Factor 1 protein, factor 2 protein and inhibitors thereof for use in treating or preventing diseases

Abstract

To provide proteins for enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or non-transformed cells.SOLUTION: The present invention provides proteins comprising amino acid sequences encoded by nucleic acids derived from a human chromosomal region C19Orf10 termed Factor 1 and / or C19Orf63 termed Factor 2, or inhibitors thereof. Further provided is use of inhibitors of Factor 1 and Factor 2 for medical use, preferably for use in treating or preventing a disease in which angiogenesis contributes to disease development or progression.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to proteins comprising an amino acid sequence encoded by a nucleic acid derived from the human chromosomal region C19Orf10 designated Factor 1 and / or C19Orf63 designated Factor 2, for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissues or cells. Inhibitors of Factor 1 and Factor 2 are also provided for medical use, particularly for use in treating or preventing diseases in which angiogenesis contributes to the development or progression of the disease. [Background technology]

[0002] Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality worldwide. In Germany alone, the incidence rate is approximately 280,000 cases per year. Treatment for patients with AMI involves reperfusion therapy to open the coronary artery blockage, combined with the administration of platelet aggregation and coagulation inhibitors to prevent reocclusion of the vessel. In addition, pulse and blood pressure can be lowered by the administration of beta-blockers and ACE inhibitors. The use of statins to lower cholesterol levels is also common. Medical approaches to directly repair the myocardium are currently limited to the experimental use of autologous bone marrow cells. Drugs with similar effects without the use of bone marrow cells are desperately needed.

[0003] Persistent tissue necrosis during AMI triggers a wound healing response in which necrotic areas are replaced by granulation tissue, ultimately resulting in a collagen-rich scar. Monocytes are recruited from the bone marrow to the infarcted myocardium and play a key role in wound healing after AMI. The monocyte response in the myocardium is temporally biphasic. Proinflammatory monocytes appear earlier and promote the digestion of infarcted tissue and the removal of necrotic debris, whereas reparative monocytes predominate later, leading to angiogenesis and repair. Cell surface expression of the chemokine receptor CXCR4 identifies reparative monocyte subsets in mice and humans. The angiogenic and prohealing effects of CXCR4+ bone marrow cells are thought to be mediated by secreted proteins acting in a paracrine manner, although the identity of these factors remains poorly understood. Therefore, we performed a bioinformatic secretome analysis using human CXCR4+ bone marrow cells to identify novel secreted proteins that may exhibit therapeutic potential and regulate infarct healing after AMI.

[0004] These studies identified two distinct polypeptides that exhibit angiogenic stimulatory and / or cytoprotective effects, which we termed Factor 1 and Factor 2 proteins.

[0005] Both factors have been described in various scientific publications that refer to biological contexts that do not involve the angiogenic stimulatory and / or cytoprotective effects of these factors in non-transformed cells or tissues, and none of the studies discloses any evidence or hint of a function for these factors or a significant correlation of one of the factors with diseases or pathologies that are solely associated with non-transformed cells or tissues.

[0006] The amino acid sequence of human factor 1 is encoded in open reading frame 10 of human chromosome 19 (C19Orf10). The protein was described in 2007 as a novel secretory factor in the synovium in a proteomic analysis of so-called fibroblast-like synoviocytes (FLS-cells). The correlation between the secretion of this protein and inflammatory diseases of the joints has been assumed without any experimental or statistical evidence (Weiler et al., Arthritis Research and Therapy 2007, "The identification and characterization of a novel protein, c19orf10, in the synovium"). A corresponding patent application claims the protein as a therapeutic agent for treating joints, and for the diagnosis of tissues undergoing proliferative changes and for monitoring tissue changes (US 2008 / 0004232 A1, "Characterization of c19orf10, a novel synovial protein"). Another scientific publication describes the enhanced secretion of the protein in hepatocellular carcinoma cells (Sunagozaka et al., International Journal of Cancer, 2010, Identification of a secretory protein c19orf10 activated in hepatocellular carcinoma). Recombinantly produced proteins demonstrated growth-enhancing effects on cultured hepatocellular carcinoma cells. It is noteworthy that C19Orf10 was originally considered an interleukin and was also called IL-25, IL-27, and IL-27W. However, the terms "IL-25" and "IL-27" have been used inconsistently in the art to refer to various different proteins. For example, US 2004 / 0185049 calls a protein IL-27 and discloses its use in regulating immune responses. This protein is structurally different from Factor 1 (compare the amino acid sequence of Factor 1 shown in SEQ ID NO: 1 with the amino acid sequence of "IL-27" according to UniProt:Q8NEV9).Similarly, EP 2 130 547 A1 discloses the use of a protein called IL-25 for the treatment of inflammation. This protein, also known in the art as IL-17E, is structurally different from Factor 1 (compare the amino acid sequence of Factor 1 shown in SEQ ID NO: 1 with the amino acid sequence of "IL-25" according to UniProt: Q9H293).

[0007] The amino acid sequence of human factor 2 is encoded by open reading frame 63 of human chromosome 19 (C19Orf63). The protein was described as a novel secreted factor, INM02, in 2009 (Wang et al., Journal of Endocrinology 2009, "Molecular cloning of a novel secreted peptide, INM02, and regulation of its expression by glucose"). The presence of the protein was demonstrated in human serum using a polyclonal antibody. Furthermore, a correlation was demonstrated between glucose concentrations in cultured MIN6 (β cells) and isolated rat pancreatic islets and in the culture medium. Correlation analysis between diabetes and INM2 expression showed no significant difference. A corresponding patent application claims the production of a polyclonal antibody against the protein and its use in the treatment of diabetes (CN 200910055490, "Novel polyclonal antibody of secretive peptide INM02 and preparation method thereof").

[0008] Another scientific publication described the protein as a novel secreted factor hHSS1 (human hematopoietic signal peptide-containing secretory 1) (Junes-Gill et al., J Neurooncol, 2011, hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33). Published data demonstrate the expression of hHSS1 in hematopoietic stem cells and suggest its function as a tumor suppressor in the development of certain brain tumors (gliomas). A corresponding patent application claims the use of hHSS1 in the treatment of brain tumors (WO2011 / 094446 A1, A method for treating brain cancer using a novel tumor suppressor gene and secreted factor). Summary of the Invention [Problem to be solved by the invention]

[0009] (Summary of the Invention) In a first aspect, the present invention provides a protein comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof or a variant having at least 80% sequence identity with SEQ ID NO: 1 for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells. [Means for solving the problem]

[0010] In a second aspect, the present invention provides a protein comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof or a variant having at least 80% sequence identity to SEQ ID NO: 3 for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells.

[0011] In a third aspect, the present invention provides nucleic acids encoding proteins of the first and second aspects for enhancing proliferation and / or curing and / or inhibiting apoptosis in non-transformed tissue or cells.

[0012] In a fourth aspect, the present invention provides a vector for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells, comprising a nucleic acid of the third aspect.

[0013] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a protein of the first and / or second aspect and / or a nucleic acid of the third aspect and / or a vector of the fourth aspect and optionally a suitable pharmaceutical excipient for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells.

[0014] In a sixth aspect, the present invention provides inhibitors of Factor 1 and Factor 2, respectively, for medical use, preferably for treating or preventing diseases in which angiogenesis contributes to the development or progression of the disease.

[0015] In a seventh aspect, the present invention provides nucleic acids encoding such inhibitors for treating or preventing diseases in which angiogenesis contributes to the development or progression of the disease.

[0016] In an eighth aspect, the present invention provides a vector comprising a nucleic acid of the seventh aspect encoding such an inhibitor for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease.

[0017] In a ninth aspect, the present invention provides a pharmaceutical composition comprising an inhibitor of the sixth aspect and / or a nucleic acid of the seventh aspect and / or a vector of the eighth aspect and optionally a suitable pharmaceutical excipient for use in treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease. The above summary does not necessarily describe all aspects of the invention. [Brief explanation of the drawings]

[0018] [Figure 1]Human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) were cultured for 24 hours in minimal medium in the absence (control) or presence of 10% FCS, human recombinant VEGF-A (R&D Systems), or various concentrations of recombinant human Factor 1 (amino acid sequence shown in SEQ ID NO: 2) or Factor 2 (amino acid sequence shown in SEQ ID NO: 4), as indicated. (A) HCAEC proliferation was measured by bromodeoxyuridine incorporation. (B) HCAEC migration was assessed after wounding a confluent endothelial cell monolayer with a pipette tip. (C) HUVEC network formation was assessed in cells cultured on growth factor-reduced Matrigel. N = 3–5 independent experiments / condition; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control. [Figure 2] Ventricular cardiomyocytes were isolated from 1- to 3-day-old Sprague-Dawley rats by Percoll density gradient centrifugation. Cardiomyocytes were exposed to 180 hours of simulated ischemia (2-deoxyglucose in glucose-free medium in a 5% CO2 / 95% N2 atmosphere) followed by 60 minutes of simulated reperfusion (return to glucose-containing medium in a 5% CO2 / 95% room air atmosphere) in the absence (control) or presence of the indicated recombinant human GDF-15 or various concentrations of recombinant mouse factor 1 (amino acid sequence shown in SEQ ID NO: 13). Cell death was assessed by in situ TdT-mediated dUTP nick end labeling (TUNEL). N=3 independent experiments / condition; *P<0.05 vs. control. [Figure 3]Murine factor 1 or factor 2 cDNAs (nucleic acids shown in SEQ ID NOS: 7 and 10) were cloned into replication-deficient adenovirus. A replication-deficient adenovirus encoding galactosidase (lacZ) was used as a control. Male C57BL / 6 mice, 10–12 weeks old, were anesthetized and ventilated with isoflurane and then underwent permanent left anterior descending coronary artery (LAD) ligation. Virus was injected into the left ventricular (LV) cavity immediately after LAD ligation. (A) Left ventricular systolic function (fractional area change, FAC) was assessed by transthoracic echocardiography 28 days after LAD ligation. (B) The density of isolectin-positive capillaries in the border zone infarct was quantified by fluorescence microscopy 28 days after LAD ligation. *P<0.05, **P<0.01 vs. Ad.lacZ control. [Figure 4] Male C57BL / 6 mice, 10-12 weeks old, were anesthetized and ventilated with isoflurane, underwent temporary left anterior descending coronary artery ligation for 1 hour, and then reperfused for 28 days. Mice received a single sc injection of recombinant murine Factor 1 (amino acid sequence shown in SEQ ID NO: 13) or Factor 2 (amino acid sequence shown in SEQ ID NO: 24) at the time of reperfusion. Recombinant Factor 1 or Factor 2 were then continuously injected sc for 7 days. Control mice were injected with PBS. (A) Left ventricular systolic function (fractional area change, FAC) was assessed by transthoracic echocardiography 28 days after reperfusion. (B) Isolectin-positive capillary density in the border zone infarct was quantified by fluorescence microscopy 28 days after reperfusion. *P<0.05, **P<0.01 vs. PBS control. [Figure 5] Male C57BL / 6 mice, 10-12 weeks old, were anesthetized and ventilated with isoflurane (1-2%), temporarily ligated for 1 hour, and then reperfused for 28 days. At the time of reperfusion, mice received a single sc injection of recombinant murine Factor 1 or Factor 2 (SEQ ID NOs: 13 and 24, respectively; control mice received PBS injections). Recombinant Factor 1 or Factor 2 were then injected sc for 7 consecutive days. Control mice received PBS injections. Mice were examined daily for 28 days to assess post-infarction survival. [Figure 6]Sequence homologies to the protein encoded by human C19Orf10 were searched using the BLASTP algorithm. From the identified sequences, examples from various vertebrate species, primarily mammals, and one each from amphibians, birds, and fish, were selected. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm. Identities between all aligned species are marked with an "*", most highly conserved amino acid positions (i.e., only showing conservative substitutions) are marked with a ":", and highly conserved amino acid positions are marked with a "." [Figure 7] Sequence homologies to the protein encoded by human C19Orf10 were searched using the BLASTP algorithm. From the identified sequences, examples from various vertebrate species, primarily mammals, and one each from amphibians, birds, and fish, were selected. The selected mammalian amino acid sequences were aligned using the CLUSTALW2 algorithm. Identities between all aligned species are marked with an "*", most highly conserved amino acid positions (i.e., only showing conservative substitutions) are marked with a ":", and highly conserved amino acid positions are marked with a "." [Figure 8] Sequence homologs of HSS1 to the protein encoded by the human C19Orf63 splice variant were searched for using the BLASTP algorithm. From the identified sequences, examples from various vertebrate species, primarily mammals, and one sequence from each of amphibians and birds were selected. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm. Identities between all aligned species are marked with an *, most highly conserved amino acid positions (i.e., only showing conservative substitutions) are marked with a :, and highly conserved amino acid positions are marked with a . [Figure 9]Sequence homologies to the protein encoded by the human C19Orf63 splice variant HSS1 were searched using the BLASTP algorithm. From the identified sequences, examples from various vertebrate species, primarily mammals, and one sequence from each amphibian and fish species were selected. The selected mammalian amino acid sequences were aligned using the CLUSTALW2 algorithm. Identities between all aligned species are marked with an *, most highly conserved amino acid positions (i.e., only showing conservative substitutions) are marked with a :, and highly conserved amino acid positions are marked with a . [Figure 10] Sequence homologies to the protein encoded by the human C19Orf63 splice variant HSM1 were searched for using the BLASTP algorithm. From the identified sequences, examples from various vertebrate species, primarily mammals, and one sequence from each of amphibians and fish, were selected. The selected mammalian amino acid sequences were aligned using the CLUSTALW2 algorithm. Identities between all aligned species are marked with an "*", most highly conserved amino acid positions (i.e., only showing conservative substitutions) are marked with a ":", and highly conserved amino acid positions are marked with a "." [Figure 11] Sequence homologies to the protein encoded by the human C19Orf63 splice variant HSM1 were searched for using the BLASTP algorithm. From the identified sequences, examples from various vertebrate species, primarily mammals, and one sequence from each of amphibians and fish, were selected. The selected mammalian amino acid sequences were aligned using the CLUSTALW2 algorithm. Identities between all aligned species are marked with an "*", most highly conserved amino acid positions (i.e., only showing conservative substitutions) are marked with a ":", and highly conserved amino acid positions are marked with a "." [Figure 12]The effects of Factor 1- and 2-specific antibodies on the proliferation of HCAECs stimulated with recombinant Factor 1 (Panel A) and recombinant Factor 2 (Panel B) are shown. Data are means ± SEM of three to six experiments. Panel A: #P<0.05, ##P<0.01 vs. unstimulated control (left column). *P<0.05, **P<0.01 vs. Factor 1 without antibody; Panel B: ##P<0.01 vs. unstimulated control (far left column). *P<0.05, **P<0.01 vs. Factor 2 without antibody.

