Factor 1 and Factor 2 proteins for use in the treatment or prevention of a disease, and inhibitors thereof

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 growth and / or healing and / or inhibiting apoptosis of non-transformed tissues or non-transformed cells. Also provided are inhibitors of factor 1 and factor 2 for medical use, in particular for use in treating or preventing diseases in which angiogenesis contributes to the development or progression of the disease.

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 coronary artery occlusion and administration of platelet aggregation inhibitors and coagulation inhibitors to prevent reocclusion of the blood vessels. Furthermore, 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 a drug that has 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 results in 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 monocyte response in the myocardium is temporally biphasic. Proinflammatory monocytes appear earlier and promote the digestion of infarcted tissue and the removal of necrotic debris, 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 a bioinformatic secretome analysis using human CXCR4+ bone marrow cells to identify novel secreted proteins that show therapeutic potential after AMI and control infarct healing.

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

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

[0006] The amino acid sequence of human factor 1 is encoded by open reading frame 10 of human chromosome 19 (C19Orf10). This protein was described as a novel secreted factor in the synovium in a proteome analysis of so-called fibroblast-like synoviocytes (FLS-cells) in 2007. The interrelationship between the secretion of this protein and joint inflammatory diseases has been postulated 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). The corresponding patent application claims this protein for use as a therapeutic agent for treating joints and for diagnosing and monitoring tissue changes in tissues undergoing proliferative changes (US 2008 / 0004232 A1, Characterization of c19orf10, a novel synovial protein). Another scientific publication describes the 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). The recombinant produced protein showed an effect of enhancing proliferation on cultured hepatocellular carcinoma cells. It is noted that C19Orf10 was also called IL-25, IL-27 and IL-27W because it was initially thought to be an interleukin. However, the terms "IL-25" and "IL-27" have been used inconsistently in the art and have been used to denote 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 amino acid sequence of factor 1 shown in SEQ ID NO: 1 with the amino acid sequence of "IL-27" according to UniProt:Q8NEV9).Similarly, EP 2 130 547 A1 refers to a protein called IL-25 and discloses its use in the treatment of inflammation. This protein is also known as IL-17E in the art 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 by open reading frame 63 of human chromosome 19 (C19Orf63). This protein was described as a novel secreted factor INM02 in 2009 (Wang et al., Journal of Endocrinology 2009, Molecular cloning of a novel secreted peptide, INM02, and regulation of its expression by glucose). The presence of this protein was demonstrated in human serum using polyclonal antibodies. Furthermore, the correlation between cultured MIN6 (β-cells) and isolated rat pancreatic islets and the glucose concentration in the medium was shown. Correlation analysis between diabetes and the expression of INM2 showed no significant difference. The corresponding patent application claims the production of polyclonal antibodies 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 this protein as a novel secreted factor hHSS1 (human hematopoietic signal peptide-containing secretion 1) (Junes-Gill et al., J Neurooncol, 2011, hHSS1: a novel secreted facto and suppressor of glioma growth located at chromosome 19q13.33). The published data show the expression of hHSS1 in hematopoietic stem cells and suggest 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). Summary of the Invention Problems to be Solved by the Invention

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

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

[0011] In a third aspect, the present invention provides a nucleic acid encoding the proteins of the first and second aspects for enhancing the growth of non-transformed tissue or non-transformed cells and / or healing 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 growth 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 for use in enhancing the growth and / or inhibiting the healing and / or apoptosis of non-transformed tissues or non-transformed cells, comprising the protein of the first and / or second aspect and / or the nucleic acid of the third aspect and / or the vector of the fourth aspect and optionally a suitable pharmaceutical excipient.

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

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

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

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

[0018]

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[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 thereto but is limited only by the claims. Unless otherwise specified, 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 practice the present invention, unless otherwise specified, 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). Further, conventional clinical cardiological 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” (and its variations “comprises” and “comprising”) is to be understood as meaning that the stated integer or step or group of integers or steps includes, but does not exclude, any other integer or step or group of integers or steps. The singular forms used in this specification and the claims form “a”, “an”, and “the”, and refer to the plural unless the context clearly requires otherwise.

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

[0024] The term "open reading frame" (ORF) refers to a nucleotide sequence that can be translated into amino acids. Typically, such an ORF contains a start codon in a given reading frame, usually followed by a subsequent region having a length of a plurality of three nucleotides but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA). Typically, an ORF occurs naturally or is constructed artificially, i.e., by genetic engineering means. An ORF encodes a protein that, when translated, forms a peptide bond chain of amino acids.

[0025] The terms "protein" and "polypeptide" are used interchangeably herein and refer to a chain bound by any peptide bond regardless of length or post-translational modification. Proteins (including protein derivatives, protein variants, protein fragments, protein moieties, protein epitopes, and protein domains) that can be used in the present invention can be further modified by chemical modification. This is because such chemically modified polypeptides contain chemical groups other than the 20 natural amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of a polypeptide can result in one or more favorable properties compared to a non-polypeptide, such as enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to variants that can be used in the present invention include, but are not limited to, the following: pegylation, glycosylation of a non-glycosylated parent polypeptide; covalent bonding with a therapeutic small molecule such as an exenatide, albiglutide, taspoglutide, DPP4 inhibitor, incretin, and glucagon-like peptide 1 agonist including liraglutide; or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications applicable to variants that can be used in the present invention can occur co-translationally (during translation) or post-translationally.

[0026] The term "amino acid" includes natural amino acids and amino acid derivatives. In the context of the present invention, a hydrophobic non-aromatic amino acid is preferably any non-aromatic amino acid having a Kyte-Doolittle hydropathy index of 0.5 or more, more preferably 1.0 or more, and even more preferably 1.5 or more. Preferably, in the context of the present invention, the hydrophobic non-aromatic amino acid is 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-translation" refers to events that occur after the translation of a nucleotide triplet into an amino acid and the formation of a peptide bond with subsequent amino acids in the sequence. Such post-translation events can occur after a complete polypeptide has been formed or can already have occurred in a portion of the polypeptide that has already been translated during the translation process. Post-translation events typically change or modify the chemical or structural properties of the resulting polypeptide. Examples of post-translation events include, but are not limited to, events such as glycosylation or phosphorylation of amino acids, or cleavage by an endopeptidase of the peptide chain.

[0028] As used herein, the term "co-translation (during translation)" refers to events that occur during the translation of a nucleotide triplet into an amino acid chain. Such events typically change or modify the chemical or structural properties of the resulting amino acid chain. Examples of co-translation events include, but are not limited to, 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 "variant" refers to a polypeptide that differs from the polypeptide or fragment thereof from which it is derived by having one or more changes in its amino acid sequence. The polypeptide from which the protein variant is derived is also known as the parent polypeptide. Similarly, the fragment from which the protein fragment variant is derived is known as the parent fragment. Typically, variants are constructed by artificial, preferably genetic engineering means. Typically, the parent polypeptide is a wild-type protein or wild-type protein domain. Furthermore, the variants that can be used in the present invention are derived from homologs, orthologs, or paralogs of the parent polypeptide, or artificially constructed variants, and have at least one biological activity of the parent polypeptide. The changes in the amino acid sequence can be amino acid substitutions, insertions, deletions, N-terminal truncations, or C-terminal truncations that can occur at one or various sites, or any combination of these changes. In a preferred embodiment, the variants that can be used in the present invention have a total of 100 or fewer (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or fewer) changes (i.e., substitutions, insertions, deletions, N-terminal truncations, and / or C-terminal truncations) in the amino acid sequence. The amino acid substitutions can be conservative and / or semi-conservative and / or non-conservative. In a preferred embodiment, the variants that can be used in the present invention differ from the protein or domain from which it is derived by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 or fewer amino acid substitutions, preferably conservative amino acid changes.

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

[0031] [Table 1]

[0032] When the novel cysteine remains as a free thiol, the change from A, F, H, I, L, M, P, V, W, or Y to C is semi-conservative. Furthermore, one of ordinary skill in the art will recognize that glycine in a sterically bulky position should not be substituted and that P should not be introduced into portions of proteins having an α-helix or β-sheet structure.

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

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

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

[0037] The similarity of nucleotide and amino acid sequences, such as the percentage of sequence identity, can be determined by sequence alignment. Such alignments can be performed using various known algorithms, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) or the CLUSTALW2 algorithm (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948.) (which 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 of 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 pair alignment option), and protein weight matrix = Gonnet, gap open = 10, gap extension = 0.20, gap distance = 5, no end 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 of Altschul et al. (1990) J. Mol. Biol. 215:403-410. The BLAST protein search is 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 described at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome (expect threshold = 10, word size = 3, maximum matches in query range = 0, matrix = BLOSUM62, gap cost = existence: 11 extension: 1, composition adjustment = factor 1 and factor 2 conditional composition score matrix adjustment with a database of non-redundant protein sequences (nr) to obtain amino acid sequences homologous to the polypeptide).

