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

Abstract

The present disclosure relates to tumor-targeted drug carriers that improve anti-tumor efficacy by effectively delivering cytotoxic agents to the tumor microenvironment. Aspects of the present disclosure relate to polypeptides comprising an albumin polypeptide or an IgG Fc domain polypeptide operably linked to a collagen binding domain. Additional aspects relate to compositions comprising the polypeptides, nucleic acids or cells of the present disclosure.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 856,468, filed on June 3, 2019, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 12, 2020, is named ARCD-P0688WO-1001126260_SL.txt, and is 71598 bytes in size. Background Art

[0005] II. Field of the Invention

[0006] The present invention generally relates to the field of medicine. More specifically, it relates to compositions and methods of nucleotide constructs, proteins and drug carriers for treating cancer.

[0007] III. Background Technology

[0008] Serum albumin (SA) is the most abundant protein in the blood. Many compounds, including small molecules, peptides, and cytokines, have been fused, conjugated, or co-formulated with SA to improve drug delivery to disease lesions. The ultra-long plasma half-life and / or hydrophilicity of SA help improve the pharmacokinetics, safety, and efficacy of drugs.

[0009] Doxorubicin (Dox) is a small molecule anticancer drug that has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of a variety of cancers. Dox is internalized in cells by passive transmembrane diffusion and interferes with DNA function, leading to the death of proliferating cells. Although Dox treatment prolongs survival in certain patient populations, the antitumor efficacy is not significant, partly due to acquired drug resistance. The poor therapeutic index of Dox also limits its therapeutic use. In fact, considerable toxicity of Dox has been reported clinically, including bone marrow suppression, excessive inflammation, and cardiotoxicity. 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 Dox maleimide derivatives with pH-sensitive, cleavable linkers (aldoxorubicin), which were developed to achieve in situ conjugation to cysteine-34 (in the human sequence) of circulating SA.

[0010] Although strategies have been developed to passively target cytotoxic drugs to tumors, there is a need in the art for more targeted therapies that can reduce nonspecific toxicity and increase the efficacy of cytotoxic agents while lowering the minimum effective dose required to achieve a therapeutic effect. Summary of the invention

[0011] Here, the inventors describe tumor-targeted drug carriers that improve anti-tumor efficacy by effectively delivering cytotoxic agents to the tumor microenvironment. Aspects of the present disclosure relate to polypeptides comprising an albumin polypeptide or an IgG Fc domain polypeptide operably linked to a collagen binding domain. Additional aspects relate to compositions comprising the polypeptides, nucleic acids, or cells of the present disclosure. Additional aspects also relate to nucleic acids encoding the polypeptides of the present disclosure. Still further aspects relate to cells comprising the nucleic acids or polypeptides of the present disclosure.

[0012] Another aspect of the disclosure relates to a method of making a polypeptide, comprising expressing a nucleic acid of the disclosure in a cell and isolating the expressed polypeptide.

[0013] Additional aspects relate to methods for treating cancer comprising administering a polypeptide, nucleic acid or composition of the present disclosure. Additional aspects relate to methods for reducing the 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.

[0014] Additional aspects relate to a method for increasing accumulation of a cytotoxic agent in a tumor in a subject, the method comprising administering to the subject a polypeptide, nucleic acid or composition of the disclosure.

[0015] Additional aspects relate to methods for targeted delivery of cytotoxic agents to tumor vasculature, the method comprising administering a polypeptide or composition of the present disclosure to a subject. Additional aspects also relate to methods for treating tumors or methods for treating tumors of a subject, the method comprising administering a polypeptide or composition of the present disclosure to a tumor or subject. In some aspects, the method is used to inhibit tumor growth or tumor progression. The inhibition may be at least, at most, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (or any range derivable therein).

[0016] In some embodiments, the polypeptide is operably linked to the cytotoxic agent. The term "operably linked" refers to covalent or non-covalent linkage. In some embodiments, the connection is covalent. In some embodiments, the connection is non-covalent. In some embodiments, the polypeptide is covalently linked to the cytotoxic agent. In some embodiments, the polypeptide is non-covalently linked to the cytotoxic agent. In some embodiments, the polypeptide is connected to the cytotoxic agent through a cleavable linker. In some embodiments, the cleavable linker includes a pH-cleavable linker. In some embodiments, the linker includes a hydrazone linker. In some embodiments, the linker is broken at a pH value of less than 7.4. In some embodiments, the linker is broken at an acidic pH. In some embodiments, the optimal breakage of the linker is at a pH value of 4.5, 5, 5.5, 6 or 6.5 (or any range therebetween). Optimal cleavage refers to a pH at which at least 75%, 80%, 85%, 90%, 95% or 99% cleavage occurs in a period of less than 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 0.5 hours or 0.25 hours (or any range derivable therein) in solution or in vitro. In some embodiments, the polypeptide is linked to the cytotoxic agent and / or collagen binding polypeptide via a bifunctional linker.

[0017] 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 adriamycin. 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.

[0018] In some embodiments, the polypeptide is covalently linked to the collagen binding domain by 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 to SEQ ID NO: 4 or SEQ ID NO: 11 to SEQ ID NO: 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 that is 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% identical to SEQ ID NO:13 or a fragment thereof. In some embodiments, the collagen binding domain comprises a polypeptide that is 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% identical to SEQ ID NO:14 or a fragment thereof.

[0019] 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% identity (or any range derivable therein) to one of SEQ ID NO:7 to SEQ ID NO:10.

[0020] In some embodiments, the polypeptide is covalently linked to an IgG Fc domain polypeptide. In some embodiments, the IgG Fc domain 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% identity (or any range derivable therein) to one of SEQ ID NO: 15 to SEQ ID NO: 18.

[0021] In some embodiments, the collagen binding domain is located at the amino terminus of an albumin polypeptide. In some embodiments, the collagen binding domain is located at the carboxyl terminus of an albumin polypeptide. The phrase "at the amino terminus" or "at the carboxyl terminus" refers to the relative position of one polypeptide to another polypeptide. For example, when a polypeptide is "at the amino terminus", it is attached to the N-terminal amino group of another polypeptide. However, there may be an intervening sequence between two polypeptides or domains. Similarly, a polypeptide "at the carboxyl terminus" refers to a polypeptide that is attached to the carboxyl terminus of another polypeptide or domain. In some embodiments, a cytotoxic agent is attached to the amino terminus of a collagen binding domain. In some embodiments, a cytotoxic agent is attached to the carboxyl terminus of a collagen binding domain. In some embodiments, a cytotoxic agent is attached to the amino terminus of an albumin polypeptide. In some embodiments, a cytotoxic agent is attached to the carboxyl terminus of an albumin polypeptide. In some embodiments, a collagen binding domain is located at the amino terminus of an IgG Fc domain polypeptide.

[0022] In some embodiments, the collagen binding domain is located at the carboxyl terminus of the IgG Fc domain polypeptide. In some embodiments, the cytotoxic agent is attached to the amino terminus of the collagen binding domain. In some embodiments, the cytotoxic agent is attached to the carboxyl terminus of the collagen binding domain. In some embodiments, the cytotoxic agent is attached to the amino terminus of the IgG Fc domain polypeptide. In some embodiments, the cytotoxic agent is attached to the carboxyl terminus of the IgG Fc domain polypeptide.

[0023] In some embodiments, the polypeptide comprises a linker between an IgG Fc domain polypeptide and a 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).

[0024] 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 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 greater than 12 (or any range derivable therein).

[0025] 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 solid support such as a microwell plate or bead. In some embodiments, the composition does not include a liposome, particle, or nanovesicle.

[0026] In some embodiments, the composition does not comprise nanoparticles or solid supports such as microplates or beads.

[0027] 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 can be linked together or at the amino terminus and carboxyl terminus of the albumin polypeptide or IgG Fc domain polypeptide.

[0028] 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, at most, 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).

[0029] 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, at most, 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).

[0030] 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 a colon tumor. In some embodiments, the subject has a cancer described herein or a tumor from a cancer described herein. In some embodiments, the cancer or tumor includes a solid tumor. In some embodiments, hematologic tumors or cancers are excluded.

[0031] In some embodiments, the non-specific toxicity is reduced compared to the toxicity of the same cytotoxic agent linked to albumin and not linked to the collagen binding domain. For example, the non-specific toxicity of a polypeptide comprising a collagen binding domain may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the same polypeptide without the 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 the collagen binding domain. In some embodiments, the increase is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0032] In some embodiments, the non-specific toxicity is reduced compared to the toxicity of the same cytotoxic agent connected to the IgG Fc domain and not connected to the collagen binding domain. For example, the non-specific toxicity of the polypeptide comprising the collagen binding domain can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to the same polypeptide without the 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 connected to the IgG Fc domain and not connected to the collagen binding domain. In some embodiments, the increase is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0033] In some embodiments, the method further comprises administering another cancer therapy. In some embodiments, the subject has or will receive immunotherapy. In some embodiments, it has been determined that the subject is unresponsive to immunotherapy. In some embodiments, the subject suffers from refractory cancer. In some embodiments, the subject is an object that has experienced toxicity associated with a previous therapy or a previous immunotherapy. In some embodiments, the method further comprises administering immunotherapy. In some embodiments, immunotherapy is administered before, after, or simultaneously with the polypeptide. In some embodiments, immunotherapy includes check point inhibitor therapy. In some embodiments, check point inhibitor therapy includes single check point inhibitor therapy, which indicates that only one check point inhibitor is administered. In some embodiments, check point inhibitor therapy includes combined check point inhibitor therapy, which indicates that at least two check point inhibitors are administered, such as PD-1 inhibitors and CTLA-4 inhibitors. In some embodiments, check point inhibitor therapy comprises PD-1 antibodies. In some embodiments, check point inhibitor therapy comprises one or more than one check point inhibitor described herein.

[0034] 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 compositions of the present disclosure further comprise one or more than one immune checkpoint inhibitor. In some embodiments, the compositions of the present disclosure comprise PD-1 antibodies. In some embodiments, the compositions of the present disclosure comprise CTLA-4 antibodies. In some embodiments, the compositions of the present disclosure comprise PD-1 and CTLA-4 antibodies.

[0035] 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 within or around a tumor. In some embodiments, the polypeptide or composition is administered by a route of administration described herein.

[0036] In some embodiments, the dose of the cytotoxic agent administered is less than the minimum effective dose of the cytotoxic agent not conjugated to the collagen binding domain. In some embodiments, the dose of the cytotoxic agent administered is less than at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or any range derivable therein) of the minimum effective dose of the cytotoxic agent not conjugated to the collagen binding domain. In some embodiments, the dose of the cytotoxic agent administered is less than the minimum effective dose of the cytotoxic agent conjugated to the albumin polypeptide and not conjugated to the collagen binding domain. In some embodiments, the dose of the cytotoxic agent administered is less than at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or any range derivable therein) of the minimum effective dose of the cytotoxic agent conjugated to the albumin polypeptide and not conjugated to the collagen binding domain. In some embodiments, the dosage of cytotoxic agent is less than the minimum effective dose of cytotoxic agent conjugated to IgG Fc domain polypeptide and not conjugated to collagen binding domain. In some embodiments, the dosage of cytotoxic agent is less than at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or any range derivable therefrom) of the minimum effective dose of cytotoxic agent conjugated to IgG Fc domain polypeptide and not conjugated to collagen binding domain. In some embodiments, the object has been treated with cytotoxic agent before. In some embodiments, it has been determined that the object is unresponsive to previous treatment or wherein the object has experienced nonspecific toxicity to previous treatment. In some embodiments, in response to previous therapy, the object experiences more than 2, 3, 4 or 5 immune-related adverse events.

[0037] In some embodiments, the compositions and polypeptides of the present disclosure provide targeted delivery of cytotoxic agents. Compared to compositions comprising the same polypeptide without the collagen binding domain, such targeted delivery can reduce cardiac damage, prolong survival, reduce effective dose concentrations, increase tumor infiltrating lymphocytes, increase CD8+ cytotoxic T cells, increase natural killer cells, reduce inflammatory cytokines such as IFN-g, TNF-a, IL-5 and Il-6, or red blood cells, white blood cells, hematocrit and / or hemoglobin concentrations without adverse reductions.

[0038] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to a gene product.

[0039] 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 a laboratory test animal such as a fruit fly, zebrafish, etc.

[0040] In some embodiments, the patient has previously been treated for cancer. In some embodiments, the subject is resistant to the previous cancer treatment. In some embodiments, the subject is determined to have a poor response to the previous cancer treatment.

[0041] It is contemplated that the methods and compositions include exclusions of any of the embodiments described herein.

[0042] As used herein, the terms "or" and "and / or" are used to describe multiple components that are mutually combined or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "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.

[0043] In this application, the term "about" is used according to its plain and ordinary meaning in the art of cell biology to indicate a value that includes the standard deviation of error for the device or method being employed to determine the value.

[0044] The term "comprising," which is synonymous with "including," "comprising," or "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes any elements, steps, or ingredients not specified. The phrase "consisting essentially of" limits the scope of the subject matter to the specified materials or steps, as well as those that do not materially affect the basic and novel characteristics thereof. It is contemplated that embodiments described in the context of the term "comprising" may also be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0045] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. In addition, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or use any composition of the invention. Aspects of the embodiments described in the examples may also be embodiments implemented elsewhere in a different example or application, such as in the Summary of the Invention, Detailed Description of the Invention, Claims, and Description of the Figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented herein.

[0047] Figure 1A-H: Synthesis and characterization of Dox-CBD-SA. (A) Schematic diagram of CBD-SA-mediated drug delivery. (B) Synthesis scheme of Dox-CBD-SA. (C) Affinity of CBD-SA and SA for type I and type III collagen was detected by ELISA (Kd values ​​are shown). ND = Not determined due to low signal. The relationship between [concentration] and [signal] is shown in Figure 7. The experiment was repeated twice. (D) The Dox conjugation rate of each protein is shown. Calculated values ​​according to BCA protein quantification (protein) and absorbance at 495nm (Dox) (mean ± SD of three repeated experiments). (E) The Dox release kinetics of Dox-CBD-SA under three different pH conditions were evaluated by fluorescence (495nm excitation, 590nm emission) (n = 3, mean ± SD. The experiment was repeated twice). (F) MMTV-PyMT cells were inoculated and incubated overnight. Dox, Dox-SA or Dox-CBD-SA (red) were added. Cells were also stained with LysoTracker (green). Scale bar = 20 μm. Representative images are shown. The experiment was repeated twice. (G, H) Cytotoxicity of Dox variants on MMTV-PyMT cells or MC38 cells in vitro (n = 6, mean ± SEM). The experiment was repeated twice.

[0048] Figure 2A-D :Dox-CBD-SA showed plasma pharmacokinetics comparable to Dox-SA and higher tumor accumulation than adriamycin and Dox-SA. (A) Adriamycin, Dox-SA or Dox-CBD-SA (5 mg / kg based on Dox) were administered to tumor-free FVB mice by tail vein injection (iv). Plasma was collected at the indicated time points. Plasma concentrations of Dox were measured by fluorescence (mean ± SEM, n = 4 for adriamycin, n = 5 for Dox-SA and Dox-CBD-SA). (B) The plasma half-life of Dox was calculated using a two-phase exponential decay: MFI (t) = Ae -αt +Be -βt. t1 / 2, α, rapid elimination half-life; t1 / 2, β, slow elimination half-life. (Mean ± SEM, n = 4 for subacyl doxorubicin, n = 5 for Dox-SA and Dox-CBD-SA). (C) MMTV-PyMT tumor-bearing mice were treated with subacyl doxorubicin, Dox-SA or Dox-CBD-SA (based on Dox at 4.16 mg / kg). Tumors were harvested at the indicated time points, and the amount of Dox in the tumor was quantified (mean ± SEM. n = 5 per group for 2 hours, n = 7 per group for 24 hours). (D) 100 μg DyLight 488-labeled SA or equimolar amounts of DyLight 488-labeled CBD-SA were intravenously injected into MMTV-PyMT tumor-bearing mice. One hour after injection, tumors were harvested and fluorescence was analyzed by confocal microscopy. Tissues were also stained with DAPI and anti-CD31 antibodies. Scale bar = 100 μm. Representative images of 3 tumors. The experiment was repeated twice. Statistical analysis was performed using ANOVA with Tukey's test. *p<0.05; **p<0.01; NS=not significant.