[0019] (Detailed Description of the Invention) Before describing the present invention in detail, it should be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as such may vary. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only, and that the scope of the present invention is not limited thereto, but rather is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] (definition) Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0021] The present invention is carried out using conventional chemical, biochemical, cell biological and recombinant DNA techniques as described in the literature of the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).Furthermore, conventional clinical cardiology methods as described in the literature of the art (see, for example, Braunwald's Heart Disease. A Textbook of Cardiovascular Medicine, 9th Edition, edited by P. Libby et al., Saunders Elsevier Philadelphia, 2011) are used.

[0022] Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" and its variations "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of steps but not the exclusion of any other integer or step or group of steps. As used in this specification and the claims, the singular forms "a," "an," and "the" refer to the plural unless the context clearly requires otherwise.

[0023] A nucleic acid molecule is understood to be a polymeric macromolecule based on nucleotide monomers. A nucleotide monomer consists of a nucleic acid base, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed by phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0024] The term "open reading frame" (ORF) refers to a nucleotide sequence that can be translated into amino acids. Typically, such an ORF contains a start codon in a given reading frame, followed by a region usually several 3 nucleotides in length, but no stop codon (TAG, TAA, TGA, UAG, UAA, or UGA). Typically, ORFs are naturally occurring or artificial, i.e., constructed by genetic engineering means. ORFs encode proteins that are translated into peptide-linked chains of amino acids.

[0025] The terms "protein" and "polypeptide" are used interchangeably herein to refer to any peptide-bonded chain, regardless of length or post-translational modification. Proteins (including protein derivatives, protein variants, protein fragments, protein portions, protein epitopes, and protein domains) that can be used in the present invention can be further modified by chemical modification, meaning that such chemically modified polypeptides contain chemical groups other than the 20 naturally occurring amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of polypeptides can confer advantageous properties over non-polypeptides, such as one or more of enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to variants that can be used in the present invention include, but are not limited to, pegylation, glycosylation of a non-glycosylated parent polypeptide; covalent conjugation to a therapeutic small molecule such as exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretins, and glucagon-like peptide 1 agonists, including liraglutide; or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications applicable to variants that can be used in the present invention can occur co-translationally (during translation) or post-translationally.

[0026] The term "amino acid" includes natural amino acids and amino acid derivatives. A hydrophobic non-aromatic amino acid in the context of the present invention is preferably any non-aromatic amino acid with a Kyte-Doolittle hydropathic index of 0.5 or more, more preferably 1.0 or more, and even more preferably 1.5 or more. Preferably, a hydrophobic non-aromatic amino acid in the context of the present invention is selected from the group consisting of the amino acids alanine (Kyte Doolittle hydropathic index 1.8), methionine (Kyte Doolittle hydropathic index 1.9), isoleucine (Kyte Doolittle hydropathic index 4.5), leucine (Kyte Doolittle hydropathic index 3.8), and valine (Kyte Doolittle hydropathic index 4.2), or derivatives thereof with the above Kyte Doolittle hydropathic indexes.

[0027] As used herein, the term "post-translational" refers to events that occur after the translation of a nucleotide triplet into an amino acid and the formation of a peptide bond with the subsequent amino acid in the sequence. Such post-translational events can occur after the complete polypeptide is formed or can occur already on a portion of the polypeptide that has already been translated during the translation process. Post-translational events typically change or modify the chemical or structural properties of the resulting polypeptide. Examples of post-translational events include, but are not limited to, events such as glycosylation or phosphorylation of an amino acid, or cleavage of a peptide chain by endopeptidases, etc.

[0028] As used herein, the term "co-translational" refers to an event that occurs during the process of translation of nucleotide triplets into amino acid chains. The event typically changes or modifies the chemical or structural properties of the resulting amino acid chain. Examples of co-translational events include, but are not limited to, events that can stop the translation process entirely or prevent peptide bond formation, resulting in two separate translation products.

[0029] As used herein, the term "variant" refers to a polypeptide that differs in one or more changes in its amino acid sequence from the polypeptide or fragment thereof from which it is derived. The polypeptide from which a protein variant is derived is also known as the parent peptide. Similarly, the fragment from which a protein fragment variant is derived is known as the parent fragment. Typically, variants are constructed artificially, preferably by genetic engineering means. Typically, the parent polypeptide is a wild-type protein or wild-type protein domain. Furthermore, the variants that can be used in the present invention are derived from a homolog, ortholog, or paralog of the parent polypeptide, or an artificially constructed variant, and retain at least one biological activity of the parent polypeptide. The amino acid sequence changes can be amino acid substitutions, insertions, deletions, N-terminal truncations, or C-terminal truncations, which can occur at one or various sites, or any combination of these changes. In preferred embodiments, variants useful in the present invention have a total of 100 or fewer (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or fewer) changes (i.e., substitutions, insertions, deletions, N-terminal truncations, and / or C-terminal truncations) in the amino acid sequence. The amino acid substitutions can be conservative and / or semi-conservative and / or non-conservative. In preferred embodiments, variants useful in the present invention differ from the protein or domain from which they are derived by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 or fewer amino acid substitutions, preferably conservative amino acid changes.

[0030] Typical substitutions are made with aliphatic amino acids, amino acids with aliphatic hydroxyl side chains, amino acids with acidic residues, amide derivatives, amino acids with basic residues, or amino acids with aromatic residues. Typical semi-conservative and conservative substitutions are as follows:

[0031] [Table 1]

[0032] If the new cysteine ​​remains a free thiol, then a change from A, F, H, I, L, M, P, V, W, or Y to C is semi-conservative. Furthermore, one skilled in the art will recognize that glycines in sterically bulky positions should not be substituted and that P should not be introduced into portions of proteins with alpha helix or beta sheet structure.

[0033] Alternatively or additionally, a "variant" as used herein can be characterized by a degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention has at least 80% sequence identity with its parent polypeptide. Preferably, the polypeptide and reference polypeptide exhibit sequence identity over a contiguous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids or over the entire length of the reference polypeptide. Preferably, the polynucleotide and reference polynucleotide exhibit sequence identity over a contiguous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides or over the entire length of the reference polypeptide.

[0034] The term "at least 80% sequence identity" is used throughout this specification with respect to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the respective reference polypeptide or respective reference polynucleotide.

[0035] A fragment of a protein comprises an amino acid deletion, which may be an N-terminal truncation, a C-terminal truncation, or an internal deletion, or any combination thereof. Such variants comprising an N-terminal truncation, a C-terminal truncation, and / or an internal deletion are referred to as "fragments" in the context of this application. Fragments can be natural (e.g., splice variants) or artificially constructed, preferably by genetic engineering means. Preferably, a fragment (or deletion variant) has a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or fewer amino acids at its N-terminus and / or its C-terminus and / or internally, preferably at its N-terminus, its N- and C-terminus, or its C-terminus, compared to the parent polypeptide.

[0036] When comparing two sequences and no reference sequence is specified to calculate the percentage sequence identity, the sequence identity should be calculated based on the longer of the two sequences being compared, unless otherwise specified.

[0037] Nucleotide and amino acid sequence similarity, eg, percentage sequence identity, can be determined by sequence alignment. Such alignments can be performed using various known algorithms, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80) or the CLUSTALW2 algorithm (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. (2007). Clustal W and Clustal X version 2.0). Bioinformatics, 23, 2947-2948.) (available, for example, at http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html).Preferably, the CLUSTALW2 algorithm at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html is used with the default parameters set out at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html (alignment type=slow, protein weight matrix=Gonnet, gap open=10, gap extension=0,1 (for the slow vs alignment option), and protein weight matrix=Gonnet, gap open=10, gap extension=0,20, gap distance=5, no end gaps=none, output options: format=Aln w / numbers, order=aligned).

[0038] The grade of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST protein searches are performed using the BLASTP program, available at, for example, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. Preferred algorithm parameters used are the default parameters found at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome (expectation threshold=10, word size=3, max matches in query range=0, matrix=BLOSUM62, gap cost=presence:11 extension:1, composition adjustment=factor 1 and factor 2 conditional composition score matrix adjustment with a database of non-redundant protein sequences (nr) to obtain amino acid sequences homologous to the polypeptide).

[0039] To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis can be complemented with established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62) or Markov random fields. When sequence identity percentages are referred to in this application, the percentages are calculated over the entire length of the longer sequence unless otherwise specified.

[0040] As used herein, the term "host cell" refers to a cell that harbors a nucleic acid (e.g., a plasmid or virus) of the invention. Such a host cell can be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a cardiac, plant, or animal cell). The cell can be transformed or untransformed. The cell can be, for example, an isolated cell in a cell culture, or part of a tissue that can itself be isolated, or part of a more complex tissue structure such as an organ or individual.

[0041] The terms "Factor 1," "Factor 1 protein," or "Factor 1 polypeptide" are used interchangeably and refer to the protein (human homolog) set forth in NCBI reference sequence NM_019107.3, and its mammalian homologs (particularly from mouse or rat). The amino acid sequence of the human homolog is encoded in open reading frame 10 of human chromosome 19 (C19Orf10). Preferably, Factor 1 protein refers to a protein comprising, consisting essentially of, or consisting of the core portion of human Factor 1 having the amino acid sequence set forth in SEQ ID NO:1. In a more preferred embodiment, Factor 1 protein has the amino acid sequence set forth in SEQ ID NO:2.

[0042] The terms "Factor 2," "Factor 2 protein," or "Factor 2 polypeptide" are used interchangeably and refer to the protein (human homolog) set forth in NCBI reference sequence NM_175063.4 and its mammalian homologs (particularly from mouse or rat). The amino acid sequence of human Factor 2 is encoded by open reading frame 63 of human chromosome 19 (C19Orf63). Preferably, Factor 2 protein refers to a protein comprising, consisting essentially of, or consisting of the core portion of human Factor 2 having the amino acid sequence set forth in SEQ ID NO:3. In more preferred embodiments, the Factor 2 protein has the amino acid sequences shown in SEQ ID NOs: 4 and 5, respectively. In the most preferred embodiment, the Factor 2 protein is in a secreted form, preferably having the amino acid sequence shown in SEQ ID NO: 4.

[0043] The term "non-transformed tissue" or "non-transformed cell" refers to tissues and cells that exhibit physiological parameters of an equivalent non-cancerous or oncogenic cell or tissue. Such parameters include, but are not limited to, cell cycle regulation, cell division rate, contact inhibition, anchorage-independent growth, or metabolism. The equivalent non-cancerous or oncogenic cell or tissue may be healthy, damaged, or diseased.

[0044] The term "healing" includes the regeneration and repair of living cells, tissues, organs, and entire biological systems, and the partial or complete restoration of normal function. In the case of tissues, organs, or entire biological systems, the term includes the regeneration and repair process in which cells in the body reduce the size of damaged or necrotic areas and replace them with new, living tissue. This replacement can occur, for example, through regeneration, in which necrotic cells are replaced by new cells that form tissue similar to that originally present, or through repair, in which damaged tissue is replaced with scar tissue. Regeneration is a preferred variant of the healing process, as it is a process that results in the partial or complete restoration of normal function. Therefore, in the context of the present invention, the term healing includes all processes, and those skilled in the art will combine this term, but preferably, regeneration processes should be promoted instead of processes that result in the production of non-functional tissue, such as scar tissue. In the context of the present invention, the term healing preferably refers to the promotion of proliferation, migration, network formation, and angiogenesis.