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

[0040] As used herein, the term "host cell" refers to a cell having a nucleic acid of the invention (e.g., a plasmid or virus). Such host cells can be prokaryotic cells (e.g., bacterial cells) or eukaryotic cells (e.g., cardiomyocytes, plant, or animal cells). The cell may or may not be transformed. The cell can be, for example, an isolated cell in cell culture, or a part of a tissue that can be isolated by itself, or a part 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 and refer to the protein (human homolog) shown in NCBI reference sequence NM_019107.3, and its mammalian homologs (especially from mouse or rat). The amino acid sequence of the human homolog is encoded by open reading frame 10 (C19Orf10) of human chromosome 19. Preferably, the Factor 1 protein refers to a protein that comprises, consists essentially of, or consists of the core portion of human Factor 1 having the amino acid sequence shown in SEQ ID NO: 1. In a more preferred embodiment, the Factor 1 protein has the amino acid sequence shown in SEQ ID NO: 2.

[0042] The terms "Factor 2", "Factor 2 protein", or "Factor 2 polypeptide" are used interchangeably and refer to the protein (human homolog) shown in NCBI reference sequence NM_175063.4 and its mammalian homologs (particularly from mouse or rat). The amino acid sequence of human Factor 2 is encoded by open reading frame 63 (C19Orf63) on human chromosome 19. Preferably, the Factor 2 protein refers to a protein that comprises, consists essentially of, or consists of the core portion of human Factor 2 having the amino acid sequence shown in SEQ ID NO: 3. In a more preferred embodiment, the Factor 2 proteins have 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 terms "non-transformed tissue" or "non-transformed cell" refer to tissues and cells that exhibit physiological parameters of equivalent non-cancerous or precancerous cells or tissues. Such parameters include, for example but not limited to, cell cycle regulation, cell division rate, contact inhibition, anchorage-independent growth, or metabolism. Equivalent non-cancerous or precancerous cells or tissues can be healthy, damaged, or diseased.

[0044] The term "to heal" includes the regeneration and repair of living cells, tissues, organs, and entire living systems, and the partial or complete restoration of normal function. In the case of a tissue, organ, or entire living system, the term includes the processes of regeneration and repair in which cells in the body reduce the size of damaged or necrotic areas and replace them with new living tissue. Such replacement can occur by regeneration, for example, where necrotic cells are replaced with new cells that form a tissue similar to what was originally there, or by repair where 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 variant of the healing process. Thus, the term "to heal" in the context of the present invention includes all processes, and those skilled in the art will combine with this term, but preferably, the regeneration process should be promoted instead of processes that result in the production of non-functional tissue such as scar tissue. The term "to heal" in the context of the present invention preferably aims at promoting proliferation, migration, network formation, and angiogenesis.

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

[0046] The term "to inhibit apoptosis" refers to the ability of the protein, nucleic acid, vector, or pharmaceutical composition of the present invention to prevent a cell or cell population from undergoing apoptosis under conditions where a control cell or cell population would undergo apoptosis. For example, methods of measuring whether a cell undergoes apoptosis, such as by TUNEL assay, are well known to those skilled in the art. The description of this aspect further includes the definitions and explanations of terms used throughout this specification. These explanations and definitions are effective throughout the present application unless otherwise specified.

[0047] (Aspect) The elements of the present invention will be described below. Although the elements are described with specific embodiments, it should be understood that any number of them can be combined in any manner to generate further embodiments. The various described examples and preferred embodiments should not be construed as being limited only to the explicitly described embodiments. This specification should be understood to support and include combinations of the explicitly described embodiments with any number of disclosed and / or preferred elements. Further, unless the context indicates otherwise, any substitution and combination of all the elements described in this application should be considered to be disclosed in the specification of this application.

[0048] In a first aspect, the present invention provides a protein for use in enhancing the growth of, and / or healing, and / or inhibiting apoptosis in non-transformed tissue or non-transformed cells, in particular for enhancing the growth of non-transformed tissue, enhancing the growth of non-transformed cells, inhibiting the apoptosis of non-transformed tissue, or inhibiting the apoptosis of non-transformed cells, preferably comprising a Factor 1 protein having the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof or a variant having at least 80% sequence identity with SEQ ID NO: 1, consisting essentially of, or consisting of. In a particularly preferred embodiment of the present invention, the protein comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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. A preferred fragment of SEQ ID NO: 2 is the N-terminal signal sequence MAAPSGGWNGVGASLWAALLLGAVALRPAEA (SEQ ID NO: 35)is lacking. A person skilled in the art can, without undue burden, determine which positions in the parent polypeptide can mutate 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 that can be identified, aligned, and analyzed by bioinformatics methods well known in the art. Such analysis is exemplified in Example 7 and the results are shown in FIGS. 6 and 7. Mutations are preferably introduced into regions of the protein that are not completely conserved between species, preferably mammals, i.e., one or more of the amino acid positions mutate (not marked with a "*"). In a more preferred embodiment, only amino acids that are neither completely conserved (indicated by a "*") nor less conserved (indicated by a ":" or a ".") are changed. In a particularly preferred embodiment of the invention, the Factor 1 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 2. Such mutations may be present in the full-length protein represented by SEQ ID NO: 2 or in the protein lacking the N-terminal signal sequence represented by SEQ ID NO: 1.

[0050] The N-terminal deletion variant may have one or more amino acids deleted from amino acid positions 32 to 55 (based on SEQ ID NO: 2), i.e., from the N-terminal conserved region, in addition to the N-terminal signal. Thus, the N-terminal of the deletion factor 1 protein may be further at position 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 alternatively, the deletion factor 1 protein may have one or more of amino acid positions 146 to 173 (based on SEQ ID NO: 2) deleted from the C-terminal conserved region. Thus, the C-terminal of the deletion factor 1 protein may be at position 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, additional amino acid sequences for stabilizing or purifying the obtained protein. Examples of such amino acids include His6-tags (SEQ ID NO: 36), myc-tags, or FLAG-tags.

[0051] In such an embodiment, it is preferred to mutate the protease cleavage site within the protein of the first aspect of the present invention to stabilize the protein (see Segers et al. Circulation 2007, 2011). Those skilled in the art know methods for determining potential proteolytic cleavage sites within a protein. For example, the protein sequence can be submitted to websites that provide 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 cleavage sites with low frequency (less than 10):

[0052] [Table 2]

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

[0054] Factor 1 and Factor 2 showed growth-enhancing activity, particularly in enhancing angiogenesis. In a preferred embodiment, the protein of the first aspect is used to enhance the growth, preferably angiogenesis, of non-transformed tissue or non-transformed cells. Thus, it is convenient to use Factor 1 or Factor 2 in the treatment of diseases in which enhancing angiogenesis may be beneficial. Examples of such diseases are further illustrated below.

[0055] Enhancement of growth includes any degree of enhancement of cells or tissues compared to control cells or control tissues that do not receive the protein of the present invention in the context of the present invention. Cell growth can be measured, for example, by bromodeoxyuridine incorporation as described in Example 2. Enhancement of tissue growth can be determined, for example, by measuring the increase in weight or size of each tissue and histological methods. Such methods are well known in the art, and many of them are standard methods for clinical application.

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

[0057] In another preferred embodiment, the protein of the first aspect is used to inhibit apoptosis of non-transformed tissues or non-transformed cells. Thus, the protein of the present invention exhibits anti-apoptotic potential and protects cells or tissues from cell death by apoptosis. In this context, "protect" or "cytoprotective effect" means that the degree of cell death by apoptosis in cells treated with the factor 1 protein of the present invention is 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% reduced 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 are, for example, fragmented genomes that can be tested 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 an experimental heart failure animal model. For example, mice with post-ischemic systolic dysfunction can be treated with the protein, and the degree of apoptotic cardiomyocytes in the heart tissues of the treated mice and control mice can be evaluated. The peptide can be preferably administered parenterally, for example, intraperitoneally, intravenously, or subcutaneously. In a particularly preferred embodiment, the protein of the present invention exhibits the above functions, i.e., enhancement and healing of the growth of non-transformed tissues or non-transformed cells, and inhibition of apoptosis, preferably all of them. Fragments and variants of the factor 1 protein included in the present invention having at least 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90% and more preferably at least 100% of the amino acid sequence shown by 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 cell death by apoptosis.

