Factor 1 protein and factor 2 protein and inhibitors thereof for use in treatment or prevention of diseases

Abstract

To provide novel drugs for diseases such as acute myocardial infarction capable of direct repair of heart muscle without using autologous myeloid cells.SOLUTION: Disclosed is the use of a protein containing a specific amino acid sequence, fragment or variant thereof for producing a pharmaceutical to enhance the proliferation and / or to heal and / or to inhibit apoptosis of non-transformed tissue or non-transformed cell, where the fragment or variant of the protein of the specific amino acid sequence has at least 90% sequence identity to the specific amino acid sequence, and exhibits anti-apoptotic potential to protect cells or tissues from apoptotic cell death where the anti-apoptotic potential is at least 50% of that of the protein of the specific amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Acute myocardial infarction (AMI) is a major cause of worldwide morbidity and mortality. In Germany alone, the incidence is approximately 280,000 cases / year. Treatment of patients with AMI includes a combination of reperfusion therapy to open the coronary artery occlusion and administration of platelet aggregation inhibitors and coagulation inhibitors to prevent reocclusion of the blood vessels. Furthermore, the pulse and blood pressure can be lowered by administration of β-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. There is a need for drugs that have a similar effect without using bone marrow cells.

[0003] The tissue necrosis that persists during AMI triggers a wound healing response in which the necrotic area is replaced by granulation tissue and ultimately a collagen-rich scar. Monocytes are replenished from the bone marrow to the infarcted myocardium and play an important role during wound healing after AMI. The response of monocytes in the myocardium is temporally biphasic. Proinflammatory monocytes appear earlier and promote the digestion of infarcted tissue and the removal of necrotic debris, while reparative monocytes become dominant later and bring about angiogenesis and repair. Cell surface expression of the chemokine receptor CXCR4 identifies a subset of reparative monocytes in mice and humans. The angiogenesis-stimulating and healing-promoting effects of CXCR4+ bone marrow cells are thought to be mediated by secreted proteins acting in a paracrine manner, but the identity of these factors is mostly unknown. Therefore, the inventors performed bioinformatics secretome analysis using human CXCR4+ bone marrow cells to identify novel secretory proteins that demonstrate therapeutic potential after AMI and control infarct healing.

[0004] These tests identified two distinct polypeptides that exhibit angiogenic stimulating and / or cytoprotective effects, which the inventors named Factor 1 and Factor 2 proteins.

[0005] Both factors have been described in various scientific publications that refer to a biological context that does not include the angiogenic stimulating and / or cytoprotective effects of these factors in non-transformed cells or tissues. None of the studies have disclosed any evidence or hint of a significant correlation between the function of these factors or any disease or condition associated solely with non-transformed cells or tissues and any one of these factors.

[0006] The amino acid sequence of human factor 1 is encoded in the open reading frame 10 of human chromosome 19 (C19Orf10). This protein was described in 2007 as a novel secretory factor in the synovium in proteomic analysis of so-called fibroblast-like synovial cells (FLS- cells). The correlation between the secretion of this protein and inflammatory joint diseases has been hypothesized 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 joint treatment and for the diagnosis and monitoring of tissue changes in tissues undergoing proliferative changes (US 2008 / 0004232 A1, Characterization of c19orf10, a novel synovial protein). Another scientific publication describes enhanced secretion of this protein in hepatocellular carcinoma cells (Sunagozaka et al., International Journal of Cancer, 2010, Identification of a secretory protein c19orf10 activated in hepatocellular carcinoma). Recombinant-produced proteins showed a proliferation-enhancing effect on cultured hepatocellular carcinoma cells. It is noteworthy that C19Orf10 was initially thought to be an interleukin and was therefore also called IL-25, IL-27, and IL-27W. However, the terms "IL-25" and "IL-27" have been used inconsistently in the field and have been used to refer to various different proteins. For example, US 2004 / 0185049 refers to a certain protein as IL-27 and discloses its use in the regulation of immune responses. This protein is structurally different from factor 1 (compare the factor 1 amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence of "IL-27" according to UniProt:Q8NEV9).Similarly, EP 2 130 547 A1 refers to a certain protein as IL-25 and discloses its use in the treatment of inflammation. This protein is also known in the field as IL-17E and 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 in open reading frame 63 of human chromosome 19 (C19Orf63). This protein was described in 2009 as a novel secreted factor, INM02 (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 this protein was demonstrated in human serum using a polyclonal antibody. Furthermore, correlations were shown between cultured MIN6 (β-cells) and isolated rat pancreatic eyelets and glucose concentrations in the culture medium. Correlation analysis between diabetes and INM2 expression did not show a significant difference. The corresponding patent application claims the production of a polyclonal antibody against this 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 signaling peptide-containing secretion 1) (Junes-Gill et al., J Neurooncol, 2011, hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33). The published data shows hHSS1 expression in hematopoietic stem cells and suggests its function as a tumor suppressor in the development of certain brain tumors (gliomas). The 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). [Overview of the project] [Problems that the invention aims to solve]

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

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

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

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

[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 pharmaceutically acceptable excipient, for use in enhancing the proliferation of non-transformed tissue or non-transformed cells and / or in promoting healing and / or inhibiting apoptosis.

[0014] In the 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 is a contributing factor to the onset or progression of the disease.

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

[0016] In the eighth aspect, the present invention provides a vector comprising a nucleic acid from the seventh aspect that encodes such an inhibitor for treating or preventing diseases in which angiogenesis is a contributing factor to the onset or progression of the disease.

[0017] In the 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 pharmaceutically acceptable excipient for use in the treatment or prevention of diseases in which angiogenesis is a contributing factor to the onset or progression of the disease. The above abstract does not necessarily describe all aspects of the present invention. [Brief explanation of the drawing]

[0018] [Figure 1] Human coronary artery endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were cultured for 24 hours in minimal medium in the absence (control) or presence (control) 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 shown. (A) HCAEC proliferation was measured by bromodeoxyuridine uptake. (B) HCAEC migration was evaluated after a monolayer of confluent endothelial cells was injured with a pipette tip. (C) HUVEC network formation was evaluated in cells cultured on growth factor-reduced Matrigel. N=3–5. Independent experiments / conditions; *P<0.05, **P<0.01, ***P<0.001 vs. control. [Figure 2] Ventricular cardiomyocytes were isolated from 1-3 day old Sprague-Dawley rats by Percoll density gradient centrifugation. Cardiomyocytes were exposed to simulated ischemia (2-deoxyglucose-containing glucose-free medium in a 5% CO2 / 95% N2 atmosphere) for 180 hours, followed by simulated reperfusion (returning to glucose-containing medium in a 5% CO2 / 95% room air atmosphere) in the absence (control) or presence of recombinant human GDF-15 or various concentrations of recombinant mouse factor 1 (amino acid sequence shown in SEQ ID NO: 13), either in the absence of the indicated recombinant human GDF-15 or in the presence of recombinant mouse factor 1 (amino acid sequence shown in SEQ ID NO: 13), followed by 60 minutes of simulated reperfusion (returning to glucose-containing medium in a 5% CO2 / 95% room air). Cell death was assessed by in situ TdT-mediated dUTP nick-end labeling (TUNEL). N=3 independent experiments / conditions; *P<0.05 vs control. [Figure 3] Mouse factor 1 or factor 2 cDNAs (nucleic acids shown in SEQ ID NOs: 7 and 10) were cloned into replication-deficient adenoviruses. A replication-deficient adenovirus encoding galactosidase (lacZ) was used as a control. Male C57BL / 6 mice aged 10–12 weeks were anesthetized and ventilated with isoflurane, followed by permanent left anterior descending coronary artery (LAD) ligation. The virus was injected into the left ventricular (LV) cavity immediately after LAD ligation. (A) Left ventricular systolic function (differential area change, FAC) was evaluated by transthoracic echocardiography 28 days after LAD ligation. (B) Isolectin-positive capillary density in borderline infarcts 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 aged 10–12 weeks were anesthetized and ventilated with isoflurane, temporarily ligated the left anterior descending coronary artery for one hour, and then reperfused for 28 days. At the time of reperfusion, the mice received a single sc injection of recombinant mouse factor 1 (amino acid sequence shown in SEQ ID NO: 13) or factor 2 (amino acid sequence shown in SEQ ID NO: 24). Recombinant factor 1 or factor 2 was then continuously infused via sc for 7 days. Control mice were injected with PBS. (A) Left ventricular systolic function (differential area change, FAC) was evaluated by transthoracic echocardiography 28 days post-reperfusion. (B) Isolectin-positive capillary density in borderline infarcts was quantified by fluorescence microscopy 28 days post-reperfusion. *P<0.05, **P<0.01 vs. PBS control. [Figure 5] Male C57BL / 6 mice aged 10–12 weeks were anesthetized and ventilated with isoflurane (1–2%), temporarily ligated the left anterior descending coronary artery for 1 hour, and then reperfused for 28 days. At the time of reperfusion, mice were given a single sc injection of recombinant mouse factor 1 or factor 2 (sequence numbers 13 and 24, respectively) (control mice were injected with PBS). Recombinant factor 1 or factor 2 was then continuously infused with sc for 7 days. Control mice were injected with PBS. Mice were examined daily for 28 days to assess post-infarction survival. [Figure 6] Sequence homologs to proteins encoded by human C19Orf10 were investigated using the BLASTP algorithm. From the identified sequences, examples were selected from various vertebrate species, mainly mammals, and one each from amphibians, birds, and fish. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm. All aligned interspecies identities were marked with "*", highly conserved amino acid positions (i.e., only conserved substitutions) were marked with ":", and highly conserved amino acid positions were marked with ".". [Figure 7] Sequence homologs to proteins encoded by human C19Orf10 were investigated using the BLASTP algorithm. From the identified sequences, examples were selected from various vertebrate species, mainly mammals, and one each from amphibians, birds, and fish. The amino acid sequences of the selected mammals were aligned using the CLUSTALW2 algorithm. All aligned interspecies identities were marked with "*", highly conserved amino acid positions (i.e., only indicating conserved substitutions) were marked with ":", and highly conserved amino acid positions were marked with ".". [Figure 8] The sequence homolog HSS1 of the protein encoded by the human C19Orf63 splice mutant was investigated using the BLASTP algorithm. From the identified sequences, one sequence was selected from various vertebrate species, mainly mammals, as well as amphibians and birds. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm. All aligned interspecies identities were marked with "*", highly conserved amino acid positions (i.e., only conserved substitutions) were marked with ":", and highly conserved amino acid positions were marked with ".". [Figure 9] The sequence homologs with the protein encoded by the human C19Orf63 splice variant HSS1 were examined by the BLASTP algorithm. From the identified sequences, examples from various vertebrate species, mainly mammals, and one sequence from amphibian and fish species were selected. The selected mammalian amino acid sequences were aligned using the CLUSTALW2 algorithm. The identity between all aligned species was marked with "*", the almost highly conserved amino acid positions (i.e., those showing only conservative substitutions) were marked with ":", and the highly conserved amino acid positions were marked with ".