[0049] Figure 3A-L: Dox-CBD-SA showed enhanced anti-tumor efficacy and lymphocyte infiltration into tumors in the MMTV-PyMT breast cancer model. (A) 5x10 5 MMTV-PyMT cells were inoculated into FVB mice. Adriamycin, Dox-SA or Dox-CBD-SA (based on Dox at 5 mg / kg) were injected intravenously on day 7. The tumor volume before the first mouse death is depicted (mean ± SEM). (B) Survival rate. (CF) Tumor individual growth curve. CR indicates complete response frequency. The experiment was repeated three times. (GL) 5x10 5 MMTV-PyMT cells. Adriamycin, Dox-SA or Dox-CBD-SA (Dox at 5 mg / kg) were intravenously injected on day 7. Lymphocytes in the tumor were extracted on day 14 and then analyzed by flow cytometry. (G) CD45 per mg tumor weight is shown in the figure. + CD8 + CD3 + T cells, (H)CD45 + CD4 + CD3 + T cells and (I)CD45 + NK1.1 + CD3 - The number of NK cells. Bars represent mean ± SEM. (JL) The figure shows: (J) [CD45 + CD8 + CD3+ T cells], (K) [CD45 + CD4 + CD3 + T cells] or (L) [CD45 + NK1.1 + CD3 - The number of NK cells] was compared with [tumor weight]. The experiment was repeated twice. Statistical analysis was performed using (A, H, I) ANOVA and Tukey test or (G) Kruskal-Wallis test followed by Dunn test, or (B) log-rank (Mantel-Cox) test. *p<0.05; **p<0.01; NS=not significant.

[0050] Figure 4A-G : Dox-CBD-SA treatment shows reduced toxicity. Tumor-free FVB mice were administered 20 mg / kg of adriamycin or Dox-CBD-SA (based on Dox) by tail vein injection on day 0. (AD) Plasma cytokine concentrations on day 3. (E) Red blood cell counts on day 6. (F) White blood cell counts on day 3. (G) Spleen weight on day 16. Data represent mean ± SEM. The experiment was repeated twice. Statistical analysis was performed using ANOVA with Tukey's test. *p<0.05;

[0051] **p<0.01; NS=not significant.

[0052] Figure 5A-H : Dox-CBD-SA treatment combined with anti-PD-1 checkpoint inhibitors completely eradicated established MC38 tumors. 5 MC38 cells. Mice were intravenously injected with 5 mg / kg (based on Dox) adriamycin or Dox-CBD-SA on days 6, 9, and 12. αPD-1 was also injected intraperitoneally on days 10 and 13. (A) Experimental timeline. (B) The figure shows the tumor volume before the death of the first mouse (mean ± SEM), CR = complete response. (C) Survival rate. (DG) Tumor individual growth curve. CR indicates the frequency of complete response. (H) On day 60, survivors after Dox-CBD-SA + αPD-1 treatment were injected subcutaneously with 5×10 5 MC38 cells were re-challenged. Untreated mice were also challenged with the same number of cells as the control group. The number of mice that developed palpable tumors is shown. The experiment was repeated twice. Survival curves were statistically analyzed using the log-rank (Mantel-Cox) test. *p<0.05; **p<0.01; NS=not significant.

[0053] Figure 6 :Confirmation of CBD-SA fusion 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 ions. The experiment was repeated twice.

[0054] Figure 7A-B : Binding affinity of CBD-SA to type I and type III collagen. The affinity of CBD-SA to (A) type I collagen and (B) type III collagen was determined by ELISA (n=4, mean ± SD). The relationship between [concentration] and [signal] is shown. The experiment was repeated twice.

[0055] Figure 8 : SDS-PAGE analysis of mouse SA and CBD-SA conjugated with Dox. Dox-SA and Dox-CBD-SA were analyzed by Coomassie blue stained SDS-PAGE. R was reduced; NR was not reduced. Representative images are shown. The experiment was repeated twice.

[0056] Figure 9A-B : Hydrodynamic size. (A) The size of unconjugated CBD-SA, Dox-CBD-SA and Dox-CBD-SA reconstituted after lyophilization was detected by DLS. (B) The size of unconjugated SA and Dox-SA was also detected. The experiment was repeated twice.

[0057] Fig.10 : Binding interface between type III collagen and the von Willebrand factor A3 domain. Crystal structure of the von Willebrand factor (CBD) A3 domain in complex with type III collagen (PDB 4DMU). Images were processed using UCSF Chimera. Lysines are indicated in blue.

[0058] Fig.11 : In vitro release kinetics of Dox from Dox-SA. The release kinetics of Dox from Dox-SA under three different pH conditions were evaluated by fluorescence method (495 nm excitation, 590 nm emission, n=3, mean ± SD. The experiment was repeated twice.

[0059] Figure 12A-B : Plasma pharmacokinetics of SA and CBD-SA labeled with DyLight 800. 200 μg of SA or CBD-SA labeled with DyLight 800 was administered to tumor-free FVB mice by tail vein injection (iv). Plasma was collected at the indicated time points. (A) The signal intensity of each sample was normalized by the average signal intensity of the samples collected 1 minute after injection (mean ± SEM, n = 4). (B) The plasma half-life of labeled SA and CBD-SA was calculated using a two-phase exponential decay: MFI (t) = Ae -t +Be -βt . t1 / 2, β, slow elimination half-life. (mean ± SEM, n = 4). The experiment was repeated once.

[0060] Figure 13A-B : Changes in hematological values ​​in mice receiving 20 mg / kg doxorubicin or Dox-CBD-SA. (A) Hematocrit and (B) hemoglobin concentration on day 6 after injection. The experiment was repeated twice. Statistical analysis was performed using analysis of variance with Tukey's test. **p<0.01; NS=not significant.

[0061] Fig.14 : Histological analysis of major organs after Dox-CBD-SA treatment. Tumor-free FVB mice received Dox-CBD SA (20 mg / kg) on ​​day 0. On day 16, the heart, liver, kidney, and lung were removed and processed to obtain tissue sections (untreated n=7, Dox-CBD-SA treated n=5). Scale bar = 200 μm. Blind evaluation of histology by H&E staining showed no significant abnormalities observed after Dox-CBD-SA treatment. Representative images are shown. The experiment was repeated twice.

[0062] Figures 15A-15B: MC38 tumor re-challenge and weight changes of MC38 tumor-bearing mice during treatment. (A) The figure shows the tumor size (mean ± cell stimulation) of survivors treated with Dox CBD SA + αPD-1 who were re-challenged with MC38 cells. Untreated mice were also challenged with the same number of cells as the control group. The number of mice with palpable tumors is shown. (B) Figure 5 shows the weight changes of mice during treatment (mean ± SEM). The line represents 85% of the initial body weight. The experiment was repeated twice. DETAILED DESCRIPTION

[0063] Since small molecule anticancer drugs are widely distributed in tissues and cause systemic side effects, attempts have been made to modify the drugs to improve their pharmacokinetics and biodistribution. Actively targeting tumor-specific or tumor-associated antigens for drug delivery is a therapeutic strategy. However, this essentially limits the scope of application of cancer and may also lead to acquired drug resistance due to antigen-selective cell targeting and killing, which may be lost due to mutations. Here, the inventors designed CBD-SA (collagen binding domain-serum albumin) to overcome these problems. Unlike other active targeting strategies, CBD-SA does not require prior study of the expression of tumor-associated antigens because collagen is almost ubiquitous in tumors, and CBD enters the tumor stroma through abnormal vascular structures in the tumor microenvironment. Subsequently, CBD-SA binds to exposed collagen and converts the tumor stroma into a reservoir for chemotherapeutic drugs.

[0064] Cardiotoxicity is the main disadvantage of Dox, which limits the lifetime cumulative dose of Dox. Surprisingly, it was found that even 20 mg / kg of Dox-CBD-SA administration did not show any signs of cardiac damage. This shows that Dox pre-conjugated with CBD-SA is less cardiotoxic than free Dox, which causes irreversible damage to cardiac tissue at a cumulative dose of 15 mg / kg in a mouse model. Importantly, the cumulative dose of 15 mg / kg is almost equivalent to the maximum cumulative dose in humans. The efficacy and non-specific toxicity of the conjugate are somewhat surprising and unexpected, because one might assume that CBD-SA might accumulate in undesirable parts of the body, such as the liver, kidneys, and wounds, where collagen might be exposed through porous or leaky endothelium. However, the inventors did not observe pathological damage to the liver and kidneys after administering 20 mg / kg Dox-CBD-SA. Therefore, the present disclosure describes a new strategy for targeting cytotoxic agents to the tumor environment.

[0065] I. Targeting Peptides

[0066] A. Collagen binding domain

[0067] Von Willebrand factor (vWF) is a coagulation factor that binds to type I and type III collagen and the adhesion receptor GPIb on platelets. Upon injury, collagen beneath endothelial cells is exposed to plasma, and the binding of vWF to collagen initiates the thrombotic cascade. The vWF A domain has the highest affinity for collagen among reported proteins / peptides of non-bacterial origin.

[0068] In some embodiments, the polypeptide comprises a collagen binding domain from decorin. In some embodiments, the collagen binding domain comprises a decorin peptide, such as LRELHLNNNC derived from bovine (SEQ ID NO: 11) or LRELHLDNNC derived from human (SEQ ID NO: 12).

[0069] In some embodiments, the collagen binding domain comprises a peptide fragment from human decorin represented by the following amino acid sequence:

[0070]

[0071] In some embodiments, the collagen binding peptide is a peptide from von Willebrand Factor (vWF). The sequence of human vWF comprises:

[0072]

[0073]

[0074] In some embodiments, the peptide is from the vWF A3 domain. The VWF A3 domain is derived from the human sequence, residues 1670 to 1874 (907 to 1111 of mature VWF), and has the following sequence:

[0075] In some embodiments, the ECM-peptide comprises all or a fragment of vWF A3, which is represented by the following amino acid sequence:

[0076]

[0077] In some embodiments, the collagen binding domain comprises a polypeptide having the following sequence:

[0078]

[0079] In some embodiments, the polypeptide comprises a collagen binding domain albumin polypeptide having the sequence:

[0080]

[0081] Typical peptides include all or part of any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 11 to SEQ ID NO: 14. The 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% identity (or any range derivable therein) to a polypeptide of the present disclosure, e.g., to SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 11 to SEQ ID NO: 14.

[0082] B. Linker

[0083] 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 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,

[0084] 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, the linker is located between the cytotoxic agent and the collagen binding domain or the albumin polypeptide. In some embodiments, the linker is located 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 located between the albumin polypeptide and the collagen binding domain. In some embodiments, the linker is located between the IgG Fc domain polypeptide and the collagen binding domain. In some embodiments, the linker includes a bifunctional linker. Linkers such as amino acid or peptide mimetic sequences can be inserted between peptide and / or antibody sequences. The linker can have one or more than one properties, including a flexible conformation, an inability to form an ordered secondary structure, or a hydrophobic or charged property, which can promote or interact with any domain. Examples of amino acids commonly found in flexible protein regions can include Gly, Asn, and Ser. For example, a suitable peptide linker can 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 nearly neutral amino acids, such as Thr and Ala, can also be used in linker sequences. The length of the linker sequence can be varied without significantly affecting the function or activity of the fusion protein (see, e.g., U.S. Patent No. 6,087,329). In a specific aspect, the linkers can be at least, at most, 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 amino acid residues (or any range derivable therein).Embodiments of linkers may also include chemical moieties and conjugating agents such as sulfo-succinimide derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), and disuccinimidyl tartrate (DST). Embodiments of linkers also include linear carbon chains such as C. N (where N = 1 to 100 carbon atoms). In some embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker, or a maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. In some embodiments, the linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Conjugation of sulfo-smcc occurs through a maleimide group that reacts with a sulfhydryl (thiol, -SH), while its sulfo-NHS ester reacts with a primary amine (as seen at lysine and the N-terminus of a protein or peptide). In addition, the linker can be a maleimidocaproyl (mc). In some embodiments, a covalent bond can be achieved by using Traut's reagent.

[0085] C. Albumin

[0086] In some embodiments, the albumin polypeptide is from a mouse. In some embodiments, the albumin polypeptide is from a human.

[0087] In some embodiments, the albumin polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the sequence:

[0088]

[0089] In some embodiments, the albumin polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the sequence:

[0090]

[0091] In some embodiments, the albumin polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the sequence:

[0092]

[0093] In some embodiments, the albumin polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the sequence:

[0094]

[0095] D. Fc domain from human IgG

[0096] Similar to albumin, the Fc domain from human IgG is used to extend drug half-life because the Fc domain also has a cellular recycling system like albumin. In some embodiments, the albumin polypeptide is from human.

[0097] In some embodiments, the hIgG1 Fc polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the following sequence (IGHG1, 99 to 330):

[0098]

[0099] In some embodiments, the hIgG2 Fc polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the following sequence (IGHG2, 99-326):

[0100]

[0101] In some embodiments, the hIgG3 Fc polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the following sequence (IGHG3, 99-376):

[0102]

[0103] In some embodiments, the hIgG4 Fc polypeptide may comprise a polypeptide or fragment 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 (or any range derivable therein) to a polypeptide having the following sequence (IGHG4, 99 to 327):

[0104]

[0105] II. Cytotoxic Agents

[0106] Embodiments of the present disclosure relate to albumin-collagen binding domain conjugates linked to cytotoxic agents. Embodiments of the present disclosure relate to IgG Fc domain-collagen binding domain conjugates linked to cytotoxic agents. Cytotoxic agents include enzyme inhibitors, such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, anthracyclines, vinca drugs, mitomycin, bleomycin, cytotoxic nucleosides, pteridines, diacetylenes, podophyllotoxin, dolastatin, maytansinoid alkaloids, differentiation inducers, and paclitaxel.

[0107] Members of these classes include, for example, paclitaxel, aminopterin, methotrexate, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytarabine, melphalan, vinblastine, leucine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, carminomycin, aminopterin, talithromycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including paclitaxel, taxol retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, enediynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemipteratin, maytansines (including DM1), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and maytansine alkaloids (DM4) and their analogs.

[0108] Cytotoxic agents also include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin, maytansinoids and calicheamicin, hemiacetylcholine. Toxins can exert their cytotoxic and cytostatic effects through mechanisms such as tubulin binding, DNA binding or topoisomerase inhibition.

[0109] Cytotoxic agents such as maytansinoids, dolastatins, auristatins, trichothecenes, calicheamicins, and CC1065, and derivatives of these toxins having toxin activity, may also be used. Other cytotoxic agents include BCNU, streptozotocin, vincristine, and 5-fluorouracil, the family of agents collectively referred to as LL-E33288 complexes described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicins (U.S. Pat. No. 5,877,296). Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, Dianthus caryophyllus proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcumin, crotonin, saponin, gelonin, mitogenins, restrictocin, benomycin, enomycin and trichothecenes. See, e.g., WO 93 / 21232 published on October 28, 1993. In some embodiments, the cytotoxic agent comprises a chemotherapeutic agent as described herein.

[0110] III. Alternative Therapies

[0111] A. Immunotherapy

[0112] In some embodiments, the method includes administering cancer immunotherapy. Cancer immunotherapy (sometimes referred to as immuno-oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapy can be divided into active, passive or hybrid (active and passive). These methods take advantage of the fact that cancer cells often have molecules on their surfaces that can be detected by the immune system, called tumor-associated antigens (TAA); they are usually proteins or other macromolecules (such as carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAA. Passive immunotherapy can enhance existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapy that can be used for the methods of the present disclosure is described below.

