Endostatin peptides for the treatment of tumors, fibrosis, and acute lung injury - Patents.com

Abstract

The present invention provides compositions and methods for the treatment or prevention of tumor growth, fibrosis, acute lung injury, or a combination thereof. In one embodiment, the composition comprises a peptide derived from the C-terminal region of endostatin.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 241,274, filed September 7, 2021, the contents of which are incorporated by reference in their entirety herein. [Background technology]

[0002] The essence of many medical procedures and procedures involves the removal or destruction of harmful or unwanted tissue. Examples of such important procedures include the surgical removal of cancerous growths, the destruction of metastatic tumors by chemotherapy, and the reduction of glandular (e.g., prostate) hyperplasia.

[0003] There is clearly a need for effective agents that destroy harmful or unwanted cells and tissues, thus facilitating their removal or inhibiting their further growth, but which would have a primarily local effect with minimal or no systemic toxicity.

[0004] Endostatin, a 183 amino acid proteolytic cleavage fragment corresponding to the C-terminus of collagen 18, has antitumor activity without toxic side effects (O'Reilly et al. (1997) Cell, 88:277-285; Kisker et al. (2001) Cancer Res, 61:7669-7674; Dhanabal et al. (1999) Cancer Res, 59:189-197; Yoon et al. (1999) Cancer Res, 59:6251-6256; Folkman and Kalluri, (2003) Cancer Medicine, 6th edition, pp. 161-194. Hamilton: BC Decker Inc.). Several antiangiogenic activities have been reported for this protein, such as inhibition of endothelial cell proliferation, migration, and tube formation. This activity has been localized to the N-terminal region of endostatin. Endostatin also inhibits vascular endothelial growth factor (VEGF)-induced vascular permeability (Takahashi et al. (2003) Faseb J, 17:896-898). Endostatin inhibits endothelial cell migration by inhibiting focal adhesion kinase phosphorylation via binding to α5β1 integrin (Wickstrom et al. (2002) Cancer Res, 62:5580-5589). It has also been shown that cell surface glypicans are low affinity endostatin receptors (Karumanchi et al. (2001) Mol Cell, 7:811-822).Endostatin down-regulates c-myc activity (Shichiri and Hirata (2001) Faseb J, 15:1044-1053), cyclin-D1 activity (Hanai et al. (2002) J Biol Chem, 277:16464-16469), and RhoA activity (Wickstrom et al. (2003) J Biol Chem, 278:37895-37901), blocks VEGF signaling (Hajitou et al. (2002) Faseb J, 16:1802-1804, Kim et al. (2002) J Biol Chem, 277:27872-27879), and inhibits the wnt signaling pathway (Hanai et al. (2002) J Cell Biol, 158:529-539). In addition, endostatin has been shown to bind and inactivate metalloproteinases (Kim et al. (2000) Cancer Res, 60:5410-5413; Nyberg et al. (2003) J Biol Chem, 278:22404-22411; Lee et al. (2002) FEBS Lett, 519:147-152) and regulate a spectrum of genes that suppress angiogenesis (Abdollahi et al. (2004) Mol Cell, 13:649-663).

[0005] The crystal structures of both mouse and human endostatin have been solved (Hohenester et al. (1998) Embo J, 17:1656-1664; Ding et al. (1998) Proc Natl Acad Sci USA, 95:10443-10448) and show a non-covalently held dimer at the high concentrations required for crystallization (Ding et al. (1998) Proc Natl Acad Sci USA, 95:10443-10448). The presence of two disulfide bonds results in a highly folded structure. Endostatin binds one atom of zinc per monomer via three histidine residues (histidine residues 1, 3, and 11) and aspartic acid residue 76 at the N-terminus of the molecule. The heparin-binding property of endostatin is mediated by nonadjacent arginine residues clustered on the three-dimensional globular surface of the molecule (Sasaki et al. (1999) Embo J, 18:6240-6248).

[0006] Oligomeric endostatin (NC1 and dimer) has been shown to associate primarily with laminin in basement membranes (Javaherian et al. (2002) J Biol Chem, 277:45211-45218). This association may be important for some of the biological functions exhibited by endostatin. On the other hand, the heparin-binding properties of endostatin are manifested in its interaction with the cell surface. It is likely that several biological functions of endostatin are mediated by different regions of the protein.

[0007] Thus, there is a need in the art for novel, non-toxic compositions and methods for treating tumor growth, fibrosis, and acute lung injury. The present invention fulfills this unmet need. Summary of the Invention

[0008] In one embodiment, the present invention provides a composition comprising a therapeutic agent having anti-tumor activity, anti-fibrotic activity, anti-lung injury activity, or a combination thereof, wherein the agent is a C-terminal endostatin-derived peptide, an isolated nucleic acid encoding a C-terminal endostatin-derived peptide, or a variant, derivative, mutant, or fragment thereof.

[0009] In one embodiment, the C-terminal endostatin-derived peptide comprises an amino acid sequence selected from the group consisting of at least one of SEQ ID NOs: 8-27, fragments thereof, and variants thereof.

[0010] In one embodiment, the C-terminal endostatin derived peptide variant comprises at most five amino acid substitutions.

[0011] In one embodiment, the C-terminal endostatin derived peptide variant comprises consecutive amino acids.

[0012] In one embodiment, the C-terminal endostatin-derived peptide fragment comprises at least 8 consecutive amino acids of SEQ ID NOs:8-27.

[0013] In one embodiment, the therapeutic agent further comprises a second agent, wherein the second agent is an anti-cancer agent.

[0014] In one embodiment, the therapeutic agent further comprises a second agent, wherein the second agent is an anti-fibrotic agent.

[0015] The present invention also provides a method of treating or preventing a disease or disorder in a subject in need thereof. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an agent, wherein the agent is selected from the group consisting of a C-terminal endostatin derived peptide, an isolated nucleic acid encoding a C-terminal endostatin derived peptide, and a variant, derivative, mutant, or fragment thereof.

[0016] In one embodiment, the disease or disorder is cancer. In one embodiment, the cancer is prostate cancer, lung cancer, breast cancer, liver cancer, ovarian cancer, endometrial cancer, bladder cancer, colon cancer, lymphoma, skin cancer, pancreatic cancer, gastric cancer, myeloma, or glioma. In one embodiment, the method further comprises administering a second agent, and the second agent is an anti-cancer agent.

[0017] In one embodiment, the disease or disorder is a fibrotic disease or disorder or a fibrosis-associated disease or disorder. In one embodiment, the fibrotic disease or disorder or a fibrosis-associated disease or disorder is cardiac fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, familial pulmonary fibrosis, radiation-induced pulmonary fibrosis, coal workers' pneumoconiosis, asbestosis, bleomycin lung, sarcoidosis, silicosis, acute lung injury, ARDS, hypertrophic scars, keloid scars, liver cirrhosis, systemic scleroderma, localized scleroderma, morphea, vascular fibrosis, renal fibrosis, graft-versus-host disease, pulmonary fibrosis ... The primary fibrosis and / or fibrosis-related diseases include fibrosis as a result of chronic myelopathy (e.g., fibrosis as a result of chronic myelopathy (e.g., fibrosis caused by chronic myelopathy ...

[0018] In one embodiment, the disease or disorder is acute lung injury. In one embodiment, the acute lung injury is acute respiratory distress syndrome (ARDS), viral-induced acute lung injury, SARS, COVID-19, influenza-induced acute lung injury, acute lung injury due to sepsis, acute lung injury due to pneumonia, acute lung injury due to aspiration, acute lung injury due to trauma, acute lung injury due to blood transfusion, acute lung injury due to smoke, acute lung injury due to toxic gas inhalation, acute lung injury due to pancreatitis, acute lung injury due to drug overdose, acute lung injury due to burn, and ventilator-associated lung injury manifested with inflammation. [Brief description of the drawings]

[0019] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0020] [Figure 1] 1 shows the results of an exemplary experiment demonstrating descriptive statistics for viable tumor volume over time in control (n=4) and BioE4 groups of mice (n=5, days 0-5; n=4, days 7-15). Volume measurements are in mm.

[0021] [Figure 2A] 2A and 2B show the results of an exemplary experiment demonstrating mouse-specific tumor growth trajectories separated for control and BioE4 groups. [Figure 2B] 2A and 2B show the results of an exemplary experiment demonstrating mouse-specific tumor growth trajectories separated for control and BioE4 groups.

[0022] [Diagram 3] 1 shows the results of an exemplary experiment demonstrating parameter estimates for the general linear mixed model. BioE4 is an indicator variable, with 1 representing the BioE4 group and 0 representing the control group.

[0023] [Figure 4] Results of an exemplary experiment using the above model are shown, in which a direct comparison was made between BioE4 and control for mean tumor volume on day 15 and mean percentage change in tumor volume on day 15. Model-based estimates on day 15 show that mean tumor volume was significantly (p=0.0001) greater in the control group compared to the BioE4 group, and the mean percentage increase in volume on day 15 was also significantly (p=0.02) greater in the control group compared to the BioE4 group.

[0024] [Diagram 5]1 shows the results and descriptive statistics of an exemplary experiment for viable tumor volume over time in PBS (n=5), Bio96 (n=5), and BioE4-03 (n=4) groups of mice. Volume measurements are in mm.

[0025] [Figure 6A] 6A-6C show the results of an exemplary experiment demonstrating mouse-specific tumor growth trajectories separated for control, Bio96, and BioE4-03 groups. [Figure 6B] 6A-6C show the results of an exemplary experiment demonstrating mouse-specific tumor growth trajectories separated for control, Bio96, and BioE4-03 groups. [Figure 6C] 6A-6C show the results of an exemplary experiment demonstrating mouse-specific tumor growth trajectories separated for control, Bio96, and BioE4-03 groups.

[0026] [Figure 7] 1 shows the results of an exemplary experiment demonstrating parameter estimates for a general linear mixed model comparing PBS to Bio96 and Bio96E4-03.

[0027] [Figure 8]Shown are the results of an exemplary experiment using the above model, where direct comparisons were made between PBS control and Bio96, and between PBS and BioE4-03, for mean tumor volume on day 21 and mean percent change in tumor volume on day 21. Model-based estimates at day 21 show that mean tumor volume was significantly larger in the PBS control group compared to the Bio96 group (871.4 mm3 vs. 1492.1 mm3, p=0.003), and mean tumor volume was significantly larger in the PBS control group compared to the BioE4-03 group (767.7 mm3 vs. 1492.1 mm3, p=0.0009). On day 21, the mean rate of increase in volume was significantly greater in the PBS control group when compared to the Bio96 group (90.6 mm3 / day vs. 151.1 mm3 / day, p=0.004), and the mean rate of increase in volume was significantly greater in the PBS control group when compared to the BioE4-03 group (76.0 mm3 / day vs. 151.1 mm3 / day, p=0.0008).

[0028] [Figure 9] An exemplary histological analysis of tumors using hematoxylin and eosin (H&E) staining is shown.

[0029] [Figure 10] An exemplary histological analysis of tumors using Masson's Trichrome staining is shown.

[0030] [Figure 11-01] 1 shows an exemplary analysis of gene expression in A549 cells (human lung adenocarcinoma cells). [Figure 11-02] 1 shows an exemplary analysis of gene expression in A549 cells (human lung adenocarcinoma cells). [Figure 12-01] 1 shows an exemplary analysis of gene expression in HCT116T cells (human colon carcinoma cells). [Figure 12-02] 1 shows an exemplary analysis of gene expression in HCT116T cells (human colon carcinoma cells). [Figure 13] 1 shows an exemplary analysis of protein expression in normal lung fibroblasts using different endostatin peptides.

[0031] [Figure 14] An exemplary analysis of gene expression in fibroblasts from lung tissue of a normal donor treated with Bio96-17 is shown. Alpha smooth muscle actin RNA was measured by qRT-PCR in fibroblasts 48 hours after treatment with TGF-beta in the presence or absence of peptide Bio96-17.

[0032] [Figure 15] 1 shows an exemplary analysis of gene expression in fibroblasts derived from the lungs of a patient with systemic sclerosis (SSc) treated with Bio96-17.

[0033] [Figure 16] 1 shows an exemplary analysis of gene expression in fibroblasts derived from the lungs of a patient with SSc treated with BioE4-03.

[0034] [Figure 17] 1 shows an exemplary analysis of gene expression in fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis (IPF) treated with BioE4-03 or Bio96-17.

[0035] [Figure 18] 1 shows an exemplary analysis of protein expression in normal lung fibroblasts treated with increasing concentrations of E96-87.

[0036] [Figure 19] FIG. 1 shows an exemplary analysis of matrix metalloprotease (MMP)-1 gene expression in systemic sclerosis pulmonary fibrosis lung tissue in organ culture treated with different peptide fragments.

[0037] [Figure 20]1 shows an exemplary analysis of hydroxyproline levels in lung tissue of patients with SSc in organ culture treated with BioE4-03.

[0038] [Figure 21] 1 shows an exemplary analysis of hydroxyproline levels in lung tissue of patients with IPF in organ culture treated with BioE4-03.

[0039] [Figure 22] 1 shows an exemplary analysis of hydroxyproline levels in normal donor lung tissue in organ culture treated with BioE4-03.

[0040] [Diagram 23] 1 shows an exemplary analysis of hydroxyproline levels in normal donor lung tissue in organ culture treated with Bio96-17.

[0041] [Figure 24] 1 shows an exemplary analysis of secreted Col1A1 protein levels in normal donor lung tissue in organ culture treated with Bio96-17 following induction of fibrosis using TGF-beta.

[0042] [Diagram 25] 1 shows an exemplary analysis of Col1A1 and fibronectin (FN) gene expression in skin tissue from TGFbeta-induced fibrosis donors treated with BioE4-03.