[0058] In a second aspect, the present invention provides a protein comprising, consisting essentially of, or consisting of a Factor 2 protein having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof or a variant having at least 80% sequence identity with SEQ ID NO: 3 for use in enhancing the growth and / or healing of non-transformed tissue or non-transformed cells. In a preferred embodiment of the present invention, the Factor 2 protein comprises the amino acid sequence of SEQ ID NO: 3, a fragment thereof or a variant having at least 80% identity with SEQ ID NO: 3. Those skilled in the art can, without undue burden, determine which positions in the polypeptide can be mutated to what extent and which positions must be maintained 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%, 99% sequence identity with SEQ ID NO: 3.

[0059] The protein of the second aspect of the present invention may further comprise, for example, additional amino acid sequences for stabilizing or purifying the obtained protein.

[0060] In one aspect, it is preferred to mutate the protease cleavage site within the protein of the first aspect of the present invention to stabilize the protein. Suitable proteolytic cleavage sites 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, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4, a fragment thereof, or a variant having at least 80% identity with SEQ ID NO: 4. Preferred fragments are the N-terminal signal sequence MAAASAGATRLLLLLLMAVAA PSRARG’(SEQ ID NO: 37)is lacking. A person skilled in the art can, without undue burden, determine which positions in the polypeptide can be mutated to what extent and which positions must be maintained 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 exemplified in Example 8 and the results are shown in Figures 8 and 9. Mutations are preferably introduced only into regions of the protein that are not completely conserved between species (preferably mammals), i.e., one or more of the amino acid positions not marked with "*" are mutated. In a more preferred embodiment, only the amino acids that are neither completely conserved (indicated by "*") nor conserved to a lesser extent (indicated by ":" or ".") are changed. In a particularly preferred embodiment of the present invention, the factor 2 protein comprises the amino acid sequence of SEQ ID NO: 4 or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4.

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

[0063] In another preferred embodiment of the present invention, the protein of the second aspect of the present invention comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5, a fragment thereof, or a variant having at least 80% identity with SEQ ID NO: 5. Preferred fragments lack the N-terminal signal sequence MAAASAGATRLLLLLLMAVAAPSRARG (SEQ ID NO: 37). One of ordinary skill in the art can, without undue burden, determine which positions in the polypeptide can be mutated to what extent and which positions must be maintained 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 illustratively described in Example 9, and the results are shown in FIGS. 10 and 11. Mutations are preferably introduced only into regions of the protein that are not completely conserved between species (preferably mammals), i.e., one or more of the amino acid positions not marked with "*" are mutated. In a more preferred embodiment, only amino acids that are not completely conserved (indicated by "*") or conserved to a lesser extent (indicated by ":" or ".") are changed. In a particularly preferred embodiment of the present invention, the Factor 2 protein comprises the amino acid sequence of SEQ ID NO: 5 or a fragment thereof. Preferably, the protein has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 5.

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

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

[0066] Fragments and variants of the protein of the second aspect of the present invention having at least 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90%, more preferably at least 100% of that of the protein having the amino acid sequence shown by 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 protein of the second aspect is used to enhance the growth of non-transformed tissue or non-transformed cells. The enhancement of growth includes any grade of enhancement compared to control cells or tissues not administered the protein. The experimental methods for measuring growth are described above. Each growth measurement value for the protein of the second aspect of the present invention evaluated by bromodeoxyuridine incorporation is also described in Example 2.

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

[0069] In another aspect of the present invention, the protein of aspect 1 and / or 2 is administered in vivo, ex vivo, or in vitro, preferably in vivo. In a typical embodiment of administering the protein of the first aspect of the present invention and / or the protein of the second aspect of the present invention ex vivo or in vitro, growth is enhanced and / or healed and / or apoptosis is inhibited for tissue engineering to generate tissue for transplantation into an individual. The cells used for tissue engineering can be derived from the same individual as well as from another individual of the same or a different species. The method of removing the cells or tissue and the transplantation of the new tissue into an individual are not included in the present invention.

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

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

[0072] In a preferred embodiment, the non-transformed cell or the non-transformed tissue is a muscle cell or muscle tissue. Muscle includes all types of muscle known to those skilled in the art. Such muscle is, 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 non-transformed cell or the non-transformed tissue is an epithelial cell or epithelial tissue. In another preferred embodiment, the non-transformed cell or the non-transformed tissue is a nerve cell or nerve tissue.

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

[0074] In another preferred embodiment, the non-transformed cell or the non-transformed tissue belongs to or is derived from a specific part or organ of the body of an individual selected from the group comprising skin, bone, heart, cartilage, blood vessels, esophagus, stomach, intestine, gland, liver, kidney, lung, brain, and spleen. In a particularly preferred embodiment, the non-transformed cell or the non-transformed tissue belongs to or is derived from the heart.

[0075] In the case of damaged non-transformed cells or non-transformed tissue, it is further preferred that the damage results from a genetic / hereditary disease or an acquired disease resulting from, for example, ischemia, reperfusion injury, inflammation, infection, trauma, mechanical load, 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 diseased or damaged cells or tissues, it is preferred that the damage results from a disease associated with atrophy, hypoplasia, inflammation, injury, or trauma. It is particularly preferred that the disease is associated with injury. It is also particularly preferred that the disease is associated with trauma.

[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, muscular atrophy, myositis, central core disease, nemaline (rod) myopathy, centronuclear myopathy, myotubular myopathy, centronuclear myotubular myopathy, ophthalmoplegia, and mitochondrial myopathy. Muscular dystrophy can 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 can be selected from the group consisting of myositis ossificans, fibromyositis, idiopathic inflammatory myopathy (e.g., dermatomyositis, polymyositis, and inclusion body myositis), and pyomyositis.

[0078] In another preferred embodiment of the present invention, the disease is primary or secondary cardiomyopathy. Primary cardiomyopathy is selected from genetic cardiomyopathy and cardiomyopathy caused by spontaneous mutation. Cardiomyopathy includes, for example, but is not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular myocardial non-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 ischemic cardiomyopathy caused by atherosclerosis or other coronary artery diseases, cardiomyopathy caused by myocardial infections or poisoning, hypertensive heart disease caused by pulmonary hypertension and / or arterial hypertension, and heart valve diseases, and ischemic cardiomyopathy caused by atherosclerosis or other coronary artery diseases is particularly preferred.

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

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

[0082] The above Factor 1 and Factor 2 proteins, fragments, or variants are used to treat or ameliorate atrophy; hypogenesis; inflammation, injury; trauma, ischemia; reperfusion injury; inflammation; infectious diseases; trauma; mechanical load; poisoning; primary or secondary cardiomyopathy, preferably genetic cardiomyopathy and cardiomyopathy caused by spontaneous mutation. Cardiomyopathy includes, for example but not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular myocardial non-compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), tako-tsubo cardiomyopathy, Loeffler endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; myocardial infarction or for improvement of left ventricular systolic function. The above Factor 1 and Factor 2 proteins, fragments, or variants can also be used in the treatment methods of the described pathological conditions and diseases respectively.

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

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

[0085] The nucleic acid sequence can be optimized to enhance expression in a host cell. Parameters to be considered include C:G content, preferred codons, and avoidance of inhibitory secondary structures. These factors can be variously combined to obtain a nucleic acid sequence with enhanced expression in a specific host (see, for example, Donnelly et al., International Publication No. WO 97 / 47358). Enhancing the ability of a specific sequence to be expressed in a specific host requires several empirical experiments. Such experiments involve measuring the expression of the expected nucleic acid sequence and varying the sequence if necessary. A number of different coding nucleic acid sequences can be obtained starting from the known degeneracy of the genetic code and a specific amino acid sequence. The degeneracy of the genetic code results from the fact that almost all amino acids are encoded by various combinations of nucleotide triplets, i.e., "codons". The translation of a specific codon into a specific amino acid is well known in the art (see, for example, Lewin GENES IV, p. 119, Oxford University Press, 1990).

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

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

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

[0089] Polyadenylation signals that can be used as part of a gene expression cassette include the minimal rabbit [β] globin polyadenylation signal and the bovine growth hormone polyadenylation (BGH) (Xu et al., Gene 272:149-156, 2001, Post et al., U.S. Patent U.S.5,122,458). Examples of additional regulatory elements useful for enhancing or regulating gene expression that may be present or for 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). The enhancer region can be associated with a promoter.

