected. Plasma samples diluted in acidified isopropanol (75 mM HCl, 10% water, 90% isopropanol) were loaded into a 96-well black plate (100 μL / well). Fluorescence was measured as described above. Plasma samples were also collected from uninjected mice, diluted in acidified isopropanol, and measured to generate a standard curve of background fluorescence. Exponential two-phase decay (Y = Ae -αt + Be - αt ) fits were used to calculate plasma half-life. Fast clearance half-life: t 1 / 2,□ , slow clearance half-life: t 1 / 2 , α. Data were analyzed using Prism software (v7, GraphPad).

[0202] 14. Plasma pharmacokinetics of SA and CBD-SA SA and CBD-SA were labeled with DyLight 800 NHS ester (Thermo fisher scientific) according to the manufacturer's instructions. Unreacted dye was removed by Zeba spin desalting column as described above. After labeling, 200 μg of each protein was injected intravenously into female FVB mice. Blood samples were collected in EDTA-coated tubes at 1 min, 1 h, 4 h, 24 h, 74 h, and 120 h after injection. Blood samples were stored at 4° C. until the end of sample collection. Samples were centrifuged (2000×g, 5 min) and plasma was collected. Blood samples were diluted in PBS and loaded into a 96-well black plate (100 μL / well). The concentration of each protein in plasma was measured with a LI-COR Infrared Odyssey Imager (Li-COR Biosciences). The method of curve fitting and calculation of plasma half-life were described above.

[0203] 15. MMTV-PyMT Tumor Inoculation and Treatment The MMTV-PyMT mouse breast cancer model was prepared as previously described (9). A total of 5 × 10 5 MMTV-PyMT cells were injected subcutaneously into the right mammary gland of each mouse. Mice were treated with aldoxorubicin, Dox-SA or Dox-CBD-SA (5 mg / kg) by tail vein injection on day 7. Tumors were measured with digital calipers at the indicated time points and volumes were calculated as ellipsoids, where V = 4 / 3 × 3.14 × depth / 2 × width / 2 × height / 2. For tumor volumes of 500 mm 3 Mice were sacrificed when the tumor volume exceeded 100 μg / kg / day or when active ulceration was observed. For treatment experiments, FVB mice from Charles River were used. For tumor infiltrating lymphocyte (TIL) analysis, FVB mice from both Jackson Laboratory and Charles River were used. The proportion of mice from the different suppliers was equalized between all groups.

[0204] 16. MC38 Tumor Inoculation and Treatment The MC38 mouse colon carcinoma model was prepared similarly to the B16F10 melanoma model as previously described (9). A total of 5 × 10 5 MC38 cells were injected intradermally into the left side of the back of each C57BL / 6 mouse. Mice were injected iv with aldoxorubicin, Dox-SA or Dox-CBD-SA (5 mg / kg) on ​​days 6, 9 and 12. Mice were also treated ip with 100 μg of anti-PD-1 (clone 29F.1A12, Bio X Cell) on days 10 and 13. Tumor growth was monitored as above. Tumors with a volume of 600 mm were 3 Mice were sacrificed when the tumor volume exceeded 100 μg / kg or when active ulceration was observed. On day 60, naïve C57BL / 6 mice or tumor-free survivors were cultured with 5 × 10 5 The mice were rechallenged by intradermal injection of 10 MC38 cells.

[0205] 17. Tumor accumulation research We referred to a previous report on polypeptide-Dox nanoparticles (40). Aldoxorubicin, Dox-SA, or Dox-CBD-SA were injected at 4.16 mg / kg via the tail vein into FVB mice bearing established tumors. Tumors were collected 2 or 24 h after injection, weighed, and placed on ice. Tumor tissues were suspended in 1 mL of acidified isopropanol and homogenized for 40 s at 5000 beats / min using Lysing Matrix D and FastPrep-24 5G (MP Biomedical). After homogenization, samples were protected from light and incubated overnight at 4 °C. Samples were centrifuged (5000 × g, 5 min), and the supernatants were loaded into 96-well black plates (100 μL / well, triplicates). Fluorescence was measured to quantify the amount of Dox in tissue extracts as described above. Tumors from untreated mice were also processed and serial dilutions of tissue extracts were measured to obtain a standard curve of tissue-derived autofluorescence.

[0206] 18. Histological analysis of intratumoral injected CBD-SA Mouse SA (Sigma-Aldrich) and CBD-SA were conjugated with NHS-DyLight 488 according to the manufacturer's instructions. Unreacted dye was removed by Zeba spin desalting columns, and then the fluorescent protein solution was stored at 4°C until use. 100 μg of fluorescently labeled SA or CBD-SA labeled with equimolar dyes was intravenously injected into MMTV-PyMT tumor-bearing mice. One hour after injection, tumors were harvested and frozen in dry ice with OCT compound. 10 μm tissue slices were obtained by cryosectioning. Tissues were fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. After washing with PBS-T, tissues were blocked with 2% BSA in PBS-T for 1 h at room temperature. Tissues were stained with biotin-labeled anti-mouse CD31 antibody (1:100, Biolegend) and Alexa Fluor 647 streptavidin (1:1000, Biolegend). Tissues were washed three times and then covered with ProLong Gold antifade mounting medium containing DAPI (Thermo Fisher Scientific). An IX83 microscope (Olympus) was used for imaging at 60x magnification. Images were processed using ImageJ software (NIH).