[0045] The term "enhancing proliferation" refers to an increase in the cell division rate of a cell or group of cells compared to a cell or group of cells not treated with a protein, nucleic acid, vector, or pharmaceutical composition of the invention. Methods for measuring the cell division rate of a cell, for example, by counting mitotic cells using FACS, are well known in the art.

[0046] The term "inhibiting apoptosis" refers to the ability of a protein, nucleic acid, vector, or pharmaceutical composition of the invention to prevent a cell or group of cells from undergoing apoptosis under conditions in which a control cell or group of cells undergoes apoptosis. Methods for determining whether a cell undergoes apoptosis, such as by TUNEL assay, are well known to those skilled in the art. The description of the embodiments further includes definitions and explanations of terms used throughout the specification. These explanations and definitions are valid throughout the entire application unless otherwise specified.

[0047] (Aspect) The elements of the present invention are described below. While the elements are described with specific embodiments, it should be understood that they can be combined in any number and in any way to create further embodiments. The various described examples and preferred embodiments should not be construed as being limited to only the explicitly described embodiments. The specification should be understood to support and include embodiments that combine any number of the disclosed and / or preferred elements with the explicitly described embodiments. Furthermore, unless the context indicates otherwise, all permutations and combinations of all elements described herein should be considered to be disclosed in the specification of this application.

[0048] In a first aspect, the present invention provides a protein comprising, consisting essentially of, or consisting of Factor 1 protein, preferably having the amino acid sequence set forth in SEQ ID NO: 1, or a fragment thereof or a variant having at least 80% sequence identity with SEQ ID NO: 1, for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or non-transformed cells, in particular for use in enhancing proliferation of non-transformed tissue, enhancing proliferation of non-transformed cells, inhibiting apoptosis of non-transformed tissue, or inhibiting apoptosis of non-transformed cells. In a particularly preferred embodiment of the present invention, the protein comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.

[0049] In a preferred embodiment of this aspect of the invention, the protein comprises the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, or a variant having at least 80% sequence identity with SEQ ID NO: 2. A preferred fragment of SEQ ID NO: 2 includes an N-terminal signal sequence MAAPSGGWNGVGASLWAALLLGAVALRPAEA (SEQ ID NO: 35)The parent polypeptide lacks the amino acid sequence of SEQ ID NO: 1. Those skilled in the art can determine, without undue burden, which positions in the parent polypeptide can be mutated and to what extent, and which positions must be maintained to preserve the function of the polypeptide. Such information can be obtained, for example, from homologous sequences, which can be identified, aligned, and analyzed by bioinformatics methods well known in the art. Such analysis is exemplarily described in Example 7, and the results are shown in Figures 6 and 7. Mutations are preferably introduced in regions of the protein that are not completely conserved among species, preferably mammals; i.e., one or more of the amino acid positions are mutated (not marked with an "*"). In more preferred embodiments, only amino acids that are not completely conserved (indicated with an "*") or to a lesser extent conserved (indicated with a ":" or ".") are changed. In particularly preferred embodiments of the present invention, the Factor 1 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2, or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 2. Such mutations may be present in the full-length protein shown in SEQ ID NO: 2 or in the protein lacking the N-terminal signal sequence shown in SEQ ID NO: 1.

[0050] N-terminal deletion mutants may have, in addition to the N-terminal signal, one or more amino acids deleted from amino acid positions 32-55 (based on SEQ ID NO:2), i.e., the N-terminal conserved region. Thus, the N-terminus of the deletion factor 1 protein may further be positions 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56. Alternatively, deletion factor 1 proteins may have one or more amino acid deletions from the C-terminal conserved region, i.e., positions 146-173 (based on SEQ ID NO:2). Thus, the C-terminus of the deletion factor 1 protein can be at positions 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, or 172. Proteins of the first aspect of the invention can further comprise additional amino acid sequences, for example, to stabilize or purify the resulting protein. Examples of such amino acids include His6-tags (SEQ ID NO: 36), myc-tags, or FLAG-tags.

[0051] In such embodiments, it is preferred to mutate protease cleavage sites within the protein of the first aspect of the invention to stabilize the protein (see Segers et al. Circulation 2007, 2011). Those skilled in the art know how to determine potential proteolytic cleavage sites within a protein. For example, protein sequences can be submitted to websites that provide such analyses (e.g., http: / / web.expasy.org / peptide_cutter / or http: / / pmap.burnham.org / proteases). Submitting the protein sequence of SEQ ID NO: 2 to http: / / web.expasy.org / peptide_cutter / determined the following low frequency (less than 10) cleavage sites:

[0052] [Table 2]

[0053] These sites can be altered to remove each identified protease recognition / cleavage sequence to increase the serum half-life of the protein.

[0054] Factor 1 and Factor 2 have demonstrated growth-enhancing activity, particularly in enhancing angiogenesis. In a preferred embodiment, the proteins of the first aspect are used to enhance the proliferation, preferably angiogenesis, of non-transformed tissues or cells. Therefore, Factor 1 or Factor 2 are advantageously used in the treatment of diseases in which enhanced angiogenesis may be beneficial. Examples of such diseases are further illustrated below.

[0055] Enhanced proliferation, in the context of the present invention, includes any degree of enhancement of cell or tissue proliferation compared to control cells or tissues not administered with a protein of the present invention. Cell proliferation can be measured, for example, by bromodeoxyuridine incorporation as described in Example 2. Enhanced tissue proliferation can be determined, for example, by measuring the increase in weight or size of each tissue and by histological methods. Such methods are well known in the art, and many are standard methods for clinical applications.

[0056] In another preferred embodiment, the protein of the first aspect is used to heal non-transformed tissue or cells. In another preferred embodiment, the protein of the first aspect is used to enhance the proliferation and heal non-transformed tissue or cells. In a particularly preferred embodiment, the protein of the first aspect is used to enhance the proliferation and heal non-transformed tissue or cells, and to inhibit apoptosis.

[0057] In another preferred embodiment, the proteins of the first aspect are used to inhibit apoptosis in non-transformed tissues or cells. Thus, the proteins of the present invention exhibit anti-apoptotic potential and protect cells or tissues from apoptotic cell death. In this context, "protect" or "cytoprotective effect" means that the extent of apoptotic cell death in cells treated with a Factor 1 protein of the present invention is reduced by at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50%, and most preferably at least 60% compared to controls. Those skilled in the art can assess cell death, for example, by in situ TdT-mediated dUTP nick end labeling (TUNEL) as described in Example 3. Other indicators of apoptosis are fragmented genomes, which can be tested, for example, by DNA laddering (Liu et al., 2005, Circulation 111:90-96), cytochrome-c release, or caspase 3 activity (Most et al., 2003, J. Biol. Chem. 278:48404-48412). The anti-apoptotic effect of peptides can be evaluated in vivo using an experimental animal model of heart failure. For example, mice with postischemic systolic dysfunction can be treated with the protein, and the degree of apoptotic cardiomyocytes in the cardiac tissue of treated and control mice can be evaluated. The peptides can be preferably administered parenterally, for example, intraperitoneally, intravenously, or subcutaneously. In particularly preferred embodiments, the proteins of the present invention exhibit two, preferably all, of the above functions: enhancing proliferation and healing of non-transformed tissues or cells, and inhibiting apoptosis. Fragments and variants of Factor 1 protein included in the present invention that are at least 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90% and more preferably at least 100% of that of the protein having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, more preferably SEQ ID NO: 1, exhibit anti-apoptotic potential and protect cells or tissues from apoptotic cell death.

[0058] In a second aspect, the present invention provides a protein comprising, consisting essentially of, or consisting of Factor 2 protein having the amino acid sequence set forth in SEQ ID NO: 3, or a fragment thereof, or a variant having at least 80% sequence identity with SEQ ID NO: 3, for use in enhancing the growth and / or curing non-transformed tissues or cells. In a preferred embodiment of the present invention, the Factor 2 protein comprises the amino acid sequence of SEQ ID NO: 3, a fragment thereof, or a variant having at least 80% sequence identity with SEQ ID NO: 3. Those skilled in the art can determine, without undue burden, which positions in the polypeptide can be mutated and to what extent, and which positions must be maintained, to preserve the function of the polypeptide. Such information can be obtained, for example, from homologous sequences, which can be identified, aligned, and analyzed by bioinformatics methods well known in the art. In a particularly preferred embodiment of the present invention, the protein of the second aspect of the present invention comprises the amino acid sequence of SEQ ID NO: 3, or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3.

[0059] The proteins of the second aspect of the invention may further comprise additional amino acid sequences, for example to stabilise or purify the resulting protein.

[0060] In some embodiments, it may be preferable to mutate protease cleavage sites within the proteins of the first aspect of the invention to stabilize the protein. Suitable proteolytic cleavage sites may be identified as described above.

[0061] In another preferred embodiment of the invention, the protein of the second aspect of the invention comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4, a fragment thereof, or a variant having at least 80% identity to SEQ ID NO: 4. Preferred fragments include an N-terminal signal sequence MAAASAGATRLLLLLLMAVAA PSRARG' (SEQ ID NO: 37)The Factor 2 protein lacks the amino acid sequence of SEQ ID NO: 4. Those skilled in the art can determine, without undue burden, which positions in the polypeptide can be mutated and to what extent, and which positions must be maintained, to preserve the function of the polypeptide. Such information can be obtained, for example, from homologous sequences, which can be identified, aligned, and analyzed by bioinformatics methods well known in the art. Such analysis is exemplarily described in Example 8, and the results are shown in Figures 8 and 9. Mutations are preferably introduced only in regions of the protein that are not completely conserved between species (preferably mammalian), i.e., one or more amino acid positions not marked with an "*" are mutated. In more preferred embodiments, only amino acids that are neither completely conserved (indicated by an "*") nor to a lesser extent conserved (indicated by a ":" or ".") are altered. In a particularly preferred embodiment of the present invention, the Factor 2 protein comprises the amino acid sequence of SEQ ID NO: 4, or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4.

[0062] Such mutations can be present in the full-length protein set forth in SEQ ID NO:4 or in the protein lacking the N-terminal signal sequence. N-terminal deletion mutants can lack one or more amino acids from amino acid positions 27-73 (based on SEQ ID NO:4), i.e., the N-terminal conserved region, in addition to the N-terminal signal. Thus, the N-terminus of a deletion Factor 2 protein can be at position 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56. Additionally or alternatively, a deletion Factor 1 protein may lack one or more amino acid positions 190-254 (based on SEQ ID NO:4, i.e., from the C-terminal conserved region). Furthermore, the C-terminus of the deletion factor 2 protein is 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 , 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, or 253.

[0063] In another preferred embodiment of the present invention, the protein of the second aspect of the present invention comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:5, a fragment thereof, or a variant having at least 80% identity to SEQ ID NO:5. A preferred fragment lacks the N-terminal signal sequence MAAASAGATRLLLLLLMAVAAPSRARG (SEQ ID NO:37). Those skilled in the art can determine, without undue burden, which positions in the polypeptide can be mutated and to what extent, and which positions must be maintained, to preserve the function of the polypeptide. Such information can be obtained, for example, from homologous sequences, which can be identified, aligned, and analyzed by bioinformatic methods well known in the art. Such analysis is illustratively described in Example 9, and the results are shown in Figures 10 and 11. Mutations are preferably introduced only in regions of the protein that are not completely conserved between species (preferably mammalian), i.e., one or more amino acid positions not marked with a "*" are mutated. In a more preferred embodiment, only amino acids that are neither completely conserved (indicated by a "*") nor to a lesser extent (indicated by a ":" or ".") are changed. In particularly preferred embodiments of the invention, the Factor 2 protein comprises the amino acid sequence of SEQ ID NO: 5 or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.

[0064] Such mutations may be present in the full-length protein shown in SEQ ID NO:5, or in the protein shown in SEQ ID NO:5 lacking the N-terminal signal sequence.

[0065] N-terminal deletion mutants may lack one or more amino acids from amino acid positions 27-73 (based on SEQ ID NO:4), i.e., the N-terminal conserved region, in addition to the N-terminal signal. Furthermore, the N-terminus of a deletion Factor 2 protein may be at positions 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56. Additionally or alternatively, deletion Factor 1 proteins may lack one or more amino acid positions 190-262 (based on SEQ ID NO:4), i.e., the C-terminal conserved region. Thus, the C-terminus of the deletion factor 2 protein is 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, The amino acid sequence may be at position 3, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, or 261.

[0066] Fragments and variants of the protein of the second aspect of the invention that are at least 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90%, more preferably at least 100% of that of the protein having the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, most preferably SEQ ID NO:3, exhibit anti-apoptotic potential and protect cells or tissues from apoptotic cell death.

[0067] In a preferred embodiment, the proteins of the second aspect are used to enhance proliferation of non-transformed tissues or cells. Enhanced proliferation includes any grade of enhancement compared to control cells or tissues not administered the protein. Experimental methods for measuring proliferation are described above. Respective proliferation measurements for proteins of the second aspect of the invention, assessed by bromodeoxyuridine incorporation, are also described in Example 2.