[0090] The expression of the 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. The regulatory steps that are contemplated include, for example, but are not limited to, transcription initiation, promoter clearance, transcription elongation, splicing, export from the nucleus, mRNA stability, translation initiation, translation efficiency, translation elongation, and protein holding. Other regulatory steps that affect the concentration of factor 1 or factor 2 polypeptide inside the cell affect the half-life of the protein. Such regulatory steps include, for example, regulation of protein denaturation. Since the protein of the present invention includes a secreted protein, the protein can be directed to the secretory pathway of the host cell. Secretion efficiency regulates the concentration of each protein outside the cell, along with the regulatory steps for expression and protein stability. The outside of the cell can refer to, for example, but is not limited to, the culture medium, tissue, intracellular matrix or lumen, or body fluid, such as blood or lymph fluid. The regulation of the above regulatory steps can 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 related to the transcription of nucleic acid. This transcriptional regulation can be achieved by the use of a cell type or tissue type specific promoter sequence. The results of this cell type or tissue type specific regulation can 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 the expression is limited to each cell or tissue type. Cell or tissue type specific promoter sequences are well known in the art and are available for a wide range of cell or tissue types.

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

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

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

[0094] Nucleic acids encoding the above Factor 1 and Factor 2 proteins, fragments, or variants are preferably used to treat or ameliorate atrophy; hypogenesis; inflammation, injury; trauma, ischemia; reperfusion injury; inflammation; infectious diseases; trauma; mechanical load; poisoning; primary or secondary cardiomyopathy, preferably genetic cardiomyopathy and cardiomyopathy caused by spontaneous mutations. Cardiomyopathies include, for example, but are not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular myocardial non-compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), tako-tsubo cardiomyopathy, Loeffler endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; those in the improvement of myocardial infarction or left ventricular systolic function.

[0095] In a fourth aspect, the present invention provides a vector comprising the nucleic acid or expression system 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.

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

[0097] As used herein, the term "vector" refers to a protein and / or polynucleotide or a mixture thereof that introduces a protein and / or nucleic acid contained therein into a cell or into which they can be introduced. In the context of the present invention, the target gene encoded by the introduced polynucleotide is preferably expressed in the host cell when the vector(s) is introduced. Examples of suitable vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores.

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

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

[0100] The nucleic acid 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 a host cell, tissue, or individual using a vector suitable for therapeutic administration. Suitable vectors can preferably supply the nucleic acid into the 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 an individual after myocardial infarction, and the healing may include improvement of left ventricular systolic function and may be associated with an increase in capillary density in the border zone infarction. Furthermore, the use may reduce the mortality rate after myocardial infarction. Methods for measuring parameters such as improvement of left ventricular systolic function, increase in capillary density in the border zone infarction, and reduction of mortality rate after myocardial infarction are well known in the art and are typically described in Examples 4 to 6.

[0102] Vectors containing nucleic acids encoding the above Factor 1 and Factor 2 proteins, fragments, or variants are preferably used to treat or ameliorate atrophy; hypogenesis; inflammation, injury; trauma, ischemia; reperfusion injury; inflammation; infectious diseases; trauma; mechanical load; poisoning; primary or secondary cardiomyopathy, preferably hereditary cardiomyopathy and cardiomyopathy caused by spontaneous mutations. Cardiomyopathies include, for example, but are not limited to, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular myocardial non-compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), takotsubo cardiomyopathy, Loeffler endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or obesity-related cardiomyopathy; those in improving myocardial infarction or left ventricular systolic function.

[0103] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the protein of the first and / or second aspect and / or the nucleic acid of the third aspect and / or the vector of the fourth aspect and optionally a carrier for use in enhancing the growth and / or healing and / or inhibiting apoptosis of non-transformed tissue or non-transformed cells. The terms "enhancing the growth and / or healing and / or inhibiting apoptosis of non-transformed tissue or non-transformed cells" have the meanings and preferred meanings as described above.

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

[0105] "Pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

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

[0107] The term "active ingredient" refers to a substance in a pharmaceutical composition or formulation that is biologically active, i.e., that confers a pharmaceutical value. In the context of the present invention, the active ingredient represents the protein of the first and / or second aspect and / or the nucleic acid of the third aspect and / or the vector of the fourth aspect. A pharmaceutical composition can contain one or more active ingredients that can act independently of one another or in cooperation. 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 salts of a polypeptide, for example, but not limited to. 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 (such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid). Further, when the peptide has an acidic moiety, suitable pharmaceutically acceptable salts thereof include alkali metal salts (such as sodium or potassium salts); alkaline earth metal salts (such as calcium or magnesium salts); and salts formed with suitable organic ligands (such as ammonium, quaternary ammonium, and amine cations formed using a counteranion, such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkylsulfonate, and arylsulfonate).Specific examples of pharmaceutically acceptable salts include, but are not limited to: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, acid tartrate, borate, bromide, butyrate, calcium edetate, camphor, camphorsulfonate, cantharate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylic acid, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinol, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfonate, mokate, 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, theocrylates, tosylate, triethiodide, undecanoate, valerate, etc. (see, e.g., S.M. Berge et al., "Pharmaceutical salts", J. Pharm. Sci., 66, pp. 1-19 (1977)).

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

[0110] In a preferred embodiment of the present invention, the pharmaceutical composition is customized for treating a disease or disorder. As used herein, "treating" a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing symptoms characteristic of the disorder being treated; (c) suppressing the worsening of 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) reducing the mortality rate after the occurrence of a disease or disorder; (g) curing; and (h) preventing the disease. As used herein, "preventing" 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, cure by the pharmaceutical composition of the present invention includes treating an individual after myocardial infarction, and the cure includes improvement of left ventricular systolic function and may be associated with an increase in capillary density in the border zone infarction. Furthermore, the cure can reduce the mortality rate after myocardial infarction. Methods that can be used to measure parameters such as improvement of left ventricular systolic function, increase in capillary density in the border zone infarction, and reduction of mortality rate after myocardial infarction are well known in the art and are typically described in Examples 4 to 6.

[0112] The pharmaceutical composition contemplated by the present invention can be formulated by various methods well known to those skilled in the art. For example, the pharmaceutical composition of the present invention can be in liquid form, such as in the form of a solution, emulsion, or suspension. Preferably, the pharmaceutical composition of the present invention is formulated for parenteral administration, preferably intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intranasal, intraperitoneal, intracoronary, intracardiac administration, or administration via mucosa, preferably intravenous, subcutaneous, or intraperitoneal administration. Formulations for oral or rectal administration are also possible. Preferably, the pharmaceutical composition of the present invention is in the form of a sterile aqueous solution that may contain other substances, such as salts or glucose sufficient to make the solution isotonic with blood. The aqueous solution should be appropriately buffered as necessary (preferably pH 3-9, more preferably pH 5-7). The pharmaceutical composition is preferably in unit dosage form. In such form, the pharmaceutical composition is subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form can be a packaged formulation, a package containing a discrete amount of the pharmaceutical composition, such as a vial or an ampoule. Administration of the pharmaceutical composition is preferably via intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intranasal, intraperitoneal, intracoronary, or intracardiac routes, but other routes of administration known in the art are also included.

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

[0114] It is more preferred that the pharmaceutical composition be administered one 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 period for administering the medicine 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 one or more bolus injections and / or each dosing period administered as an infusion.

[0116] Aspects 1 to 5 of the present invention are based on the inventors' finding that Factor 1 and Factor 2 are potent angiogenesis-stimulating molecules in vitro and in vivo. Thus, the above proteins, nucleic acids, and vectors are expected to be used ex vivo, for example, as therapeutic agents for stimulating the above cells ex vivo or for use in cell culture applications.

[0117] However, based on the finding of the angiogenesis-stimulating activities of Factor 1 and 2, the inventors examined whether Factor 1 and Factor 2 are targets for anti-angiogenesis therapy, respectively. The inventors succeeded in inhibiting angiogenesis by inhibiting Factor 1 or Factor 2.