[0207] 19. Flow Cytometry and Antibodies MMTV-PyMT models were prepared as described above. Mice were treated with aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg / kg) on ​​day 7. Mice were sacrificed on day 14. Cell suspensions were obtained from each tumor as previously described (9). Tumors were harvested and digested for 30 min at 37°C in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2% FBS, 2 mg / mL collagenase D, and 40 μg / mL DNase I (Roche). Single cell suspensions were obtained by gentle disruption of organs with a 70 μm cell strainer. Red blood cells were lysed with ACK lysis buffer (Quality Biological). Fixable live / dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer's instructions. After a washing step, cells were stained with specific antibodies for 20 min on ice before fixation. Cells were stained with the following antibodies: CD3 (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), CD8α (53-6.7, BD Biosciences), CD45 (30-F11, BD Biosciences), and NK1.1 (PK136, BD Biosciences). All flow cytometric analyses were performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0208] 20. Toxicity Profile Tumor-free FVB mice were administered 20 mg / kg aldoxorubicin or Dox-CBD-SA by intravenous injection. Blood samples were collected from each mouse by submandibular bleeding into EDTA-coated tubes on days 3 and 6 after injection for plasma cytokine and hematological analysis. The weight of each mouse was measured at the indicated time points. On day 16, mice were sacrificed and organs were harvested. Spleens were weighed and other organs were used for histological analysis. Mice were sacrificed when a loss of more than 15% of the initial body weight was observed.

[0209] 21. Hematological analysis Blood samples were analyzed using a COULTER Ac·T 5diff CP hematology analyzer (Beckman Coulter) according to the manufacturer's instructions.

[0210] 22. Measurement of plasma cytokines Plasma was collected from whole blood samples as described above and stored at −20°C until use. Cytokine concentrations in plasma were measured using a Ready-SET-Go! ELISA kit (eBioscience) and Can Get Signal solution (TOYOBO) according to the manufacturer's instructions.

[0211] 23. Histological analysis of heart, liver, kidneys, and lungs Organs were fixed overnight in 2% paraformaldehyde in PBS. After embedding in paraffin, blocks were cut into 5 μm sections followed by H&E staining.

[0212] 24. Statistical analysis Statistically significant differences between experimental groups were determined using Prism software (v7, GraphPad) as previously described (9). Variances between groups were found to be similar by Brown-Forsythe test when using one-way ANOVA followed by Tukey's HSD post-hoc test. For nonparametric data (Figure 3G), the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. Survival curves were analyzed by using the log-rank (Mantel-Cox) test. Symbols * and ** indicates P values ​​less than 0.05 and 0.01, respectively; NS, not significant.

[0213] 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.

[0214] References The following references and publications referenced throughout the specification, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007680375000017.tif63160TIFF0007680375000018.tif238160TIFF00076803750 00019.tif245160TIFF0007680375000020.tif245160TIFF0007680375000021.tif114160

Claims

1. A pharmaceutical composition for reducing non-specific toxicity of a cancer treatment comprising a chemotherapeutic agent in a subject having cancer, the pharmaceutical composition comprising a polypeptide comprising an albumin polypeptide operably linked to a collagen binding domain having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4, the polypeptide being further linked to a chemotherapeutic agent through a pH-cleavable linker, wherein the non-specific toxicity is reduced compared to the toxicity of the same chemotherapeutic agent linked to albumin and not linked to the collagen binding domain.

2. The pharmaceutical composition of claim 1, wherein the pH-cleavable linker comprises a hydrazone linker.

3. The pharmaceutical composition of claim 2, wherein the pH-cleavable linker is cleaved at a pH of less than 7.

4.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the collagen binding domain is at the amino terminus of the albumin polypeptide.

5. The pharmaceutical composition of any one of claims 1 to 3, wherein the polypeptide comprises a linker between the albumin polypeptide and the collagen binding domain.

6. The pharmaceutical composition of claim 5, wherein the linker between the albumin polypeptide and the collagen binding domain comprises glycine and serine amino acid residues.

7. The linker between the albumin polypeptide and the collagen binding domain, wherein the linker is GGGS (SEQ ID NO: 19), (GGGS) n , where n is 2, 3, 4, 5, 6, 7, 8, 9, or 10, or (GGGS) 2 The pharmaceutical composition of claim 6, comprising (SEQ ID NO:5).

8. The pharmaceutical composition of any one of claims 1 to 3, wherein the polypeptide comprises at least two collagen binding domains.

9. A pharmaceutical composition according to any one of claims 1 to 3, wherein the chemotherapeutic agent comprises doxorubicin.

10. A pharmaceutical composition described in any one of claims 1 to 3, which does not contain a liposome or a nanovesicle.

11. a. the cancer involves a solid tumor; and / or b. the cancer includes breast or colon cancer; A pharmaceutical composition according to any one of claims 1 to 3.

12. a. the pharmaceutical composition is administered in combination with one or more additional cancer therapies, and / or b. the subject is undergoing or will undergo immunotherapy, and / or c. the pharmaceutical composition is administered in combination with immunotherapy, which is administered before, after, or simultaneously with the pharmaceutical composition; and / or d. the pharmaceutical composition is administered systemically, and / or e. the subject has been previously treated with a chemotherapeutic agent and the subject has been determined to be non-responsive to the previous treatment or the subject is experiencing non-specific toxicity to the previous treatment; A pharmaceutical composition according to any one of claims 1 to 3.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is administered in combination with immunotherapy, the immunotherapy comprising checkpoint inhibitor therapy.

14. The pharmaceutical composition of claim 13, wherein the checkpoint inhibitor therapy includes a PD-1 antibody.

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