Complement modulators

Abstract

The present invention relates to a modulator protein comprising (i) a modulator module that modulates the classical pathway of complement activation and / or the lectin pathway of complement activation, and (ii) a binding module comprising, preferably consisting of, CCP domain 19 and CCP domain 20 of Factor H; for use in the prevention and / or treatment of a disease caused and / or exacerbated by activation of the classical pathway of complement activation and / or the lectin pathway, and to modulator proteins, methods and kits related thereto.

Description

[0001] This invention relates to modulatory proteins comprising: (i) modulatory modules regulating the classical pathway of complement activation and / or the lectin pathway of complement activation, and (ii) binding modules comprising, preferably, CCP domains 19 and 20 of H factor; for the prevention and / or treatment of diseases caused and / or aggravated by activation of the classical pathway of complement activation and / or the lectin pathway, and to modulatory proteins, methods, and kits thereof.

[0002] Even in primitive organisms, the innate immune system is highly conserved. Cellular effectors in this branch primarily include neutrophils, monocytes, and macrophages, while soluble innate immune effectors, in addition to other effectors such as acute-phase proteins or pore-forming peptides, are mainly composed of the complement system (Parkin & Cohen (2001) The Lancet 357: 1777–89). The complement system consists of components in serum, which Paul Ehrlich describes as “complementing” the antibody response against bacteria. Some of these components are thermally unstable. Other functions of the complement system include opsonization of microbial invaders, immune complexes, debris, apoptotic cells, and necrotic cells to support their effective clearance via uptake by phagocytes (Ricklin et al. (2010) Nature Immunology 11: 785–797). The complement system is activated via three pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP).

[0003] CP activation typically occurs in an antibody-dependent manner via complement component C1q, which acts as a pattern recognition molecule (PRM). Following a series of proteolytic activation events, the CP C3 convertase (C4bC2a; following conventional nomenclature; some researchers have proposed naming the CP / LP convertase C4b2b to unify complement nomenclature, where 2b represents the larger cleavage fragment containing enzymatic activity of C2) cleaves complement component C3 (which is central to all three complement activation pathways) into C3a (anaphylatoxin) and C3b (opsonin). This cleavage results in a conformational change, and the previously internal thioester bond extends onto the protein surface of C3b. This active, and short-lived, thioester bond can covalently bind to the hydroxyl and amino groups of molecules located on the cell surface, or it can be hydrolyzed (“quenched”). Therefore, if C3 convertase is not downregulated, opsonization of the cell can occur through numerous C3b molecules. The production of large numbers of C3b molecules promotes the activation of C5 by C5 convertase. C5 convertase cleaves C5 into C5a (the most potent anaphylatoxin) and C5b. C5b interacts with complement factor C6-9 to form the membrane attack complex (MAC), which assembles pores in the cell membrane to lyse and kill the cell.

[0004] The lectin pathway (LP) is organized similarly to the CP pathway. It is activated by recognizing pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Within the LP, PAMPs or DAMPs can be detected by several pattern recognition molecules homologous to C1q (the pattern recognition molecule of CP): mannose-binding lectin (MBL), as well as various types of collagen lectins and fibrinolytic proteins. Upon binding to PAMP or DAMP, MBL undergoes a conformational change and then binds to the MBL-associated serine protease (MASP). MBL is structurally and functionally homologous to C1q. Similar to CP, MASP2 activates C2 to C2a and C2b via proteolysis and activates C4 to C4a and C4b via proteolysis. The activated components can construct the C3 convertase C4b2a, which is identical to that in CP and cleaves C3 into C3a and C3b. Similar to CP, in the absence of strict regulation of the C3 convertase, the production of more C3b molecules promotes C5 activation via the C5 convertase. C5 proteolytic activation is the terminus of C5b-induced MAC formation and the starting point of the complement cleavage pathway.

[0005] The alternative pathway (AP) is activated by a self-activation process at low levels. This process is known as “tick-over” activation of C3. The C3 molecule has an inherently metastable conformation. At all times, a small fraction of the C3 molecule undergoes a spontaneous conformational change (activation), which exposes the previously internal thioester module. The thioester can be quenched by water or indiscriminately attached (to its own or foreign) to nucleophiles on the cell surface. This “self-activated” C3 is called C3(H2O) and is structurally similar to the C3b molecule. C3b or C3(H2O) exposes new protein surfaces hidden within C3. These new surfaces bind factor B, another complement factor for AP. When factor B binds to C3b or C3(H2O), it can be cleaved by protease D into Ba and Bb. Bb remains bound to C3(H2O) (or C3b) and constitutes the C3 convertase of AP, C3b(H2O)Bb (or C3bBb). Similar to CP and LP C3 convertases, C3bBb can generate C3b and C3a molecules by cleaving C3. Protein properdin, a positive regulator of AP, plays a crucial role by stabilizing protein-protein interactions of AP C3 convertases. If unregulated, any C3b generated by the alternative, classical, or lectin pathways can build more AP C3 convertases, further amplifying the number of C3b molecules produced in the positive feedback loop. This step is known as the "amplification loop" of AP. Therefore, these three activation pathways converge at the C3 activation level and, if unregulated, accumulate to form MAC.

[0006] The classical and lectin pathways are inactive until they are specifically activated by sensing pathogens or endogenous danger molecules. Conversely, AP remains active at low levels and indiscriminately produces C3b (or initially C3(H2O)) molecules. More than a dozen different regulatory proteins are known within the complement system. Some regulators inhibit CP and LP at the initiation levels; however, the most tightly controlled parts of the cascade are the convertases and C3b. Convertases act as amplifiers of the activation signal, and C3b forms the platform for the formation of C3 convertase and the inflammatory C5 convertase. There are also several regulators that specifically control the cleavage of MAC.

[0007] Regulatory proteins can be categorized into decay accelerators, which destabilize C3 convertases and cause them to decay more rapidly. Another group involves proteins involved in the degradation of C3b and / or C4b. This is achieved through factor I; however, factor I requires cofactors to cleave C3b and / or C4b; cofactors are also needed to prevent non-specific degradation by soluble protease factor I. Therefore, inactivation of C3b or C4b requires the presence of cofactor proteins that bind to the target and recruit factor I (e.g., factor H, CR1, or C4b-binding protein (C4BP)). Another group of regulators inhibits MAC formation.

[0008] The complement regulatory protein (CCP) domain is a peptide sequence containing approximately 60 to 70 amino acids, including a conserved tryptophan and four conserved cysteine ​​residues forming two disulfide bonds, with considerable sequence variation among the remaining amino acids. In addition to binding complement proteins C3b and / or C4b, the CCP domain has been found to mediate further activities, including decay-accelerating activity and factor I cofactor activity. The CCP domain has been reviewed, for example, by Schmidt et al. (2008), Clin Exp Immunol. 151(1):14-24.

[0009] Many diseases, especially hereditary ones, are associated with complement dysfunction, particularly complement overactivation. Therefore, to provide artificial regulators of the complement system, the monoclonal antibody eculizumab (Hillmen et al. (2006), NEJM355(12):1233) was developed to specifically bind to complement protein C5 and inhibit its terminal activation. In a similar developmental direction, the C5 inhibitor rEV576 (coversin) was developed (Romay-Penabad et al. (2014), Lupus 23(12):1324). Furthermore, a protein termed “mini-FH” (WO 2013 / 142362 A1) was obtained, linking complement factor H to complement regulatory protein repeat sequences (CCP domains) 1–4 and 19–20 via a linker. Mini-FH was described as having increased complement regulatory activity and, in several in vitro and in vitro assays, outperforming FH by tenfold in regulatory activity against the complement bypass pathway.

[0010] Nevertheless, there remains a need in the art for improved complement inhibitors to avoid the shortcomings of existing technologies. This problem is addressed through the means and methods disclosed herein.

[0011] Therefore, the present invention relates to modulatory proteins comprising (i) modulatory modules that regulate the classical pathway of complement activation and / or the lectin pathway of complement activation, and (ii) binding modules comprising, preferably, CCP domains 19 and 20 of H factors.

[0012] Generally, the terms used herein should be given their common and conventional meanings to those skilled in the art, and should not be limited to specific or customary meanings unless otherwise stated. As used below, the terms “having,” “comprising,” or “including,” or any variation thereof, are used in a non-exclusive manner. Thus, these terms can refer either to a situation where no other features exist in the entity described herein besides the features introduced by these terms, or to a situation where one or more other features exist. For example, the expressions “A has B,” “A contains B,” and “A includes B” can refer either to a situation where no other element exists in A besides B (i.e., A is uniquely and exclusively composed of B), or to a situation where entity A contains one or more other elements besides B, such as element C, element D, or even other elements. Furthermore, as will be understood by those skilled in the art, the expressions “comprising one” and “comprising one” preferably mean “comprising one or more,” that is, equivalent to “comprising at least one.” Therefore, a statement referring to one of multiple items, unless otherwise stated, preferably refers to at least one such item, more preferably to multiple such items; thus, for example, identifying "one / type of cell" involves identifying at least one / type of cell, preferably involving identifying multiple / type of cell. Furthermore, the term "multiple / type" refers to many / types, preferably at least two / types, more preferably at least three / types, and still more preferably at least four / types of the indicated items.