[0043] [Figure 26] Figure 1 shows the effect of BioE4 given via oral gavage on tumor size (Y-axis) in mice. Treatment days are shown on the X-axis. Peptides were given twice a week. The red line indicates untreated tumor-bearing mice, and the blue line indicates BioE4-treated mice.

[0044] [Figure 27]Figure 1 shows the effect of orally administered E4 on bone marrow infiltration of CD138+ myeloma plasma cells 49 days after injection of bortezomib-resistant multiple myeloma cells (MM.1S BzR). E4 or vehicle was first administered 14 days after injection of MM.1S BzR cells and then twice weekly. The number of CD138+ myeloma plasma cells in the bone marrow was used as an index of tumor burden and peptide efficacy.

[0045] [Figure 28A] Figures 28A and 28B show the results of an exemplary experiment demonstrating the effect of BioE4-03 and E4-03 on IL6. Figure 28A shows the effect of biotinylated E4-03 on IL-6 mRNA in human lung adenocarcinoma cells (A549). [Figure 28B] Figures 28A and 28B show the results of an exemplary experiment demonstrating the effect of BioE4-03 and E4-03 on IL6. Figure 28B shows the effect of non-biotinylated E4-03 on IL-6 mRNA and protein expression in A549 cells. A549 cells were treated with BioE4-03 or E4-03 for 48 hours followed by qPCR analysis for mRNA expression and ELISA analysis for protein expression after 72 hours of treatment.

[0046] [Figure 29-01] LC-MS / MS results of the top of the pepsin digestion of BioE4-03 (SEQ ID NO: 2) are shown. The X-axis of each chart, indicated by the SEQ ID NOs listed in Table 1 of Example 4 below, indicates the number of peptide spectrum matches for each treatment group indicated on the Y-axis: "Feed" indicates undigested BioE4-03 diluted in 0.1% formic acid, "WKSL" indicates undigested BioE4-03 diluted in 0.1% formic acid aliquoted with a ZipTip®, TO indicates BioE4-03 aliquoted immediately after the addition of pepsin (0 min digestion), T15 indicates BioE4-03 aliquoted 15 min after pepsin digestion, and T45 indicates BioE4-03 aliquoted 45 min after pepsin digestion (T45). [Figure 29-02]LC-MS / MS results of the top of the pepsin digestion of BioE4-03 (SEQ ID NO: 2) are shown. The X-axis of each chart, indicated by the SEQ ID NOs listed in Table 1 of Example 4 below, indicates the number of peptide spectrum matches for each treatment group indicated on the Y-axis: "Feed" indicates undigested BioE4-03 diluted in 0.1% formic acid, "WKSL" indicates undigested BioE4-03 diluted in 0.1% formic acid aliquoted with a ZipTip®, TO indicates BioE4-03 aliquoted immediately after the addition of pepsin (0 min digestion), T15 indicates BioE4-03 aliquoted 15 min after pepsin digestion, and T45 indicates BioE4-03 aliquoted 45 min after pepsin digestion (T45). [Figure 29-03] LC-MS / MS results of the top of the pepsin digestion of BioE4-03 (SEQ ID NO: 2) are shown. The X-axis of each chart, indicated by the SEQ ID NOs listed in Table 1 of Example 4 below, indicates the number of peptide spectrum matches for each treatment group indicated on the Y-axis: "Feed" indicates undigested BioE4-03 diluted in 0.1% formic acid, "WKSL" indicates undigested BioE4-03 diluted in 0.1% formic acid aliquoted with a ZipTip®, TO indicates BioE4-03 aliquoted immediately after the addition of pepsin (0 min digestion), T15 indicates BioE4-03 aliquoted 15 min after pepsin digestion, and T45 indicates BioE4-03 aliquoted 45 min after pepsin digestion (T45). [Figure 29-04] LC-MS / MS results of the top of the pepsin digestion of BioE4-03 (SEQ ID NO: 2) are shown. The X-axis of each chart, indicated by the SEQ ID NOs listed in Table 1 of Example 4 below, indicates the number of peptide spectrum matches for each treatment group indicated on the Y-axis: "Feed" indicates undigested BioE4-03 diluted in 0.1% formic acid, "WKSL" indicates undigested BioE4-03 diluted in 0.1% formic acid aliquoted with a ZipTip®, TO indicates BioE4-03 aliquoted immediately after the addition of pepsin (0 min digestion), T15 indicates BioE4-03 aliquoted 15 min after pepsin digestion, and T45 indicates BioE4-03 aliquoted 45 min after pepsin digestion (T45). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] In one aspect, the present invention is directed to methods and compositions for the treatment, inhibition, prevention, or reduction of tumor growth. In another aspect, the present invention is directed to methods and compositions for the treatment, inhibition, prevention, or reduction of fibrosis or fibrosis-related diseases or disorders. In one aspect, the present invention is directed to methods and compositions for the treatment, inhibition, prevention, or reduction of tumor growth in combination with fibrosis or fibrosis-related diseases or disorders. In another aspect, the present invention is directed to methods and compositions for the treatment, inhibition, prevention, or reduction of acute lung injury. In one embodiment, the composition comprises an agent derived from the C-terminal region of endostatin, e.g., an isolated nucleic acid, isolated peptide, small molecule, peptidomimetic, etc. In one embodiment, the composition comprises a fragment derived from the C-terminal region of full-length endostatin. In one embodiment, the composition comprises a recombinant fragment derived from the C-terminal region of endostatin. In one embodiment, the composition comprises a fragment of endostatin that can be generated via natural digestion in vivo.

[0048] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0049] As used herein, each of the following terms has the meaning associated with it in this section.

[0050] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0051] As used herein, when referring to a measurable value, e.g., amount, duration, etc., "about" is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0052] The term "abnormal" when used in the context of an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time, etc.) from an organism, tissue, cell, or component thereof that exhibits the "normal" (expected) respective characteristic. A characteristic that is normal or expected in one cell or tissue type may be abnormal in a different cell or tissue type.

[0053] A disease or disorder is "alleviated" if the severity of the signs or symptoms of the disease or disorder, the frequency with which the patient experiences such signs or symptoms, or both, are reduced. "Alleviating" certain cancers and / or their pathologies includes breaking down the structural integrity or connective tissue of a tumor, e.g., so that the tumor size is reduced when compared to the tumor size before treatment. "Alleviating" the metastasis of cancer includes reducing the rate at which the cancer spreads to other organs.

[0054] As used herein, "autologous" refers to biological material derived from the same individual into which the material is subsequently reintroduced.

[0055] As used herein, "allogenic" refers to biological material derived from a genetically distinct individual of the same species as the individual into which the material is to be introduced.

[0056] The terms "cell" and "population of cells" are used interchangeably and refer to a plurality of cells, i.e., two or more cells. The population may be a pure population containing one cell type. Alternatively, the population may contain two or more cell types. The present invention does not limit the number of cell types that a cell population may contain.

[0057] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in preventing the development of tumors in the first place.

[0058] As used herein, the term "cancer" is defined as a disease characterized by the abnormal growth of aberrant cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, gastric cancer, myeloma, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, sarcoma, etc.

[0059] A "disease" is a condition in the health of an animal where the animal is unable to maintain homeostasis and where, if the disease is not ameliorated, the animal's health continues to deteriorate.

[0060] In contrast, a "disorder" in an animal is a state in which the animal is able to maintain homeostasis, but in which the animal's health status is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration in the animal's health.

[0061] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide (e.g., gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0062] An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind or deliver the compound.

[0063] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0064] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0065] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0066] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0067] As used herein, the terms "fibrotic disease", "fibrotic disorder", "fibrosis-associated disease" and "fibrosis-associated disorder" refer to conditions involving fibrosis in one or more tissues. As used herein, the term "fibrosis" refers to the formation of fibrotic tissue as a repair or reactive process, rather than as a normal component of an organ or tissue. Fibrosis is characterized by fibroblast accumulation and collagen deposition that exceeds normal deposition in any particular tissue. As used herein, the term "fibrosis" is used synonymously with "fibroblast accumulation and collagen deposition."

[0068] As used herein, the term "anti-fibrotic" activity refers to the ability of an active agent to prevent excessive pathological accumulation of collagen scar or connective tissue in various body structures and organs (usually caused by some injury, allergy, infection, or by some inherited genetic abnormality), or to promote the non-surgical removal or biological dissolution of existing excessive pathological accumulations of fibrotic collagen tissue.

[0069] "Homology" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is performed when the two sequences are aligned to obtain maximum homology.

[0070] As used herein, the term "inhibit" means to suppress or block an activity or function by at least about 10 percent compared to a control value. In some examples, the activity is suppressed or blocked by 50%, 75%, 90%, or 95% compared to a control value.

[0071] As used herein, "instructional material" includes publications, records, drawings, or any other medium of expression that can be used to communicate the utility of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be affixed to a container containing the nucleic acids, peptides, and / or compositions of the invention or may be shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compounds are used cooperatively by the recipient.

[0072] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment (such as, for example, a host cell).

[0073] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0074] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0075] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0076] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0077] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™) and by synthetic means. Furthermore, the "polynucleotide" or "nucleic acid" of the present invention includes both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules.

[0078] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0079] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0080] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate the specific transcription of a polynucleotide sequence.

[0081] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0082] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0083] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if an inducer corresponding to the promoter is present in the cell.

[0084] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0085] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is preferably a mammal, such as non-primates (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys and humans), and most preferably a human.

[0086] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of a pathology, with the intent of reducing or eliminating those signs.

[0087] As used herein, "treating a disease or disorder" means reducing the frequency with which a patient experiences a symptom of the disease or disorder.

[0088] The phrase "therapeutically effective amount," as used herein, refers to an amount sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, reduce, or reverse) a disease, disorder, or condition, e.g., alleviate the symptoms of such disease.

[0089] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0090] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0091] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a fixed limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as each individual number within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose each individual number within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This is true regardless of the breadth of the range.

[0092] explanation The present invention is based in part on the discovery that peptides derived from the C-terminal region of endostatin can be used to treat or prevent tumor growth, fibrosis, and acute lung injury. Thus, in one embodiment, the present invention includes compositions comprising C-terminal endostatin peptides, polypeptides, fragments, etc., having anti-tumor activity, anti-fibrotic activity, anti-lung injury activity, or a combination thereof. In one embodiment, nucleotides encoding these peptides, host cells transformed with the nucleotides, and methods of using these peptides and nucleotides are included in the present invention.

[0093] In one embodiment, the present invention provides compositions for inhibiting tumor growth, fibrosis, acute lung injury, or a combination thereof.For example, the present invention is based in part on the identification of regions in endostatin that inhibit tumor growth, fibrosis, and acute lung injury.Endostatin fragments and peptides derived from endostatin thereof are demonstrated herein to mimic endostatin in inhibiting tumor growth and fibrosis.Furthermore, endostatin fragments and peptides derived from endostatin thereof are demonstrated herein to reduce the level of IL-6, which is increased in lung tissue of subjects with acute lung injury and promotes cytokine storm.

[0094] In certain embodiments, the endostatin derived peptide is 96: ATGQASSLL (SEQ ID NO: 1), E4-03: SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 2), E4: SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT (SEQ ID NO: 3), 96-17: ATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 4), 96-87: ATGQASSLLGGRLLGQ (SEQ ID NO: 5), 91-96: SYCETWRTEAPSATGQASSLL (SEQ ID NO: 6), 91-97, SYCETWRTEAPSATGQASSLLGGRLLGQ (SEQ ID NO: 7), or a variant or fragment thereof.

[0095] In certain embodiments, the endostatin-derived peptide comprises the amino acid sequence of any one of SEQ ID NOs: 8-27, or a variant or fragment thereof.

[0096] In one embodiment, the present invention provides a method for treating or preventing tumor growth. The method can be used to treat or prevent tumors, for example, in the lung, breast, stomach, pancreas, prostate, bladder, bone, ovary, skin, kidney, paranasal sinuses, colon, intestine, stomach, rectum, esophagus, blood, brain and its coverings, spinal cord and its coverings, muscle, connective tissue, adrenal gland, parathyroid gland, thyroid gland, uterus, testes, pituitary gland, reproductive organs, liver, gallbladder, eye, ear, nose, throat, tonsils, mouth, lymph nodes and lymphatic system, and other organs.

[0097] In one embodiment, the present invention provides a method for treating or preventing fibrosis, which can be used to treat or prevent fibrosis in, for example, the lung, breast, stomach, pancreas, prostate, bladder, bone, ovaries, skin, kidneys, sinuses, colon, intestines, stomach, rectum, esophagus, blood, brain and its coverings, spinal cord and its coverings, muscle, connective tissue, adrenal glands, parathyroid glands, thyroid gland, uterus, testes, pituitary gland, reproductive organs, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes and lymphatic system, and other organs.

[0098] In one embodiment, the present invention provides a method for treating or preventing acute lung injury. It is demonstrated herein that endostatin fragments and its endostatin-derived peptides reduce IL-6 secretion from lung-derived cells. IL-6 levels are increased in lung tissues of patients with acute lung injury, promoting cytokine storm. Thus, the present invention provides a method for treating acute respiratory distress syndrome, including acute lung injury, ARDS, virus-induced acute lung injury, SARS, COVID-19, influenza-induced acute lung injury, sepsis, pneumonia, aspiration, trauma, blood transfusion, smoke, toxic gas inhalation, pancreatitis, drug overdose, acute lung injury due to burns, and ventilator-associated lung injury manifested with other lung injury or inflammation.

