Antiangiogenic Agents and Methods of Using Such Agents

Abstract

The antiangiogenic agent or polypeptide has an amino acid segment substantially similar to domain 1 of CD2. The polypeptide has a β-sheet formed by the two segments. Methods of using such agents and polypeptides are also included. The agent or polypeptide has a terminal glycine.

Description

[Technical Field]

[0001] Government License Rights This invention was made with United States government support under Grant No. CA118113 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0002] Previous related application data This application claims the benefit of U.S. Provisional Patent Application No. 63 / 484,968, filed February 14, 2023, which is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to inhibiting or preventing angiogenesis to control or treat angiogenesis-dependent conditions characterized by or dependent on blood vessel proliferation. Additionally, the present disclosure relates to the use of anti-angiogenic agents in combination with chemotherapeutic agents. [Background technology]

[0004] Angiogenesis is the process by which new blood vessels are formed from existing capillaries, while vasculogenesis involves the proliferation of blood vessels derived from endothelial progenitor cells. Angiogenesis is a combinatorial process regulated by the balance between pro-angiogenic and anti-angiogenic molecules. Angiogenic stimuli (e.g., hypoxia or inflammatory cytokines) result in the induced expression and release of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF). These growth factors stimulate mesothelial cells (ECs) in existing vasculature to proliferate, migrate through tissues, and form new endothelialized channels. Angiogenesis involves the proliferation of endothelial cells.

[0005] Inappropriate or pathological angiogenesis contributes to the development of atherosclerotic plaques, diabetic retinopathy, degenerative maculopathy, postrenal fibrosis, idiopathic pulmonary fibrosis, acute adult respiratory distress syndrome, and asthma. Furthermore, tumor progression is associated with neovascularization, which promotes nutrient delivery to progressively growing tumor tissue. Although the idea of ​​slowing cancer progression by targeting cancer blood supply was proposed more than 30 years ago, angiogenesis inhibitors have only recently become the focus of cancer treatment. Therefore, there is a continuing need for new methods and agents for reducing pathological angiogenesis. The present disclosure aims, among other things, to address this need.

[0006] Thus, there is a continuing need for new methods and agents for reducing pathological angiogenesis, and the present disclosure is directed to, among other things, addressing this need. [Brief explanation of the drawings]

[0007] [Figure 1A] 1 shows a schematic diagram of an anti-angiogenic agent, showing two short strands of the anti-parallel β-sheet of the protein. [Figure 1B] FIG. 1 shows another schematic diagram of an anti-angiogenic agent, showing two short strands of an anti-parallel β-sheet of the protein with the hydrophobic surface facing outwards. [Figure 1C] FIG. 1 shows another schematic diagram of an anti-angiogenic agent, showing two short strands of an anti-parallel β-sheet of the protein with the hydrophobic surface facing inward. [Figure 2] NMR spectra of folded (top) and denatured (bottom) antiangiogenic agent and host protein are shown. [Figure 3] A shows a proliferation assay comparing an anti-angiogenic agent to a prior art drug (Anginex) using HUVEC cells. B shows a proliferation assay comparing an anti-angiogenic agent to a prior art drug (Anginex) using M4A4 cancer cells. [Figure 4A] 1 shows a graph demonstrating that tumor volume remained relatively constant during treatment with antiangiogenic agents (treatment began after 8 days). [Figure 4B]1 shows a graph demonstrating that tumor volume remained relatively constant during treatment with antiangiogenic agents (treatment began after 22 days). [Figure 5] 1 shows a graph illustrating the substantial difference in tumor weight after the first treatment with an anti-angiogenic agent. [Figure 6] 1 shows a graphical representation demonstrating the slower rate of tumor growth in mice treated with anti-angiogenic agents compared to mice treated with buffer and host proteins. [Figure 7] 1 shows the results of a blood vessel density study in mice treated with anti-angiogenic agents. [Figure 8] No significant changes in body weight were observed in mice in any treatment group. [Figure 9] 1 shows how cell viability can vary with dose. [Figure 10] Tumor growth curves over 14 days of treatment with different doses of anti-angiogenic agents are shown. [Figure 11] 1 shows a comparison of tumor growth curves between Avastin® and rProAgio-PEG. [Figure 12] A graph showing tumor weight at the end of a 14-day treatment course is shown. [Figure 13] 1 shows a graph depicting survival rates for various examples of anti-angiogenic agents.

[0008] definition The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

[0009] The term "angiogenesis" refers to the proliferation, development, and remodeling of vascular beds with the goal of improving tissue oxygenation and nutrient delivery. This process includes the formation of new capillaries by sprouting from pre-existing vessels, as well as the enlargement, maturation, and modification of existing vessels with respect to orientation and flow properties, ultimately optimizing blood perfusion of tissues.

[0010] The term "amino acid" refers to natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Naturally encoded 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), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid, i.e., by way of example only, an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, yet still maintain the same basic chemical structure as a natural amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0011] The term "conservatively modified variants" applies to both natural and non-natural amino acids, as well as natural and non-natural nucleic acid sequences, and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to natural and non-natural nucleic acids that encode identical or essentially identical natural and non-natural amino acid sequences, or, if the natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, to essentially identical sequences. For example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described herein without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Thus, by way of example, any natural or non-natural nucleic acid sequence herein that encodes a natural or non-natural polypeptide also represents every possible silent variation of the natural or non-natural nucleic acid. Those skilled in the art will recognize that each codon (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) of a natural or non-natural nucleic acid can be altered to yield a functionally identical molecule. Accordingly, each silent variation of natural and non-natural nucleic acids that encode natural and non-natural polypeptides is implicit in each described sequence.

