Integrin-targeting protein and methods of use thereof

Abstract

To provide pharmaceutical compositions that may be effective in treatment of medical symptoms associated with fibrosis.SOLUTION: A pharmaceutical composition for preventing or reducing excessive accumulation of fibrous material within an extracellular matrix in injured or damaged tissue of a subject, wherein the pharmaceutical composition comprises a polypeptide that binds specifically to integrins αvβ3, the polypeptide comprising a variant of domain 1 of cell adhesion protein (D1-CD2) having an amino acid sequence of at least about 75% similar to the sequence of wild-type D1-CD2. The polypeptide specifically binds to integrin αvβ3 at βA domain in the region of α2 helix, B-C loop and α2-α3 loop. The excessive accumulation of the fibrous material is not retrolental Fibroplasia, idiopathic pulmonary fibrosis, neurofibroma or angiofibroma.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] [Related Applications] This application is a continuation of U.S. Provisional Patent Application No. 62 / 129,499, filed March 6, 2015. No. 6,399,623, filed on Oct. 23, 2003, which is incorporated herein by reference in its entirety.

[0002] [Government Interests] This invention was made with government support under CA175122 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] [Field of the Invention] The present application relates to compounds or proteins that may be effective in treating medical conditions associated with fibrosis. The present disclosure generally relates to v β 3 A polypeptide that specifically binds to integrins The present disclosure further relates to methods for reducing angiogenesis, cell proliferation, and fibrosis, and Polypeptides for treating cancer, portal hypertension, and fibrosis-related diseases and conditions This article explains how to use the code. [Background technology]

[0004] Typically, solid tumors grow to a diameter of 3-4 mm without developing their own blood supply. Therefore, the construction of tumor blood vessels is essential for cancer metastasis. do.

[0005] Anti-angiogenic drugs such as Avastin® are manufactured to inhibit angiogenesis in solid tumors. However, clinical studies have not shown any significant improvement in patient survival with these antiangiogenic drugs. The benefits have not been shown to be significant. Furthermore, the development of antiangiogenic drugs has been driven primarily by VEGF / VEGFR signaling plays a role in promoting epithelial cell proliferation and metastasis Efforts are focused on strategies to block other RTK pathways.

[0006] Integrins are heterodimers, i.e., combinations of different α and β subunits. Integrins are cell surface receptors that mediate the attachment of cells to the extracellular matrix ("ECM"). They not only play an important role in cell adhesion but also regulate cell activity in response to many biological cues. Bidirectional inside-out and outside-in signaling molecules that enable activation and It functions as a v β 3 Integrins have specific expression patterns and Nevertheless, most current attempts at developing therapeutics have focused on Emphasis on integrin ligand binding. Target site(s) and action Limitations in the mechanism(s) have hindered successful drug development of these treatments. Summary of the Invention

[0007] The present disclosure does not target integrin ligand binding, and in particular, A TA-1 inhibitor that inhibits cellular angiogenesis without targeting GFR or any other RTK pathways. One aspect of the present disclosure provides an α v β 3 Binds to a novel site on integrin With respect to anti-angiogenic proteins, the proteins include variants of D1-CD2.

[0008] Another aspect of the present disclosure is a v β 3 Anti-angiogenic protein that binds to a novel site on integrins The method comprises administering a therapeutically effective amount of an anti-angiogenic protein. wherein the anti-angiogenic protein comprises a variant of D1-CD2.

[0009] According to a further aspect, the present patent invention provides a method for treating a subject in need of reducing or preventing fibrosis, or a method of reducing or preventing fibrosis in a subject at risk for fibrosis. The method is directed to the α2 helix, the BC loop, and the α2-α3 loop regions. βA domain in α v β 3 Isolated polypeptides that specifically bind to integrins - Patents.com The method includes administering

[0010] According to a further aspect, the present patent invention relates to a polypeptide that specifically binds to an integrin. and a subject in need of reducing or preventing fibrosis or at risk of fibrosis. The present invention is directed to a method of reducing or preventing fibrosis in a subject having an α2 helix, α in the BC loop, and βA domain in the region of the α2-α3 loop v β 3 Integrate The method includes administering to a subject an isolated polypeptide that specifically binds to phosphorus.

[0011] As disclosed herein, in some embodiments, v β 3 Integrin The specific binding polypeptide is domain 1 (D1- A variant of D1-CD2 having an amino acid sequence at least about 75% similar to the sequence of D1-CD2 The polypeptide comprises an α2 helix, a BC loop, and an α2-α3 loop. α in the βA domain in the α region v β 3 Binds specifically to integrins. In some embodiments, the dissociation constant between the proteins is less than about 1 μM. The amino acid substitutions for the variants with hydrophilicity values ​​were compared with the original amino acid of wild-type D1-CD2. The amino acid ranges from about -2 to about +2.

[0012] Further, in some embodiments, the variant comprises (original residue:substitution): (Ala:G ly, Ser), (Arg:Lys), (Asn:Gin, His), (Asp:Glu , Cys, Ser), (Gin:Asn), (Glu:Asp), (Gly:Ala), (His:Asn, Gin), (He:Leu, Val), (Leu:He, Val), (Lys:Arg), (Met:Leu, Tyr), (Ser:Thr), (Thr:S er), (Tip:Tyr), (Tyr:Trp, Phe), and (Val:He, L eu). In some embodiments, the variant is L94N, E95D, K96V, I97C, F 98N, D99F, L100A, K101S, and I102R. In certain embodiments, the variant comprises at least one amino acid substitution at L9 4N, E95D, K96V, I97C, F98N, D99F, L100A, K101S, and I102R. In some embodiments, the variant comprises a sequence having a substitution of: At least one substitution selected from the group consisting of I97Y, F98D, and D99Y. In certain embodiments, the sequence includes the sequences having the substitutions I97Y, F98D, and D99Y. In some embodiments, the variant comprises the sequence D99N, I102V, Q103I , E104I, E8T, T9V, W10Q, G11M, A12K In certain embodiments, the variant comprises at least one substitution selected from the group consisting of D99N, I 102V, Q103I, E104I, E8T, T9V, W10Q, G11M, A12K Includes sequences with substitutions.

[0013] Furthermore, in some embodiments, the variant is a v β 3 Small interactions with integrins In some embodiments, the variant has at least one intermolecular interaction in the βA groove. In α v β 3 In certain embodiments, the variant binds to integrins. QER is α v In certain embodiments, the variant is , FNEEVKKQ β 3 In certain other embodiments, So, the variant is β in FNEEVKKQ. 3 Cross-linked to integrins. In some embodiments, the variant is PEGylated. The ant is PEGylated with polyethylene glycol. The ethylene glycol is PEG-20kDa.

[0014] In another aspect of the presently disclosed subject matter, a pharmaceutical composition is provided. The composition comprises a therapeutically effective amount of a polypeptide as disclosed above. In the form, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.

[0015] In another aspect of the present disclosure, a subject having or suffering from a disease associated with apoptosis is Methods are provided for inducing apoptosis in a subject at risk for apoptosis. The method includes administering a therapeutically effective amount of a pharmaceutical composition as disclosed above.

[0016] Yet another aspect of the presently disclosed subject matter is a method for treating an injury or damaged tissue in a subject. The present invention provides a method for preventing or reducing excessive accumulation of fibrous material in the extracellular matrix in The method reduces the accumulation of fibrous material in the extracellular matrix in injured or damaged tissue. A step of identifying a target to be prevented or reduced, and α2 helix, BC loop , and α in the βA domain in the region of the α2-α3 loop. v β 3 Integrin specific administering to a subject an isolated polypeptide as described herein that binds to and thereby determining whether or not the fibrous material in the extracellular matrix of the subject's tissue is defective. In some embodiments, the subject is diagnosed with liver fibrosis, pancreatic fibrosis, or The patient has at least one of the following symptoms: breast fibrosis, mammary fibrosis, or other type of fibrosis.

[0017] In some embodiments, a subject having or at risk for a fibrotic disease is A method is provided for treating a fibrotic disease in a subject. The method comprises administering to a subject a therapeutically effective amount of the ... The method includes administering a therapeutically effective amount of a pharmaceutical composition as disclosed herein. Non-limiting examples of diseases include liver fibrosis and pancreatic fibrosis.

[0018] In some embodiments, the present disclosure provides methods of preventing and treating a tumor in a subject. The method comprises administering a therapeutically effective amount of a pharmaceutical composition as disclosed herein. This includes:

[0019] In some embodiments, the pharmaceutical compositions as disclosed herein are administered topically. Suitable for injectable, injectable, oral, or sustained release administration.

[0020] In some embodiments of the presently disclosed subject matter, the subject is a mammal. In some embodiments, the subject is a human.

[0021] Furthermore, in some embodiments, the present disclosure provides a method for the preparation of a nucleotide sequence comprising the steps of: and α in the βA domain in the region of the α2-α3 loop. v β 3 Specific binding to integrins In some embodiments, specific binding is provided. K D In another embodiment, the K of specific binding is less than about 1 μM. D is 10 -8 ~ 10 -9 It's M.

[0022] [Definition] "Angiogenesis" is defined as the formation of new vascular systems effective in tissue perfusion. Formation of new blood vessels by sprouting endothelial cells from existing blood vessels, or improving blood perfusion in tissues This involves remodeling existing blood vessels to alter their size, maturation direction, or flow characteristics.

[0023] The terms "subject," "individual," "patient," and "host" are used interchangeably herein. It is used in the same sense as above, specifically to refer to any vertebrate animal, such as any mammal, most Specifically, this includes human subjects, livestock, and mammalian pets. Although not required by law, they may be under the supervision of a medical professional such as a physician or veterinarian and Therapeutic treatment with the indicated compositions may be required.

[0024] The term "fibrotic" disease, disorder, or condition is used herein to refer to any disease, disorder, or condition that is related to the including acute and chronic, clinically or clinically evident fibrosis-related biology and pathology. The fibrotic disease, disorder, or condition further includes symptoms of fibrosis. Excessive production of fibrous substances or abnormal, non-functional, and and / or excessive production of fibrous material, including replacement of normal tissue elements by excessive accumulation Fibrotic diseases include diseases, disorders, or conditions that are characterized in whole or in part by The disease, disorder, or condition may be characterized, for example, by fibrosis-related biology or or pathology.

[0025] As used herein, the term "therapeutically effective amount" refers to a therapeutically effective amount for the disease, disorder, or condition being treated. Treat or at least partially halt the progression of one or more of the symptoms of a disease or condition. or a patient who already has a disease, condition, or disorder sufficient to provide some relief. The term refers to the amount of a composition containing a modified non-natural amino acid polypeptide administered to a subject. The effectiveness of such compositions may include, but is not limited to, the severity and progression of a disease, disorder, or condition. the course of treatment, past treatments, the patient's general health and response to the drug, and the treating physician's judgment. By way of example only, a therapeutically effective amount may include, but is not limited to, This can be determined by routine experimentation including dose escalation clinical trials.