Figure 10

Figure 11

Figure 12

[0019] (Detailed Description of the Invention) Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein and may vary. The terms used herein are for the purpose of describing particular embodiments only and it should be understood that the scope of the present invention is not limited thereby, but 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] (Definitions) Preferably, the terms used herein are defined as described 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] To carry out the present invention, unless otherwise specified, the conventional chemical, biochemical, cell biological methods and recombinant DNA techniques described in the literature of the art are used (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, the conventional clinical cardiovascular methods described in the literature of the art are used (see, for example, Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, 9th edition, edited by P. Libby et al, Saunders Elsevier Philadelphia, 2011).

[0022] Throughout this specification and the claims, unless the context requires otherwise, the term “comprise” (“comprises” and its variations “comprising”) is understood to mean “comprise” the integer or process or set of integers or processes indicated, but not to exclude any other integer or process or set of integers or processes. The singular forms used herein and in the claims form “a,” “an,” and “the” and refer to the plural unless the context clearly requires otherwise.

[0023] Nucleic acid molecules are understood as polymeric macromolecules based on nucleotide monomers. Nucleic acid monomers consist of a nucleic acid base, a pentose sugar (e.g., ribose or 2'-deoxyribose, but not limited to these), and one to three phosphate groups. Typically, polynucleotides are formed by phosphodiester bonds between individual nucleotide monomers. In the context of this 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) represents a nucleotide sequence that can be translated into amino acids. Typically, such an ORF contains a starting codon, a trailing region usually consisting of several 3-nucleotide lengths, but no stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) within a given reading frame. Typically, ORFs occur naturally or are constructed artificially, i.e., by genetic means. ORFs encode proteins that, when translated, result in amino acids forming peptide chains.

[0025] The terms “protein” and “polypeptide” are used interchangeably herein and refer to any chain linked by peptide bonds, regardless of length or post-translational modifications. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein moieties, protein epitopes, and protein domains) can be further modified by chemical modification. Such chemically modified polypeptides contain other chemical groups besides the 20 native 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 result in one or more advantageous properties compared to non-polypeptides, such as enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to variants usable in the present invention include, but are not limited to, the following: pegylation; glycosylation of non-glycosylated parent polypeptides; covalent bonding with therapeutic small molecules such as glucagon-like peptide 1 agonists including exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretins, and liraglutide; or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications applicable to variants usable in the present invention may occur co-translation (at translation) or post-translation.

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

[0027] As used herein, the term “post-translational” refers to events that occur after the translation of a nucleotide triplet into amino acids and the formation of peptide bonds with subsequent amino acids in the sequence. Such post-translational events may occur after the formation of a complete polypeptide or may occur during the translation process in a portion of the polypeptide that has already been translated. Post-translational events typically alter or modify the chemical or structural properties of the resulting polypeptide. Examples of post-translational events include, but are not limited to, the glycosylation or phosphorylation of amino acids, or the cleavage of a peptide chain by endopeptidases, etc.

[0028] As used herein, the term “cotranslation (at translation)” refers to an event that occurs during the translation process of a nucleotide triplet into an amino acid chain. This event typically alters or modifies the chemical or structural properties of the resulting amino acid chain. Examples of cotranslation events, though not limited to those mentioned above, include events that can completely halt the translation process or prevent peptide bond formation, resulting in two separate translation products.

[0029] As used herein, the term “mutant” refers to a polypeptide that differs from the polypeptide or fragment from which a polypeptide is derived by having one or more changes in its amino acid sequence. The polypeptide from which a protein mutant is derived is also known as the parent peptide. Similarly, the fragment from which a protein fragment mutant is derived is known as the parent fragment. Typically, mutants are constructed artificially, preferably by genetic means. Typically, the parent polypeptide is the wild-type protein or wild-type protein domain. Furthermore, the mutants usable in the present invention are derived from homologs, orthologues, or paralogs of the parent polypeptide, or from artificially constructed mutants, and possess at least one of the biological activities of the parent polypeptide. Amino acid sequence changes can be amino acid substitutions, insertions, deletions, N-terminal cleavage, or C-terminal cleavage, or any combination thereof, which can occur at one or various sites. In a preferred embodiment, the mutants usable 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 cleavage, and / or C-terminal cleavage) in their amino acid sequence. The amino acid substitutions may be conserved and / or semi-conservative and / or non-conservative. In a preferred embodiment, the mutants usable in the present invention differ from the protein or domain from which it is induced in having 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 conserved amino acid changes.

[0030] Typical substitutions occur in 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 novel cysteine ​​remains a free thiol, the conversion from A, F, H, I, L, M, P, V, W, or Y to C is semi-conservative. Furthermore, those skilled in the art will recognize that glycine in sterically bulky positions should not be substituted, and P should not be introduced into portions of proteins having an α-helical or β-sheet structure.

[0033] Alternatively, the “mutant” as used herein can be characterized by a degree of sequence identity with respect to the parent polypeptide or parent polynucleotide from which it is induced. More precisely, in the context of the present invention, a protein mutant has at least 80% sequence identity with its parent polypeptide. Preferably, the polypeptide and reference polypeptide exhibit sequence identity shown over a continuous 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 shown over a continuous 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 for polypeptide and polynucleotide sequence comparisons. This expression preferably represents 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 for each reference polypeptide or each reference polynucleotide.

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

[0036] When comparing two sequences and calculating the sequence identity percentage, if a reference sequence is not specified, the sequence identity should be calculated based on the longer of the two sequences being compared, unless otherwise specified.

[0037] The similarity of nucleotide and amino acid sequences, such as the sequence identity percentage, can be determined by sequence alignment. Such alignment can be performed using various known algorithms, preferably the Karlin and Altschul mathematical algorithm (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). This can be done using Bioinformatics, 23, 2947-2948. (These are 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 when the parameters used are the default parameters described 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 Slow vs. Alignment option), and Protein weight matrix = Gonnet, Gap open = 10, Gap extension = 0,20, Gap distance = 5, No terminal gap = None, Output option: 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). Similar algorithms are incorporated into the BLASTN and BLASTP programs in Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST protein searches are performed using the BLASTP program, available, for example, at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. The preferred algorithm parameters to use are the default parameters listed at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome (expected threshold=10, word size=3, maximum match in query range=0, matrix=BLOSUM62, gap cost=present:11 extension:1, composition adjustment=conditional composition score matrix adjustment with a database of non-redundant protein sequences (nr) to obtain homologous amino acid sequences with factor 1 and factor 2 polypeptides).

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

[0040] As used herein, the term “host cell” refers to a cell having the nucleic acid (e.g., plasmid or virus) of the present invention. Such host cells may be prokaryotic cells (e.g., bacterial cells) or eukaryotic cells (e.g., cardiac muscle, plant, or animal cells). The cells may or may not be transformed. The cells may be, for example, a cell culture, a portion of tissue that can be isolated by itself, or isolated cells in a portion of a more complex tissue structure such as an organ or an individual.

[0041] The terms “Factor 1,” “Factor 1 protein,” or “Factor 1 polypeptide” are used interchangeably to refer to the protein (human homolog) shown in the NCBI reference sequence NM_019107.3, and its mammalian homolog (particularly mouse or rat-derived). The amino acid sequence of the human homolog is encoded in the open reading frame 10 (C19Orf10) of human chromosome 19. Preferably, the Factor 1 protein refers to a protein that contains, substantially consists of, or comprises the core portion of human Factor 1 having the amino acid sequence shown in Sequence ID No. 1. In a more preferred embodiment, the Factor 1 protein has the amino acid sequence shown in Sequence ID No. 2.

[0042] The terms “Factor 2,” “Factor 2 protein,” or “Factor 2 polypeptide” are used interchangeably to refer to the protein (human homolog) shown in NCBI reference sequence NM_175063.4 and its mammalian homologs (particularly mouse or rat-derived). The amino acid sequence of human factor 2 is encoded in open reading frame 63 (C19Orf63) of human chromosome 19. Preferably, the term “Factor 2 protein” refers to a protein that contains, substantially consists of, or comprises the core portion of human factor 2 having the amino acid sequence shown in Sequence ID No. 3. In a more preferred embodiment, 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 preferably a secreted form having the amino acid sequence shown in SEQ ID NO: 4.

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

[0044] The term “heal” 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 a damaged or necrotic area and replace it with new living tissue. This replacement may occur, for example, by regeneration, in which necrotic cells are replaced by new cells that form tissue similar to what was originally there, or by repair, in which damaged tissue is replaced with scar tissue. Since regeneration is a process that results in the partial or complete restoration of normal function, it is a preferred variation of the healing process. Therefore, in the context of the present invention, the term “heal” includes all processes and will be combined with this term by those skilled in the art, but preferably, the regeneration process should be promoted instead of a process that results in the generation of non-functional tissue, such as scar tissue. In the context of the present invention, the term “heal” preferably refers to the promotion of proliferation, migration, network formation, and angiogenesis.

