Complement anaphylatoxin binders and their use in treatment of subject having ocular wound and / or fibrosis

Abstract

To provide treatment of a subject having an ocular wound and / or fibrosis.SOLUTION: The subject matter of the present invention is a binder, e.g. protein or protein fragment, binding to complement-anaphylatoxin C5a and / or C3a and / or C4a, thereby inhibiting the activity of C5a and / or C3a and / or C4a for use in the treatment of a subject having an ocular wound and / or fibrosis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The subject of the present invention is a binding agent, such as a protein or protein fragment or peptide, for use in the treatment of subjects with ocular wounds and / or fibrosis, which binds to the complement-anaphylatoxins C5a and / or C3a and / or C4a, thereby inhibiting the activity of C5a and / or C3a and / or C4a. [Background technology]

[0002] prior art Degenerative eye diseases associated with severe loss of vision are very often the result of faulty angiogenesis or wound healing / fibrosis (Friedlander M. J Clin Invest. 2007).

[0003] Although the treatment of vascular eye diseases has improved significantly with in-depth research and the introduction of anti-VEGF therapeutics (vascular endothelial growth factor, VEGF) (Lim LS et al. Lancet 2012; Feigl B. Prog Retin Eye Res 2009; Joussen AM et al. FASEB J 2004), the treatment of fibrotic eye diseases is still lacking in therapeutic approaches.

[0004] Improper wound healing and fibrosis are particularly relevant in the cornea. Corneal fibrosis can result in a loss of optical transparency, substantially impairing vision and potentially resulting in blindness in the affected eye. Corneal scarring can occur due to persistent corneal edema caused by herpes simplex infection, microbial keratitis, mechanical or chemical lesions, stromal keratopathy, endothelial decompensation, or corneal graft failure. Today, penetrating corneal transplantation is the only treatment option for restoring vision in most cases. In this regard, the number of corneal transplants performed and the number of severe corneal complications associated with corneal fibrosis due to contact lens or corneal laser refractive surgery are increasing. Despite the above, the lifetime risk of suffering from associated ocular trauma with corneal pathology is 20% (Ljubimov AV et al. Prog Retin Eye Res 2015). Current therapeutic options for inhibiting ocular fibrosis are very limited and primarily involve corticosteroids and cyclosporine A (CSA). Both substances have nonspecific efficacy and are associated with various side effects. In this regard, corticosteroids not only induce the development of cataracts and elevated intraocular pressure, but also cause systemic adverse events, such as Cushing's syndrome and changes in blood parameters (glucose). CSA is not suitable for acute treatment because its onset of action is slow and usually too slow to prevent fibrosis. Its topical administration is associated with stinging and redness of the eye and also causes systemic adverse events, particularly arterial hypertension.

[0005] However, this therapeutic dilemma is not limited to the cornea, as noted in the examples above. Misguided wound healing and scarring in ocular diseases are also associated with tissue fibrosis in various conditions, including ocular fibroblasts and myofibroblasts, which occur in the conjunctiva, sclera, iris, trabecular meshwork, vitreous, retina, choroid, and optic nerve head. Furthermore, the fundamental pathophysiological processes involved in fibrosis and scarring, related to fibroblast activation and / or differentiation, are similarly associated with fibrotic diseases of the lung, liver, kidney, pancreas, heart, skin, and vasculature. Against this background, the establishment of novel therapeutic options for the treatment of ocular fibrosis and other fibrotic conditions is of considerable clinical importance.

[0006] Physiological wound healing involves several tissue processes and follows a sequence of cell migration and / or transformation, proliferation, and regulation of the extracellular matrix (Ljubimov AV et al. Prog Retin Eye Res 2015); whereas activated fibroblasts and myofibroblasts are key mediators (Gabbiani G., J Pathol 2003). During the normal course of wound healing, reversible protein deposition accumulates within the extracellular matrix (Wynn TA et al. NAT Med 2012). However, in the context of fibrotic remodeling caused by dysregulation of pro- and anti-fibrotic cascades, persistent myofibroblast activation emerges, which can lead to the constant, irreversible deposition of matrix proteins, such as collagen, fibronectin, and proteoglycans (Medzhitov R. Cell 2010; Wynn TA, J Pathol. 2008).

[0007] Based on the above, the inhibition of myofibroblasts and their activation may selectively redirect the wound healing process toward normal clearance mechanisms, thereby preventing tissue fibrosis and scarring. However, when it comes to inhibiting ocular myofibroblasts, the anatomical peculiarities of the eye must be taken into account. First, the blood-ocular barrier hinders the effectiveness of systemically applied inhibitors / modulators, especially those based on proteins / peptides. Second, direct administration (e.g., topical administration in the form of eye drops) requires the inhibitor / modulator to penetrate the tissue intended to be treated. Therefore, the inhibitor / modulator must be small enough to penetrate the conjunctiva, sclera, iris, trabecular meshwork, vitreous body, retina, choroid, and even the optic nerve head. Proteins with molecular weights between 28 and 67 kDa can penetrate the cornea and enter the anterior chamber when the corneal epithelium is intact, whereas proteins with molecular weights between 60 and 90 kDa can penetrate the cornea and enter the anterior chamber after the corneal epithelium has been removed (Thiel MA et al. Clin Exp Immunol 2002). Conventional therapeutic approaches using specific inhibitors, such as monoclonal antibodies (anti-VEGF antibody, bevacizumab: 149 kDa), do not meet these conditions. Summary of the Invention

[0008] It was an object of the present invention to provide a treatment for subjects with ocular wounds or fibrosis that overcomes the drawbacks of prior art methods.

[0009] It is therefore an object of the present invention to provide a substance which inhibits the process of fibroblast / myofibroblast activation and / or transdifferentiation, i.e. which at least essentially inhibits the process of fibroblast / myofibroblast activation and / or transdifferentiation, and which preferably has a molecular weight of less than 90 kDa, preferably 80 kDa or less, preferably 70 kDa or less, more preferably 60 kDa or less, more preferably 50 kDa or less, more preferably 45 kDa or less, more preferably 40 kDa or less, even more preferably 35 kDa or less, even more preferably 30 kDa or less, even more preferably 25 kDa or less, even more preferably 20 kDa or less, even more preferably 15 kDa or less, and even more preferably 10 kDa or less.

[0010] The subject of the present invention is a binding agent, in particular a protein or protein fragment, for use in the treatment of subjects with ocular wounds and / or fibrosis, which binds to the complement-anaphylatoxins C5a and / or C3a and / or C4a, preferably thereby inhibiting the activity of C5a and / or C3a and / or C4a.

[0011] Inhibiting the activity of C5a and / or C3a and / or C4a means essentially inhibiting the activity of C5a and / or C3a and / or C4a by binding to C5a and / or C3a and / or C4a.

[0012] A subject of the present invention is a binding agent for use in treating a subject with an ocular wound or fibrosis, wherein said binding agent is administered to promote wound healing, in particular corneal wound healing.

[0013] The binding agent may be selected from the group comprising proteins or fragments thereof, peptides, non-IgG scaffolds, in particular aptamers, oligonucleotides, antibodies or antibody-like proteins, peptidomimetics.

[0014] Such antibodies, antibody-like proteins, or binding agents may bind to several overlapping peptide fragments of complement component C5a protein (e.g., several overlapping fragments of human C5a protein having the amino acid sequence set forth in SEQ ID NO:20 or SEQ ID NO:21). Here, overlap refers to the overlap of a particular peptide fragment with the target amino acid sequence of the antibody, antibody-like protein, or binding agent. The antibody, antibody-like protein, or binding agent may also bind to human C5a only at epitopes within or overlapping with fragments of a protein having the amino acid sequence set forth in SEQ ID NOs:22-34 (see, e.g., Cooke et al. (2010) Acta Cryst D66:190-197, and as described in U.S. Patent Application Publication No. 2016 / 0159892). Furthermore, the antibody, antibody-like protein, or binding agent may also bind to the epitope of C5a formed by the amino acid sequence according to SEQ ID NO: 35 to 40 (SEQ ID NO: 35: X1X2ETCEX3RX4, SEQ ID NO: 36: X5X6KX7X8X9L, and SEQ ID NO: 37: X5X6KX7X8X9I). X is selected from the group consisting of H and N; X is selected from the group consisting of D, N, H, P, and G; X is selected from the group consisting of M, L, I, and V; and X is selected from the group consisting of Q, L, and I (U.S. Patent Application No. 2012 / 0231008, U.S. Patent Application No. 2017 / 0002067, WO 2011 / 063980, and U.S. Patent No. 8,802,096).

[0015] Such antibodies, antibody-like proteins, or binding agents may bind to several overlapping peptide fragments of complement component C3a protein (e.g., several overlapping fragments of human C3a protein having the amino acid sequence set forth in SEQ ID NO: 43). The antibodies, antibody-like proteins, or binding agents may also bind to human C3a only at epitopes within or overlapping with fragments of proteins having the amino acid sequences according to SEQ ID NOs: 44-47 (see, e.g., Hugli TE. J Biol Chem. 1975; Hugli TE et al. PNAS 1977; Payan D et al. J. Exp Med. 1982).

[0016] Such antibodies, antibody-like proteins, or binding agents may bind to several overlapping peptide fragments of complement component C4a protein (e.g., several overlapping fragments of human C4a protein having the amino acid sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49). The antibodies, antibody-like proteins, or binding agents may also bind to human C4a only at epitopes within or overlapping with fragments of a protein having the amino acid sequence according to SEQ ID NO: 50 (see, e.g., Yu CY et al. EMBO J. 1986; Nettesheim DG et al. PNAS 1988).

[0017] A peptide is defined as a compound consisting of at least two amino acids in which the carboxyl group of one acid is bonded to the amino group of the other, and can be produced by peptide synthesis. Thus, as defined for the present invention, a peptide may have from 2 to 50 amino acids. By definition for the present invention, a protein contains more than 50 amino acids.

[0018] A protein is defined as a macromolecule consisting of one or more chains of amino acids or peptides linked by peptide bonds, which can be produced by proteolytic ligation of two or more peptides, recombinant expression, or protein biosynthesis.

[0019] A protein fragment is defined as a section of amino acid sequence derived from a protein that served as a template.

[0020] Antibodies according to the present invention are proteins comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as various immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 kDa or 214 amino acids in length. Full-length immunoglobulin heavy chains are generally about 50 kDa or 446 amino acids in length. The light chain is encoded by a variable region gene (approximately 110 amino acids in length) at the NH2-terminus and a kappa or lambda constant region gene at the COOH-terminus. The heavy chain is similarly encoded by a variable region gene (approximately 116 amino acids in length) and one of the other constant region genes.

[0021] The basic structural unit of an antibody is generally a tetramer of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain, in which the variable regions of the light and heavy chains bind antigen and the constant regions mediate effector functions. Immunoglobulins also exist, for example, in Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105,1987; Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hood et al., Immunology, Benjamin, NY, 2nd ed., 1984; Hunkapiller and Hood, Nature 323:15-16,1986). An immunoglobulin light or heavy chain variable region comprises a framework region interrupted by three hypervariable regions, also called complementarity-determining regions (CDRs) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al., US Department of Health and Human Services, 1983). As mentioned above, the CDRs are primarily responsible for binding to an epitope of an antigen. The immune complex is an antibody, such as a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody, or a functional antibody fragment, that specifically binds to the antigen.

[0022] Chimeric antibodies are constructed by genetic engineering, typically by constructing light and heavy chain genes from immunoglobulin variable and constant region genes belonging to different species. For example, variable segments from genes derived from a mouse monoclonal antibody can be linked to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of a variable region or antigen-binding region derived from a mouse antibody and a constant region or effector region derived from a human antibody, although other mammals may be used, or variable regions may be engineered by molecular techniques. Methods for producing chimeric antibodies are well known in the art; see, for example, U.S. Pat. No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin containing human framework regions and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is designated the "donor," and the human immunoglobulin providing the framework is designated the "acceptor." In one embodiment, all CDRs of a humanized immunoglobulin are derived from the donor immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody containing a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. Humanized antibodies or other monoclonal antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conservative substitutions include gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.Humanized immunoglobulins can be constructed by genetic engineering (see, e.g., U.S. Pat. No. 5,585,089). Human antibodies are antibodies in which the light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells secreting the desired antibody. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display methods (see, e.g., Dower et al., PCT Publication No. WO 91 / 17271; McCafferty et al., PCT Publication No. WO 92 / 001047; and Winter, PCT Publication No. WO 92 / 20791) or selected from human combinatorial monoclonal antibody libraries (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (see, e.g., Lonberg et al., PCT Publication No. WO 93 / 12227; and Kucherlapati, PCT Publication No. WO 91 / 10741).

[0023] Thus, antibodies according to the invention may have any format known in the art, such as a human antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, or a CDR-grafted antibody. In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody, e.g., an IgG (typical full-length immunoglobulin) or antibody fragment, comprising at least the F variable region of the heavy and / or light chain, e.g., a chemically coupled antibody (fragment antigen binding), including, but not limited to, e.g., a Fab minibody, a single-chain Fab antibody, a monovalent Fab antibody with an epitope tag, e.g., Fab-V5Sx2; a bivalent Fab (minibody) dimerized at the CH3 domain; a bivalent Fab or multivalent Fab formed, e.g., via multimerization with the aid of heterologous domains, e.g., via dimerization of dHLX domains, e.g., Fab-dHLX-FSx2; F(ab')2-fragments, scFv fragments, multimerized multivalent and / or multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T-cell engagers), trifunctional antibodies, multivalent antibodies, e.g., from classes different from G; single domain antibodies, e.g., nanobodies derived from camel or fish immunoglobulins, and many others.

[0024] In addition to antibodies, other biopolymer scaffolds are known in the art to conjugate target molecules and have been used to generate highly target-specific biopolymers, examples being aptamers, spiegelmers, anticalins, and conotoxins.

[0025] In a preferred embodiment, the antibody format is selected from the group comprising Fv fragments, scFv fragments, Fab fragments, scFab fragments, (Fab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group comprising scFab fragments, Fab fragments, scFv fragments, and their bioavailability-optimized conjugates, e.g., PEGylated fragments. One particular format is the scFab format.

[0026] Non-Ig scaffolds can be protein scaffolds, which can be used as antibody mimetics because they can bind to a ligand or antigen. Non-Ig scaffolds include tetranectin-based non-Ig scaffolds (e.g., as described in U.S. Patent Application No. 2010 / 0028995), fibronectin scaffolds (e.g., as described in European Patent No. 1266025); lipocalin-based scaffolds (e.g., as described in WO 2011 / 154420), ubiquitin scaffolds (e.g., as described in WO 2011 / 073214), metastasis scaffolds (e.g., as described in U.S. Patent Application No. 2004 / 0023334), protein A scaffolds (e.g., as described in European Patent No. 2231860), ankyrin repeat-based scaffolds (e.g., as described in WO 2010 / 060748), microprotein, preferably cystine-knot forming microprotein, scaffolds (e.g., as described in European Patent No. 2314308), Fyn They may be selected from the group comprising SH3 domain-based scaffolds (e.g., as described in WO 2011 / 023685), EGFR-A-domain-based scaffolds (e.g., as described in WO 2005 / 040229), and Kunitz domain-based scaffolds (e.g., as described in EP 1941867).

[0027] Non-immunoglobulin (IgG) scaffolds are defined as small antibody alternatives. Aptamers are defined as molecules that bind to specific targets and may be composed of RNA and / or DNA and / or amino acids (peptides).

[0028] Aptamers may be associated with nucleic acid molecules comprised of RNA and / or DNA, for example as disclosed in SEQ ID NO: 41 (5'-GCGAU G(dU)GGU GGU(dG)(dA) AGGGU UGUUG GG(dU)G(dU) CGACG CA(dC)GC-3), and as described in US Patent Application No. 2012 / 0065254, are capable of binding to C5a, wherein the binding site for C5a comprises the C5a amino acid sequence comprising SEQ ID NO: 42 (see Yatime L. et al. NAT Commun. 2015).

[0029] One embodiment of the inventive antibody according to the present invention can be produced as follows: Balb / c mice were immunized with 100 μg of antigen-peptide-BSA conjugate (BSA = bovine serum albumin) on days 0 and 14 (emulsified in 100 μl of complete Freund's adjuvant) and 50 μg on days 21 and 28 (in 100 μl of incomplete Freund's adjuvant).

[0030] Splenocytes from immunized mice and cells of the myeloma cell line SP2 / 0 were fused with 1 ml of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded into 96-well cell culture plates. Hybrid clones were selected by growth in HAT medium (RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 20% fetal bovine serum and HAT medium supplement). After two weeks, the cells were passaged three times in HAT medium before being returned to normal cell culture medium.