[0113] 1. Checkpoint inhibitors and combination therapy

[0114] Embodiments of the present disclosure may include the administration of immune checkpoint inhibitors (also referred to as checkpoint inhibitor therapy), which are further described below.

[0115] a. PD-1, PD-L1, and PD-L2 inhibitors

[0116] PD-1 can play a role in the tumor microenvironment where T cells encounter infection or tumor. Activated T cells upregulate PD-1 and continue to express it in peripheral tissues. Cytokines such as IFN-γ induce the expression of PD-L1 on epithelial cells and tumor cells. PD-L2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of peripheral effector T cells and prevent excessive damage to tissues during immune responses. The inhibitors disclosed herein can block one or more than one function of PD-1 and / or PD-L1 activity.

[0117] 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.

[0118] In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 inhibitor is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, a PD-L2 inhibitor is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The inhibitor can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Typical 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, for example, as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0119] 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 nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or 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, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambolizumab, and SCH-900475, are anti-PD-1 antibodies 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. Other PD-1 inhibitors include MEDI0680 (also known as AMP-514) and REGN2810.

[0120] 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 some aspects, the immune checkpoint inhibitor is a PD-L2 inhibitor, such as rHIgM12B7.

[0121] 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 with the above-mentioned antibodies for binding and / or binding to the same epitope on PD-1, PD-L1 or PD-L2. In another embodiment, the antibody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% (or any range derivable therein) of variable region amino acid sequence identity with the above-mentioned antibodies.

[0122] b. CTLA-4, B7-1 and B7-2

[0123] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The gene bank accession number of the complete cDNA sequence of human CTLA-4 is L15006. CTLA-4 is present on the surface of T cells and acts as a "close" switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA-4 is a member of the immunoglobulin superfamily, expressed on the surface of helper T cells, and transmits inhibitory signals to T cells. CTLA-4 is similar to the T cell co-stimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also present in regulatory T cells and may be important for their function. Activation of T cells through T cell receptors and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitors disclosed herein can block one or more than one function of CTLA-4, B7-1 and / or B7-2 activity. In some embodiments, the inhibitor blocks the interaction between CTLA-4 and B7-1. In some embodiments, the inhibitor blocks the interaction between CTLA-4 and B7-2.

[0124] 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.

[0125] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be produced using methods known in the art. Alternatively, anti-CTLA-4 antibodies known in the art can be used. For example, anti-CTLA-4 antibodies are disclosed in: US 8119129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675206, also known as tremelimumab; formerly known as tesimumab), U.S. Patent No. 6207156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the above-mentioned published patents are incorporated herein by reference. Antibodies that compete with any of these antibodies known in the art 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 U.S. Patent No. 8,017,114; all of which are incorporated herein by reference.

[0126] Another anti-CTLA-4 antibody used as a checkpoint inhibitor in the methods and compositions of the present disclosure is ipilimumab (also known as 10D1, MDX-010, MDX-101, and ) or antigen-binding fragments and variants thereof (see, e.g., WO0 1 / 14424).

[0127] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tesimumab or ipilimumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tesimumab or ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of tesimumab or ipilimumab. In another embodiment, the antibody competes with the above-mentioned antibodies for binding and / or binding to the same epitope on PD-1, B7-1, or B7-2. In another embodiment, the antibody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any range derivable therein) of variable region amino acid sequence identity with the above-mentioned antibodies.

[0128] 2. Inhibition of co-stimulatory molecules

[0129] In some embodiments, immunotherapy includes inhibitors of costimulatory molecules. In some embodiments, inhibitors include 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.

[0130] 3. Dendritic cell therapy

[0131] Dendritic cell therapy stimulates an anti-tumor response by having dendritic cells present tumor antigens to lymphocytes, which activate dendritic cells, prompting them to kill other cells presenting the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment, they help target cancer antigens. An example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0132] One way to induce dendritic cells to present tumor antigens is to vaccinate with autologous tumor lysates or short peptides (small parts of proteins that correspond to protein antigens on cancer cells). These peptides are often combined with adjuvants (highly immunogenic substances) to increase immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0133] Dendritic cells can also be activated in vivo by causing tumor cells to express GM-CSF. This can be achieved by genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0134] Another strategy is to remove dendritic cells from the patient's blood and activate them in vitro. 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 broken down tumor cells). These cells (with optional adjuvants) are injected and elicit an immune response.

[0135] 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 and can induce dendritic cells to mature and provide immunity to tumors.

[0136] 4. CAR-T cell therapy

[0137] A chimeric antigen receptor (CAR, also called a chimeric immunoreceptor, chimeric T-cell receptor, or artificial T-cell receptor) is an engineered receptor that attaches new specificities to immune cells to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. These receptors are called chimeras because they are made of a fusion of parts from different sources. CAR-T cell therapy refers to treatments that use such transformed cells for cancer treatment.

[0138] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen binding and T cell activation functions. The basic premise of CAR-T cells is to artificially generate T cells that target cancer cell markers. Scientists can take T cells from a person, genetically modify them, and then put them back into the patient to allow them to attack cancer cells. Once a T cell is engineered into a CAR-T cell, it acts as a "living drug." CAR-T cells establish a connection between an extracellular ligand recognition domain and an intracellular signaling molecule, which in turn activates the T cell. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important 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 depends on the choice of targeting molecule.

[0139] Typical CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtageneciloleucel (Yescarta). In some embodiments, CAR-T therapies target CD19.

[0140] 5. Cytokine therapy

[0141] Cytokines are proteins produced by various cells present in tumors. They can modulate immune responses. Tumors often use them to grow and reduce immune responses. These immunomodulatory effects make them useful as drugs to stimulate immune responses. Two commonly used cytokines are interferons and interleukins.

[0142] Interferons are produced by the immune system. They are usually involved in antiviral responses but can also be used to treat cancer. They are divided into three types: type I (IFNα and IFNβ), type II (IFNγ), and type III (IFNλ).

[0143] Interleukins have a certain effect on the immune system. IL-2 is a typical interleukin cytokine therapy.

[0144] 6. Adoptive T cell therapy

[0145] Adoptive T cell therapy is a form of passive immunization through the infusion of T cells (adoptive cell transfer). They are present in the blood and tissues and are usually activated when they detect a foreign pathogen. Specifically, T cells become activated when their surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These cells can be infected cells or antigen presenting cells (APCs). They are present in normal tissues and tumor tissues, where they are called tumor infiltrating lymphocytes (TILs). They are activated by APCs, such as dendritic cells, which present tumor antigens. Although these cells can attack tumors, the environment within tumors is highly immunosuppressive, preventing immune-mediated tumor death.

[0146] A variety of methods have been developed to generate and obtain tumor-targeted T cells. T cells specific for tumor antigens can be removed from tumor samples (TILs) or filtered from the blood. They are then activated and incubated in vitro and re-infused. Activation can be done by gene therapy or by exposing T cells to tumor antigens.

[0147] It is contemplated that cancer treatment may exclude any cancer treatment described herein. In addition, embodiments of the present disclosure include patients who have previously been treated with a therapy described herein, patients who are currently being treated with a therapy described herein, or patients who have not yet been treated with a therapy described herein. In some embodiments, the patient is a patient who has been determined to be resistant to a therapy described herein. In some embodiments, the patient is a patient who has been determined to be sensitive to a therapy described herein.

[0148] B. Oncolytic viruses

[0149] In some embodiments, additional therapies include oncolytic viruses. An oncolytic virus is a virus that preferentially infects and kills cancer cells. When infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought to not only directly destroy tumor cells, but also stimulate the host's anti-tumor immune response for long-term immunotherapy.

[0150] C. Polysaccharide

[0151] In some embodiments, the additional therapy includes polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can upregulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophages, NK cells, T cells, and immune system cytokines, and have been studied in clinical trials as immune adjuvants.

[0152] D. Neoantigens

[0153] In some embodiments, additional therapies include administration of neoantigens. Many tumors express mutations. These mutations may generate new targetable antigens (neoantigens) for T cell immunotherapy. + The presence of T cells is higher in tumors with a high mutational burden. In many human tumors, the levels of transcripts associated with the cytolytic activity of natural killer cells and T cells are positively correlated with mutational burden.

[0154] E. Chemotherapy

[0155] In some embodiments, the additional therapy comprises chemotherapy. Suitable chemotherapeutic drug classes include (a) alkylating agents, such as nitrogen mustards (e.g., chloroethylamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimine and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorzoxazone, streptozotocin) and triazines (e.g., dacarbazine), (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine In some embodiments, the present invention relates to chemotherapeutic agents 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) other agents such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylethylenediazine derivatives (e.g., procarbazine) and adrenocortical inhibitors (e.g., paclitaxel and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0156] 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 cannot be absorbed orally and must therefore be administered by other routes, such as intravenous, subcutaneous, intratumoral, or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and effective doses used in clinical applications include about 15 mg / m every three weeks. 2 About 20 mg / m 2 This lasts for 5 days, with a total of three courses contemplated in certain embodiments.In some embodiments, the amount of cisplatin delivered to a cell and / or subject with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount delivered when cisplatin is used alone.

[0157] Other suitable chemotherapeutic agents include anti-microtubule agents such as paclitaxel and doxorubicin hydrochloride. The combination of the Egr-1 promoter / TNF1 construct delivered by an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and / or TNF-α, indicating that combination therapy of the construct and doxorubicin overcomes resistance to doxorubicin and TNF-α.

[0158] Doxorubicin is poorly absorbed and is preferably administered intravenously. In certain embodiments, an appropriate intravenous dose for adults includes about 60 mg / m 2 Up to about 75 mg / m 2, about 21 days apart, or about 25 mg / m 2 To about 30mg / m 2 , for 2 or 3 consecutive days, repeated at intervals of about 3 to about 4 weeks, or about 20 mg / m 2 , once a week. Elderly patients should use the lowest dose when previous chemotherapy or tumor bone marrow infiltration has caused previous bone marrow suppression, or when used in combination with other myelosuppressive drugs.

[0159] Nitrogen mustards are another suitable chemotherapeutic agent that can be used in the methods of the present disclosure. Nitrogen mustards can include, but are not limited to, ketamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-myosin), and chlorambucil. Cyclophosphamide ( Available from Mead Johnson, (available from Adria) is another suitable chemotherapeutic agent. Suitable oral dosages for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous dosages include, for example, about 40 mg / kg to about 50 mg / kg taken in divided doses over a period of about 2 days to about 5 days, or about 10 mg / kg to about 15 mg / kg about every 7 days to about every 10 days, or about 3 mg / kg to about 5 mg / kg twice a week, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse gastrointestinal reactions, intravenous routes are preferred. The drug is sometimes also administered intramuscularly, infiltrated, or into a body cavity.

[0160] Additional suitable chemotherapeutic agents include pyrimidine analogs such as cytarabine (Ara-C), 5-fluorouracil (5-FU), and floxuridine (fluorodeoxyuridine; FudR). 5-FU can be administered at about 7.5 mg / m 2 About 1000 mg / m 2 Any dose between 5 and 10 is administered to a subject. In addition, the 5-FU administration regimen may be for a variety of time periods, such as up to six weeks, or as determined by a person of ordinary skill in the present invention.

[0161] Gemcitabine diphosphate ( Eli Lilly & Co., "gemcitabine") is another suitable chemotherapeutic agent that is recommended for the treatment of advanced and metastatic pancreatic cancer and therefore will also be useful in these cancers as disclosed herein.

[0162] The amount of the chemotherapeutic agent delivered to the patient can be variable. In a suitable embodiment, when chemotherapy is applied together with the construct, the amount of the chemotherapeutic agent applied can effectively cause the host's cancer to stop or disappear. In other embodiments, the amount of the chemotherapeutic agent applied can be less than 2 times to 10000 times of the effective dose of the chemotherapeutic agent. For example, the amount of the chemotherapeutic agent applied may be less than about 20 times, less than about 500 times, or even less than about 5000 times the effective dose of the chemotherapeutic agent. The desired therapeutic activity of the chemotherapeutic agent of the present disclosure combined with the construct can be tested in vivo, and the effective dose can be determined. For example, such compounds can be tested in a suitable animal model system before human testing, including but not limited to rats, mice, chickens, cattle, monkeys, rabbits, etc. In vitro testing can also be used to determine suitable combinations and dosages, as described in the embodiments.

[0163] F. Radiotherapy

[0164] In some embodiments, the additional therapy or prior therapy includes radiation, such as ionizing radiation. As used herein, "ionizing radiation" refers to radiation containing particles or photons that have sufficient energy or can generate sufficient energy through nuclear interactions to produce ionization (gain or loss of electrons). Typical and preferred ionizing radiation is x-radiation. Methods of delivering x-radiation to target tissues or cells are well known in the art.

[0165] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 26 Gy, 27 Gy, 18 Gy, 19 Gy, 30 Gy, 31 Gy, 32 Gy, 33 Gy, 34 Gy, 35 Gy, 36 Gy, 37 Gy, 38 Gy, 39 Gy, 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, or 40 Gy (or any range derivable therein). In some embodiments, ionizing radiation is administered for at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any range derivable therein). When more than one dose is administered, the doses may be spaced about 1 hour, 4 hours, 8 hours, 12 hours, or 24 hours, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, or 16 weeks, or any range derivable therein.

[0166] In some embodiments, the amount of IR can be expressed as a total dose of IR, which is then 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 Gy to 90 Gy, administered in 20 to 60 fractionated doses, each of 2 Gy to 3 Gy each. In some embodiments, the total dose of IR is at least, up to, 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 fractionated doses of at least, at most, or exactly 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 12 Gy, 14 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, or 50 Gy (or any range derivable therein).In some embodiments, at least, at most, 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 , 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 divided doses (or any range derivable therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 divided doses (or any range derivable therein) are administered daily. In some embodiments, at least, at most, 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 divided doses (or any range derivable therein) are administered weekly.

[0167] G. Surgery

[0168] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, 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 combination with other therapies, such as the treatment of the present embodiment, 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 also include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0169] After removing part or all of the cancer cells, tissues or tumors, a cavity can be formed in the body. Treatment can be completed by perfusion, direct injection or local application of other anticancer therapies. Such treatments can be repeated, for example, every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, or every 1 week, 2 weeks, 3 weeks, 4 weeks and 5 weeks or every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. These treatments can also have different dosages.

[0170] H. Other drugs

[0171] It is expected that other agents can be used in combination with certain aspects of this embodiment to improve the therapeutic effect of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferation cells to apoptosis inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-overproliferation effect on neighboring overproliferation cell groups. In other embodiments, cell inhibitors or differentiation agents can be used in combination with certain aspects of this embodiment to improve the anti-overproliferation efficacy of treatment. Cell adhesion inhibitors are expected to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is also expected that other agents that increase the sensitivity of overproliferation cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of this embodiment to improve the therapeutic effect.

[0172] IV. Nucleic Acids

[0173] In certain embodiments, there is a recombinant nucleic acid encoding a polypeptide described herein.

[0174] As used in the present application, the term "polynucleotide" refers to a recombinant or isolated nucleic acid molecule that does not contain total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids with a length of 100 or less than 100 residues), recombinant vectors including, for example, plasmids, cosmids, phages, viruses, etc. In some aspects, polynucleotides include regulatory sequences that are substantially separated from their naturally occurring genes or protein coding sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA or synthetic), its analogs or its combination. Other coding or non-coding sequences can but not necessarily be present in polynucleotides.