[0012] As used herein, the term "effective amount" refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms, or pathogenesis of a disease, or any other desired alteration of a biological system. By way of example, the administered agent or compound can include, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, augmentative, and / or therapeutic treatments. An appropriate "effective" amount in any individual case can be determined using techniques, such as dose escalation studies.

[0013] As used herein, the term "nucleic acid sequence" refers to the order and identity of the nucleotides that comprise a nucleic acid.

[0014] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0015] The term "terminal glycine" refers to a glycine amino acid residue located at the end of a polypeptide chain. In protein chemistry, the term "terminal" can apply to either the N-terminus (amino terminus) or the C-terminus (carboxyl terminus) of a chain. Thus, a "terminal glycine" can be the first amino acid residue at the N-terminus or the last residue at the C-terminus of a peptide or protein. In one example, the terminal glycine can be at the N-terminus. In another example, the terminal glycine can be at the C-terminus.

[0016] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0017] A particular nucleic acid sequence also implicitly encompasses "splice variants." Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any proteins encoded by splice variants of that nucleic acid. A "splice variant," as the name suggests, is a product of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced ​​such that different (alternative) nucleic acid splice products encode different polypeptides. Mechanisms for the generation of splice variants vary but include alternative splicing of exons. Alternative polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.

[0018] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and to non-naturally occurring amino acid polymers.

[0019] As used herein, the term "pharmaceutically acceptable" refers to a substance, including but not limited to, a salt, carrier, or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the substance may be administered to an individual without causing undesired biological effects or interacting adversely with any of the components of the composition in which it is contained.

[0020] As used herein, the term "prophylactically effective amount" refers to the amount of a composition comprising at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide that is administered prophylactically to a patient to thereby alleviate to some extent one or more symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, etc. It is considered well within the skill of one of ordinary skill in the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, dose escalation clinical trials.

[0021] The phrase "substantially similar" in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have at least 75%, preferably at least 85%, more preferably at least 90%, 95%, or more, nucleotide or amino acid residue identity, or any integer value therebetween, as determined, for example, using a sequence comparison algorithm such as those described below or by visual inspection. Preferably, substantial identity exists over a region of the sequences that is at least about 10, preferably about 20, more preferably about 40-60 residues in length, or any integer value therebetween, preferably greater than 60-80 residues, more preferably at least about 90-100 residues; most preferably, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of nucleotide sequences.

[0022] As used herein, the term "synergistic" refers to a combination of prophylactically or therapeutically effective agents that is more effective than the additive effects of any two or more single agents. The synergistic effect of a combination of prophylactic or therapeutic agents may allow for the use of lower dosages and / or less frequent administration of one or more of the agents in a subject with a particular disease or condition. In some cases, the synergistic effect of a combination of prophylactic or therapeutic agents may be used to avoid or reduce adverse or unwanted side effects associated with the use of either agent alone.

[0023] As used herein, the term "therapeutically effective amount" refers to the amount of a composition comprising at least one unnatural amino acid polypeptide and / or at least one modified unnatural amino acid polypeptide administered to a patient already suffering from a disease, condition, or disorder sufficient to cure or at least partially arrest or alleviate to some extent one or more of the symptoms of the disease, disorder, or condition being treated. The effectiveness of such compositions depends on factors including, but not limited to, the severity and course of the disease, disorder, or condition, previous treatments, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including, but not limited to, a dose escalation clinical trial. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present application provides antiangiogenic agents and methods for inhibiting angiogenesis, wherein angiogenesis-dependent conditions in a mammal are addressed by administering the antiangiogenic agent to the mammal in a therapeutically effective amount and frequency. The administration is intended to achieve regression or cessation of the condition without causing significant toxicity. Conditions that can be treated include various neoplasms, such as breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, central nervous system tumors, glioblastoma multiforme, and solid tumor neoplasms such as melanoma. While not an exhaustive list, antiangiogenic agents can induce regression or cessation in most, if not all, solid tumors. The mammal being treated can be a human.

[0025] In specific embodiments, antiangiogenic agents include polypeptides derived from domain 1 of CD2, sourced from both human and non-human sources. Altering the structure and function of domain 1 of CD2 creates novel polypeptides with enhanced stability and activity. Illustratively, the modifications can generate at least two short strands of an antiparallel β-sheet that emulate the active sites of various endogenous antiangiogenic polypeptides, such as PF4, IL8, TSP-1, endostatin, and other synthetic peptides. These antiangiogenic agents or polypeptides can be characterized by a β-sheet structure with two segments in an antiparallel configuration, an inward-facing hydrophobic surface, an outward-facing hydrophilic surface, and a segment containing at least four amino acids (although six or more amino acids per segment may be required, and eight or more amino acids per segment may be required). Amino acid residues can alternate between hydrophilic and hydrophobic. The antiangiogenic agent can have a terminal glycine.

[0026] Antiangiogenic agents can be used in in vivo, in vitro, and ex vivo applications and exhibit antiangiogenic effects in both clinical and preclinical settings. Methods for systematically varying polypeptide sequences through directed evolution are well established. The methods of the present disclosure can be applied to develop non-CD2 polypeptides characterized by at least two short strands of antiparallel β-sheet.

[0027] Antiangiogenic agents can be prepared using rational polypeptide design to identify and predict the influence of specific amino acid residues on polypeptide properties. This method can include creating a training set of polypeptide variants, deriving an activity model to predict effects based on amino acid modifications, and using the model to identify amino acids at specific positions that confer desired activity and properties.