[0031] Cell culture supernatants were initially screened for antigen-specific IgG antibodies 3 weeks after fusion. Positively testing microcultures were transferred to 24-well plates for expansion. After retesting, selected cultures were cloned and recloned using limiting dilution, and their isotypes were determined. (See also Lane, RD (1985). A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas. J. Immunol. Meth. 81: 223-228; Ziegler, B. et al. (1996). Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies. Horm. Metab. Res. 28: 11-15.)

[0032] Antibodies can be generated by phage display according to the following procedure: The human naive antibody gene library HAL7 / 8 was used to isolate recombinant single-chain Fv variable regions (scFv) against the peptide. The antibody gene library was screened using a panning strategy involving the use of peptides containing a biotin tag linked to the peptide sequence via two different spacers. A mixture of panning rounds using nonspecifically binding antigens and streptavidin-binding antigens was used to minimize background from nonspecific binders. Phages eluted from the third panning round were used to generate monoclonal scFv expressing E. coli strains. Supernatants from the cultures of these clonal lines were used directly for antigen ELISA tests (Hust, M., Meyer, T., Voedisch, B., Ruelker, T., Thie, H., El-Ghezal, A., Kirsch, M. I., Schuette, M., Helmsing, S., Meier, D., Schirrmann, T., Duebel, S., 2011. A human scFv antibody generation pipeline for proteome research. Journal of Biotechnology 152, 159-170; Schuette, M., Thullier, P., Pelat, T., Wezler, X., Rosenstock, P., Hinz, D., Kirsch, M. I., Hasenberg, M., Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Duebel, S., 2009. Identification of a putative Crf splice variant and generation of recombinant antibodies). for the specific detection of Aspergillus fumigatus. See PLoS One 4, e6625)

[0033] Humanization of the mouse antibody was carried out according to the following procedure: To humanize a mouse-derived antibody, the antibody sequence is analyzed for structural interactions between the framework region (FR) and the complementarity-determining region (CDR) and the antigen. Based on structural modeling, appropriate human-derived FRs are selected, and the mouse CDR sequences are grafted onto the human FRs. Amino acid sequence variations in the CDR or FR can be introduced to restore structural interactions that are abolished by species switching of the FR sequence. This restoration of structural interactions can be achieved by a random approach using a phage display library or by a directed approach guided by molecular modeling (see Almagro JC, Fransson J., 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33).

[0034] In a preferred embodiment, the antibody format is selected from the group comprising Fv fragments, scFv fragments, Fab fragments, scFab fragments, F(ab)2 fragments, and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group comprising scFab fragments, Fab fragments, scFv fragments, and their bioavailability-optimized conjugates, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0035] In one embodiment of the invention, the binding agent, such as a protein according to the invention or a protein fragment thereof, binds to C5a and C3a, thereby inhibiting the activity of C5a and C3a.

[0036] In one embodiment of the invention, the binding agent, such as a protein according to the invention or a protein fragment thereof, binds to C5a and C4a, thereby inhibiting the activity of C5a and C4a.

[0037] In one embodiment of the invention, the binding agent, such as a protein according to the invention or a protein fragment thereof, binds to C3a and C4a, thereby inhibiting the activity of C3a and C4a.

[0038] In one embodiment of the invention, the binding agent, such as a protein according to the invention or a protein fragment thereof, binds to C5a, C3a and C4a, thereby inhibiting the activity of C5a, C3a and C4a.

[0039] In one particular embodiment of the invention, the binding agent, e.g., protein or protein fragment, is a soluble complement receptor protein or protein fragment. In one particular embodiment of the invention, the protein or protein fragment / peptide is a recombinant soluble complement receptor protein or a synthetic protein fragment / peptide.

[0040] Soluble receptors are defined as the extracellular portion of a receptor (Fischer DG. Science 1993). In the case of C3a, this is the extracellular portion of the C3a anaphylatoxin chemotactic receptor (C3aR1), and in the case of C5a, this is the extracellular portion of the C5a anaphylatoxin chemotactic receptor 1 and / or 2 (C5aR1 / CD88 and C5aR2 / C5L2). Because no distinct specific C4a receptor is known, in the case of C4a, this is the extracellular portion of the C3a anaphylatoxin chemotactic receptor (C3aR1) and / or C5a anaphylatoxin chemotactic receptor 1 and / or 2 (C5aR1 / CD88 and C5aR2 / C5L2).

[0041] In one embodiment of the invention, the binding agent, such as a protein according to the invention or a protein fragment thereof, specifically binds to complement-anaphylatoxins C5a and / or C3a and / or C4a.

[0042] The receptor / ligand binding affinities of anaphylatoxin chemotactic receptors (C3aR1, C5aR1 / CD88, and C5aR2 / C5L2) for their primary ligands (C3a and C5a, respectively) and their cross-reactivities for all other anaphylatoxins (C3a, C4a, C5a) are known in the art (Cain SA. et al. J Biol Chem. 2002, Kalant D. et al. J Biol Chem 2003, Okinaga S. et al. Biochemistry 2003). Relevant ligand-binding sites within the amino acid sequences of anaphylatoxin chemotactic receptors, primarily those contributing to the extracellular and transmembrane domains, have been studied and are therefore known in the art.

[0043] Regarding C3aR1, studies have shown that the large extracellular loop 2 domain plays an important role in ligand binding; in addition, the charged transmembrane residues Arg161, Arg340, and Asp417 are essential for ligand-effector binding and / or signal coupling (Sun J. et al. Protein Sci. 1999).

[0044] The amino acid sequence shown in SEQ ID NO: 17 covers amino acids 332 to 341, a fragment of the large extracellular loop 2 containing Arg340 of human C3aR1 (SEQ ID NO: 3), which has 90% identity with the corresponding amino acid sequence of mouse C3aR1 (SEQ ID NO: 6).

[0045] The receptor-binding site of human C3a has been thoroughly studied and is summarized by Sun et al. (Sun J et al. Protein Sci. 1999): Human C3a consists of 77 amino acids. The three-dimensional structure of C3a consists of a large globular core of four tightly packed alpha helices covalently linked by three disulfide bonds, with a flexible irregular structure at the C-terminus (Huber R et al. Hoppe Seyler's Z Physiol Chem. 1980). The C-terminal region of C3a folds into a pseudo-beta turn and is stabilized by adjacent alpha-helical segments, according to NMR studies (Chazin WJ et al. Biochemistry 1988). A C-terminal 21-residue fragment of C3a (i.e., C3a 57-77) has been shown to retain all the biological activity of the native molecule (Lu ZX et al. J Biol Chem. 1984, Ember JA et al. Biochemistry 1991). Synthetic peptide analogs of C3a indicated that the major effector binding site of C3a resides in the disordered C-terminal region (LGLAR sequence) (Caporale LH et al. J Biol Chem. 1980, Unson CG et al. Biochemistry 1984).

[0046] In one embodiment, the binding agent that is the subject of the present invention is capable of binding to the C-terminal 21 residue fragment of disordered C3a.

[0047] Regarding C5aR1 / CD88, the extracellular N-terminus plays a crucial role in ligand binding, and five aspartic acid residues within amino acids 2–22 are essential for ligand-effector binding, thereby contributing at least 45% of the total binding energy of C5a (DeMartino JA. J Biol Chem. 1994). The extracellular loops 2 and 3 domains are involved in ligand-effector binding by interacting with the C-terminus of C5a (Siciliano SJ et al. PNAS. 1994, Monk PN et al. J Biol Chem. 1995). Furthermore, Tyr11 and Tyr14 are posttranslationally sulfated, which is important for C5aR1 binding to C5a (Farzan M et al. J Exp Med. 2001). The known binding sites, functions, and structures of the C5a anaphylatoxin chemotactic receptor have been summarized in a comprehensive review (Monk PN et al. Br J Pharmacol. 2007).

[0048] The amino acid sequence shown in SEQ ID NO: 15 covers amino acids 19 to 27, which is an N-terminal fragment containing two aspartic acids of human C5aR1 (SEQ ID NO: 2), and similarly, the amino acid sequence shown in SEQ ID NO: 16 covers amino acids 18 to 26, which is an N-terminal fragment containing two aspartic acids of mouse C5aR1 (SEQ ID NO: 5).

[0049] The receptor binding site of human C5a was studied in detail and summarized by Monk et al. as follows (Monk PN et al. Br J Pharmacol. 2007): Human C5a consists of 74 amino acids, including Asn64, which bears an N-linked carbohydrate moiety that is not essential for biological activity but very likely regulates C5a activity in vivo. The solution structure of human C5a (Zhang X et al. Proteins 1997; Zuiderweg ER and Fesik SW. Biochemistry 1989; Zuiderweg ER et al. Biochemistry 1989) shows an antiparallel four-helix bundle (residues 1–63), stabilized by three disulfide bonds (Cys21–Cys47, Cys22–Cys54, Cys34–Cys55), and four distinct helical segments (4–12, 18–26, 32–39, 46–63) connected by loop segments 13–17, 27–33, and 40–45. The 63-residue helical bundle fragment is highly cationic, conferring high affinity to cell surfaces. The C-terminal residues 69–74 also form a bulky helical turn connected to the four-helix bundle by a short loop. Reducing the disulfide bond preceding the N-terminal disulfide of C5a 1-74 or selectively removing residues significantly reduces function. The fragment C5a 1-69, lacking the C-terminal pentapeptide, binds to cells but lacks agonist activity, consistent with the N-terminal helical bundle that confers affinity, but only the C-terminus is the receptor activation domain. Loop 1 (residues C5a 12-20, including four Lys residues 12, 14, 19, and 20), loop 3 (C5a 39-46), and the C-terminal 6-8 residues (especially Arg 74) are important for binding to the C5a receptor (C5aR) and agonist efficacy. Neutralizing antibodies against C5a indicate that the Lys 20-Arg 37 region is important for receptor binding.

[0050] In one embodiment, the binding agent that is the subject of the present invention is capable of binding to the Lys20 to Arg37 region of C5a as described above.

[0051] Regarding C5aR2 / C5L2, studies have shown that (similar to C5aR1 / CD88) the extracellular N-terminus, containing a sulfated Tyr residue adjacent to the amino acid sequence, plays an important role in ligand binding. Furthermore, both receptors (C5aR1 / CD88 and C5aR2 / C5L2) share similar charged and hydrophobic residues in their extracellular and transmembrane domains, suggesting similar ligand binding modes (Farzan M et al. J Exp Med. 2001, Okinaga S. et al. Biochemistry 2003, Gao H et al. FASEB J. 2005, Scola AM. J Biol Chem. 2007). C5L2 can bind C3a and C4a with similar affinity to C3aR1, but at different binding sites for C5a, allowing C5L2 to simultaneously bind different complement-anaphylatoxins.

[0052] The amino acid sequence shown in SEQ ID NO:7 covers amino acids 46-59, a fragment of transmembrane domain 1 of human C5aR2 (SEQ ID NO:1), which has 79% identity with the corresponding amino acids 48-61 and includes Gly51, Asn55, and Val58, which are thought to play important roles in receptor / ligand binding of human C5aR1 (SEQ ID NO:2) (Monk PN et al. Br J Pharmacol. 2007).

[0053] The amino acid sequence shown in SEQ ID NO:8 covers amino acids 79-88, a fragment of transmembrane domain 2 of human C5aR2 (SEQ ID NO:1), which has 70% identity with the corresponding amino acids 81-90 and includes Asp68, which is thought to play an important role in receptor / ligand binding of human C3aR1 (SEQ ID NO:3) (Sun J. et al. Protein Sci. 1999).

[0054] The amino acid sequence shown in SEQ ID NO:9 covers amino acids 118-126, a fragment of transmembrane domain 3 of human C5aR2 (SEQ ID NO:1), which has 89% identity with the corresponding amino acids 120-128 and includes Ser123 and Leu126, which are thought to play important roles in receptor / ligand binding of human C5aR1 (SEQ ID NO:2) (Monk PN et al. Br J Pharmacol. 2007).

[0055] The amino acid sequence shown in SEQ ID NO: 10 covers amino acids 161-169, a fragment of transmembrane domain 4 of human C5aR2 (SEQ ID NO: 1), which has 89% identity with the corresponding amino acids 163-171 and includes Leu166, Thr168, Val169, Pro170, and Ser171, which are thought to play important roles in receptor / ligand binding of human C5aR1 (SEQ ID NO: 2) (Monk PN et al. Br J Pharmacol. 2007).

[0056] The amino acid sequence shown in SEQ ID NO: 11 covers amino acids 242-249, a fragment of transmembrane domain 6 of human C5aR2 (SEQ ID NO: 1), which shares 63% identity with the corresponding amino acids 251-258. It includes Phe251, which is thought to play an important role in receptor / ligand binding of human C5aR1 (SEQ ID NO: 2) (Monk PN et al. Br J Pharmacol. 2007), and shares 75% identity with the corresponding amino acids 386-393, adjacent to His394, which is thought to play an important role in receptor / ligand binding of human C3aR1 (SEQ ID NO: 3) (Sun J. et al. Protein Sci. 1999).

[0057] The amino acid sequence shown in SEQ ID NO: 12 covers amino acids 98-103, a fragment of the extracellular loop 1 domain of human C5aR2 (SEQ ID NO: 1), which has 67% identity with the corresponding amino acids 100-105. It also contains Trp102, Phe104, and Gly105, which are thought to play important roles in receptor / ligand binding of human C5aR1 (SEQ ID NO: 2) (Monk PN et al. Br J Pharmacol. 2007), and has 83% identity with the corresponding amino acids 86-91, a fragment of the extracellular loop 1 domain of human C3aR1 (SEQ ID NO: 3).

[0058] The amino acid sequence shown in SEQ ID NO: 13 covers amino acids 13-23, a fragment of the extracellular N-terminal domain of human C5aR2 (SEQ ID NO: 1), which has 82% identity with the corresponding amino acids 33-43 (SEQ ID NO: 14) of mouse C5aR2 (SEQ ID NO: 4), including Tyr14, which is important for receptor / ligand binding.

[0059] The term "specific binding" is defined as a protein-ligand binding affinity with a dissociation constant of 1 mM or less, preferably 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less, and even more preferably 10 nM or less, as measured by radioligand binding assays (Cain SA, Monk PN, J Biol Chem. 2002) or surface plasmon resonance (BIAcore) (as described in Colley CS et al. MAbs. 2018, U.S. Patent Application No. 2012 / 0065254), or an ELISA-based binding assay (Michelfelder S., J Am Soc Nephrol. 2018). The radioligand binding assay may be a radiolabeled ligand competitive receptor binding assay, as described in Kalant et al. J Biol Chem 2003, which determines the binding affinity between complement receptor C5aR1 (referred to as CD88 in Kalant et al. J Biol Chem 2003), C3aR, or C5L2 (SEQ ID NOs: 1, 2, and 3 of the present invention) and the anaphylatoxins C3a, C4a, or C5a in a culture system. In the above assays, receptor-bound radiolabeled C3a, C4a, or C5a was competitively displaced using increasing concentrations of unlabeled C3a, C4a, or C5a.Those skilled in the art will appreciate that unlabeled compounds that differ from unlabeled C3a, C4a, or C5a can be tested for displacement of receptor-bound radiolabeled C3a, C4a, or C5a, including using the binding agents of the present invention.

[0060] The term "inhibit activity" in relation to a protein or protein fragment / peptide, non-IgG scaffold, aptamer, oligonucleotide, antibody or antibody-like protein, peptidomimetic, or fragment thereof according to the invention refers to the property of inhibiting the activation and / or transdifferentiation process of fibroblasts / myofibroblasts in the presence of C5a and / or C3a and / or C4a stimulation. To this end, the effectiveness of fibroblasts (e.g., human corneal keratinocytes) incubated with C3a and / or C4a and / or C5a at a concentration of 0.1 μg / ml in DMEM (Dulbecco's Modified Eagle's Medium) growth medium without fetal bovine serum for 24 hours ("stimulation control") is tested in comparison with fibroblasts incubated under identical conditions but with the protein or protein fragment / peptide, non-IgG scaffold, aptamer, oligonucleotide, antibody or antibody-like protein, peptidomimetic, or fragment thereof according to the invention ("inhibition control"). After stimulation, the proportion of myofibroblasts (expressed as a percentage) in the monolayer fibroblast cultures was determined by alpha-smooth muscle actin (aSMA) immunocytochemical staining using an anti-aSMA antibody. This revealed myofibroblasts as cells that stained positive for aSMA in the cytoplasm. A protein or protein fragment / peptide, non-IgG scaffold, aptamer, oligonucleotide, antibody or antibody-like protein, peptidomimetic, or fragment thereof according to the present invention is defined as being effective in "inhibiting the activity" of myofibroblast activation if it can reduce the proportion of myofibroblasts in the "inhibition control" by preferably at least 10%, more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, even more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, and even more preferably at least 65%, compared to the proportion of myofibroblasts in the "stimulation control," taking into account optimal conditions and concentrations.