[0175] In this regard, the term "gene", "polynucleotide" or "nucleic acid" is used to refer to a coded protein, polypeptide or peptide (including correct transcription, post-translational modification, or positioning). As will be appreciated by those skilled in the art, the term includes genomic sequences, expression cassettes, cDNA sequences and smaller engineered nucleic acid fragments that express or can be suitable for expressing proteins, polypeptides, domains, peptides, fusion proteins and mutants. Nucleic acids encoding all or part of a polypeptide may comprise a continuous nucleic acid sequence encoding all or part of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid sequence comprising a slightly different, but still encoding the same or essentially similar protein (see above).

[0176] In a specific embodiment, there is an isolated nucleic acid fragment and a recombinant vector comprising a nucleic acid sequence encoding a polypeptide (e.g., a polymerase, an RNA polymerase, one or more truncated polymerase domains or an interacting component as a polypeptide), which, when the interacting components interact, drives gene transcription dependent on the polymerase activity of the polymerase domain. The term "recombinant" can be used in conjunction with the name of a polypeptide or a specific polypeptide, which generally refers to a polypeptide produced by a nucleic acid molecule that has been manipulated in vitro or a replication product of the molecule.

[0177] Regardless of the length of the coding sequence itself, the nucleic acid fragment can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding fragments, etc., so that their total length can vary greatly. It is therefore expected that nucleic acid fragments of almost any length can be used, and the total length is preferably limited by the ease of preparation and use in the expected recombinant nucleic acid scheme. In some cases, the nucleic acid sequence can encode a polypeptide sequence with additional heterologous coding sequences, such as to allow purification, transportation, secretion, post-translational modification of the polypeptide, or for therapeutic benefits (such as targeting or therapeutic efficacy). As described above, tags or other heterologous polypeptides can be added to the modified polypeptide coding sequence, wherein "heterologous" refers to a polypeptide different from the modified polypeptide.

[0178] In certain embodiments, there are polynucleotide variants with substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity compared to the polynucleotide sequences provided herein using methods described herein (e.g., BLAST analysis using standard parameters), including all values ​​and ranges therebetween. In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% and above identity to the amino acid sequence described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0179] A. Carrier

[0180] The polypeptide can be encoded by a nucleic acid molecule. The nucleic acid molecule can be in the form of a nucleic acid vector. The term "vector" is used to refer to a vector nucleic acid molecule, in which a heterologous nucleic acid sequence can be inserted to be introduced into a cell, where it can be replicated and expressed. The nucleic acid sequence can be "heterologous", which means that it is unrelated to the cell into which the vector is introduced or the nucleic acid incorporated therein, including sequences homologous to sequences in the cell or nucleic acid, but located at a position not usually found in the host cell or nucleic acid. 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 have the ability to construct vectors by standard recombinant techniques (e.g., Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). Vectors can be used in host cells to produce polymerases, RNA polymerases, one or more than one truncated polymerase domains, or fusions, attachments, or interactions components connected to one or more than one truncated RNA polymerase domains.

[0181] The term "expression vector" refers to a vector comprising a nucleic acid sequence encoding at least a portion of a gene product that is capable of transcription. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide or peptide. An expression vector may contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possible translation of an operably linked coding sequence in a specific host organism. In addition to control sequences for transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that have other functions and are described herein.

[0182] B. Cells

[0183] The present disclosure provides methods for modifying a target RNA of interest, particularly in prokaryotic cells, eukaryotic cells, tissues, organs or organisms, more particularly in mammalian cells, tissues, organs or organisms. The target RNA may be contained in a nucleic acid molecule within a cell. In some embodiments, the target RNA is in a eukaryotic cell, such as a mammalian cell or a plant cell. Mammalian cells are mostly cells of humans, non-human primates, cattle, pigs, rodents or mice. The cell may be a non-mammalian eukaryotic cell, such as poultry, fish or shrimp. The plant cell may be a crop plant, such as cassava, corn, sorghum, wheat or rice. The plant cell may also be algae, trees or vegetables. The regulation of RNA induced in cells by the methods, systems and compositions of the present disclosure may cause cells and the offspring of cells to be altered to improve the production of biological products such as antibodies, starch, alcohol or other desired cell products. The regulation of RNA induced in cells may cause cells and the offspring of cells to include changes in the biological products produced by the changes.

[0184] Mammalian cells can be human or non-human mammals, such as cells of primates, cattle, sheep, pigs, dogs, rodents, Leporidae such as monkeys, cattle, sheep, pigs, dogs, rabbits, rats or mice. Cells can be non-mammalian eukaryotic cells, such as cells of poultry (e.g., chickens), vertebrates (e.g., salmon) or shellfish (e.g., oysters, clams, lobsters, shrimp). Cells can also be plant cells. Plant cells can be monocots or dicots or crops or cereals, such as cassava, corn, sorghum, soybeans, wheat, oats or rice. The plant cell can also be an algae, a tree, or a production plant, a fruit or a vegetable (e.g., a citrus tree, such as 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 nightshade; a Brassica; a lettuce; a spinach; a pepper; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, cocoa, etc.).

[0185] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably. All of these terms also include their progeny, i.e., any and all progeny. It should be understood that all progeny may not be identical due to intentional or unintentional 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 a vector. Host cells can and have been used as receptors for vectors or viruses. Host cells can be "transfected" or "transformed," which refers to the process by which exogenous nucleic acids, such as recombinant protein coding sequences, are transferred or introduced into host cells. Transformed cells include the primary subject cell and its progeny.

[0186] Some vectors may employ control sequences that allow them to be replicated and / or expressed in prokaryotic and eukaryotic cells. Those skilled in the art will further appreciate the conditions for incubating all of the above host cells to maintain them and allow for vector replication. Techniques and conditions that allow for large-scale production of vectors and production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins or peptides are also understood and known.

[0187] C. Expression System

[0188] There are many up to systems that at least comprise some or all of the above-mentioned compositions discussed above. Prokaryotic and / or eukaryotic systems can be used together with embodiments to produce nucleic acid sequences, or their homologous polypeptides, proteins and peptides. For example, vectors, fusion proteins, RNA hairpin binding proteins, RNA targeting molecules, RNA regulatory domains and accessory proteins disclosed herein can utilize expression systems, such as inducible or constructed expression systems. Many such systems are widely available commercially.

[0189] Insect cell / baculovirus systems can produce high levels of protein expression of heterologous nucleic acid fragments, such 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 used, for example, under the name 2.0 From Purchased and sold under the name BACPACKGEN TM BACULOVIRUSEXPRESSION SYSTEMfrom Buy.

[0190] In addition to the disclosed expression systems, other examples of expression systems include A COMPLETE CONTROL inducible mammalian expression system, which involves a synthetic ecdysone inducible receptor, or its pET expression system, an E. coli expression system. Another embodiment of the inducible expression system can be obtained from Get it, it carries T-REX TM (tetracycline regulated expression) system, which is an inducible mammalian expression system using the full-length CMV promoter. Also provided is 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 pastoris. One skilled in the art will know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein or peptide.

[0191] V. Protein Composition

[0192] The polypeptides or polynucleotides of the present disclosure, e.g., polypeptides or polynucleotides comprising or encoding an albumin polypeptide linked to a collagen binding domain, can include 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 acids or nucleic acid substitutions or substitutions with SEQ ID NOs: 1 to SEQ ID NOs: at least, or at most, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 6, 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 99, 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 or 2000 or more or any range derivable therein of at least 60%, 61% or more of consecutive amino acids or nucleic acids. %, 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.

[0193] The polypeptides or polynucleotides of the present disclosure, such as polypeptides or polynucleotides comprising or encoding an albumin polypeptide linked to a collagen binding domain, may include SEQ ID NO: 1 to SEQ ID NO: 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 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162 62, 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 6, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000, 1500, 2000 or more than 2000 consecutive amino acids or any range derivable therein.

[0194] In some embodiments, the polypeptide comprises SEQ ID NO: 1 to SEQ ID NO: 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 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 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 6、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573 73, 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).

[0195] In some embodiments, the polypeptide comprises SEQ ID NO: 1 to 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 17, 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 (or any range derivable therein) consecutive amino acids.

[0196] In some embodiments, the polypeptide comprises SEQ ID NO: 1 to SEQ ID NO: 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, 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 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 , 164, 165, 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 (or any range derivable therein) consecutive amino acids, and starting from SEQ ID NO: 1 to SEQ ID NO: 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 4, 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 6, 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, 1 92、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 1, 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 93, 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.

[0197] In some embodiments, the polypeptide comprises SEQ ID NO: 1 to SEQ ID NO: NO:18 of 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 2, 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, 18, 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 (or any range derivable therein) consecutive amino acids, which are at least, at most, or exactly the same as SEQ ID NO: 1 to SEQ ID NO: Any one of NO:18 is 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.

[0198] The polypeptides of the present disclosure can include at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 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、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 6, 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 65、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) permutations.

[0199] The substitution may be SEQ ID NO: 1 to SEQ ID 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th 8th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th 39th, 140th, 141st, 142nd, 143rd, 144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th,180th, 181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 194th, 195th, 196th, 197th, 198th, 199th, 200th 201st, 202nd, 203rd, 204th, 205th, 206th, 207th, 208th, 209th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 218th, 219th, 220th, 221st 1st, 222nd, 223rd, 224th, 225th, 226th, 227th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 255th, 256th, 257th, 258th, 259th, 260th, 261st, 262nd, No. 263, No. 264, No. 265, No. 266, No. 267, No. 268, No. 269, No. 270, No. 271, No. 272, No. 273, No. 274, No. 275, No. 276, No. 277, No. 278, No. 279, No. 280, No. 281, No. 282, No. 283 284th, 285th, 286th, 287th, 288th, 289th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th 4th, 305th, 306th, 307th, 308th, 309th, 310th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 319th, 320th, 321st, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th, 336th, 337th, 338th, 339th, 340th, 341st, 342nd, 343rd, 344th, 345th,No. 346, No. 347, No. 348, No. 349, No. 350, No. 351, No. 352, No. 353, No. 354, No. 355, No. 356, No. 357, No. 358, No. 359, No. 360, No. 361, No. 362, No. 363, No. 364, No. 365, No. 366 367th, 368th, 369th, 370th, 371st, 372nd, 373rd, 374th, 375th, 376th, 377th, 378th, 379th, 380th, 381st, 382nd, 383rd, 384th, 385th, 386th, 387th 7th, 388th, 389th, 390th, 391st, 392nd, 393rd, 394th, 395th, 396th, 397th, 398th, 399th, 400th, 401st, 402nd, 403rd, 404th, 405th, 406th, 407th, 408th, 409th, 410th, 411th, 412th, 413th, 414th, 415th, 416th, 417th, 418th, 419th, 420th, 421st, 422nd, 423rd, 424th, 425th, 426th, 427th, 428th, No. 429, No. 430, No. 431, No. 432, No. 433, No. 434, No. 435, No. 436, No. 437, No. 438, No. 439, No. 440, No. 441, No. 442, No. 443, No. 444, No. 445, No. 446, No. 447, No. 448, No. 449 450th, 451st, 452nd, 453rd, 454th, 455th, 456th, 457th, 458th, 459th, 460th, 461st, 462nd, 463rd, 464th, 465th, 466th, 467th, 468th, 469th, 470th 0th, 471st, 472nd, 473rd, 474th, 475th, 476th, 477th, 478th, 479th, 480th, 481st, 482nd, 483rd, 484th, 485th, 486th, 487th, 488th, 489th, 490th, 491st, 492nd, 493rd, 494th, 495th, 496th, 497th, 498th, 499th, 500th, 501st, 502nd, 503rd, 504th, 505th, 506th, 507th, 508th, 509th, 510th, 511th,512th, 513th, 514th, 515th, 516th, 517th, 518th, 519th, 520th, 521st, 522nd, 523rd, 524th, 525th, 526th, 527th, 528th, 529th, 530th, 531st, 532nd, 533rd, 534th, 535th, 536th, 537th, 538th, 539th, 540th, 541st, 542nd, 543rd, 544th, 545th, 546th, 547th, 548th, 549th, 550th, 551st, 552nd, 553rd, 554th, 555th, 556th, 557th, 558th, 559th, 560th, 561st, 562nd, 563rd, 564th, 565th, 566th, 567th, 568th, 569th, No. 570, No. 571, No. 572, No. 573, No. 574, No. 575, No. 576, No. 577, No. 578, No. 579, No. 580, No. 581, No. 582, No. 583, No. 584, No. 585, No. 586, No. 587, No. 588, No. 589, No. 590, No. 591, No. 592, No. 593, No. 594, No. 595, No. 596, No. 597, No. 598, The amino acid or nucleic acid at position 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).

[0200] 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 , 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、400, 500, 550, 1000 or more amino acids (or any range derivable therein).

[0201] Substitution variants generally include an exchange of one amino acid with another amino acid at one or more than one site within a protein, and can be designed to modulate one or more than one property of a polypeptide, with other functions or properties retained or lost. The substitution can be conservative, i.e., one amino acid is replaced with an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine 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, thereby affecting the function or activity of the polypeptide.Non-conservative changes generally involve replacing a residue with a chemically different residue, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa.

[0202] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also contemplated that bacteria containing such variants can be implemented in compositions and methods. Thus, there is no need to isolate the protein.

[0203] The term "functionally equivalent codons" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also to codons that encode biologically equivalent amino acids.

[0204] It is also understood that the amino acid and nucleic acid sequences may include additional residues, such as additional N or C terminal amino acids, or 5' or 3' sequences, respectively, but are substantially as listed below one of the sequences disclosed herein, as long as the sequence meets the above criteria, including the maintenance of biological protein activity involved in protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, include various non-coding sequences located on both sides of the 5' or 3' end of the coding region.

[0205] The following is a discussion based on changing the amino acids of a protein to create an equivalent or even improved second generation molecule. For example, certain amino acids can replace other amino acids in a protein structure without significantly losing the ability to bind to the interaction. Structures such as enzyme catalytic domains or interacting components can have amino acid substitutions to maintain this function. Since it is the interaction ability and properties of a protein that determine the biological functional activity of the protein, certain amino acid substitutions can be made in the protein sequence and its potential DNA coding sequence, but still produce proteins with similar properties. Therefore, the inventors expect that various changes can be made in the DNA sequence of a gene without significantly losing its biological utility or activity.

[0206] In other embodiments, the function of the polypeptide is changed by introducing one or more than one substitution. For example, certain amino acids can replace other amino acids in the protein structure to change the interaction binding ability of the interaction component. Structures such as protein interaction domains, nucleic acid interaction domains, and catalytic sites can have substituted amino acids to change this function. Since the interaction ability and properties of the protein determine the biological function activity of the protein, certain amino acid substitutions can be made in the protein sequence and its potential DNA coding sequence, but proteins with different characteristics will still be produced. Therefore, the inventors expect that various changes can be made in the DNA sequence of the gene, and significantly change its biological utility or activity.

[0207] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathicity of amino acids contributes to the generation of the secondary structure of a protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0208] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. U.S. Pat. No. 4,554,101 (incorporated herein by reference) indicates that the greatest local average hydrophilicity of a protein (controlled by the hydrophilicity of its neighboring amino acids) correlates with the biological properties of the protein. It is understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still produce a biologically and immunologically equivalent protein.

[0209] As noted above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Typical substitutions that take into account various of the foregoing characteristics are well known and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0210] In specific embodiments, all or part of the proteins described herein may also be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and may be used according to known protocols. For example, see Stewart and Young (1984); Tam et al., (1983); Merrifield (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference. Alternatively, recombinant DNA techniques may be employed in which a nucleotide sequence encoding a peptide or polypeptide is inserted into an expression vector, transformed or transfected into a suitable host cell and incubated under conditions suitable for expression.

[0211] One embodiment includes using gene transfer to cells, including microorganisms, to produce and / or present proteins. The gene for the target protein can be transferred into a suitable host cell, and the cell is then incubated under suitable conditions. Nucleic acids encoding almost any polypeptide can be used. The production of recombinant expression vectors and the elements contained therein are discussed herein. Alternatively, the protein to be produced can be an endogenous protein that is typically synthesized by the cells used for protein production.