[0045] The term "enhances proliferation" refers to an increase in the rate of cell division of cells or cell populations compared to cells or cell populations not treated with the protein, nucleic acid, vector, or pharmaceutical composition of the present invention. For example, methods for measuring the rate of cell division of cells by counting mitotic cells using FACS are well known in this field.

[0046] The term "inhibits apoptosis" refers to the ability of the protein, nucleic acid, vector, or pharmaceutical composition of the present invention to prevent cells or cell groups from undergoing apoptosis under conditions in which control cells or cell groups undergo apoptosis. Methods for measuring whether cells undergo apoptosis, for example, by the 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 this specification. These definitions and explanations are effective throughout this application unless otherwise specified.

[0047] (Appearance) The elements of the present invention are described below. These elements are described in conjunction with specific embodiments, but it should be understood that any number of them can be combined in any way to produce further embodiments. The various described examples and preferred embodiments should not be construed as being limited only to the embodiments explicitly described. This specification should be understood as supporting and including embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, it should be considered that any substitution and combination of all elements described herein is disclosed in this specification.

[0048] In a first aspect, the present invention provides a factor 1 protein or a fragment thereof having the amino acid sequence shown in SEQ ID NO: 1, or a variant having at least 80% sequence identity with SEQ ID NO: 1, for use in enhancing the proliferation and / or healing and / or inhibiting apoptosis of non-transformed tissue or non-transformed cells, particularly for use in enhancing the proliferation of non-transformed tissue, enhancing the 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 or a fragment thereof of SEQ ID NO: 1. 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 present 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. The preferred fragment of SEQ ID NO: 2 is the N-terminal signal sequence MAAPSGGWNGVGASLWAALLLGAVALRPAEA (Sequence ID 35)It lacks. Those skilled in the art can determine, without undue burden, which positions in the parent polypeptide can mutate 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 described exemplary 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 fully conserved between species, preferably mammals, i.e., one or more of the amino acid positions are mutated (not marked with "*"). In a more preferred embodiment, only amino acids that are neither fully conserved (indicated with "*") nor less conserved (indicated with ":" or ".") are altered. In a particularly preferred embodiment of the present invention, the Factor 1 protein comprises, substantially consists of, or comprises the amino acid sequence or fragment thereof of SEQ ID NO: 2. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2. Such mutations may be present in the full-length protein represented by SEQ ID NO: 2 or in proteins lacking the N-terminal signal sequence represented by SEQ ID NO: 1.

[0050] In addition to the N-terminal deletion signal, N-terminal deletion mutants may also have one or more amino acids deleted from the N-terminal conserved region, i.e., amino acid positions 32–55 (based on SEQ ID NO: 2), in addition to the N-terminal signal. Therefore, the N-terminus of the deletion factor 1 protein may also have one or more amino acids deleted from 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, or the deletion factor 1 protein may also have one or more amino acids deleted from the C-terminal conserved region, i.e., amino acid positions 146–173 (based on SEQ ID NO: 2). Therefore, the C-terminus of the deletion factor 1 protein may 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. The protein of the first aspect of the present invention may further include, for example, further amino acid sequences for stabilizing or purifying 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 preferable to stabilize the protein by mutating the protease cleavage sites within the protein of the first aspect of the present invention (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, a protein sequence can be submitted to a website that provides such analysis (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 / determines the following low-frequency (less than 10) cleavage sites:

[0052] [Table 2]

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

[0054] Factors 1 and 2 exhibited proliferation-enhancing activity, particularly in enhancing angiogenesis. In a preferred embodiment, the first-phase protein is used to enhance the proliferation of non-transformed tissue or non-transformed cells, preferably angiogenesis. Therefore, it is convenient to use Factor 1 or Factor 2 in the treatment of diseases in which enhanced angiogenesis may be beneficial. Further examples of such diseases are given below.

[0055] In the context of this invention, enhanced proliferation includes any degree of enhancement of cells or tissues compared to control cells or tissues not administered with the protein of the present invention. Cell proliferation can be measured, for example, by the uptake of bromodeoxyuridine 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 application.

[0056] In another preferred embodiment, the first phasor protein is used for healing non-transformed tissue or non-transformed cells. In another preferred embodiment, the first phasor protein is used for enhancing proliferation and healing non-transformed tissue or non-transformed cells. In a particularly preferred embodiment, the first phasor protein is used for enhancing proliferation and healing non-transformed tissue or non-transformed cells, and for inhibiting apoptosis.

[0057] In another preferred embodiment, the first-phase protein is used to inhibit apoptosis in non-transformed tissue or non-transformed cells. Thus, the protein of the present invention exhibits anti-apoptotic potential and protects cells or tissues from apoptotic cell death. In this context, “protect” or “cytoprotective effect” means that in cells treated with the factor 1 protein of the present invention, the degree of apoptotic cell death is reduced by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%, and most preferably at least 60% compared to the control. Those skilled in the art can evaluate cell death, for example, by in situ TdT-mediated dUTP nick-end labeling (TUNEL) as described in Example 3. Other indicators of apoptosis include 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 the peptide can be evaluated in vivo using experimental animal models of heart failure. For example, mice with post-ischemic systolic dysfunction can be treated with the protein, and the degree of apoptotic cardiomyocytes in the cardiac tissue of treated mice and control mice can be evaluated. The peptide can be administered parenterally, for example, intraperitoneally, intravenously, or subcutaneously. In a particularly preferred embodiment, the protein of the present invention exhibits two, preferably all, of the above functions: enhancement and healing of the proliferation of non-transformed tissue or non-transformed cells, and inhibition of apoptosis. Fragments and variants of the Factor 1 protein included in the present invention, which are at least 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90%, and more preferably at least 100% of a protein having the amino acid sequence shown 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 for use in enhancing the proliferation and / or healing of non-transformed tissues or non-transformed cells, comprising, substantially comprising, a fragment thereof, or a variant having at least 80% sequence identity with SEQ ID NO: 3, having the amino acid sequence shown in SEQ ID NO: 3. 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% 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 preserved, in order to preserve the function of the polypeptide. Such information can be obtained, for example, from homologous sequences that 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 protein in the second aspect of the present invention may further include, for example, a further amino acid sequence for stabilizing or purifying the obtained protein.

[0060] In one embodiment, it is preferable to stabilize the protein by mutating the protease cleavage site within the protein of the first aspect of the present invention. A suitable proteolytic cleavage site can be identified as described above.

[0061] In another preferred embodiment of the present invention, the protein of the second aspect of the present invention comprises, substantially comprises, the amino acid sequence of SEQ ID NO: 4, a fragment thereof, or a variant having at least 80% identity with SEQ ID NO: 4. A preferred fragment is the N-terminal signal sequence. MAAASAGATRLLLLLLMAVAA PSRARG' (Sequence ID 37)It lacks. 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 preserved, in order to preserve the function of the polypeptide. Such information can be obtained, for example, from homologous sequences that can be identified, aligned, and analyzed by bioinformatics methods well known in the art. Such analysis is described exemplary 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 mammals), i.e., one or more amino acid positions not marked with "*" are mutated. In a more preferred embodiment, only amino acids that are neither completely conserved (indicated with "*") nor less conserved (indicated with ":" or ".") are altered. In a particularly preferred embodiment of the present invention, the factor 2 protein comprises the amino acid sequence or fragment thereof of SEQ ID NO: 4. 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 may be present in the full-length protein shown in SEQ ID NO: 4, or in the protein lacking the N-terminal signal sequence. N-terminal deletion mutants may lose one or more amino acids from positions 27–73 (based on SEQ ID NO: 4), i.e., from the N-terminal conserved region, in addition to the N-terminal signal. Therefore, the N-terminus of the 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. Furthermore, the deletion factor 1 protein may also lack one or more amino acids from 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 Possible positions are 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, substantially comprises, or consists of the amino acid sequence of SEQ ID NO: 5, a fragment thereof, or a variant having at least 80% identity with SEQ ID NO: 5. The 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 preserved, in order to preserve the function of the polypeptide. Such information can be obtained, for example, from homologous sequences that can be identified, aligned, and analyzed by bioinformatics methods well known in the art. Such analysis is described exemplary 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 mammals), i.e., one or more amino acid positions not marked with "*" are mutated. In a more preferred embodiment, only amino acids that are neither completely conserved (indicated with "*") nor less conserved (indicated with ":" or ".") are altered. In a particularly preferred embodiment of the present invention, the factor 2 protein comprises the amino acid sequence or a fragment thereof of SEQ ID NO: 5. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with 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 that lacks the N-terminal signal sequence.

[0065] N-terminal deletion mutants may lack one or more amino acids from the N-terminal conserved region, i.e., amino acid positions 27–73 (based on SEQ ID NO: 4), in addition to the N-terminal signal. Furthermore, the N-terminus of the 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, the deletion factor 1 protein may lack one or more amino acids from the C-terminal conserved region, i.e., amino acid positions 190–262 (based on SEQ ID NO: 4). Therefore, 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, 22 Possible positions are 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 present invention, which are at least 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90%, and more preferably at least 100% of that of a protein having the amino acid sequence shown 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 second aspect protein is used to enhance the proliferation of non-transformed tissue or non-transformed cells. Enhancement of proliferation includes any grade of enhancement compared to control cells or tissues without protein administration. Experimental methods for measuring proliferation are described above. Each growth measurement for the second aspect protein of the present invention, evaluated by bromodeoxyuridine uptake, is also described in Example 2.

[0068] In another preferred embodiment, the second-faced protein is used for the healing of non-transformed tissue or non-transformed cells. In a particularly preferred embodiment, the second-faced protein of the present invention exhibits both of the above functions, namely, the enhancement of proliferation and healing of non-transformed tissue or non-transformed cells.

[0069] In another embodiment of the present invention, the proteins of phase 1 and / or 2 are administered in vivo, ex vivo, or in vitro, preferably in vivo. In a typical embodiment of administering the proteins of phase 1 and / or phase 2 of the present invention ex vivo or in vitro, proliferation is enhanced and / or healing is promoted and / or apoptosis is inhibited for tissue engineering to produce tissue for transplantation into an organism. The cells used for tissue engineering may be from the same organism, as well as from the same species or from a different organism of a different species. Methods for removing such cells or tissue and transplanting the new tissue into an organism are not included in the present invention.