[0068] In another preferred embodiment, the protein of the second aspect is used in the healing of non-transformed tissue or cells. In a particularly preferred embodiment, the protein of the second aspect of the invention exhibits both of the above functions, i.e., enhanced proliferation and healing of non-transformed tissue or cells.

[0069] In another embodiment of the invention, the proteins of Aspects 1 and / or 2 are administered in vivo, ex vivo, or in vitro, preferably in vivo. In typical embodiments, proteins of the first aspect of the invention and / or proteins of the second aspect of the invention are administered ex vivo or in vitro to enhance proliferation and / or healing and / or inhibit apoptosis for tissue engineering to generate tissue for transplantation into an individual. Cells used for tissue engineering can be from the same individual, as well as from another individual of the same or different species. Methods for removing the cells or tissue and transplanting the new tissue into an individual are not included in the present invention.

[0070] In a preferred embodiment of the present invention, the non-transformed cells are stem cells. Such stem cells can be embryonic stem cells or adult stem and progenitor cells. The present invention includes totipotent stem cells and pluripotent stem cells.

[0071] In a preferred embodiment, the non-transformed cells or the non-transformed tissue are diseased. In another preferred embodiment, the non-transformed cells or the non-transformed tissue are damaged. In another preferred embodiment, the non-transformed cells or the non-transformed tissue are damaged and diseased.

[0072] In a preferred embodiment, the non-transformed cells or the non-transformed tissue are muscle cells or muscle tissue. Muscles include all types of muscles known to those skilled in the art. Such muscles are, for example, skeletal muscles, smooth muscles, or cardiac muscles. In a more particularly preferred embodiment, the muscle is cardiac muscles. In another preferred embodiment, the non-transformed cells or the non-transformed tissue are epithelial cells or epithelial tissue. In another preferred embodiment, the non-transformed cells or the non-transformed tissue are nerve cells or nerve tissue.

[0073] In a preferred embodiment, said non-transformed cells or said non-transformed tissue belong to the circulatory system of an individual. In another preferred embodiment, the non-transformed cell or the non-transformed tissue belongs to or is derived from a specific system of the individual's body selected from the group including the digestive system, endocrine system, excretory system, immune system, integumentary system, muscular system, nervous system, reproductive system, respiratory system, or skeletal system. In another preferred embodiment of the invention, the cell belongs to 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the listed systems of the individual.

[0074] In another preferred embodiment, the non-transformed cells or tissues belong to or are derived from a specific part or organ of an individual's body selected from the group consisting of skin, bone, heart, cartilage, blood vessels, esophagus, stomach, intestines, glands, liver, kidney, lung, brain, and spleen. In a particularly preferred embodiment, the non-transformed cells or tissues belong to or are derived from the heart.

[0075] In the case of damaged non-transformed cells or non-transformed tissues, it is more preferred that the damage is caused by genetic / hereditary diseases or acquired diseases, such as ischemia, reperfusion injury, inflammation, infection, trauma, mechanical stress, poisoning, or surgery. In a particularly preferred embodiment, the damage is caused by ischemia. In another particularly preferred embodiment, the damage is caused by reperfusion injury.

[0076] In the context of the present invention, in the case of pathological or damaged cells or tissues, it is preferred that the damage results from atrophy, degeneration, inflammation, injury, or a disease associated with a wound. It is particularly preferred that the disease is associated with an injury. It is also particularly preferred that the disease is associated with a wound.

[0077] In a preferred embodiment of the present invention, the disease is a skeletal muscle disorder selected from the group consisting of muscular dystrophy, muscle weakness, muscle atrophy, myositis, central core disease, nemaline (rod) myopathy, centronuclear myopathy, myotubular myopathy, centronuclear myotubular myopathy, ophthalmoplegia, and mitochondrial myopathy. The muscular dystrophy may be selected from the group consisting of Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, peripheral muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. The myositis may be selected from the group consisting of myositis ossificans, fibromyositis, idiopathic inflammatory myopathies (eg, dermatomyositis, polymyositis, and inclusion body myositis), and pyomyositis.

[0078] In another preferred embodiment of the present invention, the disease is a primary or acquired cardiomyopathy. Primary cardiomyopathy is selected from hereditary cardiomyopathy and cardiomyopathy caused by spontaneous mutation. Cardiomyopathy includes, but is not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), takotsubo cardiomyopathy, Loeffler's endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, and obesity-related cardiomyopathy.

[0079] In the context of the present invention, said acquired cardiomyopathy is preferably selected from ischemic cardiomyopathy caused by atherosclerosis or other coronary artery disease, cardiomyopathy caused by infection or poisoning of the myocardium, hypertensive heart disease caused by pulmonary arterial hypertension and / or arterial hypertension, and valvular heart disease, with ischemic cardiomyopathy caused by atherosclerosis or other coronary artery disease being particularly preferred.

[0080] In the most preferred embodiment of the present invention, the non-transformed cells or tissues damaged by ischemia or reperfusion injury belong to the heart. Therefore, the resulting disease to be treated is preferably selected from the group consisting of myocardial infarction, angina pectoris, and heart failure, with myocardial infarction being particularly preferred. The term "myocardial infarction" used in the context of the present invention includes acute myocardial infarction (AMI).

[0081] In particularly preferred embodiments, the use of a protein according to the first aspect of the invention or a protein according to the second aspect of the invention involves application to an individual after myocardial infarction, where the healing includes improvement in left ventricular systolic function and may be associated with an increase in capillary density in the border zone infarct. Furthermore, the protein according to the first aspect of the invention or the protein according to the second aspect of the invention may reduce mortality after myocardial infarction. Methods that can be used to measure parameters such as improvement in left ventricular systolic function, increase in capillary density in the border zone infarct, and reduction in mortality after myocardial infarction are well known in the art and are typically described in Examples 4 to 6.

[0082] The Factor 1 and Factor 2 proteins, fragments, or variants can be used to treat or improve atrophy, hypoplasia, inflammation, injury, wound, ischemia, reperfusion injury, inflammation, infection, trauma, mechanical stress, poisoning, and primary or acquired cardiomyopathies, preferably hereditary cardiomyopathies and cardiomyopathies caused by spontaneous mutations. Examples of cardiomyopathies include, but are not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), takotsubo cardiomyopathy, Loeffler's endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, and obesity-related cardiomyopathy; myocardial infarction; or improvement of left ventricular systolic function. The Factor 1 and Factor 2 proteins, fragments, or variants can also be used in methods for treating the conditions and diseases described above.

[0083] In a third aspect, the present invention provides nucleic acids encoding proteins of the first and second aspects for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells.

[0084] The term "enhancing proliferation and / or curing and / or inhibiting apoptosis of non-transformed tissue or cells" has the above meaning and preferred meaning.

[0085] Nucleic acid sequences can be optimized to enhance expression in host cells. Parameters to consider include C:G content, preferred codons, and avoidance of inhibitory secondary structures. These factors can be combined in various ways to obtain a nucleic acid sequence with enhanced expression in a particular host (see, e.g., Donnelly et al., International Publication No. WO 97 / 47358). The ability of a particular sequence to enhance expression in a particular host requires some empirical experimentation. Such experimentation involves measuring the expected expression of a nucleic acid sequence and, if necessary, altering the sequence. Many different encoding nucleic acid sequences can be obtained, starting with a particular amino acid sequence and the known degeneracy of the genetic code. The degeneracy of the genetic code arises because almost all amino acids are coded for by various combinations of nucleotide triplets or "codons." The translation of specific codons into specific amino acids is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990).

[0086] In a preferred embodiment of the present invention, the nucleic acid further comprises a transcriptional regulatory element or expression control sequence positioned to regulate the expression of the protein. Such nucleic acids, together with the regulatory elements, are often referred to as expression systems. As used herein, the term "expression system" refers to a system designed to produce one or more gene products of interest. Typically, such systems are "artificially," i.e., engineered by genetic engineering means that can be used to produce the gene products of interest in vivo, in vitro, or ex vivo. The term "expression system" further encompasses the expression of the gene products of interest, including transcription of a polynucleotide, mRNA splicing, translation into a polypeptide, co-translational and post-translational modification of the polypeptide or protein, and targeting of the protein to one or more compartments within the cell, secretion from the cell, and uptake of the protein in the same or another cell. This overview refers to expression systems for use in eukaryotic cells, tissues, or organisms. Expression systems for prokaryotic systems can vary, and methods for constructing prokaryotic expression systems are well known in the art.

[0087] The regulatory elements present in a gene expression cassette generally include: (a) a promoter transcriptionally linked to the nucleotide sequence encoding the polypeptide; (b) a 5' ribosome binding site operably linked to the nucleotide sequence; (c) a terminator operably linked to the 3' end of the nucleotide sequence; and (d) a 3' ribosome binding site operably linked to the nucleotide sequence. Polyadenylation signal. Additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing may also be present. A promoter is a genetic element that is recognized by RNA polymerase and mediates the transcription of downstream regions. Preferred promoters are strong promoters that result in increased transcription levels. Examples of strong promoters include the immediate early human cytomegalovirus promoter (CMV) and CMV containing intron A (Chapman et al., Nucl. Acids Res. 19:3979-3986, 1991). Further examples of promoters include naturally occurring promoters, such as the EF1α promoter, the murine CMV promoter, the Rous sarcoma virus promoter, and the SV40 early / late promoter, and the [β]-actin promoter; and artificial promoters, such as synthetic muscle-specific promoters and chimeric muscle-specific / CMV promoters (Li et al., Nat. Biotechnol. 17:241-245, 1999; Hagstrom et al., Blood 95:2536-2542, 2000).

[0088] The ribosome binding site is located at or near the start codon. Examples of preferred ribosome binding sites include CCACCAUGG, CCGCCAUGG, and ACCAUGG (where AUG is the start codon) (Kozak, Cell 44:283-292, 1986). The polyadenylation signal is involved in cleavage of the transcribed RNA and addition of a poly(A) tail to the RNA. Polyadenylation signals in higher eukaryotes contain an AAUAAA sequence approximately 11 to 30 nucleotides from the polyadenylation addition site. The AAUAAA sequence is involved in signaling RNA cleavage (Lewin, Genes IV, Oxford University Press, NY, 1990). The poly(A) tail is important for mRNA processing, nuclear export, translation, and stabilization.

[0089] Polyadenylation signals that can be used as part of the gene expression cassette include the minimal rabbit [β]globin polyadenylation signal and the bovine growth hormone polyadenylation signal (BGH) (Xu et al., Gene 272:149-156, 2001; Post et al., U.S. Patent US5,122,458). Examples of additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing include enhancers, leader sequences, and operators. Enhancer regions increase transcription. Examples of enhancer regions include the CMV enhancer and the SV40 enhancer (Hitt et al., Methods in Molecular genes 7:13-30, 1995; Xu et al., Gene 272:149-156, 2001). Enhancer regions can be associated with promoters.

[0090] The expression of the proteins of the second aspect of the present invention or the proteins of the second aspect of the present invention can be regulated. Such regulation can be achieved at many steps in gene expression. Possible regulatory steps include, but are not limited to, transcription initiation, promoter clearance, transcription elongation, splicing, nuclear export, mRNA stability, translation initiation, translation efficiency, translation elongation, and protein folding. Other regulatory steps that affect the concentration of Factor 1 or Factor 2 polypeptides inside the cell affect the half-life of the protein. Such regulatory steps include, for example, regulation of protein denaturation. Because the proteins of the present invention include secreted proteins, the proteins may be directed into the secretory pathway of the host cell. Secretion efficiency, along with regulatory steps on expression and protein stability, regulates the concentration of each protein outside the cell. The outside of the cell can refer to, for example, but is not limited to, the culture medium, tissue, intracellular matrix or cavity, or body fluids such as blood or lymph. Regulation of the above regulatory steps can be, for example, cell-type or tissue-type independent or cell-type or tissue-type specific. In particularly preferred embodiments of the present invention, regulation of the regulatory steps is cell-type or tissue-type specific. Such cell-type or tissue-type specific regulation is preferably achieved by regulating the transcription of the nucleic acid. This transcription regulation can be achieved by using a cell-type or tissue-type specific promoter sequence. The result of this cell-type or tissue-type specific regulation can have different degrees of specificity. This means that the expression of each polypeptide is enhanced in each cell or tissue compared to other cell or tissue types, or that the expression is limited to each cell or tissue type. Cell- or tissue-type specific promoter sequences are well known in the art and are available for a wide range of cell or tissue types.

[0091] In another preferred embodiment, the expression is not cell-type or tissue-type specific, but depends on physiological conditions. Such conditions include, for example, inflammation or wounding. Such physiological condition-specific expression can also be achieved by regulating all of the above-mentioned regulatory steps. A preferred method for regulating physiological condition-specific expression is transcriptional regulation. For this purpose, wound- or inflammation-specific promoters can be used. Each promoter contains a natural sequence, which may be derived from a gene specifically expressed during, for example, immune response and / or wound tissue regeneration. Another possibility is the use of an artificial promoter sequence, constructed, for example, by combining two or more natural sequences. In another preferred embodiment, the regulation is cell-type or tissue-type specific and physiological condition specific. In a particularly preferred embodiment, the expression is cardiac-specific. In another particularly preferred embodiment, the expression is cardiac-specific and wound-specific.