[0028] Alternatively, agents can be developed by introducing mutations into the host polypeptide or domain 1 of CD2. One skilled in the art can create specific antiangiogenic agents using recombinant techniques, including using site-directed mutagenesis to generate desired polypeptides from the sequences identified as SEQ ID NOS: 1-12. Mutated nucleic acid sequences, accounting for the degeneracy of the genetic code, can be subcloned into an appropriate expression vector for production in a host such as yeast or E. coli, followed by standard purification processes. Antiangiogenic agents can have a terminal glycine.

[0029] In one embodiment, the agent has a terminal glycine. The agent set forth in SEQ ID NO: 12 has a terminal glycine. Glycine is the smallest of the 20 common amino acids and can affect the solubility, stability, and bioavailability of peptide-based therapeutics.

[0030] In one embodiment, the agent can incorporate N-linked glycosylation, a process in eukaryotes that affects polypeptide folding, solubility, and circulatory duration, which often requires the consensus sequence Asn-X-Ser / Thr, particularly within the loop region of the peptide, as observed at position N65 in the yeast Pichia expression system.

[0031] Specific embodiments of antiangiogenic agents aim to control or inhibit angiogenesis, which is understood to prevent new blood vessel formation or the differentiation of circulating stem cells into endothelial cells. Furthermore, these agents can induce apoptosis in activated endothelial cells, thereby inhibiting angiogenesis, which may involve destroying blood vessels near or within tumors activated by angiogenic factors. The inhibitory mechanism is believed to involve the regulation of apoptosis, and the developed polypeptides have demonstrated potent activity in inducing endothelial cell apoptosis in vitro without affecting epithelial cells and fibroblasts. They also exhibit minimal effects on the tube structures formed by HUVEC cells and low toxicity to existing normal blood vessels. Survival factors involved include vascular endothelial growth factors or mitogens, as well as factors that support cellular recovery after injury.

[0032] These anti-angiogenic agents may be incorporated into methods of treating mammals by inhibiting angiogenesis comprising administering the anti-angiogenic agent, hi certain embodiments, the anti-angiogenic polypeptides have demonstrated increased circulation time compared to small molecule and short peptide agents.

[0033] Specific embodiments provide methods for inhibiting angiogenesis and treating angiogenesis-related diseases. In other embodiments, the present invention provides methods for inhibiting or reducing tumor growth and treating individuals suffering from cancer. These methods comprise administering to the individual a therapeutically effective amount of one or more polypeptide therapeutics described above. These methods are particularly intended for therapeutic and prophylactic treatment of animals, more particularly humans.

[0034] As described herein, angiogenesis-associated diseases include, but are not limited to, solid tumors, blood-borne tumors such as leukemia, and tumor metastasis; benign tumors, such as hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas; inflammatory disorders such as immune and non-immune inflammation; rheumatoid arthritis and psoriasis; ocular angiogenic diseases, such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrorenal fibrosis, and Rube's disease; Osler-Weber syndrome; myocardial angiogenesis, plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; and wound granulation and wound healing; and angiogenesis-dependent cancers, including telangiectasia, psoriasis, scleroderma, pyogenic granulomas, coronary collaterals, limb ischemia, angiogenesis, corneal diseases, rubeosis, arthritis, diabetic neovascularization, bone fractures, angiogenesis, and hematopoiesis.

[0035] One potential advantage of combining an antiangiogenic agent with a chemotherapeutic agent may be improved treatment and control of angiogenesis-dependent conditions by reducing the dose of the chemotherapeutic agent. The combination may be administered for a longer period of time, or optionally, due to the increased effectiveness of the combination, a shorter treatment period may be administered.

[0036] Depending on the nature of the combination therapy, administration of the polypeptide therapeutic of the present invention may continue while the other therapy is being administered and / or thereafter. Administration of the polypeptide therapeutic may be in a single dose or multiple doses. In some instances, administration of the polypeptide therapeutic is initiated at least several days before the administration of the conventional therapy, while in other instances, administration is initiated either immediately before or at the time of administration of the conventional therapy.

[0037] In one embodiment, the anti-angiogenic agent may be administered in combination with chemotherapy and other therapeutic agents, as well as radiation therapy. The chemotherapy agent may be selected from the group consisting of vinca alkaloids, camptothecins, taxanes, or platinum analogs, including vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, docetaxel, 5FU, cisplatin, carboplatin, irinotecan, topotecan, or cyclophosphamide. The chemotherapy agent may be administered in a low-dose regimen in combination with the anti-angiogenic agent due to the antitumor effect of the anti-angiogenic agent. The chemotherapy agent may be administered at a dose less than the maximum tolerated dose.