[0118] Using anti-angiogenesis strategies, treat cancer conditions and other disorders, such as age-related macular degeneration, in which angiogenesis is involved in the progression of the disease (see, for example, Ferrara N and Kerbel RS (2005) Nature:438:967-974 or Potente M, et al. (2011) Cell. 146(6):873-887). Further, in a further aspect, the present invention relates to the anti-angiogenic properties of inhibitors of Factor 1 and Factor 2. In these aspects, the above definitions are equally applicable to the terms defined. Further, the specific definitions described in Aspects 1 to 5 and, for example, the description of the terms "vector" and preferred embodiments of preferred vectors are also applicable 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 an inhibitor of Factor 1 and / or 2 protein in the treatment or prevention of a disease, preferably a disease in which angiogenesis contributes to the development or progression of the disease, for medical use. The term "inhibitor" refers to a compound that interferes with the angiogenesis-stimulating activity of Factor 1 or Factor 2. The inhibitor prevents the production, circulation, and / or secretion of Factor 1 or Factor 2 in the cells by acting on the transcription and / or translation of the mRNA encoding Factor 1 or Factor 2, and / or at the site of disease development or progression. Further, the inhibitor can act by specifically binding to Factor 1 or Factor 2 protein or a cellular protein to which Factor 1 or Factor 2 protein specifically binds, preferably its cell receptor. Such binding can prevent or interfere with the natural interaction of Factor 1 or Factor 2 with other cellular proteins, preferably their respective cell receptors. Those skilled in the art are well aware of methods for interfering with the binding of a receptor to its agonist, and this knowledge can be used to design appropriate inhibitors of Factor 1 and Factor 2. Additionally, the inhibitor can be derived from Factor 1 or Factor 2 protein itself by deleting or mutating portions of Factor 1 and Factor 2 proteins that exhibit angiogenesis-promoting functions of Factor 1 and Factor 2, respectively. Such inactivated mutants or deleted Factor 1 or Factor 2 will compete with wild-type Factor 1 and Factor 2 for their natural binding partners. Compounds that interfere with the angiogenesis-stimulating activity of Factor 1 or Factor 2 reduce the activity by at least 20%, preferably at least 30%, more preferably at least 40%. In the context of an inhibitor that specifically binds to Factor 1 or Factor 2, or consists essentially of or consists of mutants or fragments of Factor 1 or Factor 2, respectively, they preferably exhibit inhibition of Factor 1 protein or Factor 2 protein at this level at equimolar concentrations. A preferred assay that can be used to measure the inhibition of angiogenesis-stimulating activity is described in Example 10 of this specification.To determine whether a certain inhibitor has this activity in equimolar amounts, the molar amounts of Factor 1 and 2 and each inhibitor must be measured. Factor 1, represented by SEQ ID NO: 1, has an MW of 15.84 kD, Factor 2, represented by SEQ ID NO: 3, has an MW of 21.57 kD, and the molecular weight of IgG is approximately 150 kD. Thus, 100 ng of Factor 1 and 947 ng of Factor 1-specific IgG are approximately equimolar, and 100 ng of Factor 2 and 695 ng of Factor 2-specific IgG are approximately equimolar. It is clear from Panels A and B of Figure 12, respectively, that the Factor 1- and Factor 2-specific antibodies described herein are inhibitors of the Factor 1 protein or Factor 2 protein in that sense. In the context of an inhibitor that interferes with the transcription and / or translation of the mRNA encoding Factor 1 or Factor 2, the level of inhibition is preferably measured based on the protein produced in cells that naturally produce Factor 1 or Factor 3. Those skilled in the art are well aware of many methods for measuring the amounts of mRNA encoding Factor 1 or Factor 3 and Factor 1 or Factor 2 protein and can be used to evaluate the ability of a compound to interfere with the transcription and / or translation of the mRNA encoding Factor 1 or Factor 2. Preferably, the Factor 1 protein comprises, consists essentially of, or consists of the amino acid sequence represented by SEQ ID NO: 1, and Factor 2 comprises, consists essentially of, or consists of the amino acid sequence represented by SEQ ID NO: 3, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 1 or 3.

[0120] In the context of the present invention, the inhibitor is preferably a protein comprising, consisting essentially of, or consisting of an inhibitory fragment or inhibitory mutant of the amino acid sequence represented by SEQ ID NO: 1 or 3, or a variant thereof having at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 3. It is well known in the art that a protein whose function is exhibited by protein-protein interaction via a receptor contains a domain necessary for binding to the receptor and a domain that stimulates the receptor to transmit a signal intracellularly. Thus, those skilled in the art are well aware of methods for producing inhibitory fragments or mutants of such receptor-binding proteins. For example, a series of N- and / or C-terminal truncated Factor 1 or Factor 2 proteins can be produced and their angiogenesis-stimulating activity can be tested in the assay described in Example 2. Then, the ability of those fragments that no longer exhibit angiogenesis-stimulating activity to inhibit the angiogenesis-stimulating activity of Factor 1 or 2 protein can be tested in the assay described in Example 10. Mutants of Factor 1 and 2 can be generated, for example, by alanine scanning mutagenesis as known in the art. In alanine scanning, a series of mutants are generated in which each mutant contains one, two, three or more amino acids that have been mutated to alanine (so-called cassette), and the position of the cassette within Factor 1 or 2 is different. Factor 1 or 2 mutants that have lost angiogenesis-stimulating activity can be re-identified 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 a variant having at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 3, or Factor 1 or 3, or a variant having at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 3. The term "ligand" represents a chemical moiety 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 can be a peptidomimetic.

[0122] As used herein, the term "immunoglobulin (Ig)" refers to a glycoprotein that confers immunity of the immunoglobulin superfamily. "Surface immunoglobulin" includes, but is 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. It binds to the membrane of effector cells through the transmembrane region. Typically, the term "antibody" as used herein refers to a secreted immunoglobulin that lacks a transmembrane region and can be released into the bloodstream and body cavities. Antibodies are classified into various isotypes based on the heavy chains 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 the antibody, and these chains play different roles and are found in IgA, IgD, IgE, IgG, and IgM antibodies that bring about appropriate immune responses against different types of antigens. The individual heavy chains differ in size and composition, with α and γ containing approximately 450 amino acids, while μ and ε have 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 that are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) that incorporate more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the heavy and light chains can be determined as known in the art. For example, the following set of rules can be used to find the CDRs within each antibody light chain sequence and heavy chain sequence: Light chain CDR-1: Start: about residue 24, the residue before CDR-1 is always Cys, and the residue after CDR1 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 the residue before Val-Tyr, Ile-Lys, Ile-Phe, length is always 7 residues; Light chain CDR-3: Start: Always at the 33-residue compound after the end of CDR-2; always the residue before Cys, always the residue after Phe-Gly-XXX-Gly, length: 7 - 11 residues Heavy chain CDR-1: Start: Always about 4 residues after Cys, residue 26 (based on the Chothia AbM definition, the Kabat definition starts after 5 residues); always the residue before Cys-XXX-XXX-XXX; always the residue after Trp. Typically Trp-Val, and Trp-Ile, Trp-Ala, length: 10 - 12 residues [AbM definition, the Chothia definition excludes the last 4 residues); Heavy chain CDR-2: Start: Always 15 residues after the end of the Kabat / AbM definition, of the heavy chain CDR-1; typically the residue before Leu-Glu-Trp-Ile-Gly (many variations), the residue after Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala, Length Kabat definition 16 - 19 residues (definition according to AbM; the Chothia definition ends 7 residues earlier) Heavy chain CDR-3: Start: Always 33 residues after the end of the heavy chain CDR-2 (always 2 amino acid residues after Cys); always the residue before Cys-XXX-XXX (typically Cys-Ala-Arg); always the residue after Trp-Gly-XXX-Gly; length: 3 - 25 residues. This set of rules is known to those skilled in the art and can also be found at http: / / www.bioinf.org.uk / abs / #cdrid.

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

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

[0125] The term "antigen-binding fragment" refers to an antibody fragment that retains the ability to specifically bind to an antigen or antigenic protein but lacks some or all of the other structural features or portions of the antibody, including artificial constructs. 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 (sdAbs), nanobodies, single-chain Fv, divalent single-chain variable fragments (di-scFvs), tandem scFvs, diabodies, single-chain diabodies These include scDBs, triabodies, bispecific T cell engagers (BiTEs), or dual affinity retargeting molecules (DART molecules).

[0126] "Fab fragments" (also called "Fab portion" or "Fab region") each contain a single antigen-binding site and a remaining "Fc fragment" (also called "Fc portion" or "Fc region"), the name reflecting its ability to crystallize readily. "Fab' fragment" refers to a Fab fragment that also contains the hinge region of an Ig molecule, and "F(ab')2 fragments" are fragments that are chemically or disulfide-linked to the Fab fragment. It is understood that a "single-chain Fv (scFv)" fragment comprises two Fab' fragments bound together by a single VH domain. sdAb (Desmyter et al. 1996) and "nanobodies" contain only a single VH domain, whereas "single-chain Fv (scFv)" fragments comprise a short linker. 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 made by joining two scFvs (scFvA-scFvB). This can be done by producing a single peptide chain with two VH and two VL regions, resulting in "tandem scFvs" (VHA-VLA-VHB-VLB). Another possibility is to make scFvs with a linker that is too short to fold the two variable regions together, forcing the scFvs to dimerize. Usually, linkers of 5 residues in length are used to make these dimers. This type is known as "diabodies". Even shorter linkers (1 or 2 amino acids) between the VH and VL domains result in the formation of monospecific trimers, so-called "triabodies" or "tribodies". Bispecific diabodies are formed by expressing chains with the sequences VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. A single-chain diabody (scDb) contains VHA-VLB and VHB-VLA fragments linked by a linker peptide (P) of 12 to 20 amino acids, preferably 14 amino acids (VHA-VLB-P-VHB-VLA). "Bispecific T cell engagers (BiTEs)" are fusion proteins consisting of two scFvs of different antibodies, where one scFv binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. 2004). Bispecific affinity retargeting molecules ("DART" molecules) are diabodies further stabilized by a C-terminal disulfide bridge.