[0092] Another possibility for regulating the expression of a protein of the second aspect of the present invention or a protein of the second aspect of the present invention is conditional regulation of the gene expression. To achieve conditional regulation, an operator sequence can be used. For example, a Tet operator sequence can be used to repress gene expression. Conditional regulation of gene expression by a Tet operator sequence together with a Tet repressor is well known in the art, and many respective systems have been established for a wide range of prokaryotes and eukaryotes. Those skilled in the art know how to select an appropriate system and adapt it to the specific requirements of each application.

[0093] In a particularly preferred embodiment, the use of the nucleic acid of the present invention includes application to an individual after myocardial infarction, and the healing includes improvement of left ventricular systolic function and may be associated with an increase in capillary density in the border zone infarct. Furthermore, the use of the nucleic acid may reduce the mortality rate after myocardial infarction. Methods that can be used to measure parameters such as improvement of left ventricular systolic function, increase in capillary density in the border zone infarct, and reduction in mortality rate after myocardial infarction are well known in the art and are typically described in Examples 4 to 6.

[0094] Nucleic acids encoding the Factor 1 and Factor 2 proteins, fragments, or variants are preferably used to treat or improve atrophy, hypoplasia, inflammation, injury, wound, ischemia, reperfusion injury, inflammation, infection, trauma, mechanical stress, poisoning, primary or acquired cardiomyopathies, preferably hereditary cardiomyopathies and cardiomyopathies caused by spontaneous mutations, such as, but not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), takotsubo cardiomyopathy, Loeffler's endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; myocardial infarction; or improvement of left ventricular systolic function.

[0095] In a fourth aspect, the present invention provides a vector comprising a nucleic acid or expression system of the third aspect for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells.

[0096] The term "enhancing proliferation and / or curing and / or inhibiting apoptosis of non-transformed tissue or cells" has the above meaning and preferred meaning.

[0097] As used herein, the term "vector" refers to a protein or polynucleotide or mixture thereof that introduces or can introduce proteins and / or nucleic acids contained therein into cells. In the context of the present invention, it is preferred that the gene of interest encoded by the introduced polynucleotide is expressed in a host cell upon introduction of the vector(s). Examples of suitable vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors, e.g., lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores.

[0098] In a preferred embodiment of the present invention, the vector is a viral vector. Suitable viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors.

[0099] In a particularly preferred embodiment of the invention, the vector is an adenovirus or adeno-associated virus (AAV) vector.

[0100] Nucleic acids encoding one or more proteins of the first aspect of the invention and one or more proteins of the second aspect of the invention may be introduced into a host cell, tissue or individual using a vector suitable for therapeutic administration. A suitable vector is preferably capable of delivering the nucleic acid into target cells without producing unacceptable side effects.

[0101] In a particularly preferred embodiment, the use of the vector of the present invention includes application to an individual after myocardial infarction, and the healing includes improvement in left ventricular systolic function and may be associated with an increase in capillary density in the border zone infarct. Furthermore, the use may reduce the mortality rate after myocardial infarction. Methods that can be used to measure parameters such as improvement in left ventricular systolic function, increase in capillary density in the border zone infarct, and reduction in mortality rate after myocardial infarction are well known in the art and are typically described in Examples 4 to 6.

[0102] Vectors containing nucleic acids encoding the Factor 1 and Factor 2 proteins, fragments, or variants are preferably used to treat or improve atrophy, hypoplasia, inflammation, injury, wound, ischemia, reperfusion injury, inflammation, infection, trauma, mechanical stress, poisoning, primary or acquired cardiomyopathies, preferably hereditary cardiomyopathies and cardiomyopathies caused by spontaneous mutations, such as, but not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), takotsubo cardiomyopathy, Loeffler's endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; myocardial infarction; or improvement of left ventricular systolic function.

[0103] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a protein of the first and / or second aspect and / or a nucleic acid of the third aspect and / or a vector of the fourth aspect and optionally a carrier for use in enhancing proliferation and / or curing and / or inhibiting apoptosis of non-transformed tissue or cells. The term "enhancing proliferation and / or curing and / or inhibiting apoptosis of non-transformed tissue or cells" has the meaning and preferred meaning given above.

[0104] As used herein, the term "carrier" refers to a pharmacologically inert substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer solution, or vehicle with which a therapeutically active ingredient is administered. Such pharmaceutical carriers can be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions in water or oils (including, but not limited to, those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Saline solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" (E.W. Martin). In a preferred embodiment of the present invention, the carrier is a suitable pharmaceutical excipient. Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc. Such suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

[0105] "Pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0106] The term "composition" is intended to include formulations of active compounds that include an encapsulating material as a carrier to provide a capsule in which the active ingredient, with or without other carriers, is surrounded by the carrier (i.e., associated with the active ingredient).

[0107] The term "active ingredient" refers to a substance in a pharmaceutical composition or formulation that is biologically active, i.e., that provides pharmaceutical value. In the context of the present invention, the active ingredient refers to a protein of the first and / or second aspect and / or a nucleic acid of the third aspect and / or a vector of the fourth aspect. A pharmaceutical composition may contain one or more active ingredients that may act independently or in concert with each other. The active ingredients may be formulated in neutral or salt form. The salt form is preferably a pharmaceutically acceptable salt.

[0108] The term "pharmaceutically acceptable salt" refers to salts of, for example, but not limited to, polypeptides. Suitable pharmaceutically acceptable salts include acid addition salts, which may be formed, for example, by mixing a solution of a polypeptide of the invention with a solution of a pharmaceutically acceptable acid (e.g., hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid). Furthermore, if the peptide contains an acidic moiety, suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with appropriate organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed with counter anions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates, and arylsulfonates).Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphor, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinol, hydrabamine, hydrobromide, hydrochloride, hydroiodide, -Hydroxyethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfonate, mocate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, acetate, succinate, tannate, tartrate, theocrylate, tosylate, triethiodide, undecanoate, valerate, etc. (e.g., SM See Berge et al., "Pharmaceutical salts," J. Pharm. Sci., 66, pp. 1-19 (1977)).

[0109] An effective amount of the active ingredient is administered to a cell, tissue, or individual. An "effective amount" is the amount of active ingredient sufficient to achieve its intended purpose. The active ingredient may be a therapeutic agent. The effective amount of a given active ingredient will vary depending on parameters such as the nature of the ingredient, the route of administration, the size and species of the individual receiving the active ingredient, and the purpose of administration. The effective amount in a particular case can be determined empirically by one skilled in the art using methods established in the art. "Administering" as used in the context of the present invention includes in vivo administration to an individual and direct administration to a cell or tissue in vitro or ex vivo.

[0110] In a preferred embodiment of the present invention, the pharmaceutical composition is customized for treating a disease or disorder. As used herein, "treat" (treating, treating, or treatment) of a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing symptoms characteristic of the disorder being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting or preventing the recurrence of the disorder in patients who previously had the disorder; (e) limiting or preventing the recurrence of symptoms in patients who previously had symptoms of the disorder; (f) reducing mortality after the onset of the disease or disorder; (g) curing; and (h) preventing the disease. As used herein, "prevent" (preventing, preventing, prevention, or prophylaxis) of a disease or disorder means preventing such a disease or disorder from occurring in a patient.

[0111] In a particularly preferred embodiment of the present invention, treatment or healing with the pharmaceutical composition of the present invention includes treating an individual after myocardial infarction, and the healing may include improvement in left ventricular systolic function and may be associated with an increase in capillary density in the border zone infarct. Furthermore, healing may reduce mortality after myocardial infarction. Methods that can be used to measure parameters such as improvement in left ventricular systolic function, increase in capillary density in the border zone infarct, and reduction in mortality after myocardial infarction are well known in the art and are typically described in Examples 4 to 6.

[0112] Pharmaceutical compositions contemplated by the present invention can be formulated in a variety of ways well known to those skilled in the art. For example, pharmaceutical compositions of the present invention can be in liquid form, such as a solution, emulsion, or suspension. Preferably, pharmaceutical compositions of the present invention are formulated for parenteral administration, preferably intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary, or intracardiac administration, or transmucosal administration, preferably intravenous, subcutaneous, or intraperitoneal administration. Formulations for oral or anal administration are also possible. Preferably, pharmaceutical compositions of the present invention are in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or glucose to make the solution isotonic with blood. The aqueous solution should be suitably buffered, if necessary (preferably pH 3-9, more preferably pH 5-7). The pharmaceutical composition is preferably in unit dosage form. In such form, the pharmaceutical composition is subdivided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form can be a packaged preparation, a package containing discrete quantities of the pharmaceutical composition, such as vials or ampoules. Administration of the pharmaceutical compositions is preferably by intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary, or intracardiac routes, but other routes of administration known in the art are also included.

[0113] In this case, the pharmaceutical composition is used as a treatment for an individual, and the use of the pharmaceutical composition can replace or be administered in addition to standard treatment for each disease or condition.When the pharmaceutical composition is further used, the pharmaceutical composition can be administered before, after, or simultaneously with the standard treatment.In a preferred embodiment, the standard treatment is reperfusion therapy, and the pharmaceutical composition can be administered before, after, or simultaneously with the reperfusion therapy.

[0114] It is more preferred that the pharmaceutical composition be administered once or more than once. This includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 times. The period for administering the pharmaceutical is not limited. Preferably, the administration does not exceed 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0115] A single dose of the pharmaceutical composition may not depend on the total amount of the dose administered and the duration of each dose administered as one or more bolus injections and / or infusions.

[0116] The first through fifth aspects of the present invention are based on the inventors' discovery that Factor 1 and Factor 2 are potent angiogenic stimulatory molecules in vitro and in vivo. Thus, the proteins, nucleic acids, and vectors are also contemplated for ex vivo use, where they may be therapeutic agents, for example, for stimulating the cells ex vivo or for use in cell culture applications.

[0117] However, knowledge of the angiogenesis-stimulating activity of Factors 1 and 2 led us to investigate whether Factor 1 and Factor 2, respectively, are targets for antiangiogenic therapy. We have successfully inhibited angiogenesis using either Factor 1 inhibition or Factor 2 inhibition.

[0118] Antiangiogenic strategies are used to treat cancer conditions and other disorders in which angiogenesis plays a role in disease progression, such as age-related macular degeneration (see, e.g., Ferrara N and Kerbel RS (2005) Nature:438:967-974 or Potente M, et al. (2011) Cell. 146(6):873-887). In yet further aspects, the present invention relates to the antiangiogenic properties of inhibitors of Factor 1 and Factor 2. In these aspects, the definitions set forth above in the definitions section apply equally. Furthermore, specific definitions set forth in the first to fifth aspects and, for example, in the description of preferred embodiments of the term "vector" and preferred vectors, also apply to the following aspects of the present invention, unless the context of their use clearly dictates otherwise.

[0119] In a sixth aspect, the present invention provides inhibitors of Factor 1 and / or Factor 2 proteins for medical use, preferably in the treatment or prevention of diseases in which angiogenesis contributes to the development or progression of the disease. The term "inhibitor" refers to a compound that interferes with the angiogenesis-stimulating activity of Factor 1 or Factor 2. Inhibitors prevent their cellular production and secretion into the circulation and / or into sites of disease development or progression by affecting the transcription and / or translation of mRNA encoding Factor 1 or Factor 2. Inhibitors may also act by specifically binding to Factor 1 or Factor 2 protein or a cellular protein to which Factor 1 or Factor 2 protein specifically binds, preferably its cellular receptor. Such binding may prevent or disrupt the natural interaction of Factor 1 or Factor 2 with other cellular proteins, preferably their respective cellular receptors. Those skilled in the art are familiar with methods for interfering with the binding of receptors and their agonists and can use this knowledge to design appropriate inhibitors of Factor 1 and Factor 2. Furthermore, inhibitors can be derived from the Factor 1 or Factor 2 proteins themselves by deleting or mutating portions of the Factor 1 and Factor 2 proteins that exhibit the pro-angiogenic functions of Factor 1 and Factor 2, respectively. Such inactivating mutations or deletions of Factor 1 or Factor 2 will compete with wild-type Factor 1 and Factor 2 for their natural binding partners. Compounds that interfere with the angiogenesis-stimulating activity of Factor 1 or Factor 2 reduce said activity by at least 20%, preferably at least 30%, and more preferably at least 40%. In the context of inhibitors that specifically bind to Factor 1 or Factor 2 or comprise, consist essentially of, or consist of mutants or fragments of Factor 1 or Factor 2, respectively, they preferably exhibit this level of inhibition of Factor 1 or Factor 2 protein at equimolar concentrations. A preferred assay that can be used to measure inhibition of angiogenesis-stimulating activity is described in Example 10 herein.To determine whether an inhibitor has this activity at equimolar amounts, the molar amounts of Factors 1 and 2 and each inhibitor must be measured. Factor 1, as set forth in SEQ ID NO:1, has a MW of 15.84 kD, and Factor 2, as set forth in SEQ ID NO:3, has a MW of 21.57 kD; the molecular weight of IgG is approximately 150 kD. Thus, 100 ng of Factor 1 and 947 ng of Factor 1-specific IgG are approximately equimolar, and 100 ng of Factor 2 and 695 ng of Factor 2-specific IgG are approximately equimolar. It is clear from panels A and B of Figure 12 that the Factor 1- and Factor 2-specific antibodies described herein are inhibitors of Factor 1 protein or Factor 2 protein, respectively. In the context of inhibitors that interfere with the transcription and / or translation of mRNA encoding Factor 1 or Factor 2, the level of inhibition is preferably measured based on protein produced in cells that naturally produce Factor 1 or Factor 2. Those skilled in the art are familiar with numerous methods for measuring the amount of mRNA encoding Factor 1 or Factor 3 and Factor 1 or Factor 2 protein, which can be used in assessing the ability of a compound to interfere with the transcription and / or translation of mRNA encoding Factor 1 or Factor 2. Preferably, Factor 1 protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1, and Factor 2 comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 1 or 3.