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

[0071] In a preferred embodiment, the untransformed cells or untransformed tissue are pathological. In another preferred embodiment, the untransformed cells or untransformed tissue are damaged. In yet another preferred embodiment, the untransformed cells or untransformed tissue are damaged and pathological.

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

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

[0074] In another preferred embodiment, the untransformed cells or untransformed tissue belong to or originate from a specific part or organ of an individual's body selected from the group including skin, bone, heart, cartilage, blood vessels, esophagus, stomach, intestines, glands, liver, kidney, lungs, brain, and spleen. In a particularly preferred embodiment, the untransformed cells or untransformed tissue belong to or originate from the heart.

[0075] In the case of damaged non-transformed cells or non-transformed tissue, it is even more preferable that the damage results from a genetic / hereditary disorder or an acquired disorder resulting from, for example, ischemia, reperfusion injury, inflammation, infection, trauma, mechanical stress, poisoning, or surgery. In a particularly preferred embodiment, the damage results from ischemia. In another particularly preferred embodiment, the damage results from reperfusion injury.

[0076] In the context of the present invention, in the case of pathological or damaged cells or tissues, it is preferable that the damage arises from a disease associated with atrophy, degeneration, inflammation, injury, or wound. It is particularly preferable that the disease is associated with injury. It is also particularly preferable that the disease is associated with 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, nuclear myopathy, myotubular myopathy, nuclear myotubular myopathy, ophthalmoplegia, and mitochondrial myopathy. 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. Myositis may be selected from the group consisting of myositis ossificans, fibromyositis, idiopathic inflammatory myopathy (e.g., dermatomyositis, polymyositis, and inclusion body myositis), and suppurative myositis.

[0078] In another preferred embodiment of the present invention, the disease is primary or acquired cardiomyopathy. Primary cardiomyopathy is selected from hereditary cardiomyopathy and cardiomyopathy resulting from 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 endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, and obesity-related cardiomyopathy.

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

[0080] In the most preferred embodiment of the present invention, the non-transformed cells or non-transformed tissue 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. As used in the context of the present invention, the term "myocardial infarction" includes acute myocardial infarction (AMI).

[0081] In a particularly preferred embodiment, the use of the first or second phase protein of the present invention includes application to an individual after myocardial infarction, where healing includes improvement of left ventricular systolic function and may be associated with increased capillary density in borderline infarction. Furthermore, the first or second phase protein of the present invention may reduce post-myocardial infarction mortality. Methods that can be used to measure parameters such as improvement of left ventricular systolic function, increased capillary density in borderline infarction, and reduced post-myocardial infarction mortality are well known in the art and are typically described in Examples 4–6.

[0082] The Factor 1 and Factor 2 proteins, fragments, or variants described above are used to treat or improve atrophy; degeneration; inflammation, injury; wound, ischemia; reperfusion injury; inflammation; infection; trauma; mechanical stress; poisoning; primary or acquired cardiomyopathy, preferably hereditary cardiomyopathy and cardiomyopathy resulting from 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 endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; and in the improvement of myocardial infarction or left ventricular systolic function. The Factor 1 and Factor 2 proteins, fragments, or variants described above may also be used in the treatment of the conditions and diseases described, respectively.

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

[0084] The term “enhancing the proliferation of non-transformed tissue or non-transformed cells, and / or curing and / or inhibiting apoptosis” 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 nucleic acid sequences with enhanced expression in specific hosts (see, e.g., Donnelly et al., International Publication No. WO 97 / 47358). The ability of specific sequences to enhance expression in a particular host requires several empirical experiments. Such experiments involve measuring the expression of the expected nucleic acid sequence and, if necessary, altering that sequence. A large number of diverse coding nucleic acid sequences can be obtained, starting with a specific amino acid sequence and a known degeneracy of the gene code. The degeneracy of the gene code arises from the fact that almost all amino acids are encoded by various combinations of nucleotide triplets, or "codons." The translation of specific codons to specific amino acids is well known in this field (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 an expression regulatory sequence at a position that modulates the expression of the protein. Such nucleic acids, together with the regulatory element, are often referred to as an expression system. As used herein, the term “expression system” refers to a system designed to produce one or more gene products of interest. Typically, such a system is designed “artificially,” i.e., by genetic technology means that can be used to produce the gene products of interest in vivo, in vitro, or ex vivo. The term “expression system” further includes the expression of the gene products of interest, including the transcription of polynucleotides, mRNA splicing, translation into polypeptides, co-translation and post-translational modification of polypeptides or proteins, and the targeting of the protein to one or more compartments within a cell, secretion from the cell, and uptake of the protein in the same or another cell. This summary is referred to as an expression system for use in eukaryotic cells, tissues, or organisms. Expression systems for prokaryotic systems may differ, and methods for constructing expression systems for prokaryotic cells are well known in the art.

[0087] The regulatory elements present in a gene expression cassette generally include: (a) a promoter transcribed to the nucleotide sequence encoding the polypeptide; (b) a 5' ribosome binding site functionally bound to the nucleotide sequence; (c) a terminator bound to the 3' end of the nucleotide sequence; and (d) a 3' ribosome binding site functionally bound to the nucleotide sequence. Polyadenylation signals. Further regulatory elements may also exist that are useful for enhancing or regulating gene expression or polypeptide processing. Promoters are gene elements recognized by RNA polymerase that mediate transcription of downstream regions. Preferred promoters are strong promoters that result in increased transcription levels. Examples of strong promoters include the pre-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 natural promoters such as the EF1α promoter, the mouse CMV promoter, the Roussarcoma virus promoter, and the SV40 early / late promoter, and the [β]-actin promoter; and artificial promoters such as the synthetic muscle-specific promoter and the chimeric muscle-specific / CMV promoter (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). Polyadenylation signals are involved in the cleavage of transcribed RNA and the addition of a poly(A) tail to that RNA. In higher eukaryotes, polyadenylation signals contain an AAUAAA sequence approximately 11–30 nucleotides from the polyadenylation site. The AAUAAA sequence is involved in the signaling of RNA cleavage (Lewin, Genes IV, Oxford University Press, NY, 1990). The poly(A) tail is important for mRNA processing, export from the nucleus, translation, and stabilization.

[0089] Polyadenylation signals that can be used as parts of a gene expression cassette include the minimal rabbit [β]globin polyadenylation signal and bovine growth hormone polyadenylation (BGH) (Xu et al., Gene 272:149-156, 2001, Post et al., U.S. Patent US5,122,458). Examples of further regulatory elements that may be 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 a protein of the second aspect of the present invention or the protein of the second aspect of the present invention can be regulated. Such regulation can be achieved at many steps of gene expression. Possible regulatory steps include, but are not limited to, transcription initiation, promoter clearance, transcription extension, splicing, nuclear export, mRNA stability, translation initiation, translation efficiency, translation extension, and protein holding. Other regulatory steps that affect the intracellular factor 1 or factor 2 polypeptide concentration affect the protein half-life. Such regulatory steps include, for example, the regulation of protein denaturation. Since the proteins of the present invention include secretory proteins, these proteins can be directed to the secretory pathway of the host cell. Secretory efficiency, along with regulatory steps for expression and protein stability, regulates the extracellular concentration of each protein. The extracellular environment can refer to, but is not limited to, culture medium, tissue, intracellular matrix or lumen, or body fluids, such as blood or lymph. The regulation of the above regulatory steps may be, for example, cell type or tissue type independent or cell type or tissue type specific. In a particularly preferred embodiment of the present invention, the regulation of the regulatory steps is cell type or tissue type specific. Such cell-type or tissue-type specific regulation is preferably achieved by a regulatory step relating to nucleic acid transcription. This transcriptional regulation can be achieved by using cell-type or tissue-type specific promoter sequences. The results of this cell-type or tissue-type specific regulation may have different grades 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 such expression is restricted to each cell or tissue type. Cell-type 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 dependent on physiological conditions. Such conditions include, for example, inflammation or wound. Such physiological condition-specific expression can also be achieved by modulating all of the above regulatory steps. A preferred method of regulatory expression for physiological condition-specific expression is transcriptional regulation. For this purpose, wound or inflammation-specific promoters can be used. Each promoter may be a native sequence, for example, derived from a gene that is specifically expressed during an immune response and / or wound tissue regeneration. Another possibility is the use of artificial promoter sequences constructed by combining, for example, two or more native 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 yet another embodiment, the expression is cardiac-specific and wound-specific.

[0092] Another possibility for regulating the expression of a protein in the second aspect of the present invention, or for regulating a protein in the second aspect of the present invention, is the conditional regulation of said gene expression. Operator sequences can be used to achieve conditional regulation. For example, gene expression can be suppressed using a Tet operator sequence. Conditional regulation of gene expression by Tet operator sequences, along with Tet repressors, is well known in the art, and many such systems have been established for a wide range of prokaryotes and eukaryotes. Those skilled in the art know how to select a suitable 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 individuals after myocardial infarction, where healing includes improvement of left ventricular systolic function and may be associated with increased capillary density in borderline infarction. Furthermore, the use of the nucleic acid may reduce post-myocardial infarction mortality. Methods that can be used to measure parameters such as improvement of left ventricular systolic function, increased capillary density in borderline infarction, and reduced post-myocardial infarction mortality are well known in the art and are typically described in Examples 4-6.

[0094] Nucleic acids encoding the Factor 1 and Factor 2 proteins, fragments, or variants described above are preferably used to treat or improve atrophy; hypoplasia; inflammation, injury; wound, ischemia; reperfusion injury; inflammation; infection; trauma; mechanical stress; poisoning; primary or acquired cardiomyopathy, preferably hereditary cardiomyopathy and cardiomyopathy resulting from 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 endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; and in improving myocardial infarction or 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 the proliferation of non-transformed tissue or non-transformed cells and / or in healing and / or inhibiting apoptosis.