[0061] In one embodiment of the invention, the binding agent is a protein or protein fragment and is selected from the group comprising the human C5L2 protein set forth in SEQ ID NO: 1, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the human C5L2 protein set forth in SEQ ID NO: 1, the human C5aR1 protein set forth in SEQ ID NO: 2, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the human C5aR1 protein set forth in SEQ ID NO: 2, the human C3aR protein set forth in SEQ ID NO: 3, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the human C3aR protein set forth in SEQ ID NO: 3, the mouse C5L2 protein set forth in SEQ ID NO: 4, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5L2 protein set forth in SEQ ID NO: 4, the mouse C5aR1 protein set forth in SEQ ID NO: 5, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein set forth in SEQ ID NO: 5, the mouse C3aR protein set forth in SEQ ID NO: 6 and a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C3aR protein set forth in SEQ ID NO: 6.

[0062] In certain embodiments of the invention, the identity to the respective full-length amino acid sequences is at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%.

[0063] In one embodiment, full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) can be produced in wheat germ (ab153291; Abcam; Cambridge, UK).

[0064] In another embodiment, full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) located on the cell membrane can be produced in wheat germ (ab157989; Abcam; Cambridge, UK) and post-translationally modified by sulfation.

[0065] In another embodiment, full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) located on the cell membrane can be produced in wheat germ (ab152249; Abcam; Cambridge, UK) and sulfated at Tyr174.

[0066] The degree of identity between two amino acid sequences is defined as the result of a heuristic algorithm such as FASTA (Lipman DJ et al. Science 1985, Pearson WR et al. PNAS 1988) or basic local alignment search tool (BLAST) (Lobo I. Nature Education 2008). If the protein / peptide or protein fragment to be tested is identical to SEQ ID NOs: 1-17 (each has a BLAST result of 100% identity) or contains an identical fragment (each has a BLAST result of 100% identity), the identity of the protein / peptide or protein fragment to be tested to SEQ ID NOs: 1-17 is 100%.

[0067] In one embodiment of the present invention, the protein or protein fragment comprises at least one conserved region selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and proteins or fragments that are at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 17.

[0068] In one embodiment of the present invention, the protein or protein fragment comprises at least two conserved regions selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and proteins or fragments that are at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 17.

[0069] In another embodiment of the invention, the protein or protein fragment comprises at least three of the aforementioned conserved regions, or at least four of the aforementioned conserved regions, or at least five of the aforementioned conserved regions, or at least six of the aforementioned conserved regions.

[0070] In one embodiment of the invention, the conserved region exhibits at least 65%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity with any one of the aforementioned amino acids set forth in SEQ ID NOs: 1-17.

[0071] Table 1 provides a summary of the sequence identities determined by BLAST between the corresponding amino acid sequences of the conserved sequence fragments characteristic of human and mouse C5L2, C5AR1, and C3AR (SEQ ID NOs: 1-17).

[0072] [Table 1]

[0073] A subject of the present invention is a composition comprising at least one binding agent, such as a protein or protein fragment, according to the invention for use in the treatment of subjects with ocular wounds and / or fibrosis.

[0074] A subject of the present invention is a composition comprising at least two binding agents according to the invention, such as two proteins / peptides or protein fragments, for use in the treatment of subjects with ocular wounds and / or fibrosis.

[0075] The subject of the present invention is a composition comprising at least three binding agents according to the present invention, such as proteins / peptides or protein fragments, for use in treating subjects with ocular wounds and / or fibrosis. For the sake of clarity, it is understood that the word "fibrosis" in the expression "ocular wounds and / or fibrosis" refers to the general definition of the term "fibrosis" and is not limited to ocular fibrosis alone. The expressions "ocular wounds and / or fibrosis" and "fibrosis and / or ocular wounds" can be used interchangeably herein.

[0076] A single binding agent, e.g., a protein or protein fragment, may contain one or more binding sites for C5a and / or C3a and / or C4a. According to Table 1, the number of binding sites may vary depending on the number of included sequences selected from SEQ ID NOs: 1-17.

[0077] In this regard, compositions comprising one or more binding agents, e.g., proteins / peptides or protein fragments comprising SEQ ID NOs: 1-17, enhance the inhibitory effect on C3a-, C4a-, and C5a-dependent activities. In particular, combinations of proteins or protein fragments derived primarily from C3a-binding portions, e.g., SEQ ID NOs: 8, 12, and 18, with proteins or protein fragments derived primarily from C5a-binding portions, e.g., SEQ ID NOs: 7, 9, 10, 11, 13, 14, 15, and 16, are of particular interest.

[0078] A subject of the present invention is a pharmaceutical composition comprising a binding agent, such as a protein or protein fragment, according to the invention, or a composition according to the invention, for use in the treatment of subjects with ocular wounds and / or fibrosis.

[0079] The binding agents of the present invention may be PEGylated or similarly modified to modify the biological stability and / or half-life of the binding agent. PEGylation is the process of covalently and non-covalently attaching or fusing (amalgamating) polyethylene glycol (PEG, known in pharmaceuticals as macrogol) polymer chains to molecules and macrostructures, such as drugs, therapeutic proteins, or vesicles, resulting in a process called PEGylation. PEGylation is typically achieved by incubating a reactive derivative of PEG with the target molecule. Covalent attachment of PEG to a drug or therapeutic protein "masks" the agent from the host's immune system (reducing immunogenicity and antigenicity), increases its hydrodynamic size (size in solution), and extends circulation time by reducing renal clearance.

[0080] The binding agents of the invention may be subject to post-translational or post-synthetic modifications, including, among others, sugars, fatty acids, phosphate groups (phosphoryl groups, phosphorylation), hydroxyl groups, methyl groups (protein methylation), ubiquitin (protein ubiquitination), to alter the actual structure of the binding agent and enhance its function or stability. These modifications can be made to both the amino (amino terminus) and carboxyl terminus (carboxyl terminus) of the binding agent, as well as to amino acid side chains (amino acids) within proteins, and can be reversible and / or irreversible.

[0081] Also of interest are prodrugs of the binders according to the present invention. Prodrugs are drugs or compounds that are metabolized (i.e., converted in the body) into pharmacologically active drugs after administration. Inactive prodrugs are pharmacologically inactive drugs that are metabolized in the body to active forms. Instead of administering a drug directly, the corresponding prodrug can be used to improve the absorption, distribution, metabolism, and excretion methods of the drug.

[0082] In one embodiment of the invention, the pharmaceutical composition is for topical administration, i.e., administered topically.

[0083] In one embodiment of the invention, the pharmaceutical composition is for intraocular administration, i.e., is administered intraocularly.

[0084] In one embodiment of the invention, the pharmaceutical composition is for intravitreal administration, i.e., is administered intravitreally.

[0085] In one embodiment of the invention, the pharmaceutical composition is for subconjunctival administration, i.e., is administered subconjunctivally.

[0086] In one embodiment of the invention, the pharmaceutical composition is for intravascular / intravenous administration, i.e., administered into a blood vessel / vein.

[0087] One embodiment of the invention is a binding agent, such as a protein or protein fragment, according to the invention, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject suffering from a disease selected from the group comprising conjunctivitis and conjunctival scarring (including ocular pemphigoid), scleritis and episcleritis, corneal scarring and opacification due to corneal ulcers, keratoconjunctivitis, keratitis, bullous keratopathy, corneal degeneration, iridocyclitis and adhesions of the iris and ciliary body, chorioretinal inflammation or chorioretinal scarring / fibrosis due to degeneration or hemorrhage or rupture or neovascularization, fibrotic vitreoretinopathies, such as proliferative vitreoretinopathy, retinopathy of prematurity and diabetic retinopathy; choroidal neovascularization and macular degeneration, secondary glaucoma, endophthalmitis, and impaired wound healing and fibrosis after ocular surgery or trauma, including intraocular foreign bodies.

[0088] One embodiment of the invention is a binding agent, such as a protein / peptide or protein fragment, according to the invention, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject, wherein the subject is suffering from corneal fibrosis.

[0089] One embodiment of the invention is a binding agent, such as a protein / peptide or protein fragment, according to the invention, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject, wherein the subject is suffering from chorioretinal fibrosis.

[0090] One embodiment of the invention is a binding agent according to the invention, such as a protein / peptide or protein fragment, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject, wherein the subject is suffering from impaired wound healing and fibrosis after ocular surgery or trauma.

[0091] One embodiment of the invention is a binding agent, such as a protein or protein fragment, according to the invention, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject suffering from a disease selected from the group comprising (idiopathic) pulmonary fibrosis, skin keloid formation, scleroderma, myelofibrosis, renal fibrosis, pancreatic fibrosis, and cardiac fibrosis, and fibrosis in (non-)alcoholic steatohepatitis, glomerulonephritis, and (ANCA-associated) vasculitis.

[0092] One embodiment of the invention is a binding agent, such as a protein or protein fragment, according to the invention, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject, wherein the subject is suffering from pulmonary fibrosis.

[0093] One embodiment of the invention is a binding agent, such as a protein or protein fragment, according to the invention, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject, wherein the subject is suffering from fibrosis due to glomerulonephritis and / or kidney fibrosis.

[0094] One embodiment of the invention is a binding agent, such as a protein or protein fragment, according to the invention, or a composition according to the invention, or a pharmaceutical composition according to the invention, for use in treating a subject, wherein the subject is suffering from steatohepatitis and / or liver fibrosis.

[0095] The following embodiments are the subject of the present invention: 1. A binding agent that binds to complement-anaphylatoxins C5a and / or C3a and / or C4a, thereby preferably inhibiting the activity of C5a and / or C3a and / or C4a, for use in treating subjects with ocular wounds and / or fibrosis. 2. The binding agent for use in treating a subject with an ocular wound and / or fibrosis according to embodiment 1, wherein said binding agent is selected from the group comprising a protein or fragment thereof, a peptide, a non-IgG scaffold, an aptamer, an oligonucleotide, an antibody or antibody-like protein, a peptidomimetic or fragment thereof. 3. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to embodiment 1 or 2, wherein the binding agent is a protein or a fragment thereof. 4. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to any one of embodiments 1 to 3, wherein the binding agent is administered to promote wound healing, particularly corneal wound healing. 5. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of embodiments 1 to 4, wherein the binding agent binds to C5a and C3a, thereby essentially inhibiting the activity of C5a and C3a. 6. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to any one of embodiments 1 to 5, wherein the binding agent binds to C5a and C4a, thereby essentially inhibiting the activity of C5a and C4a. 7. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to any one of embodiments 1 to 6, wherein the binding agent binds to C3a and C4a, thereby essentially inhibiting the activity of C3a and C4a. 8. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of embodiments 1 to 7, wherein the binding agent binds to C5a, C3a and C4a, thereby essentially inhibiting the activity of C5a, C3a and C4a. 9. The binding agent is selected from the group consisting of the human C5L2 protein set forth in SEQ ID NO:1, a protein / peptide or fragment that is at least 60% identical to the full-length amino acid sequence of the human C5L2 protein set forth in SEQ ID NO:1, the human C5aR1 protein set forth in SEQ ID NO:2, a protein or fragment that is at least 60% identical to the full-length amino acid sequence of the human C5aR1 protein set forth in SEQ ID NO:2, the human C3aR protein set forth in SEQ ID NO:3, a protein or fragment that is at least 60% identical to the full-length amino acid sequence of the human C3aR protein set forth in SEQ ID NO:3, the mouse C5L2 protein set forth in SEQ ID NO:4, and the mouse C5L2 protein set forth in SEQ ID NO:4. 9. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of embodiments 1 to 8, wherein the binding agent is a protein or protein fragment selected from the group comprising a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein set forth in SEQ ID NO: 5, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein of SEQ ID NO: 5, a mouse C3aR protein set forth in SEQ ID NO: 6, and a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C3aR protein of SEQ ID NO: 6. 10. The binder for use in treating a subject with an eye wound and / or fibrosis according to any one of embodiments 1 to 9, wherein the binder is a protein / peptide or protein fragment and comprises at least one conserved region selected from the group comprising the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17. 11. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to embodiment 10, wherein the binding agent is a protein / peptide or protein fragment and comprises at least two conserved regions selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17. 12. A composition comprising at least two binding agents, preferably proteins or protein fragments, according to any one of embodiments 1 to 11 for use in treating a subject with ocular wounds and / or fibrosis. 13. A composition comprising at least three proteins or protein fragments according to any one of embodiments 1 to 9 for use in treating a subject with ocular wounds and / or fibrosis. 14. A pharmaceutical composition comprising the binding agent of any one of embodiments 1 to 11, or the composition of embodiment 12 or 13, for use in treating a subject with ocular wounds and / or fibrosis. 15. The pharmaceutical composition of embodiment 14, wherein the pharmaceutical composition further comprises a carrier and / or excipient and / or stabilizer. 16. A pharmaceutical composition according to embodiment 14 or 15 for topical administration. 17. A pharmaceutical composition according to embodiment 14 or 15 for intracancer administration. 18. A pharmaceutical composition according to embodiment 14 or 15 for intravitreal application. 19. The pharmaceutical composition according to embodiment 14 or 15 for subconjunctival application. 20. The above subjects are those with conjunctivitis and conjunctival scarring (including ocular pemphigoid), scleritis and episcleritis, corneal scarring and opacity due to corneal ulcers, keratoconjunctivitis, keratitis, bullous keratopathy, corneal degeneration, iridocyclitis and adhesions of the iris and ciliary body, chorioretinal inflammation or chorioretinal scarring / fibrosis due to degeneration or hemorrhage or rupture or neovascularization, fibrotic vitreoretinopathy The binding agent according to any one of embodiments 1 to 11, or the composition according to embodiment 12 or 13, or the pharmaceutical composition according to any one of embodiments 14 to 19, for use in treating a subject suffering from a disease selected from the group comprising: proliferative vitreoretinopathy, retinopathy of prematurity and diabetic retinopathy; choroidal neovascularization and macular degeneration, secondary glaucoma, endophthalmitis, and impaired wound healing and fibrosis after ocular surgery or trauma, including intraocular foreign bodies. 21. The binding agent according to any one of embodiments 1 to 11, or the composition according to embodiment 12 or 13, or the pharmaceutical composition according to any one of embodiments 14 to 19, for use in treating a subject suffering from a disease selected from the group comprising (idiopathic) pulmonary fibrosis, skin keloid formation, scleroderma, myelofibrosis, renal fibrosis, pancreatic fibrosis, and cardiac fibrosis, and fibrosis in (non-)alcoholic steatohepatitis, glomerulonephritis, and (ANCA-associated) vasculitis.