[0120] Inhibitors used in the context of the present invention are preferably proteins comprising, consisting essentially of, or consisting of an inhibitory fragment or mutant of the amino acid sequence set forth in SEQ ID NO: 1 or 3, or a variant thereof having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 3. It is well known in the art that proteins that function through receptor-mediated protein-protein interactions contain a domain required for binding to the receptor and a domain that stimulates the receptor to transmit a signal intracellularly. Therefore, those skilled in the art are familiar with methods for producing inhibitory fragments or mutants of such receptor-binding proteins. For example, a series of N- and / or C-terminally truncated Factor 1 or Factor 2 proteins can be produced and tested for their angiogenic stimulatory activity in the assay described in Example 2. The ability of those fragments that no longer exhibit angiogenic stimulatory activity to inhibit the angiogenic stimulatory activity of Factor 1 or 2 protein is then tested in the assay described in Example 10. Mutants of Factor 1 and 2 can be generated, for example, by alanine scanning mutagenesis, as known in the art. In alanine scanning, a series of mutants is generated, each containing one, two, three, or more amino acids mutated to alanine (a so-called cassette), and differing in the location of the cassette within Factor 1 or 2. Factor 1 or 2 mutants that have lost angiogenesis-stimulating activity can again be identified as described in Example 2. Inhibitory mutants can then be identified using the assay described in Example 10.

[0121] In another preferred embodiment, the inhibitor is a ligand that specifically binds to a receptor that naturally interacts with the amino acid sequence set forth in SEQ ID NO: 1 or 3, or a variant thereof having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 3, or Factor 1 or 3, or a variant thereof having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 3. The term "ligand" refers to a chemical moiety that specifically binds to a specific antigen. Preferred ligands are amino acid-based ligands, such as immunoglobulins, preferably antibodies or antigen-binding fragments thereof, and antibody-like proteins. Alternatively, the ligand can be a peptidomimetic.

[0122] As used herein, the term "immunoglobulin (Ig)" refers to immunity-conferring glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" include, but are not limited to, molecules such as B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (β2M), CD3, CD4, and CD8. It binds to the membrane of effector cells via its transmembrane region. Typically, the term "antibody" as used herein refers to a secretory immunoglobulin that lacks a transmembrane region and can be released into the bloodstream and body cavities. Antibodies are classified into various isotypes based on the heavy chain they possess. There are five types of human Ig heavy chains, designated by the Greek letters α, δ, ε, γ, and μ. The type of heavy chain present defines the antibody class, and these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, each performing different functions and mediating the appropriate immune response to different types of antigens. Distinct heavy chains vary in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). Antibodies contain four polypeptide chains: two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy and light chains can be determined as known in the art. For example, the following set of rules can be used to find the CDRs within each antibody light chain sequence and heavy chain sequence: Light chain CDR-1: Start: approximately residue 24, the residue before CDR-1 is always Cys, the residue after CDR1 is always Trp. Typically, Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu; length: 10-17 residues Light chain CDR-2: Start: always 16 residues after the end of L1, generally Ile-Tyr, and residues before Val-Tyr, Ile-Lys, Ile-Phe, always 7 residues in length; Light chain CDR-3: Start: always 33 residues after the end of CDR-2; always before Cys, always after Phe-Gly-XXX-Gly; Length: 7-11 residues Heavy chain CDR-1: Start: always approximately residue 26, 4 residues after Cys (based on the Chothia AbM definition; the Kabat definition starts 5 residues later); always residues before Cys-XXX-XXX-XXX; always residues after Trp, typically Trp-Val, Trp-Ile, and Trp-Ala; length: 10-12 residues [AbM definition; the Chothia definition excludes the last 4 residues]; Heavy chain CDR-2: Start: Always 15 residues after the end of the Kabat / AbM definition of heavy chain CDR-1; typically the residue before Leu-Glu-Trp-Ile-Gly (many variations exist), or the residue after Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala, length 16-19 residues by Kabat definition (AbM definition; Chothia definition ends 7 residues earlier) Heavy chain CDR-3: Start: always 33 residues after the end of heavy chain CDR-2 (always 2 amino acid residues after Cys); always residues before Cys-XXX-XXX (typically Cys-Ala-Arg); always residues after Trp-Gly-XXX-Gly; length: 3 to 25 residues. This set of rules is known to those skilled in the art and can also be found at http: / / www.bioinf.org.uk / abs / #cdrid.

[0123] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs may include, for example, amino acid residues in the CDRs not encoded by human germline immunoglobulin sequences (e.g., mutations generated by random or site-specific mutagenesis in vitro or somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include "humanized antibodies" in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) are grafted onto human FR sequences. Human antibodies also include antibodies isolated from human immunoglobulin libraries or from transgenic animals directed against one or more human immunoglobulins that do not express endogenous immunoglobulins.

[0124] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. In some embodiments, monoclonal antibodies are produced by hybridomas comprising a B cell obtained from a non-human animal (e.g., a mouse) fused with an immortalized cell. As used herein, the term "recombinant antibody" includes any antibody produced, expressed, produced, or isolated by recombinant means, including: (a) antibodies isolated from, or hybridomas produced by, animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes; (b) antibodies isolated from host cells (e.g., transfectomas) transformed to express the antibody; (c) antibodies isolated from a recombinant combinatorial antibody library; and (d) antibodies produced, expressed, produced, or isolated by any other means, including splicing of immunoglobulin gene sequences with other DNA sequences. As used herein, a "heterologous antibody" is defined in terms of the transgenic organism producing such an antibody. This term refers to antibodies that have amino acid sequences or coding nucleic acid sequences that correspond to those found in organisms not comprised in transgenic organisms, generally from species other than the transgenic organism. As used herein, a "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain combined with a murine light chain is a heterohybrid antibody.

[0125] The term "antigen-binding fragment" refers to an antibody fragment that retains the function of specifically binding to an antigen or antigenic protein, but lacks some or all other structural features of an antibody or an artificial construct comprising a portion of an antibody. Preferred examples of antigen-binding fragments include, but are not limited to, the following: Fab fragment, Fc fragment, Fab' fragment, F(ab')2, single domain antibody (sdAb), nanobody, single chain Fv, bivalent single chain variable fragment (di-scFvs), tandem scFvs, diabody, single chain de These include scDBs, triabodies, bispecific T cell engagers (BiTEs), or dual affinity retargeting molecules (DART molecules).

[0126] "Fab fragments" (also called "Fab portions" or "Fab regions") each contain a single antigen-binding site and a remaining "Fc fragment" (also called "Fc portion" or "Fc region") (the name reflects its ability to crystallize readily). "Fab' fragments" refer to Fab fragments that also contain the hinge region of an Ig molecule, and "F(ab')2 fragments" are fragments that are chemically or disulfide-bonded. sdAb (Desmyter et al. 1996) and "nanobodies" contain only a single VH domain, whereas "single-chain Fv (scFv)" fragments contain a short link. It contains a heavy chain variable domain connected to a light chain variable domain via a linker peptide (Huston et al. 1988). Di-scFvs can be generated by combining two scFvs (scFvA-scFvB). This can be done by producing a single peptide chain with two VH and two VL domains, resulting in a "tandem scFvs" (VHA-VLA-VHB-VLB). Another possibility is to create scFvs with a linker that is too short to fold the two variable domains together, forcing the scFvs to dimerize. Typically, a linker five residues in length is used to generate these dimers. This type is known as a "diabody." An even shorter linker (one or two amino acids) between the VH and VL domains leads to the formation of monospecific trimers, so-called "triabodies" or "tribodies." Bispecific diabodies are formed by expressing chains with the sequences VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. Single-chain diabodies (scDbs) comprise VHA-VLB and VHB-VLA fragments linked by a linker peptide (P) of 12–20 amino acids, preferably 14 amino acids (VHA-VLB-P-VHB-VLA). Bispecific T cell engagers (BiTEs) are fusion proteins consisting of two scFvs from different antibodies, where one scFv binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. 2004). Dual affinity retargeting molecules (DART molecules) are diabodies further stabilized by a C-terminal disulfide bridge.

[0127] The term "antibody-like protein" refers to a protein with antibody-like properties that binds to antigens or antigenic proteins without necessarily possessing the structural characteristics of antibodies. Antibody-like proteins can occur naturally or be artificially engineered, e.g., biotechnologically. Examples of natural antibody-like proteins include, but are not limited to, antigen-binding proteins such as the lipocalin family, which typically represent a broad family of proteins responsible for storing or transporting biologically important compounds. They share a conserved barrel of eight antiparallel beta strands as their central folding motif, with six hypervariable loops at one end of this barrel structure that connect each pair of beta strands. These loops form the entrance to a binding pocket. Structural diversity among members of the lipocalin family reflects differences in the shape and chemical properties of their binding partners. Thus, although they consist of a single polypeptide chain and are much smaller than immunoglobulins, they have great potential for binding to antigens with different specificities. Examples of artificially designed antibody-like proteins include scaffold-based proteins, which are produced by fusing a peptide with a known affinity for a certain target or by inserting the peptide into a scaffold protein to combine the binding properties of the peptide with the desired beneficial properties of the scaffold carrier. Those skilled in the art are familiar with such scaffold-based proteins. As used herein, the term "scaffold protein" refers to a protein that has structural rigidity, i.e., folds into a stable tertiary structure. The amino acids of a scaffold protein appear to occupy specific three-dimensional positions within the scaffold protein. Therefore, when one or more amino acids of a scaffold protein are replaced with a polypeptide of appropriate length, the polypeptide will occupy a similar position to the replaced one. This allows a specific polypeptide to be positioned in a specific three-dimensional position and / or orientation within the scaffold protein.Furthermore, scaffold proteins can be used as an alternative to antibodies for molecular recognition (see, for example, Skerra A.: (2007) Curr. Opin. Biotechnol. 2007, 18:295-304, or Skerra A. (2000) J. Mol. Recognit. 2000, 13:167-187). An example of such a scaffold protein is the Fyn SH3 domain, which contains two domains that can be mutated to impart new binding specificity to the SH3 domain. Methods for selecting Fyn SH3 domains that specifically bind to a certain antigen are described, for example, in WO2000 / 072742 or WO2008 / 022759.

[0128] In the context of the present invention, the term "peptidomimetic" refers to any molecule whose essential elements (active groups) resemble those of natural peptides or proteins in three-dimensional space, retain the ability to interact with biological targets, and produce the same biological effect. Peptidomimetics typically include small protein-like chains designed to resemble peptides, which can be obtained by modifying existing peptides or designing similar systems (e.g., peptoids and β-peptides) that mimic peptides. Regardless of the approach, chemical structural changes are designed to favorably adjust molecular properties, such as increasing or decreasing stability or biological activity. Therefore, modifications include non-naturally occurring peptide changes, including, but not limited to, backbone changes and the incorporation of unnatural amino acids.

[0129] The term "specific binding" or "specifically binding" to an antigen, e.g., Factor 1 or Factor 2, refers to the ability of a ligand to bind to an antigenic determinant of the antigen with high affinity. In that context, "high affinity" refers to an interaction with a Kd of 1 x 10 -5 Less than M, preferably 1 x 10 -6 Less than M, more preferably 1 x 10 -7 Less than M, and even more preferably 1 x 10 -8 Less than M, most preferably 1 x 10-9 This means that it is less than M.

[0130] A preferred antibody for use in the context of the sixth aspect of the present invention is a monoclonal antibody, preferably a human or humanized antibody. In another preferred embodiment, the inhibitor is a nucleic acid that inhibits or prevents the transcription and / or translation of an mRNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 or 3, or a variant thereof having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 3. Those skilled in the art are familiar with methods for determining the sequence of such a nucleic acid based on a genomic sequence encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 or 3. An example of such an inhibitory nucleic acid is an siRNA specific to the mRNA encoding Factor 1 or Factor 2.