[0212] VI. Combination therapy

[0213] The compositions and related methods of the present disclosure, in particular the administration of polypeptides comprising an albumin polypeptide or an IgG Fc domain polypeptide linked to a collagen binding domain, may also be administered in combination with additional therapies, such as those described herein or in combination with other conventional therapies known in the art.

[0214] Therapeutic compositions and treatments disclosed herein can be implemented at intervals of several minutes to several weeks, before another treatment or medicament, simultaneously with another treatment or medicament and / or after another treatment or medicament. In embodiments where medicaments are applied to cells, tissues or organisms respectively, it is generally ensured that the time is not expired between each delivery, so that the therapeutic agent can still play a favorable combination effect on cells, tissues or organisms. For example, in such a case, it is expected that two, three, four or more than four medicaments or treatment methods can be used to contact cells, tissues or organisms substantially simultaneously (that is, in less than about one minute). In other aspects, one or more therapeutic agents or treatments may be 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, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours before and / or after administration of another therapeutic agent or treatment. , 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 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.

[0215] Various combinations of therapeutic agents and treatments can be used. Non-limiting examples of such combinations are shown below, where the therapeutic agent (such as a composition disclosed herein) is the "composition" and the second agent (such as an additional agent, chemotherapeutic agent, or checkpoint inhibitor as described herein or known in the art) is the "additional agent".

[0216] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0217] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0218] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0219] In some embodiments, more than one course of treatment may be employed. It is contemplated that multiple courses of treatment may be administered.

[0220] VII. Treatment Methods

[0221] The current methods and compositions are directed to methods of treating cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is a non-lymphoid cancer. In some embodiments, the cancer is breast cancer or colon cancer.

[0222] The compositions of the present disclosure can be used for in vivo, in vitro or ex vivo administration. The administration route 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 administration. In some embodiments, the composition is administered directly into cancerous tissue or lymph nodes.

[0223] As used herein, "tumor" 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 disease," "proliferative disease," and "tumor" are not mutually exclusive herein.

[0224] Cancers suitable for treatment include, but are not limited to, tumors of all types, locations, sizes, and characteristics. The methods and compositions disclosed herein are suitable for treating, for example, pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendix cancer, astrocytoma, cerebellar or brain basal cell carcinoma in children, bile duct cancer, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma brain tumor, brain astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive tumor neuroectodermal tumor brain tumor, visual pathway and hypothalamic glioma, breast cancer, specific breast cancers such as ductal carcinoma in situ, invasive ductal carcinoma, tubular breast carcinoma, medullary breast cancer carcinoma, mucinous breast carcinoma, papillary carcinoma breast, cribriform breast carcinoma, invasive lobular carcinoma, inflammatory breast cancer in situ, lobular carcinoma in situ, male breast cancer, Paget's disease of the nipple, phyllodes tumor of the breast, recurrent and / or metastatic breast cancer, carcinoma, luminal A or B breast cancer, triple negative / basal-like breast cancer, and HER2-enriched breast cancer, lymphoma, bronchial adenoma / carcinoid, tracheal carcinoma, Burkitt's lymphoma, carcinoid, childhood carcinoid, gastrointestinal cancer of unknown primary, central nervous system lymphoma, primary cerebellar astrocytoma, childhood brain astrocytoma / glioblastoma, childhood cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, , cutaneous T-cell lymphoma, , desmoplastic small round cell tumor, , endometrial cancer, , ependymoma, , esophageal cancer, , Ewing's, , extragonadal germ cell tumors in children, , extrahepatic bile duct cancer, eye cancer, retinoblastoma, gallbladder cancer, gastric cancer (gastric cancer), gastrointestinal carcinoma-like tumors, gastrointestinal stromal tumors (GIST), germ cell tumors: extracranial, extragonadal, or ovarian, gestational trophoblastic tumor, brain stem glioma, glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart disease, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood intraocular melanoma tumors, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphocytic leukemia, acute myeloid (also called acute myeloid leukemia) leukemia, chronic lymphocytic (leukemia, chronic myeloid leukemia, hairy cell lip and oral cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (old classification of all lymphomas except Hodgkin's disease) lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma,Medulloblastoma in children, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma in adults, mesothelioma in children, metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia in adults, acute myeloid leukemia in children, multiple myeloma, chronic myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant tumors, fibrohistiocytoma of bone, ovarian cancer, epithelial ovarian cancer (surface epithelial stromal tumor), ovarian germ cell tumor, ovarian tumor of low malignant potential, pancreatic cancer, islet cell sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma, and supratentorial primitive Neuroectodermal tumors, childhood pituitary adenoma, plasmacytoma / multiple myeloma, pleuropulmonary 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, childhood salivary gland carcinosarcoma, Ewing family tumors, Kaposi sarcoma, soft tissue sarcoma, cervical syndrome sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer, Merkel cell small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck with occult primary, metastatic gastric cancer, supratentorial primitive neuroectodermal tumor, childhood T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma, childhood thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, childhood vulvar cancer and Wilms tumor (kidney cancer). ,

[0225] VIII. Pharmaceutical Compositions and Methods

[0226] In some embodiments, a pharmaceutical composition is administered to a subject. Various aspects relate to administering an effective amount of a composition to a subject. In some embodiments, a composition comprising an inhibitor may be administered to a subject or patient to treat cancer or reduce the size of a tumor. In addition, such compounds may be administered in combination with other cancer therapies.

[0227] The composition can be formulated for parenteral administration, for example, for injection by intravenous, catheter injection, intra-arterial injection, intramuscular, subcutaneous or even intraperitoneal routes. Typically, such compositions can be prepared as injections, such as liquid solutions or suspensions; they can also be prepared in solid forms, which are used to prepare solutions or suspensions to which liquids are added before injection; and the preparations can also be emulsified. According to the present disclosure, the preparation of such preparations is known to those skilled in the art. Other routes of administration include intratumoral, peritumoral, intralymphatic, injection into cancerous tissue, and injection into lymph nodes. In some embodiments, administration is systemic.

[0228] Other routes of administration are also contemplated. For example, the construct and the agent can be administered in combination with a carrier. In some embodiments, the carrier is a nanoparticle or microparticle. In some embodiments, the nanoparticle or microparticle is a tumor-directed nanoparticle or microparticle. For example, the carrier may also include a targeting portion that directs the carrier to the tumor. The targeting portion may be a binding agent (e.g., an antibody, including scFv, etc. or other antigen binding agents) that specifically recognizes tumor cells. In some embodiments, the construct is encapsulated in a carrier. In some embodiments, the construct is covalently or non-covalently attached to the carrier surface. In some embodiments, the carrier is a liposome. In other embodiments, carrier molecules described herein are excluded.

[0229] Particles can have structures of variable size and are referred to as microspheres, microparticles, nanoparticles, nanospheres or liposomes. Such particle preparations can be formed by covalent or non-covalent conjugation of constructs to particles. In some embodiments, particles described herein are excluded.

[0230] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid for easy syringability. It should also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0231] The carrier can also be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using a surfactant to maintain. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferably included, such as sugar or sodium chloride. Using delayed absorption agents, such as aluminum monostearate and gelatin, the absorption of the injection composition can be prolonged.

[0232] Sterile injectable solutions are prepared by mixing the active compound with the various other ingredients listed above in the required amounts in a suitable solvent and then sterilizing by filtration. Generally, the various sterilized active ingredients are mixed into a sterile carrier containing a basic dispersion medium and the required other ingredients listed above. For the preparation of sterile powders for sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying techniques, which will produce a powder of the active ingredient and any additional required ingredients obtained from the previously sterile filtered solution.

[0233] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of humans and animals within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction, or other problems and complications commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

[0234] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods.

[0235] Depending on the condition of the object, some changes will inevitably occur in dosage. In any case, the person responsible for administration will determine the appropriate dosage for the individual object. The effective amount of the therapeutic or preventive composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for an object, each unit containing a predetermined amount of the composition, calculated to produce the desired response associated with its administration discussed above, i.e., appropriate approach and scheme. Depending on the number of treatments and the unit dose, the amount of administration depends on the desired effect. The precise amount of the composition also depends on the judgment of the practitioner and varies from person to person. Factors affecting dosage include the physical and clinical state of the object, the route of administration, the intended goal of treatment (symptom relief and cure), and the efficacy, stability and toxicity of the specific composition.

[0236] After formulation, the solution will be administered in a therapeutically or prophylactically effective amount and in a manner compatible with the dosage formulation. The formulation is easily administered in a variety of dosage forms, such as the type of injection solution described above.

[0237] Typically, for an adult (weighing about 70 kg), about 0.1 mg to about 3000 mg (including all values ​​and ranges therebetween) of the compound, or about 5 mg to about 1000 mg (including all values ​​and ranges therebetween) of the compound, or about 10 mg to about 100 mg (including all values ​​and ranges therebetween) of the compound are administered. It should be understood that these dosage ranges are only examples and can be adjusted according to factors known to the skilled person.

[0238] In certain embodiments, about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6 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, 1 3.5, 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 4, 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, 12 0, 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、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、350 0, 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 / minute or mg / kg / min or micrograms / kg / hour or mg / kg / hour, or any range derivable therein.

[0239] The dose may be administered as needed or once every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, or 24 hours (or any range derivable therein) or once, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or more than 9 times a day (or any range derivable therein). The dose may be administered for the first time before or after the onset of the condition. In some embodiments, the first dose of a regimen of once in 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours (or any range derivable therein) is administered to the patient or the first dose is administered to the patient 1, 2, 3, 4, or 5 days after the patient experiences or exhibits signs or symptoms of the condition (or any range derivable therein). The patient can be treated for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more than 10 days (or any range derivable therein) or until the symptoms of the disorder disappear or are alleviated, or the patient can be treated 6 hours, 12 hours, 18 hours or 24 hours or 1 day, 2 days, 3 days, 4 days or 5 days after the symptoms of infection disappear or are alleviated.

[0240] IX. Examples

[0241] The following examples are included to demonstrate preferred embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present disclosure and therefore can be considered to constitute preferred modes of its practice. However, it will be appreciated by those skilled in the art based on the present disclosure that many changes may be made to the disclosed specific embodiments and still obtain close or similar results without departing from the spirit and scope of the present disclosure.

[0242] Example 1 - Engineered collagen binding serum albumin as a drug conjugation carrier for cancer treatment

[0243] Serum albumin (SA) is used as a carrier to deliver cytotoxic agents to tumors through passive targeting. To further improve the tumor targeting ability of SA, the inventors sought to develop a method to retain SA-drug conjugates in tumors through a combination of passive and active targeting. Due to tumor vascular permeability, SA is recombinantly fused to the collagen binding domain (CBD) of von Willebrand factor to bind to the tumor matrix after extravasation. Doxorubicin (Dox) is conjugated to CBD-SA via a pH-sensitive linker. In a breast cancer mouse model, Dox-CBD-SA treatment significantly inhibited tumor growth compared with Dox-SA and doxorubicin treatment. Dox-CBD-SA effectively stimulates host anti-tumor immunity and can completely eradicate MC38 colon cancer when used in combination with anti-PD-1 checkpoint inhibitors. Compared with doxorubicin, Dox-CBD-SA reduces adverse events. Therefore, engineered CBD-SA can be a widely used, clinically relevant drug conjugated carrier protein for the treatment of solid tumors.

[0244] A. Introduction

[0245] Doxorubicin (Dox) is a small molecule anticancer drug that has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of a variety of cancers. Dox is internalized in cells through passive transmembrane diffusion and interferes with DNA function, leading to cell death. Although Dox treatment prolongs the survival of certain patient groups, the antitumor efficacy is not significant, partly due to acquired drug resistance. The poor therapeutic index of Dox also limits its therapeutic use. In fact, considerable toxicity of Dox has been reported clinically, including bone marrow suppression, excessive inflammation, and cardiotoxicity (13, 14). In order to improve the efficacy, Dox is often used in combination with other chemotherapeutic drugs. Here, the inventors designed a recombinant mouse SA (CBD-SA), in which the N-terminus was fused to the C-terminus of the VWF A3 domain, and adriamycin was conjugated to CBD-SA through a pH-dependent cleavable hydrazone bond before injection (i.e., Dox-CBD-SA) (21). The inventors evaluated the engineered CBD-SA as a tumor-targeted drug carrier to improve the antitumor efficacy by effectively delivering Dox to the tumor microenvironment.

[0246] B. Results

[0247] 1. CBD-SA binds to collagen and can be conjugated with Dox

[0248] The inventors synthesized Dox-CBD-SA conjugates to target the tumor microenvironment (Figure 1A, Figure 1B). The inventors first studied the binding ability of CBD-SA to recombinant collagen in vitro. SA was recombinantly expressed, and CBD was located at the N-terminus of mouse SA, using a (GGGS)2 linker (SEQ ID NO: 5). The molecular weight of CBD-SA was analyzed by MALDI-TOF MS ( Figure 6 ). Strong binding affinity of CBD-SA to type I and type III collagen (nM range dissociation constant (Kd) values) was observed (Figure 1C, Figure 7). For Dox conjugation, the inventors first used 2-iminothiolane (also known as Traut's reagent) to thiolate the lysine residues of CBD-SA. Then, adriamycin was covalently conjugated to CBD-SA. Unmodified SA was also conjugated to adriamycin in the same manner (Dox-SA). SDS-polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions showed that the purified Dox-SA and Dox-CBD-SA were monomers ( 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 monomeric form and that Dox conjugation does not change this property even after freeze-drying / reconstitution cycles. Approximately 3 Dox molecules were conjugated to each SA molecule and each CBD-SA molecule (Figure 1D). It is worth noting that the inventors' conjugation method does not affect the ability of CBD-SA to bind to collagen because there are no cysteine ​​or lysine residues at the binding interface between the VWF A3 domain and human collagen III ( Fig.10 , PDB: 4DMU (22). This interface is also distal to the C-terminal fusion site to the SA domain.

[0249] 2. Dox release under acidic pH conditions

[0250] Since Dox is linked to SA via a pH-sensitive cleavable linker, the release kinetics of Dox from the conjugate under different pH conditions were examined (Figure 1E). After 48 h of incubation, Dox release from Dox-CBD-SA at pH 5.0 and pH 6.5 (reported tumor microenvironment conditions) reached a maximum. In contrast, at pH 7.4, only about 20% of Dox was released after 48 h. Dox-SA showed a similar release profile ( Fig.11 These data showed pH-dependent release of Dox from the conjugate, consistent with previously reported release kinetics of small chemicals linked via hydrazone bonds (21).

[0251] 3. Dox conjugates are taken up by cancer cells and remain cytotoxic

[0252] The inventors compared the intracellular localization of Dox conjugates and free drugs using confocal laser scanning microscopy by detecting the fluorescence of Dox. Since Dox is the main drug for the treatment of breast cancer (23), the inventors chose mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse breast cancer as an experimental model. MMTV-PyMT cells were incubated in the presence of Dox or Dox conjugates, and their intracellular uptake was then evaluated (Figure 1F). After 1 hour of incubation, free Dox was detected in the cytoplasm, intracellular acidic organelles, and preferably in the nucleus, indicating that its delivery was mediated by passive transmembrane diffusion. In contrast, 1 hour after the addition of Dox-SA or Dox-CBD-SA, the cytoplasm did not show strong fluorescence compared to unconjugated Dox. Instead, punctate fluorescence was observed, and some dots were co-localized with lysosomes, indicating that both Dox-SA and Dox-CBD-SA were internalized by endocytosis. 24 hours after adding Dox conjugate, Dox-derived fluorescence was also observed in the nucleus, indicating that the acidic pH of intracellular organelles induced the release of drugs from the conjugate. The inventors then examined the in vitro cytotoxicity of different Dox forms. MMTV-PyMT cells or MC38 colon cancer cells were inoculated and incubated for 3 days in the presence of Dox forms. Viability tests showed that all three Dox forms had similar cytotoxicity in vitro (Figure 1G, Figure 1H).