[0127] The term "antibody-like protein" refers to a protein that has properties similar to an antibody that binds to an antigen or antigenic protein that does not necessarily have the structural characteristics of an antibody. Antibody-like proteins can occur naturally or can be designed artificially, for example, by biotechnology. Examples of natural antibody-like proteins include, but are not limited to, antigen-binding proteins such as the lipocalin family, which typically represents 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-strands as their central folding motif and contain six hypervariable loops that bind to each pair of β-strands at one end of this barrel structure. These loops form the entrance to the binding pocket. The structural diversity among members of the lipocalin family reflects the differences in the shape and chemical properties of their binding partners. Thus, although consisting of a single polypeptide chain and being 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 a peptide having a known affinity for a certain target or by inserting the peptide into a scaffold protein to combine the binding properties of the peptide with the desirable beneficial properties of the scaffold carrier. Those skilled in the art are familiar with such scaffold-based proteins. As used herein, the term "scaffold protein" refers to a protein that has structural rigidity, i.e., folds into a stable tertiary structure. The amino acids of the scaffold protein appear to occupy specific three-dimensional positions within the scaffold protein. Thus, substituting one or more of the amino acids of the scaffold protein with a polypeptide of appropriate length, the polypeptide will occupy a position similar to that of the substituted one. This allows for the placement of a specific polypeptide at a specific three-dimensional position and / or orientation within the scaffold protein.Furthermore, scaffold proteins can be used as alternatives to antibodies for molecular recognition (see, for example, Skerra A.: (2007) Curr. Opin. Biotechnol. 2007, 18:295-304, or Skerra A. (2000) J. Mol. Recognit. 2000, 13:167-187). Examples of such scaffold proteins include the Fyn SH3 domain, which contains two domains that can be mutated to confer novel binding specificities to the SH3 domain. Methods for selecting a Fyn SH3 domain that specifically binds to a certain antigen are described, for example, in WO2000 / 072742 or WO2008 / 022759.

[0128] In the context of the present invention, the term "peptide mimetic" refers to any molecule whose essential elements (active groups) are spatially very similar to those of a natural peptide or protein, retain the ability to interact with a biological target, and produce the same biological effect. Peptide mimetics typically include small protein-like chains designed to resemble peptides, which can be obtained, for example, by modifying existing peptides or designing similar systems (such as peptoids and β-peptides) that are similar to peptides. Regardless of the approach, chemical structure changes are designed to conveniently adjust molecular properties, such as increasing or decreasing stability and biological activity. Thus, modifications include, but are not limited to, unnatural peptide changes that include changes in the backbone and incorporation of non-natural amino acids.

[0129] The terms "specifically bind" or "specifically binding" with respect to an antigen, such as Factor 1 or Factor 2, refer to the ability of a ligand to bind to an epitope of the antigen with high affinity. In that context, "high affinity" means that the Kd of the interaction is less than 1 x 10 -5 M, preferably less than 1 x 10 -6 M, more preferably less than 1 x 10 -7 M, even more preferably less than 1 x 10 -8 M, and most preferably less than 1 x 10-9 means 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 an mRNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 or 3 or a variant having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 3. Those skilled in the art are well aware of methods for determining the sequence of such nucleic acids based on the genomic sequence encoding the protein comprising the amino acid sequence of SEQ ID NO: 1 or 3. Examples of such inhibitory nucleic acids include siRNAs specific for the mRNA encoding Factor 1 or Factor 2.

[0131] When the inhibitor of 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. Thus, in a seventh aspect, the present invention provides a nucleic acid encoding an inhibitor of the sixth aspect of the present invention for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease. The nucleic acid can further comprise any of the elements described in the context of the third aspect of the present invention.

[0132] Accordingly, in an eighth aspect, the present invention provides a vector comprising a nucleic acid of the sixth aspect of the present invention for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease. The term "vector" in the context of the seventh aspect has the same meaning as that 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, adenoviral vectors, adeno AccompanyViral (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retrovirus vectors, and lentivirus vectors are included. Metastasis Further, in a ninth aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease in which angiogenesis contributes to the development or progression of the disease, the inhibitor of the sixth aspect, the nucleic acid of the seventh aspect, or the vector of the eighth aspect, and optionally a suitable pharmaceutical excipient. This pharmaceutical composition may also include any of the components described in the fifth aspect of the present invention.

[0134] The term "disease in which angiogenesis contributes to the development or progression of the disease" used in the sixth to ninth aspects of the present invention refers to a disease in which the proliferation of cells that form blood vessels occurs during the onset, occurrence, and / or progression of the disease. Cells that can form and proliferate blood vessels include endothelial cells that line the inside of blood vessels and smooth muscle cells that form the blood vessel wall. Endothelial cells and smooth muscle cells do not proliferate in healthy blood vessels. These cells proliferate, for example, in response to injury or chemical signals (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 neovascularization diseases, abnormal blood vessel formation is the cause of the disease, and in some diseases such as benign or malignant tumors, angiogenesis occurs during the progression of the disease, supplying oxygen and nutrients to the growing tumor mass. In this disease, angiogenesis is not the cause of the disease, but promotes the progression of the disease. Neovascularization of malignant tumors also contributes to metastasis by providing a route for tumor cells to escape from the tumor mass.

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

[0136] The inventors have confirmed that antibodies against certain epitopes of Factor 1 and Factor 2 interfere with the angiogenesis-stimulating functions of Factor 1 and Factor 2, respectively, i.e., they are antagonist antibodies. Thus, in a tenth aspect, the present invention is directed to ligands of Factor 1 and Factor 2 that inhibit the angiogenesis-stimulating activities of Factor 1 and Factor 2, respectively. Antibodies or fragments thereof that inhibit the angiogenesis-stimulating activities of Factor 1 and Factor 2, respectively, are particularly preferred. The inventors have succeeded in generating examples of such inhibitory antibodies by producing antibodies that specifically bind to domains exposed on the surfaces 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 described in the general definitions section are applied equally to the specific definitions in the context of the sixth aspect of the present invention.

[0137] Accordingly, 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 that specifically binds to a region of another factor 1 protein corresponding to this epitope, preferably an antibody or a fragment thereof, or 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 to 195 of SEQ ID NO: 3, or a ligand that specifically binds to a region of another factor 2 protein corresponding to this epitope, preferably an antibody or a fragment thereof, or 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" represents, for example, an amino acid sequence derived from another factor 1 or 2 protein that aligns with the indicated amino acid sequence of factor 1 or 2 using standard alignment means such as ClustalW and the above standard parameters. Figures 6 to 11 show various such alignments of factor 1 and 2 proteins. One 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. (Example)

[0140] The present invention is further illustrated by examples to assist in better understanding. The examples do not limit the scope of the present invention in any way.

Example 1

[0141] In a multi-site placebo-controlled clinical trial, the effect of intracoronary injection of autologous bone marrow cells in AMI patients was tested by one of the inventors (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+-enriched 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, 4000 expressed sequence tags (ESTs) most strongly expressed by CXCR4+ bone marrow cells in the microarray were tested. Using a series of bioinformatics tools, putative secreted factors characterized by the deletion of an N-terminal signal peptide, a mitochondrial or nuclear signal peptide, an endoplasmic reticulum retention sequence, and a transmembrane domain were identified from among these ESTs. A total of 283 putative secreted factors were identified, and 117 of them 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. After culturing the transfected HEK cells in serum-free medium for 30 hours, a conditioned culture supernatant was obtained. The conditioned HEK cell supernatant was individually tested for angiogenic stimulating effects in a microvascular angiogenesis assay and for cytoprotective effects in a cardiomyocyte death assay. This screening led to the identification of two secreted proteins; "Factor 1" showed angiogenic stimulating effects and cytoprotective effects in the above assays; "Factor 2" showed angiogenic stimulating effects in the above assays.

[0142] The sequences identified by screening and each mouse or human homolog are as follows: Factor 1: Human Factor 1 was identified by 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 NCBI reference sequence: NM_019107.3 (SEQ ID NO: 6). The amino acids of human factor 1 are shown in FIG. 6 (SEQ ID NO: 2).

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

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

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

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

[0147] The secreted mouse homolog was used in Examples 4-6 and Figures 3-5: Mus musculus hematopoietic signal peptide-containing secretory 1 (2310044H10Rik) mRNA, complete cds, or spliced. 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 secreted form of mouse factor 2 is shown in Figure 8 (SEQ ID NO: 24).