[0131] Where the inhibitor of the sixth aspect of the invention is a protein that can be encoded by a nucleic acid, it is contemplated that the inhibitor will be administered by providing a nucleic acid encoding the inhibitor. Thus, in a seventh aspect, the present invention provides a nucleic acid encoding an inhibitor of the sixth aspect of the invention for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease. The nucleic acid may further comprise any of the elements described in the context of the third aspect of the invention.

[0132] Thus, in an eighth aspect, the present invention provides a vector comprising a nucleic acid of the sixth aspect of the invention for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease. In the context of the seventh aspect, the term "vector" has the same meaning as described in the context of the fourth aspect of the invention.

[0133] In a preferred embodiment of this aspect of the present invention, the vector is a viral vector. Suitable viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors. Furthermore, in a ninth aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease, comprising an inhibitor of the sixth aspect, a nucleic acid of the seventh aspect, or a vector of the eighth aspect, and optionally a suitable pharmaceutical excipient. The pharmaceutical composition may also include any of the components described in the fifth aspect of the present invention.

[0134] The term "diseases in which angiogenesis contributes to the development or progression of the disease" used in the sixth to ninth aspects of the present invention refers to diseases in which proliferation of cells that form blood vessels occurs at the onset, during development, and / or during the progression of the disease. Cells capable of forming and proliferating blood vessels include endothelial cells, which line the interior of blood vessels, and smooth muscle cells, which form the blood vessel walls. Endothelial cells and smooth muscle cells do not proliferate in healthy blood vessels. These cells proliferate, for example, in response to injury or chemical cues (e.g., VEGF). Angiogenesis, also known as neovascularization, is characterized by the process of forming new blood vessels from pre-existing vessels. In some diseases, such as ocular neovascular diseases, abnormal blood vessel formation is the cause of the disease. In other diseases, such as benign or malignant tumors, angiogenesis occurs during the progression of the disease to provide oxygen and nutrients to the growing tumor mass. In these diseases, angiogenesis is not the cause of the disease but promotes its progression. Neovascularization in malignant tumors also contributes to metastasis by providing tumor cells with an escape route from the tumor mass.

[0135] Preferably, the disease in which angiogenesis contributes to the development or progression of the disease is a proliferative disease. Preferred proliferative diseases are selected from the group consisting of benign tumors, malignant tumors, rheumatoid arthritis, psoriasis, ocular neovascular diseases, Osier-Webber syndrome, plaque angiogenesis, restenosis after transplantation and angioplasty, telangiectasia, hemophilic arthropathy, angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, scleroderma, hypertrophic scars, cat scratch disease, and ulcers, especially macular degeneration, adrenocortical carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, head and neck cancer, laryngeal cancer, liver cancer, lung cancer, melanoma, myeloproliferative diseases, cervical cancer, non-melanoma skin cancer, ovarian cancer, prostate cancer, benign prostatic hyperplasia, pancreatic cancer, rectal cancer, and testicular cancer. Preferred diseases to treat are benign tumors, malignant tumors, and ocular neovascular diseases.

[0136] The inventors have determined that antibodies directed against certain epitopes of Factor 1 and Factor 2 interfere with the angiogenesis-stimulating functions of Factor 1 and Factor 2, respectively, i.e., are antagonist antibodies. Thus, in a tenth aspect, the present invention is directed to ligands of Factor 1 and Factor 2, respectively, that inhibit the angiogenesis-stimulating activity of Factor 1 and Factor 2, respectively. Antibodies or fragments thereof that inhibit the angiogenesis-stimulating activity of Factor 1 and Factor 2, respectively, are particularly preferred. The inventors have succeeded in providing examples of such inhibitory antibodies by generating antibodies that specifically bind to surface-exposed domains of Factor 1 and Factor 2. In a preferred embodiment of this aspect, the present invention relates to ligands capable of specifically binding to these fragments of Factor 1 and Factor 2, respectively. In the context of this aspect of the invention, the definitions set forth in the General Definitions section and the specific definitions in the context of the sixth aspect of the invention apply equally.

[0137] Thus, in a preferred embodiment, the present invention relates to a ligand, preferably an antibody or fragment thereof, or an antibody-like protein, that specifically binds to an epitope of human Factor 1 protein contained in or consisting of amino acids 61 to 76 of SEQ ID NO: 1, or to a region of another Factor 1 protein that corresponds to this epitope.

[0138] In a further preferred embodiment, the present invention relates to a ligand, preferably an antibody or fragment thereof, or an antibody-like protein, that specifically binds to an epitope of human Factor 2 protein contained in or consisting of amino acids 181 to 195 of SEQ ID NO: 3, or to a region of another Factor 2 protein that corresponds to this epitope.

[0139] In the context of the above-described preferred aspects of the present invention, the term "region of another Factor 1 or 2 protein corresponding to this epitope" refers to an amino acid sequence from another Factor 1 or 2 protein that aligns with the indicated amino acid sequence of Factor 1 or 2 using standard alignment means, such as ClustalW and the standard parameters described above. Figures 6-11 show various such alignments of Factor 1 and 2 proteins. One skilled in the art can readily identify a fragment of SEQ ID NO: 2 having the amino acid sequence CTIWRPQGKSYLYFTQ (SEQ ID NO: 38) and determine the corresponding amino acid in another Factor 1 protein. (Example)

[0140] Examples are provided to further illustrate and facilitate a better understanding of the present invention and are not intended to limit the scope of the invention in any way. Example 1

[0141] One of the present inventors tested the effects of intracoronary infusion of autologous bone marrow cells in patients with AMI in a multicenter, placebo-controlled clinical trial (BOOST-2, controlled clinical trial identification number ISRCTN17457407). In this clinical trial, bone marrow aspirates were obtained from patients with AMI for research purposes. CXCR4+ bone marrow cells were isolated by magnetic cell sorting (MiniMACS®, Miltenyi Biotec). After two subsequent purification steps, a CXCR4+ enriched cell population was obtained (purity >95% confirmed by flow cytometry). RNA was then isolated from these cells and used for microarray analysis (Affymetrix GeneChip HG_U133 Plus 2.0). In subsequent bioinformatic analysis, 4000 expressed sequence tags (ESTs) most strongly expressed by CXCR4+ bone marrow cells in the microarray were examined. Using a series of bioinformatic tools, we identified putative secreted factors from these ESTs, characterized by the absence of an N-terminal signal peptide, a mitochondrial or nuclear signal peptide, an endoplasmic reticulum retention sequence, and a transmembrane domain. A total of 283 putative secreted factors were identified, 117 of which were found to have mouse homologs in NCBI Blast analysis. cDNAs for the human homologs were cloned into expression plasmids and then individually transfected into human embryonic kidney (HEK) cells. Transfected HEK cells were cultured in serum-free medium for 30 hours, after which conditioned culture supernatants were obtained. The conditioned HEK cell supernatants were individually tested for their angiogenic stimulatory effects in a microangiogenesis assay and their cytoprotective effects in a cardiomyocyte death assay. This screening resulted in the identification of two secreted proteins: "Factor 1" showed angiogenic and cytoprotective effects in the above assays; and "Factor 2" showed angiogenic stimulatory effects in the above assays.

[0142] The sequences identified in the screen and their respective mouse or human homologs are as follows: Factor 1: Human Factor 1 was identified in the screen and used in Example 2 and Figure 1: Homo sapiens chromosome 19 open reading frame 10 (C19orf10) The nucleic acid sequence encoding human Factor 1 is available as NCBI reference sequence: NM_019107.3 (SEQ ID NO: 6). The amino acid sequence of human Factor 1 is shown in Figure 6 (SEQ ID NO: 2).

[0143] The mouse homologues were used in Examples 3-6 and Figures 2-5: Mus musculus (mouse) DNA fragment, Chr17, Wayne State University 104 (expression) (D17Wsu104e) The nucleic acid sequence encoding mouse factor 1 is available as NCBI reference sequence: NM_080837.2 (SEQ ID NO: 7). The amino acid sequence of mouse factor 1 is shown in Figure 6 (SEQ ID NO: 13).

[0144] Factor 2: A secreted form of human Factor 2 was identified in the screen and used in Example 2 and Figure 1: Homo sapiens chromosome 19 open reading frame 63 (C19orf63), transcript variant HSS1. The nucleic acid sequence encoding human Factor 2 is available as NCBI reference sequence: NM_175063.4 (SEQ ID NO: 8). The amino acid sequence of the secreted form of human Factor 2 is shown in Figure 8 (SEQ ID NO: 4).

[0145] Transmembrane human factor 2: Homo sapiens chromosome 19 open reading frame 63 (C19orf63), transcript variant HSM1. The nucleic acid sequence encoding the transmembrane form of human factor 2 is available as NCBI reference sequence: NM_206538.2 (SEQ ID NO: 9). The amino acid sequence of the transmembrane form of human factor 2 is shown in Figure 10 (SEQ ID NO: 5).

[0146] The amino acid sequence of the transmembrane variant of human Factor 2 is available at GenBank: AY358710.1 (SEQ ID NO: 34).

[0147] The secreted murine homologues were used in Examples 4-6 and Figures 3-5: Mus musculus hematopoietic signal peptide-containing secreted 1 (2310044H10Rik) mRNA, complete cds, or spliced. The nucleic acid sequence encoding the transmembrane form of mouse factor 2 is available at GenBank: AY761096.1 (SEQ ID NO: 10). The amino acid sequence of the secreted form of mouse factor 2 is shown in Figure 8 (SEQ ID NO: 24).

[0148] The transmembrane mouse homologue: Mus musculus RIKEN cDNA 2310044H10 gene (2310044H10Rik). The nucleic acid sequence encoding the transmembrane form of mouse factor 2 is available as NCBI reference sequence: NM_197991.2 (SEQ ID NO: 11). The amino acid sequence of the transmembrane form of mouse factor 2 is shown in Figure 10 (SEQ ID NO: 29). Example 2

[0149] To confirm the angiogenic stimulatory activity observed in the screening, both factors (human homologs encoded by the nucleic acid sequences shown in SEQ ID NOS: 6 and 8) were produced as His-tagged recombinant proteins in COS7 cells. As shown in Figure 1, recombinant Factor 1 and Factor 2 promoted angiogenic stimulatory activity in cultured human endothelial cells in a dose-dependent manner. Human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) were purchased from Provitro (Berlin, Germany). Cells were cultured for 24 hours in minimal medium containing 10% fetal calf serum (FCS), human recombinant VEGF-A (R&D Systems), or various concentrations of the indicated recombinant human Factor 1 (SEQ ID NOS: 2) or Factor 2 (SEQ ID NOS: 4) in the presence or absence (control). (A) HCAEC proliferation was measured by bromodeoxyuridine incorporation. (B) HCAEC migration was assessed after wounding a confluent endothelial cell monolayer with a pipette tip. (C) HUVEC network formation was assessed in cells cultured in growth factor-reduced Matrigel. N=3-5 independent experiments / conditions; *P<0.05, **P<0.01, ***P<0.001 vs. control (see Figure 1). Example 3

[0150] To confirm the cardioprotective effects of Factor 1 observed in the screening study, neonatal rat ventricular cardiomyocytes were subjected to simulated ischemia-reperfusion injury in the presence or absence of recombinant Factor 1. Factor 1 (the murine homolog encoded by the nucleic acid sequence shown in SEQ ID NO: 7) was produced as a His-tagged recombinant protein in COS7 cells. As shown in Figure 2, recombinant Factor 1 promoted dose-dependent anti-apoptotic effects in cultured cardiomyocytes. Ventricular cardiomyocytes were isolated from 1- to 3-day-old Sprague-Dawley rats by Percoll density gradient centrifugation. Cardiomyocytes were subjected to 180 minutes of simulated ischemia (glucose-free medium containing 2-deoxyglucose in a 5% CO2 / 95% N2 atmosphere) and then subjected to 60 minutes of simulated reperfusion (returned to glucose-containing medium in 5% CO2 / 95% room air) in the presence or absence (control) of recombinant human GDF-15 (R&D Systems, a known anti-apoptotic cytokine) or various concentrations of the indicated recombinant mouse Factor 1. Cell death was assessed by in situ TdT-mediated dUTP nick end labeling (TUNEL). N=3 independent experiments / condition; *P<0.05 vs. control. Example 4

[0151] To investigate the therapeutic potential of Factor 1 and Factor 2 in the setting of AMI, adenoviruses encoding the murine homologs of Factor 1 or Factor 2 (nucleic acid sequences shown in SEQ ID NOS: 7 and 10) were generated and tested in a mouse model of AMI. Adenoviral expression of both factors resulted in improved left ventricular systolic function 28 days after infarction. This was associated with increased capillary density in the border zone infarct (Figure 3).