[0026] As used herein, the term "vector" refers to a vector that is used to deliver an exogenous gene to a host cell. The term "DNA molecule" refers to a molecule that functions as a medium that can stably transport DNA. For this purpose, a vector is a system capable of replicating and introducing itself into a host cell. The vector should have a gene encoding the desired vector and carry a selectable marker.

[0027] The term "amino acid" refers to natural and unnatural amino acids, as well as naturally occurring amino acids. This refers to amino acid analogs and amino acid mimetics that function in a similar manner to amino acids. The coded amino acids are the 20 common amino acids (alanine, arginine, asparagine, Aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, Isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenium Amino acid analogs include, by way of example only, hydrogen, carboxyl, Naturally occurring amino acids, such as an alpha carbon bonded to an R group, an amino group, and an R group. Analogs refer to compounds having the same basic chemical structure. Such analogs may have modified R groups (e.g., or norleucine) or may have a naturally occurring amino acid The peptide may have a modified backbone while still retaining the same basic chemical structure. Non-limiting examples of acid analogs include homoserine, norleucine, methionine sulfoxide, methionine, and methionine methylsulfonium.

[0028] As used herein, the term "effective amount" refers to an amount of a substance that is effective for treating the disease or condition being treated. A drug or compound administered in an amount sufficient to alleviate to some extent one or more of the symptoms A result is a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or By way of example, the administered drug or chemical may be The compounds include, but are not limited to, natural amino acid polypeptides, non-natural amino acid polypeptides, The polypeptides may include modified natural amino acid polypeptides or modified non-amino acid polypeptides. Such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, Compositions containing amino acid polypeptides or modified non-natural amino acid polypeptides The substances may be administered for prophylactic, enhancing, and / or therapeutic treatments. An appropriate "effective" amount may be determined using techniques, such as a dose escalation study.

[0029] As used herein, the term "cell or population of cells" refers to a cell or population of cells excised from a tissue. isolated cells or multiple cells grown in vitro by tissue culture techniques Most specifically, a population of cells refers to in vivo cells within an animal or human tissue. Refers to the cell.

[0030] As used herein, the term "contacting a cell or population of cells" refers to the The peptide or probe according to the invention is administered to an isolated or cultured cell or population of cells. or delivering the probe in a suitable pharma- ceutically acceptable carrier to an animal or human. Administration refers to administration to a target tissue, including, but not limited to, intravenous delivery, intraperitoneal delivery, , intramuscular delivery, subcutaneously, or by any other method known in the art. One effective method is to deliver the drug directly to the blood vessels that lead directly to the target organ or tissue, such as the pancreas. , thereby reducing dilution of the probe in the general circulatory system.

[0031] As used herein, the term "pharmaceutically acceptable carrier" refers to any If the dimer probe is administered and approved by a federal or state government regulatory agency, or the United States Pharmacopoeia, or more specifically other commonly recognized The term "diluents, adjuvants, excipients, or vehicles" refers to any of the substances listed in the Pharmacopoeias prescribed by the Pharmaceuticals and Medical Devices Agency (PMA). Chemical carriers are of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. The pharmaceutical carrier may be a liquid such as water and oil, including water and oil of any suitable concentration. It can be cassia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the heterodimeric probe and the pharma- ceutically acceptable carrier are sterile. Water is an effective carrier when the heterodimeric probe is administered intravenously. Saline and aqueous dextrose and glycerol solutions are also suitable as liquids, particularly for injections. Suitable pharmaceutical carriers also include glucose, lactose, sucrose, and the like. Cerose, glycerol monostearate, sodium chloride, glycerol, propylene The compositions of the present invention also include excipients such as glycols, water, ethanol, etc. The compositions of the present invention, if used, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may be advantageously provided in the form of a solution, emulsion, sustained release formulation, or any other suitable form for use. It is possible.

[0032] The terms "polypeptide," "peptide," and "protein" refer to a sequence of amino acid residues. are used interchangeably herein to refer to polymers of

[0033] The term "variant" refers to a peptide or polynucleotide that differs from a reference peptide or polynucleotide, but A peptide or polynucleotide that retains essential properties. A variant differs in amino acid sequence from another reference peptide. Generally, the difference is The sequences of the reference peptide and the variants are generally very similar and have many The variant and reference peptides are restricted to be identical in the region. one or more modifications (e.g., substitutions, additions, and / or deletions) in the amino acid sequence. Variants of a peptide may be conservatively modified variants of the original sequence (e.g. For example, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, and about 99% The substituted or inserted amino acid residues are not included in the genetic code. Variants of the peptide may or may not be encoded by can be naturally occurring, such as an allelic variant, or can be non-naturally occurring. It may be an unknown variant.

[0034] As used herein, the term "target" refers to a peptide that is desired to be detected. The target peptide may be on the surface of a cell, which may be a isolated from an animal host, cultured cells, or a cell or population of cells within an animal tissue It is being done.

[0035] The present disclosure includes peptides that can be derived from the sequences of naturally occurring peptides. , can be obtained by fragmenting a naturally occurring sequence, or the sequence of amino acids present in or the sequence of the genetic material (DNA or RNA) that codes for it "Derived from a naturally occurring amino acid sequence" if the amino acid sequence can be synthesized based on knowledge of the These are said to be "derivatives" of peptides. Such "derivatives" or "variants" are included within the scope of the present disclosure. share substantial similarity with a similarly sized fragment of a peptide or peptide and It can function with the same biological activity as tides.

[0036] As used herein, the term "specifically binds" or "specific binding" refers to the binding of a binding protein to a given protein (e.g., an integrin). The affinity of is the dissociation constant (K D ) is defined in terms of the binding protein K D About 10 - 8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, or even less than this, about 10 -7 Specific for proteins when less than M and binds to at least 100-fold less, for example at least 1,000-fold less, Its affinity for binding to non-specific antigens (e.g., BSA) is reduced, e.g., 10,000-fold Both are less than 10 times K D The present invention binds to a given protein with an affinity corresponding to that of the present invention. When used in the fine print, "K D " or "K d The term " The dissociation constant refers to the dissociation constant of a specific protein-protein binding interaction such as that found in a protein-protein complex.

[0037] The derivatives of the present disclosure contain sequences that are substantially similar to the sequences of naturally occurring peptides. In addition, one or more additional Similarly, the present disclosure includes fragments that may contain amino acids of the sequence of naturally occurring peptides. contains a sequence substantially similar to those found naturally in peptides, and / or may lack one or more additional amino acids at their carboxy termini. Includes peptide fragments.

[0038] As used herein, the term "prophylactically effective amount" refers to the amount of a substance that is effective to treat the disease, condition, or condition being treated. A therapeutic agent that is administered prophylactically to a patient to relieve to some extent one or more of the symptoms of a condition or disorder. At least one non-natural amino acid polypeptide or at least one modified non-natural In such prophylactic applications, the amino acid polypeptide is administered in a dose-dependent manner. The amount administered may depend on the patient's health status, weight, etc., and may be determined by, but is not limited to, dose escalation clinical trials. Determining such a prophylactically effective amount is within the skill of the art, including by routine experimentation. is often considered.

[0039] The phrase "substantially similar" in the context of two nucleic acids or polypeptides includes, e.g. As determined using sequence comparison algorithms, such as those described below. , or by visual inspection, when compared and aligned against maximum correspondence, At least 75%, preferably at least 85%, more preferably at least 90%, % or more, or any integer value therebetween, of nucleotide or amino acid residue identity. Preferably, at least about 10, more preferably at least about 20, more preferably at least about 30, more preferably at least about 40, more preferably at least about 50, more preferably at least about 60, more preferably at least about 70, more preferably at least about 80, more preferably at least about 90, more preferably at least about 10 ... is about 20, more preferably about 40-60 residues in length or any integer value therebetween. Over a region of the sequence, preferably a region longer than 60-80 residues, more preferably There is substantial identity over at least about 90-100 residues, most preferably The sequence may be, for example, a sequence of the coding region of a nucleotide sequence over the entire length of the sequence being compared. are substantially identical.

[0040] As used herein, the term "synergistic" refers to the interaction of any two or more single agents. It refers to a prophylactically or therapeutically effective combination of drugs that has a more effective than additive effect. A synergistic effect of a combination of preventive or therapeutic agents is achieved by reducing the severity of one or more of the agents. Dosage and / or more frequent administration of the drug to subjects with a particular disease or condition In some cases, it may be possible to administer a combination of prophylactic or therapeutic agents at a lower dose. The synergistic effect can be used to reduce adverse side effects or undesirable effects associated with the use of any single treatment. Undesirable side effects may be avoided or reduced.

[0041] As used herein, the term "therapeutically effective amount" refers to a therapeutically effective amount for the disease, disorder, or condition being treated. Treat or at least partially halt the progression of one or more of the symptoms of a disease or condition. or a patient who already has a disease, condition, or disorder sufficient to provide some relief. at least one non-natural amino acid polypeptide and / or at least The term "modified non-natural amino acid polypeptide" refers to the amount of a composition that contains at least one modified non-natural amino acid polypeptide. The effectiveness of the composition may include, but is not limited to, the severity and progression of the disease, disorder, or condition, past symptoms, and / or past symptoms. symptoms, including the treatment of the condition, the patient's health condition and response to medications, and the judgment of the treating physician. By way of example only, a therapeutically effective amount may include, but is not limited to, a dose escalation regimen. This can be determined by routine experimentation, including clinical trials.

[0042] Unless otherwise specified, a particular nucleic acid sequence also includes conservatively modified variants thereof (e.g., For example, degenerate codon substitutions) and complementary sequences, as well as sequences explicitly shown, Specifically, the third codon of one or more selected (or all) codons is and generating sequences in which positions A, B, C, and D are replaced with mixed groups and / or deoxyinosine residues. Degenerate codon substitutions can be achieved by the following: Used interchangeably with mRNA, oligonucleotide, and polynucleotide do.