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

Example 2

[0149] To confirm the angiogenesis-stimulating activity observed in screening, both factors (human homologs encoded by the nucleic acid sequences shown in SEQ ID NOs: 6 and 8) were produced as His-tagged recombinant proteins in COS7 cells. As shown in Figure 1, recombinant factor 1 and recombinant factor 2 promoted angiogenesis-stimulating effects in a dose-dependent manner in cultured human endothelial cells. Human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) 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 calf serum (FCS), human recombinant VEGF-A (R&D Systems), or various concentrations of the indicated recombinant human factor 1 (SEQ ID NO: 2) or factor 2 (SEQ ID NO: 4). (A) HCAEC proliferation was measured by bromodeoxyuridine incorporation. (B) HCAEC migration was evaluated after wounding a confluent endothelial cell monolayer 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 (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 (mouse homolog encoded by the nucleic acid sequence shown in SEQ ID NO: 7) was produced as a His-tagged recombinant protein in COS7 cells. As shown in Figure 2, recombinant factor 1 promoted an anti-apoptotic effect in a dose-dependent manner in cultured cardiomyocytes. Ventricular cardiomyocytes were isolated from 1 - 3-day-old Sprague-Dawley rats by Percoll density gradient centrifugation. The cardiomyocytes were exposed to simulated ischemia for 180 minutes (5% CO 2 / 95% N2 atmosphere in glucose-free medium containing 2-deoxyglucose), then in the presence or absence of recombinant human GDF-15 (R&D Systems, a known anti-apoptotic cytokine) or various concentrations of the indicated recombinant mouse factor 1 for 60 minutes, followed by simulated reperfusion (5% CO 2 / returned to glucose-containing medium in 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 examine the therapeutic potential of factor 1 and factor 2 in the setting of AMI, recombinant adenoviruses encoding the murine homologs of factor 1 or factor 2 (nucleic acid sequences shown in SEQ ID NOs: 7 and 10) were generated and tested in a mouse model of AMI. Adenoviral expression of both factors resulted in improved left ventricular systolic function 28 days after infarction. This was associated with an increase in capillary density in the border zone infarct (Figure 3).

[0152] Mouse factor 1 or factor 2 cDNA was cloned into replication-deficient adenoviruses using the AdEasy XL vector system (Stratagene). A replication-deficient adenovirus encoding galactosidase (lacZ) was used as a control. Viruses were purified using an Adeno-X virus purification kit (BD Biosciences). Male C57BL / 6 mice, 10 - 12 weeks old, were anesthetized and ventilated with isoflurane (1 - 2%), and then permanent left anterior descending coronary artery (LAD) ligation was performed. Viruses (5x10 9The p.f.u. 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 the border zone infarct 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 examine the therapeutic potential of Factor 1 and Factor 2 when used as recombinant proteins in a setting of reperfused AMI (closely mimicking the clinical situation of AMI patients who received reperfusion therapy), male C57BL / 6 mice, 10 - 12 weeks old, were subjected to 1-hour coronary artery ligation (ischemia) and then reperfused for 28 days. The mice were treated s.c. with both factors for the first 7 days after reperfusion. Factor 1 and Factor 2 (mouse homologs; amino acid sequences corresponding to SEQ ID NOs: 13 and 24) were generated as His-tagged recombinant proteins in HEK293 cells. Treatment with recombinant Factor 1 or recombinant Factor 2 resulted in a significant improvement in left ventricular systolic function 28 days after infarction. This was associated with a significant increase in capillary density in the border zone infarct (Figure 4). Male C57BL / 6 mice, 10 - 12 weeks old, were anesthetized and ventilated with isoflurane (1 - 2%), and temporary left anterior descending coronary artery ligation was performed for 1 hour, followed by reperfusion for 28 days. The mice were injected s.c. once with recombinant Factor 1 or Factor 2 (10 μg each) at the time of reperfusion (control mice were injected with PBS). Subsequently, recombinant Factor 1 or Factor 2 was continuously infused s.c. for 7 days using an Alzet minipump (10 μg / day). Control mice were infused with PBS. (A) Left ventricular systolic function (fractional area change, FAC) was evaluated by transthoracic echocardiography 28 days after reperfusion (N = 10 - 13 mice / group). (B) Isolectin-positive capillary density in the border zone infarct 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 examine whether the use of Factor 1 and Factor 2 as recombinant proteins can increase survival after reperfused AMI, coronary artery ligation was performed on mice for 1 hour and then reperfused for 28 days. Both factors were s.c. injected into the mice for the first 7 days after reperfusion. Factor 1 and Factor 2 (mouse homologs; SEQ ID NOs: 7 and 10) were generated as His-tagged recombinant proteins in HEK293 cells. Treatment with recombinant Factor 1 or recombinant Factor 2 significantly improved survival during the first 28 days after occlusion (Figure 5). Male C57BL / 6 mice at 10 - 12 weeks of age were anesthetized and ventilated with isoflurane (1 - 2%), and temporary left anterior descending coronary artery ligation was performed for 1 hour and then reperfused for 28 days. The mice were given a single s.c. injection of recombinant Factor 1 or Factor 2 (10 μg each) at the time of reperfusion (PBS was injected into control mice). Then, recombinant Factor 1 or Factor 2 was continuously s.c. infused for 7 days (10 μg / day) using an Alzet mini pump. PBS was infused into control mice. The mice were examined daily for 28 days to evaluate post-infarct survival. N = 29 PBS-treated mice; N = 25 Factor 1-treated mice; N = 15 Factor 2-treated mice.

Example 7

[0155] Homologous sequences to the protein encoded by human C19Orf10 were identified 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. SEQ ID NO: 2 was used as the matrix. The parameters used were default parameters: 11 extension: 1, composition adjustment = conditional composition score matrix adjustment with the non-redundant protein sequence (nr) database. From the identified sequences, one sequence was selected from examples from various vertebrates, mainly mammals, and one sequence from amphibians, birds, and fish. Each sequence is listed in Table 1. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. Each parameter used was the default parameter: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0.20, gap separation = 5, no end gaps = no, output option: format = Aln w / numbers, order = aligned. The 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 to the protein encoded by human C19Orf63 splice variant 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. SEQ ID NO: 4 was used as the matrix. The parameters used were the default parameters: 11 extension: 1, composition adjustment = conditional composition score matrix adjustment with the database of non-redundant protein sequences (nr). From the identified sequences, one sequence was selected from examples from various vertebrate species, mainly mammals, and one sequence from amphibians and fish. Each sequence is shown in Table 2. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. Each parameter used was the default parameter: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0.20, gap separation = 5, no end gap = none, output option: format = Aln w / numbers, order = aligned. Multiple alignments obtained for all sequences are shown in Figure 8, and those related to mammalian sequences are shown in Figure 9.

[0158]

Table 4

Example 9

[0159] Homologous sequences to the protein encoded by the human C19Orf63 splice variant 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. SEQ ID NO: 5 was used as the matrix.