[0013] Furthermore, as used below, the terms “preferred,” “more preferably,” “most preferably,” “particularly,” “more particularly,” “specifically,” “more specifically,” or similar terms are used in combination with optional features without limiting further possibilities. Therefore, features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As those skilled in the art will recognize, the invention can be carried out by using alternative features. Similarly, features introduced by “in an embodiment” or similar expressions are intended to be optional features, without any limitation on other embodiments of the invention, without any limitation on the scope of the invention, and without any limitation on the possibility of combining features introduced in this way with other optional or non-optional features.

[0014] The methods described below are preferably in vitro methods. In principle, the method steps can be performed in any arbitrary order that a person skilled in the art would deem suitable, but are preferably performed in the indicated order; moreover, one or more, preferably all, of the steps can be assisted or performed by automated equipment. Furthermore, these methods may include steps other than those explicitly mentioned above.

[0015] As used herein, unless otherwise specified, the term “about” refers to an indication of technical precision generally accepted in the relevant art, preferably ±20%, more preferably ±10%, and most preferably ±5%. Furthermore, the term “substantially” indicates the absence of deviations that affect the indicated results or uses, i.e., potential deviations that would cause the indicated results to deviate from the specified outcome by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, “substantially composed of” or “substantially constitutes” means including the specified components but excluding other components, except for materials present as impurities, unavoidable materials present due to the process used to provide the components, and components added to achieve a purpose different from the technical effects of the present invention. For example, compositions defined using the phrase “substantially composed of” or “substantially constitutes” encompass any known and acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition substantially composed of one set of components will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of non-specified components. As mentioned herein, if in doubt, database entries refer to the current entries as of the date of filing of this application.

[0016] As used herein, the term "polynucleotide" refers to a linear or circular nucleic acid molecule. The polynucleotides of the present invention should preferably be provided as isolated polynucleotides (i.e., isolated from their natural background) or in a genetically modified form, preferably containing at least one heterologous sequence. The term polynucleotide encompasses single-stranded as well as partially or fully double-stranded polynucleotides. Preferably, the polynucleotide is a DNA polynucleotide, which may also be referred to as "DNA". Furthermore, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified derivatives such as biotinylated polynucleotides, locked nucleic acids, peptide nucleic acids, etc. In light of the description herein, template DNA, for example for PCR, primers, such as sequencing or amplification primers, and probe oligonucleotides are all included in the term polynucleotide.

[0017] Unless otherwise explicitly stated, specific polynucleotides mentioned herein preferably include polynucleotide variants. As used herein, the term "polynucleotide variant" refers to a variant of the polynucleotide mentioned herein that comprises a nucleic acid sequence characterized in that the sequence can be derived from the specific nucleic acid sequence by at least one nucleotide substitution, addition, and / or deletion, wherein the polynucleotide variant should have the function and / or activity specified for the specific polynucleotide. Thus, a polynucleotide variant can be, for example, an ortholog, paralog, or other homolog of the specific polynucleotide; the polynucleotide variant can also be a mutant of the specific polynucleotide, preferably a naturally occurring mutant, such as a mutant identified in cancer cells. It is also preferred that the polynucleotide variant is or is derived from a non-naturally occurring allele of the specific polynucleotide. Further polynucleotide variants include polynucleotides comprising a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity with a specifically indicated nucleic acid sequence, and / or encoding a polypeptide having an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity with a specifically indicated amino acid sequence. The percentage identity value is preferably calculated over the entire nucleic acid sequence region, preferably as specified elsewhere herein. The polynucleotides of the present invention consist of, are substantially composed of, or contain the above-described nucleic acid sequence. Therefore, they may also contain other nucleic acid sequences.

[0018] The term "protein" is as understood by those skilled in the art; preferably, a protein comprises at least one amino acid chain, i.e., a polypeptide as specified below, and more preferably, multiple polypeptides. Thus, a protein can be a polymer, such as a dimer, trimer, etc., wherein the polypeptides in the polymer can be covalently linked, for example, through disulfide bridging, or non-covalently linked, for example, through ionic interactions, hydrophobic interactions, and / or van der Waals interactions. A protein can be composed of the same polypeptide, for example, a homodimer, or can contain at least two different polypeptides, for example, a heterodimer. More preferably, as specified, a protein contains all the structural components indicated in a continuous covalent polypeptide chain; therefore, a protein is preferably a polypeptide or contained within a polypeptide, such as a fusion polypeptide.

[0019] As used herein, the term "peptide" refers to a molecule comprising a number of amino acids covalently linked together by peptide bonds. A peptide consisting of fewer than 20 amino acids covalently linked by peptide bonds may also be called a "peptide". Preferably, a peptide comprises 100 to 1000 amino acids, more preferably 150 to 1000, even more preferably 200 to 500, and most preferably 250 to 400 amino acids. Peptides may also be included in fusion peptides, i.e., may contain amino acid sequences other than those specifically indicated. Furthermore, peptides may contain additional non-peptide structures, such as at least one glycosylation, lipid conjugation, etc. Therefore, unless explicitly stated otherwise, specific peptides mentioned herein preferably include peptide variants.

[0020] As used herein, the term "peptide variant" refers to any chemical molecule comprising at least one polypeptide as specified herein, which is structurally different from the specifically indicated polypeptide. Preferably, the peptide variant comprises a polypeptide having a continuous amino acid sequence corresponding to at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% of the amino acid sequence of the specifically indicated polypeptide. Furthermore, it should be understood that the peptide variant as referred to in the invention should have an amino acid sequence that differs due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant still preferably has at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity with the amino acid sequence of the specific polypeptide. The degree of identity between the two amino acid sequences can be determined by algorithms known in the art, as described elsewhere herein. The peptide variants mentioned above can be allelic variants or any other species-specific homologs, paralogs, or orthologs. Furthermore, the peptide variants mentioned herein include fragments of specific peptides or peptide variants of the types described above, preferably provided that these fragments and / or variants possess the specified activity. Such fragments may be, or can be derived from, for example, degradation products of peptides or splice variants. Further, variants differ due to post-translational modifications such as phosphorylation, glycosylation, ubiquitination, SUMOylation, or myristylation, by the inclusion of non-natural amino acids, and / or by being peptide mimics. The “activity” of the peptide as specified herein is preferably retained in the peptide variant; as those skilled in the art will understand from the description herein, the activity of a peptide does not necessarily have to reflect its principal natural function, but may be other activities relevant in the context of the claimed invention. Strictly based on the example, the p53 peptide is active in cell cycle regulation, but p53, and especially its mutants, is also immunogenic. Therefore, p53 mutants, including fragments thereof, are p53 variants as referred to herein, preferably possessing immunogenic activity. The foregoing is applied to both peptide variants and protein variants with necessary modifications.

[0021] The term "fraction" is used broadly herein to refer to any sub-region, preferably a subdomain, of a biomacromolecule, containing the indicated sequence, structure, and / or activity. Thus, the term includes sub-regions resulting from actual fragmentation of a biomacromolecule, but also sub-regions derived from the corresponding biomacromolecule in an abstract manner, such as in computer simulations. Therefore, as used herein, an Fc or Fab fragment of an immunoglobulin, but also, for example, a single-chain antibody, bispecific antibody, and nanobody, can be referred to as a fragment of an immunoglobulin. Furthermore, a disease epitope can be a fragment of a disease antigen.

[0022] Unless otherwise explicitly stated, the compounds specified herein, particularly polynucleotides and peptides, may be contained within larger structures, such as those covalently or non-covalently linked to other sequences, adjuvants, carrier molecules, retardants, and other excipients. In particular, the specified peptides may be contained within fusion peptides comprising other peptides, which may serve, for example, as tags for purification and / or detection, as linkers, or for extending the in vivo half-life of the compound. The term "detectable tag" refers to a segment of amino acids added to or introduced into a fusion peptide; preferably, the tag is added to the C-terminus or N-terminus of the fusion peptide. This segment of amino acids preferably allows detection of the peptide by an antibody that specifically recognizes the tag; or it preferably allows for the formation of a functional conformation, such as a chelating agent; or it preferably allows for visualization, such as in the case of a fluorescent tag. Preferred detectable tags are Myc tags, FLAG tags, 6-His tags, HA tags, GST tags, or fluorescent protein tags, such as GFP tags. These tags are well known in the art. Preferably, the peptides included in the fusion polypeptide contain other amino acids or other modifications that can act as mediators of secretion, mediators of blood-brain barrier channels, cell-penetrating peptides, and / or immunostimulants. Other polypeptides or peptides that the polypeptide can fuse with are signaling and / or transport sequences, such as IL-2 signaling sequences, and adapter sequences.