[0043] As used herein, the term "subject" refers to the target of administration of a pharmaceutical composition. The subject of the methods disclosed herein may be a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the methods disclosed herein may be a human or other vertebrate. Thus, in accordance with the presently disclosed subject matter, veterinary Therapeutic uses are provided. Thus, the presently disclosed subject matter is mammals, such as the reptile and non-human primates, as well as endangered species, such as the Siberian tiger economically important animals, such as those raised on farms for human consumption, which are important due to animals that are important to humans and / or kept as pets or zoo animals The present invention provides for administration to these mammals that are of social importance. Examples of such animals include: Including, but not limited to, carnivores such as cats and dogs, pigs, domestic pigs, and wild boars. Pigs, cattle, bulls, sheep, giraffes, deer, goats, bison, and camels, rabbits, guinea pigs Threatened species include ruminants and / or ungulates such as rats, rats, and mice. These bird species, as well as poultry, are kept in captivity and / or in zoos. Specifically, domesticated poultry, such as turkeys, chickens, and chicks, which are also economically important to humans, Avian treatment is also provided, including treatment for birds, ducks, geese and guinea fowl. Therefore, it is important to note that the following animals are not limited to domestic pigs, ruminants, ungulates, horses (competitive animals), etc. Treatment of livestock, including horses, poultry, etc. is also provided. The term is used to refer to animals of a particular age or It does not indicate gender or gender identity.

[0044] The present disclosure also provides that such variants have substantially the same activity as the derivatives described above. It may have minor amino acid substitutions (thus altering the natural sequence) provided that it has activity. the amino acid sequence of the fragments described above) includes obvious variants. Examples of obvious or obvious substitutions include a single basic residue substitution of one hydrophobic residue for another (i.e., Arg for Lys), substitutions for one aromatic residue (i.e., Leu for He) or for another aromatic residue (i.e., Phe for Tyr), and the like.

[0045] The detailed description is given with reference to the accompanying drawings, in which the left-most digit(s) of a reference number The reference number(s) identifies the figure in which the reference number first appears. The use of symbols indicates similar or identical items or features. [Brief description of the drawings]

[0046] [Figure 1A] 1 is a depiction of D1-CD2 merging into the βA groove in accordance with the present disclosure. [Figure 1B] 1 is a depiction of D1-CD2 merging into the βA groove in accordance with the present disclosure. [Figure 1C] 1 is a depiction of D1-CD2 merging into the βA groove in accordance with the present disclosure. [Figure 1D] 1 is a depiction of D1-CD2 merging into the βA groove in accordance with the present disclosure. [Diagram 2] 1 is a tubular representation illustrating the intermolecular energies of D1-CD2 variants according to the present disclosure. [Diagram 3] 1 shows a H-NMR readout illustrating that ProAgio has structural characteristics similar to those of D1-CD2, in accordance with the present disclosure. [Figure 4A] 1 is a graphical representation illustrating ProAgio interaction with integrins in accordance with the present disclosure. [Figure 4B] 1 is a graphical representation illustrating ProAgio interaction with integrins in accordance with the present disclosure. [Figure 4C] 1 is a tubular representation illustrating ProAgio interaction with integrins in accordance with the present disclosure. [Diagram 5] 1 shows αvβ3 expression in HUVEC cells in accordance with the present disclosure. [Figure 6] 1 is a graphical representation illustrating the attachment of HUVEC cells to ProAgio in accordance with the present disclosure. [Figure 7] 1 shows exogenous αvβ3 expression in CHO and COS-7 cells in accordance with the present disclosure. [Figure 8] 1 is a graphical representation illustrating the attachment of αvβ3-expressing CHO and COS-7 cells to ProAgio-coated plates in accordance with the present disclosure. [Figure 9] 1 is a graphical representation illustrating the lack of adhesion of αIIBβ3 integrin-expressing CHO cells to ProAgio-coated plates in accordance with the present disclosure. [Figure 10]1 shows co-immunoprecipitation of ProAgio with β3 integrin in HUVEC cell extracts in accordance with the present disclosure. [Figure 11] 1 is a graphical representation illustrating that conjugation of RGD does not prevent attachment of HUVEC cells to ProAgio in accordance with the present disclosure. [Figure 12] 1 shows confirmation, in accordance with the present disclosure, that conjugation of RGD does not prevent adhesion of HUVEC cells to ProAgio. [Figure 13A] 1 shows cross-linking of ProAgio to αv integrin in accordance with the present disclosure. [Figure 13B] 1 shows cross-linking of ProAgio to αv integrin in accordance with the present disclosure. [Figure 13C] 1 shows cross-linking of ProAgio with bis(sulfosuccinimidyl)-glutaric acid in accordance with the present disclosure. [Figure 14] 1 shows cross-linking of ProAgio with glutaraldehyde in accordance with the present disclosure. [Figure 15A] 1 shows cross-linking of ProAgio with glutaraldehyde in accordance with the present disclosure. [Figure 15B] 1 shows cross-linking of ProAgio with glutaraldehyde in accordance with the present disclosure. [Figure 16] In accordance with the present disclosure, we show that mutations to β3 integrin reduced adhesion of cells expressing the mutant to ProAgio. [Figure 17] In accordance with the present disclosure, it is shown that the mutations reduced adhesion of CHO cells expressing the mutants. [Figure 18] 1 is a graphical representation illustrating that the mutations reduced adhesion of CHO cells expressing the mutants to ProAgio in accordance with the present disclosure. [Figure 19] In accordance with the present disclosure, it is shown that the mutation abolishes co-immunoprecipitation of PrоAgio and β3 integrin. [Figure 20]In accordance with the present disclosure, we show that actin filament stress fibers were reduced and disappeared in HUVEC cells treated with ProAgio. [Figure 21] In accordance with the present disclosure, it is shown that ProAgio treatment results in the inactivation of FAK that is activated by attachment to the ECM. [Figure 22] 1 is a graphical representation illustrating that ProAgio induces apoptosis of HUVEC cells in accordance with the present disclosure. [Diagram 23] 1 is a graphical representation illustrating that ProAgio is more effective at inducing apoptosis than other anti-angiogenic agents in accordance with the present disclosure. [Figure 24] 1 is a graphical representation illustrating that ProAgio induces apoptosis of HUVEC and COS-7 cells expressing exogenous integrins in accordance with the present disclosure. [Diagram 25] 1 is a graphical representation illustrating that ProAgio induces apoptosis of HUVEC and COS-7 cells expressing exogenous integrins in accordance with the present disclosure. [Figure 26] In accordance with the present disclosure, we demonstrate that ProAgio treatment does not result in floating / detachment of HUVEC cells. [Figure 27] 1 shows the effect of ProAgio treatment on caspase-8, caspase-9, caspase-7, and caspase-3 in accordance with the present disclosure. [Figure 28] 1 is a graphical representation illustrating the effect of caspase 8 and caspase 9 inhibitors on ProAgio in accordance with the present disclosure. [Figure 29] 1 shows the effect of caspase 8 and caspase 9 inhibitors on mutual activation in accordance with the present disclosure. [Diagram 30] 1 shows the effect of Cilengitide on the recruitment of caspase 8 to the integrin β3 cytoplasmic domain in accordance with the present disclosure. [Diagram 31] 1 shows PEGylation of ProAgio in accordance with the present disclosure. [Diagram 32]1 is a graphical representation illustrating the effect and dose dependency of ProAgio-PEG on tumor growth inhibition in accordance with the present disclosure. [Diagram 33] 1 is a graphical representation illustrating the effect and dose dependency of ProAgio-PEG on tumor growth inhibition in accordance with the present disclosure. [Diagram 34] 1 is a graphical representation illustrating the effect of ProAgio-PEG when administered at a late stage of tumor growth in accordance with the present disclosure. [Diagram 35] 1 is a graphical representation illustrating the effect of ProAgio-PEG when administered at a late stage of tumor growth in accordance with the present disclosure. [Diagram 36] 1 shows CD31 immunostaining of tissue sections prepared from harvested tumors to analyze the effect of ProAgio-PEG on tumor vasculature in accordance with the present disclosure. [Figure 37] 1 is a tubular representation illustrating the effect of ProAgio-PEG on tumor vasculature in accordance with the present disclosure. [Figure 38] 1 is a graphical representation illustrating the efficacy of ProAgio-PEG compared to Avastin® and Endostar® in accordance with the present disclosure. [Figure 39] 1 is a graphical representation illustrating the efficacy of ProAgio-PEG compared to Avastin® and Endostar® in accordance with the present disclosure. [Diagram 40] 1 is a graphical representation illustrating the efficacy of ProAgio-PEG compared to Avastin® and Endostar® in accordance with the present disclosure. [Diagram 41] 1 is a graphical representation illustrating the efficacy of ProAgio-PEG compared to Avastin® and Endostar® in accordance with the present disclosure. [Diagram 42] 1 is a graphical representation illustrating the effect of ProAgio-PEG on tumor growth in accordance with the present disclosure. [Diagram 43]1 is a graphical representation illustrating the effect of ProAgio-PEG on tumor growth in accordance with the present disclosure. [Diagram 44] 1 shows the effect of ProAgio-PEG on tissues in accordance with the present disclosure. [Diagram 45] 1 is a graphical representation illustrating the effect of ProAgio-PEG on weight gain or loss in accordance with the present disclosure. [Figure 46] 1 shows the effect of ProAgio on livers from mice with fibrosis in accordance with the present disclosure. [Figure 47] 1 shows the effect of ProAgio on livers from mice with fibrosis in accordance with the present disclosure. [Figure 48] 1 is a table showing the effect of ProAgio on livers from mice with fibrosis in accordance with the present disclosure. [Figure 49] 1 is a table showing the effect of ProAgio on livers from mice with fibrosis in accordance with the present disclosure. [Figure 50] 1 is a graphical representation illustrating the effect of ProAgio on apoptosis of primary human hepatic stellate cells in accordance with the present disclosure. [Figure 51] 1 shows the effect of ProAgio on livers from mice with fibrosis in accordance with the present disclosure. [Figure 52] 1 is a graphical representation illustrating the effect of ProAgio on mouse body weight in accordance with the present disclosure. [Diagram 53] 1 is a graphical representation illustrating the effect of ProAgio on fibrotic mouse liver in accordance with the present disclosure. [Fig. 54A.54B] 1 is a graphic representation illustrating the effect of ProAgio concentration on liver fibrosis. [Fig. 55A-55D] Showing results of ProAgio as a treatment for breast cancer. [Figure 56] ProAgio has been shown to reduce and prevent fibrosis in pancreatic cancer, which is caused by cirrhotic stromal and desmoplastic cells. [Fig. 57A-57C] Showing results of ProAgio as a treatment for liver cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The present disclosure provides anti-angiogenic or anti-fibrotic agents and methods for inhibiting angiogenesis. Conditions that depend on angiogenesis or fibrosis in mammals are related to ProAgio and The antiangiogenic drugs described herein are capable of causing regression or halting the progression of the condition without significant toxicity. The mammal is treated by administering the compound to the mammal in a therapeutically effective amount and frequency. Conditions that depend on angiogenesis include breast cancer, lung cancer, prostate cancer, colon cancer, prostate cancer, and egg cancer. Includes fossa carcinoma, neuroblastoma, central nervous system tumor, neuroblastoma, glioblastoma multiforme, or melanoma , and solid tumor neoplasms. Although it is not possible to reverse or halt the progression of most, if not all, solid tumors, Anti-angiogenic drugs can be used to provide the desired effect. It can be.