[0038] It is contemplated that the antiangiogenic agent may be used in combination with an anti-neoplastic agent, including the following anti-neoplastic agents: acivicin; aclarubicin; acodazole hydrochloride; AcrQnine; adozelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacytidine; azetepa; azotomycin; batimastat; benzdepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carbopra cin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cilolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexorumaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomitine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride Hydrochloride); Estramustine; Estramustine phosphate sodium; Etanidazole; Ethiodized oil I 131; Etoposide; Etoposide phosphate; Etoprine; Fadrozole hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine phosphate; Fluorouracil; Flurocitabine; Foskidone; Fostriecin sodium; Gemcitabine; Gemcitabine hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Ilmofosine; Interferon alpha-2a;Interferon alpha-2b; Interferon alpha-n1; Interferon alpha-n3; Interferon beta-Ia; Interferon gamma-Ib; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide acetate; Liarozole hydrochloride; Lometrexol sodium; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytansine; Mechlorethamine hydrochloride; Megestrol acetate; Melengesterol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitochromin; Mitodilin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole Benzalkonium; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofurin; Ribopurin; Rogletimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium strontium hydrochloride 89;Sulofenur;Talisomycin;Taxane;Taxoid;Tecogalan sodium;Tegafur;Teroxantrone hydrochloride;Temoporfin;Teniposide;Teroxirone;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Tiazofurin;Tirapazamine;Topotecan hydrochloride;Toremifene citrate;Trestorone acetate;Triciribine phosphate;Trimetrexate;Trimetrexate glucuronate;Triptorelin;Tubrozole hydrochloride;Uracil mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglycinate sulfate; Vinleurosin sulfate; Vinorelbine tartrate; Vinrosidine sulfate; Vinzolidine sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin hydrochloride.

[0039] Other antitumor compounds include 20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecipenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, atorusacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsal morphogenetic polypeptide Do1, antiandrogen, prostate cancer, antiestrogens, antineoplastons, antisense oligonucleotides, aphidicolin glycinate, apoptotic gene regulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzyl benzoate Nzoylstaurosporine, beta-lactam derivatives, beta-arretin, betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisazilidinylspermine, bisnafide, bistraten A, bizelesin, brefurate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, CaRestM3, CARN700, cartilage-derived inhibitors, carzelle Syn, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorins (chlorlns), chloroquinoxaline sulfonamides, cicaprost, cis-porphyrin, cladribine, clomiphene analogs, clotrimazole, colismycin A, colismycin B, combretastatin A4, combretastatin analogs, conagenin, crambecidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentaquinone, cycloplatam, sipemycin,Cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexphosphamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, 9-, dioxamycin, diphenylspiromustine, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocanicin SA, ebselen, ecomustine, edelfosine, edrecolomab, ecolomab Flornithine, elemene, emiteflu, epirubicin, epristeride, estramustine analogues, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride (fmasteride), flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunornithine hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, Gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immunostimulating peptides, insulin-like growth factor 1 receptor inhibitors, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, irinotecan, ilopract, irsog Razin, isobengazole, isohomohalichondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprolide + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogs, lipophilic disaccharide peptides, lipophilic platinum compounds, rissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine,losoxantrone, lovastatin, losoxoribine, lurtotecan, lutetium texaphyrin, lisofylline, lytic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, mervalone, meterelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, millimostim, mismatched double-stranded RNA, mitoguazone, mitolactol, mitomycin analogs, mitonafide, mitotoxin fibroblast growth factor -Saporin, mitoxantrone, mofalotene, molgramostim, monoclonal antibodies, human chorionic gonadotropin, monophosphoryl lipid A + myobacterium cell wall SK, mopidamol, multidrug-resistant genie inhibitors, multitumor suppressive 1-based therapy, mustard anticancer drugs, mycaperoxide B, mycobacterial cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagressip, naloxone + pentazocine, napavine, naphterpine, nartograstim, nedaplatin, nemorubicin, neridronic acid, Endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitric oxide antioxidants, nitrulline, O6-benzylguanine, octreotide, oxenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin, osateron, oxaliplatin, oxaunomycin, paclitaxel analogs, paclitaxel derivatives, paclitaxel amine, palmitoylrhizoxin, pamidronate, panaxytriol, panomyphen, parabactin, pazelliptin, pegasparga azepam, perdecin, pentosan polysulfate sodium, pentostatin, penttrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum-triamine complexes, porfimer sodium, porfiromycin, propylbis-acridone, prostaglandin J2, proteasome inhibitors, protein A system immunomodulators,Protein kinase C inhibitors, protein kinase C inhibitors, microalgae, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridines, pyridoxylated hemoglobin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors, demethylated leteriptin, rhenium Re 186 etidronate, rhizoxin, ribozyme, RII retinamide, rogletimide, rohitukin, romurtide, roquinimex, rubiginone B1, ruboxil, safingol, saintpin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetic, semustine, senescence-derived inhibitory factor 1, sense oligonucleotide, signal transduction inhibitor, signal transduction modulator, single-chain antigen binding protein, sizofiran, sobuzoxane, borocaptate sodium, sodium phenylacetate, sorberol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipiramide, stromelysin inhibitor, sulfmosine, super-acting vasoactive intestinal peptide antagonist, suramin, swainsonine, synthetic glycosaminoglycan, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium tegafur, tellapyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, saliblastin, thalidomide, thiocoraline, thrombopoietin, thrombopoietin mimetic drugs, thymalfasin, thymopoietin receptor agonists, thymotrin, thyroid-stimulating hormone, ethyl etiopurinse, tirapazamine, titanocene dichloride, topotecan, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonists,Vapreotide, variolin B, vector systems, erythrocyte gene therapy, veraresol, veramine, verudine, verteporfin, vinorelbine, vinxartin, vitaxin, vorozole, zanoteron, zeniplatin, zilascorb, zinostatin stimalamer. Those skilled in the art will recognize many other compounds within this category of drugs that are useful in combination with antiangiogenic agents.