[0023] The degree of identity (e.g., expressed as "% identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined using algorithms known in the art. Preferably, the degree of identity is determined by comparing two best-aligned sequences within a comparison window, wherein the sequence fragment in the comparison window may contain additions or deletions (e.g., gaps or protrusions) to achieve optimal alignment compared to the sequence it is comparing with. The percentage is calculated by determining the number of positions in both sequences where the same residues occur, preferably over the entire length of the polynucleotide or polypeptide as specified herein, to produce a number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the sequence identity percentage. The best alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), computerized implementations of these algorithms (e.g., BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection. Assuming two sequences for comparison have been identified, GAP and BESTFIT are preferably used to determine their best alignment and thus the degree of identity. Preferably, a default value of 5.00 gap weight and 0.30 gap weight length is used. More preferably, the basic local alignment search tool (BLAST) is used, employing the default parameter values ​​for alignment. In the context of biological sequences mentioned herein, the term "substantially identical" means a % identity value of at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%. As will be understood, the term substantially identical includes 100% identity. The above content, after necessary modifications, applies to the term "basically complementary".

[0024] The term "complement regulatory protein repeat domain," which may also be referred to herein as "complement regulatory protein repeat sequence," "CCP domain," or "CCP," and is also known in the art as "short complement-like repeat sequence," "short common repeat sequence," or "SCR," is known in principle to those skilled in the art, as described above. CCP domains with specific activities are known in the art and have been described in the context of the corresponding activities.

[0025] The three main pathways of complement activation—the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP)—are well known in the art and have been described above.

[0026] The term "H factor," as known to those skilled in the art, serves as a cofactor in the inactivation of C3b via I factor and is used to increase the dissociation rate of the C3bBb complex (C3 convertase) and the C3bBb3b complex (C5 convertase) in the alternative complement pathway. In addition to these complement regulatory functions, the CCP domain of the H factor or its extensions may have other biological functions that enhance complement regulation in vivo, such as glycosaminoglycan binding, binding to sialic acid, or binding to C3 activation / inactivation fragments such as C3b, iC3b, C3dg, and C3d. Preferably, the H factor is a mammalian H factor, more preferably a human H factor. Most preferably, the H factor is a human H factor having the amino acid sequence specified in Genbank Acc. No. NP_000177.2, Uniprot Acc No. P08603 (CFAH_HUMAN). CCP domains 19 to 20 of the H factor preferably comprise, and preferably consist of, the amino acid sequence shown in SEQ ID NO:1 or a sequence having at least 70% identity with it. More preferably, CCP domains 19 to 20 of the H factor comprise, and preferably consist of, the amino acid sequence shown in SEQ ID NO:2 or a sequence having at least 70% identity with it.

[0027] The term "C4b-binding protein," which may be abbreviated as "C4BP," is known to those skilled in the art as a regulatory molecule of the classical / lectin pathway for complement activation. It binds as a cofactor to C4b and weakly to C3b, serving to inactivate complement proteins C4b (and C3b) through proteolytic hydrolysis by serum protease I. C4BP also increases the rate of dissociation of the C4b2a complex (C3 convertase) and the C4b2a3b complex (C5 convertase) in the classical / lectin complement pathway. Preferably, C4BP is mammalian C4BP, more preferably human C4BP. Most preferably, C4BP is a human C4BP having the amino acid sequence specified in Genbank Acc. No: AAA36507.1 (α chain, Uniprot Acc. No. P04003 (C4BPA_HUMAN)); and / or Genbank Acc. No. AAA35615.1 (β chain, Uniprot Acc. No. P20851 (C4BPB_HUMAN)).

[0028] The term "modulation" is known to those skilled in the art. Preferably, the term refers to any effect applied to the system that causes at least one target parameter to change to a desired value. Thus, modulating complement activation includes every activity that causes a change in the complement activation state, i.e., modulation of complement activation. This modulation can be activation, i.e., resulting in enhanced activation; or the modulation can be inhibition, i.e., resulting in reduced activation. The modulation is preferably inhibition, i.e., complement activation is preferably reduced by modulation as referred to herein.

[0029] As used herein, the term "regulatory module" refers to a module, i.e., a substructure, of a modulatory protein containing a polypeptide having regulatory activity in mediating the classical and / or lectin pathways of complement activation. Regulatory modules that regulate the classical and / or lectin pathways of complement activation are known in the art and have been described elsewhere herein. Preferably, the regulatory module does not regulate, in particular, inhibit, alternative pathways of complement activation. More preferably, the modulatory protein does not regulate, in particular, inhibit, alternative pathways of complement activation.

[0030] Preferably, the regulatory module comprises a module of effector proteins of the complement-activated classical pathway and / or lectin pathway, preferably an invertase decay-accelerating module and / or invertase, preferably a C4b-based specific cofactor module, or a C4b and C3b-based specific cofactor module. Therefore, the regulatory module preferably comprises at least one, preferably at least three, more preferably at least four CCPs, and more preferably has invertase decay-accelerating activity for invertases of the complement-activated classical pathway and / or lectin pathway, wherein the term "decay-accelerating activity" preferably refers to the module, such as a CCP domain or CCP domain extension, mediating the decay, preferably inactivation, of C3 invertases, i.e., C4bC2a, of the complement-activated classical pathway and lectin pathway. Alternatively, the regulatory properties of the module, such as a CCP domain or CCP domain extension, may be cofactor activity mediating factor I-mediated degradation of C4b, or degradation of C4b and C3b. Another possibility is that the modulatory properties of modules, such as CCP domains or CCP domain extensions, simultaneously include activities that mediate decay acceleration and cofactor activity against classical and lectin pathways, or classical, lectin, and bypass pathways.

[0031] Preferably, the decay-accelerating activity of the modulator module is determined by surface plasmon resonance (SPR). Preferably, the modulator module comprises, and preferably consists of, an effector module of: a C4b-binding protein, a decay-accelerating protein (CD55), a membrane cofactor protein (CD46), H factor, and / or complement receptor 1 (CR1; CD35, preferably comprising the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and / or SEQ ID NO: 12), wherein the term "effector module" refers to a module, such as a domain, of a protein that mediates the indicated effect. Therefore, the effector module is preferably a substructure of the indicated protein having activity in regulating the classical pathway and / or the lectin pathway of complement activation. Preferably, the C4b-binding protein is a C4b-binding protein, the decay-accelerating protein is CD55, the membrane cofactor protein is CD46, and / or the CR is CR1. Preferably, the effector module of the C4b-binding protein comprises CCP domains 1 to 4 of the α chain; more preferably, the effector module of the decay accelerator protein (CD55) comprises CCP domains 1 to 4, preferably comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 70% identity with it; more preferably, the effector module of the membrane cofactor protein (CD46) comprises CCP domains 1 to 4, preferably comprising the amino acid sequence of SEQ ID NO: 9 or a sequence having at least 70% identity with it; and even more preferably, the effector module of complement receptor 1 (CR1) comprises CCP domains 1 to 3, CCP domains 8 to 10, CCP domains 15 to 17 or combinations thereof; preferably comprising the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and / or SEQ ID NO: 12 or a sequence having at least 70% identity with it.

[0032] Preferably, the modulator module comprises CCP domains 1 to 4 of C4BP, that is, preferably comprises the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 70% identity with it, more preferably composed of the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 70% identity with it. Even more preferably, the modulator module comprises the amino acid sequence of SEQ ID NO: 3, more preferably composed of the amino acid sequence of SEQ ID NO: 3.

[0033] As used herein, the term "binding module" refers to a module of a modulator protein containing CCP domain 19 and CCP domain 20 of the H factor. Preferably, in the above-described amino acid sequence of the binding module, the aspartic acid at position 1119 is replaced with glycine. Therefore, the binding module preferably comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 70% identity with it, more preferably composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 70% identity with it. Also preferably, the binding module comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, more preferably composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. As described in more detail in the embodiments herein, it has been surprisingly found that the binding module as specified has activity in binding complement factor C4b and its proteolytic derivative C4d. Therefore, the binding module preferably mediates binding with at least one of the C4b peptide, iC4b peptide, C4dg peptide and C4d peptide.