[0048] The present disclosure generally relates to a v β 3 The present invention relates to a polypeptide that specifically binds to an integrin. More specifically, the present disclosure relates to a method for the production of wild-type cell adhesion proteins. Domain 1 (D1-CD2 ) array : 1 KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKT SDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQ DIYKVSIYDTKGKNVLEKIFDLKIQER 106 (SEQ ID NO: 1) and a variant of D1-CD2 having an amino acid sequence at least about 75% similar to The present disclosure further relates to methods for treating diseases and conditions associated with angiogenesis, cell proliferation, cancer, and fibrosis. The present disclosure also relates to methods of using the polypeptides in the treatment of α2 helix, B in the βA domain in the region of the -C loop and the α2-α3 loop v β 3 Integri Examples of such polypeptides include isolated polypeptides that specifically bind to Pr Exemplary sequences of ProAgio include: KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSD KKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDI YKVSIYDTKGKNVNDVCNFASRQER (SEQ ID NO:2) and KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSD KKKIAQFRKEKETFKEKD TYKLFKNGTLKIKHLKTDDQD IYKVSIYDTKGKNVLEKYDYLKIQER (SEQ ID NO: 3) Includes:

[0049] As disclosed herein, ProAgio is a novel cellular endothelial cell line that is capable of regulating host proteins and cell adhesion. ProAgio contains domain 1 of the CD2 protein ("D1-CD2"). The sites are the α2 helix, the BC loop, and the groove in the α2-α3 loop region (" The βA groove is the integrin that targets αvβ3 integrin. β integrins and is relatively close to the RGD binding site in the βA domain. Thus, it is disclosed that ProAgio can target the βA groove of αvβ3 integrin. However, one of skill in the art can use ProAgio to synthesize any βA groove containing It should be appreciated that the βA groove of an integrin may be targeted.

[0050] One embodiment is a method for determining whether or not a subject has a fibrous substance in an extracellular matrix in an injured or damaged tissue. A method for preventing or reducing excessive accumulation, comprising the steps of: and identifying a target for the targeting of the wild-type cell adhesion protein domain 1 (D1-C D1-CD2 barrier having an amino acid sequence at least about 75% similar to the sequence of D2 Including a v β 3 A method for administering a polypeptide that specifically binds to an integrin to a subject. and a polypeptide comprising an α2 helix, a BC loop and an α2-α3 loop. In the βA domain, α v β 3 Methods for specifically binding to integrins In the method of claim 22, the subject has liver fibrosis. The subject has pancreatic fibrosis. They may have liver fibrosis, or breast fibrosis.

[0051] Exemplary fibrotic diseases, disorders and conditions include, for example, scleroderma (localized scleroderma, systemic scleroderma, localized scleroderma, or linear scleroderma), renal fibrosis (glomerulosclerosis, tubulointerstitial fibrosis, fibrosis, progressive renal disease, or diabetic nephropathy), cardiac fibrosis (e.g., myocardial fibrosis), Pulmonary fibrosis (e.g. glomerulosclerotic pulmonary fibrosis, idiopathic pulmonary fibrosis, silicosis, asbestosis, interstitial lung disease) lung disease, interstitial fibrotic lung disease, and chemotherapy / radiation-induced pulmonary fibrosis), oral fibrosis, cardiac Endomyocardial fibrosis, deltoid fibrosis, pancreatitis, inflammatory bowel disease, Crohn's disease, nodular fasciitis, acidophilia Bulbar fasciitis, a condition characterized by varying degrees of replacement of normal muscle tissue with fibrous tissue. General fibrosis syndrome, retroperitoneal fibrosis, liver fibrosis, liver cirrhosis, chronic renal failure, myelofibrosis (bone marrow fibrosis), drug-induced ergotine toxicity, glioblastoma in Li-Fraumeni syndrome, diffuse glioblastoma, myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative syndrome, These include gynecological cancers, Kaposi's sarcoma, leprosy, collagenous colitis, and acute fibrosis. Fibrosis can be either chronic or acute. Fibrotic symptoms include dysfunction, and potentially an excess of extracellular matrix in tissues that can form tissues that result in organ failure. These include excessive amounts of fibrous tissue, including the accumulation of fibrous tissue.

[0052] The present disclosure provides a method for inhibiting tumor growth and inhibiting VEGF / VEGFR or any other RTK mediated pathway. These compounds are active in inducing angiogenic endothelial cell apoptosis without targeting the vascular pathway. It contains a non-toxic protein, later called ProAgio.

[0053] While not being bound by any single theory, ProAgio 3 The cytoplasmic domain of By recruiting and activating spase-8, it efficiently inhibits α v β 3 Integrin-expressing cells It is believed that this drug specifically induces apoptosis in cancer-associated pancreatic stellate cells (CAPaS). C) and angiogenic endothelial cells both have high levels of α v β 3 To express integrins, ProAgio depletes CAPaSCs and promotes the proliferation of new cells in and around tumors or other tissues. This may result in the removal of new blood vessels.

[0054] ProAgio induces apoptosis with or without ligand binding. Since ProAgio binding does not interfere with RGD binding, These results suggest that the effect of ProAgio is independent of ligand binding. The mechanism is different from that of v β 3 Induce integrin-mediated cell apoptosis. Thus, ProAgio does not necessarily compete with strong integrin-ligand interactions. It is not necessary to have a single integrin, but rather to have a vascular morphology involving interactions between multiple integrin pairs and multiple types of ECM. There is no need to induce anoikis, which may not occur due to adult endothelial cell-ECM adhesion .

[0055] One embodiment is a method for the apoptosis of activated hepatic and pancreatic stellate cells and myofibroblasts. These proteins are involved in the induction of cis-transferase activity in the liver and other They are thought to be the main source of extracellular matrix in organs. Stellate cells and myofibroblasts It reacts with various growth factors and cytokines present in the liver and other organs, Some of them also cause liver fibrosis. Induction of apoptosis of stellate cells, matrix degradation Promoting hepatic fibrosis by promoting hepatic apoptosis or promoting stellate cell apoptosis reduces hepatic fibrosis. In hepatic stellate cells, dedifferentiated sinusoidal endothelial cells (LSECs) express high levels of α v β 3 in The effect of ProAgio on killing dedifferentiated LSECs was enhanced by portal vein hypertension. Reduce hyperactivity.

[0056] Modifications and variations may be made in the structure of the peptides of the present disclosure, and Molecules can be obtained that have similar characteristics (eg, conservative amino acid substitutions) to the tides. For example, certain amino acids can be substituted for other amino acids in a sequence without significant loss of activity. It is the interactive capabilities of a peptide that define its biological functional activity. Because of their power and nature, certain amino acid sequence substitutions are made in peptide sequences. It is possible, nevertheless, to obtain peptides with similar properties.

[0057] 1A-19 show the design of ProAgio binding to the βA groove. The ProAgio featured is α v β 3 It can bind to the βA groove of integrins One of skill in the art can easily recognize that the βA groove-containing integrin is capable of binding to any integrin that contains the βA groove as described above. It should be understood that the above may be combined.

[0058] The drug improves vascular function by inhibiting or controlling apoptosis and fibrosis. The developed polypeptides are believed to inhibit or prevent the formation of Induction of endothelial cell apoptosis without effect on epithelial cells and fibroblasts in In addition, the developed polypeptides demonstrated strong activity in existing normal The survival factor was a growth factor or myeloid cell growth factor (MCG) for vascular endothelial cells. It does not appear to have a direct growth stimulating effect, but does have a protective effect against cell damage. These factors allow the body to recover from

[0059] These agents include administering anti-angiogenic and anti-fibrotic agents. In certain embodiments, the present invention may be incorporated into a method of treating a mammal by inhibiting Antiangiogenic or antifibrotic polypeptides have been shown to be more effective than small molecule and short peptide drugs. and showed prolonged circulation time.

[0060] Certain embodiments relate to methods of inhibiting angiogenesis or fibrosis, and to methods of treating angiogenesis-related or In another embodiment, the present invention provides a method for treating a tumor, comprising administering to the patient a therapeutically effective amount of ... fibrotic or fibrotic disease. Methods for inhibiting or reducing proliferation and for treating individuals suffering from cancer - Patents.com These methods include administering a therapeutically effective amount of one or more of the compounds described above. These methods include administering to an individual the above polypeptide therapeutic agents. Specifically, it is particularly intended for the therapeutic and prophylactic treatment of humans.

[0061] As described herein, angiogenesis-related or fibrotic diseases include, but are not limited to, For example, solid tumors, vascular infections such as leukemia, and tumor metastases, but not limited to these, may be involved in the development of vascular Plasma-dependent cancers, benign tumors, such as hemangiomas, acoustic neuromas, neurofibromas, trachoma, and Inflammatory diseases such as pyogenic granulomas, immune and non-immune inflammation, chronic rheumatoid arthritis and psoriasis Scabies, hair angiogenesis disorders, e.g. diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplants Rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, Osler-Weber syndrome syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophilic arthropathy, vascular fibrosis Tumors, granulation and wound healing, telangiectasias, psoriasis, scleroderma, pyogenic granulomas , coronary collateral circulation, ischemic limb angiogenesis, corneal disease, rubeosis, arthritis, neovascularization in diabetes , bone fracture, angiogenesis, and hematopoiesis, including angiogenesis-dependent cancers.

[0062] One potential benefit of combining antiangiogenic or antifibrotic agents with chemotherapy drugs The present invention relates to improved treatment and control of angiogenesis-dependent conditions with reduced doses of chemotherapy agents. The combination may be administered for a continuous period of time, or optionally for a shorter duration. treatment may be administered due to the increased effectiveness of the combination.

[0063] Depending on the nature of the combination therapy, administration of the polypeptide therapeutic agent of the invention may be continued. Concurrently and / or subsequently administered with other therapeutic agents. Administration of the agent may be in a single dose or in multiple doses. Administration of the tidotherapy agent is initiated at least several days prior to conventional therapy, and in other instances, Administration is commenced immediately prior to or simultaneously with administration of the type of treatment.