[0040] It is contemplated that anti-angiogenic agents may be used in combination with anti-cancer adjunctive potentiators, including the following adjunctive potentiators: tricyclic antidepressants (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline), non-tricyclic antidepressants (e.g., sertraline, trazodone, and citalopram), Ca.sup.++ antagonists (e.g., verapamil, nifedipine, nitrendipine, and caroverine), calmodulin inhibitors (e.g., prenylamine, trifluoroperazine, and clomipramine), amphotericin B, triparanol analogs (e.g., tamoxifen), antiarrhythmics (e.g., quinidine), antihypertensives (e.g., reserpine), thiol-depleting agents (e.g., buthionine and sulfoximine), and multidrug resistance reducers such as Cremophor EL. The compounds of the present invention may also be administered with cytokines, such as granulocyte colony-stimulating factor. Those skilled in the art will recognize many other compounds within this category of drugs that are useful in combination with antiangiogenic agents.

[0041] One embodiment also includes a kit for treating an angiogenesis-dependent condition in a mammal, comprising an anti-angiogenic agent and a chemotherapeutic agent. The combination of agents is provided to allow for administration in a therapeutically effective amount and frequency to cause the presence or regression of angiogenesis. In certain embodiments, the anti-angiogenic agent and / or polynucleotide is administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the antiangiogenic agents of the present invention include corticosteroids (e.g., betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin). ), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino 4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpyramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

[0042] Pharmaceuticals include the following categories and specific examples. The categories are not intended to be limited by the specific examples. One of ordinary skill in the art will be able to readily identify pharmaceuticals that have utility outside the central nervous system. One of ordinary skill in the art will also recognize many other compounds that fall within the categories and are useful according to the present invention.

[0043] In some embodiments, it may be desirable to increase the solubility and blood circulation time of the antiangiogenic agent. To increase the solubility and blood circulation time of the polypeptide, the polypeptide of the present invention may be derivatized with polyethylene glycol, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidone), polyoxomer, polysorbate, and poly(vinyl alcohol), with PEG polymers being particularly preferred. The PEG polymer has a molecular weight of about 100 to about 40,000. In addition to the examples exemplified above, other suitable hydrophilic polymers will be readily apparent to those skilled in the art based on this disclosure. Generally, the polymer used may include polymers that can be conjugated to the polypeptide of the present invention via alkylation or acylation reactions. In one example, the antiangiogenic agent is PEGylated with a 20 kDa PEG chain.

[0044] The polyethylene glycol molecule (or other chemical moiety) should be attached to the polypeptide taking into consideration its effect on the polypeptide's functional or antigenic domains. Several attachment methods are available to those skilled in the art. For example, polyethylene glycol can be covalently attached through amino acid residues via reactive groups, such as free amino or carboxyl groups. The reactive groups are those to which activated polyethylene glycol molecules can be attached. Examples of amino acid residues with free amino groups include lysine residues and N-terminal amino acid residues, while examples of amino acid residues with free carboxyl groups include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Sulfhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. For therapeutic purposes, attachment via an amino group, e.g., via the N-terminus or lysine group, is preferred. Polypeptides chemically modified at the N-terminus may be particularly desirable. When polyethylene glycol is used as an example of the present composition, various polyethylene glycol molecules (based on molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to polypeptide molecules in the reaction mixture, the type of PEGylation reaction performed, and the method for obtaining the selected N-terminally PEGylated polypeptide may be selected. Under appropriate reaction conditions, substantially selective derivatization at the N-terminus of the polypeptide with a carbonyl group-containing polymer is achieved.

[0045] A variety of administration routes are available. The particular method selected may depend on the antiangiogenic agent, the particular condition being treated, and the dosage required for efficacy. These methods may be carried out using any medically acceptable method of administration, i.e., any method that produces an effective level of immune response without causing clinically unacceptable side effects. A particular administration method is the parenteral route.

[0046] Certain specific embodiments also provide pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an active ingredient (e.g., an antiangiogenic agent, an antiangiogenic agent and a chemotherapeutic agent, or an antiangiogenic agent and an anti-inflammatory agent) and a pharmaceutically acceptable carrier. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories, using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions contain a therapeutically effective amount of an anti-angiogenic agent together with an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should be suited to the method of administration.

[0047] The amount of an anti-angiogenic agent effective in treating (e.g., see Figure 9), inhibiting, and preventing a disease or disorder associated with aberrant expression and / or activity of a therapeutic polypeptide can be determined by standard clinical techniques. Additionally, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0048] More specifically, the agent or pharmaceutical composition can be tested in vitro and then in vivo for the desired therapeutic or prophylactic activity before use in humans. For example, in vitro assays for demonstrating the therapeutic or prophylactic usefulness of a compound or pharmaceutical composition include the effect of the compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and / or tissue sample can be determined using techniques known in the art, including, but not limited to, rosette formation assays and cytolytic assays. According to the present invention, in vitro assays that can be used to determine whether administration of a particular compound is desirable include in vitro cell culture assays, in which a patient tissue sample is grown in culture and exposed to or otherwise administered a compound, and the effect of the compound on the tissue sample is observed.

[0049] It is contemplated that the antiangiogenic agent can be formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also contain a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate, in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0050] Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, and construction of nucleic acids as part of retroviral or other vectors, are known and can be used to administer the compounds of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (e.g., oral, rectal, and intestinal mucosa), or may be administered together with other bioactive agents. Administration may be systemic or local. Furthermore, it may be desirable to introduce the pharmaceutical agents or pharmaceutical compositions of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter.

[0051] In a specific embodiment, it may be desirable to administer antiangiogenic agents locally to the area that needs treatment.This can be achieved, for example, by local infusion during surgery, by topical application, for example, in combination with wound dressing after surgery, by injection, by catheter, by suppository, or by implant, but is not limited to, said implant being an implant of porous, non-porous, or gelatinous material, including membranes such as sialastic membranes, or fibers.When administering polypeptide, care must be taken to use materials that polypeptide does not absorb.