[0034] As used herein, the term "modulator protein" refers to any chemical molecule containing at least one polypeptide module as specified. It should be understood that the chemical linkages between modules do not necessarily have to be peptide bonds. The invention also contemplates that the chemical bonds between modules are ester bonds, disulfide bonds, or any other suitable covalent bonds known to those skilled in the art. Non-covalent bonds with such low dissociation constants that a module dissociates from other modules only to a negligible degree are also contemplated. Preferably, the dissociation constant of said non-covalent bond is less than 10. -5 M (e.g., in the case of Strep-tactin binding), less than 10 -6 M (e.g., in the case of Strep-label II: Strep-Tactin binding), less than 10 -8 M, less than 10 -10 M or less than 10 -12 M (e.g., streptavidin: biotin binding). Methods for determining the dissociation constant are well known to those skilled in the art, including, for example, spectroscopic titration, surface plasmon resonance measurements, equilibrium dialysis, etc. Furthermore, it is envisioned that the binding between modulator protein modules is indirect via affinity pairing, for example, the binding module containing a tag with affinity for biotin, while the modulator module is covalently coupled to biotin; or one module is coupled to a bacteriocin, and the second module is coupled to a corresponding immunoprotein. However, preferably, the chemical link between the modules is a peptide bond, i.e., preferably, the modulator protein is a modulator polypeptide comprising or composed of the described modules. Preferably, the modulator protein consists of components as described herein, preferably as a fusion polypeptide.

[0035] Modulatory proteins may also contain other modules such as peptides; such other modules may be, for example, modules that inactivate or regulate alternative pathways or classical pathways and lectin pathways or all complement activation pathways together, modules that increase serum stability, etc. Such modules are known in the art.

[0036] Modulator proteins may contain the indicated modules in any order deemed appropriate by those skilled in the art; however, particularly when the modulator protein is a regulatory peptide, the order is as indicated. Therefore, the regulatory peptide preferably contains these modules in the order of N-terminus-regulator module-binding module-C-terminus. Preferably, at least two modules of the modulator protein are linked by a "connector" peptide. Suitable connector peptides are known in principle in the art. Preferred connector peptides contain or preferably consist of glycine, alanine, and / or proline residues. More preferably, the connector peptide is a polyglycine connector peptide. Most preferably, the connector peptide is a connector containing, preferably, 5 to 15 glycine residues. Preferably, it is also contemplated to link the two modules by exchanging the last amino acid of the N-terminal module and / or the first amino acid of the C-terminal module for G residues. Preferably, the modulating protein comprises the amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 7, or a sequence having at least 70% identity with it, more preferably composed of the amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 7, or a sequence having at least 70% identity with it. Also preferably, the modulating protein comprises the amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 7, more preferably composed of the amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 7. Preferably, the modulating protein comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence having at least 70% identity with it, more preferably composed of the amino acid sequence of any one of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence having at least 70% identity with it. Also preferably, the modulating protein comprises the amino acid sequence of any one of SEQ ID NO: 4 or SEQ ID NO: 5, more preferably composed of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0037] The modulatory proteins mentioned herein possess two activities defined by their two modules: regulatory activity conferred by the regulatory module and binding activity conferred by the binding module. As will be understood by those skilled in the art, the regulatory activity of the regulatory module preferably requires the binding of the regulatory module to its regulated target, such as C4BP binding to C4b for regulation. Therefore, the modulatory proteins preferably have activities that regulate, preferably inhibit, the classical pathway of complement activation and / or the lectin pathway of complement activation, and have activities that bind to C4b peptides, iC4b peptides, C4dg peptides, and / or C4d peptides, preferably when one or more of these peptides are bound to the cell surface. Since C4b / d are deposited on the cell surface, C4b molecules mark them as targets of the complement system's membrane attack complex (MAC), thus the modulatory proteins preferably have activities that protect cells from the harmful effects of complement activation, preferably in vitro and / or in vivo. Preferably, the modulatory proteins have activities that inhibit the activation of both the classical pathway and the lectin pathway of complement activation. More preferably, the modulator protein comprises a module, preferably an additional module different from the modulator module, wherein the module inhibits the activation of the alternative pathway for complement activation; in this case, the modulator protein preferably has activity in inhibiting the alternative pathway, classical pathway and lectin pathway for complement activation.

[0038] Preferably, the host surface of the object is any internal surface present in the object, wherein the internal surface may be natural or artificial. Artificial surfaces particularly include the surface of implants. Natural surfaces are known to those skilled in the art and particularly include any surface of the lumen of the object, such as the inner surface of blood vessels, mucosa, glomerular basement membrane, Bruch's membrane, proteoglycans, glycans, extracellular matrix, cells, or other surfaces of adjacent interstitial and / or skin layers.

[0039] The modulatory protein preferably binds to cell and / or host surfaces on which the complement-activated classical pathway and / or lectin pathway is activated, wherein the term "cell and / or host surface on which the complement-activated classical pathway and / or lectin pathway is activated" refers to a cell and / or host surface having at least one of a C4b polypeptide, an iC4b polypeptide, a C4dg polypeptide, and a C4d polypeptide attached thereto. Preferably, the cells are neurons, muscle cells, endothelial cells, kidney cells, endothelial cells, blood cells, or epithelial cells. Preferably, the kidney cells are cells in the glomeruli or renal tubules. Also preferably, the host surface of the object is any internal surface present in the object, wherein the internal surface may be natural or artificial. Artificial surfaces particularly include the surface of implants. Natural surfaces are known to those skilled in the art and particularly include any surface of the lumen of the object, such as the inner surface of blood vessels, mucosa, glomerular basement membrane, Bruch's membrane, surfaces containing at least one of proteoglycans, glycans, extracellular matrix, or other surfaces, particularly surfaces adjacent to the interstitial and / or skin layers. Preferably, the endothelial cells or other host surfaces include cells in the ocular vasculature and / or host surfaces, such as in the choroid, including the Bruch membrane. Preferably, the blood cells are red blood cells, platelets, or white blood cells.

[0040] Advantageously and surprisingly, in the work upon which this invention is based, it was discovered that the extended domain of factor H exhibits binding activity to C4b and C4d, which are specific regulators of the alternative pathway of complement activation, and which are products of the classical and lectin pathways. Therefore, factor H, and particularly its CCP domains 19 and 20, have been newly and surprisingly discovered to potentially protect cells from activation by the classical and / or lectin pathways of complement activation.

[0041] The definitions above, with necessary modifications, apply hereinafter. Other definitions and interpretations made hereinafter, with necessary modifications, also apply to all embodiments described in this specification.

[0042] Preferably, the modulator protein is contained in the pharmaceutical composition. Therefore, the present invention also relates to pharmaceutical compositions comprising the modulator protein described herein.

[0043] The terms “drug” and “pharmaceutical composition” are used interchangeably herein and are known in principle to those skilled in the art. As mentioned herein, the term preferably refers to any composition of substances comprising one or more designated active agents as one or more pharmaceutically active compounds and optionally one or more excipients. One or more pharmaceutically active compounds may be present in liquid or dry form, such as lyophilized form. It will be understood that the form and properties of pharmaceutically acceptable excipients, such as carriers or diluents, are determined by the amount of the active ingredient in combination with them, the route of administration, and other well-known variables. One or more excipients must be acceptable in the sense of compatibility with the other components of the formulation and harmlessness to the recipient. The excipients used may include solids, gels, or liquids. Examples of solid carriers are lactose, anhydrous terra alba, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, etc. Examples of liquid carriers include phosphate-buffered saline solutions, physiological saline, Ringer's solution, dextrose solution, Hank's solution, syrups, oils, water, emulsions, and various types of wetting agents. Similarly, carriers or diluents may include time-delaying materials known in the art, such as glyceryl monostearate or glyceryl distearate alone or with waxes. Suitable carriers include those described above and other carriers known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. One or more excipients are selected to not affect the bioactivity of the combination.

[0044] The drug can be administered via any route deemed appropriate, such as by a medical practitioner, preferably at a therapeutically effective dose. Topical administration is preferred, or more preferably systemic administration. Suitable routes of administration conventionally used for drug administration are local, intravenous, or parenteral administration, as well as inhalation. Preferably, administration is systemic, more preferably intravenous. However, depending on the nature and mode of action of the specific compound being administered and the clinical situation, the drug composition may also be administered via other routes. Furthermore, the drug composition may be administered in combination with other additional active compounds, either in a common drug composition or as separate drug compositions, which may be provided as kits.

[0045] Therapeutic effective dose refers to the amount of an active compound that prevents, improves, or cures symptoms accompanying the diseases or conditions mentioned in this specification. The therapeutic efficacy and toxicity of a drug can be determined through standard pharmaceutical procedures in cell cultures or laboratory animals, such as ED50 (the therapeutically effective dose in 50% of the population) and LD50 (the lethal dose in 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as a ratio, LD50 / ED50. Dosing regimens will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose used for any given patient can depend on many factors, including the type and severity of the disease, the patient's body size, age, the specific formulation of the drug to be administered, sex, time and route of administration, general health condition, and other concurrent medications. Drugs mentioned herein are preferably administered at least once, for example, as a bolus injection. However, drugs may be administered more than once, and preferably at least twice, for example, permanently or periodically after a defined time window. Progress can be monitored through periodic assessments. Dosing recommendations can be indicated in the prescriber's or user's instructions for adjustment based on the expected dose for the recipient under consideration. The pharmaceutical products according to the present invention may contain other active agents besides the one or more active agents described above. Furthermore, it should be understood that the formulation of the pharmaceutical composition is preferably carried out under GMP-standardized conditions or similar conditions to ensure the quality, safety, and efficacy of the pharmaceutical product.