[0064] In silico and in situ analyses showed that α v β 3 It can bind integrins Remarkably, the β 3 βA is involved in ligand binding and induction Affects integrin signaling. D1-C from both humans and rats D2 is α v β 3 It exhibits rather weak affinity for known integrin binding sites. D 1-CD2 is a novel site, i.e., several regions of the βA domain containing the βA groove in various orientations. However, it does not bind to residues in the βA groove (Figure 1A-1D). Due to the lack of strong contacts, docking of D1-CD2 into the βA groove is restricted by the corresponding residues in the βA groove. By mutating residues of D1-CD2 at contact positions to better match This improves the structural integrity of the wild-type β-stimulator by overriding the potential structural hindrances caused by the previously mentioned mutations. Further mutations were introduced into D1-CD2 to maintain the integrity of the integument. The energy represented by the combination of these D1-CD2 variants on the green is Compared to the original D1-CD2, it was substantially increased (shown in FIG. 2). The binding energy was significantly reduced when the corresponding residues in the integrin were mutated. , thereby preventing the designed contact.

[0065] The D1-CD2 variant was expressed in E. coli and then purified. gio was analyzed by H-NMR (shown in Figure 3), far-UV CD, and fluorescence spectroscopy. The structural features of the potential D1-CD2 protein are similar to those of the D1-CD2 protein, as demonstrated by the analysis. This indicates that ProAgio is efficiently folded. Furthermore, BIACORE binding analysis demonstrated that ProAgio binds α v β 3 Integrin and ProAgio also demonstrated that the two other pairs of It weakly interacts with integrins and is v β 3 It has been shown to specifically interact with integrins. This suggests that the IL-11 pathway may be involved in the regulation of IL-11 expression (as shown in Figures 4A-4C).

[0066] Cell attachment assays using ProAgio-coated plates were performed using ProAgio io / α v β 3 The interaction of integrins was examined. High levels of α v β 3 Has expression The HUVEC cells (shown in Figure 5) were strongly adherent to the ProAgio-coated plates. The force was applied (shown in Figure 6). v β 3CHO and COS-7 cells that do not express The cells did not attach to the ProAgio-coated plates. v β 3 Also, CHO and exogenously expressed in COS-7 cells (shown in FIG. 7 ) and described above. The same cell adhesion assay was performed on these cells. v β 3 Expression CHO and COS- 7 cells attached to the ProAgio coated plates (as shown in FIG. 8). As a reference group, α v β 3 Integrin-expressing CHO cells were incubated with ProAgio-coated pregelatinocytes. These cell attachment assays showed that the cells did not adhere to the cellulose membrane (Figure 9). Alpha on the surface v β 3 1 shows ProAgio interaction with integrins.

[0067] The interaction of ProAgio with integrins was confirmed by the β 3 Intel This was further verified by co-immunoprecipitation of ProAgio with glyrin (shown in Figure 10). (I).

[0068] Since ProAgio binds to the βA groove, ProAgio and RGD bind to the same site. Since no binding was observed, ProAgio binding does not compete with RGD binding. Then, HUVEC cells were incubated with RGD, followed by the attachment of ProAgio. This experiment demonstrated that both RGD and ProAgio were able to inhibit HU without interference. Binding to VEC cells has been demonstrated (shown in FIG. 11).

[0069] Bis(sulfosuccinimidyl)-glutaric acid (BS2G) as a crosslinker (Figure 12 and Chemical cross-linking with ProAgio (shown as a βA groove site) demonstrated that ProAgio binds to the βA groove site. This cross-linking followed by trypsin digestion and LC-MS analysis demonstrated that TEMKQER ProAgio WEKTS cross-linked to integrin ay at (aa 116-122) DKK (aa 35-43) are shown (shown as Figures 13A-13C). roAgio and β 3 Integrin interactions are due to steric hindrance, i.e. , was not detected due to BS2G resulting from the bulk volume of cross-linking reactive groups.

[0070] Accordingly, glutaraldehyde (shown in Figure 14) was used as a cross-linking agent. ProAgio NLKVII (aa 99-105) is a β-Glutaraldehyde 3 Integrin FNEEVKKQ(aa 203-210) (shown in Figures 15A-15B). This also indicates that ProAgio

[0106] crosslinks to α at the βA groove site. v β 3 It has been shown to interact with integrins.

[0071] The binding of ProAgio to the βA groove was further verified by mutational analysis. β at 58A, K233A, and K234A 3 Mutations on integrins can cause The adhesion of mutant expressing cells to ProAgio was reduced (shown in FIG. 16). The computer calculations showed that the mutation is ProAgio / α v β 3 Attenuating integrin binding The mutations also demonstrated that the mutants expressing CHO cells (shown in FIG. 17) ) to ProAgio (shown in FIG. 18), and β 3 Co-immunoprecipitation of integrins was abolished (shown as FIG. 19).

[0072] Figures 20 to 30 show β 3 Recruitment of caspase-8 to the cytoplasmic domain of integrins and The effect of ProAgio on cell apoptosis due to activation of ProA Actin filament stress responses of HUVEC cells in the presence and absence of gio Analyzing the formation of BAR and focal adhesion complexes, we investigated the effect of ProAgio treatment on integrin activity The effect of oxidizing the actin filament stress fibers was evaluated. The stress was reduced and disappeared when treated with Agio (as shown in FIG. 20). Accumulation of vinculin at the fiber tips was also observed in ProAgio-induced HUVEC cell proliferation. This was reduced upon treatment (not shown), indicating dissociation of focal adhesion complexes.

[0073] Furthermore, ProAgio treatment inhibited focal adhesion kinase activated by adhesion to the ECM. 21. Thus, ProAgi o abolishes the function of integrins in endothelial cells.

[0074] ProAgio also induces apoptosis in HUVEC cells. oAgio inhibited HUVEC cell apoptosis with an EC50 of 1.4 μM after 10 h of treatment. induced cytosis (as shown in FIG. 22).

[0075] ProAgio was compared with several anti-angiogenic drugs using HUVEC cells. Angiogenesis drugs include Avastin®, Endostar® (available in China) LM609 (registered trademark), an endostatin derivative approved for the treatment of lung cancer α v β 3 Monoclonal antibodies targeting the ligand-binding sites of integrins, and Cil engitide® (RGD-based peptidomimetic). It was more effective than other antiangiogenic agents in inducing apoptosis (shown in Figure 23). (I).

[0076] Further studies were carried out to determine the effect of ProAgio on apoptosis induction by α v β3I We determined whether the effect of α v β 3 Exogenous expression of integrins Several cells, namely HUVEC, PC-3, HEK, and Similar cell apoptosis assays were performed using HUVEC and COS-7 cells. High level of alpha v β 3 and PC-3 cells express a marginal level of α v β 3 Integrin expression HEK cells express α v expresses β 3 COS-7 cells do not express α v β 3 ProAgio does not express However, ProAgio does not induce apoptosis. v β 3 Integrin Induces apoptosis in exogenously expressed HUVEC and COS-7 cells (Figure 24 and Apoptosis was also induced when PC-3 cells were treated with high concentrations of ProAg. o, i.e., observed when treated with >30 μM (not shown). Therefore, the effect of ProAgio in inducing apoptosis is v β 3 depends on.

[0077] To verify the anti-angiogenic activity, an endothelial tube formation assay was performed using HUVEC cells. ProAgio almost completely destroyed the endothelial tubes.

[0078] In contrast to the effects of cilengitide and other integrin-targeting molecules, Pro Agio treatment did not result in floating / detachment of HUVEC cells (as shown in Figure 26). These results suggest that Ikis induces apoptosis through a mechanism different from that of Ikis.

[0079] Activation of various caspases in HUVEC cells upon ProAgio treatment was This study provides evidence of the molecular mechanism by which io induces apoptosis in endothelial cells. Strong activation of caspase-9, as well as activation of caspase-7 and caspase-3 (shown in FIG. 27).

[0080] ProAgio apoptosis to the initiation of activation of caspase-8 and / or caspase-9 The cis-induction dependency was also examined using caspase-8 and caspase-9 inhibitors. Caspase 9 inhibitors do not abolish the effect of ProAgio in inducing apoptosis However, the caspase 8 inhibitor largely abolished the effect of ProAgio (shown in FIG. 28). On the other hand, caspase-9 inhibitors did not abolish the activation of caspase-8, but The activation of caspase-9 was largely suppressed by a caspase-8 inhibitor (Fig. 29). As a result, activation of caspase 8 plays a key role in the induction of apoptosis. This suggests that the effect was mediated.

[0081] Untethered β3 integrin is known as integrin-mediated death ("IMD"). mechanism, inhibiting cellular apoptosis by directly recruiting caspase 8 to its cytoplasmic domain. ProAgio is similar to the IMD mechanism and can cause thrombocytosis. It may induce endothelial cell apoptosis by a mechanism different from that of other integrin antagonists. ProAgio treatment inhibits β- 3 Ca-receptor binding to the cytoplasmic site of integrins We then tested whether direct recruitment and activation of caspase-8 occurs. During ProAgio treatment, 3 As a control, caspase was co-immunoprecipitated with integrin. 8, without ProAgio treatment, 3 It did not co-precipitate with integrins. tide, under the same conditions, 3 Recruitment of caspase-8 to the integrin cytoplasmic domain (shown in FIG. 30).

[0082] Figures 31 to 43 show the efficacy of ProAgio in inhibiting tumor growth and reducing tumor blood vessels. ProAgio has shown efficacy by forming linear PEG chains at the introduced Cys residues. ("ProAgio-PEG") was used to synthesize poly(Ag-PEG) PEGylated with ethylene glycol (as shown in Figure 31). In vitro studies using HUVEC cells showed that the IL-16 receptor agonist (IL-16) was introduced into the IL-16 receptor agonist (IL-16) at a site not involved in integrin contact. , demonstrating that PEGylation of ProAgio does not result in a significant decrease in activity.

[0083] A xenograft model of PC-3 was generated. Tumor-bearing mice were treated with various doses of P roAgio-PEG and PEGylated D1-CD2, or buffered saline were administered daily. The treatments were administered intraperitoneally at a single dose for 20 days starting on day 5 after tumor inoculation. ProAgio-PEG was administered at a dose of either 10 or 20 mg / kg. Inhibits tumor growth in the 20 and 10 groups (2 and 1 tumors, respectively) At the same time, tumors disappeared completely in the 100 mg / kg dose group. In mice treated with either CD1 or CD2, the IL-1 markers proliferated at normal rates.

[0084] The effect of ProAgio-PEG on tumor growth inhibition is dose-dependent. However, the dose-dependence was less dramatic above 10 mg / kg (Figure 32 and and 33).

[0085] ProAgio-PEG is also effective when administered at a later stage of tumor growth (Figure 1). 34 and 35).

[0086] To analyze the effect of ProAgio-PEG treatment on tumor vasculature, Immunostaining of the endothelial marker CD31 was performed on tissue sections prepared from the tumor ( (Figure 36) ProAgio reduced blood vessel density, branching, and length. (shown in FIG. 37).