[0052] In specific embodiments in which the antiangiogenic agent is a nucleic acid encoding a polypeptide, the nucleic acid can be administered in vivo to promote expression of the encoded polypeptide by constructing it as part of an appropriate nucleic acid expression vector and administering it intracellularly, for example, by using a retroviral vector, or by direct injection, or by using biolistics, or by coating it with lipids or cell surface receptors or transfecting agents, or by administering it in association with a homeobox-like peptide known to enter the nucleus, etc. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the host cell DNA for expression by homologous recombination.

[0053] Other embodiments relate to vectors comprising polynucleotides encoding antiangiogenic agents, host cells, and production of antiangiogenic agents by synthetic and recombinant techniques. Vectors can be, for example, phage, plasmid, viral, or retroviral vectors. Retroviral vectors can be replication-competent or replication-deficient. In the latter case, viral propagation will generally occur only in complementary host cells. Polynucleotides encoding antiangiogenic agents can be ligated to vectors containing selectable markers for propagation within the host. Generally, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. The polynucleotide insert should be operably linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA, and tac promoters, the SV40 early and late promoters, and retroviral LTR promoters, to name a few. Other suitable promoters will be known to those skilled in the art. Expression constructs further include sites for transcription initiation, transcription termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs preferably includes a translation initiation codon at the beginning of the polypeptide to be translated and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, expression vectors preferably include at least one selectable marker.

[0054] It is contemplated that regulatory genes and sequences can be used to express and replicate antiangiogenic agents. The nature of regulatory sequences for gene expression may vary between species or cell types, but will generally include, as necessary, 5' untranscribed and 5' untranslated sequences involved in initiation of transcription and translation, respectively, e.g., TATA box, capping sequence, CAAT sequence, etc. Promoters can be constitutive or inducible. Regulatory sequences can also include enhancer sequences or upstream activator sequences, as desired.

[0055] In one embodiment, a polynucleotide encoding an anti-angiogenic agent may be fused to a polynucleotide encoding a signal sequence that directs localization of the polypeptide to a specific compartment of a prokaryotic or eukaryotic cell and / or directs secretion of the polypeptide. For example, in E. coli, it may be desirable to direct expression of the protein to the periplasmic space. Several vectors are commercially available for constructing fusion proteins that direct protein localization.

[0056] One specific embodiment provides a stent comprising a generally tubular structure (e.g., comprising a helical shape), the surface of which is coated with an anti-angiogenic agent as described above. The stent may be a generally cylindrically shaped scaffolding that can be inserted into a body passageway (e.g., a bile duct) or portion of a body passageway that has been narrowed, irregularly contoured, blocked, or obstructed by a disease process (e.g., tumor ingrowth) to prevent closure or reclosure of the passageway.

[0057] A specific embodiment also provides for the use of anti-angiogenic agents in a wide variety of surgical procedures. For example, within one aspect of the invention, anti-angiogenic proteins (e.g., in the form of a spray or film) may be utilized to coat or spray the area of ​​a tumor prior to removal to separate surrounding normal tissue from malignant tissue and / or to prevent the spread of disease to surrounding tissue. Within yet another aspect of the invention, surgical meshes coated with anti-angiogenic proteins may be utilized in any procedure in which a surgical mesh may be utilized.

[0058] The following examples are set forth to aid in the understanding of the present invention, but are not intended to, and should not be construed as, limiting the scope of the invention in any way. [Example]

[0059] Example 1: Expression of anti-angiogenic agents The antiangiogenic agent was expressed from the bacterium Escherichia coli and purified. To ensure that the designed polypeptide was still properly folded, the structure was confirmed by 1H-NMR analysis. The NMR spectra of the antiangiogenic agent (120 μM) and CD2-D1 (120 μM) were compared. As shown in Figure 2, the NMR spectrum of the antiangiogenic agent was nearly identical to that of CD2-D1 (top), whereas the denatured polypeptide (by organic solvent) showed a completely different spectrum (bottom). The resulting polypeptide exhibited a high affinity for both the host protein and the developed protein. 1 They exhibited very similar structural features as evidenced by the similarity of their 1 H-NMR, CD, and fluorescence spectra.

[0060] Example 2: Endothelial cells and apoptosis To determine the effects of antiangiogenic agents on endothelial cells, we performed a cell viability assay using HUVEC cells. The cells were treated with various concentrations of an antiangiogenic agent, anginex, and the host polypeptide from which the antiangiogenic agent was derived. As shown in Figure 3A, the antiangiogenic agent was more effective at inducing apoptosis in HUVEC cells (Figure 2A). We further tested whether the effect was specific to endothelial cells. To this end, cell proliferation assays were performed using HUVEC and M4A4 cells in the presence of 5 μM or 10 μM of the antiangiogenic agent, anginex, and the host protein. As shown in Figures 3B and 3C, a strong inhibition of cell proliferation by the agent was evident in HUVEC cells, whereas no effect was observed in epithelial M4A4 cells. These observations indicated that the effect of the antiangiogenic agent was endothelial cell-specific.

[0061] Example 3: Inhibition of tumor growth of PC-3 cell xenografts The potent activity of the antiangiogenic agent in inhibiting proliferation and induction of apoptosis on HUVEC cells was evident. A xenograft model of PC-3 cells was prepared using immunodeficient mice. Tumor-bearing mice (6 mice per group) were treated with daily doses of the antiangiogenic agent (10 mg / kg), PEGylated antiangiogenic agent (10 mg / kg), host protein (10 mg / kg), and buffered saline for 2 weeks. Treatment began 7 days after tumor inoculation. Tumors were measured either by volume or by tumor cell bioluminescence.