[0046] Furthermore, the present invention relates to polynucleotides encoding the modulator proteins described herein, preferably encoding the modulator polypeptides described herein.

[0047] The present invention also relates to modulator proteins, pharmaceutical compositions and / or polynucleotides described elsewhere herein for pharmaceutical use; and the present invention relates to the use of modulator proteins described herein in the preparation of pharmaceuticals.

[0048] This invention also relates to modulatory proteins, pharmaceutical compositions, and / or polynucleotides described herein for the prevention and / or treatment of cell or host surface damage caused by components of the complement-activated classical pathway and / or lectin pathway.

[0049] Furthermore, this invention relates to the modulatory proteins, pharmaceutical compositions, and / or polynucleotides described herein for the prevention and / or treatment of diseases caused and / or exacerbated by activation of the complement-activated classical pathway and / or lectin-dependent pathway. Additionally, this invention relates to the use of the modulatory proteins described herein in the preparation of medicaments for the treatment and / or prevention of cell or host surface damage caused by components of the complement-activated classical pathway and / or lectin pathway, and / or for the prevention and / or treatment of diseases caused and / or exacerbated by activation of the complement-activated classical pathway and / or lectin pathway.

[0050] Therefore, the present invention relates to modulatory proteins comprising (i) modulatory modules that regulate the classical pathway of complement activation and / or the lectin pathway of complement activation, and (ii) binding modules comprising, preferably, CCP domain 19 and CCP domain 20 of H factor; for the prevention and / or treatment of diseases caused and / or aggravated by activation of the classical pathway of complement activation and / or the lectin pathway.

[0051] Those skilled in the art will understand the term "disease caused and / or aggravated by activation of the classical pathway and / or lectin pathway of complement activation"; the corresponding diseases are reviewed, for example, by Ricklin et al. (2017), Mol Immunol. 89:10-21. Preferably, the term includes each disease in which activation of the classical pathway and / or lectin pathway of complement activation results in the severity of at least one disease symptom. Preferably, the disease is one in which complement activation via the classical pathway and / or disease pathway causes damage to the cells of the subject and / or at least one host surface. Preferably, the cells of the subject are blood-accessible cells of the subject, such as cells lining blood vessels. Also preferably, the host surface of the subject is any internal surface present in the subject, wherein the internal surface may be natural or artificial. Artificial surfaces particularly include the surface of implants. Natural surfaces are those known to those skilled in the art and particularly include any surface of the lumen of the subject, preferably as specified above. Thus, the host surface may be, in particular, the basement membrane or glycocalyx, such as the basement membrane or glycocalyx of blood vessels, glomeruli, or renal tubules. Therefore, the aforementioned cell or host surface damage can be tissue damage and / or organ damage. Damage caused by complement activation via the classical and / or lectin pathways preferably includes, more preferably, features of deposition of C4b polypeptide, iC4b polypeptide, C4dg polypeptide, and / or C4d polypeptide on the cell or host surface. Methods for identifying such deposition are known in the art. In view of the above, the disease is preferably pathogenic microbial infection, systemic lupus erythematosus, lupus nephritis, antiphospholipid syndrome, catastrophic antiphospholipid syndrome, platelet-ineffective transfusion, IgA nephropathy, transplant-associated microangiopathy, hematopoietic stem cell transplant-associated microangiopathy, antibody-mediated rejection, ischemia-reperfusion injury, secondary membranous nephropathy, xenotransplantation, acute kidney injury, or Guillain-Barré syndrome.

[0052] Preferably, the diseases are selected from: adhesive capsulitis, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, allergic reactions, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atrial fibrosis, atypical hemolytic uremic syndrome, anterior subcapsular cataract, autoimmune diseases (including, for example, autoimmune hemolytic anemia (AIHA); warm antibody-type AIHA). AIHA; Mixed AIHA; Progressive lipodystrophy, Behçet's disease, British amyloid angiopathy, bronchiectasis, bullous pemphigoid, Buerger's disease, C3 glomerulonephritis and other glomerulonephritis, chronic kidney disease, Clq nephropathy, cancer, catastrophic antiphospholipid syndrome, cirrhosis, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Kreutzfeld-Jacob disease, Crohn's disease, cryoglobulinemia vasculitis, cystic fibrosis, boxing dementia, Lewy body dementia (DLB), diffuse neurofibrillary tangles with calcification, discoid lupus erythematosus, Down syndrome, Dupuytren's contracture, Evan's syndrome, endometriosis, endocardial myocardial fibrosis, excessive wound healing, focal segmental glomerulosclerosis, formal thought disorder. Disorders, frontotemporal dementia (FTD), chromosome 17-related frontotemporal dementia with Parkinson's syndrome, frontotemporal degeneration, Gerstmann-Straussler-Scharinkel disease, Guillain-Barré syndrome, Hallewarden-Scpatz disease, hemolytic uremic syndrome, hereditary angioedema, hypophosphatase disease, IgA nephropathy, idiopathic pulmonary fibrosis, idiopathic pneumonia syndrome, immune complex diseases, inclusion body myositis, infectious diseases, inflammatory diseases, ischemia / reperfusion injury, keloids, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency (MoCD), type I membranoproliferative glomerulonephritis (MPGN), type II membranoproliferative glomerulonephritis (M... PGN (dense deposit disease), membranous nephropathy, multiple infarct dementia, lupus (e.g., systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuronopathy, multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, myelofibrosis, neuromyelitis optica, Niemann-Pick disease type C, non-alcoholic fatty liver disease, non-Guam motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Peyronie's disease, Pick's disease, post-encephalitis Parkinson's syndrome, polymyositis, prion amyloid angiopathy, progressive subcortical gliosis.gliosis), progressive supranuclear paralysis, posterior capsule opacification, psoriasis, sepsis, Shigella-producing Escherichia coli (STEC)-Hus, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, scleroderma, systemic sclerosis, tangled dementia, transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegner's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, primary mixed cryoglobulinemia, type II mixed cryoglobulinemia, type III mixed cryoglobulinemia, nephritis, drug-induced thrombocytopenia, lupus nephritis, acquired bullous epidermolysis, delayed hemolytic transfusion reaction, hypocomplement-induced urticarial vasculitis syndrome, pseudolens bullous keratopathy, valvular fibrosis, and platelet transfusion ineffectiveness. In a preferred embodiment, atypical hemolytic uremic syndrome may also be referred to as "complement-mediated thrombotic microangiopathy".

[0053] As used herein, the term "subject" refers to an animal with a complement system, preferably a mammal. More preferably, the subject is a cow, pig, sheep, horse, cat, dog, mouse, monkey, or rat, and most preferably a human. Preferably, the subject is known or suspected of having a disease caused and / or aggravated by activation of the classical pathway and / or lectin pathway of complement activation, or is known or suspected that the subject will develop such a disease within the next 6 months, preferably 4 weeks, more preferably 7 days, and most preferably 24 hours.

[0054] The term "treatment" refers to a significant improvement in the disease or disorder mentioned herein or its associated symptoms; as used herein, the term includes prevention of the worsening of the disease, disorder, or associated symptoms. Treatment may also include complete recovery of health with respect to the disease or disorder mentioned herein. It should be understood that the term "treatment," as used herein, may not be effective in all subjects. However, the term should require, preferably requires, that a statistically significant portion of the subjects suffering from the disease or disorder mentioned herein be successfully treated. Whether a portion is statistically significant can be determined without doubt by those skilled in the art using various well-known statistical evaluation tools, such as the determination of confidence intervals, p-values, Student's t-test, Mann-Whitney test, etc. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. Preferred p-values ​​are 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment is effective for at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population.

[0055] The term "prevention" refers to maintaining the health of a subject against the disease or disorder mentioned herein for a period of time. It will be understood that this period of time may depend on the amount of pharmaceutical compound administered and individual factors of the subject discussed elsewhere in this specification. It should be understood that prevention may not be effective in all subjects treated with the compounds according to the invention. However, the term requires, preferably requires, that a statistically significant portion of a cohort or population of subjects be effectively prevented from suffering from the disease or disorder mentioned herein or its accompanying symptoms. Preferably, the cohort or population of subjects envisioned in this context is typically, i.e., those who would develop the disease or disorder mentioned herein without the preventative measures according to the invention. Whether a portion is statistically significant can be determined without doubt by those skilled in the art using various well-known statistical evaluation tools discussed elsewhere in this specification.