[0096] The following embodiments are the subject of the present invention: 1. A binding agent that binds to complement-anaphylatoxins C5a and / or C3a and / or C4a, thereby preferably inhibiting the activity of C5a and / or C3a and / or C4a, for use in treating subjects with ocular wounds and / or fibrosis. 2. The binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claim 1, wherein the binding agent is selected from the group comprising a protein or fragment thereof, a peptide, a non-IgG scaffold, an aptamer, an oligonucleotide, an antibody or antibody-like protein, a peptidomimetic or fragment thereof. 3. A binder for use in treating a subject with an ocular wound and / or fibrosis according to claim 1 or 2, wherein the binder is a protein or a fragment thereof. 4. A binder for use in treating a subject with an ocular wound and / or fibrosis according to any one of claims 1 to 3, wherein the binder is administered to promote wound healing, particularly corneal wound healing. 5. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 4, wherein the binding agent binds to C5a and C3a, thereby essentially inhibiting the activity of C5a and C3a. 6. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 5, wherein the binding agent binds to C5a and C4a, thereby essentially inhibiting the activity of C5a and C4a. 7. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 6, wherein the binding agent binds to C3a and C4a, thereby essentially inhibiting the activity of C3a and C4a. 8. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 7, wherein the binding agent binds to C5a, C3a and C4a, thereby essentially inhibiting the activity of C5a, C3a and C4a. 9. A binding agent for use in treating a subject with eye wounds and / or fibrosis according to any one of claims 1 to 6 or 8, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C5a protein having the amino acid sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, where overlap means overlapping of the target amino acid sequences of an antibody, antibody-like protein, or binding agent and a particular peptide fragment. 10. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claim 9, wherein the binding agent is capable of binding to C5a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO: 22-34. 11. The binder is selected from SEQ ID NOs: 35 to 40 (SEQ ID NO: 35: X1X2ETCEX3RX4, SEQ ID NO: 36: X5X6KX7X8X9L, and SEQ ID NO: 37: X5X6KX7X8X9I), wherein X1 is selected from the group consisting of N, H, D, F, K, Y, and T; X2 is selected from the group consisting of D, L, Y, and H; X3 is selected from the group consisting of Q, E, and K; X4 is selected from the group consisting of A, V, and L; X5 is selected from the group consisting of S, H, P, and N; X6 is selected from the group consisting of H and N; X7 is selected from the group consisting of D, N, H, P, and G; X8 is selected from the group consisting of M, L, I, and V; and X9 is selected from the group consisting of Q, L, and I. 12. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to any one of claims 1 to 5, 7, or 8, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C3a protein having the amino acid sequence set forth in SEQ ID NO:43. 13. A binding agent for use in treating a subject with eye wounds and / or fibrosis as described in claim 12, wherein the binding agent is also capable of binding to human C3a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO: 44-47. 14. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 4 or 6 to 8, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C4a protein having the amino acid sequence set forth in SEQ ID NO:48 or SEQ ID NO:49. 15. A binding agent for use in treating a subject with eye wounds and / or fibrosis as described in claim 13, wherein the binding agent is also capable of binding to human C4a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO: 50. 16. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claims 1 to 15, wherein the binding agent is an antibody or antibody-like protein. 17. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claims 1 to 15, wherein the binding agent is an aptamer. 18. A binding agent for use in treating a subject with an eye wound and / or fibrosis as described in claim 17, wherein the binding agent is an aptamer, and wherein the aptamer can be associated with a nucleic acid molecule consisting of RNA and / or DNA such as disclosed in SEQ ID NO:41. 19. A binding agent for use in treating a subject with an eye wound and / or fibrosis as described in claim 18, wherein the binding agent is an aptamer, wherein the aptamer may be associated with a nucleic acid molecule consisting of RNA and / or DNA as disclosed in SEQ ID NO:41, and wherein the aptamer binds to a binding site on C5a consisting of SEQ ID NO:42. 20. The binding agent is selected from the group consisting of the human C5L2 protein set forth in SEQ ID NO: 1, a protein / peptide or fragment that is at least 60% identical to the full-length amino acid sequence of the human C5L2 protein set forth in SEQ ID NO: 1, the human C5aR1 protein set forth in SEQ ID NO: 2, a protein or fragment that is at least 60% identical to the full-length amino acid sequence of the human C5aR1 protein set forth in SEQ ID NO: 2, the human C3aR protein set forth in SEQ ID NO: 3, a protein or fragment that is at least 60% identical to the full-length amino acid sequence of the human C3aR protein set forth in SEQ ID NO: 3, the mouse C5L2 protein set forth in SEQ ID NO: 4, and the mouse C5L2 protein set forth in SEQ ID NO: 4. 20. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 19, wherein the binding agent is a protein or protein fragment selected from the group comprising: a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein set forth in SEQ ID NO: 5, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein of SEQ ID NO: 5, a mouse C3aR protein set forth in SEQ ID NO: 6, and a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C3aR protein of SEQ ID NO: 6. 21. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 20, wherein the binding agent is a protein / peptide or protein fragment and comprises at least one conserved region selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17. 22. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to claim 21, wherein the binding agent is a protein / peptide or protein fragment and comprises at least two conserved regions selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17. 23. A binder for use in treating a subject with eye wounds and / or fibrosis according to any one of claims 1 to 22, wherein the binder-mediated inhibition of C3a and / or C4a and / or C5a can be determined by a cell activation assay, preferably a fibroblast / myofibroblast activation and / or transdifferentiation assay. 24. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 23, wherein the binding agent-mediated inhibition of C3a and / or C4a and / or C5a can be determined by a cell activation assay, preferably a fibroblast / myofibroblast activation and / or transdifferentiation assay, and wherein the binding agent selected from a protein or protein fragment / peptide, a non-IgG scaffold, an aptamer, an antibody or fragment thereof is effective by inhibiting myofibroblast activation activity by preferably at least 10%, more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, even more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, and even more preferably at least 65%. 25. A binder for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 24, wherein the binder at least essentially inhibits the process of fibroblast / myofibroblast activation and / or transdifferentiation and has a molecular weight of preferably less than 90 kDa, preferably 80 kDa or less, preferably 70 kDa or less, more preferably 60 kDa or less, more preferably 50 kDa or less, more preferably 45 kDa or less, more preferably 40 kDa or less, even more preferably 35 kDa or less, even more preferably 30 kDa or less, even more preferably 25 kDa or less, even more preferably 20 kDa or less, even more preferably 15 kDa or less, and even more preferably 10 kDa or less. 26. A composition comprising at least two binding agents, preferably proteins or protein fragments, according to any one of claims 1 to 25, for use in treating a subject with an ocular wound and / or fibrosis. 27. A composition comprising at least three binding agents, preferably proteins or protein fragments, according to any one of claims 1 to 26, for use in treating a subject with an ocular wound and / or fibrosis. 28. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 25, or the composition of claim 26 or 27, for use in treating a subject with ocular wounds and / or fibrosis. 29. The above subjects are those with conjunctivitis and conjunctival scarring (including ocular pemphigoid), scleritis and episcleritis, corneal scarring and opacity due to corneal ulcers, keratoconjunctivitis, keratitis, bullous keratopathy, corneal degeneration, iridocyclitis and adhesions of the iris and ciliary body, chorioretinal inflammation or chorioretinal scarring / fibrosis due to degeneration or hemorrhage or rupture or neovascularization, fibrotic vitreoretinopathy 29. The binding agent of any one of claims 1 to 25, or the composition of claim 25 or 26, or the pharmaceutical composition of claim 28, for use in treating a subject suffering from a disease selected from the group comprising: proliferative vitreoretinopathy, retinopathy of prematurity and diabetic retinopathy; choroidal neovascularization and macular degeneration, secondary glaucoma, endophthalmitis, and impaired wound healing and fibrosis after ocular surgery or trauma, including intraocular foreign bodies. 30. The binding agent of any one of claims 1 to 25, or the composition of claim 26 or 27, or the pharmaceutical composition of claim 28, for use in treating a subject suffering from a disease selected from the group comprising (idiopathic) pulmonary fibrosis, skin keloid formation, scleroderma, myelofibrosis, renal fibrosis, pancreatic fibrosis, and cardiac fibrosis, and fibrosis in (non-)alcoholic steatohepatitis, glomerulonephritis, and (ANCA-associated) vasculitis. 31. The binding agent of any one of claims 1 to 25, the composition of claim 26 or 27, or the pharmaceutical composition of claim 28, wherein the subject suffers from pulmonary fibrosis. 32. The binding agent of any one of claims 1 to 25, the composition of claim 26 or 27, or the pharmaceutical composition of claim 28, wherein the subject suffers from corneal fibrosis. 33. The binding agent of any one of claims 1 to 25, the composition of claim 26 or 27, or the pharmaceutical composition of claim 28, wherein the subject suffers from chorioretinal fibrosis. 34. The binding agent of any one of claims 1 to 25, or the composition of claim 26 or 27, or the pharmaceutical composition of claim 28, wherein the subject suffers from fibrosis due to glomerulonephritis and / or renal fibrosis. 35. The binding agent of any one of claims 1 to 25, or the composition of claim 26 or 27, or the pharmaceutical composition of claim 28, wherein the subject suffers from steatohepatitis and / or liver fibrosis.

[0097] The following embodiments are the subject of the present invention: 1. A binding agent that binds to complement-anaphylatoxins C5a and / or C3a and / or C4a, thereby preferably inhibiting the activity of C5a and / or C3a and / or C4a, for use in treating subjects with ocular wounds and / or fibrosis. 2. The binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claim 1, wherein the binding agent is selected from the group comprising a protein or fragment thereof, a peptide, a non-IgG scaffold, an aptamer, an oligonucleotide, an antibody or antibody-like protein, a peptidomimetic or fragment thereof. 3. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claim 1 or 2, wherein the binding agent is administered to promote wound healing, particularly corneal wound healing. 4. A binding agent for use in treating a subject with eye wounds and / or fibrosis according to any one of claims 1 to 3, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C5a protein having the amino acid sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, where overlap means overlapping of the target amino acid sequences of an antibody, antibody-like protein, or binding agent and a particular peptide fragment. 5. A binding agent for use in treating a subject with ocular wounds and / or fibrosis according to claim 4, wherein the binding agent is capable of binding to C5a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NOs: 22-34. 6. The binder is selected from SEQ ID NOs: 35 to 40 (SEQ ID NO: 35: X1X2ETCEX3RX4, SEQ ID NO: 36: X5X6KX7X8X9L, and SEQ ID NO: 37: X5X6KX7X8X9I), wherein X1 is selected from the group consisting of N, H, D, F, K, Y, and T; X2 is selected from the group consisting of D, L, Y, and H; X3 is selected from the group consisting of Q, E, and K; X4 is selected from the group consisting of A, V, and L; X5 is selected from the group consisting of S, H, P, and N; X6 is selected from the group consisting of H and N; X7 is selected from the group consisting of D, N, H, P, and G; X8 is selected from the group consisting of M, L, I, and V; and X9 is selected from the group consisting of Q, L, and I. 7. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 3, wherein the binding agent is capable of binding to several overlapping peptide fragments of the complement component C3a protein having the amino acid sequence set forth in SEQ ID NO:43. 8. A binding agent for use in treating a subject with eye wounds and / or fibrosis as described in claim 7, wherein the binding agent is also capable of binding to human C3a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO: 44-47. 9. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 3, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C4a protein having the amino acid sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49. 10. A binding agent for use in treating a subject with eye wounds and / or fibrosis as described in claim 9, wherein the binding agent is also capable of binding to human C4a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO: 50. 11. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claims 1 to 10, wherein the binding agent is an antibody or antibody-like protein. 12. A binding agent for use in treating a subject with an ocular wound and / or fibrosis according to claims 1 to 10, wherein the binding agent is an aptamer. 13. A binding agent for use in treating a subject with an ocular wound and / or fibrosis as described in claim 12, wherein the binding agent is an aptamer, and wherein the aptamer can be associated with a nucleic acid molecule consisting of RNA and / or DNA such as disclosed in SEQ ID NO:41. 14. A binding agent for use in treating a subject with an eye wound and / or fibrosis as described in claim 13, wherein the binding agent is an aptamer, wherein the aptamer may be associated with a nucleic acid molecule consisting of RNA and / or DNA as disclosed in SEQ ID NO:41, and wherein the aptamer binds to a binding site on C5a consisting of SEQ ID NO:42. 15. The binding agent is selected from the group consisting of the human C5L2 protein set forth in SEQ ID NO: 1, a protein / peptide or fragment that is at least 60% identical to the full-length amino acid sequence of the human C5L2 protein set forth in SEQ ID NO: 1, the human C5aR1 protein set forth in SEQ ID NO: 2, a protein or fragment that is at least 60% identical to the full-length amino acid sequence of the human C5aR1 protein set forth in SEQ ID NO: 2, the human C3aR protein set forth in SEQ ID NO: 3, a protein or fragment that is at least 60% identical to the full-length amino acid sequence of the human C3aR protein set forth in SEQ ID NO: 3, the mouse C5L2 protein set forth in SEQ ID NO: 4, and the mouse C5L2 protein set forth in SEQ ID NO: 4. 15. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 14, wherein the binding agent is a protein or protein fragment selected from the group comprising: a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein set forth in SEQ ID NO: 5, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein of SEQ ID NO: 5, a mouse C3aR protein set forth in SEQ ID NO: 6, and a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C3aR protein of SEQ ID NO: 6. 16. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 15, wherein the binding agent is a protein / peptide or protein fragment and comprises at least one conserved region selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17. 17. The binding agent for use in treating a subject with an eye wound and / or fibrosis according to claim 16, wherein the binding agent is a protein / peptide or protein fragment and comprises at least two conserved regions selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17. 18. A binding agent for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 15 to 17, wherein the binding agent is a protein / peptide or protein fragment and comprises at least one conserved region selected from the group comprising the amino acid sequence set forth in SEQ ID NO: 18 and the amino acid sequence set forth in SEQ ID NO: 19. 19. A composition comprising at least two binding agents, preferably proteins or protein fragments, according to any one of claims 1 to 18, for use in treating a subject with an ocular wound and / or fibrosis. 20. A composition comprising at least three proteins or protein fragments according to any one of claims 1 to 19 for use in treating a subject with ocular wounds and / or fibrosis. 21. A pharmaceutical composition comprising a binding agent according to any one of claims 1 to 18 or a composition according to claim 19 or 20 for use in treating a subject with an ocular wound and / or fibrosis. 22. The above subjects are those with conjunctivitis and conjunctival scarring (including ocular pemphigoid), scleritis and episcleritis, corneal scarring and opacity due to corneal ulcers, keratoconjunctivitis, keratitis, bullous keratopathy, corneal degeneration, iridocyclitis and adhesions of the iris and ciliary body, chorioretinal inflammation or chorioretinal scarring / fibrosis due to degeneration or hemorrhage or rupture or neovascularization, fibrotic vitreoretinopathy 22. The binding agent of any one of claims 1 to 18, or the composition of claim 19 or 20, or the pharmaceutical composition of claim 21, for use in treating a subject suffering from a disease selected from the group comprising: proliferative vitreoretinopathy, retinopathy of prematurity and diabetic retinopathy; choroidal neovascularization and macular degeneration, secondary glaucoma, endophthalmitis, and impaired wound healing and fibrosis after ocular surgery or trauma, including intraocular foreign bodies. 23. The binding agent according to any one of claims 1 to 18, or the composition according to claim 19 or 20, or the pharmaceutical composition of claim 21, for use in treating a subject suffering from a disease selected from the group comprising (idiopathic) pulmonary fibrosis, skin keloid formation, scleroderma, myelofibrosis, renal fibrosis, pancreatic fibrosis, and cardiac fibrosis, and fibrosis in (non-)alcoholic steatohepatitis, glomerulonephritis, and (ANCA-associated) vasculitis. [Brief explanation of the drawings]