[0087] The following supplementation and augmentation drugs: anticancer supplementation and augmentation drugs, tricyclic antidepressants (e.g., imipramine, dexamethasone, Cypramine, Amitriptyline, Clomipramine, Trimipramine, Doxapine, Nortol liptiline, protriptyline, amoxapine and maprotiline), non-tricyclic antidepressants ( e.g., sertraline, trazodone and citalopram), Ca.sup.++ antagonists ( For example, verapamil, nifedipine, nitrendipine and caroverine), calmoduli antidepressants (e.g., prenylamine, trifluoperazine, and clomipramine), amphotericin inhibitors (e.g., prenylamine, trifluoperazine, and clomipramine), Tericin B, triparanol analogues (e.g., tamoxifen), antiarrhythmics (e.g., , quinidine), antihypertensives (e.g., reserpine), thiol scavenger (e.g., buthionine and Anticancer supplements, including multidrug resistance reducing agents such as sulphoximines and sulfoximines, and Cremafil EL It is contemplated that anti-angiogenic agents may be used in conjunction with the enhancing agents. In combination with high FI drugs, cytokines such as granulocyte colony stimulating factor may be administered. Numerous other compounds that fall within this category of drugs are effective.

[0088] One embodiment also provides a method for treating vascular endothelial cell death in a mammal comprising administering an antiangiogenic agent and a chemotherapeutic agent. The present invention also includes a kit for treating angiogenesis-dependent conditions, comprising: The combination of drugs is provided to allow for administration in a therapeutically effective amount and frequency. In certain embodiments, the anti-fibrotic agent and / or polynucleotide alone or in combination with an anti-inflammatory agent. Anti-inflammatory drugs that may be used include, but are not limited to, corticosteroids (e.g., betamethasone, Desonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, rednisolone, prednisone, and triamcinolone), nonsteroidal noninflammatory drugs (e.g. For example, diclofenac, diflunisal, etodolac, fenoprofen, flocctaphene , flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofen Fenamate, mefinamic acid, meloxicam, nabumetone, naproxen, oxaprozin , phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin), as well as antihistamines, aminoarylcarboxylic acid derivatives, aryl Arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acid derivatives, arylpropionic acid derivatives , pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamides Midcaproic acid, S-methionine, 3-amino-4-hydroxybutyric acid, amixetrine, Bendazac, Benzindamine, Buchrome, Difenpyramide, Ditazol, Emorph Azone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, Paranyline, Perisoxal, Pifoxime, Proquazone, Proxazole, and Tetanol. Nidap included.

[0089] Pharmaceutical agents include the following categories and specific examples. The categories are further defined by specific examples: It is not intended to be limiting. Those of skill in the art will recognize that certain compounds have utility outside the central nervous system, Such pharmaceutical agents can be easily identified. Numerous other compounds are useful in accordance with the invention.

[0090] In some embodiments, the solubility and blood circulation time of the anti-angiogenic agent may be increased. In order to increase the solubility and blood circulation time of the polypeptide, it may be desirable to use a poly(ethylene glycol) Poly(ethylene glycol) may be used to derivatize the polypeptides of the present invention, e.g., poly(ethylene glycol). Poly(ethylene glycol) (PEG), poly(vinylpyrrolidone), poloxamer, polysorbate Poly(vinyl alcohol), and poly(vinyl alcohol), with PEG polymers being particularly preferred. The polymer is a PEG polymer having a molecular weight of about 100 to about 40,000. In addition to those listed above, other suitable hydrophilic polymers will be readily apparent to those of skill in the art based on this disclosure. Generally, the polymers used are those which have been modified by alkylation or acylation reactions. The invention also includes a polymer that can be attached to the polypeptide. In one example, the anti-angiogenic agent is PEGylated with 0 kDa PEG-chains.

[0091] Polyethylene glycol molecules (or other chemical moieties) may be used to modify the functionality or structure of a polypeptide. In view of the effect on the antigenic domain, it should be attached to the polypeptide. There are a number of attachment methods available. For example, polyethylene glycol can be used to attach free amino or can be covalently attached through amino acid residues via reactive groups such as carboxyl groups. The group is one to which an activated polyethylene glycol molecule can be attached. The amino acid residues bearing the group may include lysine residues and the N-terminal amino acid residue. Those with carboxyl groups include aspartic acid residues, glutamic acid residues, and C-terminal The sulfhydryl groups may also be used to attach polyethylene glycol molecules. At amino groups, such as at the N-terminus or at lysine groups, can be used as reactive groups for attachment. Attachment of the polypeptide is preferred for therapeutic purposes. Polypeptides chemically modified at the N-terminus are particularly Using polyethylene glycol as an illustration of the compositions of the present invention, various polyethylene glycols may be used. Polyethylene glycol molecules (depending on molecular weight, branching, etc.) in the reaction mixture The ratio of choline molecules to polypeptide (polypeptide) molecules, the PEGylation reaction being carried out and a method for obtaining a selected N-terminally PEGylated polypeptide. Under suitable reaction conditions, the polypeptide can be attached at the N-terminus by a carbonyl group-containing polymer. A qualitatively selective derivatization is achieved.

[0092] A variety of routes of administration are available. The particular mode selected will depend on the anti-angiogenic agent, therapeutic These may depend on the particular condition being treated and the dosage required for efficacy. The method is capable of generating an effective level of immune response without producing clinically unacceptable adverse effects. The present invention may be practiced using any mode of administration that is pharmacologic and / or pharmacokinetically acceptable, meaning any mode that results in a therapeutically acceptable effect. One mode of administration is the parenteral route.

[0093] Certain particular embodiments also provide pharmaceutical compositions. Such compositions are useful for therapeutic purposes. An effective amount of the active ingredients (e.g., an anti-angiogenic agent, an anti-angiogenic agent plus a chemotherapeutic or anti-angiogenic agent) The pharmaceutical composition includes a vascular activating agent plus an anti-inflammatory agent, and a pharma- ceutical acceptable carrier. Carriers may be water or oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The liquid may be sterile, such as water and oil, including water and oils. Water is preferred when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions are also particularly suitable. Suitable pharmaceutical excipients include starch, glutaraldehyde, glyceryl stearate, sorbitol, sorbitol hydrate, sorbitol glycerol, sorbitol hydrate ... Cocosme, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk powder, silica gel, Sodium stearate, glycerol monostearate, talc, sodium chloride, dry These include skim milk, glycerol, propylene, glycol, water, ethanol, etc. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, etc. The compositions may take the form of conventional binders such as triglycerides and sustained release formulations. Oral formulations may be formulated as a suppository containing pharmaceutical grade mannitol, Lactose, starch, magnesium stearate, sodium saccharin, cellulose Standard carriers such as sucrose, magnesium carbonate, etc. may be included in such compositions. Such compositions are suitable for administration to patients. A therapeutically effective amount of an antiangiogenic agent is added together with a suitable amount of carrier to provide a form for administration. The formulation should suit the mode of administration.

[0094] More specifically, a drug or pharmaceutical composition may be formulated to have the desired therapeutic or prognostic effect prior to use in humans. For example, compounds may be tested in vitro and then in vivo for preventive activity. In vitro assays for demonstrating the therapeutic or prophylactic utility of a pharmaceutical composition include The effect of the compound on a cell line or a patient tissue sample. and / or effects on tissue samples, including but not limited to rosette formation assays and This can be determined using techniques known to those skilled in the art, including cytolytic assays. Thus, an index that can be used to determine whether administration of a particular compound is indicated. In vitro assays involve growing patient tissue samples in culture, subjecting them to a compound, or In vitro cell culture assays are performed in which the compound is administered to tissue samples and the effect of the compound on the tissue samples is observed. Includes the massage.

[0095] For example, in the α2 helix, the “BC” loop, and the “α2-α3” loop, v β 3 administering a composition comprising a polypeptide that specifically binds to an integrin. (1) fibrous material; (2) excessive production of fibrous material within the extracellular matrix; and and / or (3) due to abnormal, non-functional, and / or excessive accumulation of matrix-associated components. Fibrosis, characterized in whole or in part by replacement of normal tissue elements with fibrosis. and have or are suspected of having various fibrotic diseases, disorders, or conditions, if The present invention relates to a method for treating various types of cancer, including methods for treating subjects prone to or at risk for cancer. Treat and / or prevent, in whole or in part, certain diseases, disorders, and conditions Specific embodiments for

[0096] Antiangiogenic agents are routinely available as pharmaceutical compositions adapted for intravenous administration to humans. It is contemplated that compositions for intravenous administration may be formulated according to the process. Typically, compositions for intravenous administration are sterile. The solution is in an isotonic aqueous buffer. Optionally, the composition also contains a solubilizing agent and an injection agent. It may contain a local anesthetic, such as lignocaine, to relieve pain at the site. may be, for example, a lyophilized powder or in a sealed container such as an ampoule or sachet indicating the quantity of active agent. are supplied as anhydrous concentrates, either individually or mixed together, in unit dosage form. If the product is to be administered by infusion, the solution should contain sterile water or saline of pharmaceutical grade. When the composition is administered by injection, it may be dispensed in a dropper bottle. An ampoule of saline can be provided so that the ingredients can be mixed prior to administration.

[0097] For example, encapsulation in liposomes, microparticles, microcapsules, and expressing compounds Recombinant cells, receptor-mediated endocytosis, retroviruses or other Various delivery systems are known, such as the construction of a nucleic acid as part of a vector, and the like, and are The method of introduction can be used to administer the compound, including but not limited to intradermal, intramuscular, The compound or composition may be administered by any of the following routes: intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral. The agent is administered, for example, by infusion or bolus injection, to the epithelium or lining of the skin and mucous membrane (e.g., oral cavity Administered by any convenient route, such as by absorption through the rectal, intestinal and rectal mucosa. Administration may be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention can be administered by any suitable method, including intraventricular and intrathecal injection. It may be desirable to introduce the drug into the central nervous system by a suitable route, and intraventricular injection may be used to It may be utilized by an intracatheter.

[0098] In certain embodiments, administering the anti-angiogenic agent locally to the area in need of treatment. This may be desirable, for example and not by way of limitation, during surgery. by topical infusion, for example in conjunction with wound dressings after surgery, This can be achieved by means of a suppository or by means of an implant, The embedding may be porous, non-porous, or gel, including membranes such as silastic membranes or fibers. When administering a polypeptide, it is necessary to use a substance that the polypeptide does not absorb. Care should be taken to ensure that

[0099] In certain embodiments, the anti-angiogenic agent is a nucleic acid encoding a polypeptide, The nucleic acid can be expressed by constructing it as part of a suitable nucleic acid expression vector and expressing it in, for example, a retrovirus. By the use of vectors, by direct injection, or by the use of irradiation with microparticles or by application of lipids or cell surface receptors or transducing agents, or into the nerve nucleus. Binding to and administering homeobox-like peptides known to mediate drug entry By administering the encoded polypeptide so that it is intracellular, such as by Alternatively, the nucleic acid can be administered in vivo to promote expression of the peptide. It can be introduced into host cell DNA for expression by homologous recombination.