[0056] In the context of treatment and prevention specified herein, the modulatory modulator preferably further modulates the alternative pathway of complement activation. More preferably, in this context, the modulatory protein further modulates, in particular, the alternative pathway of complement activation.

[0057] Therefore, the present invention also relates to modulatory proteins comprising (i) modulatory modules that regulate the classical pathway and / or the complement-activated lectin pathway, and optionally the alternative pathway of complement activation; and (ii) binding modules comprising, preferably, CCP domain 19 and CCP domain 20 of the H factor; for the prevention and / or treatment of diseases caused and / or aggravated by activation of the classical pathway and / or the lectin pathway of complement activation.

[0058] The present invention also relates to methods for treating and / or preventing cell damage in subjects caused by components of the complement-activated classical pathway and / or lectin pathway, and / or for preventing and / or treating diseases in subjects caused and / or exacerbated by activation of the complement-activated classical pathway and / or lectin-dependent pathway, said methods comprising...

[0059] (a) Applying the modulator protein as described herein to the subject; and

[0060] (b) thereby treating and / or preventing cell and / or host surface damage caused by components of the complement-activated classical and / or lectin pathways in the subject and / or for the prevention and / or treatment of diseases caused and / or aggravated by activation of the complement-activated classical and / or lectin-dependent pathways in the subject.

[0061] The method of the present invention for treatment and / or prevention is preferably an in vivo method; however, the method or its steps may also be performed in vitro, for example by pretreating the organ graft according to step (a) prior to implantation. As understood by those skilled in the art, the modulator protein can be applied to the organ graft by: (i) applying the modulator protein to the donor subject prior to explantation, (ii) perfusing the organ graft with the modulator protein in vitro, preferably after explantation from the donor subject and before implantation into the recipient subject, and / or (iii) applying the modulator protein to the recipient subject, preferably before, during, and / or after transplantation.

[0062] The present invention also relates to a method for preventing damage to host cells and / or surfaces caused by components of the classical pathway and / or lectin pathway activated by complement, preferably an in vitro method, the method comprising:

[0063] (A) Contacting the host cell and / or surface with the modulated protein as described herein; and

[0064] (B) thereby preventing damage to the host cells and / or surface.

[0065] In the context of methods used for prevention, the term "host cell" encompasses every type of living cell. Preferably, the cells are susceptible to damage from components of the classical pathway and / or lectin pathway of the complement system. Thus, the cells can be, in particular, eukaryotic cells. Preferably, the cells are cultured cells, i.e., preferably cultured in vitro, and more preferably in a culture medium containing blood or blood-derived products such as serum.

[0066] In the context of methods used for prevention, the term "surface" includes any surface known or suspected to be susceptible to damage from components of the classical pathway and / or lectin pathway of the complement system. Thus, a surface can be the surface of a cell layer or the surface of extracellular material derived from such a cell layer, can be a functional surface, such as the surface of a bead or biosensor, or can be a porous material, such as a filter like a hemodialysis filter or any other biomaterial. Preferably, the surface is planned or placed at risk of contact with a composition of substances containing blood or blood-derived products, such as serum, or other biological fluids containing active complement components, such as cerebrospinal fluid (CSF) or synovial fluid (SF).

[0067] The present invention also relates to devices comprising the modulated proteins and / or pharmaceutical compositions described herein.

[0068] As used herein, the term "device" refers to a system comprising at least the aforementioned components, preferably operably connected to each other and / or to additional components to allow administration of a specified compound or composition. Preferred components for administering the modulating protein are well known in the art and described above. How these components are operably connected will depend on the type of components included in the device and the intended type of administration. Preferably, in this case, these components are included in a single device. The device may accordingly include a delivery unit for administering the modulating protein, and optionally a storage unit for storing the modulating protein until administration. However, in such embodiments, it is also contemplated that the components of the invention may be presented as separate devices, and preferably packaged together as a kit. Those skilled in the art will recognize without question how these components can be connected. Preferred devices are those that can be applied without requiring special knowledge of a skilled technician. In a preferred embodiment, the device is a syringe comprising the modulating protein, more preferably with a needle. More preferably, the device is an intravenous infusion (IV) device comprising the modulating protein. Preferably, the device is a tube or endoscope containing a modulating protein for flushing the application site, such as the heart, or includes a needle for topical application of the compound or composition.

[0069] The present invention also relates to kits comprising modulatory proteins as described herein and / or pharmaceutical compositions as described herein, wherein the modulatory proteins as described herein and / or pharmaceutical compositions as described herein are included in a housing.

[0070] As used herein, the term "kit" refers to a collection of the aforementioned compounds, components, or reagents, which may or may not be packaged together. The components of the kit may be included in or provided in individual vials (i.e., as separate parts of the kit), for example, as the compositions specified above. In embodiments, the kit housing allows for the translocation of the kit's compounds, particularly co-translocation; therefore, the housing may be a transportable container, particularly including all specified components. Furthermore, it should be understood that the kit of the present invention can be used to practice the methods mentioned above. It is preferably envisioned that all components be provided in a ready-to-use manner for practicing the methods mentioned above. Further, the kit preferably includes instructions for carrying out the methods. The instructions may be provided in paper or electronic form as a user manual. For example, the manual may contain explanations of the results obtained when the kit is used to perform the methods described above. Preferably, the kit is suitable for the methods of the present invention, more preferably suitable for including all reagents required to perform one or more of the methods described above. Preferably, the kit includes other components, such as administration components and / or other complement activation inhibitors.

[0071] The present invention also relates to the use of modulatory proteins and / or pharmaceutical compositions as defined herein for the inhibition of complement activation via the classical and / or lectin pathways on host cells or surfaces, wherein said use is preferably in vitro.

[0072] The present invention also relates to a method for detecting activation of the classical complement pathway and / or the lectin complement pathway in a sample, comprising (i) contacting the sample with a detection reagent comprising a binding module comprising, preferably, CCP domains 19 and 20 of a factor, and the binding module binding to a detector compound, and

[0073] (ii) Detecting the binding of the test reagent to the sample, and thereby

[0074] (iii) Detect activation of the classical complement pathway and / or lectin complement pathway in the sample.

[0075] The preferred method for detecting activation is an in vitro method.

[0076] In view of the above, the following implementation plan is specifically proposed:

[0077] Implementation Scheme 1: A modulator protein comprising (i) a modulator module that regulates the classical pathway of complement activation and / or the lectin pathway of complement activation, and (ii) a binding module comprising, preferably, a CCP domain 19 and a CCP domain 20 of an H factor.

[0078] Implementation Scheme 2: The modulator protein according to Implementation Scheme 1, wherein in the amino acid sequence of the binding modulator, the aspartic acid at position 1119 is replaced with glycine.

[0079] Implementation Scheme 3: The modulator protein according to Implementation Scheme 1 or 2, wherein the binding modulator comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 70% identity with it.

[0080] Implementation Scheme 4: A modulator protein according to any one of Implementation Schemes 1 to 3, wherein the binding modulator consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 70% identity with it.

[0081] Implementation Scheme 5: A modulator protein according to any one of Implementation Schemes 1 to 4, wherein the binding modulator comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0082] Implementation Scheme 6: A modulator protein according to any one of Implementation Schemes 1 to 5, wherein the binding modulator consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0083] Implementation Scheme 7: A modulator protein according to any one of Implementation Schemes 1 to 6, wherein the modulator module comprises a module of an effector protein of the classical pathway of complement activation and / or the lectin pathway, preferably an invertase decay acceleration module and / or an invertase or C4b (or C4b and C3b) specific cofactor activity module.

[0084] Implementation Scheme 8: A modulator protein according to any one of Implementation Schemes 1 to 7, wherein the modulator module comprises, preferably, an effector module consisting of: a C4b binding protein, a decay-accelerating protein, a membrane cofactor protein, and / or a complement receptor (CR).

[0085] Implementation Scheme 9: The modulator protein according to Implementation Scheme 8, wherein the C4b binding protein is a C4b binding protein, wherein the decay accelerating protein is CD55, wherein the membrane cofactor protein is CD46, and / or wherein the CR is CR1.

[0086] Implementation Scheme 10: The modulator protein according to Implementation Scheme 8 or 9, wherein the modulator module comprises an amino acid sequence of at least one of SEQ ID NO: 3 and SEQ ID NO: 8 to SEQ ID NO: 12 or a sequence having at least 70% identity with it.

[0087] Implementation Scheme 11: A modulator protein according to any one of Implementation Schemes 1 to 10, wherein the modulator module comprises the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 90% identity with it.

[0088] Implementation Scheme 12: A modulator protein according to any one of Implementation Schemes 1 to 11, wherein the modulator module consists of the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 70% identity with it.

[0089] Implementation Scheme 13: A modulator protein according to any one of Implementation Schemes 1 to 12, wherein the modulator module comprises the amino acid sequence of SEQ ID NO: 3.