[0098] [Figure 1] FIG. 1 shows the inhibitory effect of human C5L2 protein fragment (hC5L2) on C3a-mediated activation of myofibroblasts using human keratocytes. [Figure 2] FIG. 2 shows the inhibitory effect of human C5L2 protein fragment (hC5L2) on C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 3] FIG. 3 shows the inhibitory effect of human C5L2 protein fragment (hC5L2) on C5a- and C3a-mediated activation of myofibroblasts using human keratocytes. [Figure 4]FIG. 4 shows the inhibitory effect of mouse C5L2 protein fragment (mC5L2) on C3a-mediated activation of myofibroblasts using human keratocytes. [Figure 5] FIG. 5 shows the inhibitory effect of mouse C5L2 protein fragment (mC5L2) on C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 6] FIG. 6 shows the inhibitory effect of mouse C5L2 protein fragment (mC5L2) on C5a- and C3a-mediated activation of myofibroblasts using human keratocytes. [Figure 7] FIG. 7 shows the effect of human C5L2 protein fragment concentration on myofibroblasts in the presence of fetal calf serum (FCS) using human keratocytes. [Figure 8] FIG. 8 shows the effect of mouse C5L2 protein fragment concentration on myofibroblasts in the presence of fetal calf serum (FCS) using human keratocytes. [Figure 9] FIG. 9 shows the effect of human C5L2 protein fragment concentration on myofibroblasts in the absence of fetal bovine serum using human keratocytes. [Figure 10] FIG. 10 shows the effect of mouse C5L2 protein fragment concentration on myofibroblasts in the absence of fetal bovine serum using human keratocytes. [Figure 11] FIG. 11 shows the inhibitory effect of human C5L2 protein fragment (hC5L2) on C3a-mediated activation of myofibroblasts using human alveolar basal epithelial cells. [Figure 12] FIG. 12 shows the inhibitory effect of human C5L2 protein fragment (hC5L2) on C5a-mediated activation of myofibroblasts using human alveolar basal epithelial cells. [Figure 13] FIG. 13 shows the inhibitory effect of human C5L2 protein fragment (hC5L2) on C5a- and C3a-mediated activation of myofibroblasts using human alveolar basal epithelial cells. [Figure 14]FIG. 14 shows the effect of human C5L2 protein fragment concentration on myofibroblasts in the presence of fetal calf serum (FCS) using human alveolar basal epithelial cells. [Figure 15] FIG. 15 shows the effect of inhibiting C3a-mediated myofibroblast activation by full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) using human keratocytes. [Figure 16] FIG. 16 shows the effect of inhibiting C5a-mediated myofibroblast activation by full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) using human keratocytes. [Figure 17] FIG. 17 shows the effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) on inhibiting C3a- and C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 18] FIG. 18 shows the effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) concentration on myofibroblasts in the presence of fetal calf serum (FCS) using human keratocytes. [Figure 19] FIG. 19 shows the effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) concentration on myofibroblasts in the absence of fetal calf serum (FCS) using human keratocytes. [Figure 20] FIG. 20 shows the effect of inhibiting C3a-mediated myofibroblast activation by full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) using human keratocytes. [Figure 21] FIG. 21 shows the effect of inhibiting C5a-mediated myofibroblast activation by full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) using human keratocytes. [Figure 22]FIG. 22 shows the effect of inhibiting C3a- and C5a-mediated activation of myofibroblasts by full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) using human keratocytes. [Figure 23] FIG. 23 shows the effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) concentration on myofibroblasts in the presence of fetal calf serum (FCS) using human keratocytes. [Figure 24] FIG. 24 shows the effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) concentration on myofibroblasts in the absence of fetal calf serum (FCS) using human keratocytes. [Figure 25] FIG. 25 shows the effect of inhibiting C3a-mediated myofibroblast activation by full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) using human keratocytes. [Figure 26] FIG. 26 shows the effect of inhibiting C5a-mediated myofibroblast activation by full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) using human keratocytes. [Figure 27] FIG. 27 shows the effect of full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) on inhibiting C3a- and C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 28] FIG. 28 shows the effect of full-length recombinant human C3a anaphylatoxin chemotactic receptor 1 (rhC3AR) concentration on myofibroblasts in the presence of fetal calf serum (FCS) using human keratocytes. [Figure 29] FIG. 29 shows the effect of full-length recombinant human C3a anaphylatoxin chemotactic receptor 1 (rhC3AR) concentration on myofibroblasts in the absence of fetal calf serum (FCS) using human keratocytes. [Figure 30] FIG. 30 shows the effect of inhibiting C3a-mediated myofibroblast activation by an RNA / DNA aptamer that binds to human C5a using human keratocytes. [Figure 31] FIG. 31 shows the effect of inhibiting C5a-mediated myofibroblast activation by an RNA / DNA aptamer that binds to human C5a, using human keratocytes. [Figure 32] FIG. 32 shows the effect of inhibiting C3a- and C5a-mediated activation of myofibroblasts by an RNA / DNA aptamer that binds to human C5a, using human keratocytes. [Figure 33] FIG. 33 shows the effect of RNA / DNA aptamer concentration that binds human C5a on myofibroblasts in the presence of fetal calf serum (FCS) using human keratocytes. [Figure 34] FIG. 34 shows the effect of RNA / DNA aptamer concentration that binds human C5a on myofibroblasts in the absence of fetal calf serum (FCS) using human keratocytes. [Figure 35] FIG. 35 shows the effect of an antibody that binds to human C5a (antibody 250565) on inhibiting C3a, C5a, or C3a and C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 36] FIG. 36 shows the effect of an antibody that binds to human C5a (antibody 308733) on inhibiting C3a, C5a, or C3a and C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 37] FIG. 37 shows the effect of an antibody that binds to human C3a (antibody sc28294) on inhibiting C3a-, C5a-, or C3a- and C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 38] FIG. 38 shows the effect of an antibody that binds to human C3a (antibody HM1072) on inhibiting C3a-, C5a-, or C3a- and C5a-mediated activation of myofibroblasts using human keratocytes. [Figure 39] FIG. 39 shows fibrosis assessment scores in a mouse corneal alkali burn model 20 days after corneal alkali burn in the presence or absence of mouse C5L2 protein fragment (mC5L2). [Figure 40] FIG. 40 shows the Cowell fibrosis score in a mouse corneal alkali burn model 20 days after corneal alkali burn in the presence or absence of mouse C5L2 protein fragment (mC5L2). DETAILED DESCRIPTION OF THE INVENTION

[0099] Example 1 Human C5L2 protein fragments cause inhibition of C3a-activated myofibroblasts

[0100] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO:18 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C3a-activated myofibroblasts. Human keratocytes were stimulated with human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 1). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours were used. As shown in Figure 1, 0.1 μg / ml of C3a induced significant myofibroblast activation (measured as aSMA-positive cells, 74 ± 22%) compared to the reference group (serum-free: 10 ± 11%; FCS: 16 ± 14%; p < 0.001 and p < 0.001, respectively). A list of genes with differential expression levels (fold change: ≥2 or ≤-2) after 24 hours of incubation with 0.1 μg / ml human C3a and DMEM growth medium without fetal bovine serum (serum-free control), derived from human keratocytes and generated from gene expression Clariom S human microarrays, is shown in Table 2. Compared to C3a-activated myofibroblasts, incubation in the presence of human C3a and human C5L2 protein fragments resulted in a significant decrease (hC5L2 0.1 μg / ml: 16±9%; hC5L2 0.2 μg / ml: 17±11%; hC5L2 0.3 μg / ml: 8±7%) (p<0.001, p<0.001, and p<0.001, respectively). Table 5 shows a list of genes with different expression levels (fold change: ≥2 or ≤-2) after 24 hours of incubation with 0.1 μg / ml of human C3a and 0.3 μg / ml of the human C5L2 protein fragment according to SEQ ID NO: 18, obtained from human keratocytes and generated from gene expression Clariom S human microarrays. Thus, the human C5L2 protein fragment was responsible for causing the inhibition of C3a-activated myofibroblasts. Bars = standard error of the mean.

[0101] Example 2 Human C5L2 protein fragments cause inhibition of C5a-activated myofibroblasts

[0102] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO:18 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a-activated myofibroblasts. Human keratocytes were stimulated with human C5a for 24 hours and evaluated for activated myofibroblasts (Figure 2). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours were used. As shown in Figure 2, 0.1 μg / ml of C5a induced significant myofibroblast activation (measured as aSMA-positive cells, 77 ± 23%) compared to the reference group (serum-free: 10 ± 11%; FCS: 16 ± 14%; p < 0.001 and p < 0.001, respectively). Table 3 lists genes with differential expression levels (fold change: ≥2 or ≤-2) after 24 hours of incubation with 0.1 μg / ml human C5a and DMEM growth medium without fetal bovine serum (serum-free control), derived from human keratocytes and generated from gene expression Clariom S human microarrays. Compared to C5a-activated myofibroblasts, incubation in the presence of human C5a and human C5L2 protein fragments resulted in a significant reduction in activated myofibroblasts (hC5L2 0.1 μg / ml: 41 ± 22%; hC5L2 0.2 μg / ml: 26 ± 26%; hC5L2 0.3 μg / ml: 7 ± 7%) (p = 0.001, p < 0.001, and p < 0.001, respectively). Table 6 shows a list of genes with different expression levels (fold change: ≥2 or ≤-2) after 24 hours of incubation with 0.1 μg / ml of human C5a and 0.1 μg / ml of human C5a with 0.3 μg / ml of the human C5L2 protein fragment according to SEQ ID NO: 18, obtained from human keratocytes and generated from gene expression Clariom S human microarrays. Thus, the human C5L2 protein fragment was responsible for causing the inhibition of C5a-activated myofibroblasts. Bars = standard error of the mean.

[0103] Example 3 Human C5L2 protein fragments cause inhibition of C5a- and C3a-activated myofibroblasts

[0104] To explore the potential functional role of the human C5L2 protein fragment according to SEQ ID NO:18 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a / C3a-activated myofibroblasts. Human keratocytes were stimulated with human C5a and human C3a, respectively, for 24 hours and evaluated for activated myofibroblasts (Figure 3). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS). As shown in Figure 3, C5a and C3a, both at a concentration of 0.1 μg / ml, caused significant myofibroblast activation (87 ± 11% as measured by aSMA-positive cells) compared to the control group (serum-free: 10 ± 11%; FCS: 16 ± 14%; p < 0.001 and p < 0.001, respectively). Table 4 lists genes with differential expression levels (fold change: ≥ 2 or ≤ -2) after 24 hours of incubation with human C3a and human C5a, both at a concentration of 0.1 μg / ml, and DMEM growth medium without fetal bovine serum (serum-free control), generated from a Clariom S human gene expression microarray obtained from human keratocytes. Incubation in the presence of human C3a, C5a, and human C5L2 protein fragments resulted in a significant reduction in activated myofibroblasts compared to C5a and C3a-activated myofibroblasts (hC5L2 0.1 μg / ml: 23±14%; hC5L2 0.2 μg / ml: 16±12%; hC5L2 0.3 μg / ml: 6±6%) (p<0.001, p<0.001, and p<0.001, respectively). Table 7 lists genes with different expression levels (fold change: ≥2 or ≤-2) after 24-hour incubation with human C3a and C5, both at 0.1 μg / ml, and human C3a and C5, both at 0.1 μg / ml, with 0.3 μg / ml of the human C5L2 protein fragment according to SEQ ID NO: 18, obtained from human keratocytes and generated from gene expression Clariom S human microarrays.Thus, human C5L2 protein fragments were responsible for causing inhibition of myofibroblasts activated by C5a and C3a. Bars = standard error of the mean.

[0105] Example 4 Murine C5L2 protein fragments cause inhibition of C3a-activated myofibroblasts

[0106] To explore the potential functional role of the murine C5L2 protein fragment (mC5L2) according to SEQ ID NO:19 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C3a-activated myofibroblasts. Human keratocytes were stimulated with human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 4). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours were used. As shown in Figure 4, 0.1 μg / ml of C3a induced significant myofibroblast activation (measured as aSMA-positive cells, 74 ± 22%) compared to the reference group (serum-free: 10 ± 11%; FCS: 16 ± 14%; p < 0.001 and p < 0.001, respectively). Compared to C3a-activated myofibroblasts, incubation in the presence of C3a and mouse C5L2 protein fragments resulted in a significant reduction in activated myofibroblasts (mC5L2 0.1 μg / ml: 31 ± 13%; mC5L2 0.2 μg / ml: 16 ± 10%; mC5L2 0.3 μg / ml: 21 ± 13%) (p < 0.001, p < 0.001, and p < 0.001, respectively). Thus, mouse C5L2 protein fragments were responsible for the inhibition of C3a-activated myofibroblasts. Bars = standard error of the mean.

[0107] Example 5 Murine C5L2 protein fragments inhibit C5a-activated myofibroblasts

[0108] To explore the potential functional role of the murine C5L2 protein fragment (mC5L2) according to SEQ ID NO:19 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a-activated myofibroblasts. Human keratocytes were stimulated with human C5a for 24 hours and evaluated for activated myofibroblasts (Figure 5). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours were used. As shown in Figure 5, 0.1 μg / ml of C5a induced significant myofibroblast activation (measured as aSMA-positive cells, 77 ± 23%) compared to the reference group (serum-free: 10 ± 11%; FCS: 16 ± 14%; p < 0.001 and p < 0.001, respectively). Compared to C5a-activated myofibroblasts, incubation in the presence of C5a and mouse C5L2 protein fragments resulted in a significant reduction in activated myofibroblasts (mC5L2 0.1 μg / ml: 33±18%; mC5L2 0.2 μg / ml: 20±19%; mC5L2 0.3 μg / ml: 20±10%) (p<0.001, p<0.001, and p<0.001, respectively). Thus, mouse C5L2 protein fragments were responsible for the inhibition of C5a-activated myofibroblasts. Bars = standard error of the mean.

[0109] Example 6 Murine C5L2 protein fragments cause inhibition of C5a- and C3a-activated myofibroblasts

[0110] To explore the potential functional role of the murine C5L2 protein fragment (mC5L2) according to SEQ ID NO:19 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a / C3a-activated myofibroblasts. Human keratocytes were stimulated with human C5a and human C3a, respectively, for 24 hours and evaluated for activated myofibroblasts (Figure 6). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS). As shown in Figure 6, both C5a and C3a at a concentration of 0.1 μg / ml induced significant activation of myofibroblasts (87 ± 11% as measured by aSMA-positive cells) compared with the control group (serum-free: 10 ± 11%; FCS: 16 ± 14%; p < 0.001 and p < 0.001, respectively). Incubation in the presence of human C3a, C5a, and mouse C5L2 protein fragments resulted in a significant reduction in activated myofibroblasts compared with C5a and C3a-activated myofibroblasts (mC5L2 0.1 μg / ml: 17 ± 10%; mC5L2 0.2 μg / ml: 11 ± 12%; mC5L2 0.3 μg / ml: 13 ± 11%) (p < 0.001, p < 0.001, and p < 0.001, respectively). Thus, the murine C5L2 protein fragment was responsible for causing inhibition of myofibroblasts activated by C5a and C3a. Bars = standard error of the mean.

[0111] Example 7 Effect of human C5L2 protein fragment concentration on myofibroblasts in the presence of fetal bovine serum

[0112] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO:18 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium containing 10% fetal calf serum (FCS) and different concentrations of human C5L2 protein fragments (Figure 7). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. Human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum were used as reference groups (serum-free: 10±11%; FCS: 16±14%). As shown in Figure 7, human C5L2 protein fragments were found to have a slight positive effect on myofibroblast activation at low concentrations (hC5L2 0.05 μg / ml: 19±15%; hC5L2 0.1 μg / ml: 24±21%; hC5L2 0.2 μg / ml: 16±12%), whereas inhibition of myofibroblast activity was observed at higher concentrations (hC5L2 0.3 μg / ml: 11±10%). Nevertheless, the differences remained small compared to human keratocytes cultured in 10% FCS (p=0.554, p=0.136, p=0.918, and p=0.345, respectively).

[0113] Example 8 Effect of mouse C5L2 protein fragment concentration on myofibroblasts in the presence of fetal bovine serum

[0114] To explore the potential functional role of the mouse C5L2 protein fragment (mC5L2) according to SEQ ID NO:19 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium containing 10% fetal calf serum (FCS) and different concentrations of the mouse C5L2 protein fragment (Figure 8). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (serum-free: 10±11%; FCS: 16±14%). As shown in Figure 8, mouse C5L2 protein fragments were found to have a slight positive effect on myofibroblast activation at low concentrations (mC5L2 0.05 μg / ml: 11±6%; mC5L2 0.1 μg / ml: 19±15%; mC5L2 0.2 μg / ml: 11±12%), whereas inhibition of myofibroblast activity was observed at higher concentrations (mC5L2 0.3 μg / ml: 11±7%). Nevertheless, the differences remained small compared to human keratocytes cultured in 10% FCS (p=0.101, p=0.580, p=0.293, and p=0.277, respectively).

[0115] Example 9 Effect of human C5L2 protein fragment concentration on myofibroblasts in the absence of fetal bovine serum

[0116] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO: 18 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium without fetal bovine serum and with different concentrations of the human C5L2 protein fragment (Figure 9). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal bovine serum (FCS) (serum-free: 10±11%; FCS: 16±14%). As shown in Figure 9, human C5L2 protein fragments were found to have a slight positive effect on myofibroblast activation at low concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (hC5L2 0.05 μg / ml: 23 ± 15%; hC5L2 0.1 μg / ml: 19 ± 11%; p = 0.005 and p = 0.039, respectively), whereas at higher concentrations, myofibroblast inhibition was observed and showed no difference compared to the serum-free control (hC5L2 0.2 μg / ml: 17 ± 16%; hC5L2 0.3 μg / ml: 9 ± 8%; p = 0.150 and p = 0.755, respectively). A list of genes with different expression levels (fold change: ≧2 or ≦−2) after 24 hours of incubation with 0.3 μg / ml of human C5L2 protein fragment according to SEQ ID NO: 18 and DMEM growth medium without fetal bovine serum (serum-free control) obtained from human keratocytes and generated from gene expression Clariom S human microarrays is shown in Table 8.