[0099] composition In one aspect, the present invention provides a composition comprising an agent derived from the C-terminal region of endostatin having anti-tumor activity, anti-fibrotic activity, or a combination thereof. Exemplary agents include, but are not limited to, isolated nucleic acids, vectors, isolated peptides, peptidomimetics, small molecules, etc.

[0100] In one embodiment, the composition of the present invention comprises an isolated peptide derived from the C-terminal region of endostatin, or a biologically functional fragment thereof. The composition may comprise any isoform of the C-terminal endostatin peptide of the present invention, including, for example, endostatin from any organism.

[0101] In certain embodiments, the isolated peptide of the composition comprises: 96: ATGQASSLL (SEQ ID NO: 1), E4-03: SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 2), E4: SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT (SEQ ID NO: 3), 96-17: ATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 4), 96-87: ATGQASSLLGGRLLGQ (SEQ ID NO: 5), 91-96: SYCETWRTEAPSATGQASSLL (SEQ ID NO: 6), 91-97, SYCETWRTEAPSATGQASSLLGGRLLGQ (SEQ ID NO: 7), or a variant or fragment thereof.

[0102] In certain embodiments, the endostatin-derived peptide comprises the amino acid sequence of any one of SEQ ID NOs: 8-27, or a fragment or variant thereof.

[0103] In one embodiment, the peptide is biotinylated at the N-terminus, amidated at the C-terminus, or a combination thereof.

[0104] In certain examples, as described herein, N-terminally biotinylated peptides are presented with the prefix "Bio". Thus, in one embodiment, Bio96 refers to SEQ ID NO: 1, which is biotinylated at its N-terminus. In one embodiment, BioE4-03 refers to SEQ ID NO: 2, which is biotinylated at its N-terminus. In one embodiment, BioE4 refers to SEQ ID NO: 3, which is biotinylated at its N-terminus. In one embodiment, Bio96-17 refers to SEQ ID NO: 4, which is biotinylated at its N-terminus. In one embodiment, Bio96-87 refers to SEQ ID NO: 5, which is biotinylated at its N-terminus. In one embodiment, Bio91-96 refers to SEQ ID NO: 6, which is biotinylated at its N-terminus. In one embodiment, Bio91-97 refers to SEQ ID NO: 7, which is biotinylated at its N-terminus. In certain embodiments, the biotinylated peptides are amidated at the C-terminus, as described herein.

[0105] In one embodiment, the composition comprises a fragment of full-length endostatin derived from the m region of endostatin. Fragments of full-length C-terminal region endostatin peptides include, but are not limited to, fragments of SEQ ID NOs: 1-27 that contain at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 consecutive amino acids of the full-length C-terminal region endostatin peptides set forth in SEQ ID NOs: 1-27. In one embodiment, the composition comprises a recombinant C-terminal fragment of endostatin.

[0106] In one embodiment, the composition comprises a fragment of any one of SEQ ID NOs: 1-27. For example, in certain embodiments, the composition comprises a fragment or variant of any one of SEQ ID NOs: 1-27, wherein the fragment or variant is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acid residues in length.

[0107] In one embodiment, the present invention comprises C-terminal endostatin polypeptides and variants thereof. In one embodiment, the endostatin derived peptide comprises a fragment of endostatin that mimics the ability of endostatin to inhibit tumor growth, fibrosis, or a combination thereof. In one embodiment, the endostatin derived peptide comprises a fragment of endostatin that reduces, treats, or prevents acute lung injury. In one embodiment, the endostatin derived peptide comprises a derivative of an endostatin fragment.

[0108] In one embodiment, the endostatin fragment comprises one or more peptides that may be derived as a result of endostatin digestion. In one embodiment, the digestion occurs naturally in a mammalian stomach. In one embodiment, the digestion occurs in vitro via an enzyme protein. In one embodiment, the enzyme protein comprises pepsin. In one embodiment, the digested endostatin comprises BioE4-03 (SEQ ID NO:2).

[0109] In one embodiment, the endostatin fragment or endostatin-derived peptide comprises a peptide that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more peptides selected from the group consisting of SEQ ID NOs:8-27 shown in Table 1 of Example 4 below. In one embodiment, the endostatin fragment comprises a peptide that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of one or more peptides selected from the group consisting of SEQ ID NOs:8-27. In one embodiment, the endostatin fragment comprises a peptide at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more peptides that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of one or more peptides selected from the group consisting of SEQ ID NOs: 8-27. In one embodiment, the endostatin fragment comprises one or more peptides selected from the group consisting of SEQ ID NOs: 8-27.

[0110] In one embodiment, the composition comprises a fragment of any one of SEQ ID NOs: 8-27. For example, in certain embodiments, the composition comprises a fragment or variant of any one of SEQ ID NOs: 8-27, wherein the fragment or variant is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acid residues in length.

[0111] In some embodiments, the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 8-27, or a fragment or variant thereof. In some embodiments, the peptide comprises a fragment that is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 consecutive amino acids of any one of SEQ ID NOs: 8-27. In some embodiments, the peptide comprises any one of SEQ ID NOs: 8-27 with at most 1, 2, 3, 4, or 5 amino acid substitutions. In one embodiment, the peptide comprises a fragment that is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids of any one of SEQ ID NOs: 8-27 having at most 1, 2, 3, 4, or 5 amino acid substitutions.

[0112] The peptide of the present invention can be produced using chemical methods.For example, peptide can be synthesized by solid phase technology (Roberge JY et al (1995) Science 269:202-204), cleaved from resin, and purified by preparative high performance liquid chromatography.Automatic synthesis can be achieved, for example, using ABI 431 A Peptide Synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0113] In one embodiment, the present invention includes any form of peptide that includes amino acids derived from the C-terminal region of endostatin protein, but does not include full-length endostatin protein or the N-terminal region of endostatin protein. In one embodiment, the present invention includes an amino acid sequence that has substantial homology to the C-terminal endostatin or C-terminal endostatin derived peptides disclosed herein. In certain embodiments, a peptide that is "substantially homologous" is about 50% homologous, about 70% homologous, about 80% homologous, about 85% homologous, about 90% homologous, about 91% homologous, about 92% homologous, about 93% homologous, about 94% homologous, about 95% homologous, about 96% homologous, about 97% homologous, about 98% homologous, or about 99% homologous to the amino acid sequence of the C-terminal region of endostatin.

[0114] Alternatively, the peptides may be produced by recombinant means or by cleavage from a longer polypeptide. The composition of the peptide may be confirmed by amino acid analysis or sequencing.

[0115] Variants of the peptides according to the invention may be (i) those in which one or more of the amino acid residues are replaced with a conserved or non-conserved amino acid residue, which may or may not be encoded by the genetic code, (ii) those in which one or more modified amino acid residues are present, e.g., residues modified by attachment of a substituent group, (iii) those in which the peptide is an alternative splice variant of the peptide of the invention, (iv) fragments of the peptide, and / or (v) those in which the peptide is fused to another peptide, such as a leader sequence or secretion sequence, or a sequence used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag or immunoglobulin Fc region). Fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of the skilled artisan from the teachings herein.

[0116] As known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes to the sequence of a second polypeptide. A variant is defined to include a peptide sequence that differs from the original sequence, preferably by less than 40% of the residues per segment of interest, more preferably by less than 25% of the residues per segment of interest, more preferably by less than 10% of the residues per segment of interest, and most preferably by only a few residues per segment of interest, and at the same time is sufficiently homologous to the original sequence to preserve the functionality of the original sequence and / or its ability to inhibit tumor growth, fibrosis, or a combination thereof. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0117] The peptides of the present invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, phosphorylation, protein folding, and proteolytic processing. Some modifications or processing events require the introduction of additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation can be examined by adding dog microsomal membranes or Xenopus egg extracts to standard translation reactions (US Pat. No. 6,103,489).

[0118] The peptides of the invention can include unnatural amino acids, formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[0119] The peptide or protein of the present invention can be combined with other molecules, such as proteins, to prepare fusion proteins. This can be achieved, for example, by synthesis of N-terminal or C-terminal fusion proteins, provided that the resulting fusion protein retains the functionality of the antitumor activity, antifibrotic activity, or a combination thereof, of the C-terminal endostatin peptide of the present invention. In one embodiment, the peptide or protein of the present invention can be fused to biotin.

[0120] The peptides or proteins of the invention may be phosphorylated using conventional methods, such as those described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0121] Cyclic derivatives of the peptides of the invention are also part of the invention. Cyclization may allow the peptide to adopt a more favorable conformation for association with other molecules. Cyclization can be achieved using techniques known in the art. For example, a disulfide bond can be formed between two appropriately spaced components with free sulfhydryl groups, or an amide bond can be formed between an amino group of one component and a carboxyl group of another component. Cyclization can also be achieved using azobenzene-containing amino acids, as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The bond-forming component may be the side chain of an amino acid, a non-amino acid component, or a combination of the two. In certain embodiments of the invention, the cyclic peptide may include a beta turn at the correct position. A beta turn can be introduced into the peptides of the invention by adding the amino acid Pro-Gly at the correct position.

[0122] It may be desirable to produce cyclic peptides that are more flexible than those containing peptide bond linkages as described above. More flexible peptides can be prepared by introducing cysteines at the right and left positions of the peptide and forming disulfide bridges between the two cysteines. The two cysteines are positioned so as not to distort the beta sheets and turns. The peptide is more flexible as a result of the length of the disulfide linkages and the fewer number of hydrogen bonds in the beta sheet portion. The relative flexibility of the cyclic peptides can be determined by molecular dynamics simulations.

[0123] The present invention also relates to peptides comprising amino acids of the C-terminal region of endostatin protein, but not including the full-length endostatin protein or the N-terminal region of endostatin protein, whereby the peptides may be fused or incorporated to a targeting protein and / or a targeting domain that can direct the chimeric protein to a desired cellular component or cell type or tissue. The chimeric protein may also include additional amino acid sequences or domains. The chimeric protein is recombinant in the sense that the various components are from different sources and therefore are not found together in nature (i.e., heterologous).

[0124] In one embodiment, the targeting domain can be a transmembrane domain, a membrane-binding domain, or a sequence that directs the protein to associate with, for example, a vesicle or the nucleus. In one embodiment, the targeting domain can target the peptide to a specific cell type or tissue. For example, the targeting domain can be an antibody against a cell surface ligand or a cell surface antigen (e.g., a tumor antigen) of the target tissue. The targeting domain can target the peptide of the present invention to a cellular component.

[0125] The peptides of the present invention can be synthesized by conventional techniques. For example, the peptides or chimeric proteins can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods use either solid phase synthesis or liquid phase synthesis methods (see, for example, JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2001 for a review of solid phase synthesis techniques). ndEd., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and RB Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254; and for conventional solution synthesis see M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984 and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1). By way of example, the peptides of the invention can be synthesized by directly incorporating phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry.

[0126] N- or C-terminal fusion proteins comprising the peptide or chimeric protein of the present invention linked to another molecule can be prepared by fusing the N- or C-terminus of the peptide or chimeric protein with the sequence of a selected protein or selectable marker having a desired biological function via recombinant techniques. The resulting fusion protein contains a C-terminal endostatin peptide or endostatin-derived peptide fused to a selected protein or marker protein as described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc. For example, the polypeptide can be linked to the CH1, CH2, and / or CH3 domains of a heavy chain. If the constant region is from a light chain, it can be from a kappa or lambda light chain. If the constant region is from a heavy chain, it can be from any one of the following classes of antibodies: IgG, IgA, IgE, IgD, and IgM. The IgG can be IgG1, IgG2, IgG3, or IgG4. The constant domain can be an Fc fragment. The constant domain can be derived from a mammalian antibody, such as a human antibody. Soluble receptor-IgG fusion proteins are common immunological reagents, and methods for their construction are known in the art (see, for example, U.S. Pat. Nos. 5,225,538, 5,726,044, 5,707,632, 750,375, 5,925,351, 6,406,697, and Bergers et al. Science 1999 284:808-12). In one example, the immunoglobulin is the constant portion of the heavy chain of human IgG, particularly IgG1, where dimerization between the two heavy chains occurs at the hinge region. It is recognized that inclusion of the CH2 and CH3 domains of the Fc region as part of a fusion polypeptide increases the in vivo circulatory half-life of a polypeptide comprising the Fc region, and the in vivo circulatory half-life of oligomers or dimers comprising the polypeptide.

[0127] The peptides of the present invention can be developed using biological expression systems. The use of these systems allows the creation of large libraries of random peptide sequences and screening of these libraries for peptide sequences that bind to specific proteins. Libraries can be created by cloning synthetic DNA encoding random peptide sequences into appropriate expression vectors (see Christian et al 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries can also be constructed by simultaneous synthesis of overlapping peptides (see U.S. Patent No. 4,708,871).

[0128] The peptides and chimeric proteins of the present invention can be converted into pharmaceutical salts by reaction with inorganic acids, such as, for example, hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, or organic acids, such as, for example, formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0129] In one embodiment, the present invention provides a composition comprising an isolated nucleic acid encoding a C-terminal endostatin, an endostatin-derived peptide, or a biologically functional fragment thereof.

[0130] In one embodiment, the isolated nucleic acid sequence encodes a C-terminal endostatin. In various embodiments, the isolated nucleic acid sequence is 96: ATGQASSLL (SEQ ID NO: 1), E4-03: SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 2), E4: SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT (SEQ ID NO: 3), 96-17: ATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 4), 96-87: ATGQASSLLGGRLLGQ (SEQ ID NO: 5), 91-96: SYCETWRTEAPSATGQASSLL (SEQ ID NO: 6), 91-97, encoding an endostatin-derived peptide comprising the amino acid sequence of SYCETWRTEAPSATGQASSLLGGRLLGQ (SEQ ID NO: 7), or a variant or fragment thereof. In one embodiment, the peptide is biotinylated at the N-terminus, amidated at the C-terminus, or a combination thereof.