[0152] Mouse factor 1 or factor 2 cDNA was cloned into replication-deficient adenovirus using the AdEasy XL vector system (Stratagene). A replication-deficient adenovirus encoding galactosidase (lacZ) was used as a control. Virus was purified using the Adeno X virus purification kit (BD Biosciences). Male C57BL / 6 mice aged 10–12 weeks were anesthetized and ventilated with isoflurane (1–2%), followed by permanent left anterior descending coronary artery (LAD) ligation. Virus (5x10 9 pfu) was injected into the left ventricular (LV) cavity immediately after LAD ligation. (A) Left ventricular systolic function (fractional area change, FAC) was assessed by transthoracic echocardiography (Visualsonics) 28 days after LAD ligation (N = 10–12 mice / group). (B) Isolectin-positive capillary density in the border zone infarct was assessed by fluorescence microscopy 28 days after LAD ligation (N = 3 mice / group). *P < 0.05, **P < 0.01 vs. Ad.lacZ control. Example 5

[0153] To investigate the therapeutic potential of Factor 1 and Factor 2 as recombinant proteins in the setting of reperfused AMI (which closely resembles the clinical situation of AMI patients undergoing reperfusion therapy), 10- to 12-week-old male C57BL / 6 mice underwent 1 hour of coronary artery ligation (ischemia) followed by 28 days of reperfusion. Mice were treated sc with both factors for the first 7 days after reperfusion. Factor 1 and Factor 2 (murine homologs; amino acid sequences corresponding to SEQ ID NOS: 13 and 24) were produced as His-tagged recombinant proteins in HEK293 cells. Treatment with recombinant Factor 1 or Factor 2 resulted in a significant improvement in left ventricular systolic function 28 days after infarction. This was associated with a significant increase in capillary density in the border zone of the infarct (Figure 4). Male C57BL / 6 mice, 10- to 12-week-old, were anesthetized and ventilated with isoflurane (1-2%) and underwent temporary left anterior descending coronary artery ligation for 1 hour, followed by 28 days of reperfusion. Mice received a single sc injection of recombinant Factor 1 or Factor 2 (10 μg each) at the time of reperfusion (control mice were injected with PBS). Recombinant Factor 1 or Factor 2 was then continuously infused sc (10 μg / day) for 7 days using an Alzet minipump. Control mice were infused with PBS. (A) Left ventricular systolic function (fractional area change, FAC) was assessed by transthoracic echocardiography 28 days after reperfusion (N = 10-13 mice / group). (B) Isolectin-positive capillary density in the border zone infarct was assessed by fluorescence microscopy 28 days after reperfusion (N = 6 mice / group). *P < 0.05, **P < 0.01 vs. PBS control. Example 6

[0154] To determine whether Factor 1 and Factor 2, when used as recombinant proteins, could increase survival after reperfused AMI, mice underwent coronary artery ligation for 1 hour and then reperfused for 28 days. Mice were injected sc with both factors for the first 7 days after reperfusion. Factor 1 and Factor 2 (murine homologs; SEQ ID NOS: 7 and 10) were produced as His-tagged recombinant proteins in HEK293 cells. Treatment with recombinant Factor 1 or recombinant Factor 2 significantly improved survival during the first 28 days after occlusion (Figure 5). Male C57BL / 6 mice, 10–12 weeks old, were anesthetized and ventilated with isoflurane (1–2%), underwent temporary left anterior descending coronary artery ligation for 1 hour, and then reperfused for 28 days. Mice received a single sc injection of recombinant Factor 1 or Factor 2 (10 μg each) at the time of reperfusion (control mice were injected with PBS). Recombinant Factor 1 or Factor 2 were then continuously infused sc for 7 days (10 μg / day) using an Alzet minipump. Control mice were infused with PBS. Mice were examined daily for 28 days to assess postinfarction survival. N = 29 PBS-treated mice; N = 25 Factor 1-treated mice; N = 15 Factor 2-treated mice. Example 7

[0155] Homologous sequences to the protein encoded by human C19Orf10 were identified with the BLASTP algorithm at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. SEQ ID NO: 2 was used as the matrix. The parameters used were the default parameters: 11 extension: 1, composition adjustment = condition composition score matrix adjustment together with a database of non-redundant protein sequences (nr). From the sequences identified, examples were selected from various vertebrates, primarily mammals, and one sequence from each of amphibians, birds, and fish. Each sequence is listed in Table 1. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm (http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html). The default parameters were used: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0, 20, gap spacing = 5, no end gap = none, and output options: format = Aln w / numbers, order = aligned. The multiple alignments obtained for all sequences are shown in Figure 6 and for the mammalian sequences in Figure 7.

[0156] [Table 3] Example 8

[0157] Homologous sequences to the protein encoded by the human C19Orf63 splice variant HSS1 were searched with the BLASTP algorithm at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. SEQ ID NO: 4 was used as the matrix. The parameters used were the default: 11 extension: 1, composition adjustment = condition composition score matrix adjustment with a database of non-redundant protein sequences (nr). From the sequences identified, examples from various vertebrate species were selected, primarily mammals, with one sequence each from amphibians and fish. Each sequence is shown in Table 2. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html, using the default parameters: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0, 20, gap spacing = 5, no end gap = none, and output options: format = Aln w / numbers, order = aligned. The multiple alignments obtained for all sequences are shown in FIG. 8 and for the mammalian sequences in FIG.

[0158] [Table 4] Example 9

[0159] Homologous sequences to the protein encoded by the human C19Orf63 splice variant HSM1 were searched with the BLASTP algorithm at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. SEQ ID NO: 5 was used as the matrix.

[0160] The parameters used were the default: 11 extension: 1, composition adjustment = condition composition score matrix adjustment with a database of non-redundant protein sequences (nr). From the sequences identified, examples from various vertebrate species were selected, primarily mammals, with one sequence each from amphibians and fish. Each sequence is listed in Table 3. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm (http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html). The default parameters were used: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0, 20, gap spacing = 5, no end gap = none, and output options: format = Aln w / numbers, order = aligned. The multiple alignments obtained for all sequences are shown in Figure 10 and for the mammalian sequences in Figure 11.

[0161] [Table 5] Example 10

[0162] Human coronary artery endothelial cells (HCAECs, Provitro) were grown in EGM-2 medium (Lonza) supplemented with 10% FCS (Biochrom) in a T75 flask. Cells from passages 3 to 6 were used. Before stimulation with various drugs, the cells were cultured overnight in MCDB131 (Life Technologies) containing 2% FCS. HCAECs were then plated in a 96-well plate (5x10 3Cells were seeded onto 1000-well plates and stimulated with recombinant human Factor 1, recombinant human Factor 2, or VEGF (positive control) for 16 hours in the presence or absence of various concentrations of rabbit anti-Factor 1 antibody, rabbit anti-Factor 2 antibody, or control IgG. Antibodies were produced by Eurogentec and raised against polypeptides contained in human Factor 1 (CTIWRPQGKSYLYFTQ, SEQ ID NO:38, i.e., amino acids 61-76 of SEQ ID NO:1) or Factor 2 (CEQAQKAKNPQEQKSF; SEQ ID NO:39, i.e., amino acids 181-195 of SEQ ID NO:3 plus an N-terminal Cys). Cell proliferation was measured by a colorimetric BrdU incorporation immunoassay (Roche). Data are shown in Figure 12 (Panel A: Factor 1; Panel B: Factor 2; data are the mean ± SEM of 3-6 experiments).

Claims

1. A protein comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof or a variant having at least 80% sequence identity with SEQ ID NO: 1 for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissues or cells.

2. A protein comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof or a variant having at least 80% sequence identity with SEQ ID NO: 3 for use in enhancing the growth and / or curing non-transformed tissue or cells.

3. The protein of claim 2, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 4 or SEQ ID NO:

5.

4. 5. The protein of any one of claims 1 to 4, wherein the non-transformed tissue or cell is healthy, diseased or damaged.

5. The cell or tissue is (i) a muscle tissue cell, a connective tissue cell, an epithelial tissue cell, or a nervous tissue cell, or muscle tissue, connective tissue, epithelial tissue, or nervous tissue; and / or (ii) belonging to or derived from the circulatory system, digestive system, endocrine system, excretory system, immune system, integumentary system, muscular system, nervous system, reproductive system, respiratory system, or skeletal system; and / or (iii) A protein according to any one of claims 1 to 5, which belongs to or is derived from the heart, skin, bone, cartilage, blood vessels, esophagus, stomach, intestines, glands, liver, kidney, lung, brain, and spleen.

6. The protein of claim 5, wherein the damage results from a genetic / hereditary disease or an acquired disease resulting from ischemia, reperfusion injury, inflammation, infection, trauma, mechanical stress, poisoning, or surgery, preferably the resulting disease is selected from the group consisting of myocardial infarction, angina pectoris, and heart failure.

7. 7. The protein of claim 5 or 6, wherein the disease is associated with atrophy, degeneration, inflammation, injury, or wounding.

8. 8. The protein according to claim 6 or 7, wherein the disease is primary, preferably hereditary cardiomyopathies and cardiomyopathies caused by spontaneous mutations, or acquired cardiomyopathies, preferably ischemic cardiomyopathies caused by atherosclerosis or other coronary artery diseases, cardiomyopathies caused by infection or poisoning of the myocardium, hypertensive heart diseases caused by pulmonary arterial hypertension and / or arterial hypertension, and heart valve diseases.

9. A nucleic acid encoding a protein according to any one of claims 1 to 8 for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissues or cells.

10. 10. A vector comprising the nucleic acid of claim 9 for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells.

11. The vector of claim 10, wherein the vector is selected from the group consisting of: a plasmid vector; a cosmid vector; a phage vector, such as a lambda phage, a filamentous phage vector; a viral vector, preferably an adenoviral vector, an adeno-associated viral (AAV) vector, an alphavirus vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, a vesicular stomatitis virus vector, a retroviral vector and a lentiviral vector; a virus-like particle; and a bacterial spore.

12. A pharmaceutical composition comprising a protein according to any one of claims 1 to 8, a nucleic acid according to claim 9, or a vector according to claim 10 or 11, and optionally a suitable pharmaceutical excipient, for use in enhancing proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or cells.

13. 13. The pharmaceutical composition of claim 12, which is administered by oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronary route.

14. 14. The pharmaceutical composition according to claim 12 or 13, which is administered before, after or simultaneously with reperfusion therapy.

15. 15. The pharmaceutical composition of claim 14, wherein said administration is by one or more bolus injections and / or infusions.

16. Inhibitors of factor 1 and / or 2 proteins for medical use.

17. 17. The inhibitor of factors 1 and / or 2 according to claim 16, wherein the medical use is the treatment or prevention of a disease in which angiogenesis contributes to the onset or progression of the disease.

18. The inhibitor described in claim 17, wherein the Factor 1 protein comprises the amino acid sequence shown in SEQ ID NO: 1 and the Factor 2 protein comprises the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3.

19. The inhibitor described in claim 17 or 18, which is selected from the group consisting of a protein comprising an inhibitory fragment of SEQ ID NO: 1 or 3 or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3; a ligand that specifically binds to the amino acid shown in SEQ ID NO: 1 or 3 or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3; and a nucleic acid that inhibits or prevents the transcription and / or translation of mRNA encoding a protein comprising the amino acid shown in SEQ ID NO: 1 or 3 or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3.

20. 20. The inhibitor of claim 19, wherein the ligand is selected from the group consisting of an antibody or fragment thereof, an antibody-like protein, or a peptidomimetic.

21. A nucleic acid encoding the inhibitor of any one of claims 17 to 20 for use in treating or preventing a disease in which angiogenesis contributes to the onset or progression of the disease.

22. A vector comprising the nucleic acid of claim 22 for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease.

23. A pharmaceutical composition comprising an inhibitor described in any one of claims 17 to 20, a nucleic acid described in claim 21, or a vector described in claim 22, and optionally a suitable pharmaceutical excipient, for use in treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease.

24. The inhibitor according to any one of claims 17 to 20, the nucleic acid according to claim 21, the vector according to claim 22, and the pharmaceutical composition according to claim 23, wherein the disease in which angiogenesis contributes to the onset or progression of the disease is a proliferative disease.

25. The proliferative diseases include benign tumors, malignant tumors, rheumatoid arthritis, psoriasis, ocular neovascular diseases, Osier-Webber syndrome, plaque angiogenesis, restenosis after transplantation and angioplasty, telangiectasia, hemophilic arthropathy, angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, scleroderma, hypertrophic scars, cat scratch disease, and ulcers, particularly macular degeneration, adrenocortical carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, uterine cancer, and the like. The inhibitor of any one of claims 17 to 20, the nucleic acid of claim 21, the vector of claim 22, or the pharmaceutical composition of claim 23, which is selected from the group consisting of cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, head and neck cancer, laryngeal cancer, liver cancer, lung cancer, melanoma, myeloproliferative disorders, cervical cancer, non-melanoma skin cancer, ovarian cancer, prostate cancer, benign prostatic hyperplasia, pancreatic cancer, rectal cancer, and testicular cancer.

26. A ligand that specifically binds to factor 1 or factor 2 and inhibits the angiogenesis-stimulating activity of factor 1 or factor 2.

27. 27. The ligand of claim 26, which specifically binds to an epitope of human Factor 1 protein contained in or consisting of amino acids 61 to 76 of SEQ ID NO: 1, or a region of another Factor 1 protein corresponding to this epitope.

28. 27. The ligand of claim 26, which specifically binds to an epitope of human Factor 2 protein contained in or consisting of amino acids 181 to 195 of SEQ ID NO: 3, or a region of another Factor 2 protein corresponding to this epitope.

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