[0253] 4. Dox-CBD-SA exhibits similar plasma pharmacokinetics to doxorubicin and accumulates in tumors

[0254] Subramycin reacts rapidly with endogenous SA after intravenous (iv) administration, so it has a longer plasma half-life compared to Dox (18). The inventors tested the plasma pharmacokinetics of pre-conjugated subramycin with or without SA and CBD-SA using tumor-free FVB mice. After intravenous injection, similar plasma half-lives of subramycin, Dox-SA and Dox-CBD-SA were observed (Figure 2A, Figure 2B). The inventors also tested the plasma pharmacokinetics of fluorescently labeled SA and CBD-SA with pH-insensitive linkers (Figure 12). The results showed that the half-life of each protein conjugated to Dox or the dye was similar, indicating that Dox is not released from the conjugate in the blood circulation.

[0255] The inventors then hypothesized that the fusion of CBD and SA would increase the amount of Dox in the tumor by actively targeting collagen in the tumor microenvironment. In order to verify this hypothesis, the inventors measured the amount of Dox in the tumor tissue after a single intravenous injection. 2 hours after administration, Dox-CBD-SA showed significantly higher Dox tumor accumulation (Fig. 2C) compared with adriamycin and Dox-SA. After 24 hours of injection, the highest Dox tumor accumulation was also achieved by conjugation with CBD-SA, showing a significant increase compared with adriamycin. Histological analysis showed that fluorescently labeled CBD-SA co-localized with CD31 staining in tumor tissue, indicating that CBD-SA targets tumor vasculature (Fig. 2D). These data show that the fusion of SA conjugated with CBD and Dox enables Dox to target tumors, resulting in enhanced tumor accumulation of Dox.

[0256] 5. Dox-CBD-SA showed good efficacy in the MMTV-PyMT mouse breast cancer model

[0257] Inspired by plasma pharmacokinetics and tumor accumulation studies, the inventors evaluated the in vivo antitumor effect of Dox-CBD-SA. MMTV-PyMT orthotopic tumor-bearing mice received a single intravenous injection of Dox form (5 mg / kg, based on Dox) through the tail vein. Dox-SA and Dox-CBD-SA significantly inhibited tumor growth, while adriamycin did not (Figure 3A, Figure 3C to Figure 3F). This suggests that pre-conjugation of Dox with SA will provide a higher therapeutic effect compared to in situ conjugation of adriamycin with endogenous SA. It is worth noting that Dox-CBD-SA showed better therapeutic effects than Dox-SA. Dox-CBD-SA treatment significantly prolonged survival compared to all other groups (Figure 3B) and induced complete tumor remission in 2 of 12 mice. These data indicate that CBD-fused SA can be used as an excellent Dox carrier compared to unmodified SA in terms of anti-tumor efficacy.

[0258] 6. Dox-CBD-SA enhances tumor infiltration of lymphocytes

[0259] Dox has been reported to induce ICD, thereby stimulating an immune response against necrotic cell antigens (15). Indeed, ICD increases the number of tumor-infiltrating lymphocytes (TILs), a marker of good prognosis in a variety of cancers, such as colorectal and breast cancer (24, 25). The inventors analyzed TILs, specifically 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 various Dox forms. Dox-CBD-SA, but not adriamycin or Dox-SA, significantly increased the number of CD8 + T cells, CD4 + In particular, Dox-CBD-SA treatment increased the number of CD8 + The number of T cells increased more than two-fold compared to the other treatment groups (Figure 3G). The plot of individual tumor size and TIL cell number showed that Dox-CBD-SA indeed induced a small tumor size with tumor-infiltrating CD8 + T cells, CD4 + These data suggest that lymphocytes, especially CD8 + The enhanced infiltration of cytotoxic T cells may contribute to the excellent antitumor effect of Dox-CBD-SA.

[0260] 7. Dox-CBD-SA shows reduced toxicity

[0261] Because conjugated adriamycin is only released very slowly from SA at physiological pH (Figure 1E), the inventors hypothesized that Dox-CBD-SA exhibits reduced toxicity compared to adriamycin. The inventors used tumor-free FVB mice to evaluate adverse events after a single injection of adriamycin or Dox-CBD-SA (20 mg / kg, based on Dox). Taking adriamycin increased the plasma concentrations of inflammatory cytokines such as IFN-γ, TNF-α, IL-5 and IL-6, while Dox-CBD-SA did not (Figures 4A-D). Adriamycin treatment also caused a significant decrease in red blood cell (RBC) counts, white blood cell (WBC) counts, hematocrit and hemoglobin concentrations (Figure 4E, Figure 4F, Figure 13). In contrast, the adverse effects of Dox-CBD-SA on blood values ​​were mild. Compared with the untreated group, only a significant decrease in WBC counts was observed. Administration of adriamycin induced splenomegaly, whereas Dox-CBD-SA treatment did not (Figure 4G). Histological analysis showed that Dox-CBD-SA treatment did not cause observable damage to the heart, liver, kidney, or lung ( Fig.14). These data suggest that pre-conjugation of Dox to CBD-SA reduces toxicity in various aspects.

[0262] 8. Dox-CBD-SA combined with anti-PD-1 antibody (αPD-1) can eradicate MC38 tumors

[0263] Based on the observation of increased TILs induced by Dox-CBD-SA treatment (Figures 3G-3L), the inventors hypothesized that the combination of Dox-CBD-SA and CPI would show better therapeutic efficacy compared to the combination of adriamycin and CPI. To test this hypothesis, the inventors selected αPD-1 as the most widely used CPI in the clinic (26). Importantly, αPD-1 is used in combination with Dox in clinical trials (e.g., NCT02648477). The inventors used the MC38 colon cancer model to examine the antitumor effects of the combination of adriamycin and Dox-CBD-SA with αPD-1, which is immunogenic (27) but cannot be cured by Dox monotherapy (28). C57BL / 6 mice were subcutaneously inoculated with 5×10 5 MC38 cells. The treatment scheme is shown in Figure 5A. Mice were administered adriamycin or Dox-CBD-SA 6, 9, and 12 days after tumor inoculation. Since Dox-CBD-SA increased the number of TILs, the inventors injected 100 μg of αPD-1 twice (days 10 and 13) one day after Dox treatment. Dox-CBD-SA plus αPD-1 treatment completely eradicated established MC38 tumors (average tumor volume on day 6 was approximately 100 mm 3 , Figure 5B, Figure 5G), and significantly prolonged the survival of mice compared with all other groups (Figure 5C). In other treatment groups, a small number of mice failed to show a complete response, and the average tumor size gradually increased (Figure 5B, Figure 5D-5F). Among the survivors treated with Dox-CBD-SA+αPD-1, mice that were re-challenged with MC38 cells without additional treatment did not develop palpable tumors, indicating that they had acquired strong immune anti-tumor memory (Figure 5H, Figure 15A). During the treatment, no mice showed greater than 15% weight loss (Figure 15B). These data indicate that Dox-CBD-SA acts synergistically with αPD-1 by inducing ICD, further showing anti-tumor effects that cannot be achieved by the same dose of aldosterone + αPD-1.

[0264] C. Discussion

[0265] Since small molecule anticancer drugs are widely distributed in tissues and cause systemic side effects, attempts have been made to modify the drugs to improve their pharmacokinetics and biodistribution. Nanoparticle formulations (17) or SA responsiveness (18, 19) Doxorubicin exhibits improved pharmacokinetics and accumulation within tumors, partly due to its pathologically abnormal vasculature (5). However, due to the heterogeneity of human cancers, this effect may not always be effective in human cancers (29). Therefore, drugs that rely solely on passive targeting can still have room for improvement. Active targeting of tumor-specific or tumor-associated antigens for drug delivery is another therapeutic strategy. However, this inherently limits the scope of application for cancer and may also lead to acquired drug resistance due to antigen-selective cell targeting and killing, which may be lost due to mutations (30). Here, the inventors designed CBD-SA to overcome these problems. Unlike other active targeting strategies, CBD-SA does not require prior investigation of the expression of tumor-associated antigens because collagen is almost ubiquitous in tumors and CBD enters the tumor stroma through abnormal vascular structures in the tumor microenvironment (6). Subsequently, CBD-SA binds to exposed collagen (Figure 1C, Figure 7) and converts the tumor stroma into a reservoir for chemotherapeutic drugs. Compared with adriamycin and Dox-SA, the accumulation of Dox in tumor tissue was significantly higher after conjugation of Dox with CBD-SA (Figure 2C). After Dox-CBD-SA accumulates in tumor tissue, the hydrazone bond can be cleaved under the slightly acidic conditions in the tumor microenvironment (Figure 1E)(21), enabling the sustained release of Dox from CBD-SA. At the same time, tumor cells are known to take up SA (1). Notably, CBD fusion did not alter the cellular uptake of SA (Figure 1F), indicating that Dox-CBD-SA can also be delivered intracellularly as effectively as Dox-SA. Therefore, part of the Dox release may occur in the tumor stroma while Dox-CBD-SA remains 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 ) is conducive to the spread of tumor tissue (32).

[0266] In terms of anti-tumor efficacy, compared with adriamycin and Dox-SA, Dox-CBD-SA significantly inhibited the growth of MMTV-PyMT breast cancer and prolonged the survival of mice (Figures 3A-3F). Since Dox-CBD-SA has the highest accumulation in tumor tissue in vivo, Dox-CBD-SA should more effectively induce tumor cell death by inhibiting tumor cell proliferation. In addition to this effect, a single injection of Dox-CBD-SA can still bring lasting therapeutic effects despite its faster plasma clearance half-life (Figure 2A, Figure 2B). This can be explained by the inventor's observation that Dox-CBD-SA treatment induced a higher number and higher density of TILs (Figures 3G-3L) compared with Dox-SA and adriamycin treatment. Therefore, the anti-tumor mechanism of Dox-CBD-SA may not only be direct cell killing, but also stimulate host anti-tumor immunity. Since Dox-CBD-SA accumulates effectively in tumors, it can more effectively induce ICD and tumor antigen exposure to the immune system than adriamycin and Dox-SA. Therefore, Dox-CBD-SA combined with αPD-1 synergistically eradicated MC38 colon cancer (Figure 5B, Figure 5G). The improved therapeutic efficacy of Dox-SA and Dox-CBD-SA compared with adriamycin (Figures 3A-3F) also suggests that pre-conjugation of Dox before injection will provide higher anti-tumor efficacy. In addition to its rapid clearance from the blood circulation, in situ conjugation of adriamycin with other thiol compounds in plasma (such as cysteine, glutathione, fibronectin, or α1-antitrypsin) (18) is also a possible reason for the low therapeutic efficacy of adriamycin.

[0267] Cardiotoxicity is a major drawback of Dox, which limits the lifetime cumulative dose of Dox (13). Histological analysis showed that even administration of 20 mg / kg of Dox-CBD-SA did not show any signs of cardiac damage ( Fig.14 ). This suggests that Dox pre-conjugated with CBD-SA is less cardiotoxic than free Dox, which caused irreversible damage to cardiac tissue at a cumulative dose of 15 mg / kg in a mouse model (34). Importantly, the cumulative dose of 15 mg / kg is almost equivalent to the maximum cumulative dose in humans (35).

[0268] In terms of the manufacturing process, the inventors used Traut's reagent to conjugate Dox, which can precisely control the drug conjugation rate (36). Since there are no lysine residues on the binding interface between the VWF A3 domain and collagen, this method has little risk of CBD binding to collagen ( Fig.10)(22). In addition, SA contains approximately 7 times the number of lysine residues as the CBD sequence, which also indicates a lower risk of adverse conformational changes in CBD due to conjugation. Traut's reagent has also been used in an ADC targeting CD70 (MDX-1203, Bristol-Myers Squibb)(37), indicating its applicability. Since CBD-SA is produced in high yield (approximately 70 mg / L to 100 mg / L HEK293 cell incubation), the inventors proposed to pre-conjugate Dox with CBD-SA to produce high anti-tumor efficacy in a simple and translatable preparation method.

[0269] The reduction in nonspecific toxicity was unexpected because one might expect CBD-SA to accumulate in unwanted sites in the body, such as the liver, kidneys, and wounds, where collagen might be exposed through a porous or leaky endothelium. However, the inventors observed no pathological damage to the liver and kidneys after administration of 20 mg / kg Dox-CBD-SA ( Fig.14 ). Furthermore, the increased efficacy of the current peptides was somewhat unexpected, as previous work in the field has shown that chemical conjugation generally decreases the half-life of SA. Methotrexate conjugates have been reported to accelerate the clearance of methotrexate-SA conjugates from the circulation in a drug:protein ratio-dependent manner (38). Therefore, it is surprising that even though the half-lives of Dox-SA and Dox-CBD-SA are shorter than those reported for untreated mouse SA (t 1 / 2 , β=35(h))(39), the treatment efficiency is still improved.

[0270] In summary, Dox-CBD-SA accumulated in tumors and activated host antitumor immunity. Therefore, monotherapy with Dox-CBD-SA suppressed the growth of orthotopic MMTV-PyMT breast tumors and prolonged survival. More importantly, Dox-CBD-SA combined with αPD-1 immune checkpoint inhibition completely eradicated tumors in the immunogenic MC38 model. CBD fusion provides active targeting capabilities to SA, which is usually used as a passive targeted drug carrier that can effectively deliver drugs from the systemic circulation to tumors. CBD-SA is expected to be non-immunogenic and biologically acceptable because it is composed of two proteins (VWF A3 domain and SA) that are naturally present in the blood. In addition, CBD-SA is independent of tumor type-specific antigens, thus providing broad applicability as a drug carrier for various types of solid tumors. Therefore, CBD-SA may have the potential for clinical translation into cancer treatment as an anti-tumor drug carrier.

[0271] D. Materials and Methods

[0272] 1. Study Design

[0273] This study aimed to validate the strategy of delivering anticancer drugs to tumors via engineered collagen-bound SA as a drug conjugation carrier. Specifically, the inventors tested whether the antitumor efficacy of Dox-CBD-SA against breast cancer and colon cancer mouse models was improved compared with the unmodified form. The side effects of Dox-CBD-SA were also tested using tumor-free mice. The inventors measured multiple aspects of tumor growth, anticancer immune response, and post-treatment toxicity. No statistical methods were used to predetermine the required sample size, but the sample size was determined based on estimates from pilot experiments and previously published results so that appropriate statistical tests could produce significant results. CBD-SA was prepared by multiple people to ensure reproducibility. All experiments were repeated at least twice, except for Figure 12 (once). For animal studies, mice were randomly assigned to treatment groups within cages before the first injection of Dox-CBD-SA and treated in the same manner. According to animal care guidelines, samples were excluded from analysis only if the animals had health problems for reasons unrelated to treatment. When the tumor size of the MMTV-PyMT model exceeded 500mm 3 And the tumor size of MC38 model exceeds 600mm 3 Survival endpoints were reached when 1% of patients were treated with 1% leukemia / drug therapy. The n values ​​used to calculate the statistics are indicated in the figures or legends. Administration and pathological analysis were performed in a blinded manner. Statistical methods are described in the “Statistical Analysis” section.

[0274] 2. Cell Incubation

[0275] Mouse mammary tumor virus-polyomavirus intermediate T antigen (MMTV-PyMT) cells were obtained from spontaneously developed mammary carcinomas of FVB-Tg (MMTV-PyMT) transgenic mice as described previously (9). The MC38 colon cancer cell line was kindly provided by the laboratory of R. Weichselbaum (University of Chicago). Both cell lines were grown in DMEM (Gibco) supplemented with 110 mg / L sodium pyruvate, 10% heat-inactivated FBS, and 1% penicillin / streptomycin. The cell lines were checked for mycoplasma contamination by the IMPACT I pathogen test (IDEXX BioResearch).