[0100] Other embodiments include a method for the preparation of ... Vectors and the production of anti-angiogenic agents by synthetic and recombinant techniques are covered. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors can be replication competent or replication defective. In this case, viral proliferation would generally occur only in complementing host cells. The polynucleotide encoding the agent may contain a selectable marker for proliferation in the host. Generally, plasmid vectors can be conjugated to vectors containing the same vector. The polynucleotide insert is introduced into the nucleic acid in a precipitate or in a complex with a charged lipid. to name a few, the phage lambda PL promoter, E. coli lac, Trp, p The hoA and tac promoters, the SV40 early and late promoters, and the retrovirus The promoter should be operably linked to a suitable promoter, such as the promoter of the LTR of the ribosomal RNA. Suitable promoters will be known to those of skill in the art. The expression construct may contain promoters for transcription initiation, termination, and transcriptional regulation. It would further contain a site for translation and a ribosome binding site for translation in the non-transcribed region. The coding portion of the transcript expressed by the construct preferably encodes a polypeptide to be translated. Initiation and termination codons (UAA, UGA or UA As indicated, the expression vector preferably contains a translation initiation codon at position G. Typically, the gene will contain at least one selectable marker.

[0101] Regulatory genes and sequences may be used in conjunction with the expression and replication of anti-angiogenic agents. The nature of the regulatory sequences for gene expression may vary between species or cell types, but ATA box, capping sequence, CAAT sequence, etc., which are involved in the initiation of transcription and translation, respectively. The promoter may optionally include 5' non-transcribed and 5' translated sequences associated with the The regulatory sequence may also be an enhancer sequence or an inducible sequence, if desired. The nucleic acid sequence may include a flow activator sequence.

[0102] In one embodiment, the polynucleotide encoding the anti-angiogenic agent is derived from a eukaryotic or prokaryotic cell. Orienting the localization of a polypeptide to a particular compartment of a cell and / or regulating the distribution of the polypeptide. The polypeptide may be fused to a polynucleotide encoding a signal sequence that directs secretion of the polypeptide. In bacteria, it may be desirable to direct expression of a protein to the periplasmic space. Vectors are commercially available for the construction of fusion proteins that direct protein localization. do.

[0103] Certain embodiments provide a stent that includes a generally tubular structure (e.g., including a helical shape). The surface of the substrate is coated with an anti-angiogenic drug as described above. Stents are used to prevent closure or reclosure of the passageway by the disease process (e.g., (in a tumor growth) narrowed, irregularly contoured, blocked, or obstructed A usually tubular form that can be inserted into a body passage (e.g., a bile duct) or part of a body passage It can be a scaffold for.

[0104] Certain embodiments also provide for the use of anti-angiogenic agents in a wide range of surgical procedures. For example, within one embodiment of the invention, anti-angiogenic proteins (e.g., spray or in the form of a film) to isolate normal surrounding tissue from malignant tissue and / or It is applied or sprayed on the site of the tumor prior to removal to prevent spread of the disease to the surrounding tissues. In yet another aspect of the invention, the anti-angiogenic protein can be used to The coated surgical mesh can be utilized in any process in which a surgical mesh can be utilized. It can be used.

[0105] The examples which follow are presented to aid in the understanding of the invention, but are not to be construed as limiting the scope of the invention in any manner whatsoever. It is not intended, and should not be construed, as limiting the scope of the invention. stomach. EXAMPLES

[0106] ProAgio-PEG vs. Avastin® with PC-3 xenografts and to compare the effects of ProAgio-PEG versus Endostar® ProAgio-PEG was compared with Avastin® and Endo Compared with α-glucose phosphate phosphate (registered trademark), it had a higher tumor growth suppression effect (Figure 38- (shown in Figure 41).

[0107] In addition, ProAgio-PEG treatment is a combination of Avastin® and Endo This resulted in a greater degree of vascular reduction than that of star® (not shown).

[0108] Tumor angiogenesis is generally influenced by the microenvironment surrounding the tumor. An orthotopic model was performed using immunocompetent mice. Specifically, Balb / c mice were used. Experiments were performed with mouse breast 4T-1 cells using ProAgio-PEG or D1 -Treat tumor-bearing mice with the same regimen of either CD2-PEG Treatment was started 5 days after tumor inoculation. There was a significant difference in tumor growth between the groups (shown in Figures 42 and 43). These results show that it is effective in inhibiting tumor growth in a topical model.

[0109] Figures 44 and 45 demonstrate the non-toxicity of ProAgio to normal tissues and organs. A good anti-angiogenic drug will have minimal toxicity to existing blood vessels in normal tissues / organs. To evaluate this attribute of ProAgio, Tissue sections from the liver, lung, heart, and kidney of treated mice were prepared. Tissue sections were analyzed by phosphorus and eosin ("H&E") staining and immunostaining. H&E staining showed that ProAgio showed obvious abnormalities or damage to the cellular structure of the tissue, e.g. It was revealed that the anti-CD31 antibody did not cause any changes or necrosis (Figure 44). Immunofluorescence staining using ProAgio-PEG demonstrated that ProAgio-PEG inhibited the expression of PEG in the liver, lung, heart, or liver. PEGylated D1-CD2 or buffered saline, and was less disruptive or less effective at blocking blood vessels. In addition, ProAgio showed no abnormal weight gain (Figure 44). or no loss (as shown in FIG. 45).

[0110] To further analyze the toxicity of ProAgio, healthy CD-I mice were treated with Mice were administered three doses of 60 mg / kg ProAgio-PEG by intravenous infusion. The mice were observed for 14 days. All mice behaved normally. Blood and urine samples were collected. Plasma AST / ALT, TnT, creatinine, and urinary albumin were measured after drug administration. The study was conducted 48 hours after administration of ProAgio-PEG at these doses. The study showed that the drug did not cause damage to the liver, kidneys, or heart (not shown).

[0111] Figures 46-50 show the use of ProAgio to treat fibrotic and other disorders. Figures 46 to 48 show the effect of Propionib in mice with hepatic fibrosis (Balb / c mice). The effect of Agio was shown in mice administered 250 mg / kg twice a week in the drinking water. Liver fibrosis was induced using 10% alcohol over a period of 6 weeks. Immediately after induction of inflammatory bowel disease, mice were intraperitoneally injected with ProAgio (10 mg / kg) or buffer ten times. The first four doses were administered daily, and the subsequent six doses were administered once every other day. Figure 46 shows the treated liver of a fibrotic mouse. FIG. 48 shows a magnified view of the treated liver of the alcohol and buffer or alcohol. The liver weights of mice treated with alcohol and ProAgio are shown. SD , standard deviations, and β values ​​were calculated using unpaired two-tailed t-tests. , The liver size and weight of the ProAgio-treated group were smaller than those of the buffer-treated group. were lighter (average 35%-45%).

[0112] FIG. 49 shows the results of treatment with alcohol and buffer or alcohol and ProAgio. TIMP1 levels (ng) in liver extracts prepared from livers harvested from mice / g liver sample). TIMP1 levels in liver extracts from ProAgio treatment groups. was approximately 5-fold lower than that in the buffer-treated group. MMP2 levels in liver extracts were approximately 30% greater than those in the buffer-treated group (see table). SD is the standard deviation, and β values ​​were calculated using unpaired two-tailed t-test. I calculated it.

[0113] In an experiment not shown, mice were given 250 mg / kg of 10% arginine twice weekly for 11 weeks. Liver fibrosis was induced by administering cholangiocarcinoma treatment at 10 mg / kg. g of either ProAgio or buffered saline was administered intraperitoneally from the first feeding treatment. Starting at 12 weeks, the first 3 doses will be administered daily, followed by 7 doses every other day Four mice in the buffer-treated group died, and none in the ProAgio-treated group. The liver size and weight of the ProAgio-treated group were 100% larger than those of the buffer-treated group. They were smaller and lighter (50%-60% on average).

[0114] In separate experiments not shown, TIMP1 and MMP2 measurements were determined. The TIMP1 levels in liver extracts from Agio-treated animals were approximately 7-fold lower than those in the buffer-treated animals. Furthermore, MMP2 levels in liver extracts from ProAgio-treated subjects were significantly higher than those in the buffer-treated subjects. This was approximately 25% of those in the treatment group.

[0115] Cell experiments were also performed. The main cause of liver fibrosis is activation of hepatic stellate cells. Figure 50 shows the main The effect of ProAgio on apoptosis of human hepatic stellate cells ("hHSCs") is shown. h by culturing for 8 days in uncoated plastic culture plates. HSCs were activated. Non-activated hHSCs were cells on day 1 of culture. Non-activated or activated hHSCs were incubated with 5 μM ProAgio (black bars) or buffer ( The apoptosis was measured by the apoptosis kit. % apoptosis was calculated by defining non-activated cells not treated with Treatment of activated and non-activated hHSCs with ProAgio Agio effectively induces apoptosis of activated hHSCs, whereas ProAgio effectively induces apoptosis of non-activated hHSCs. Error bars indicate no effect on SCs across five independent experiments. Standard deviations are shown.

[0116] Figures 51-53 show experiments demonstrating the effect of ProAgio on mice with liver fibrosis. The study included 250 mg / kg TAA + 10% alcohol administered twice weekly for 12 weeks. Liver fibrosis was induced using a steroid-delivery treatment. The treatment was then administered intraperitoneally. Three treatment doses were administered daily, followed by six treatment doses administered once every other day. The treatment groups were 10 mg / kg ProAgio (8 mice per group), 20 mg / kg Esbr IET® (a newly approved drug for the treatment of pulmonary fibrosis) (8 animals per group) , and buffered saline (8 mice per group). One mouse in the buffer group, and Esb One mouse in the Riet® died.

[0117] Figures 54A-54B show the effect of ProAgio on fibrotic liver. ProAgio is thought to be the main source of extracellular matrix in the liver. Figure 54A shows the effect of treatment on blood flow in the fibrotic liver. The blood flow through the portal vein was measured by Doppler imaging and was per mm 3 The control group was the measurement of mice without induction of fibrosis. The values ​​are the standard deviations of measurements from six mice. ProAgio induced apoptosis in visceral sinusoidal endothelial cells (LSECs). Human primary LSECs were activated in culture without VEGF. Activated LSECs were treated with ProAgio at the concentrations shown for 10 hours. The control group was LSECs without activation. Apoptosis was presented as % apoptosis. Error bars represent standard deviations from five independent experiments. Not only did it reduce fibrosis but it also normalized liver vasculature.