[0117] Example 10 Effect of mouse C5L2 protein fragment concentration on myofibroblasts in the absence of fetal bovine serum

[0118] To explore the potential functional role of the mouse C5L2 protein fragment (mC5L2) according to SEQ ID NO: 19 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium without fetal bovine serum and with different concentrations of the mouse C5L2 protein fragment (Figure 10). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal bovine serum (FCS) (serum-free: 10±11%; FCS: 16±14%). As shown in Figure 10, mouse C5L2 protein fragments were found to have a slight positive effect on myofibroblast activation at low concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (mC5L2 0.05 μg / ml: 23±13%; mC5L2 0.1 μg / ml: 22±17%; p=0.003 and p=0.009, respectively), whereas at higher concentrations, myofibroblast inhibition was observed and showed no difference compared to the serum-free control (hC5L2 0.2 μg / ml: 18±10%; hC5L2 0.3 μg / ml: 9±7%; p=0.064 and p=0.647, respectively).

[0119] Example 11 Human C5L2 protein fragments cause inhibition of C3a-activated myofibroblasts

[0120] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO: 18 in the treatment of subjects with pulmonary fibrosis, we investigated the effect of its presence on C3a-activated myofibroblasts. Human alveolar basal epithelial cells (A549 cells) were stimulated with human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 11). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human alveolar basal epithelial cells (A549 cells) incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS). As shown in Figure 11, C3a at 0.1 μg / ml caused significant activation of myofibroblasts (measured as aSMA-positive cells, 87 ± 6%) compared to the reference group (serum-free: 16 ± 16%; FCS: 39 ± 21%; p < 0.001 and p < 0.001, respectively). Incubation in the presence of human C3a and human C5L2 protein fragments resulted in a significant reduction in C3a-activated myofibroblasts (hC5L2 at 0.1 μg / ml: 55 ± 19%; hC5L2 at 0.2 μg / ml: 5 ± 6%; hC5L2 at 0.3 μg / ml: 8 ± 12%) (p = 0.001, p < 0.001, and p < 0.001, respectively). As shown in Figure 11, the specific effect of human C5L2 protein fragments on myofibroblast activation showed no difference compared to serum-free controls (hC5L2 0.3 μg / ml: 9±11%; p=0.250). Thus, human C5L2 protein fragments were responsible for causing inhibition of C3a-activated myofibroblasts. Bars = standard error of the mean.

[0121] Example 12 Human C5L2 protein fragments cause inhibition of C5a-activated myofibroblasts

[0122] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO: 18 in the treatment of subjects with pulmonary fibrosis, we investigated the effect of its presence on C5a-activated myofibroblasts. Human alveolar basal epithelial cells (A549 cells) were stimulated with human C5a for 24 hours and evaluated for activated myofibroblasts (Figure 12). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human alveolar basal epithelial cells (A549 cells) incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS). As shown in Figure 12, C5a at 0.1 μg / ml caused significant activation of myofibroblasts (measured as aSMA-positive cells, 83 ± 10%) compared to the reference group (serum-free: 16 ± 16%; FCS: 39 ± 21%; p < 0.001 and p < 0.001, respectively). Incubation in the presence of human C5a and human C5L2 protein fragments resulted in a significant decrease (hC5L2 at 0.1 μg / ml: 3 ± 4%; hC5L2 at 0.2 μg / ml: 11 ± 13%; hC5L2 at 0.3 μg / ml: 10 ± 10%) compared to C5a-activated myofibroblasts (p < 0.001, p < 0.001, and p < 0.001, respectively). As shown in Figure 12, the specific effect of human C5L2 protein fragments on myofibroblast activation showed no difference compared to serum-free controls (hC5L2 0.3 μg / ml: 9±11%; p=0.250). Thus, human C5L2 protein fragments were responsible for causing inhibition of C5a-activated myofibroblasts. Bars = standard error of the mean.

[0123] Example 13 Human C5L2 protein fragments cause inhibition of C5a- and C3a-activated myofibroblasts

[0124] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO: 18 in the treatment of subjects with pulmonary fibrosis, we investigated the effect of its presence on C5a / C3a-activated myofibroblasts. Human alveolar basal epithelial cells (A549 cells) were stimulated with human C5a and human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 13). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human alveolar basal epithelial cells (A549 cells) incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS). As shown in Figure 13, both C5a and C3a at a concentration of 0.1 μg / ml caused significant activation of myofibroblasts (measured as aSMA-positive cells, 90 ± 10%) compared to the control group (serum-free: 16 ± 16%; FCS: 39 ± 21%; p < 0.001 and p < 0.001, respectively). Incubation in the presence of human C3a, C5a, and human C5L2 protein fragments significantly reduced C5a- and C3a-activated myofibroblasts (hC5L2 0.1 μg / ml: 44 ± 37%; hC5L2 0.2 μg / ml: 21 ± 25%; hC5L2 0.3 μg / ml: 16 ± 14%) (p = 0.006, p < 0.001, and p < 0.001, respectively). As shown in Figure 13, the specific effect of human C5L2 protein fragments on myofibroblast activation showed no difference compared to serum-free controls (hC5L2 0.3 μg / ml: 9±11%; p=0.250). Thus, human C5L2 protein fragments were responsible for causing inhibition of myofibroblasts activated by C5a and C3a. Bars = standard error of the mean.

[0125] Example 14 Human C5L2 protein fragments cause myofibroblast inhibition in the presence of fetal bovine serum

[0126] To explore the potential functional role of the human C5L2 protein fragment (hC5L2) according to SEQ ID NO: 18 in the treatment of subjects with pulmonary fibrosis, we investigated the effect of its concentration on myofibroblasts. Human alveolar basal epithelial cells (A549 cells) were incubated for 24 hours in DMEM growth medium containing 10% fetal calf serum (FCS) and different concentrations of human C5L2 protein fragment (Figure 14). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human alveolar basal epithelial cells (A549 cells) incubated for 24 hours in DMEM growth medium with and without fetal calf serum (serum-free: 16±16%; FCS: 39±21%). As shown in Figure 14, human C5L2 protein fragments were found to have a slight positive effect on myofibroblast activation at low concentrations (hC5L2 0.05 μg / ml: 30±34% and hC5L2 0.1 μg / ml: 22±18%), whereas inhibition of myofibroblast activity was observed at higher concentrations (hC5L2 0.2 μg / ml: 14±9% and hC5L2 0.3 μg / ml: 10±15%). Nevertheless, the differences remained small compared to human keratocytes cultured in 10% FCS (p=0.268, p=0.360, p=0.693, and p=0.390, respectively). As shown in Figure 14, the intrinsic effect of human C5L2 protein fragments on myofibroblast activation showed no difference compared to serum-free controls (hC5L2 0.3 μg / ml: 9±11%; p=0.250).

[0127] Example 15 Full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) protein causes inhibition of C3a-activated myofibroblasts

[0128] To explore the potential functional role of the rhC5L2 protein according to SEQ ID NO:1 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C3a-activated myofibroblasts. Human keratocytes were stimulated with human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 15). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, we used human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours. As shown in Figure 15, 0.1 μg / ml of C3a caused significant activation of myofibroblasts (measured as aSMA-positive cells, 74±22%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C3a-activated myofibroblasts, incubation in the presence of human C3a and human rhC5L2 protein resulted in a significant decrease (rhC5L2 0.1 μg / ml: 13±17%; rhC5L2 0.2 μg / ml: 20±9%; rhC5L2 0.3 μg / ml: 24±21%; rhC5L2 0.5 μg / ml: 34±20%) (p<0.001, p<0.001, and p<0.001, respectively). Thus, rhC5L2 protein was responsible for the inhibition of C3a-activated myofibroblasts. Bars = standard error of the mean.

[0129] Example 16 Full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) protein causes inhibition of C5a-activated myofibroblasts

[0130] To explore the potential functional role of the rhC5L2 protein according to SEQ ID NO:1 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a-activated myofibroblasts. Human keratocytes were stimulated with human C5a for 24 hours and evaluated for activated myofibroblasts (Figure 16). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, we used human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours. As shown in Figure 16, 0.1 μg / ml of C5a caused significant activation of myofibroblasts (measured as aSMA-positive cells, 77±23%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C5a-activated myofibroblasts, incubation in the presence of human C5a and human rhC5L2 protein resulted in a significant decrease (rhC5L2 0.1 μg / ml: 11±7%; rhC5L2 0.2 μg / ml: 24±11%; rhC5L2 0.3 μg / ml: 26±14%; rhC5L2 0.5 μg / ml: 32±15%) (p<0.001, p<0.001, and p<0.001, respectively). Thus, rhC5L2 protein was responsible for the inhibition of C5a-activated myofibroblasts. Bars = standard error of the mean.

[0131] Example 17 Full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) protein causes inhibition of myofibroblasts activated by C5a and C3a

[0132] To explore the potential functional role of the rhC5L2 protein according to SEQ ID NO:1 in the treatment of subjects with ocular wounds or fibrosis, the effect of its presence on C5a / C3a-activated myofibroblasts was studied. Human keratocytes were stimulated with human C5a and human C3a for 24 hours and evaluated for activated myofibroblasts (FIG. 17). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker for the extracellular matrix. As a reference group, human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS) were used. As shown in Figure 17, both C5a and C3a at a concentration of 0.1 μg / ml caused significant activation of myofibroblasts (measured as aSMA-positive cells, 88±11%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C5a- and C3a-activated myofibroblasts, incubation in the presence of human C3a, C5a, and human rhC5L2 protein resulted in a significant decrease (rhC5L2 0.1 μg / ml: 24±15%; rhC5L2 0.2 μg / ml: 26±18%; rhC5L2 0.3 μg / ml: 33±23%; rhC5L2 0.5 μg / ml: 40±16%) (p<0.001, p<0.001, and p<0.001, respectively). Thus, rhC5L2 protein was responsible for the inhibition of C5a- and C3a-activated myofibroblasts. Bars = standard error of the mean.

[0133] Example 18 Effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) protein concentration on myofibroblasts in the presence of fetal bovine serum

[0134] To explore the potential functional role of rhC5L2 protein according to SEQ ID NO:1 in the treatment of subjects with ocular wounds or fibrosis, the effect of its concentration on myofibroblasts was studied. Human keratocytes were incubated for 24 hours in DMEM growth medium containing 10% fetal calf serum (FCS) and different concentrations of human rhC5L2 protein (Figure 18). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. Human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum were used as reference groups (serum-free: 11±14%; FCS: 20±19%). As shown in Figure 18, human rhC5L2 protein was found to have a positive effect on myofibroblast activation at all concentrations (rhC5L2 0.1 μg / ml: 33±22%; rhC5L2 0.2 μg / ml: 20±24%; rhC5L2 0.3 μg / ml: 41±30%; rhC5L2 0.5 μg / ml: 48±33%). Compared to human keratocytes cultured in 10% FCS, the differences were significant at rhC5L2 concentrations of 0.1 μg / ml and 0.5 μg / ml (p=0.046, p=0.118, p=0.070, and p=0.033, respectively).

[0135] Example 19 Effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 2 (rhC5AR2 / rhC5L2) protein concentration on myofibroblasts in the absence of fetal bovine serum

[0136] To explore the potential functional role of rhC5L2 protein according to SEQ ID NO:1 in the treatment of subjects with ocular wounds or fibrosis, the effect of its concentration on myofibroblasts was studied. Human keratocytes were incubated for 24 hours in DMEM growth medium without fetal bovine serum and with different concentrations of human rhC5L2 protein (FIG. 19). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. Human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal bovine serum were used as reference groups (serum-free: 11±14%; FCS: 20±19%). As shown in Figure 19, human rhC5L2 protein was found to have a positive effect on myofibroblast activation at all concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (rhC5L2 0.1 μg / ml: 14±17%; rhC5L2 0.2 μg / ml: 28±38%; rhC5L2 0.3 μg / ml: 36±14%; rhC5L2 0.5 μg / ml: 39±24%). Compared to serum-free control human keratocytes, the differences were significant at rhC5L2 concentrations of 0.3 μg / ml and 0.5 μg / ml (p=0.501, p=0.224, p<0.001, and p=0.007, respectively).

[0137] Example 20 Full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) protein causes inhibition of C3a-activated myofibroblasts

[0138] To explore the potential functional role of the rhC5AR1 protein according to SEQ ID NO:2 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C3a-activated myofibroblasts. Human keratocytes were stimulated with human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 20). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, we used human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours. As shown in Figure 20, 0.1 μg / ml of C3a caused significant myofibroblast activation (measured as aSMA-positive cells, 74±22%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C3a-activated myofibroblasts, incubation in the presence of human C3a and human rhC5AR1 protein resulted in a significant decrease (rhC5AR1 0.1 μg / ml: 3±7%; rhC5AR1 0.2 μg / ml: 3±4%; rhC5AR1 0.3 μg / ml: 36±14%; rhC5AR1 0.5 μg / ml: 42±24%) (p<0.001, p<0.001, p<0.001, and p=0.005, respectively). Thus, rhC5AR1 protein was responsible for the inhibition of C3a-activated myofibroblasts. Bars = standard error of the mean.

[0139] Example 21 Full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) protein causes inhibition of C5a-activated myofibroblasts

[0140] To explore the potential functional role of the rhC5AR1 protein according to SEQ ID NO:2 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a-activated myofibroblasts. Human keratocytes were stimulated with human C5a for 24 hours and evaluated for activated myofibroblasts (Figure 21). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours were used. As shown in Figure 21, 0.1 μg / ml of C5a caused significant activation of myofibroblasts (measured as aSMA-positive cells, 77±23%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C5a-activated myofibroblasts, incubation in the presence of human C5a and human rhC5AR1 protein resulted in a significant decrease (rhC5AR1 0.1 μg / ml: 3±4%; rhC5AR1 0.2 μg / ml: 5±7%; rhC5AR1 0.3 μg / ml: 18±23%; rhC5AR1 0.5 μg / ml: 39±29%) (p<0.001, p<0.001, p<0.001, and p=0.001, respectively). Thus, rhC5AR1 protein was responsible for the inhibition of C5a-activated myofibroblasts. Bars = standard error of the mean.

[0141] Example 22 Full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) protein causes inhibition of myofibroblasts activated by C5a and C3a

[0142] To explore the potential functional role of the rhC5AR1 protein according to SEQ ID NO:2 in the treatment of subjects with ocular wounds or fibrosis, the effect of its presence on C5a / C3a-activated myofibroblasts was studied. Human keratocytes were stimulated with human C5a and human C3a for 24 hours and evaluated for activated myofibroblasts (FIG. 22). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker for the extracellular matrix. As a reference group, human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS) were used. As shown in Figure 22, C5a and C3a, both at a concentration of 0.1 μg / ml, caused significant activation of myofibroblasts (88±11% as measured by aSMA-positive cells) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C5a- and C3a-activated myofibroblasts, incubation in the presence of human C3a, C5a, and human rhC5AR1 protein resulted in a significant decrease (rhC5AR1 0.1 μg / ml: 5±7%; rhC5AR1 0.2 μg / ml: 18±21%; rhC5AR1 0.3 μg / ml: 33±19%; rhC5AR1 0.5 μg / ml: 38±24%) (p<0.001, p<0.001, p<0.001, and p<0.001, respectively). Thus, rhC5AR1 protein was responsible for the inhibition of C5a- and C3a-activated myofibroblasts. Bars = standard error of the mean.

[0143] Example 23 Effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) protein concentration on myofibroblasts in the presence of fetal bovine serum

[0144] To explore the potential functional role of the rhC5AR1 protein according to SEQ ID NO:2 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium containing 10% fetal calf serum (FCS) and different concentrations of human rhC5AR1 protein (Figure 23). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. Human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum were used as reference groups (serum-free: 11±14%; FCS: 20±19%). As shown in Figure 23, human rhC5AR1 protein was found to have a positive effect on myofibroblast activation at all concentrations (rhC5AR1 0.1 μg / ml: 60±29%; rhC5AR1 0.2 μg / ml: 50±23%; rhC5AR1 0.3 μg / ml: 54±27%; rhC5AR1 0.5 μg / ml: 64±24%). Compared to human keratocytes cultured in 10% FCS, the differences were significant (p=0.003, p<0.001, p<0.001, and p<0.001, respectively).

[0145] Example 24 Effect of full-length recombinant human C5a anaphylatoxin chemotactic receptor 1 (rhC5AR1) protein concentration on myofibroblasts in the absence of fetal bovine serum

[0146] To explore the potential functional role of the rhC5AR1 protein according to SEQ ID NO:2 in treating subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium without fetal bovine serum and with different concentrations of human rhC5AR1 protein (Figure 24). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as an extracellular matrix marker. Human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal bovine serum were used as reference groups (serum-free: 11±14%; FCS: 20±19%). As shown in Figure 24, the human rhC5AR1 protein was found to have a positive effect on myofibroblast activation at all concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (rhC5AR1 0.1 μg / ml: 37±26%; rhC5AR1 0.2 μg / ml: 34±22%; rhC5AR1 0.3 μg / ml: 43±20%; rhC5AR1 0.5 μg / ml: 52±11%). The differences were significant compared to the serum-free control human keratocytes (p=0.017, p=0.012, p<0.001, and p<0.001, respectively).