[0131] Additionally, the present invention encompasses isolated nucleic acids encoding peptides having substantial homology to the C-terminal endostatin or endostatin-derived peptides disclosed herein. In certain embodiments, the isolated nucleic acid sequence encodes a C-terminal endostatin or endostatin-derived peptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-7.

[0132] In one embodiment, the isolated nucleic acid encoding a peptide having substantial homology to a C-terminal endostatin or endostatin-derived peptide comprises a nucleic acid sequence encoding a peptide at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more peptides selected from the group consisting of SEQ ID NOs:8-27 shown in Table 1 of Example 4 below. In one embodiment, the isolated nucleic acid comprises a nucleic acid sequence encoding a peptide at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of one or more peptides selected from the group consisting of SEQ ID NOs:8-27. In one embodiment, the isolated nucleic acid comprises a nucleic acid sequence encoding a peptide at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more peptides that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of one or more peptides selected from the group consisting of SEQ ID NOs: 8-27. In one embodiment, the isolated nucleic acid comprises a nucleic acid sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs: 8-27.

[0133] Isolated nucleic acid sequences encoding C-terminal endostatin or endostatin-derived peptides can be obtained using any of a number of recombinant methods known in the art, such as by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to contain the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0134] The isolated nucleic acid may include any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition includes an isolated DNA molecule, including, for example, an isolated cDNA molecule encoding C-terminal endostatin or endostatin-derived peptide, or a functional fragment thereof. In one embodiment, the composition includes an isolated RNA molecule encoding C-terminal endostatin or endostatin-derived peptide, or a functional fragment thereof.

[0135] The nucleic acid molecules of the invention may be modified to improve stability in serum or growth medium for cell culture. Modifications can be added to enhance the stability, functionality, and / or specificity of the nucleic acid molecules of the invention and minimize immunostimulatory properties. For example, to enhance stability, the 3'-residues may be stabilized against degradation, e.g., they may be selected to consist of purine nucleotides, in particular adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogs, e.g., substitution of uridine by 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0136] In one embodiment of the invention, the nucleic acid molecule may contain at least one modified nucleotide analogue. For example, the termini may be stabilized by incorporating modified nucleotide analogues.

[0137] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., containing modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA can be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides, the phosphoester group attached to the adjacent ribonucleotide is replaced with a modified group, e.g., a phosphothioate group. In preferred sugar-modified ribonucleotides, the 2'OH-group is replaced with a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0138] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine, are suitable. It should be noted that the above modifications can be combined.

[0139] In some examples, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modifications of one or more nucleotides. In certain embodiments, the nucleic acid molecule of the present invention may have enhanced resistance to nucleases. For increased nuclease resistance, the nucleic acid molecule may comprise, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) may be modified or replaced with a number of different "oxy" or "deoxy" substituents. For increased nuclease resistance, the nucleic acid molecule of the present invention may comprise 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene bridged nucleic acids, and certain nucleobase modifications, e.g., 2-amino-A, 2-thio (e.g., 2-thio-U), and G-clamp modifications, may also increase binding affinity to the target.

[0140] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0141] In certain embodiments, the nucleic acid molecules of the invention preferably have one or more of the following properties: Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA, as well as RNA and DNA modified, for example, to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the components of nucleic acid, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those naturally occurring, preferably in the human body. Rare or unusual but naturally occurring RNAs are referred to in the art as modified RNAs, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often referred to as modified RNAs, are typically the result of post-transcriptional modifications and are within the scope of the term unmodified RNA as used herein. Modified RNAs, as used herein, refer to molecules in which one or more of the components of nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those naturally occurring, preferably those that are in the human body. Although they are referred to as "modified RNAs", they of course include molecules that are not strictly speaking RNAs due to the modifications. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with non-ribophosphate constructs that allow the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of the ribophosphate backbone).

[0142] Modifications of the nucleic acids of the invention can be at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0143] The invention also includes vectors into which an isolated nucleic acid of the invention has been inserted. The art is replete with suitable vectors useful in the present invention.

[0144] In brief summary, expression of natural or synthetic nucleic acid encoding C-terminal endostatin or endostatin-derived peptides is typically achieved by operably linking the nucleic acid encoding C-terminal endostatin or endostatin-derived peptides or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and optionally integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0145] The vector of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0146] The isolated nucleic acid of the present invention can be cloned into several types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. The vectors of the present invention include any vector suitable for expression in eukaryotes, including plants, animals, and fungi, and prokaryotes, including archaea and bacteria.

[0147] Furthermore, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584, WO 01 / 29058, and U.S. Patent No. 6,326,193).

[0148] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0149] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term stable uptake of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In one embodiment, the composition comprises a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors have several characteristics that make them ideally suited for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0150] In one embodiment, a replication-deficient adenovirus can be used. In one embodiment, a replication-deficient serotype 5 adenovirus can be used.

[0151] In certain embodiments, the vector also comprises conventional control elements operably linked to the transgene in a manner that allows transcription, translation and / or expression of the transgene in cells transfected with a plasmid vector produced according to the invention or infected with a virus produced according to the invention. As used herein, "operably linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences acting in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, transcription termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product, if desired. Numerous expression control sequences, including promoters that are natural, constitutive, inducible, and / or tissue-specific, are known in the art and can be utilized.

[0152] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but some promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved even when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp, beyond which activity begins to decrease. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0153] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of achieving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the Elongation Growth Factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of the polynucleotide sequence to which it is operably linked when expression of the polynucleotide sequence is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0154] Enhancer sequences found in vectors also regulate the expression of genes contained therein. Typically, enhancers bind to protein factors to enhance the transcription of genes. Enhancers can be located upstream or downstream of the gene they regulate. Enhancers can also be tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vectors of the present invention contain one or more enhancers to boost the transcription of genes present in the vector.

[0155] To assess the expression of C-terminal endostatin or endostatin-derived peptides, the expression vector introduced into the cells can also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that are desired to be transfected or infected through the viral vector. In other embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0156] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that encode a polypeptide that is not present in or expressed by the recipient organism or tissue and whose expression is manifested by some easily detectable property (e.g., enzymatic activity). Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or may be obtained commercially. In general, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0157] Methods for introducing and expressing genes in cells are known in the art. In relation to expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0158] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0159] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0160] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0161] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained or complexed with micelles, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist as micelles or in bilayer structures with a "collapsed" structure. They may also simply be scattered in the solution, and may form aggregates that are not uniform in size or shape.Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids.For example, lipids include the lipid droplets that naturally occur in cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0162] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO, dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that have structures in solution that differ from normal vesicular structures are also encompassed. For example, lipids may adopt micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0163] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, RT-qPCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blot) or by the assays described herein to identify agents that fall within the scope of the present invention.

[0164] In one embodiment, the present invention provides a delivery vehicle comprising C-terminal endostatin or endostatin-derived peptides or nucleic acid molecules encoding C-terminal endostatin or endostatin-derived peptides. Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymersomes, liposomes, and micelles. For example, in certain embodiments, the delivery vehicle is loaded with C-terminal endostatin or endostatin-derived peptides or nucleic acid molecules encoding C-terminal endostatin or endostatin-derived peptides. In certain embodiments, the delivery vehicle provides controlled, delayed, or continuous release of its loaded cargo. In certain embodiments, the delivery vehicle comprises a targeting moiety that targets the delivery vehicle to a treatment site.

[0165] The present invention also provides a scaffold or matrix composition comprising C-terminal endostatin or endostatin-derived peptide, a nucleic acid molecule encoding C-terminal endostatin or endostatin-derived peptide, a cell producing C-terminal endostatin or endostatin-derived peptide, or a combination thereof. In another embodiment, C-terminal endostatin or endostatin-derived peptide, a cell producing C-terminal endostatin or endostatin-derived peptide, a nucleic acid molecule encoding C-terminal endostatin or endostatin-derived peptide, or a combination thereof is applied to the surface of the scaffold. The scaffold of the present invention may be of any type known in the art. Non-limiting examples of such scaffolds include hydrogels, electrospun scaffolds, foams, meshes, sheets, patches, and sponges.

[0166] The present invention also provides pharmaceutical compositions comprising one or more of the compositions described herein.The formulations can be used in admixture with conventional excipients, i.e., pharma-ceutically acceptable organic or inorganic carrier substances suitable for administration to wounds or treatment sites.The pharmaceutical compositions can be sterilized and, if desired, can be mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for affecting osmotic buffers, coloring agents, and / or aromatic agents.They can also be combined with other active agents, such as other analgesics, if desired.

[0167] Administration of the compositions of the invention can be by, for example, parenteral, intravenous, intratumoral, subcutaneous, intramuscular, intratracheal or intraperitoneal injection, inhalation, infusion, or by any other acceptable systemic method.

[0168] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrating agents; binders; lubricants; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0169] The compositions of the present invention may contain a preservative at about 0.005% to 2.0% of the total weight of the composition. Preservatives are used to prevent spoilage when exposed to contaminants in the environment. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0170] In some embodiments, the composition includes an antioxidant and a chelating agent that inhibits degradation of one or more components of the composition. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol, and ascorbic acid in the preferred range of about 0.01% to 0.3% by weight of the total weight of the composition, more preferably BHT in the range of 0.03% to 0.1% by weight. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total weight of the composition. Particularly preferred chelating agents include edetate (e.g., disodium edetate) and citric acid in the range of about 0.01% to 0.20% by weight of the total weight of the composition, more preferably in the range of 0.02% to 0.10% by weight. Chelating agents are useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. BHT and disodium edetate are particularly preferred antioxidants and chelating agents, respectively, for some compounds, although other suitable and equivalent antioxidants and chelating agents that may be known to those of skill in the art may be substituted.

[0171] Liquid suspensions can be prepared using conventional methods to achieve the suspension of the composition of the present invention in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, fish oil, and mineral oils such as liquid paraffin. Liquid suspensions can further include one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweetening agents. Oily suspensions can further include a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids, long chain aliphatic alcohols, partial esters derived from fatty acids and hexitols, or partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-parahydroxybenzoates, ascorbic acid, and sorbic acid.

[0172] Methods for Treating Tumors In one embodiment, the C-terminal endostatin or endostatin-derived peptides of the present invention reduce the production of extracellular matrix proteins by fibroblasts in fibrotic lung and skin. Cancer associated fibroblasts (CAFs) also cause fibrosis. Thus, in various embodiments, the C-terminal endostatin or endostatin-derived peptides of the present invention can be used to treat or prevent cancer, fibrotic diseases, or a combination thereof.

[0173] In one embodiment, the present invention provides a method of treating or preventing tumor growth in a subject in need thereof. Exemplary conditions that may be treated or prevented by the present invention include, but are not limited to, tumors of the lung, breast, stomach, pancreas, prostate, bladder, bone, ovaries, skin, kidney, sinuses, colon, intestine, stomach, rectum, esophagus, blood, brain and its coverings, spinal cord and its coverings, muscle, connective tissue, adrenal gland, parathyroid gland, thyroid gland, uterus, testes, pituitary gland, reproductive organs, liver, gallbladder, eye, ear, nose, throat, tonsils, mouth, lymph nodes and lymphatic system, and other organs.

[0174] In one embodiment, the present invention provides a method for preventing metastasis of malignant tumors or other cancerous cells and for slowing the rate of tumor growth. The method comprises administering an effective amount of one or more of the disclosed compounds to a subject diagnosed with or having a malignant tumor or cancerous cell. In one embodiment, the method comprises administering to the subject a composition comprising a C-terminal endostatin or endostatin-derived peptide, or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide, as described herein.

[0175] In one embodiment, the endostatin peptide of the present invention is used to treat or prevent lung cancer or skin cancer.However, the present invention is not limited to the treatment of lung cancer.The following are non-limiting examples of cancers that can be treated or prevented by the disclosed methods and compositions: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system atypical teratoma / rhabdoid tumor, central nervous system embryonal tumor, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytoma / malignant glioma , cervical cancer, childhood visual pathway tumors, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family tumors, extracranial cancer, extragonadal germ cell tumors, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, mycosis fungoides, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor stromal tumor, gist), germ cell tumor, cancer of pregnancy, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, bone malignant fibrous histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myeloid leukemia leukemia, myeloid leukemia, myeloma, myeloproliferative disorder, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancercancer), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovary, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, airway carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyoblastoma tumor, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0176] Methods for Treating Fibrosis One aspect of the present invention provides a method of treating or preventing fibrosis, a fibrosis-associated disease or disorder, or a cardiovascular disease or disorder, comprising administering to a subject a composition comprising a C-terminal endostatin or endostatin-derived peptide, or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide, as described herein. In one embodiment, the fibrosis-associated disease or disorder includes cardiac fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, familial pulmonary fibrosis, radiation-induced pulmonary fibrosis, coal workers' pneumoconiosis, asbestosis, bleomycin lung, sarcoidosis, silicosis, acute lung injury, ARDS, wound healing disease or disorder, hypertrophic scar, keloid scar, liver cirrhosis, systemic scleroderma, morphea, including but not limited to, vascular fibrosis, renal fibrosis, pulmonary ... These include, but are not limited to, fibrosis, fibrosis as a result of graft versus host disease (GVHD), subepithelial fibrosis, endomyocardial fibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, post-operative scarring, asthma, glomerulonephritis, multifocal fibrosclerosis, diabetic nephropathy, rheumatoid arthritis, atherosclerosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, systemic sclerosis, hepatitis, and Sjogren's syndrome.