[0062] As shown in Figures 4A and 4B, antiangiogenic agents and antiangiogenic agent-PEG inhibited tumor growth. Figure 4A graphically illustrates that tumor volume remained relatively constant during treatment with the antiangiogenic agent when treatment began after 8 days. Figure 4B graphically illustrates that there was a dose-dependent effect and tumor volume remained relatively constant during treatment with the antiangiogenic agent when treatment began after 22 days. As controls, tumors grew at a normal rate in mice treated with buffer and host protein. At the end of the treatment course, tumors from each treatment group were excised and weighed.

[0063] As shown in Figure 5, there was a substantial difference in tumor weight between the antiangiogenic and control groups. This time, tumors were grown at 2 x 10 6 The tumors were reprogrammed with 1000 cells. Treatment began 22 days after tumor implantation. The antiangiogenic agent completely inhibited tumor growth.

[0064] As shown in Figure 6, in the control group, tumors grew at a normal rate in mice treated with buffer and host protein (CD2). From the image, it can be seen that tumors remained stable when treated with the exemplary antiangiogenic agent. For reference purposes only, the protein M1WT may be referred to as Agio1, and M1PEG may be referred to as Agio2.

[0065] Example 4: Vascular Density After Treatment Figure 7 shows that vascular density, monitored using immunofluorescence staining of tumor tissue sections collected from treated mice, was dramatically reduced after treatment with the PEGylated antiangiogenic agent compared to the PEGylated host protein and buffer treatment groups. To determine whether the effects of antiangiogenic agent treatment actually extended to tumor vasculature, tumors were harvested after treatment. Tissue slides were prepared from the harvested tumors. The slides were immunostained with an antibody against CD31, a molecular marker specific to endothelial cells. The immunostaining of the tumor tissue slides was visualized by confocal microscopy. These results indicated that the antiangiogenic agent had a specific effect on tumor angiogenesis.

[0066] Example 5: Toxicity and immunogenicity of antiangiogenic agents The toxicity of the parent CD2 domain 1 protein was previously analyzed and found to be nontoxic in mice. To ensure that the new design did not alter the toxicity of the protein, the toxicity of the antiangiogenic polypeptide was examined using CD-1 mice. First, the body weight of tumor-bearing nude mice was carefully monitored over the course of 14 days of treatment. As shown in Figure 8, no significant changes in mouse body weight were observed in any of the treatment groups. Furthermore, toxicity was tested in normal CD-1 mice. Three groups of mice (7 mice per group) were intravenously injected with 100 μl of the polypeptide (100 mg / kg, 20 doses used) at one, two, and three doses, with a three-day interval between each injection. The animals were returned to their cages for 30 days. No deaths were observed among the mice tested. All animals behaved normally (no changes in eating habits, no abnormal weight gain or loss, and no abnormal fur appearance).

[0067] Toxicity tests showed that the antiangiogenic agent and the antiangiogenic agent combined with PEG had no acute toxicity at at least three doses, which were approximately 20 times higher than the doses used in tumor treatment in mice. Furthermore, the inventors also tested whether there was any liver, kidney, or cardiovascular damage during treatment with the antiangiogenic agent and the antiangiogenic agent combined with PEG. Histological analysis showed no damage to the organs of the treated animals.

[0068] Example 6: N-linked glycosylation The polypeptide shown in SEQ ID NO: 11 was expressed and purified from a Pichia yeast expression system. Expression of this polypeptide in the yeast Pichia was achieved by both intracellular and secretory expression. The polypeptide was further purified using an ion exchange column. The polypeptide was expressed as a His-tagged polypeptide. The His-tag was removed by thrombin cleavage. The glycosylated protein expressed and purified from the yeast Pichia was found to be an anti-angiogenic agent and to have N-linked glycosylation. As shown in Figure 13, cells treated with various examples of anti-angiogenic agents showed strong survival rates.

[0069] Example 7: Terminal Glycine The polypeptide shown in SEQ ID NO: 12 was expressed and purified from an expression system. Expression of this polypeptide in the yeast Pichia was achieved by both intracellular and secretory expression. The polypeptide was further purified using an ion exchange column. Glycine-terminal proteins are anti-angiogenic agents. Cells were treated with various methods, and the polypeptide shown in SEQ ID NO: 12 had strong survival potential.

[0070] Example 8: Sequences SEQ ID NO: 1 is the amino acid sequence of domain 1 of CD2 from rat (WT rat CD2-D1): [ka]

[0071] SEQ ID NO: 2 is the amino acid sequence of domain 1 of CD2 from human (WT human CD2-D1): KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQER

[0072] SEQ ID NO: 3, designated M1WT or Agio1, is the amino acid sequence of variant domain 1 of CD2, derived from SEQ ID NO: 1 by the mutations W7Q, G8M, A9K, D94N, R96K, I97V, L98I, and E99I: [ka]

[0073] SEQ ID NO: 4 is the amino acid sequence of variant domain 1 of CD2 derived from SEQ ID NO: 1 by the mutations E41I, K43V, K45L, M46G, K47S, P48V, and G53L: [ka]

[0074] SEQ ID NO: 5 is the amino acid sequence of variant domain 1 of CD2 derived from SEQ ID NO: 1 by the mutations E41N, M46Q, and F49S: [ka]

[0075] SEQ ID NO: 6, called ProAgio-PEG or Agio2, is the amino acid sequence of variant domain 1 of CD2, derived from SEQ ID NO: 3 by the mutation M23C: [ka]