[0147] Example 25 Full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) protein causes inhibition of C3a-activated myofibroblasts

[0148] To explore the potential functional role of the rhC3AR protein according to SEQ ID NO:3 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C3a-activated myofibroblasts. Human keratocytes were stimulated with human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 25). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, we used human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours. As shown in Figure 25, 0.1 μg / ml of C3a caused significant activation of myofibroblasts (measured as aSMA-positive cells, 74±22%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C3a-activated myofibroblasts, incubation in the presence of human C3a and human rhC3AR protein resulted in a significant decrease (rhC3AR 0.1 μg / ml: 13 ± 19%; rhC3AR 0.2 μg / ml: 36 ± 14%; rhC3AR 0.3 μg / ml: 50 ± 22%; rhC3AR 0.5 μg / ml: 68 ± 22%) (p < 0.001, p < 0.001, p = 0.023, and p = 0.547, respectively). Thus, rhC3AR protein at concentrations of 0.1 μg / ml, 0.2 μg / ml, and 0.3 μg / ml was responsible for the inhibition of C3a-activated myofibroblasts. Bars = standard error of the mean.

[0149] Example 26 Full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) protein causes inhibition of C5a-activated myofibroblasts

[0150] To explore the potential functional role of the rhC3AR protein according to SEQ ID NO:3 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a-activated myofibroblasts. Human keratocytes were stimulated with human C5a for 24 hours and evaluated for activated myofibroblasts (Figure 26). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of extracellular matrix. As a reference group, human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours were used. As shown in Figure 26, 0.1 μg / ml of C5a caused significant activation of myofibroblasts (measured as aSMA-positive cells, 77±23%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C5a-activated myofibroblasts, incubation in the presence of human C5a and human rhC3AR protein resulted in a significant decrease (rhC3AR 0.1 μg / ml: 44 ± 20%; rhC3AR 0.2 μg / ml: 43 ± 19%; rhC3AR 0.3 μg / ml: 60 ± 28%; rhC3AR 0.5 μg / ml: 70 ± 18%) (p = 0.001, p = 0.001, p = 0.0103, and p = 0.460, respectively). Thus, rhC3AR protein at concentrations of 0.1 μg / ml and 0.2 μg / ml was responsible for the inhibition of C5a-activated myofibroblasts. Bars = standard error of the mean.

[0151] Example 27 Full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) protein causes inhibition of myofibroblasts activated by C5a and C3a

[0152] To explore the potential functional role of the rhC3AR protein according to SEQ ID NO:3 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a / C3a-activated myofibroblasts. Human keratocytes were stimulated with human C5a and human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 27). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker for the extracellular matrix. As a reference group, human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours were used. As shown in Figure 27, both C5a and C3a at a concentration of 0.1 μg / ml caused significant activation of myofibroblasts (88±11% as measured by aSMA-positive cells) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C5a and C3a-activated myofibroblasts, incubation in the presence of human C3a, C5a, and human rhC3AR protein resulted in a significant decrease (rhC3AR 0.1 μg / ml: 34 ± 16%; rhC3AR 0.2 μg / ml: 61 ± 24%; rhC3AR 0.3 μg / ml: 61 ± 23%; rhC3AR 0.5 μg / ml: 67 ± 24%) (p < 0.001, p = 0.012, p = 0.006, and p = 0.044, respectively). Thus, rhC3AR protein was responsible for the inhibition of C5a- and C3a-activated myofibroblasts. Bars = standard error of the mean.

[0153] Example 28 Effect of full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) protein concentration on myofibroblasts in the presence of fetal bovine serum

[0154] To explore the potential functional role of rhC3AR protein according to SEQ ID NO:3 in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium containing 10% fetal calf serum (FCS) and different concentrations of human rhC3AR protein (Figure 28). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (serum-free: 11±14%; FCS: 20±19%). As shown in Figure 28, human rhC3AR protein was found to have a positive effect on myofibroblast activation at all concentrations (rhC3AR 0.1 μg / ml: 77±21%; rhC3AR 0.2 μg / ml: 77±31%; rhC3AR 0.3 μg / ml: 76±25%; rhC3AR 0.5 μg / ml: 72±19%). Compared to human keratocytes cultured in 10% FCS, the differences were significant (p<0.001, p<0.001, p<0.001, and p<0.001, respectively).

[0155] Example 29 Effect of full-length recombinant human C3a anaphylatoxin chemotactic receptor (rhC3AR) protein concentration on myofibroblasts in the absence of fetal bovine serum

[0156] To explore the potential functional role of rhC3AR protein according to SEQ ID NO:3 in treating subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium without fetal bovine serum and with different concentrations of human rhC3AR protein (Figure 29). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as an extracellular matrix marker. As a reference group, human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal bovine serum (serum-free: 11±14%; FCS: 20±19%) were used. As shown in Figure 29, the human rhC3AR protein was found to have a positive effect on myofibroblast activation at all concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (rhC3AR 0.1 μg / ml: 27±29%; rhC3AR 0.2 μg / ml: 31±35%; rhC3AR 0.3 μg / ml: 34±27%; rhC3AR 0.5 μg / ml: 50±29%). The differences were significant compared to serum-free control human keratocytes (p=0.136, p=0.114, p=0.028, and p=0.004, respectively).

[0157] Example 30 An RNA / DNA aptamer that binds to human C5a causes inhibition of C3a-activated myofibroblasts

[0158] To explore the potential functional role of the L-RNA / L-DNA aptamer (C5a aptamer) that binds to human C5a, containing the C5a binding site according to SEQ ID NO: 41 (C5a aptamer), in the treatment of subjects with ocular wounds or fibrosis, the effect of its presence on C3a-activated myofibroblasts was studied. Human keratocytes were stimulated with human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 30). To detect activated myofibroblasts, an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix were used. As a reference group, human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS) were used. As shown in Figure 30, C3a at 0.1 μg / ml caused significant activation of myofibroblasts (measured as aSMA-positive cells, 74 ± 22%) compared to the reference group (serum-free: 11 ± 14%; FCS: 20 ± 19%; p < 0.001 and p < 0.001, respectively). Incubation with human C3a and C5a aptamer resulted in a significant reduction in C3a-activated myofibroblasts (C5a aptamer 1 μg / ml: 65 ± 20%; C5a aptamer 2 μg / ml: 55 ± 31%; C5a aptamer 3 μg / ml: 47 ± 25%; C5a aptamer 5 μg / ml: 51 ± 16%) (p = 0.356, p = 0.112, p = 0.017, and p = 0.017, respectively). Thus, the C5a aptamer was responsible for causing inhibition of C3a-activated myofibroblasts at concentrations of 3 μg / ml and 5 μg / ml. Bars = standard error of the mean.

[0159] Example 31 An RNA / DNA aptamer that binds to human C5a causes inhibition of C5a-activated myofibroblasts

[0160] To explore the potential functional role of the L-RNA / L-DNA aptamer (C5a aptamer) that binds to human C5a, containing the C5a binding site according to SEQ ID NO: 41 (C5a aptamer), in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a-activated myofibroblasts. Human keratocytes were stimulated with human C5a for 24 hours and evaluated for activated myofibroblasts (Figure 31). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS). As shown in Figure 31, C5a at 0.1 μg / ml caused significant activation of myofibroblasts (measured as aSMA-positive cells, 77±23%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Incubation in the presence of human C5a and the C5a aptamer resulted in a significant reduction in C5a-activated myofibroblasts (C5a aptamer 1 μg / ml: 31±33%; C5a aptamer 2 μg / ml: 33±35%; C5a aptamer 3 μg / ml: 29±27%; C5a aptamer 5 μg / ml: 34±27%) (p<0.001, p<0.001, p<0.01, and p<0.01, respectively). Thus, the C5a aptamer was responsible for causing the inhibition of C5a-activated myofibroblasts. Bars = standard error of the mean.

[0161] Example 32 An RNA / DNA aptamer that binds to human C5a causes inhibition of C5a- and C3a-activated myofibroblasts

[0162] To explore the potential functional role of the L-RNA / L-DNA aptamer (C5a aptamer) that binds to human C5a, containing the C5a binding site according to SEQ ID NO: 41 (C5a aptamer), in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its presence on C5a / C3a-activated myofibroblasts. Human keratocytes were stimulated with human C5a and human C3a for 24 hours and evaluated for activated myofibroblasts (Figure 32). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (FCS) were used. As shown in Figure 32, both C5a and C3a at a concentration of 0.1 μg / ml caused significant activation of myofibroblasts (88±11% as measured by aSMA-positive cells) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p<0.001 and p<0.001, respectively). Compared to C5a and C3a-activated myofibroblasts, incubation in the presence of human C3a, C5a, and human rhC3AR protein resulted in a significant decrease (C5a aptamer 1 μg / ml: 84 ± 13%; C5a aptamer 2 μg / ml: 84 ± 13%; C5a aptamer 3 μg / ml: 62 ± 21%; C5a aptamer 5 μg / ml: 49 ± 33%) (p = 0.519, p = 0.495, p = 0.005, and p = 0.007, respectively). Thus, the C5a aptamer was responsible for the inhibition of C5a- and C3a-activated myofibroblasts. Bars = standard error of the mean.

[0163] Example 33 Effect of RNA / DNA aptamer concentration binding to human C5a on myofibroblasts in the presence of fetal bovine serum

[0164] To explore the potential functional role of the L-RNA / L-DNA aptamer (C5a aptamer) that binds to human C5a, containing the C5a binding site according to SEQ ID NO: 41 (C5a aptamer), in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium containing 10% fetal calf serum (FCS) and different concentrations of the C5a aptamer (Figure 33). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal calf serum (serum-free: 11±14%; FCS: 20±19%). As shown in Figure 33, the C5a aptamer was found to have a positive effect on myofibroblast activation at all concentrations (C5a aptamer 1 μg / ml: 38±14%; C5a aptamer 2 μg / ml: 41±19%; C5a aptamer 3 μg / ml: 51±32%; C5a aptamer 5 μg / ml: 73±34%). The differences were significant compared to human keratocytes cultured in 10% FCS (p=0.005, p=0.001, p=0.020, and p=0.001, respectively).

[0165] Example 34 Effect of RNA / DNA aptamer concentration that binds human C5a on myofibroblasts in the absence of fetal bovine serum

[0166] To explore the potential functional role of the L-RNA / L-DNA aptamer (C5a aptamer) that binds to human C5a, containing the C5a binding site according to SEQ ID NO: 41 (C5a aptamer), in the treatment of subjects with ocular wounds or fibrosis, we investigated the effect of its concentration on myofibroblasts. Human keratocytes were incubated for 24 hours in DMEM growth medium without fetal bovine serum and with different concentrations of the C5a aptamer (Figure 34). To detect activated myofibroblasts, we used an aSMA antibody and a vimentin antibody as a marker of the extracellular matrix. As a reference group, we used human keratocytes incubated for 24 hours in DMEM growth medium with and without 10% fetal bovine serum (serum-free: 11±14%; FCS: 20±19%). As shown in Figure 34, the C5a aptamer was found to have a slight positive effect on myofibroblast activation at all concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (C5a aptamer 1 μg / ml: 17±14%; C5a aptamer 2 μg / ml: 13±10%; C5a aptamer 3 μg / ml: 11±8%; C5a aptamer 5 μg / ml: 5±4%). However, the differences were not significant compared to serum-free control human keratocytes (p=0.219, p=0.629, p=0.983, and p=0.270, respectively).

[0167] Example 35 Antibodies that bind to human C5a cause inhibition of myofibroblasts activated by C5a, but not by C3a or the combination of C3a and C5a.

[0168] To explore the potential functional role of an antibody that binds human C5a (C5a Ab) in the treatment of subjects with ocular wounds or fibrosis, we studied the effect of its presence on C3a, C5a, and C5a / C3a-activated myofibroblasts. Additionally, we also studied the effect of its concentration on myofibroblasts with and without fetal bovine serum.

[0169] The antibodies studied were polyclonal rabbit immunoglobulin G antibody 250565 (Abbiotec; San Diego, USA), raised against a sequence within amino acids 700-755 of human complement C5 isoform 1 preproprotein (accession number: NP_001726), corresponding to the sequence within amino acids 23-74 of SEQ ID NO: 20; and polyclonal rabbit immunoglobulin G antibody 308733 (Biorbyt; Cambridge, United Kingdom), raised against a sequence within amino acids 1275-1290 of human complement C5 isoform 1 preproprotein (accession number: NP_001726).

[0170] Human keratocytes were stimulated with human C3a, human C5a, and a combination of human C5a / C3a for 24 hours and evaluated for activated myofibroblasts (Figures 35 and 36). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker for the extracellular matrix. Human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours served as reference groups. As shown in Figures 35 and 36, C3a, C5a, and C5a / C3a caused significant activation of myofibroblasts (measured by aSMA-positive cells, C3a 0.1 μg / ml 74±22%; C5a 0.1 μg / ml: 77±23%; C5a 0.1 μg / ml and C3a 0.1 μg / ml: 88±11%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p-value<0.001). Compared to C3a-activated myofibroblasts, incubation with human C3a and C5a antibodies did not result in a significant decrease (C5a Ab (250565) 5 μg / ml: 78±14%; C5a Ab (308733) 5 μg / ml: 50±42%) (p=0.645, p=0.155, respectively). Compared to C5a-activated myofibroblasts, incubation with human C5a and C5a antibodies resulted in a significant decrease (C5a Ab (250565) 5 μg / ml: 30±31%; C5a Ab (308733) 5 μg / ml: 29±31%) (p<0.001, p<0.001, respectively). Compared with C5a- and C3a-activated myofibroblasts, incubation in the presence of human C3a, C5a, and C5a antibodies did not result in a significant decrease (C5a Ab (250565) 5 μg / ml: 95±6%; C5a Ab (308733) 5 μg / ml: 76±30%) (p=0.079, p=0.294, respectively).As shown in Figures 35 and 36, the C5a antibody was found to have a positive effect on myofibroblast activation in the presence of 10% FCS (C5a Ab (250565) 5 μg / ml: 49±29%; C5a Ab (308733) 5 μg / ml: 53±23%). The difference was significant compared to human keratocytes cultured with 10% FCS (p<0.001, p=0.002, respectively). As shown in Figures 35 and 36, the C5a antibody was found to have a positive effect on myofibroblast activation at all concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (C5a Ab (250565) 5 μg / ml: 32±25%; C5a Ab (308733) 5 μg / ml: 54±42%). The differences were significant compared to serum-free control human keratocytes (p = 0.034 and p = 0.016, respectively). Thus, the C5a antibodies 250565 (Abbiotec) and 308733 (Biorbyt) at a concentration of 5 μg / ml were responsible for the inhibition of myofibroblasts activated by C5a, but not by C3a or the combination of C3a and C5a. Bars = standard error of the mean.

[0171] Example 36 Antibodies that bind to human C3a cause inhibition of myofibroblasts activated by C3a, but not by C5a, or the combination of C3a and C5a.

[0172] To explore the potential functional role of an antibody that binds human C3a (C3a mAb) in the treatment of subjects with ocular wounds or fibrosis, we studied the effects of its presence on C3a, C5a, and C5a / C3a-activated myofibroblasts. Additionally, we also studied the effects of its concentration on myofibroblasts with and without fetal bovine serum.

[0173] The antibodies studied were a monoclonal mouse immunoglobulin G1 (kappa light chain) antibody sc28294 (Santa Cruz Biotechnology; Dallas, USA) raised against a sequence within amino acids 541 to 840 of the human complement C3 preproprotein (accession number: NP_000055.2), covering SEQ ID NO: 43; and a monoclonal mouse immunoglobulin G1 (kappa light chain) antibody raised against a sequence of the mouse C5 protein (reference specification by Mastellos D et al. Mol Immunol 2004). 2a The antibody was HM1072 (Hycult Biotech; Uden, The Netherlands).