[0177] In one embodiment, fibrosis-related diseases or disorders include, but are not limited to, cardiac fibrosis. In one embodiment, cardiac fibrosis results from cardiac injury. For example, in one embodiment, cardiac fibrosis results from injury, including, but not limited to, myocardial infarction, aortic stenosis, restrictive cardiomyopathy, systemic and pulmonary hypertension, or carcinoid heart disease. In one embodiment, interstitial lung diseases include, but are not limited to, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, coal workers' pneumoconiosis, asbestosis, acute lung injury, and ARDS. In one embodiment, wound healing diseases and disorders include, but are not limited to, hypertrophic scar, keloid scar.

[0178] In one embodiment, fibrosis involves the formation or development of excess fibrous connective tissue in an organ or tissue as a repair or reactive process distinct from the formation of fibrous tissue as a normal component of the organ or tissue. The skin and lungs are prone to fibrosis.

[0179] In some instances, fibrotic diseases are characterized by fibroblast activation, increased collagen and fibronectin production, and transdifferentiation into contractile myofibroblasts. This process usually takes months and years and can result in organ dysfunction or death. Fibrosis-related diseases and disorders represent one of the largest groups of disorders for which there is no effective therapy, and therefore represent a major unmet medical need. In many cases, the only remedy for patients with fibrosis is organ transplantation, but because the supply of organs is insufficient to meet the demand, patients often die while waiting to receive a suitable organ. Pulmonary fibrosis alone can be the main cause of death in scleroderma, lung disease, idiopathic pulmonary fibrosis, radiation- and chemotherapy-induced pulmonary fibrosis, and conditions caused by occupational inhalation of dust particles.

[0180] The present invention can be carried out in any subject diagnosed with or at risk of developing fibrosis. Fibrosis is associated with many diseases and disorders. Subjects can be diagnosed with or at risk of developing interstitial lung disease, including idiopathic pulmonary fibrosis, scleroderma, radiation-induced pulmonary fibrosis, bleomycin lung, sarcoidosis, silicosis, familial pulmonary fibrosis, autoimmune disease, or any disorder in which alveolar septal rupture with one or more fibroproliferative matrix molecule deposition, enhanced pathological collagen accumulation, apoptosis, and honeycombing occurs. Subjects can be identified as having or at risk of developing fibrosis due to exposure to asbestos, crushed stone, silica, and metal dust, by administration of drugs such as bleomycin, busulfone, feitoin, and nitrofurantoin, which are risk factors for developing fibrosis, or by radiation, such as for patients with head and neck cancer who develop fibrosis of the salivary glands. It is also contemplated that the compositions and methods of the present invention may be used in the treatment of organ fibrosis secondary to allogeneic organ transplantation, such as graft fibrosis, non-limiting examples of which include kidney transplant fibrosis, heart transplant fibrosis, liver transplant fibrosis, and the like.

[0181] In certain embodiments, the methods of the invention are used to treat multiple fibrotic or fibrosis-associated diseases or disorders having underlying causes including myocardial infarction, cirrhosis, hepatitis, and the like.

[0182] The present invention can be implemented in any subject who has been diagnosed with or is at risk of developing scleroderma. Scleroderma is a chronic autoimmune disease characterized by fibrosis (or hardening), vascular changes, and autoantibodies. There are two main forms: localized systemic scleroderma and diffuse systemic scleroderma. The skin symptoms of localized systemic scleroderma affect the hands, arms, and face. Patients with this form of scleroderma often have one or more of the following complications: calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly), visceral fibrosis, and telangiectasia.

[0183] Diffuse systemic scleroderma progresses rapidly and affects large areas of the skin and one or more internal organs, often the kidneys, esophagus, heart, and / or lungs. Localized scleroderma, such as linear scleroderma and morphea, affects the skin but not the internal organs.

[0184] Scleroderma affects small blood vessels known as arterioles in all organs. First, the endothelial cells of the arteriole die by apoptosis, along with the smooth muscle cells. These cells are replaced by collagen and other fibrous material. Inflammatory cells, especially CD4+ helper T cells, infiltrate the arteriole and cause further damage.

[0185] Skin manifestations of scleroderma can be painful, can impair use of the affected area (e.g., use of hands, fingers, toes, feet, etc.), and can be disfiguring. Skin ulceration can occur, and such ulcers can be prone to infection or even gangrene. Ulcerated skin can be difficult or slow to heal. Difficulty in healing skin ulcerations can be particularly exacerbated in patients with circulatory disorders, such as those with Raynaud's phenomenon. Pulmonary involvement is the leading cause of death in scleroderma patients, which exhibit high morbidity and mortality. In certain embodiments, the compositions and methods of the present disclosure are used to treat scleroderma, e.g., cutaneous symptoms of scleroderma. In certain embodiments, treating scleroderma includes treating skin ulceration, such as digital ulcers. Administration of the C-terminal endostatin or endostatin-derived peptides of the present invention can be used to reduce fibrotic and / or inflammatory symptoms of scleroderma in affected tissues and / or organs.

[0186] In addition to skin symptoms / manifestations, scleroderma can also affect the heart, kidneys, lungs, joints, and gastrointestinal tract, in certain embodiments, treating scleroderma includes treating symptoms of the disease in any one or more of these tissues, such as by reducing fibrotic and / or inflammatory symptoms.

[0187] Lung problems are one of the most serious complications of scleroderma and are responsible for many of the morbidities associated with this disease.The two main lung conditions associated with scleroderma are pulmonary fibrosis and pulmonary hypertension.Patients with lung involvement may have either or both conditions.Scleroderma-associated lung fibrosis is one example of pulmonary fibrosis that can be treated using the peptide of the present invention.

[0188] Scleroderma involving the lungs causes scarring (pulmonary fibrosis). Such pulmonary fibrosis occurs in about 70% of scleroderma patients, but its progression is typically slow, and symptoms vary widely in severity between patients. For patients with symptoms related to pulmonary fibrosis, symptoms include dry cough, shortness of breath, and reduced ability to exercise. About 16% of patients with some level of pulmonary fibrosis develop severe pulmonary fibrosis. Patients with severe pulmonary fibrosis experience significant lung function loss and alveolitis.

[0189] In certain embodiments, the method of the present invention includes the use of the peptide of the present invention to treat scleroderma, for example, pulmonary fibrosis associated with scleroderma.The administration of the peptide of the present invention can be used to reduce the fibrotic symptoms of scleroderma in the lung.For example, the method can be used to improve lung function and / or reduce the risk of death from scleroderma.For example, the C-terminal endostatin or endostatin-derived peptide of the present invention can be used to treat scleroderma-associated interstitial lung disease.

[0190] Renal lesions are also common in scleroderma patients. Renal fibrosis associated with scleroderma is one example of renal fibrosis that can be treated by administration of C-terminal endostatin or endostatin-derived peptides of the present invention.

[0191] In certain embodiments, the method of the present invention is used to treat scleroderma, for example, renal fibrosis associated with scleroderma.Administration of C-terminal endostatin or endostatin-derived peptides of the present invention can be used to reduce fibrotic symptoms of scleroderma in the kidney.For example, the method can be used to improve kidney function, reduce protein in urine, reduce high blood pressure, and / or reduce the risk of renal crisis, which can lead to fatal kidney failure.

[0192] In one embodiment, the method comprises reducing fibrosis in a cell or subject by administering a composition comprising a C-terminal endostatin or endostatin-derived peptide, or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide, as described herein. In one embodiment, the method comprises reducing or degrading extracellular matrix proteins in a cell or subject by administering a composition comprising a C-terminal endostatin or endostatin-derived peptide, or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide, as described herein.

[0193] Methods for treating acute lung injury One aspect of the present invention provides a method for treating or preventing acute lung injury, comprising administering to a subject a composition comprising a C-terminal endostatin or endostatin-derived peptide or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide, as described herein. In one embodiment, acute lung injury includes, but is not limited to, acute respiratory distress syndrome, including ARDS viral-induced acute lung injury, SARS, COVID-19, influenza-induced acute lung injury, sepsis, pneumonia, aspiration, trauma, blood transfusion, smoke, toxic gas inhalation pancreatitis, drug overdose, burn-induced acute lung injury, and ventilator-associated lung injury manifested with other lung injury or inflammation.

[0194] Acute lung injury is associated with increased IL-6 levels in lung tissue, which promotes cytokine storm. Administration of the peptides of the present invention can be used to reduce cytokine-induced symptoms of acute lung injury. For example, the method can be used to improve lung function and / or reduce the risk of death from acute lung injury. For example, the C-terminal endostatin or endostatin-derived peptides of the present invention can be used to treat increased IL-6 levels in lung tissue associated with cytokine storm and lung injury. In one embodiment, the method includes reducing IL-6 levels in a cell or subject by administering a composition comprising a C-terminal endostatin or endostatin-derived peptide or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide as described herein.

[0195] Administration In certain embodiments, the method comprises administering an effective amount of a composition described herein to a subject diagnosed with, suspected of having, or at risk of developing cancer, a condition associated with tumor growth, fibrosis, a fibrosis-related disease or disorder, or acute lung injury. In certain aspects, the composition is contacted with a cell or tissue in which a tumor, fibrosis, or acute lung injury is present or is at risk of developing. In one embodiment, the composition is administered systemically to the subject.

[0196] The compositions of the present invention can be administered to patients or subjects in need in a variety of ways. Modes of administration include oral administration, inhalation, intraoperative intravenous, intravascular, intramuscular, subcutaneous, intracerebral, intraperitoneal, soft tissue injection, surgical placement, arthroscopic placement, and percutaneous insertion, such as direct injection, cannulation, or catheterization. Any administration can be a single application or multiple applications of the compositions of the present invention. Administration can be to a single site or to two or more sites on the individual being treated. Multiple administrations can be essentially simultaneous or separated in time.

[0197] In certain embodiments, the compositions of the invention are administered during surgical resection or debulking of a tumor or diseased tissue (e.g., fibrotic tissue). For example, in a subject undergoing surgical treatment of a diseased tissue or tumor, the compositions can be administered to the site to further treat the tumor, fibrosis, acute lung injury, or a combination thereof.

[0198] In one embodiment, the method comprises administering to a subject a scaffold comprising C-terminal endostatin or endostatin-derived peptide, or cells engineered to express C-terminal endostatin or endostatin-derived peptide.

[0199] Subjects to which administration of the compositions and pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals, including non-human primates, commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs.

[0200] The pharmaceutical composition of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the subject and the type and severity of the subject's disease, but appropriate dosages can be determined by clinical trials.

[0201] When a "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in the patient's (subject's) age, weight, type of disease, extent of disease, and condition.

[0202] Administration of the subject compositions may be performed in any convenient manner, including by inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient orally, subcutaneously, intradermally, intratumorally, intranodal, intrathecal, intramuscular, intravenous (iv) injection, or intraperitoneally. In one embodiment, the compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the compositions of the present invention are preferably administered by iv injection.

[0203] The disclosed compounds can be used to prevent, reduce, minimize, control, and / or mitigate tumor growth or metastasis in humans and animals. The disclosed compounds can also be used to slow the rate of primary tumor growth. The disclosed compounds, when administered to a subject in need of treatment, can be used to stop the spread of cancer cells. Thus, the compounds disclosed herein can be administered as part of a combination therapy with one or more drugs or other pharmaceuticals. When used as part of a combination therapy, the reduction in metastasis or reduction in primary tumor growth provided by the disclosed compounds allows for more effective and efficient use of any pharmaceutical or drug therapy used to treat the patient. In addition, the control of metastasis by the disclosed compounds gives the subject a greater ability to focus the disease at one location.

[0204] In one embodiment, the present invention provides a method for treating or preventing cancer growth or metastasis comprising treating a subject with a complementary therapy for cancer, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy, or hormonal therapy, or a combination thereof, prior to, concurrently with, or following treatment with C-terminal endostatin or endostatin-derived peptide, or a nucleic acid molecule encoding C-terminal endostatin or endostatin-derived peptide.

[0205] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucin sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha-2a recombinant, paclitaxel, teniposide, and streptozotocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylsulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulfan, carboxyphthalate platinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatin, clomesone, cyanomorpholinodoxorubicin, cyclodizotocin, anti-inflammatory drugs (e.g., cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfame, hycanthone, ifosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxylon, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864); anti-mitotic drugs (e.g., azotocin, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfame, hycanthone, ifosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxylon, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864); rocolchicine, halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine, and taxotere),Biological agents (e.g., alpha interferon, BCG, G-CSF, GM-CSF, interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, anti-inflammatory drugs (e.g., cyclosporine, menogaril, N,N-dibenzyl daunomycin, oxantrazole, rubidazone, VM-26, and VP-16), and synthetic agents (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel, and porfimer sodium).

[0206] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous drugs, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene, and roloxifene). Further examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0207] The endostatin peptide of the present invention or the nucleic acid molecule encoding the endostatin peptide can be administered alone or in combination with other anti-tumor agents, including cytotoxic / antineoplastic agents and anti-angiogenic agents. Cytotoxic / antineoplastic agents are defined as agents that attack and kill cancer cells. Some cytotoxic / antineoplastic agents are alkylating agents that alkylate genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, verstine, uracil mustard, chromafazine, and dacabazine. Other cytotoxic / antineoplastic agents are antimetabolites against tumor cells, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine. Other cytotoxic / antineoplastic agents are antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin. There are numerous liposomal formulations of these compounds available commercially. Still other cytotoxic / antineoplastic agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Miscellaneous cytotoxic / antineoplastic agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0208] Antiangiogenic agents are well known to those skilled in the art. Antiangiogenic agents suitable for use in the methods and compositions of the present disclosure include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferons, interleukin 1 (including alpha and beta), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2. Small molecules that contain topoisomerase, such as razoxane, a topoisomerase II inhibitor with antiangiogenic activity, can also be used.