[0118] Figures 55A-55D show the results of ProAgio as a treatment for breast cancer in mice. of breast cancer 4T-1 (a well-known breast cancer model) for 10 doses at 10 mg / mL kg of ProAgio. Figure 55A shows the tumor size at the end point. (One due to oversizing and pre-termination of the experiment due to IACUC regulation. (Tumor not shown). Figure 55B shows the level of myofibroblast staining in tumor tissue sections. Representative images of Sirius Red (collagen) and α-SMA are shown. C: Quantitative Sirius red staining or positive staining in each area. Figure 55D shows staining presented as a percentage of positive staining in each region. Another quantification of α-SMA is shown, presented as pixels. Error bars in the figures represent standard deviations of measurements from five mice.

[0119] FIG. 56 shows that ProAgio is a pancreatic tumor graft formed by cancer stroma and desmoplastic cells. It has been shown to reduce and prevent fibrosis within the cancer. In pancreatic cancer, high levels of α Activated fibroblasts or pancreatic stellate cells bearing vβ3 integrin. Section A The authors report that MIA-Paca-2 cells implanted with PaSCs under the treatment of the indicated drugs The growth of xenograft tumors was monitored by measuring the tumor volume every 4 days. PaSCs were isolated from mouse pancreas and detected by a commercially available hTERT kit. The tumors were 250 mm on average.3 Treatment was started when the section reached a size of Sections B, C, and D show CD31 staining of tumor tissue sections; section B shows Sirius Red staining. Color, section C, and staining, section D, quantification of vessel length, density, and branching points In section C, collagen content is shown as a function of % of collagen stained area. In Section D, positive α-SMA is presented as a positive fraction per region. The control groups in (C) and (D) were used to quantify non-cancerous pancreatic tissue. The error bars in (A), (C), and (D) are from experiments using six mice. The mean mean is the standard deviation from the experimental measurements. p values ​​were calculated. In (C) and (D), * means p<0.05, ** means P<0. 01, and *** means P<0.001. Images of (C) and (D) is the indication of the treatment group being indicated.

[0120] Figures 57A, 57B, and 57C show that ProAgio reduces fibrosis in liver cancer. These figures show that ProAgio inhibits and prevents fibrosis in liver cancer. It has been shown that IL-16 reduces and prevents liver disease, resulting in a reduction in liver tumor volume. In liver cancer, activated fibrogenic cells or hepatic stellate cells express high levels of αvβ3 integrin. Figures 57A and 57B show the expression of Propionib in liver cancer xenografts. These experiments showed the efficacy of Agio. ) and tumors initiated with 1 × 107 HepG alone or half HepG + LX-2 cells Every 2 days for 20 days, and approximately 18 days after tumor inoculation, With a single dose of 10 mg / kg (intraperitoneal), tumor volume was reduced by ProAgio. These cells (like myofibroblasts in other types of tumors) are responsible for the The researchers found that the IL-1 receptor agonist, IL-1, supported and sustained the cancer cells, and their apoptosis caused the tumor to shrink. Collagen content was quantified and presented as % of collagen stained area. 319.8×319.8mm 2 Within the field of view, quantitative measurements are calculated manually. Quantization was performed on three randomly selected regions in the four matched regions from each tumor. The control group is the result from the area of ​​non-cancerous liver tissue. The mean mean is the standard deviation from measurements in six mice. Collagen or fibers within the tumor after treatment with io. A total of 80 per animal group Three slides and three areas per slide were tested, with a control group consisting of stained normal liver tissue. The control group was colored ProAgio, which treated liver cancer.

[0121] ProAgio may be applicable to the treatment of other fibrotic diseases and to the treatment of osteoporosis. Additionally, ProAgio can be combined with other drugs for the treatment of rheumatoid arthritis. It can be used in combination.

[0122] One aspect of the present disclosure is a method for the preparation of an active site of a protein as disclosed herein, and Its particularities are as disclosed in the specification.

[0123] The specific examples disclosed herein are to be construed as merely illustrative. , and should not be construed as limiting the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one of ordinary skill in the art will be able to, based on the description herein, implement the present invention to its fullest extent. It is believed that the disclosure of the present invention can be utilized up to

[0124] The disclosed examples include methods of performing the methods and the methods disclosed and claimed herein. The present invention provides those of skill in the art with a complete disclosure and description of the compositions and methods of using the compounds. For example, to ensure accuracy of values ​​such as amounts, temperatures, etc. Efforts have been made, but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight and temperatures are in degrees Celsius; Pressure is at or near atmospheric. Standard temperatures and pressures are 20°C and It is defined as an atmosphere.

[0125] Percentages, concentrations, amounts, and other numerical data may be expressed herein in a range format. It should be noted that such range format is used for convenience and brevity. and therefore any numerical values ​​expressly recited as range limitations. Not only that, each numerical value and subrange is included within the range as if it were expressly recited. should be interpreted in a flexible manner to include all individual numerical values ​​or subranges contained in For purposes of illustration, the concentration range of "about 0.1% to about 5%" refers to about 0.1% by weight to about 5% by weight. Not only the explicitly listed concentrations of, but also, for example, within the ranges indicated, e.g. Individual concentrations such as 1%, 2%, 3%, and 4%, as well as, for example, 0.5%, 1.1%, This should be construed to include smaller ranges such as 2.2%, 3.3%, and 4.4%. The term "about" means an amount within the range of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ±17%, ±18%, ±19%, ±20%, ±21%, ±22%, ±23%, ±24%, ±25%, ±26%, ±27%, ±28%, ±29%, ±30%, ±31%, This may include 5%, ±6%, ±7%, ±8%, ±9%, or ±10% or more.

Claims

1. Preventing excessive accumulation of fibrous material within the extracellular matrix in injured or damaged tissue of a subject The method comprises administering to a subject in need of preventing or reducing said accumulation. Identifying and determining the sequence of domain 1 (D1-CD2) of a wild-type cell adhesion protein D1-CD2 variants having an amino acid sequence at least about 75% similar to a ｖ β ３ administering to the subject a polypeptide that specifically binds to an integrin; wherein the polypeptide comprises an α2 helix, a B-C loop, and an α2-α3 loop. In the βA domain in the region ｖ β ３ A method for specifically binding to an integrin.

2. The method of claim 1 , wherein the polypeptide has a dissociation constant of less than about 1 μM.

3. The amino acid substitutions for the polypeptide are The method of claim 1 having a hydrophilicity value range of about -2 to about +2.

4. 10. The method of claim 1, wherein the subject has liver fibrosis, pancreatic fibrosis, or breast fibrosis. 。

5. The variant is L94N, E95D, K96V, I97C, F98N, D99F, At least one selected from the group consisting of L100A, K101S, and I102R The method of claim 1 , comprising an amino acid substitution.

6. The variant is L94N, E95D, K96V, I97C, F98N, D99F, The sequence of claim 1 having the substitutions L100A, K101S, and I102R. How to.

7. The variant is selected from the group consisting of I97Y, F98D, and D99Y. The method of claim 1 , comprising at least one substitution.

8. The variant comprises a sequence having the following substitutions: I97Y, F98D, and D99Y. The method of claim 1.

9. The variants are D99N, I102V, Q103I, E104I, E8T, T9V , W10Q, G11M, A12K The method according to claim 1 .

10. The variants are D99N, I102V, Q103I, E104I, E8T, T9V 2. The method of claim 1, comprising a sequence having the substitutions: W10Q, G11M, A12K.

11. The variant is ｖ β ３ Has at least one molecular interaction with an integrin The method of claim 1 .

12. The variant has an α ｖ β ３ The antibody of claim 1 which binds to an integrin. How to.

13. The variant is TEMKQER and α ｖ The method of claim 1, which is cross-linked to an integrin. How to.

14. The variant is FNEEVKKQ and ３ The method according to claim 1, wherein the antibody is crosslinked to an integrin. Method of posting.

15. The variant of claim 1, wherein the variant is crosslinked to β3 integrin with FNEEVKKQ. Method of posting.

16. The method of claim 1 , wherein the variant is PEGylated.

17. 16. The method of claim 15, wherein the variant is PEGylated with polyethylene glycol. The method described.

18. The method of claim 1, wherein the polyethylene glycol is PEG-20 kDa.

19. A pharmaceutical composition comprising the α2 helix, the BC loop, and the α2-α3 loop region. In the βA domain, ｖ β ３ A therapeutically effective amount of a compound that specifically binds to an integrin. a polypeptide, thereby forming a lineage within the extracellular matrix in said tissue of said subject, A pharmaceutical composition which prevents or reduces the excessive accumulation of fibrous materials.

20. 20. The pharmaceutical composition of claim 19, further comprising a pharma- ceutically acceptable carrier.

21. A method for inducing apoptosis in a tissue of a subject comprising the steps of: administering a therapeutically effective amount of said pharmaceutical composition.

22. A method for preventing or reducing portal hypertension in the liver, comprising administering to the liver an α2 helix, a B-C helix, in the βA domain in the region of the α2-α3 loop, ｖ β ３ Integrin administering to a subject an isolated polypeptide that specifically binds to the to prevent excessive accumulation of fibrous material within an extracellular matrix in said tissue of said subject, Or ways to reduce it.

23. 23. The method of claim 22, wherein the subject has liver fibrosis.

24. 23. The method of claim 22, wherein the subject has pancreatic fibrosis.

25. 23. The method of claim 22, wherein the subject has breast fibrosis.

26. Treating diseases involving excessive accumulation of fibrous material within the extracellular matrix in a target tissue 21. A method for administering to a subject a therapeutically effective amount of the pharmaceutical composition of claim 19 or 20. The method comprises the step of administering.

27. 24. The method of claim 23, wherein the disease is liver fibrosis or pancreatic fibrosis.

28. A method for preventing and treating a tumor in a subject, comprising the therapeutic administering an effective amount of said pharmaceutical composition.

29. The pharmaceutical composition is suitable for topical application, injection, oral administration, or sustained release administration. The method according to claims 21 to 25,

30. The method of claims 21 to 26, wherein the subject is a mammal.

31. The method according to claims 21 to 27, wherein the subject is a human.

32. In the βA domain in the region of the α2 helix, the BC loop, and the α2-α3 loop Ite α ｖ β ３ An isolated polypeptide that specifically binds to an integrin.

33. K Ｄ The peptide of claim 29, wherein the concentration is about 1 μM to 1 pM.

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