[0096] The term “enhancing the proliferation of non-transformed tissue or non-transformed cells, and / or curing and / or inhibiting apoptosis” 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 a protein and / or nucleic acids contained therein into a cell, or into which they can be introduced. In the context of the present invention, it is preferable that the target gene encoded by the introduced polynucleotide is expressed in the host cell upon introduction of the vector(s). Suitable vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors such as lambda phages, 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, adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retrovirus vectors, and lentivirus vectors.

[0099] In a particularly preferred embodiment of the present 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 present invention and one or more proteins of the second aspect of the present invention can be introduced into host cells, tissues, or organisms using a vector suitable for therapeutic administration. A suitable vector can preferably deliver nucleic acids into target cells without causing unacceptable side effects.

[0101] In a particularly preferred embodiment, the use of the vector of the present invention includes application to individuals after myocardial infarction, where the healing includes improvement in left ventricular systolic function and may be associated with increased capillary density in borderline infarction. Furthermore, such use may reduce post-myocardial infarction mortality. Methods that can be used to measure parameters such as improvement in left ventricular systolic function, increased capillary density in borderline infarction, and reduced post-myocardial infarction mortality are well known in the art and are typically described in Examples 4-6.

[0102] Vectors containing nucleic acids encoding the Factor 1 and Factor 2 proteins, fragments, or variants described above are preferably used to treat or improve atrophy; degeneration; inflammation, injury; wound, ischemia; reperfusion injury; inflammation; infection; trauma; mechanical stress; poisoning; primary or acquired cardiomyopathy, preferably hereditary cardiomyopathy and cardiomyopathy resulting from 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 endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; and in improving myocardial infarction or left ventricular systolic function.

[0103] In the fifth aspect, the present invention provides pharmaceutical compositions comprising proteins of the first and / or second aspect, and / or nucleic acids of the third aspect, and / or vectors of the fourth aspect, and optionally a carrier, for use in enhancing the proliferation of non-transformed tissue or non-transformed cells and / or curing and / or inhibiting apoptosis. The terms “enhancing the proliferation of non-transformed tissue or non-transformed cells and / or curing and / or inhibiting apoptosis” have the meanings set forth above and their preferred meanings.

[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 for administering a therapeutically active ingredient together with it. Such pharmaceutical carriers may be liquid or solid. Liquid carriers include, but not limited to, sterile solutions, such as saline solutions in water or oil (though not limited to petroleum, animal, plant, or synthetic origins, 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 pharmaceutical compositions are administered intravenously. Examples of suitable pharmaceutical carriers are listed in “Remington's Pharmaceutical Sciences” (EW Martin). In preferred embodiments of the present invention, the carrier is a suitable pharmaceutical excipient. Suitable pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol. Such suitable pharmaceutically acceptable excipients are preferably pharmaceutically acceptable.

[0105] "Medically acceptable" means that it is approved by a federal or state government supervisory authority or is listed in the United States Pharmacopeia or other generally recognized animal, more specifically human, pharmacopoeias.

[0106] The term “composition” is intended to include formulations of active compounds comprising an encapsulating material as a carrier, which provides a capsule in which an active ingredient, with or without other carriers, is surrounded by a 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., provides pharmaceutical value. In the context of this invention, the active ingredient refers to a protein in its first and / or second phase and / or a nucleic acid in its third phase and / or a vector in its fourth phase. A pharmaceutical composition may contain one or more active ingredients that can act independently or cooperatively with each other. The active ingredient can be formulated in neutral or salt form. The salt form is preferably a pharmaceutically acceptable salt.

[0108] The term “pharmaceutically acceptable salt” refers to, for example, a salt of a polypeptide, but is not limited to, for example. Suitable pharmaceutically acceptable salts include, for example, acid addition salts that can be formed by mixing a solution of the polypeptide of the present 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, carbonate, or phosphoric acid). Furthermore, if the peptide has an acidic moiety, suitable pharmaceutically acceptable salts therefor 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 suitable organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed using counter anions, e.g., halido, hydroxide, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates, and aryl sulfonates).Medicinally acceptable salts include, but are not limited to, the following: acetates, adipines, alginates, ascorbicates, aspartates, benzenesulfons, benzoates, bicarbonates, bisulfates, acid tartrates, borates, bromides, butyrates, calcium edetate, camphor, camphor sulfons, cansilates, carbonates, chlorides, citrates, clavulanates, cyclopentanepropionates, diglucons, dihydrochlorides, dodecyl sulfates, edetates, edisylates, estates, esylates, ethanesulfons, formates, fumarates, gluceptates, glucoheptates, glucons, glutamates, glycerophosphates, glycolyl arsanilates, hemisulfates, heptanoates, hexanoates, hexylresorcinol, hydravamin, hydrobromide, hydrochlorides, hydroiodides, 2 -Hydroxy-ethanesulfonate, 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, basic acetate, succinate, tannate, tartrate, theoacrylate, tosylate, triethoidide, undecanoate, valerate, etc. (e.g., SM See Berge et al., "Pharmaceutical salts," J. Pharm. Sci., 66, pp. 1-19 (1977)).

[0109] An effective dose of the active ingredient is administered to cells, tissues, or an organism. “Effective dose” is the amount of the active ingredient sufficient to achieve the intended purpose. The active ingredient may be a therapeutic agent. The effective dose of a given active ingredient will vary depending on parameters such as the properties of the ingredient, the route of administration, the size and species of the organism to which the active ingredient is administered, and the purpose of administration. The effective dose in each case can be experimentally determined by those skilled in the art using methods established in the art. “Administer” as used in the context of this invention includes in vivo administration to an organism and direct administration to cells or tissues in vitro or ex vivo.

[0110] In a preferred embodiment of the present invention, the pharmaceutical composition is customized to treat a disease or disorder. As used herein, “treat, treating, or treat” a disease or disorder means achieving one or more of the following: (a) a reduction in the severity of the disorder; (b) limiting or preventing the symptoms characteristic of the disorder being treated; (c) suppressing the exacerbation of the symptoms characteristic of the disorder being treated; (d) limiting or preventing recurrence of the disorder in a patient who has previously had the disorder; (e) limiting or preventing recurrence of symptoms in a patient who has previously had symptoms of the disorder; (f) a reduction in mortality after the onset of the disease or disorder; (g) a cure; and (h) prevention of the disease. As used herein, “prevent, preventing, prevention, or prophylaxis” 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 and healing with the pharmaceutical composition of the present invention include treatment of an individual after myocardial infarction, and such healing may include improvement of left ventricular systolic function and be associated with increased capillary density in borderline infarction. Furthermore, healing may reduce the mortality rate after myocardial infarction. Methods that can be used to measure parameters such as improvement of left ventricular systolic function, increased capillary density in borderline infarction, and reduced mortality rate after myocardial infarction are well known in the art and are typically described in Examples 4-6.

[0112] The pharmaceutical compositions anticipated by the present invention can be formulated in various ways well known to those skilled in the art. For example, the pharmaceutical compositions of the present invention may be in liquid form, such as a solution, emulsion, or suspension. Preferably, the pharmaceutical compositions of the present invention are formulated for parenteral administration, preferably intravenous, intra-arterial, intramuscular, subcutaneous, percutaneous, intrapulmonary, intraperitoneal, intracoronary, intracardiac, or mucosal administration, preferably intravenous, subcutaneous, or intraperitoneal. Formulations for oral or rectal administration are also possible. Preferably, the pharmaceutical compositions of the present invention are in the form of a sterile aqueous solution that may contain sufficient salt or glucose to make an isotonic solution with other substances, such as blood. The aqueous solution should be appropriately buffered as needed (preferably pH 3 to 9, more preferably pH 5 to 7). The pharmaceutical composition is preferably in unit dosage form. In such form, the pharmaceutical composition is divided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form may be a packaged formulation, a package containing a separated amount of the pharmaceutical composition, such as a vial or ampoule. The pharmaceutical composition is preferably administered intravenously, intra-arterially, intramuscularly, subcutaneously, percutaneously, intrapulmonaryly, intraperitoneally, intracoronarily, or intracardiacly via an intravascular route, 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 may replace or be administered in addition to the standard treatment for each disease or condition. If the pharmaceutical composition is used further, it may be administered before, after, or concurrently with the standard therapy. In a preferred embodiment, the standard therapy is reperfusion therapy, and the pharmaceutical composition may be administered before, after, or concurrently with the reperfusion therapy.

[0114] The pharmaceutical composition is more preferably administered once or more times. 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 duration for administering the pharmaceutical is not limited. Preferably, the administration does not exceed 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0115] The single dose of the pharmaceutical composition may not depend on the total amount of the dose administered or on each administration period in which it is administered as one or more bolus injections and / or infusions.

[0116] The first to fifth aspects of the present invention are based on the inventors' findings that Factor 1 and Factor 2 are potent angiogenesis-stimulating molecules in vitro and in vivo. Therefore, the above proteins, nucleic acids, and vectors are also expected to have ex vivo uses, for example, as therapeutic agents for stimulating the above cells ex vivo or for use in cell culture applications.

[0117] However, based on the findings regarding the angiogenic stimulating activity of factors 1 and 2, the inventors investigated whether factors 1 and 2 are targets for anti-angiogenic therapy, respectively. The inventors succeeded in inhibiting angiogenesis using inhibition of either factor 1 or factor 2.

[0118] Anti-angiogenic strategies are used to treat cancerous conditions and other disorders in which angiogenesis is involved in disease progression, such as age-related macular degeneration (see, for example, Ferrara N and Kerbel RS (2005) Nature:438:967-974 or Potente M, et al. (2011) Cell. 146(6):873-887). Furthermore, in a further aspect, the present invention relates to the anti-angiogenic properties of factor 1 and factor 2 inhibitors. In these aspects, the above definitions apply equally to the definitions section. Furthermore, certain definitions described in the first to fifth aspects and, for example, in the description of the term “vector” and preferred embodiments of preferred vectors also apply to the following aspects of the present invention unless the context in which they are used clearly indicates otherwise.