[0276] 3. Mice

[0277] Female FVB mice aged 8 to 12 weeks were obtained from Charles River and Jackson Laboratories. Female C57BL / 6 mice aged 8 to 12 weeks were obtained from Jackson Laboratories. All animal experiments performed in this work were approved by the Institutional Animal Care and Use Committee of the University of Chicago.

[0278] 4. Production and purification of CBD-SA

[0279] The design, production, and purification of the CBD-SA protein were similar to those of the previously reported CBD protein (9). A sequence encoding the fusion of human VWFA3 domain residues Cys1670-Gly1874 (907-1111 of mature VWF) and mouse SA without the propeptide (25 to 608 amino acids of the entire SA) was synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. A sequence encoding a His tag (6His (SEQ ID NO:21)) was inserted into the C-terminus to further purify the recombinant protein. Suspension-adapted HEK-293F cells were routinely maintained in serum-free FreeStyle 293 expression medium (Gibco). On the day of transfection, cells were incubated at 1 x 10 6 Dilute to fresh medium at a density of 10 cells / mL. Add 2 μg / mL plasmid DNA, 2 μg / mL linear 25 kDa polyethyleneimine (Polysciences), and OptiPRO SFM medium (4% final concentration, Thermo Fisher Scientific). Agitate the incubation flask with orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. 7 days after transfection, collect the cell medium by centrifugation through a 0.22 μm filter. Use The culture medium was loaded into HisTrap HP 5mL column (GE Healthcare) using pure 25 (GE Healthcare). After washing the column with wash buffer (20mM imidazole, 20mM NaH2PO4, 0.5M NaCl, pH 7.4), the protein was eluted with a gradient of 500mM imidazole (in 20mM NaH2PO4, 0.5M NaCl, pH 7.4). The eluent was further purified by size exclusion chromatography using HiLoad Superdex 200PG column (GE Healthcare). All purification steps were performed at 4 ° C. Protein purity>90% was confirmed by SDS-PAGE.

[0280] 5. MALDI-TOF MS

[0281] Purified CBD-SA was analyzed by MALDI-TOF mass spectrometry (Bruker Ultraflextreme MALDITOF / TOF) as described previously (9). Bruker flexControlr was used for data acquisition and Bruker flexAnalysise was used for data processing. First, a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) was prepared with 50:50 acetonitrile:1% TFA in water as 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 then dropped onto an MTP 384 ground steel target plate. The droplet was dried in a stream of nitrogen. All samples were analyzed using a high-quality linear positive mode method with 2500 laser shots at 75% laser intensity. The measurements were externally calibrated at three points using a mixture of carbonic anhydrase, phosphorylase B, and bovine SA.

[0282] 6. Binding Affinity Experiment

[0283] The binding affinity of CBD-SA to collagen was tested as previously described (9). 96-well ELISA plates (Greiner Bio-One) were coated with collagen I or collagen III (10 μg / mL each in PBS) overnight at 37°C and then blocked with 2% BSA in PBS and 0.05% Tween 20 (PBS-T) for 1 hour at room temperature. The wells were then washed with PBS-T and further incubated with increasing concentrations of CBD-SA for 2 hours at room temperature. After washing three times with PBS-T, biotin-conjugated anti-mouse antibodies against SA were incubated for 1 hour at room temperature. After washing, bound CBD-SA was detected with tetramethylbenzidine substrate by measuring the absorbance at 450 nm 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.

[0284] 7. Synthesis of Dox Conjugates

[0285] Mouse SA or CBD-SA was dissolved in PBS containing 2mM EDTA. 4 molar equivalents of Traut's reagent dissolved in PBS containing 2mM 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 (ThermoFisher science). 15 molar equivalents of adriamycin (MedChemExpress) dissolved in 10mM sodium phosphate buffer (pH 5.9) were added and incubated at room temperature for 1 hour and incubated overnight in the dark at 4°C. To quench the reaction, 20 molar equivalents of L-cysteine ​​(Sigma-Aldrich (pharmaceutical grade) dissolved in PBS containing 2mM EDTA) against adriamycin were added. Unreacted Dox precipitate was removed by centrifugation (10000x g, 5 minutes). The supernatant was further purified by Zeba spin desalting column and then ultrafiltered using Amicon-Ultra (Merck, 10K MWCO). The concentration of Dox in the final product was quantified by absorbance at 495 nm using a molar extinction coefficient of 10650 (L·mol-1·cm -1 The concentration of protein content was measured by Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0286] 8. Dynamic Light Scattering (DLS)

[0287] The hydrodynamic size of 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.

[0288] 9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0289] SDS-PAGE was performed as described in the inventors' previous work (9). A 4% to 20% gradient gel (Bio-Rad) was used, and 0.5 μg of each Dox conjugate was loaded with or without 10 mM DTT reduction. After electrophoresis, the gel was stained using SimplyBlue SafeStain (Thermo Fisher Scientific) according to the manufacturer's instructions. Images were acquired using the ChemiDoc XRS+ system (Bio-Rad).

[0290] 10. pH-dependent release of Dox from the conjugate

[0291] The release curve of Dox from the conjugate was determined using a Slide-A-Lyzer MINI dialysis device (Thermo Fisher science, 10K MWCO). The Dox conjugate was diluted to 100 μM (based on Dox) in PBS (pH 6.5 or 7.4) or 0.1M acetate buffer (pH 5.0). 150 μL of each sample was loaded into the device and dialyzed with 50 mL of buffer. Dialysis was performed using a magnetic stirrer. The temperature of the sample stage was set to 37 ° C, and the samples were protected from light during the dialysis process. The dialysate was collected at different time points and stored at 4 ° C until the sample collection was completed. The dialysate was loaded onto a 96-well plate in two portions (90 μL / well). Fluorescence was detected using 495 nm excitation and 590 nm emission. Serial dilutions of doxorubicin hydrochloride were prepared in the same buffer to create a standard curve.

[0292] 11. Cellular Uptake of Dox Conjugates

[0293] To evaluate the cellular uptake of Dox conjugates, MMTV-PyMT cells were seeded in 96-well high-content imaging plates (Corning) at a concentration of 5000 cells / well and incubated overnight. Cells were washed with PBS and treated with free Dox, Dox-SA or Dox-CBD-SA (110 mg / L sodium pyruvate, 10% heat-inactivated FBS, 1% penicillin / streptomycin, phenol red (-)) dissolved in DMEM at a Dox concentration of 50 μM. After incubation, the cells were washed twice, treated with 75 nM Lysotracker Deep Red, and incubated for an additional 30 minutes at 37 ° C. The cells were washed twice and observed at a magnification of x 60 by an IX83 microscope (Olympus). Images were processed using ImageJ software (NIH). Scale bar; 20 μm.

[0294] 12. In vitro cytotoxicity

[0295] 3000 cells / well of MMTV-PyMT cells or MC38 cells were seeded in a 96-well tissue culture plate (BD Falcon) and incubated overnight. The cells were washed with DMEM containing 110 mg / L sodium pyruvate, 10% heat-inactivated fetal bovine serum, 1% penicillin / streptomycin, and phenol red (-), and 80 μL / well of DMEM was added. Then, serial dilutions of adriamycin, Dox-SA, or Dox-CBD-SA (20 μL / well) were added in PBS. The cells were incubated at 37°C for 3 days and the cells were cultured using CellTiter according to the manufacturer's instructions. AQueous One Solution Cell Proliferation Assay Kit (Promega) was used to detect cell viability. The survival rate of cells treated with 80 μL / well DMEM+20 μL / well PBS was 100%, while the cell-free wells using the same mixture were defined as 0% survival rate. The maximum half inhibitory concentration (IC50) ([inhibitor] vs normalized response) was obtained by nonlinear regression analysis in Prism software.

[0296] 13. Plasma pharmacokinetics of Dox conjugates

[0297] Reference was made to previous reports on peptide-Dox nanoparticles (40). To measure the pharmacokinetics of Dox, 5 mg / kg Dox-equivalent adriamycin, Dox-SA, or Dox-CBD-SA was injected into female FVB mice. Blood samples were collected in EDTA-coated tubes at 5 minutes, 30 minutes, 1 hour, 4 hours, 12 hours, 25 hours, 50 hours, and 75 hours after injection. Blood samples were stored at 4°C until the end of sample collection. The samples were centrifuged (2000 x g, 5 minutes) and plasma was collected. Plasma samples diluted in acidified isopropanol (75 mM HCl, 10% water, 90% isopropanol) were loaded onto 96-well plates (100 μL / well). Fluorescence was measured as described above. Plasma samples were also collected from mice that did not receive injections, diluted in acidified isopropanol, and measured to create a standard curve for background fluorescence. Exponential two-phase decay (Y = Ae -αt +Be -αt ) fitting is used to calculate the plasma half-life. Rapid elimination half-life: t 1 / 2 , α, slow elimination half-life: t 1 / 2 , β. Data were analyzed using Prism software (v7, GraphPad).

[0298] 14. Plasma pharmacokinetics of SA and CBD-SA

[0299] According to the manufacturer's instructions, SA and CBD-SA are labeled with DyLight 800NHS ester (Thermo Fisher Scientific). Unreacted dye is removed by Zeba spin desalting column as described above. After labeling, 200 μg of each protein is intravenously injected into female FVB mice. At 1 minute, 1 hour, 4 hours, 24 hours, 74 hours and 120 hours after injection, blood samples are collected in EDTA coated tubes. Blood samples are stored at 4 ° C until sample collection is completed. Samples are centrifuged (2000x g, 5 minutes) and plasma is collected. Plasma samples are diluted in PBS and loaded into 96-well plates (100 μL / well). The concentration of each protein in plasma is measured with LI-COR Infrared Odyssey Imager (Li-COR Biosciences). Curve fitting method and plasma half-life calculation method are as described above.

[0300] 15. MMTV-PyMT Tumor Inoculation and Treatment

[0301] The MMTV-PyMT mouse breast cancer model was prepared as described previously (9). A total of 5 × 10 5 MMTV-PyMT cells were injected subcutaneously into the mammary gland on the right side of each mouse. Mice were treated with adriamycin, Dox-SA, or Dox-CBD-SA via tail vein injection on day 7 (5 mg / kg). Tumors were measured with a digital caliper at the indicated time points, and volumes were calculated as ellipsoids, where V = 4 / 3 × 3.14 × depth / 2 × width / 2 × height / 2. Tumors were evaluated when their volume exceeded 500 mm. 3 Mice were sacrificed when active ulcers were observed. For treatment experiments, FVB mice from Charles River were used. For tumor infiltrating lymphocyte (TIL) analysis, FVB mice from Jackson Laboratory and Charles River were used. The proportion of mice from different providers was equal in all groups.

[0302] 16. MC38 Tumor Inoculation and Treatment

[0303] The MC38 mouse colon cancer model was prepared similarly to the B16F10 melanoma model described previously (9). A total of 5 × 10 5MC38 cells were injected intradermally into the left side of the back of each C57BL / 6 mouse. On days 6, 9, and 12, mice were intravenously injected with adriamycin, Dox-SA, or Dox-CBD-SA (5 mg / kg). Mice were also treated intraperitoneally. 100 μg of anti-PD-1 (Clone 29F.1A12, Bio X Cell) was added on days 10 and 13. Tumor growth was monitored as described above. When the tumor volume exceeded 600 mm 3 On day 60, mice were sacrificed by intradermal injection of 5 × 10 5 Untreated C57BL / 6 mice or tumor-free survivors were re-challenged with MC38 cells.

[0304] 17. Tumor Accumulation Study

[0305] Previous reports on peptide-Dox nanoparticles were cited (40). Adriamycin, Dox-SA, or Dox-CBD-SA were injected into FVB mice with established tumors at a dose of 4.16 mg / kg via the tail vein. Tumors were removed and weighed and placed on ice 2 h or 24 h after injection. Tumor tissues were suspended in 1 mL of acidified isopropanol and homogenized using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) at 5000 strokes / min for 40 s. After homogenization, samples were protected from light and incubated overnight at 4°C. Samples were centrifuged (5000 x g, 5 min), and the supernatant was loaded onto a 96-well plate (100 μL / well, triplicate). Fluorescence was measured to quantify the amount of Dox in tissue extracts as described above. Untreated mouse tumors were treated, and serial dilutions of tissue extracts were measured to obtain a standard curve of tissue-derived autofluorescence.

[0306] 18. Histological Analysis of Intratumoral Injection of CBD-SA

[0307] Mouse SA (Sigma-Aldrich) and CBD-SA were conjugated with NHS-DyLight 488 according to the manufacturer's instructions. Zeba centrifugal desalting columns were used to remove unreacted dyes, and the fluorescent protein solutions were then stored at 4°C for later 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 sections were obtained by cryosectioning. The tissues were fixed with PBS containing 2% paraformaldehyde for 15 minutes at room temperature. After washing with PBS-T, the tissues were blocked with 2% BSA in PBS-T for 1 hour at room temperature. The tissues were stained with biotin-labeled anti-mouse CD31 antibodies (1:100, Biolegend) and Alexa Fluor 647 streptavidin (1:1000, Biolegend). The tissues were washed three times and then covered with ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific). Images were taken with an IX83 microscope (Olympus) at a magnification of 60. Images were processed using ImageJ software (NIH).

[0308] 19. Flow cytometry and antibodies

[0309] The MMTV-PyMT model was prepared as described above. Mice were treated with adriamycin, 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 described previously (9). Tumors were harvested and digested in Dulbecco's modified Eagle medium (DMEM) supplemented with 2% FBS, 2 mg / mL collagenase D and 40 μg / mL DNase I (Roche) at 37°C for 30 minutes. Organs were gently disrupted through a 70 μm cell strainer to obtain a single cell suspension. Red blood cells were lysed with ACK lysis buffer (Quality Biological). Fixable live / dead cells were distinguished using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer's instructions. After the washing step, cells were stained with specific antibodies on ice for 20 minutes before fixation. The following antibodies were used to stain cells: 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), and all flow cytometric analyses were performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0310] 20.Toxicity profile

[0311] Tumor-free FVB mice received 20 mg / kg of adriamycin or Dox-CBD-SA by intravenous injection. On days 3 and 6 after injection, blood samples were collected from each mouse in EDTA-coated tubes by submandibular bleeding for plasma cytokine analysis and hematological analysis. The body weight of each mouse was measured at the indicated time points. On day 16, the mice were sacrificed and their organs were removed. The spleen was 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.

[0312] 21. Hematological analysis

[0313] Blood samples were analyzed using a COULTER Ac·T 5diff CP hematology analyzer (Beckmancoulter) according to the manufacturer's instructions.

[0314] 22. Measurement of Plasma Cytokines

[0315] Plasma was collected from whole blood samples as described above and stored at -20°C until use. Cytokine concentrations in plasma were measured using Ready-SET-Go! ELISA kits (eBioscience) and Can Get Signal solution (TOYOBO) according to the manufacturer's instructions.

[0316] 23. Histological analysis of heart, liver, kidney and lung

[0317] Organs were fixed overnight with 2% paraformaldehyde in PBS, embedded in paraffin, and sectioned at 5 μm for H&E staining.

[0318] 24. Statistical Analysis

[0319] Statistically significant differences between experimental groups were determined using Prism software (v7, GraphPad) as previously described (9). Differences between groups were similar by Brown-Forsythe test using one-way ANOVA and 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. The symbols * and ** indicate P values ​​less than 0.05 and 0.01, respectively; NS, no significant difference.