[0209] Other anticancer agents that can be used in combination with the disclosed compounds include acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asparin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizeresin; bleomycin sulfate; brequinar sodium; bropiperidin; Limine;Busulfan;Cactinomycin;Calsterone;Caracemide;Carbetimer;Carboplatin;Carmustine;Carubicin hydrochloride;Carzelesin;Cedefingol;Chlorambucil;Ciloremycin;Cisplatin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytarabine;Dacarbazine;Dactinomycin;Daunorubicin hydrochloride;Decitabine;Dexormaplatin;Desaguanine;Desaguanine mesylate;Diaziquone;Docetaxel;Doxorubicin;Doxorubicin hydrochloride;Droloxifene;Droloxifene citrate ;Dromostanolone propionate;Duazomycin;Edatrexate;Eflornithine hydrochloride;Elsamitrucin;Enloplatin;Enpromate;Epipropizine;Epirubicin hydrochloride;Elbrozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate;Fluorouracil;Fluorocitabine;Fosquidone;Fostriecin sodium;Gemcitabine;Hydrochloride Gemcitabine; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Irmofosine; Interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; Interferon alpha-2b; Interferon alpha-n1; Interferon alpha-n3; Interferon beta-Ia; Interferon gamma-Ib; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide acetate; Liarozole hydrochloride; Lometrexol sodium; Lomustine; Losoxantrone hydrochloride;Masoprocol;Maytansine;Mechlorethamine hydrochloride;Megestrol acetate;Melengestrol acetate;Melphalan;Menogaril;Mercaptopurine;Methotrexate;Methotrexate sodium;Metoprine;Meturedepa;Mitindomide;Mitocalcin;Mitochromine;Mitogillin;Mitomarcin;Mitomycin;Mitosper;Mitotane;Mitoxantrone hydrochloride;Mycophenolic acid;Nocodazole;Nogalamycin;Ormaplatin;Oxisuran; Paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofurin; Ribopurin; Rogletimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; S Parfosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Sulofenur;Tallysomycin;Tecogalan sodium;Tegafur;Teroxantrone hydrochloride;Temoporfin;Teniposide;Teroxylon;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Tiazofurin;Tirapazamine;Toremifene citrate;Trestorone acetate;Triciribine phosphate;Trimeto Rexate; Trimetrexate glucuronate; Triptorelin; Tubrozole hydrochloride; Uracil mustard; Uredep; Vapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Binepidine sulfate; Vinglisinate sulfate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrocidine sulfate; Vinzolidine sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin hydrochloride. Other anticancer drugs include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; Abiraterone; Aclarubicin; Acylfulvene; Adecipenol; Adzelesin; Aldesleukin; ALL-TK antagonists; Altretamine; Ambamustine; Amidox; Amifostine; Aminolevulinic acid; Amrubicin; Amsacrine; Anagrelide;Anastrozole;Andrographolide;Angiogenesis inhibitors;Antagonist D;Antagonist G;Antarelix;Anti-dorsalizing morphogenetic protein-1;Antiandrogens, prostate carcinoma;Antiestrogens;Antineoplastics;Antisense oligonucleotides;Aphidicolin glycinate;Apoptosis gene modulators;Apoptosis regulators;Apurinic acid;ara-CDP-DL-PTBA;Arginine deaminase;Asulaculin;Atamestane;Atrimustine;Axinastatin 1;Axinastatin 2;Axinastatin 3;Azasetron;Azatoxin;Azatyrosine;Baccatin III derivatives;Balanol;Batimastat;BCR / ABL anta agonists;benzochlorins;benzoylstaurosporine;beta-lactam derivatives;beta-arretin;beta-clamicin B;betulinic acid;bFGF inhibitors;bicalutamide;bisantrene;visaziridinyl spermine;bisnafide;bistratin A;bizelesin;brefurate;bropirimine;budotitanium;buthionine sulfoximine;calcipotriol;calphostin C;camptothecin derivatives;canarypox IL-2;capecitabine;carboxamide-amino-triazoles;carboxamide triazoles;CaRest M3;CARN 700;Cartilage-derived inhibitors;Carzelesin;Casein kinase inhibitor (ICOS);Castanospermine;Cecropin B;Cetrorelix;Chlorin;Chloroquinoxaline sulfonamides;Cicaprost;Cis-porphyrins;Cladribine;Clomiphene analogs;Clotrimazole;Colismycin A;Colismycin B;Combretastatin A4;Combretastatin analogs;Conagenin;Crambesidin 816;Crisnatol;Cryptophycin 8;Cryptophycin A derivatives;Cracin A;Cyclopentanthraquinone;Cycloplatam;Sipemycin;Cytarabine ocphosphate;Cytolytic factors;Cytostatin;Dacliximab;Decitabine;Dehydrodidemnin B;Deslorelin;Dexamethasone;Dexyphosphamide;Dexrazoxane;Dexyverapamil;Diaziquone;Didemnin B;Didox;Diethylnorspermine;Dihydro-5-azacytidine;Dihydrotaxol, 9-;Dioxamycin;Diphenylspiromustine;Docetaxel;Docosanol;Dolasetron;Doxifluridine;Droloxifene;Dronabinol;Duocarmycin SA;Ebselen;Ecomustine;Edelfosine;Edrecolomab;Eflornithine;Elemene;Emiteful;Epirubicin;Epristeride;Estramustine analogs;Estrogen agonists;Estrogen antagonists;Etanidazole;Etoposide phosphate;Exemestane;Fadrozole;Fazarabine;Fenretinide;Filgrastim;Finasteride;Flavopiridol;Flazelastine;Fluasterone;Fludarabine;Fluorodaunorunicin hydrochloride hydrochloride);Forfenimex;Formestane;Fostriecin;Fotemustine;Gadolinium texaphyrin;Gallium nitrate;Galocitabine;Ganirelix;Gelatinase inhibitors;Gemcitabine;Glutathione inhibitors;Hepsulfam;Heregulin;Hexamethylene bisacetamide;Hypericin;Ibandroic acid;Idarubicin;Idoxifene;Idramanton;Ilmofosine;Ilomastat;Imidazoacridone;Imiquimod;Immunostimulating peptides;Insulin-like growth factor-1 receptor inhibitors;Interferon agonists;Interferon;Interleukins;Iobenguane;Iododoxorubicin;Ipomeanol, 4-;Ilopract;Irsogladine;Isobengazole;Isohomohalichondrin B;Itasetron ;Jasplakinolide;Kahalalide F;Lamellarin-N triacetate;Lanreotide;Leinamycin;Lenograstim;Lentinan sulfate;Leptolstatin;Letrozole;Leukemia inhibitory factor;Leukocyte alpha interferon;Leuprolide + estrogen + progesterone;Leuprorelin;Levamisole;Liarozole;Linear polyamine analogs;Lipophilic disaccharide peptides;Lipophilic platinum compounds;Lisoclininamide 7;Lovaplatin;Lombricine;Lometrexol;Lonidamine;Losoxantrone;Lovastatin;Loxoribine;Rootecan;Lutetium texaphyrin;Lisophylline;Lytic peptides;Maytansine;Mannostatin A;Marimastat;Masoprocol;Maspin;Matrilysine inhibitors;Matrix metalloproteinase inhibitors;Menogaril;Melbarone;Meterelin;Methioninase;Metoclopramide;MIF inhibitors;Mifepristone;Miltefosine;Mirimostim;Mismatched double-stranded RNA;Mitoguazone;Mitolactol;Mitomycin analogs;Mitonafide;Mitotoxin fibroblast growth factor-saporin;Mitoxantrone;Mofalotene;Molgramostim;Monoclonal antibodies, human chorionic gonadotropin;Monophosphoryl lipid A+myobacterial cell wall sk;Mopidamol;Multidrug resistance gene inhibitors;Multiple tumor suppressor 1-based therapies;Mustard anticancer drugs ;Mycaperoxide B;Mycobacterial cell wall extract;Myriaporone;N-acetyldinaline;N-substituted benzamides;Nafarelin;Nagressip;Naloxone + pentazocine;Napavine;Naphterpin;Nartograstim;Nedaplatin;Nemorubicin;Neridronic acid;Neutral endopeptidase;Nilutamide;Nisamycin;Nitric oxide modulators;Nitroxide antioxidants;Nitrulline;O6-benzylguanine;Octreotide;Oxenone;Oligonucleotides;Onapristone;Ondansetron;Ondansetron;Oracin ;Oral cytokine inducers;Ormaplatin;Osateron;Oxaliplatin;Oxaunomycin;Paclitaxel;Paclitaxel analogs;Paclitaxel derivatives;Palaumin;Palmitoyl rhizoxin;Pamidronic acid;Panaxytriol;Panomyphen;Parabactin;Pazeliptin;Pegaspargase;Perdecin;Pentosan polysulfate sodium;Pentostatin;Pentrozole;Perflubron;Perphosphamide;Perillyl alcohol;Phenazinomycin;Phenyl acetate;Phosphatase inhibitors;Picibanil;Pi hydrochloride Rocarpine;Pirarubicin;Piritrexim;Prasetin A;Prasetin B;Plasminogen activator inhibitors;Platinum complexes;Platinum compounds;Platinum-triamine complexes;Porfimer sodium;Porfiromycin;Prednisone;Propyl bis-acridone;Prostaglandin J2;Proteasome inhibitors;Protein A-based immunomodulators;Protein kinase C inhibitors;Protein kinase C inhibitors, microalgae;Protein tyrosine phosphatase inhibitors;Purine nucleoside phosphorylase inhibitors;Purpurins;Pyrazoloacridines;Pyridoxylated hemoglobin polyoxyethylene conjugates;raf antagonists;raltitrexed;ramosetron;ras farnesyl protein transferase inhibitors;ras inhibitors;ras-GAP inhibitors;demethylated reterliptin;rhenium Re186 etidronate;rhizoxin;ribozyme;RII retinamide;logretimide;rohitukin;romurtide;roquinimex;rubigino; B1;Ruboxil;Safingol;Saintpin;SarCNU;Sarcophytol A;Sargramostim;Sdi1 mimetic;Semustine;Senescence derived inhibitor 1;Sense oligonucleotides;Signal transduction inhibitors;Signal transduction modulators;Single-chain antigen binding protein;Schizofuran;Sobuzoxane;Borocaptan sodium;Sodium phenylacetate;Sorberol;Somatomedin binding protein;Sonermin;Sparfos acid;Spicamycin D;Spiromus tin;splenopentin;spongiostatin 1;squalamine;stem cell inhibitors;stem cell division inhibitors;stypiamide;stromelysin inhibitors;sulfinosine;superactive vasoactive intestinal peptide antagonists;sladista;suramin;swainsonine;synthetic glycosaminoglycans;talimustine;tamoxifen methiodide;tauromustine;tazarotene;tecogalan sodium;tegafur;tellapyrylium;telomerase inhibitors;temoporfin;temozolomide; Teniposide;Tetrachlorodecaoxide;Tetrazomine;Taliblastine;Thiocoraline;Thrombopoietin;Thrombopoietin mimetics;Thymalfasin;Thymopoietin receptor agonists;Thymotrin;Thyroid-stimulating hormone;Tin ethyl etiopurpurin;Tirapazamine;Titanocene dichloride;Topsentin;Toremifene;Pluripotent stem cell factor;Translation inhibitors;Tretinoin;Triacetyluridine;Triciribine;Trimetrexate;Triptorelin;Tropi These include, but are not limited to, setron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, vector systems, red blood cell gene therapy, veraresol, veramine, verudin, verteporfin, vinorelbine, vinxartin, vitaxin, vorozole, zanoteron, zeniplatin, zilascorub, and zinostatin stimalamer. In one embodiment, the anticancer drug is 5-fluorouracil, taxol, or leucovorin.

[0210] In certain embodiments, the method of treating fibrosis or fibrosis-related diseases or disorders includes administering C-terminal endostatin or endostatin-derived peptides, or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide, as part of a treatment regimen with one or more other drugs, biologics, or therapeutic interventions appropriate for treating fibrosis. In certain embodiments, the additional drugs, biologics, or therapeutic interventions are appropriate for the particular condition associated with fibrosis. By way of example, the C-terminal endostatin or endostatin-derived peptides of the present invention can be administered as part of a treatment regimen with one or more immunosuppressants, such as methotrexate, cyclophosphamide, azathioprine, pirfenidone, nintedanib, and mycophenolate mofetil. As a further example, the C-terminal endostatin or endostatin-derived peptides of the present invention can be administered as part of a treatment regimen with one or more agents designed to increase blood flow, such as blood flow to an ulcerated finger (e.g., nifedipine, amlodipine, diltiazem, felodipine, or nicardipine). As a further example, the C-terminal endostatin or endostatin-derived peptides of the present invention can be administered as part of a treatment regimen with one or more agents intended to reduce skin fibrosis, such as d-penicillamine, colchicine, PUVA, relaxin, and cyclosporine. As a further example, the C-terminal endostatin or endostatin-derived peptides of the present invention can be administered as part of a treatment regimen with steroids or bronchodilators.

[0211] In certain embodiments, the method of treating acute lung injury includes administering C-terminal endostatin or endostatin-derived peptides, or nucleic acid molecules encoding C-terminal endostatin or endostatin-derived peptides, as part of a treatment regimen, together with one or more other drugs, biologics, or therapeutic interventions suitable for treating acute lung injury. In certain embodiments, the additional drugs, biologics, or therapeutic interventions are suitable for the particular symptoms associated with acute lung injury. Treatments for acute lung injury are limited and include prone positioning, mechanical ventilation, inhaled vasodilators such as epoprostenol or nitric oxide, ECMO, neuromuscular blockers to facilitate lung protective ventilation, and steroids.