[0119] In a sixth aspect, the present invention provides inhibitors of factor 1 and / or 2 proteins for medical use, preferably in the treatment or prevention of diseases in which angiogenesis is a contributing factor to the onset or progression of the disease. The term “inhibitor” refers to a compound that interferes with the angiogenic activity of factor 1 or factor 2. Inhibitors prevent the production, circulation, and / or secretion of factor 1 or factor 2 in cells by acting on the transcription and / or translation of the mRNA encoding factor 1 or factor 2. Inhibitors may also act by specifically binding to factor 1 or factor 2 proteins or cellular proteins to which factor 1 or factor 2 proteins specifically bind, preferably their cellular receptors. Such binding can prevent or interfere with the innate interactions between factor 1 or factor 2 and other cellular proteins, preferably their respective cellular receptors. Those skilled in the art are well familiar with methods of interfering with the binding of receptors to their agonists, and this knowledge can be used 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, respectively, that exhibit the pro-angiogenic function of Factor 1 and Factor 2. Such inactivating mutant or deleted Factor 1 or Factor 2 will compete with wild-type Factor 1 and Factor 2 for their native binding partners. Compounds that interfere with the angiogenic activity of Factor 1 or Factor 2 reduce the 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 substantially consist of mutants or fragments of Factor 1 or Factor 2, respectively, it is preferable that they exhibit this level of inhibition of the Factor 1 or Factor 2 protein at equimolar concentrations. A preferred assay that can be used to measure the inhibition of angiogenic activity is described in Example 10 of this specification.To determine whether a particular inhibitor has this activity in equimolar amounts, the molar amounts of Factor 1 and Factor 2 and each inhibitor must be measured, respectively. Factor 1, shown in SEQ ID NO: 1, has a molar mass of 15.84 kD, and Factor 2, shown in SEQ ID NO: 3, has a molar mass of 21.57 kD, with the molecular weight of IgG being approximately 150 kD. Therefore, 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 also 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, in that sense, inhibitors of the Factor 1 protein or the 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 inhibition level is preferably measured based on the protein produced in cells that naturally produce Factor 1 or Factor 3. Those skilled in the art are familiar with many methods for measuring the amount of mRNA encoding Factor 1 or Factor 3 and Factor 1 or Factor 2 protein, which can be used to evaluate the ability of compounds to interfere with the transcription and / or translation of mRNA encoding Factor 1 or Factor 2. Preferably, Factor 1 protein comprises, substantially comprises, or comprises the amino acid sequence shown in SEQ ID NO: 1, and Factor 2 comprises, substantially comprises, or 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.

[0120] In the context of the present invention, the inhibitors used are preferably proteins comprising, substantially comprising, or consisting of an inhibitory fragment or inhibitory mutant of the amino acid sequence shown in SEQ ID NO: 1 or 3, or a variant thereof having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 3. It is well known in the art that proteins that exert their function through receptor-mediated protein-protein interactions contain a domain necessary for binding to the receptor and a domain that stimulates the receptor to transmit signals into the cell. Therefore, those skilled in the art are well familiar with methods for producing inhibitory fragments or mutants of such receptor-binding proteins. For example, a series of N- and / or C-terminal cleavage factor 1 or factor 2 proteins can be produced and their angiogenic activity tested in the assay described in Example 2. The ability of those fragments that no longer exhibit angiogenic activity to inhibit the angiogenic activity of factor 1 or 2 proteins is then tested in the assay described in Example 10. Mutants of factor 1 and 2 can be produced, for example, by alanine scanning mutagenesis, as is known in the art. In alanine scanning, each mutant contains one, two, three, or more amino acids that have been mutated into alanine (a so-called cassette), and a series of mutants are created in which the position of the cassette within factor 1 or 2 differs. Factor 1 or 2 mutants that have lost angiogenic stimulating activity can be identified again as described in Example 2. Then, inhibitory mutants can 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 shown in SEQ ID NO: 1 or 3, or a variant having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 3, or a variant having at least 80% sequence identity with Factor 1 or 3, or a variant having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 3. The term “ligand” refers to a chemical portion that specifically binds to a particular 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 may be a peptide mimetic.

[0122] As used herein, the term “immunoglobulin (Ig)” refers to the immunoglobulin superfamily of glycoproteins that provide immunity. “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, β-2 microglobulin (β2M), CD3, CD4, and CD8. Its transmembrane region allows it to bind to the membrane of effector cells. Typically, the term “antibody” as used herein refers to secretory immunoglobulins that lack 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, denoted by the Greek letters α, δ, ε, γ, and μ. The type of heavy chain present defines the class of antibody, and these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, each playing a different role and resulting in an appropriate immune response against different types of antigens. The distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). An antibody contains four polypeptide chains, namely two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated 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 (hereinafter abbreviated 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), which incorporate more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the heavy and light chains can be determined as is known in the art. For example, the CDRs within each antibody light chain sequence and heavy chain sequence can be found using the following set of rules: Light chain CDR-1: Start: approximately 24 residues, the residue before CDR-1 is always Cys, and the residue after CDR-1 is always Trp. Typically Trp-Tyr-Gln, and 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, and Ile-Phe, always 7 residues long; Light chain CDR-3: Start: Always at the terminal 33 residue of CDR-2; always a residue before Cys, always a residue after Phe-Gly-XXX-Gly; Length: 7-11 residues Heavy chain CDR-1: Start: Always approximately 26 residues 4 after Cys (based on the Chothia AbM definition; Kabat definition starts after 5 residuals); always a residue before Cys-XXX-XXX-XXX; always a residue after Trp. Typically Trp-Val, and Trp-Ile, Trp-Ala, length: 10-12 residues [AbM definition; 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 (with many variations), the residue after Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala, Length: 16-19 residues according to the Kabat definition (according to the AbM definition; the 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 a residue before Cys-XXX-XXX (typically Cys-Ala-Arg); always a residue after Trp-Gly-XXX-Gly; Length: 3 to 25 residues. This set of rules is known to those skilled in the art and can 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 contain amino acid residues in the CDR that are not encoded by human germline immunoglobulin sequences (e.g., mutations resulting from random or site-directed 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 a CDR sequence derived from the germline of another mammalian species (e.g., mouse) is transplanted onto a human FR sequence. Human antibodies also include human immunoglobulin libraries or antibodies isolated from transgenic animals 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 an antibody molecule in a single-molecule composition. Monoclonal antibodies exhibit single-binding specificity and affinity for a particular epitope. In one embodiment, monoclonal antibodies are produced by hybridomas containing B cells obtained from a non-human animal (e.g., mouse) fused with immortalized cells. As used herein, the term “recombinant antibody” includes any antibody produced, expressed, generated, or isolated by recombinant means such as: (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal to an immunoglobulin gene, or hybridomas produced therefrom; (b) antibodies isolated from host cells (e.g., transfectomas) transformed to express an antibody; (c) antibodies isolated from a recombinant combined antibody library; and (d) antibodies produced, expressed, generated, or isolated by any other means, including splicing of an immunoglobulin gene sequence with other DNA sequences. As used herein, “heterologous antibody” is defined for transgenic organisms that produce such antibodies. This term refers to antibodies found in organisms that are not composed of transgenic organisms, and which generally possess amino acid sequences or coding nucleic acid sequences corresponding to those originating from non-transgenic species. As used herein, "heterohybrid antibody" refers to an antibody having light and heavy chains of different biological origins. For example, an antibody with a human heavy chain bound to a mouse 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 or parts of the antibody, which are part of an artificial construct. Preferred examples of antigen-binding fragments include, but are not limited to, the following: Fab fragments, Fc fragments, Fab' fragments, F(ab')2, single-domain antibodies (sdAb), nanobodies, single-stranded Fv, bivalent single-stranded variable fragments (di-scFvs), tandem scFvs, diabodies, and single-stranded fragments. This includes diabodies (scDBs), triabodies, bi-specific T cell engagers (BiTEs), or biaffinity retargeting molecules (DART molecules).

[0126] Each "Fab fragment" (also called a "Fab portion" or "Fab region") has a single antigen-binding site and a residual "Fc fragment" (also called an "Fc portion" or "Fc region") (its name reflects its ability to crystallize easily). A "Fab' fragment" is a Fab fragment that further contains the hinge region of the Ig molecule, and an "F(ab')2 fragment" is a fragment that is chemically bound or disulfide bonded. It is understood to contain two more bound Fab' fragments. sdAb (Desmyter et al. 1996) and the “nanobody” contain only a single VH domain, while the “single-stranded Fv (scFv)” fragment contains a short ri It contains a heavy chain variable domain linked to a light chain variable domain via a linker peptide (Huston et al. 1988). Di-scFvs can be created by linking two scFvs (scFvA-scFvB). This can be done by producing a single peptide chain with two VH and two VL regions to obtain a "tandem scFvs" (VHA-VLA-VHB-VLB). Another possibility is to force the scFvs to dimerize by creating scFvs with a linker that is too short to fold the two variable regions together. Typically, these dimers are made using a linker with a length of 5 residues. This type is known as a "diabody". Even shorter linkers (1 or 2 amino acids) between the VH and VL domains result in the formation of a single specific trimer, a so-called "tribody". The two specific diabodies are formed by expressing chains having the sequences VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. A single-chain diabody (scDb) contains 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). A "bispecific T cell engager (BiTEs)" is a fusion protein consisting of two scFvs of different antibodies, where one scFvs binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. 2004). A biaffinity retargeting molecule ("DART" molecule) is a diabody further stabilized by a C-terminal disulfide crosslink.