[0090] Implementation Scheme 14: A modulator protein according to any one of Implementation Schemes 1 to 13, wherein the modulator module consists of the amino acid sequence of SEQ ID NO: 3.

[0091] Implementation Scheme 15: A modulator protein according to any one of Implementation Schemes 1 to 14, wherein the modulator protein is a polypeptide.

[0092] Implementation Scheme 16: A modulator protein according to any one of Implementation Schemes 1 to 15, wherein the modulator protein comprises an amino acid sequence of any one of SEQ ID NO:4 to SEQ ID NO:7, or a sequence having at least 70% identity with it.

[0093] Implementation Scheme 17: A modulator protein according to any one of Implementation Schemes 1 to 16, wherein the modulator protein consists of an amino acid sequence of any one of SEQ ID NO:4 to SEQ ID NO:7, or a sequence having at least 70% identity with it.

[0094] Implementation Scheme 18: A modulator protein according to any one of Implementation Schemes 1 to 17, wherein the modulator protein comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0095] Implementation Scheme 19: A modulator protein according to any one of Implementation Schemes 1 to 18, wherein the modulator protein consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0096] Implementation Scheme 20: A modulator protein according to any one of Implementation Schemes 1 to 19, wherein the modulator protein is bound to a C4b polypeptide, an iC4b polypeptide, a C4dg polypeptide, and / or a C4d polypeptide.

[0097] Implementation Scheme 21: A modulator protein according to any one of Implementation Schemes 1 to 20, wherein the modulator protein binds to C4b polypeptide, iC4b polypeptide, C4dg polypeptide and / or C4d polypeptide bound to the cell surface.

[0098] Implementation Scheme 22: A modulator protein according to any one of Implementation Schemes 1 to 21, wherein the modulator protein binds to cells on which the classical pathway of complement activation and / or the lectin pathway is activated.

[0099] Implementation Scheme 23: A modulator protein according to any one of Implementation Schemes 1 to 22, wherein the cells on which the classical pathway and / or lectin pathway of complement activation is activated are neurons, muscle cells, endothelial cells, cells in the kidney, endothelial cells, blood cells, or epithelial cells.

[0100] Implementation Scheme 24: A modulated protein according to any one of Implementation Schemes 1 to 23, wherein the modulated protein is contained in a pharmaceutical composition.

[0101] Implementation Scheme 25: A modulator protein according to any one of Implementation Schemes 1 to 24, wherein the modulation is inhibition.

[0102] Implementation Scheme 26: A polynucleotide encoding a modulator protein according to any one of Implementation Schemes 1 to 23, preferably encoding a polypeptide according to any one of Implementation Schemes 15 to 23.

[0103] Implementation Scheme 27: A pharmaceutical composition comprising a modulator protein according to any one of Implementation Schemes 1 to 25 and / or a polynucleotide according to Implementation Scheme 26.

[0104] Implementation Scheme 28: A modulated protein according to any one of Implementation Schemes 1 to 25, a pharmaceutical composition according to Implementation Scheme 27, and / or a polynucleotide according to Implementation Scheme 26, for pharmaceutical use.

[0105] Implementation Scheme 29: Use of the modulated protein according to any one of Implementation Schemes 1 to 25 and / or the polynucleotide according to Implementation Scheme 26 for the preparation of a medicament.

[0106] Implementation Scheme 30: A modulator protein according to any one of Implementation Schemes 1 to 25, a pharmaceutical composition according to Implementation Scheme 27, and / or a polynucleotide according to Implementation Scheme 26, for the prevention and / or treatment of cell or host surface damage caused by components of the complement-activated classical pathway and / or lectin pathway.

[0107] Implementation Scheme 31: A modulator protein according to any one of Implementation Schemes 1 to 25, a pharmaceutical composition according to Implementation Scheme 27, and / or a polynucleotide according to Implementation Scheme 26, for the prevention and / or treatment of diseases caused and / or aggravated by activation of the classical pathway and / or lectin pathway by complement activation.

[0108] Implementation Scheme 32: Use of the modulator protein according to any one of Implementation Schemes 1 to 25 and / or the polynucleotide according to Implementation Scheme 26 in the preparation of medicaments for treating and / or preventing cell or host surface damage caused by components of the complement-activated classical pathway and / or lectin pathway, and / or for preventing and / or treating diseases caused and / or aggravated by activation of the complement-activated classical pathway and / or lectin-dependent pathway.

[0109] Implementation Scheme 33: The subject matter according to any one of Implementation Schemes 30 to 32, wherein the cell or host surface damage is tissue damage and / or organ damage.

[0110] Implementation Scheme 34: The subject matter according to any one of Implementation Schemes 31 to 33, wherein the disease is pathogenic microbial infection, atypical hemolytic uremic syndrome, autoimmune hemolytic anemia, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, antiphospholipid syndrome, catastrophic antiphospholipid syndrome, platelet ineffective transfusion, IgA nephropathy, transplant-associated microangiopathy, hematopoietic stem cell transplant-associated microangiopathy, antibody-mediated rejection, ischemia-reperfusion injury, secondary membranous nephropathy, xenograft, acute kidney injury, or Guillain-Barré syndrome, preferably pathogenic microbial infection, systemic lupus erythematosus, lupus nephritis, antiphospholipid syndrome, catastrophic antiphospholipid syndrome, platelet ineffective transfusion, IgA nephropathy, transplant-associated microangiopathy, hematopoietic stem cell transplant-associated microangiopathy, antibody-mediated rejection, ischemia-reperfusion injury, secondary membranous nephropathy, xenograft, acute kidney injury, or Guillain-Barré syndrome.

[0111] Implementation Scheme 35: A method for treating and / or preventing cell damage in a subject caused by components of the complement-activated classical pathway and / or lectin pathway and / or for preventing and / or treating diseases in a subject caused and / or exacerbated by activation of the complement-activated classical pathway and / or lectin-dependent pathway, the method comprising...

[0112] (a) Applying the modulated protein according to any one of embodiments 1 to 25 and / or the polynucleotide according to embodiment 26 to the subject; and

[0113] (b) thereby treating and / or preventing cell and / or host surface damage caused by components of the complement-activated classical pathway and / or lectin pathway in the subject and / or for the prevention and / or treatment of diseases caused and / or aggravated by activation of the complement-activated classical pathway and / or lectin-dependent pathway in the subject.

[0114] Implementation Scheme 36: A method for preventing damage to host cells and / or surfaces caused by components of the classical pathway and / or lectin pathway activated by complement, preferably an in vitro method, said method comprising...

[0115] (A) Contacting the host cell and / or surface with the modulated protein according to any one of embodiments 1 to 25; and

[0116] (B) thereby preventing damage to the host cells and / or surface.

[0117] Implementation Scheme 37: An apparatus comprising a modulator protein according to any one of Implementation Schemes 1 to 25, a pharmaceutical composition according to Implementation Scheme 27, and / or a polynucleotide according to Implementation Scheme 26.

[0118] Implementation Scheme 38: The apparatus according to Implementation Scheme 36, wherein the apparatus is an application component.

[0119] Implementation Scheme 39: A kit comprising a modulator protein according to any one of Implementation Schemes 1 to 25, a pharmaceutical composition according to Implementation Scheme 27, and / or a polynucleotide according to Implementation Scheme 26, all contained in a housing.

[0120] Implementation Scheme 40: The kit according to Implementation Scheme 39, wherein the kit further includes other inhibitors of complement activation.

[0121] Implementation Scheme 41: Use of the modulator protein according to any one of Implementation Schemes 1 to 25 and / or the pharmaceutical composition according to Implementation Scheme 27 for inhibiting the classical pathway and / or lectin pathway of complement activation on host cells or surfaces.

[0122] Implementation Scheme 42: The use according to Implementation Scheme 41, wherein the use is an in vitro use.

[0123] All references cited in this specification are incorporated herein by reference in their entirety and in particular as specifically mentioned herein. Attached Figure Description

[0124] Figure 1 SPR analysis of the binding affinity of the C-terminus of FH to C3d (the cleavage product of AP). Here, 3000 response units (RU) of C3d were immobilized on a CMD500m sensor chip using amine coupling. The left side (A, C) shows the sensing plots after injection of the corresponding H factor fragments (A: FH(19 to 20) 1119G mutant protein, C: FH(19 to 20) wt), and the right side (B, D) shows the concentration-response curves of the dissociation constant (KD) determined by a 1:1 steady-state affinity model (B: FH(19 to 20) 1119G mutant protein, D: FH(19 to 20) wt).

[0125] Figure 2 SPR analysis of the binding affinity of the C-terminus of FH to C4d (the cleavage product of CP). Here, 1100 response units (RU) of C4d were immobilized on the CMD500m sensor chip via amine coupling. The left side (A, C) shows the sensing plots after injection of the corresponding H factor fragments (A: FH(19 to 20) 1119G mutant protein, C: FH(19 to 20) wt), and the right side (B, D) shows the concentration-response curves of the dissociation constant (KD) determined by the 1:1 steady-state affinity model (B: FH(19 to 20) 1119G mutant protein, D: FH(19 to 20) wt).