[0320] **********

[0321] Although certain embodiments have been described above with a certain degree of particularity or with reference to one or more than one separate embodiment, a person skilled in the art may make various changes to the disclosed embodiments without departing from the scope of the invention. In addition, where appropriate, aspects of any of the embodiments described above may be combined with aspects of any other embodiments described to form other embodiments having comparable or different characteristics and solving the same or different problems. Similarly, it should be understood that the benefits and advantages described above may be related to one embodiment or may be related to several embodiments. Any reference to a patent disclosure or other disclosure is hereby specifically incorporated by reference to the disclosure of the disclosure. The claims should not be interpreted as including means plus or step plus function limitations unless such limitations are explicitly stated in a given claim using the phrases "means" or "step", respectively.

[0322] References

[0323] The following references and publications mentioned throughout the specification are specifically incorporated herein by reference, to the extent they provide exemplary procedural or other details supplementary to those set forth herein.

[0324] 1.F.Kratz,Albumin as a drug carrier:design of prodrugs,drugconjugates and nanoparticles.J.Control.Release 132,171-183(2008).

[0325] 2.J.Morales,Defining the role of insulin detemir in basal insulintherapy.Drugs 67,2557-2584(2007).

[0326] 3.R.Flisiak,I.Flisiak,Albinterferon-alfa 2b:a new treatment optionfor hepatitis C.Expert.Opin.Biol.Ther.10,1509-1515(2010).

[0327] 4.M.Green,G.Manikhas,S.Orlov,B.Afanasyev,A.Makhson,P.Bhar,M.Hawkins, a novel -free,albumin-bound particle form of paclitaxelfor the treatment of advanced non-small-cell lung cancer.Ann.Oncol.17,1263-1268(2006).

[0328] 5.Y.Matsumura,H.Maeda,A new concept for macromolecular therapeuticsin cancer chemotherapy:mechanism of tumoritropic accumulation of proteins andthe antitumor agent smancs.Cancer Res.46,6387-6392(1986).

[0329] 6.F.Danhier,O.Feron,V.PrPr concept for macromolecular therapeutint:passive and active tumor targeting of nanocarriers for anti-cancer drugdelivery.J.Control.Release 148,135-146(2010).

[0330] 7.K.Temming,D.L.Meyer,R.Zabinski,E.C.Dijkers,K.Poelstra,G.Molema,R.J.Kok,Evaluation of RGD-targeted albumin carriers for specific delivery ofauristatin E to tumor blood vessels.Bioconjug.Chem.17,1385-1394(2006).

[0331] 8.J.Prakash,L.Beljaars,A.K.Harapanahalli,M.Zeinstra-Smith,A.de Jager-Krikken,M.Hessing,H.Steen,K.Poelstra,Tumor-targeted intracellular delivery ofanticancer drugs through the mannose-6-phosphate / insulin-like growth factorII receptor.Int.J.Cancer 126,1966-1981(2010).

[0332] 9.J.Ishihara,A.Ishihara,K.Sasaki,S.S.-Y.Lee,M.Yasui,H.Abe,L.Potin,P.Hosseinchi,K.Fukunaga,M.M.Raczy,L.T.Gray,J.-M.Williford,M.Fukayama,S.J.Kron,M.A.Swartz,J.A.Hubbell,Targeted antibody and cytokine cancerimmunotherapies through collagen affinity.Sci.Transl.Med.in press.

[0333] 10.C.Addi,F.Murschel,G.De Crescenzo,Design and use of chimericproteins containing a collagen-binding domain for wound healing and boneregeneration.Tissue Eng.Part B.Rev.23,163-182(2017).

[0334] 11.P.P.Provenzano,D.R.Inman,K.W.Eliceiri,J.G.Knittel,L.Yan,C.T.Rueden,J.G.White,P.J.Keely,Collagen density promotes mammary tumorinitiation and progression.BMC Med.6,11(2008).

[0335] 12.Z.-H.Zhou,C.-D.Ji,H.-L.Xiao,H.-B.Zhao,Y.-H.Cui,X.-W.Bian,Reorganized Collagen in the Tumor Microenvironment of Gastric Cancer and ItsAssociation with Prognosis.J.Cancer 8,1466-1476(2017).

[0336] 13.R.C.Young,R.F.Ozols,C.E.Myers,The anthracycline antineoplasticdrugs.N.Engl.J.Med.305,139-153(1981).

[0337] 14.L.Wang,Q.Chen,H.Qi,C.Wang,C.Wang,J.Zhang,L.Dong,Doxorubicin-induced systemic inflammation is driven by upregulation of toll-like receptorTLR4 and endotoxin leakage.Cancer Res.76,6631-6642(2016).

[0338] 15.G.Kroemer,L.Galluzzi,O.Kepp,L.Zitvogel,Immunogenic cell death incancer therapy.Annu.Rev.Immunol.31,51-72(2013).

[0339] 16.J.Rios-Doria,N.Durham,L.Wetzel,R.Rothstein,J.Chesebrough,N.Holoweckyj,W.Zhao,C.C.Leow,R.Hollingsworth,Doxil synergizes with cancerimmunotherapies to enhance antitumor responses in syngeneic mousemodels.Neoplasia 17,661-670(2015).

[0340] 17.M.E.O’Brien,N.Wigler,M.Inbar,R.Rosso,E.Grischke,A.Santoro,R.Catane,D.Kieback,P.Tomczak,S.Ackland,Reduced cardiotoxicity and comparableefficacy in a phase III trial of pegylated liposomal doxorubicin HCl(CAELYX TM / )versus conventional doxorubicin for first-line treatment of metastaticbreast cancer.Ann.Oncol.15,440-449(2004).

[0341] 18.F.Kratz,A.Warnecke,K.Scheuermann,C.Stockmar,J.Schwab,P.Lazar,P.DrDr D.Kieback,P.Tomczak,S.Ackland,Reduced cardiotoxicity and comparable efnousserum albumin with thiol-binding doxorubicin derivatives.Improved efficacy ofan acid-sensitive doxorubicin derivative with specific albumin-bindingproperties compared to that of the parent compound.J.Med.Chem.45,5523-5533(2002).

[0342] 19.R.Graeser,N.Esser,H.Unger,I.Fichtner,A.Zhu,C.Unger,F.Kratz,INNO-206,the(6-maleimidocaproyl hydrazone derivative of doxorubicin),showssuperior antitumor efficacy compared to doxorubicin in different tumorxenograft models and in an orthotopic pancreas carcinoma model.Invest.NewDrugs 28,14-19(2010).

[0343] 20.S.P.Chawla,Z.Papai,G.Mukhametshina,K.Sankhala,L.Vasylyev,A.Fedenko,K.Khamly,K.Ganjoo,R.Nagarkar,S.Wieland,First-line aldoxorubicin vsdoxorubicin in metastatic or locally advanced unresectable soft-tissuesarcoma:a phase 2b randomized clinical trial.JAMA Oncol.1,1272-1280(2015).

[0344] 21.M.Prabaharan,J.J.Grailer,S.Pilla,D.A.Steeber,S.Gong,Amphiphilicmulti-arm-block copolymer conjugated with doxorubicin via pH-sensitivehydrazone bond for tumor-targeted drug delivery.Biomaterials 30,5757-5766(2009).

[0345] 22.T.H.C.Brondijk,D.Bihan,R.W.Farndale,E.G.Huizinga,Implications forcollagen I chain registry from the structure of the collagen von Willebrandfactor A3 domain complex.Proc.Natl.Acad.Sci.U.S.A.109,5253-5258(2012).

[0346] 23.H.M.Kuerer,L.A.Newman,T.L.Smith,F.C.Ames,K.K.Hunt,K.Dhingra,R.L.Theriault,G.Singh,S.M.Binkley,N.Sneige,Clinical course of breast cancerpatients with complete pathologic primary tumor and axillary lymph noderesponse to doxorubicin-based neoadjuvant chemotherapy.J.Clin.Oncol.17,460-469(1999).

[0347] 24.K.M.Ropponen,M.J.Eskelinen,P.K.Lipponen,E.Alhava,V.M.Kosma,Prognostic value of tumour-infiltrating lymphocytes(TILs)in colorectalcancer.J.Pathol.182,318-324(1997).

[0348] 25.S.Loi,N.Sirtaine,F.Piette,R.Salgado,G.Viale,F.Van Eenoo,G.Rouas,P.Francis,J.P.Crown,E.Hitre,Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancertrial in node-positive breast cancer comparing the addition of docetaxel todoxorubicin with doxorubicin-based chemotherapy:BIG 02-98.J.Clin.Oncol.31,860-867(2013).

[0349] 26.K.M.Hargadon,C.E.Johnson,C.J.Williams,Immune checkpoint blockadetherapy for cancer:an overview of FDA-approved immune checkpoint inhibitors.Int.Immunopharmacol.62,29-39(2018).

[0350] 27.M.Yadav,S.Jhunjhunwala,Q.T.Phung,P.Lupardus,J.Tanguay,S.Bumbaca,C.Franci,T.K.Cheung,J.Fritsche,T.Weinschenk,Predicting immunogenic tumourmutations by combining mass spectrometry and exome sequencing.Nature 515,572-576(2014).

[0351] 28.R.Kuai,W.Yuan,S.Son,J.Nam,Y.Xu,Y.Fan,A.Schwendeman,J.J.Moon,Elimination of established tumors with nanodisc-based combinationchemoimmunotherapy.Sci.Adv.4,eaao1736(2018).

[0352] 29.U.Prabhakar,H.Maeda,R.K.Jain,E.M.Sevick-Muraca,W.Zamboni,O.C.Farokhzad,S.T.Barry,A.Gabizon,P.Grodzinski,D.C.Blakey,Challenges and keyconsiderations of the enhanced permeability and retention effect fornanomedicine drug delivery in oncology.Cancer Res.73,2412-2417(2013).

[0353] 30.C.Holohan,S.Van Schaeybroeck,D.B.Longley,P.G.Johnston,Cancer drugresistance:an evolving paradigm.Nat.Rev.Cancer 13,714-726(2013).

[0354] 31.M.Yasunaga,S.Manabe,D.Tarin,Y.Matsumura,Cancer-stroma targetingtherapy by cytotoxic immunoconjugate bound to the collagen 4 network in thetumor tissue.Bioconjug.Chem.22,1776-1783(2011).

[0355] 32.A.Pluen,Y.Boucher,S.Ramanujan,T.D.McKee,T.Gohongi,E.di Tomaso,E.B.Brown,Y.Izumi,R.B.Campbell,D.A.Berk,Role of tumor–host interactions ininterstitial diffusion of macromolecules:cranial vs.subcutaneous tumors.Proc.Natl.Acad.Sci.U.S.A.98,4628-4633(2001).

[0356] 33.N.Diamantis,U.Banerji,Antibody-drug conjugates—an emerging classof cancer treatment.Br.J.Cancer 114,362-367(2016).

[0357] 34.P.W.Fisher,F.Salloum,A.Das,H.Hyder,R.C.Kukreja,Phosphodiesterase-5inhibition with sildenafil attenuates cardiomyocyte apoptosis and leftventricular dysfunction in a chronic model of doxorubicincardiotoxicity.Circulation 111,1601-1610(2005).

[0358] 35.G.Takemura,H.Fujiwara,Doxorubicin-induced cardiomyopathy:from thecardiotoxic mechanisms to management.Prog.Cardiovasc.Dis.49,330-352(2007).

[0359] 36.M.J.McCall,H.Diril,C.F.Meares,Simplified method for conjugatingmacrocyclic bifunctional chelating agents to antibodies via 2-iminothiolane.Bioconjug.Chem.1,222-226(1990).

[0360] 37.T.K.Owonikoko,A.Hussain,W.M.Stadler,D.C.Smith,H.Kluger,A.M.Molina,P.Gulati,A.Shah,C.M.Ahlers,P.M.Cardarelli,First-in-human multicenter phase Istudy of BMS-936561(MDX-1203),an antibody-drug conjugate targetingCD70.Cancer Chemother.Pharmacol.77,155-162(2016).

[0361] 38.G.Stehle,H.Sinn,A.Wunder,H.H.Schrenk,S.Schütt,W.Maier-Borst,D.L.Heene,The loading rate determines tumor targeting properties ofmethotrexate-albumin conjugates in rats.Anticancer.drugs 8,677-685(1997).

[0362] 39.C.Chaudhury,S.Mehnaz,J.M.Robinson,W.L.Hayton,D.K.Pearl,D.C.Roopenian,C.L.Anderson,The major histocompatibility complex-related Fcreceptor for IgG(FcRn)binds albumin and prolongs its lifespan.J.Exp.Med.197,315-322(2003).

[0363] 40.J.A.MacKay,M.Chen,J.R.McDaniel,W.Liu,A.J.Simnick,A.Chilkoti,Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles thatabolish tumours after a single injection.Nat.Mater.8,993-999(2009).

Claims

1. Use of a polypeptide comprising an albumin polypeptide or an IgG Fc domain polypeptide operably linked to a collagen binding domain in the preparation of a medicament for reducing non-specific toxicity of a cancer treatment comprising a cytotoxic agent, wherein the collagen binding domain has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14, wherein the polypeptide is linked to the cytotoxic agent via a pH-cleavable linker.

2. The use according to claim 1, 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.

3. The use according to claim 1, wherein the 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.

4. The use according to any one of claims 1 to 3, wherein the cancer or tumor comprises a solid tumor.

5. The use according to any one of claims 1 to 4, wherein the cancer comprises breast cancer or colon cancer, or wherein the tumor comprises a tumor in the breast or colon.

6. The use according to any one of claims 1 to 5, wherein the polypeptide is administered together with one or more than one additional cancer therapy.

7. The use according to any one of claims 1 to 6, wherein the polypeptide is administered together with immunotherapy.

8. The use according to claim 7, wherein the immunotherapy is administered before, after or simultaneously with the polypeptide.

9. The use according to claim 7 or 8, wherein the immunotherapy comprises a checkpoint inhibitor therapy.

10. The use according to claim 9, wherein the checkpoint inhibitor therapy comprises a PD-1 antibody.

11. The use according to any one of claims 1 to 10, wherein the polypeptide is administered systemically.

12. The use according to claim 11, wherein the polypeptide is administered by intravenous injection.

13. The use according to any one of claims 1 to 12, wherein the cytotoxic agent is administered at a dose that is less than the minimum effective dose of the cytotoxic agent not linked to a collagen binding domain.

14. The use according to any one of claims 1 to 13, wherein the cytotoxic agent is administered at a dose that is less than the minimum effective dose of the cytotoxic agent conjugated to an albumin polypeptide or an IgG Fc domain polypeptide and not linked to a collagen binding domain.

15. The use according to any one of claims 1 to 14, wherein the linker comprises a hydrazone linker.

16. The use according to claim 15, wherein the linker is cleaved at a pH of less than 7.

4.

17. The use according to any one of claims 1 to 16, wherein the polypeptide is linked to the cytotoxic agent and / or the collagen binding polypeptide via a bifunctional linker.

18. The use according to any one of claims 1 to 17, wherein the cytotoxic agent comprises doxorubicin.

19. The use according to any one of claims 1 to 18, wherein the polypeptide is covalently linked to the collagen binding domain via a peptide bond.

20. The use according to any one of claims 1 to 19, wherein the collagen binding domain is located at the amino terminus of the albumin polypeptide or IgG Fc domain polypeptide.

21. The use according to any one of claims 1 to 20, wherein the polypeptide comprises a linker between the albumin polypeptide or IgG Fc domain polypeptide and the collagen binding domain.

22. The use according to claim 21, wherein the linker comprises glycine and serine amino acid residues.

23. The use according to claim 21 or 22, wherein the linker comprises GGGS, (GGGS) n or (GGGS)2.

24. The use according to any one of claims 1 to 23, wherein the polypeptide is not operably linked to a particle, nanovesicle or liposome.

25. The use according to any one of claims 1 to 24, wherein the polypeptide comprises at least two collagen binding domains.

26. Use according to any one of claims 1 to 25, wherein the ratio of cytotoxic agent to albumin is 3:1.

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