[0127] The term "antibody-like protein" refers to a protein that possesses antibody-like properties when binding to an antigen or antigenic protein, but does not necessarily have the structural features of an antibody. Antibody-like proteins can occur naturally or be artificially designed, for example, biotechnologically. Examples of naturally occurring 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 that play a role in storing or transporting biologically important compounds. They share a conserved barrel of eight antiparallel β-chains as their central folding motif, and each end of this barrel structure contains six hypervariable loops that bind to each pair of β-chains. These loops form the entry points to the binding pocket. The 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 single-chain polypeptides and are much smaller than immunoglobulins, they have a great potential to bind to antigens with different specificities. Examples of artificially designed antibody-like proteins include scaffold-based proteins produced by fusing peptides with known affinity for certain targets, or by inserting such peptides into scaffold proteins to combine the binding properties of the peptides with the desirable beneficial properties of the scaffold carrier. Those skilled in the art will know of many such scaffold-based proteins. As used herein, the term “scaffold protein” refers to a protein that is structurally rigid, 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, if one or more amino acids of a scaffold protein are replaced with a polypeptide of appropriate length, the polypeptide will occupy similar positions to those of the substituted ones. This allows for the arrangement of a particular polypeptide in a specific three-dimensional position and / or orientation within the scaffold protein.Furthermore, scaffold proteins can be used as substitutes for antibodies for molecular recognition (see, e.g., 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 transmit novel binding specificity to the SH3 domain. Methods for selecting a Fyn SH3 domain that specifically binds to a particular antigen are described, for example, in WO2000 / 072742 or WO2008 / 022759.

[0128] In the context of this invention, the term “peptide mime” refers to any molecule whose essential elements (active groups) are three-dimensionally similar to those of a natural peptide or protein, which retains the ability to interact with a biological target and produces the same biological effect. Peptide mimes typically include small protein-like chains designed to closely resemble peptides, which can be obtained by modifying existing peptides or by designing similar systems that closely resemble peptides (e.g., peptoids and β-peptides). Regardless of this approach, chemical structural changes are designed to favorably adjust molecular properties, such as increasing or decreasing stability or biological activity. Thus, modifications include, but are not limited to, changes in the backbone and changes in peptides that do not occur naturally, including the incorporation of non-natural amino acids.

[0129] The term "specific binding" or "specifically binding" to an antigen, such as factor 1 or factor 2, describes the ligand's ability to bind to the antigenic determinants of the antigen with high affinity. In that context, "high affinity" means that the Kd of the interaction is 1 x 10⁻¹⁰. -5 Less than M, preferably 1 x 10 -6 Less than M, comfortable 1 x 10 -7 Less than M, more preferably 1 x 10 -8 Less than M, most preferably 1 x 10-9 This means it is less than M.

[0130] Preferred antibodies for use in the context of the sixth aspect of the present invention are monoclonal antibodies, preferably human or humanized antibodies. In another preferred embodiment, the inhibitor is a nucleic acid that inhibits or prevents the transcription and / or translation of a protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 3 or a variant mRNA having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 3. Those skilled in the art are well familiar with methods for determining the sequence of such nucleic acids based on the genomic sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 or 3. Examples of such inhibitory nucleic acids include siRNAs specific to mRNA encoding factor 1 or factor 2.

[0131] If the inhibitor in the sixth aspect of the present invention is a protein that can be encoded by a nucleic acid, the inhibitor is expected to be administered by supplying the nucleic acid encoding the inhibitor. Accordingly, in the seventh aspect, the present invention provides a nucleic acid encoding the inhibitor in the sixth aspect of the present invention for the treatment or prevention of diseases in which angiogenesis is a contributing factor to the onset 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 present invention.

[0132] Accordingly, in the eighth aspect, the present invention provides a vector comprising nucleic acids of the sixth aspect of the present invention for treating or preventing diseases in which angiogenesis is a contributing factor to the onset or progression of the disease. In the context of the seventh aspect, the term “vector” has the same meaning as it has been described in the context of the fourth aspect of the present 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, adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retrovirus vectors, and lentivirus vectors. Furthermore, in the ninth aspect, the present invention provides a pharmaceutical composition 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 pharmaceutically acceptable excipient, for treating or preventing diseases in which angiogenesis is a contributing factor to the onset or progression of the disease. The pharmaceutical composition may also include any of the components described in the fifth aspect of the present invention.

[0134] In aspects 6 to 9 of this invention, the term "diseases in which angiogenesis is a contributing factor to the onset or progression of the disease" refers to diseases in which the proliferation of blood vessel-forming cells 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 that line the inside of blood vessels and smooth muscle cells that 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 existing blood vessels. In some diseases, such as ocular neovascular disease, the abnormal formation of blood vessels is the cause of the disease, and in some diseases, such as benign or malignant tumors, angiogenesis occurs during disease progression, supplying oxygen and influence to the growing tumor mass. In these diseases, angiogenesis is not the cause of the disease, but it promotes disease progression. Neovascularization in malignant tumors also contributes to metastasis by giving tumor cells an escape route from the tumor mass.

[0135] In this context, diseases in which angiogenesis is a contributing factor to the onset or progression of the disease are called proliferative disorders. Preferred proliferative disorders are selected from the group consisting of benign tumors, malignant tumors, rheumatoid arthritis, psoriasis, ocular angiogenesis, 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, in particular 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 disorders, cervical cancer, non-melanoma skin cancer, ovarian cancer, prostate cancer, benign prostatic hyperplasia, pancreatic cancer, rectal cancer, and testicular cancer. The diseases that are preferable to treat are benign tumors, malignant tumors, and ocular neovascularization.

[0136] The inventors have confirmed that antibodies against certain epitopes of Factor 1 and Factor 2 interfere with the angiogenic stimulating function of Factor 1 and Factor 2, respectively, i.e., they are antagonist antibodies. Therefore, in the tenth aspect of the present invention, the invention is directed toward ligands of Factor 1 and Factor 2, respectively, that inhibit the angiogenic stimulating activity of Factor 1 and Factor 2. Antibodies or fragments thereof that inhibit the angiogenic stimulating activity of Factor 1 and Factor 2, respectively, are particularly preferred. The inventors have succeeded in producing examples of such inhibitory antibodies by producing antibodies that specifically bind to domains exposed on the surface of Factor 1 and 2. In a preferred embodiment of this aspect, the present invention relates to ligands that can specifically bind to these fragments of Factor 1 and 2, respectively. In the context of this aspect of the present invention, the definitions set out in the General Definitions section and the specific definitions in the context of the sixth aspect of the present invention are applied equivalently.

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

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

[0139] In the context of the above 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 derived from another factor 1 or 2 protein that aligns with the amino acid sequence shown for 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. Those skilled in the art can easily identify the fragment of SEQ ID NO: 2 having the amino acid sequence CTIWRPQGKSYLYFTQ (SEQ ID NO: 38) and determine the corresponding amino acids of another factor 1 protein. (Examples)

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

[0141] In a multicenter, placebo-controlled clinical trial, one of the inventors tested the efficacy of intra-coronary injection of autologous bone marrow cells from AMI patients (BOOST-2, controlled clinical trial identification number ISRCTN17457407). In this clinical trial, bone marrow aspirates were obtained from AMI patients for research purposes. CXCR4+ bone marrow cells were isolated by magnetic cell separation (MiniMACS®, Miltenyi Biotec). After two subsequent purification steps, a CXCR4+-rich cell population was obtained (purity >95% confirmed by flow cytometry). Next, RNA was isolated from these cells and used for microarray analysis (Affymetrix GeneChip HG_U133 Plus 2.0). In subsequent bioinformatics analysis, the 4000 expression sequence tags (ESTs) most strongly expressed by CXCR4+ bone marrow cells in the microarray were tested. Using a series of bioinformatics methods, putative secretory factors were identified from these ESTs, characterized by deletions of N-terminal signal peptides, mitochondrial or nuclear signal peptides, endoplasmic reticulum-retaining sequences, and transmembrane domains. A total of 283 putative secretory factors were identified, 117 of which were found to have mouse homologs in NCBI Blast. The cDNA of the human homologs was cloned into expression plasmids and then individually transfected into human embryonic kidney (HEK) cells. The transfected HEK cells were cultured in serum-free medium for 30 hours to obtain conditioned culture supernatant. The conditioned HEK cell supernatant was individually tested for angiogenic stimulating effects using a microangiogenic assay and cytoprotective effects using a cardiomyocyte death assay. This screening led to the identification of two secretory proteins: "Factor 1" showed angiogenic stimulating and cytoprotective effects in the above assays; and "Factor 2" showed angiogenic stimulating effect in the above assays.

[0142] The sequences identified in the screening and their respective mouse or human homologs are as follows: Factor 1: Human Factor 1 was identified through screening 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 the NCBI reference sequence: NM_019107.3 (SEQ ID NO: 6). The amino acids of human factor 1 are shown in Figure 6 (SEQ ID NO: 2).

[0143] Mouse homologs 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 the 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: Secretory human factor 2 was identified through screening and used in Example 2 and Figure 1: The Homo sapiens chromosome 19 open reading frame 63 (C19orf63) transcription mutant HSS1. The nucleic acid sequence encoding human factor 2 is available as the NCBI reference sequence: NM_175063.4 (SEQ ID NO: 8). The secreted amino acid sequence of human factor 2 is shown in Figure 8 (SEQ ID NO: 4).

[0145] Transmembrane type human factor 2: Homo sapiens chromosome 19 open reading frame 63 (C19orf63), transcription mutant HSM1. The NCBI reference sequence encoding the transmembrane form of human factor 2 is available: 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 for the transmembrane variant of human factor 2 is available in GenBank:AY358710.1 (SEQ ID NO: 34).