[0212] The present invention encompasses the administration of C-terminal endostatin or endostatin-derived peptides, or nucleic acid molecules encoding C-terminal endostatin or endostatin-derived peptides, for the treatment or prevention of diseases and disorders. To practice the methods of the present invention, a person skilled in the art will know how to formulate and administer to a subject a suitable composition of the present invention based on the disclosure provided herein. The present invention is not limited to any particular method of administration or treatment regimen.

[0213] In one embodiment, the method includes administering to a subject C-terminal endostatin or endostatin-derived peptide, a scaffold comprising C-terminal endostatin or endostatin-derived peptide, or a cell modified to express C-terminal endostatin or endostatin-derived peptide.

[0214] Dosage and Formulation (Pharmaceutical Composition) The present invention contemplates treating diseases or disorders in a mammal, such as cancer, fibrosis, fibrosis-related diseases or disorders, acute lung injury, etc., by administration of a therapeutic agent, such as C-terminal endostatin or endostatin-derived peptides, or a nucleic acid molecule encoding a C-terminal endostatin or endostatin-derived peptide.

[0215] Administration of the therapeutic agent according to the invention may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the agent of the invention may be essentially continuous over a preselected period of time, or may be in a series of spaced doses. Both local and systemic administration are contemplated. The amount administered will vary depending on a variety of factors, including, but not limited to, the selected composition, the particular disease, weight, physical condition, and age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician using animal models or other test systems well known in the art.

[0216] One or more suitable unit dosage forms having the therapeutic agents of the invention, which may optionally be formulated for sustained release (e.g., using microencapsulation) as discussed below (see WO 94 / 07529 and U.S. Pat. No. 4,962,091, the disclosures of which are incorporated herein by reference), can be administered by a variety of routes, including parenteral routes, for example, by intravenous, intraperitoneal, inhalation, and intramuscular routes, as well as by direct injection into the affected tissue. For example, the therapeutic agents or modified cells may be injected directly into a tumor. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods may include the step of bringing into association the therapeutic agents with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers, or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[0217] In various embodiments, pharmaceutical compositions useful in the methods of the present invention may be administered systemically, parenterally, or locally, for example, in oral formulations, inhalation formulations including solids or aerosols, and topical or other similar formulations. In addition to the appropriate therapeutic composition, such pharmaceutical compositions may include pharma- ceutically acceptable carriers, as well as other components known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically-based systems, may also be used to administer the appropriate modulators according to the methods of the present invention.

[0218] When the therapeutic agents of the present invention are prepared for administration, they are preferably combined with a pharma- ceutically acceptable carrier, diluent, or excipient to form a pharmaceutical formulation or unit dosage form. The total active ingredient in such a formulation comprises 0.1-99.9% by weight of the formulation. "Pharmaceutically acceptable" refers to a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation and not harmful to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, suspension, or emulsion.

[0219] Pharmaceutical formulations containing the therapeutic agents of the present invention can be prepared by procedures known in the art using well-known and readily available ingredients. The therapeutic agents of the present invention can also be formulated as solutions suitable for parenteral administration, for example, by intramuscular, subcutaneous, or intravenous routes.

[0220] Pharmaceutical formulations of the therapeutic agents of the invention may take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

[0221] Thus, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and may be provided in unit dose form in ampoules, prefilled syringes, small volume injection containers, or in multi-dose containers with added preservatives. The active ingredient may take such forms as a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from solution, for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0222] In certain embodiments, the compositions of the invention are administered by inhalation. In certain embodiments, the invention is conveniently delivered from an insufflator, nebulizer, or pressurized pack, or other convenient means of delivering an aerosol spray. Pressurized packs may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. In certain embodiments, the invention may take the form of a dry powder composition, e.g., a powder mix of the compound and a suitable powder base, such as lactose or starch. The powder composition may be provided in unit dosage form, e.g., capsules or cartridges, or e.g., gelatin or blister packs, from which the powder can be administered using an inhaler or insufflator. The powdered or aerosolized formulation, when dispersed, preferably has an average particle or droplet size in the range of about 0.1 nanometer to about 2000 micrometers, and may further comprise one or more of the additional ingredients described herein.

[0223] It will be understood that the unit content of active ingredient(s) contained in an individual aerosol dose of each dosage form need not itself constitute an effective amount for treating a particular indication or disease, since the required effective amount may be achieved by administration of multiple dosage units. Moreover, an effective amount may be achieved using less than the dose in the dosage form, either individually or in a series of administrations.

[0224] The pharmaceutical formulations of the present invention may contain, as optional ingredients, pharma- ceutically acceptable carriers, diluents, solubilizers, or emulsifiers, and salts of the type known in the art. Non-limiting examples of carriers and / or diluents useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions at pH 7.0-8.0.

[0225] The agents of the present invention can be formulated and administered to treat various disease conditions by any means that results in contact of the active ingredients with the agent's site of action in the body of an organism. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutically active ingredients or in a combination of therapeutically active ingredients. They can be administered alone, but generally will be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0226] In general, water, a suitable oil, saline, aqueous dextrose (glucose) and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents, and buffer substances, if necessary. Antioxidants such as sodium bisulfate, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium ethylenediaminetetraacetic acid (EDTA) are also used. In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl or propyl paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field.

[0227] The active ingredient of the present invention may be formulated to be suspended in a pharma- ceutically acceptable composition suitable for use in mammals, particularly humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs as described in U.S. Patent Nos. 4,082,735, 4,082,736, 4,101,536, 4,185,089, 4,235,771, and 4,406,890. Other adjuvants that are useful include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate, and dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12. Other components may include polyoxypropylene-polyoxyethylene block copolymers (Pluronic®), non-ionic surfactants, and metabolizable oils such as squalene (US Pat. No. 4,606,918).

[0228] Furthermore, standard pharmaceutical methods can be used to control the duration of action. These are well known in the art and include controlled release preparations, and can include suitable polymers, such as polymers, polyesters, polyamino acids, polyvinyls, pyrrolidones, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose, or protamine sulfate. The concentration and method of incorporation of the polymer can be adjusted to control the release. Furthermore, the agent can be incorporated into particles of polymeric materials, such as polyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylene vinyl acetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

[0229] Thus, the pharmaceutical compositions of the present invention can be delivered to various sites within a mammalian body via various routes to achieve a particular effect (see, e.g., Rosenfeld et al., 1991; Rosenfeld et al., 1991a; Jaffe et al., supra; Berkner, supra). One skilled in the art will recognize that, although more than one route can be used for administration, a particular route may provide a more rapid and effective response than another route. Local or systemic delivery can be achieved by administration including application or instillation of the formulation into a body cavity, inhalation or insufflation of an aerosol, oral administration, or by parenteral introduction, including intramuscular, intravenous, peritoneal, subcutaneous, intradermal, and topical administration.

[0230] The active ingredient of the present invention can be provided in unit dosage form, where each dosage unit, for example, teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in combination with other active agents.The term "unit dosage form" as used herein refers to a physically separate unit suitable as a unit dosage for human and mammalian subjects, each unit containing a predetermined amount of the composition of the present invention, alone or in combination with other active agents, calculated as a sufficient amount to produce the desired effect, together with a pharmaceutically acceptable diluent, carrier, or vehicle, if appropriate.The specifications of the unit dosage form of the present invention depend on the specific effect to be achieved and the specific pharmacodynamics associated with the pharmaceutical composition in a specific host.

[0231] These methods described herein are by no means all-inclusive, and additional methods adapted to particular applications will be apparent to those of skill in the art. Moreover, the effective amount of the composition can be further approximated by analogy with compounds known to exert the desired effect. EXAMPLES

[0232] The present invention will be described in more detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all modifications that become evident as a result of the teachings provided herein.

[0233] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. Thus, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0234] Example 1: Endostatin peptides for the treatment of tumors The ability of endostatin peptides to block tumor growth was evaluated. The peptides were biotinylated at the N-terminus and amidated at the C-terminus.

[0235] The peptides evaluated in the experiment were as follows: Bio96 sequence: biotin-NH2-ATGQASSLL-CONH2 (SEQ ID NO: 1); BioE4-03 sequence: Biotin-NH2-SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA-CONH2 (SEQ ID NO: 2), and BioE4 sequence: Biotin-NH2-SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT-CONH2 (SEQ ID NO:3).

[0236] statistical methods For each of the experiments, descriptive statistics (mean and standard deviation) for tumor volume were calculated for each experimental group over time (i.e., experiment 1: control vs. BioE4 (Figure 1); experiment 2: PBS, Bio96, and BioE4-03 (Figure 5)). Because tumor growth tends to be curvilinear, a general linear mixed model (GLMM) was created to estimate treatment-specific growth curves over time and to make comparisons between peptide treatments and controls. A separate GLMM was constructed for each experiment, and each GLMM included a random effect for each mouse to control for the fact that repeated volume measurements within a given mouse are correlated with each other. Tumor volume served as the dependent variable in each model, along with time (in days), time to maturity, and time to maturity. 2 , treatment, treatment x time, and treatment x time 2 were included as independent variables. These models were used to compare the estimated mean tumor volume at specific selected time points and the mean percent change in tumor volume at specific selected time points. Mice were sacrificed if their tumors grew too large. However, all tumor volumes measured before sacrifice were included in the modeling process. SAS v9.4 (SAS Institute, Cary, NC) was used for all analyses. All hypothesis tests were two-sided, and a p-value <0.05 was considered statistically significant.

[0237] Next, the experimental results will be described. Analysis of the effects of endostatin peptides on prostate tumor growth Two groups of wild-type C57BL6 mice from Jax Labs were injected with TRAMP C-2 mouse prostate tumor cells into the right flank. Tumors grew to approximately 150 mm 3 Treatment was initiated when tumors reached a size of 10 μL per week. Treatment consisted of peptide (Bio E4) dissolved in water or water as vehicle control, given twice weekly in a volume of 100 μL by oral gavage at a dose of 50 μg / dose. Tumors were measured 3 times / week.

[0238] Direct comparisons were made between BioE4 and controls for mean tumor volume on day 15 and mean percentage change in tumor volume on day 15 (Figures 1-3). Model-based estimates are in Figure 4. On day 15, mean tumor volume was significantly (p=0.0001) greater in the control group when compared to the BioE4 group, and the mean percentage increase in volume on day 15 was also significantly (p=0.02) greater in the control group when compared to the BioE4 group.

[0239] Analysis of the effects of endostatin peptides on colon adenocarcinoma tumor growth sMIC-resistant transgenic (MB481 strain) mice (Liu et al., Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis. J. Clin. Invest. 123:4410, 2013) were administered 1 × 10 5 mc-38 colon adenocarcinoma tumor cells were injected into the mice. The tumor size was approximately 75 mm 3 Treatment was initiated when tumor volume reached 100 μg / mouse. Treatment is initiated in four groups. Bio96 100 μg / mouse, BioE4-03 100 μg / mouse were given by oral gavage in a volume of 100 μL. PBS (100 μL) was given as a vehicle control. Peptides and vehicle were administered twice / week. Tumor volumes were measured 3 times a week.

[0240] Direct comparisons were made between the PBS control and Bio96, and between PBS and BioE4-03, for mean tumor volume on day 21 and the mean percent change in tumor volume on day 21 (Figures 5-7). Model-based estimates are shown in Figure 8. At day 21, the mean tumor volume was significantly larger in the PBS control group (871.4 mm) when compared to the Bio96 group. 3 1492.1mm 3 , p=0.003), and the mean tumor volume was significantly greater in the PBS control group when compared to the BioE4-03 group (767.7 mm 3 1492.1mm 3 At day 21, the mean increase in volume was significantly greater in the PBS control group (90.6 mm) compared to the Bio96 group. 3 / day vs 151.1mm 3 / day, p=0.004), and the mean rate of increase in volume was significantly greater in the PBS control group (76.0 mm) compared to the BioE4-03 group. 3 / day vs 151.1mm 3 / day, p=0.0008). These p-values ​​were not adjusted for multiple comparisons, but they may remain statistically significant even if, for example, a Bonferroni adjustment were performed.

[0241] Histological analysis of tumors Histological analysis of tumors treated with vehicle alone, or BioE4-03 or Bio96 peptides was performed. Tumors from mice treated with Bio-E4-03 had significant necrosis (H&E) (Figure 9). Tumors from mice treated with Bio-E4-03 or Bio96 had a sparser cellular arrangement (H&E) (Figure 9). Tumors from mice treated with BioE4-03 or Bio96 showed thinner collagen bundles in the tumors (Masson's trichrome staining) compared to tumors from vehicle-treated mice (Figure 10).

[0242] Effect of BioE4-03 on human tumor cells To identify potential mechanisms by which endostatin-derived peptides may reduce tumor growth, the effect of BioE4-03 on human tumor cells was examined.

[0243] The expression of several genes involved in tumorigenesis, angiogenesis, and cancer stem cell regeneration was measured in various tumor types and organs. The findings suggest that endostatin-derived peptide E4-03 reduces tumor growth through the regulation of multiple genes involved in tumorigenesis and angiogenesis in the human cancer cells tested below.

[0244] A549 cells (human lung adenocarcinoma cells): Tumor cells were treated with 10 μg / mL BioE4-03 for 48 h. RNA was extracted and analyzed by real-time PCR. N=6-8 independent experiments. Statistical analysis was performed using paired t-test. The expression of uPA, uPAR, PAI-I, LOX, PDGF-A, VEGF-A, and IL-6 was significantly decreased by BioE4-03 (Figure 11).