[0076] SEQ ID NO: 7 is the amino acid sequence of variant domain 1 of CD2 derived from SEQ ID NO: 1 by the mutations E41I, K43V, K45L, M46G, K47S, and F49S: [ka]

[0077] SEQ ID NO: 8 is the amino acid sequence of variant domain 1 of CD2: RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVANFKRKQKPSL KSGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILNKALDLRILE

[0078] SEQ ID NO: 9, designated hProAgioB or Agio3, is the amino acid sequence of variant domain 1 of CD2 derived from SEQ ID NO: 2 by the mutations E8S, T9V, W10Q, G11M, A12K, D99N, I102V, Q103I, and E104I: KEITNALSVQMKLGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYELLKNGALKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVIIR

[0079] SEQ ID NO: 10, designated hProAgio or Agio4, is the amino acid sequence of variant domain 1 of CD2, which is derived from SEQ ID NO: 9 by the mutation M30C: KEITNALSVQMKLGQDINLDIPSFQCSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYELLKNGALKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII

[0080] SEQ ID NO: 11, designated hProAgioY or Agio5, is the amino acid sequence of variant domain 1 of CD2 from yeast: KEITNALSVQMKLGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII

[0081] SEQ ID NO: 12, designated Agio6, is the amino acid sequence of variant domain 1 of CD2, which is derived from SEQ ID NO: 9 by the mutations M30C and a terminal glycine (G) or an additional terminal glycine (G): GKEITNALSVQMKLGQDINLDIPSFQCSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYELLKNGALKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII

[0082] Example 7: Tumor growth inhibition Figure 10 shows tumor growth curves over 14 days of treatment. The results show that hProAgio or Agio4, developed from the polypeptide encoding SEQ ID NO: 10, was effective in suppressing tumor growth. Experiments were performed using PC-3 xenografts using hProAgio or Agio4 (10 mg / kg, daily dose) and buffered saline as a control. Treatment began 8 days after tumor inoculation.

[0083] Example 8: Efficacy against Avastin® To further test the efficacy of antiangiogenic polypeptides, experiments were performed and analyzed using PC-3 xenografts with rProAgio-PEG or Agio2 (20 mg / kg, daily dose) and Avastin (20 mg / kg, dosed every other day). Treatment began 21 days after tumor inoculation. Figure 11 shows tumor growth curves for Avastin® and rProAgio-PEG or Agio2. Figure 12 shows a graphical representation of tumor weight at the end of a 14-day treatment course. Tumors from each treatment group were extracted and weighed. There were significant differences in tumor weight and growth between the animal groups treated with Agio2 and Avastin®.

[0084] The foregoing detailed description and accompanying drawings have been presented for purposes of illustration and description only. They are not intended to be exhaustive or to limit the scope of the invention. The embodiments have been chosen and described in order to best explain the principles of the invention and its practical application. Those skilled in the art will understand that many modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A polypeptide for reducing or inhibiting angiogenesis, comprising an amino acid segment having a mutation in domain 1 of the cluster of differentiation 2 (CD2) protein derived from human or rat, said polypeptide having greater than 95% sequence identity to SEQ ID NO: 12 and having a terminal glycine.

2. The polypeptide of claim 1, wherein the polypeptide has greater than 99% sequence identity to SEQ ID NO:

12.

3. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is identical to SEQ ID NO:

12.

4. The polypeptide of claim 1 further comprising a polyethylene glycol (PEG) moiety.

5. The polypeptide of claim 1 further comprising a glycan moiety.

6. A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide having an amino acid segment with a mutation in domain 1 of a CD2 protein derived from a human or rat, wherein the polypeptide has greater than 95% sequence identity to SEQ ID NO:

12.

7. The pharmaceutical composition of claim 6 , wherein the polypeptide is suitable for systemic administration.

8. 10. An injectable solution or suspension comprising the polypeptide of claim 1, wherein the polypeptide is in suspension.

9. 9. The injectable solution or suspension of claim 8, further comprising a pharmaceutically acceptable excipient, carrier, or diluent.

10. A method for reducing angiogenesis in an individual in need thereof, comprising administering a therapeutically effective dose of a polypeptide having an amino acid segment that varies from domain 1 of CD2 protein, said polypeptide having greater than 95% sequence identity to SEQ ID NO: 12, and wherein said polypeptide terminates in a glycine.

11. 11. The method of claim 10, wherein the amino acid sequence is identical to SEQ ID NO:

12.

12. 11. The method of claim 10, wherein the individual has a disorder selected from the group consisting of tumor growth, atherosclerosis, diabetic retinopathy, age-related maculopathy, and post-renal fibrosis.

13. 11. The method of claim 10, wherein the polypeptide is administered by a route selected from the group consisting of intravenous, in or around a solid tumor, systemic, intra-arterial, intraocular, intraperitoneal, and topical.

14. 11. The method of claim 10, further comprising administering a therapeutically effective amount of at least one other therapeutic agent.

15. 11. The method of claim 10, further comprising administering radiation therapy and a therapeutically effective or prophylactic amount of a second agent.

16. The method of claim 10, wherein the polypeptide is administered as part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

17. 11. The method of claim 10, further comprising administering radiation therapy.

18. The method of claim 10, wherein the angiogenesis is pathogenic.

19. The method of claim 10 , wherein the individual has cancer.

20. 20. The method of claim 19, wherein the cancer is breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, neuroblastoma, a central nervous system tumor, glioblastoma multiforme, or melanoma.

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