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

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

[0149] To confirm the angiogenic stimulating activity observed in screening, both factors (human homologs encoded by the nucleic acid sequences shown in SEQ ID NOs. 6 and 8) were generated as His-labeled recombinant proteins in COS7 cells. As shown in Figure 1, recombinant factor 1 and recombinant factor 2 promoted angiogenic activity in a dose-dependent manner in cultured human endothelial cells. Human coronary artery endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were purchased from Provitro (Berlin, Germany). The cells were cultured for 24 hours in minimal medium in the presence or absence (control) of 10% fetal bovine serum (FCS), human recombinant VEGF-A (R&D Systems), or recombinant human factor 1 (SEQ ID NOs. 2) or factor 2 (SEQ ID NOs. 4) at various concentrations. (A) HCAEC proliferation was measured by bromodeoxyuridine uptake. (B) HCAEC migration was evaluated after scratching a monolayer of confluent endothelial cells with a pipette tip. (C) Network formation of HUVECs was evaluated 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 effect of factor 1 observed in screening, neonatal rat ventricular cardiomyocytes were subjected to simulated ischemia-reperfusion injury in the presence or absence of recombinant factor 1. Factor 1 (a mouse homolog encoded by the nucleic acid sequence shown in SEQ ID NO: 7) was generated as a His-labeled recombinant protein in COS7 cells. As shown in Figure 2, recombinant factor 1 promoted anti-apoptotic activity in cultured cardiomyocytes in a dose-dependent manner. Ventricular cardiomyocytes were isolated from 1-3 day old Sprague-Dawley rats by Percoll density gradient centrifugation. Cardiomyocytes were exposed to simulated ischemia for 180 minutes (glucose-free medium containing 2-deoxyglucose in a 5% CO2 / 95% N2 atmosphere), and then subjected to simulated reperfusion for 60 minutes in the presence or absence of recombinant human GDF-15 (R&D Systems, a known anti-apoptotic cytokine) or recombinant mouse factor 1 at various concentrations (control), or returned to glucose-containing medium in 5% CO2 / 95% room air. Cell death was evaluated by in situ TdT-mediated dUTP nick-end labeling (TUNEL). N=3 independent experiments / conditions; *P<0.05 vs control. [Example 4]

[0151] To investigate the therapeutic potential of factors 1 and 2 in setting AMI, adenoviruses encoding 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. Adenovirus expression of both factors resulted in improved left ventricular systolic function 28 days post-infarction. This was associated with increased capillary density in borderline infarction (Figure 3).

[0152] Mouse factor 1 or factor 2 cDNA was cloned into replication-deficient adenovirus using the AdEasy XL vector system (Stratagene). Replication-deficient adenovirus encoding galactosidase (lacZ) was used as a control. The 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 ligation of the left anterior descending coronary artery (LAD). 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 evaluated by transthoracic echocardiography (Visualsonics) 28 days after LAD ligation (N=10-12 mice / group). (B) Isolectin-positive capillary density in borderline infarcts was evaluated 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 factors 1 and 2 when used as recombinant proteins in a reperfusion AMI setting (closely resembling the clinical situation of AMI patients who have undergone reperfusion therapy), 10–12-week-old male C57BL / 6 mice were subjected to 1-hour coronary artery ligation (ischemia) followed by 28 days of reperfusion. Both factors were sc-treated in the mice for the first 7 days after reperfusion. Factors 1 and 2 (mouse homologs; amino acid sequences corresponding to SEQ ID NOs. 13 and 24) were generated as His-labeled recombinant proteins in HEK293 cells. Treatment with recombinant factor 1 or recombinant factor 2 resulted in a significant improvement in left ventricular systolic function at 28 days post-infarction. This was associated with a significant increase in capillary density in borderline infarctions (Figure 4). 10–12-week-old male C57BL / 6 mice were anesthetized and ventilated with isoflurane (1–2%), and transient left anterior descending coronary artery ligation was performed for 1 hour, followed by 28 days of reperfusion. Mice were given 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). Then, recombinant factor 1 or factor 2 was continuously infused with sc for 7 days using an Alzet minipump (10 μg / day). Control mice were injected with PBS. (A) Left ventricular systolic function (differential area change, FAC) was evaluated by transthoracic echocardiography 28 days after reperfusion (N=10-13 mice / group). (B) Isolectin-positive capillary density in borderline infarcts was evaluated by fluorescence microscopy 28 days after reperfusion (N=6 mice / group). *P<0.05, **P<0.01 vs. PBS control. [Example 6]

[0154] To investigate whether the use of recombinant factors 1 and 2 could increase survival after reperfusion-induced arterial myocardial infarction (AMI), mice were subjected to coronary artery ligation for 1 hour, followed by reperfusion for 28 days. Both factors were injected via sc-injection into the mice for the first 7 days after reperfusion. Factors 1 and 2 (mouse homologs; SEQ ID NOs. 7 and 10) were generated as His-labeled 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 aged 10–12 weeks were anesthetized and ventilated with isoflurane (1–2%), temporarily ligated the left anterior descending coronary artery 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 (PBS was injected into control mice). Recombinant factor 1 or factor 2 was then continuously infused using an Alzet minipump for 7 days (10 μg / day). Control mice were injected with PBS. Mice were examined daily for 28 days to assess post-infarction survival. N=29 PBS-treated mice; N=25 factor 1-treated mice; N=15 factor 2-treated mice. [Example 7]

[0155] Homologous sequences for the human C19Orf10-encoded protein were identified using 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. Sequence ID 2 was used as the matrix. The parameters used were the default parameters: 11 extension: 1, composition adjustment = conditional composition score matrix adjustment along with a database of non-redundant protein sequences (nr). From the identified sequences, one sequence was selected from various vertebrates, primarily mammals, as well as amphibians, birds, and fish. Each sequence is listed in Table 1. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm from http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The parameters used were the default parameters: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0, 20, gap spacing = 5, no terminal gaps = none, output options: format = Aln w / numbers, order = aligned. Multiple alignments obtained for all sequences are shown in Figure 6, and those for mammalian sequences are shown in Figure 7.

[0156] [Table 3] [Example 8]

[0157] Homologous sequences for the protein encoded by the human C19Orf63 splice mutant HSS1 were searched using 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. Sequence ID 4 was used as the matrix. The parameters used were the default parameters: extension: 1, composition adjustment = conditional composition score matrix adjustment along with a database of non-redundant protein sequences (nr). From the identified sequences, one sequence was selected from various vertebrate species, mainly mammals, and from amphibians and fish. Each sequence is shown in Table 2. The selected amino acid sequence was aligned using the CLUSTALW2 algorithm from http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The parameters used were the default parameters: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0, 20, gap spacing = 5, no terminal gaps = none, output options: format = Aln w / numbers, order = aligned. Figure 8 shows multiple alignments obtained for all sequences, and Figure 9 shows those for mammalian sequences.

[0158] [Table 4] [Example 9]

[0159] Homologous sequences for the protein encoded by the human C19Orf63 splice mutant HSM1 were searched using 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. Sequence ID 5 was used as the matrix.

[0160] The parameters used were the default parameters: extension: 1, composition adjustment = conditional composition score matrix adjustment along with a database of non-redundant protein sequences (nr). From the identified sequences, one sequence was selected from various vertebrate species, mainly mammals, and from amphibians and fish. Each sequence is listed in Table 3. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm from http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The parameters used were the default parameters: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0, 20, gap spacing = 5, no terminal gaps = none, output options: format = Aln w / numbers, order = aligned. Multiple alignments obtained for all sequences are shown in Figure 10, and those for mammalian sequences are shown 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 T75 flasks. 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. Next, the HCAECs were placed in 96-well plates (5x10). 3 Cells were seeded in wells and stimulated with recombinant human factor 1, recombinant human factor 2, or VEGF (positive control) for 16 hours in or without various concentrations of rabbit anti-factor 1 antibody, rabbit anti-factor 2 antibody, or control IgG. Antibodies were generated by Eurogentec 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 + N-terminal Cys). Cell proliferation was measured by colorimetric analysis BrdU uptake immunoassay (Roche). Data are shown in Figure 12 (Panel A: Factor 1; Panel B: Factor 2; data are mean ± SEM of 3-6 experiments).

Claims

1. 1. Use of a protein comprising the amino acid sequence of SEQ ID NO: 2 having a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids at the N-terminus, or a variant of SEQ ID NO: 2 having said deletion, for the manufacture of a medicament, wherein said variant has at least 99% sequence identity with SEQ ID NO: 2 having said deletion, The medicine, (i) treating a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical stress, myocardial infarction, and heart failure; (ii) improving left ventricular systolic function; or (iii) Protecting cardiomyocytes from apoptosis Use in, for use in.

2. 2. Use of a nucleic acid encoding a protein or variant according to claim 1 for the manufacture of a medicament, comprising: The medicine, (i) treating a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical stress, myocardial infarction, and heart failure; (ii) improving left ventricular systolic function; or (iii) Protecting cardiomyocytes from apoptosis Use in, for use in.

3. Use of a vector comprising the nucleic acid according to claim 2 for the manufacture of a medicament, comprising: The medicine, (i) treating a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical stress, myocardial infarction, and heart failure; (ii) improving left ventricular systolic function; or (iii) Protecting cardiomyocytes from apoptosis Use in, for use in.

4. The use of a vector according to claim 3, wherein the vector is selected from the group consisting of: a plasmid vector; a cosmid vector; a phage vector, a viral vector and a bacterial spore.

5. (i) the viral vector is an adenovirus vector, an adeno-associated virus (AAV) vector, an alphavirus vector, a herpes virus vector, a measles virus vector, a poxvirus vector, a vesicular stomatitis virus vector, a retrovirus vector, and a lentivirus vector; or a virus-like particle; or (ii) the phage vector is a lambda phage or a filamentous phage vector; Use of the vector according to claim 4.

6. The use according to claim 2 or any one of claims 3 to 5, The medicine, (i) treating a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical stress, myocardial infarction, and heart failure; (ii) improving left ventricular systolic function; or (iii) Protecting cardiomyocytes from apoptosis Use in, for use in.

7. 7. The use of claim 6, wherein the medicament is formulated for administration via oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronary routes.

8. 8. The use according to claim 6 or 7, wherein the medicament is formulated for administration before, after or simultaneously with reperfusion therapy.

9. 9. The use according to claim 8, wherein said administration is by one or more bolus injections and / or infusions.

10. a) A protein comprising the amino acid sequence of SEQ ID NO:2 having a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids at the N-terminus, or a variant of SEQ ID NO:2 having said deletion, said variant having at least 99% sequence identity to SEQ ID NO:2 having said deletion. b) a nucleic acid encoding the protein or variant of a); or c) a vector comprising the nucleic acid of b); and suitable pharmaceutical excipients, The pharmaceutical composition comprises: (i) treating a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical stress, myocardial infarction, and heart failure; (ii) improving left ventricular systolic function; or (iii) Protecting cardiomyocytes from apoptosis The pharmaceutical composition is for use in

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