[0245] HCT116T cells (human lung and colon carcinoma cells): Tumor cells were treated with 10 μg / mL BioE4-03 for 48 hours. RNA was extracted and analyzed by real-time PCR. N=3-4 independent experiments (except for uPAR, where n=2). The expression of uPA, uPAR, PAI-I, PDGF-A, VEGF-A, and IL-6 was significantly decreased by BioE4-03 (Figure 12).

[0246] Effect of endostatin peptides on tumor growth Figure 26 shows the effect of E4 given via oral gavage on tumor size (Y-axis) in mice. Treatment days are shown on the X-axis. Peptides were given twice a week. The red line shows mice with untreated tumors and the blue line shows mice treated with E4.

[0247] MM.1S BzR cells (bortezomib-resistant multiple myeloma cells): Immunodeficiency NOD-scid IL2R gamma nullMouse, 1 x 10 6 Bortezomib-resistant multiple myeloma cells (MM.1S BzR) were injected via the lateral tail vein. 14 days later, treatment with BioE4 peptide was started, given orally at 10 mg / kg twice a week. Mice were monitored daily for qualitative signs of disease progression. Bone marrow infiltration of CD138+ myeloma plasma cells was assessed on day 49 to measure the number of CD138+ myeloma plasma cells as an index of tumor burden and peptide efficacy (Figure 27).

[0248] Example 2: Endostatin peptide domain testing Variants of Bio-E4-03 and Bio96 endostatin peptides were evaluated for their effect on gene expression in different cells. In some experiments, the peptides were biotinylated at the N-terminus and amidated at the C-terminus.

[0249] The peptides evaluated in the experiment were as follows: E4-03: SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 2) 96-17: ATGQASSLLGGRLLGQSAASCHHA (SEQ ID NO: 4) 96-87: ATGQASSLLGGRLLGQ (SEQ ID NO: 5) 91-96: SYCETWRTEAPSATGQASSLL (SEQ ID NO: 6) 91-87: SYCETWRTEAPSATGQASSLLGGRLLGQ (SEQ ID NO: 7) Gene expression in normal lung fibroblasts Normal lung fibroblasts were treated with vehicle as control (VC) or TGFbeta (T) in the presence or absence of 10 μg / mL of various peptide fragments. Extracellular matrix proteins, collagen 1A1, and fibronectin (FN) were assessed using immunoblotting. E4-03 and 96-17, but not 96-87, 91-96, and 91-87, were effective in reducing FN and Col1A1 levels induced by TGFbeta (Figure 13).

[0250] Fibroblasts were treated with TGF-beta for 48 hours with or without peptide Bio96-17. RNA was extracted and alpha smooth muscle actin (SMA) levels were measured by qRT-PCR. Bio96-17 treatment resulted in a significant reduction in TGF-beta-induced SMA levels and therefore myofibroblast differentiation in fibroblasts derived from lung tissue of normal donors (Figure 14). Furthermore, Bio96-17 resulted in a significant reduction in SMA and Coll1A2 levels in fibroblasts derived from lungs of patients with systemic sclerosis (SSc) (Figure 15).

[0251] SSc fibroblasts were treated with 10 μg / mL BioE4-03 for 72 hours. RNA was extracted and used to measure SMA and collagen 1A2. BioE4-03 results in a significant reduction in SMA and Coll1A2 levels in fibroblasts derived from the lungs of patients with SSc (Figure 16). Furthermore, BioE4-03 results in a trend toward a decrease in SMA and Coll1A2 levels in fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) (Figure 17).

[0252] Normal lung fibroblasts were treated with TGF-beta with or without increasing concentrations of 96-87. Supernatants were assessed for fibronectin levels 72 hours after treatment. Increasing concentrations of 96-87 from 10 μg / mL to 80 μg / mL did not affect TGF-beta-induced FN production (FIG. 18).

[0253] Ex vivo organ culture assay Systemic sclerosis pulmonary fibrosis Lung tissue was divided into equal sized cores. Lung tissue in organ culture was treated with different peptide fragments derived from endostatin at a final concentration of 10 μg / mL in the presence of TGF-beta for 120 hours. Induction of matrix metalloproteinase (MMP)-1 (also known as collagenase) in the medium conditioned by the lung tissue was examined as a measure of the ability of peptides to reduce fibrosis and promote extracellular matrix degradation. Two of the endostatin peptide domains, E4-03 and 96-17, induced MMP-1 production, whereas 91-96 and 91-87 had no effect (Figure 19).

[0254] BioE4-03 results in a significant reduction in hydroxyproline levels in lung tissue from patients with SSc (Figure 20) and IPF (Figure 21) maintained in organ culture for 72 hours. Furthermore, both BioE4-03 (Figure 22) and Bio96-17 (Figure 23) result in a significant reduction in hydroxyproline levels in lung tissue from normal donors after induction of fibrosis using TGF-beta. Bio96-17 also results in a significant reduction in secreted Col1A1 protein levels in lung tissue from normal donors cultured for 120 hours after induction of fibrosis using TGF-beta (Figure 24). BioE4-03 further reduces Col1A1 and fibronectin (FN) gene expression in skin tissue from donors in which fibrosis was induced with TGF-beta (Figure 25).

[0255] Example 3: Endostatin peptides for the treatment of acute lung injury Acute lung injury, such as ARDS and COVID-19-related lung injury, is associated with elevated levels of IL-6 in lung tissue, which promotes cytokine storm. Thus, there is a need for therapies that can lower IL-6 and attenuate cytokine-induced symptoms of acute lung injury.

[0256] IL-6 mRNA levels were measured by qPCR in human lung adenocarcinoma cells (A549) treated with BioE4-03 peptide for 48 hours. Peptide-treated cells significantly reduced IL-6 mRNA expression compared to vehicle control (Figure 28A). For comparison, parallel experiments were performed with non-biotinylated E4-03. E4-03 also reduced IL-6 mRNA expression after 48 hours (Figure 28B). Furthermore, IL-6 protein levels in the supernatant of A549 cells were measured by ELISA 72 hours after treatment with E4-03 peptide. IL-6 protein levels were significantly reduced in peptide-treated cells compared to vehicle control (Figure 28B).

[0257] Example 4: Pepsin-digested endostatin peptide fragments as therapeutic variants Endostatin-derived peptides are therapeutically viable as orally administered treatments. Therefore, to determine which peptide fragments are most prevalent and therefore most likely to be therapeutically suitable after native digestion, N-terminally biotinylated (long) and C-terminally amidated E4-03 (BioE4-03): Biotin-NH2-LC-SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA-CONH2 (SEQ ID NO: 2) Pepsin digestion was performed on the Peptide samples were prepared by dissolving BioE4-03 in 0.1% formic acid. Peptides were digested with pepsin at a 1:100 pepsin:peptide ratio for 0 min (aliquot removed immediately after pepsin addition), 15 min, and 45 min. Before digestion (as a negative control) and after digestion, 0.6 μL of C 18 A 20 μL aliquot was removed using a Millipore® ZipTip® pipette tip with resin. Each eluate was then dissolved in 7 μL of 2% acetonitrile / 0.2% formic acid before being injected into the Orbitrap Elite for LC-MS / MS analysis. MS1 was detected in the Orbitrap. MS2 (including the +1 precursor) was detected in the ion trap via collision-induced dissociation.

[0258] Five separate groups were analyzed via LC-MS / MS: Group A (E4-03) was undigested BioE4-03 diluted in 0.1% formic acid. Group B was undigested BioE4-03 diluted in 0.1% formic acid aliquoted with a ZipTip (NC). Group C was BioE4-03 aliquoted immediately after the addition of pepsin (T0; 0 min digestion). Group D was BioE4-03 aliquoted 15 min after pepsin digestion (T15). Group E was BioE4-03 aliquoted 45 min after pepsin digestion (T45).

[0259] Table 1 below shows the top 20 peptides with the greatest number of total peptide spectrum matches (PSMs), sorted by MH+ molecular weight in daltons from highest to lowest. The N-terminal residue shown in lower case indicates the NHS-LC-biotin modification, and the C-terminal residue shown in lower case indicates the amidation modification. These results are also shown graphically in Figure 29.

[0260] [Table 1]

[0261] Figure 29 shows the LC-MS / MS results of pepsin digestion of BioE4-03 (SEQ ID NO: 2). The X-axis of each chart, indicated by the SEQ ID NOs listed in Table 1, indicates the number of peptide spectral matches for each treatment group, indicated on the Y-axis: "Feed" indicates undigested BioE4-03 diluted in 0.1% formic acid, "WKSL" indicates undigested BioE4-03 diluted in 0.1% formic acid aliquoted with a ZipTip®, TO indicates BioE4-03 aliquoted immediately after the addition of pepsin (0 min digestion), T15 indicates BioE4-03 aliquoted 15 min after pepsin digestion, and T45 indicates BioE4-03 aliquoted 45 min after pepsin digestion (T45).

[0262] The peptides described herein that are increased in abundance after pepsin digestion have been tested for their anti-tumor and / or anti-fibrotic activity, for example, using the assays described above in Examples 1-3. Without being bound by scientific theory, it is believed that one or more of the endostatin-derived pepsin-digested peptides have anti-tumor activity, anti-fibrotic activity, or a combination thereof, for use in treating cancer or one or more fibrosis or fibrosis-related diseases or disorders, as described above.

[0263] The disclosures of each and every patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to certain embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A pharmaceutical composition for use in treating fibrosis, cancer, or acute lung injury in a subject in need thereof, said composition comprising: a) a C-terminal endostatin-derived peptide having the amino acid sequence of SEQ ID NO: 24, 14, 20, 23, 13, 12, 16, 18, 19, 10, 11, 26, 27, 15, 17, 21, 22, or 25, or a variant, derivative, mutant, or fragment thereof; or b) an isolated nucleic acid encoding a C-terminal endostatin-derived peptide having the amino acid sequence of SEQ ID NO: 24, 14, 20, 23, 13, 12, 16, 18, 19, 10, 11, 26, 27, 15, 17, 21, 22, or 25, or a variant, derivative, mutant, or fragment thereof; A pharmaceutical composition comprising an effective amount of

2. The pharmaceutical composition described in claim 1, wherein the composition is for use in the treatment of fibrosis.

3. The composition is for use in treating fibrosis in a subject having a fibrotic disease or disorder or a fibrosis-associated disease or disorder, wherein the fibrotic disease or disorder or fibrosis-associated disease or disorder is selected from the group consisting of cardiac fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, familial pulmonary fibrosis, radiation-induced pulmonary fibrosis, coal workers' pneumoconiosis, asbestosis, bleomycin lung, sarcoidosis, silicosis, acute lung injury, ARDS, hypertrophic scars, keloid scars, cirrhosis of the liver, systemic sclerosis, localized scleroderma, morphea, and morphea.

3. The pharmaceutical composition of claim 2, wherein the fibrosis is selected from the group consisting of fair, vascular fibrosis, renal fibrosis, fibrosis as a result of graft-versus-host disease (GVHD), subepithelial fibrosis, endomyocardial fibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, postoperative scarring, asthma, glomerulonephritis, multifocal fibrosclerosis, diabetic nephropathy, rheumatoid arthritis, atherosclerosis, radiation-induced fibrosis, chemotherapy-induced fibrosis, systemic sclerosis, hepatitis, and Sjogren's syndrome.

4. The pharmaceutical composition of claim 1, wherein the composition is for use in the treatment of cancer.

5. The pharmaceutical composition of claim 4, wherein the composition is for use in the treatment of cancer, and the cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, liver cancer, ovarian cancer, endometrial cancer, bladder cancer, colon cancer, lymphoma, skin cancer, pancreatic cancer, gastric cancer, myeloma, and glioma.

6. The pharmaceutical composition of claim 1, wherein the composition is administered together with a second drug, and the second drug is an anti-fibrotic agent or an anti-cancer agent.

7. The pharmaceutical composition of claim 1, wherein the composition is for use in the treatment of acute lung injury.

8. The pharmaceutical composition of claim 1, wherein the subject is a cat or dog, or a human subject.

9. The pharmaceutical composition of claim 1, wherein the C-terminal endostatin-derived peptide is biotinylated at the N-terminus, amidated at the C-terminus, or a combination thereof.

10. The pharmaceutical composition of claim 1, wherein the C-terminal endostatin-derived peptide variant comprises at most five amino acid substitutions.

11. A pharmaceutical composition described in any one of claims 1 to 9, wherein the C-terminal endostatin-derived peptide has the amino acid sequence of SEQ ID NO: 24, 14, 20, 23, 13, 12, 16, 18, 19, 10, 11, 26, 27, 15, 17, 21, 22 or 25.

12. The pharmaceutical composition described in claim 11, wherein the C-terminal endostatin-derived peptide is 20 amino acids in length and has the amino acid sequence of SEQ ID NO: 14, in which the C-terminal residue is LGGRL, or is 16 amino acids in length and has the amino acid sequence of SEQ ID NO: 24, in which the N-terminal residue is LGGRL.

13. The pharmaceutical composition of claim 11, wherein the C-terminal endostatin-derived peptide is 16, 17, 18, 19 or 20 amino acids in length and has the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 18 or SEQ ID NO: 16, respectively, and the C-terminal residue of each peptide is LGGRLLGQSAASCHHA (SEQ ID NO: 24).

14. The pharmaceutical composition described in claim 11, wherein the C-terminal endostatin-derived peptide is 16 amino acids in length and has the amino acid sequence of SEQ ID NO: 24.

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