[0174] Human keratocytes were stimulated with human C3a, human C5a, and a combination of human C5a / C3a for 24 hours and evaluated for activated myofibroblasts (Figures 37 and 38). Activated myofibroblasts were detected using an aSMA antibody and a vimentin antibody as a marker for the extracellular matrix. Human keratocytes incubated in DMEM growth medium with and without 10% fetal calf serum (FCS) for 24 hours served as reference groups. As shown in Figures 37 and 38, C3a, C5a, and C5a / C3a caused significant activation of myofibroblasts (measured by aSMA-positive cells, C3a 0.1 μg / ml 74±22%; C5a 0.1 μg / ml: 77±23%; C5a 0.1 μg / ml and C3a 0.1 μg / ml: 88±11%) compared to the reference group (serum-free: 11±14%; FCS: 20±19%; p-value<0.001). Compared to C3a-activated myofibroblasts, incubation with human C3a and C3a antibody resulted in a significant reduction (C3a mAb (sc28294) 5 μg / ml: 15±25%; C3a mAb (HM1072) 5 μg / ml: 21±23%) (p<0.001, p<0.001, respectively). Compared to C5a-activated myofibroblasts, incubation with human C5a and C53 antibody did not result in a significant reduction (C3a mAb (sc28294) 5 μg / ml: 89±14%; C3a mAb (HM1072) 5 μg / ml: 75±22%) (p=0.167, p=0.855, respectively). In comparison with C5a- and C3a-activated myofibroblasts, incubation in the presence of human C3a, C5a, and C3a antibodies did not result in a significant decrease (C3a mAb (sc28294) 5 μg / ml: 76±22%; C3a mAb (HM1072) 5 μg / ml: 94±13%) (p=0.165, p=0.301, respectively).As shown in Figures 37 and 38, the C3a antibody was found to have a positive effect on myofibroblast activation in the presence of 10% FCS (C3a mAb (sc28294) 5 μg / ml: 61±29%; C3a mAb (HM1072) 5 μg / ml: 27±16%). The difference was significant for C3a mAb (sc28294) 5 μg / ml compared to human keratocytes cultured in 10% FCS (p=0.002, p=0.241, respectively). As shown in Figures 37 and 38, the C3a antibody was found to have a positive effect on myofibroblast activation at all concentrations compared to human keratocytes incubated in DMEM without FCS (serum-free control) (C3a mAb (sc28294) 5 μg / ml: 42±35%; C3a mAb (HM1072) 5 μg / ml: 24±16%). Compared to the serum-free control human keratocytes, the differences were significant (p<0.001, p=0.007, respectively). Thus, C3a antibodies sc28294 (Santa Cruz Biotechnology) and HM1072 (Hycult Biotech) at a concentration of 5 μg / ml were responsible for the inhibition of myofibroblast activation by C3a, but not by C5a or the combination of C3a and C5a. Bars = standard error of the mean.

[0175] Example 37 Murine C5L2 protein fragment reduces corneal fibrosis formation after corneal alkali burn in mice

[0176] To explore the potential functional role of the murine C5L2 protein fragment (mC5L2) identified by SEQ ID NO:19 in the treatment of ocular wounds or fibrosis, we investigated its presence in an in vivo mouse model of corneal alkali burn. C57 / BL6 mice (6-8 weeks old) were treated under intraperitoneal general anesthesia according to a standardized mouse model of corneal alkali burn (Saika S et al. Am J Pathol 2005). Under a stereomicroscope, a 1.5 mm diameter filter paper soaked in 2 μl of 1 M NaOH (sodium hydroxide) was placed on the center of the cornea of ​​the right mouse eye for 2 minutes to induce corneal alkali burn. Immediately after corneal alkali burn, treated eyes received either phosphate-buffered saline (PBS) and 0.3% ofloxacin ointment (days 2, 4, 6, and 8) (PBS / control group) or PBS and 0.3% ofloxacin ointment (days 2, 4, 6, and 8) and 1.5 μg / ml mC5L2 eye drops five times daily (during the entire follow-up period) (PBS with mC5L2 group).

[0177] The course of wound healing in the "PBS / control" and "PBS with mC5L2" groups was examined by gene expression analysis 5, 10, and 20 days after corneal alkali burn. Tables 9 (day 5), 10 (day 10), and 11 (day 20) show differentially expressed genes between the "PBS / control" and "PBS with mC5L2" groups, generated from mouse keratocytes and gene expression Clariom S mouse microarrays. The strongest differences in gene expression were observed on day 10 after corneal alkali burn, and the 100 most significant functional annotations for the differentially expressed genes are listed in Table 12. Therefore, mouse C5L2 protein fragment (mC5L2) affected wound healing and fibrosis after corneal alkali burn in mice by influencing gene expression in extracellular matrix organization, collagen metabolic processes, cellular responses to growth factors, transforming growth factor beta (TRF) signaling, and smooth muscle cell differentiation, among other factors.

[0178] Clinical signs of corneal fibrosis 20 days after corneal alkali burn were assessed using the corneal fibrosis scoring system established according to Cowell (Cowell BA et al. ILAR J 1999), McDonald (McDonald TO et al. Eye irritation 1997, p579-582: Marzulli FN et al. Dermatotoxicology and pharmacology), and Drew (Drew AF et al., Invest Ophthalmol Vis Sci. 2000). The Cowell score is the sum of assessments of the area of ​​fibrosis (0: none, 1: 1-25%, 2: 26-50%, 3: 51-75%, 4: 76-100%), opacity density (0: clear, 1: slight cloudiness, pupil and iris details discernible, 2: cloudiness, but iris and pupil outlines remain visible, 3: cloudiness, inhomogeneous opacity, 4: homogeneous opacity), and surface regularity (0: smooth, 1: slight surface irregularity, 2: rough surface, some swelling, 3: significant swelling, crater or Descemet's membrane formation, 4: perforation or severe Descemet's membrane). The McDonald-Shadduck score is an assessment of corneal clarity (0: no visible lesion; 1: some loss of clarity, with underlying structures clearly visible under diffuse lighting; 2: moderate loss of clarity, with underlying structures barely visible under diffuse lighting but still able to be inspected and assessed; 3: severe loss of clarity, with diffuse lighting underlying structures are not visible when looking through the lesion and their assessment does not function properly). The Drew haze score is an assessment of corneal haze (0: complete clarity; 1 / 2: minimal haze; 1: mild haze; 2: significant haze; 3: complete obscuration of the anterior chamber and iris). The Cowell, McDonald, and Drew assessment scores for corneal fibrosis 20 days after corneal alkali burn in the "PBS / control" and "PBS with mC5L2" groups are shown in Figure 39.Treatment with murine C5L2 protein fragment (mC5L2) significantly reduced scores (Cowell: 4.5±1.5, McDonald: 1.4±0.5, Drew: 1.5±0.8) compared to PBS-treated controls (Cowell: 6.4±0.8, McDonald: 2.4±0.5, Drew: 2.4±0.5; p=0.007, p=0.003, and p=0.011, respectively). Regarding the Cowell score, as shown in Figure 40, treatment with mC5L2 significantly reduced the area and density of opacities (fibrosis area: 2.9 ± 1.0 vs. 3.8 ± 0.4, p = 0.035; opacity density: 1.5 ± 0.7 vs. 2.6 ± 0.8, p = 0.009), but not the surface regularity (surface regularity: 0.0 ± 0.0 vs. 0.0 ± 0.0, p = 1.000), compared with PBS-treated controls. Thus, mouse C5L2 protein fragment (mC5L2) suppressed corneal fibrosis after alkali burn in mice, resulting in reduced opacity density, reduced haze, increased corneal clarity, and a smaller fibrotic area. Bars = standard error of the mean.

[0179] Wound healing and fibrosis were examined by protein expression in the "PBS / control" and "PBS with mC5L2" groups 20 days after corneal alkali burn. Table 13 lists the differentially expressed proteins generated from the protein expression scioDiscover antibody microarray obtained from mouse corneas between the "PBS / control" and "PBS with mC5L2" groups. Table 14 lists the functional annotations of the differentially expressed proteins. Thus, mouse C5L2 protein fragment (mC5L2) affected wound healing and fibrosis after corneal alkali burn in mice by specifically affecting the expression of proteins involved in wound response, immune system processes, collagen catabolic processes, and extracellular matrix degradation and organization. In summary, mouse C5L2 protein fragment (mC5L2) suppressed fibrosis after corneal alkali burn in mice by mediating various biological processes, as shown in Tables 12 and 14. This resulted in a smaller area of ​​fibrosis and reduced opacity on the cornea.

[0180]

Table 2-1

Table 2-2

Table 2-3

[0181]

Table 3-1

Table 3-2

Table 3-3

[0182]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

[0183]

Table 5-1

Table 5-2

Table 5-3

[0184]

Table 6-1

Table 6-2

Table 6-3

[0185]

Table 7-1

Table 7-2

[0186]

Table 8-1

Table 8-2

[0187]

Table 9-1

Table 9-2

[0188]

Table 10-1

Table 10-2

Table 10-3

[0189]

Table 11

[0190]

Table 12-1

Table 12-2

Table 12-3

Table 12-4

Table 12-5

Table 12-6

Table 12-7

[0191]

Table 13-1

Table 13-2

[0192]

Table 14-1

Table 14-2

Table 14-3

Table 14-4

Claims

1. 1. A medicament for use in treating a subject with an ocular wound and / or fibrosis, comprising a binding agent that binds to complement-anaphylatoxins C5a and / or C3a and / or C4a, thereby preferably inhibiting the activity of C5a and / or C3a and / or C4a.

2. 2. The medicament for use in treating a subject with an ocular wound and / or fibrosis of claim 1, wherein the binding agent is selected from the group comprising a protein or fragment thereof, a peptide, a non-IgG scaffold, an aptamer, an oligonucleotide, an antibody or antibody-like protein, a peptidomimetic or fragment thereof.

3. 3. A medicament for use in treating a subject with an eye wound and / or fibrosis as claimed in claim 1 or 2, wherein the binding agent is administered to promote wound healing, particularly corneal wound healing.

4. A pharmaceutical for use in treating a subject with eye wounds and / or fibrosis according to any one of claims 1 to 3, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C5a protein having the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, where overlap means overlap of the target amino acid sequences of an antibody, antibody-like protein, or adsorbent and a particular peptide fragment.

5. 5. The pharmaceutical for use in treating a subject with eye wounds and / or fibrosis of claim 4, wherein the binding agent is capable of binding to C5a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO: 22-34.

6. The binding agent is SEQ ID NO: 35-40 (SEQ ID NO: 35: X 1 X 2 ETCEX 3 RX 4 , SEQ ID NO: 36: X 5 X 6 KX 7 X 8 X 9 L, and SEQ ID NO: 37: X 5 X 6 KX 7 X 8 X 9 I), wherein X 1 is selected from the group consisting of N, H, D, F, K, Y, and T; X 2 is selected from the group consisting of D, L, Y, and H; X 3 is selected from the group consisting of Q, E, and K; X 4 is selected from the group consisting of A, V, and L; X 5 is selected from the group consisting of S, H, P, and N; X 6 is selected from the group consisting of H and N; X 7 is selected from the group consisting of D, N, H, P, and G; X 8 is selected from the group consisting of M, L, I, and V; and X 9 is selected from the group consisting of Q, L, and I.

7. A pharmaceutical for use in treating a subject with eye wounds and / or fibrosis according to any one of claims 1 to 3, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C3a protein having the amino acid sequence set forth in SEQ ID NO:

43.

8. The pharmaceutical for use in treating a subject with eye wounds and / or fibrosis according to claim 7, wherein the binding agent is also capable of binding to human C3a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO: 44-47.

9. A pharmaceutical for use in treating a subject with eye wounds and / or fibrosis according to any one of claims 1 to 3, wherein the binding agent is capable of binding to several overlapping peptide fragments of complement component C4a protein having the amino acid sequence set forth in SEQ ID NO: 48 or SEQ ID NO:

49.

10. The pharmaceutical for use in treating a subject with eye wounds and / or fibrosis as described in claim 9, wherein the binding agent is also capable of binding to human C4a only at an epitope within or overlapping with a fragment of a protein having an amino acid sequence according to SEQ ID NO:

50.

11. A medicament for use in treating a subject with ocular wounds and / or fibrosis according to claims 1 to 10, wherein the binding agent is an antibody or antibody-like protein.

12. An adsorbent for use in treating a subject with ocular wounds and / or fibrosis according to claims 1 to 10, wherein the binding agent is an aptamer.

13. A pharmaceutical for use in treating a subject with eye wounds and / or fibrosis as described in claim 12, wherein the binding agent is an aptamer, wherein the aptamer can be associated with a nucleic acid molecule consisting of RNA and / or DNA as disclosed in SEQ ID NO:

41.

14. A pharmaceutical for use in treating a subject with eye wounds and / or fibrosis as described in claim 13, wherein the binding agent is an aptamer, wherein the aptamer may be associated with a nucleic acid molecule consisting of RNA and / or DNA as disclosed in SEQ ID NO: 41, and wherein the aptamer binds to a binding site on C5a consisting of SEQ ID NO:

42.

15. The binding agent is selected from the group consisting of the human C5L2 protein set forth in SEQ ID NO: 1, a protein / peptide or fragment which is at least 60% identical to the full-length amino acid sequence of the human C5L2 protein set forth in SEQ ID NO: 1, the human C5aR1 protein set forth in SEQ ID NO: 2, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the human C5aR1 protein set forth in SEQ ID NO: 2, the human C3aR protein set forth in SEQ ID NO: 3, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the human C3aR protein set forth in SEQ ID NO: 3, the mouse C5L2 protein set forth in SEQ ID NO: 4, and the mouse C5L2 protein set forth in SEQ ID NO:

4.

15. The medicament for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 14, wherein the medicament is a protein or protein fragment selected from the group comprising: a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein of SEQ ID NO: 5, a mouse C5aR1 protein of SEQ ID NO: 5, a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C5aR1 protein of SEQ ID NO: 5, a mouse C3aR protein of SEQ ID NO: 6, and a protein or fragment which is at least 60% identical to the full-length amino acid sequence of the mouse C3aR protein of SEQ ID NO:

6.

16. 16. The pharmaceutical for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 1 to 15, wherein the binding agent is a protein / peptide or protein fragment and comprises at least one conserved region selected from the group comprising the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17.

17. 17. The pharmaceutical for use in treating a subject with an eye wound and / or fibrosis according to claim 16, wherein the binding agent is a protein / peptide or protein fragment and comprises at least two conserved regions selected from the group comprising the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence set forth in SEQ ID NO: 16, the amino acid sequence set forth in SEQ ID NO: 17, and a protein or fragment at least 60% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 17.

18. 18. A pharmaceutical for use in treating a subject with an eye wound and / or fibrosis according to any one of claims 15 to 17, wherein the binding agent is a protein / peptide or protein fragment and comprises at least one conserved region selected from the group comprising the amino acid sequence set forth in SEQ ID NO: 18 and the amino acid sequence set forth in SEQ ID NO:

19.

19. A composition comprising at least two binding agents, preferably proteins or protein fragments, according to any one of claims 1 to 18 for use in treating a subject with an ocular wound and / or fibrosis.

20. A composition comprising at least three proteins or protein fragments according to any one of claims 1 to 19 for use in treating a subject with ocular wounds and / or fibrosis.

21. A pharmaceutical composition comprising an adsorbent according to any one of claims 1 to 18 or a composition according to claim 19 or 20 for use in treating a subject with an ocular wound and / or fibrosis.

22. The subject may have conjunctivitis and conjunctival scarring (including ocular pemphigoid), scleritis and episcleritis, corneal scarring and opacity due to corneal ulcers, keratoconjunctivitis, keratitis, bullous keratopathy, corneal degeneration, iridocyclitis and adhesions of the iris and ciliary body, chorioretinitis or chorioretinal inflammation or chorioretinal scarring / fibrosis due to degeneration or hemorrhage or rupture or neovascularization, fibrotic vitreoretinopathy, 22. The medicament according to any one of claims 1 to 18, or the composition according to claim 19 or 20, or the pharmaceutical composition of claim 21, for use in treating a subject suffering from a disease selected from the group comprising: proliferative vitreoretinopathy, retinopathy of prematurity and diabetic retinopathy; choroidal neovascularization and macular degeneration, secondary glaucoma, endophthalmitis, and impaired wound healing and fibrosis after ocular surgery or trauma, including intraocular foreign bodies.

23. 22. The medicament according to any one of claims 1 to 18, or the composition according to claim 19 or 20, or the pharmaceutical composition of claim 21, for use in treating a subject suffering from a disease selected from the group comprising (idiopathic) pulmonary fibrosis, skin keloid formation, scleroderma, myelofibrosis, renal fibrosis, pancreatic fibrosis, and cardiac fibrosis, and fibrosis in (non-)alcoholic steatohepatitis, glomerulonephritis, and (ANCA-associated) vasculitis.

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