[0160] The parameters used were the default parameters: extension: 11, composition adjustment = conditional composition score matrix adjustment with the non-redundant protein sequence (nr) database. From the identified sequences, examples from various vertebrate species, mainly mammals, and one sequence each from amphibians and fish were selected. Each sequence is listed in Table 3. The selected amino acid sequences were aligned using the CLUSTALW2 algorithm at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. Each parameter used was the default parameter: alignment type = slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0.20, gap separation = 5, no end gap = no, output option: format = Aln w / numbers, order = aligned. The multiple alignments obtained for all sequences are shown in Figure 10, and those related to 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 at passages 3 - 6 were used. Before stimulation with various agents, the cells were cultured overnight in MCDB131 (Life Technologies) containing 2% FCS. Next, HCAECs were seeded into 96-well plates (5 x 103 cells / well) and stimulated with recombinant human factor 1, recombinant human factor 2, or VEGF (positive control) for 16 hours in the presence or absence of various concentrations of rabbit anti-factor 1 antibody, rabbit anti-factor 2 antibody, or control IgG. The antibodies were generated by Eurogentec and raised against the 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 of SEQ ID NO: 3). Cell proliferation was measured by colorimetric BrdU incorporation immunoassay (Roche). The data are shown in Figure 12 (panel A: factor 1; panel B: factor 2; data are mean ± SEM of 3 - 6 experiments). The present invention also includes, for example, the following aspects. [Aspect 1] A protein or a fragment thereof comprising 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 inhibiting healing and / or apoptosis. [Aspect 2] A protein or a fragment thereof comprising 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 of non-transformed tissue or non-transformed cells. [Aspect 3] The protein according to aspect 2, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 4 or SEQ ID NO: 5. [Aspect 4] A protein according to any one of Aspects 1 to 4, wherein the non-transformed tissue or cell is healthy, diseased, or damaged. [Aspect 5] wherein the cell or the tissue is (i) a muscle tissue cell, a connective tissue cell, an epithelial tissue cell, or a nerve tissue cell, or muscle tissue, connective tissue, epithelial tissue, or nerve tissue; and / or (ii) belongs to or is derived from the circulatory system, digestive system, endocrine system, excretory system, immune system, integumentary system, muscular system, nervous system, reproductive system, respiratory system, or skeletal system; and / or (iii) belongs to or is derived from the heart, skin, bone, cartilage, blood vessel, esophagus, stomach, intestine, gland, liver, kidney, lung, brain, and spleen, a protein according to any one of Aspects 1 to 5. [Aspect 6] wherein the injury results from a genetic / hereditary disease or an acquired disease resulting from ischemia, reperfusion injury, inflammation, infection, trauma, mechanical load, poisoning, or surgery, preferably the resulting disease is selected from the group consisting of myocardial infarction, angina pectoris, and heart failure, a protein according to Aspect 5. [Aspect 7] a protein according to Aspect 5 or 6, wherein the disease is associated with atrophy, hypoplasia, inflammation, injury, or wound. [Aspect 8] wherein the disease is primary, preferably cardiomyopathy caused by genetic or spontaneous mutations, or acquired cardiomyopathy, preferably ischemic cardiomyopathy caused by atherosclerosis or other coronary artery diseases, cardiomyopathy caused by myocardial infection or poisoning, hypertensive heart disease caused by pulmonary hypertension and / or arterial hypertension, and heart valve disease, a protein according to Aspect 6 or 7. [Aspect 9] A nucleic acid encoding a protein according to any one of Aspects 1 to 8 for use in enhancing the proliferation and / or promoting the healing and / or inhibiting apoptosis of non-transformed tissues or non-transformed cells. [Aspect 10] A vector comprising the nucleic acid according to Aspect 9 for use in enhancing the proliferation and / or promoting the healing and / or inhibiting apoptosis of non-transformed tissues or non-transformed cells. [Aspect 11] The vector according to aspect 10, selected from the group consisting of: plasmid vector; cosmid vector; phage vector, such as lambda phage, filamentous phage vector; viral vector, preferably adenovirus vector, adeno-associated virus (AAV) vector, alphavirus vector, herpesvirus vector, measles virus vector, poxvirus vector, vesicular stomatitis virus vector, retrovirus vector and lentivirus vector; virus-like particle; and bacterial spore. [Aspect 12] A pharmaceutical composition for use in enhancing the growth and / or healing and / or inhibiting apoptosis of non-transformed tissue or non-transformed cells, comprising the protein according to any one of aspects 1 to 8, the nucleic acid according to aspect 9, or the vector according to aspect 10 or 11, and optionally a suitable pharmaceutical excipient. [Aspect 13] The pharmaceutical composition according to aspect 12, administered via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronary route. [Aspect 14] The pharmaceutical composition according to aspect 12 or 13, administered before, after, or simultaneously with reperfusion therapy. [Aspect 15] The pharmaceutical composition according to aspect 14, wherein the administration is by one or more bolus injections and / or infusions. [Aspect 16] An inhibitor of factor 1 and / or 2 protein for medical use. [Aspect 17] The inhibitor of factor 1 and / or 2 according to aspect 16, wherein the medical use is the treatment or prevention of a disease in which angiogenesis contributes to the development or progression of the disease. [Aspect 18] The inhibitor according to aspect 17, wherein the factor 1 protein comprises the amino acid sequence shown in SEQ ID NO: 1, and the factor 2 protein comprises the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3. [Aspect 19] A protein comprising an inhibitory fragment of SEQ ID NO: 1 or 3, or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3; a ligand that specifically binds to the amino acid represented by SEQ ID NO: 1 or 3 or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3; and a nucleic acid that inhibits or prevents transcription and / or translation of an mRNA encoding a protein comprising the amino acid represented by SEQ ID NO: 1 or 3 or a variant thereof having at least 80% sequence identity with SEQ ID NO: 1 or 3. The inhibitor according to embodiment 17 or 18 selected from the group consisting of [Embodiment 20] The inhibitor according to embodiment 19, wherein the ligand is selected from the group consisting of an antibody or a fragment thereof, an antibody-like protein, or a peptidomimetic. [Embodiment 21] A nucleic acid encoding the inhibitor according to any one of embodiments 17 to 20 for use in treating or preventing a disease in which angiogenesis contributes to the expression or progression of the disease. [Embodiment 22] A vector comprising the nucleic acid according to embodiment 22 for use in treating or preventing a disease in which angiogenesis contributes to the expression or progression of the disease. [Embodiment 23] An inhibitor according to any one of embodiments 17 to 20, a nucleic acid according to embodiment 21, or a vector according to embodiment 22 for use in treating or preventing a disease in which angiogenesis contributes to the expression or progression of the disease, and, optionally, a suitable pharmaceutical excipient. A pharmaceutical composition comprising [Embodiment 24] An inhibitor according to any one of embodiments 17 to 20, a nucleic acid according to embodiment 21, a vector according to embodiment 22, and a pharmaceutical composition according to embodiment 23, wherein the disease in which angiogenesis contributes to the expression or progression of the disease is a proliferative disease. [Embodiment 25] The inhibitor according to any one of aspects 17 to 20, the nucleic acid according to aspect 21, the vector according to aspect 22, or the pharmaceutical composition according to aspect 23, wherein the proliferative disease is selected from the group consisting of a benign tumor, a malignant tumor, rheumatoid arthritis, psoriasis, an ocular angiogenesis disease, Osier-Webber syndrome, plaque angiogenesis, restenosis after transplantation and angioplasty, telangiectasia, hemophilic arthropathy, angiofibroma, wound granulation, intestinal adhesion, atherosclerosis, scleroderma, hypertrophic scar, cat scratch disease, and ulcers, particularly age-related 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 disease, neck cancer, non-melanoma skin cancer, ovarian cancer, prostate cancer, benign prostatic hyperplasia, pancreatic cancer, rectal cancer, and testicular cancer. [Aspect 26] A ligand that specifically binds to factor 1 or factor 2 and inhibits the angiogenesis-stimulating activity of factor 1 or factor 2. [Aspect 27] The ligand according to aspect 26 that specifically binds to an epitope of the human factor 1 protein contained in or consisting of amino acids 61 to 76 of SEQ ID NO: 1, or a region of another factor 1 protein corresponding to this epitope. [Aspect 28] The ligand according to aspect 26 that specifically binds to an epitope of the human factor 2 protein contained in or consisting of amino acids 181 to 195 of SEQ ID NO: 3, or a region of another factor 2 protein corresponding to this epitope.

Claims

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 the variant has at least 99% sequence identity with SEQ ID NO: 2 having said deletion, wherein the medicament (i) treats a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical load, myocardial infarction, and heart failure; (ii) improves left ventricular systolic function; or (iii) protects cardiomyocytes from apoptosis for use in such treatment. Use.

2. Use of a nucleic acid encoding the protein or variant according to claim 1 for the manufacture of a medicament, wherein the medicament (i) treats a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical load, myocardial infarction, and heart failure; (ii) improves left ventricular systolic function; or (iii) protects cardiomyocytes from apoptosis for use in such treatment. Use.

3. Use of a vector comprising the nucleic acid according to claim 2 for the manufacture of a medicament, wherein the medicament (i) treats a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical load, myocardial infarction, and heart failure; (ii) improves left ventricular systolic function; or (iii) protects cardiomyocytes from apoptosis for use in such treatment. Use.

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

5. (i) The viral vector is an adenovirus vector, an adeno-associated virus (AAV) vector, an alphavirus vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, a vesicular stomatitis virus vector, a retrovirus vector and a lentivirus vector; or is 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 the use according to any one of claims 3 to 5, wherein the medicament for use in treating a disease selected from the group consisting of (i) cardiac ischemia, cardiac reperfusion injury, cardiac mechanical load, myocardial infarction, and heart failure; (ii) improving left ventricular systolic function; or (iii) protecting cardiomyocytes from apoptosis Use.

7. The use according to claim 6, wherein the medicament is formulated for administration via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronary route.

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

9. The use according to claim 8, wherein the 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, wherein the variant has at least 99% sequence identity with SEQ ID NO: 2 having said deletion, protein or variant b) a nucleic acid encoding the protein or variant of a), or c) a vector comprising the nucleic acid of b), and a suitable pharmaceutical excipient, a pharmaceutical composition, wherein the pharmaceutical composition (i) treats a disease selected from the group consisting of cardiac ischemia, cardiac reperfusion injury, cardiac mechanical load, myocardial infarction, and heart failure; (ii) improves left ventricular systolic function; or (iii) protects cardiomyocytes from apoptosis for use in this regard. Pharmaceutical composition.

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