[0126] Figure 3 SPR analysis of the binding affinity of the fusion construct with an FH C-terminus to C4d (a cleavage product of CP). Here, 1100 RU of C4d was immobilized on a CMD500m sensor chip using amine coupling. The left side (A, C, E) shows the sensing plots after injection of the corresponding proteins (A: C4BP (1 to 4), C: 2XC4BP, E: Mini C4BP), and the right side (B, D, F) shows the concentration-response curves of the dissociation constant (KD) determined by a 1:1 steady-state affinity model (B: C4BP (1 to 4), D: 2XC4BP, F: Mini C4BP). C4BP(1 to 4) = Domains 1 to 4 of C4-binding protein (regulator of CP); 2XC4BP(1 to 4) = Same as C4BP(1 to 4), except that this construct incorporates two copies of C4BP(1 to 4); mini-C4BP = C4BP(1 to 4) fused with FH(19 to 20)1119G.

[0127] Figure 4 SPR analysis of the binding affinity of the control protein to C4d (the cleavage product of CP). Here, biotinylated C4d of 487RU was immobilized on a streptavidin sensor chip. The left side (A, C) shows the sensing plots after injection of the corresponding control protein, and the right side (B, D) shows the concentration-response curves of the dissociation constant (KD) determined by a 1:1 steady-state affinity model. CR1 (15 to 17) = protein domains 15 to 17 of complement receptor 1 (A, B), which are active in all activation pathways of the complement system; FH (15 to 18) = protein domains 15 to 18 of FH (C, D), which have no specific known binding or regulatory activity.

[0128] Figure 5 SPR analysis of the binding affinity of the fusion construct with the C-terminus of FH to C4b (a cleavage product of CP). Here, 2300 RU of biotinylated C4b was immobilized on a streptavidin sensor chip. The left side (A, C, E, G) shows the sensing plots after injection of the corresponding proteins (A: FH(19 to 20) 1119G mutant protein; C: FH(19 to 20) wt; E: C4BP(1 to 4), G: MiniC4BP), and the right side (B, D, F, H) shows the concentration-response curves of the dissociation constant (KD) determined according to the 1:1 steady-state affinity model (B: FH(19 to 20) 1119G mutant protein; D: FH(19 to 20) wt; F: C4BP(1 to 4), H: MiniC4BP).

[0129] Figure 6SPR analysis of the binding affinity of the fusion construct with an FH C-terminus to C4b (a cleavage product of CP). Here, biotinylated C4b of 3127RU was immobilized on a streptavidin sensor chip. The left-hand plots (A, C, E, G, I, K) show the sensing plots after injection of the corresponding proteins, and the right-hand plots (B, D, F, H, J, L) show the concentration-response curves of the dissociation constant (KD) determined according to the 1:1 steady-state affinity model. CR1(1-3) = Protein domains 1-3 of complement receptor 1 (A, B), which are active in all activation pathways of the complement system; CR1(1-3) / FH(19-20)SL = Fusion of CR1 protein domains 1-3 and FH(19-20) without an additional linker (=SL) (C, D); CR1(1-3) / FH(19-20)LL = Fusion of CR1 protein domains 1-3 and FH(19-20) with an additional glycine linker (=LL) (E, F) ; DAF(1 to 4) = Domains 1 to 4 (G, H) of complement regulator decay accelerator factor, which is active in all activation pathways of the complement system; DAF(1 to 4) / FH(19 to 20)SL = Fusion of DAF protein domains 1 to 4 and FH(19 to 20) without additional linkers (=SL)(I, J); DAF(1 to 4) / FH(19 to 20)LL = Fusion of DAF protein domains 1 to 4 and FH(19 to 20) with additional glycine linkers (=LL)(K, L).

[0130] Figure 7Functional hemolysis assay using the fusion construct FH(19 to 20)1119G. In this hemolysis assay, red blood cells were washed and diluted to a defined cell density, then mixed with a defined percentage of serum + / - analyte and incubated. After incubation, the remaining cells were centrifuged, and the supernatant was measured at 405 nm. When this value was compared to a lysis control (completely permeable to water for lysis), the absorbance indicated how much hemoglobin was released and the corresponding number of cells lysed, expressed as a percentage. In this particular assay, sheep red blood cells (which do not lyse in human serum via complement-mediated lysis) were loaded with antibodies to specifically activate CP. Samples in which serum was supplemented with EDTA (blocking any complement-mediated lysis), EGTA (blocking CP-mediated lysis), or PBS instead of the inhibitor served as controls. HA = hemolysis assay; NHS = normal human serum; Eculizumab = antibody approved for the treatment of complement-mediated diseases, which binds to terminal protein C5; Mini-C4BP has the amino acid sequence of SEQ ID NO: 4; 2xC4BP (1 to 4) have the amino acid sequence of SEQ ID NO: 6, and Fc-Mini-C4BP has the amino acid sequence of SEQ ID NO: 7.

[0131] The following examples are for illustrative purposes only. They should not be construed as limiting the scope of the invention in any way.

[0132] Example 1: Ligands modulated and immobilized by random orientation

[0133] Before use, the CMD500m chip was placed at room temperature for 20 to 30 minutes and inserted into a Reichert SR7500DC system equilibrated in PBS-T (0.005% Tween 20) as the standard run buffer. The flow rate was typically set at 25 µl / min. First, the chip was conditioned by five repeated washes with 50 mM sodium hydroxide (NaOH) to expose the dextran and attached carboxyl groups required for the next reaction. The reactive groups were then activated by a 1:1 mixture of 0.1 M sulfonyl-N-hydroxysulfosuccinimide (s-NHS / sulpho-NHS) dissolved in water and 0.1 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) dissolved in 5 mM MES buffer at pH 6.0. The former resulted in a more stable amine reactive intermediate and higher coupling efficiency, while the latter acted as a zero-length crosslinking agent.

[0134] The reaction mixture activates the carboxyl groups of the carboxymethyl dextran hydrogel on the CMD500m chip, allowing the activated carboxyl groups to covalently bind to the primary amine groups of the protein to be immobilized on the chip. In this way, the ligands are covalently immobilized. Immobilization of all ligands is performed only in the left flow cell, while the right flow cell is activated without exposure to the ligands, thus generating a reference flow cell. Finally, an additional quenching step with 1M ethanolamine at pH 8.5 is performed in both flow cells to react with any remaining activated carboxyl groups. The immobilization unit of the ligand is obtained by subtracting the signal from the reference cell from the signal from the immobilization cell. After each ligand immobilization, a positive control test is performed by injecting an analyte known to bind the attached or immobilized ligand. Once a good quality binding signal is detected, the target analyte is injected in serial dilutions.

[0135] The precise concentrations of each immobilized ligand and the pH of the buffer solution are shown in the table below (Table 1).

[0136] Table 1: Different ligands immobilized with random orientation using the CMD500m sensor chip

[0137]

[0138] Example 2: Regulation and immobilization of ligands with a "physiological orientation"

[0139] In this embodiment, the thioester moieties of ligands C4b and C4d were biotinylated to covalently bind to the avidin surface of the sensor chip. C4b (Comptech, USA) and C4d were generated and biotinylated using the NHS-PEG4-Biotin kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions.

[0140] Table 2: Different ligands immobilized using SAP sensor chips with physiological orientation; running buffer was PBS-T (0.005% Tween 20).

[0141]

[0142] Example 3: Binding procedures of different produced proteins

[0143] To evaluate the binding characteristics of different C4BP and other related proteins, a 1:2 (i.e., 1+1) dilution series of each protein was prepared, and injections were performed from the lowest concentration to the highest. The highest concentration of each analyte was injected twice (repeatedly) to demonstrate experimental reproducibility. The chip was washed twice with run buffer before analyte injection to generate a baseline curve. At the end of each injection cycle, residual bound molecules were forcibly eluted by repeated injections of regeneration buffer (1M NaCl). Response units were plotted against analyte concentrations, and affinity was fitted to a 1:1 steady-state affinity model to obtain the given KD values.

[0144] Example 4: Hemolysis assay using the classic route with 5% NHS

[0145] Sheep red blood cells (shRBCs) were washed three times with Dulbecco phosphate-buffered saline (DPBS) to remove stock solution (2500 g, 3 min). A partial cell density of 1.3 was obtained at A405 nm when 10 µl of cells were lysed in 140 µl H2O and the absorbance of 100 µl of the lysate was measured. Those cells were then diluted in PBS++ (1 x PBS + 0.15 mM CaCl2 + 0.5 mM MgCl2, as the final assay concentration).

[0146] Subsequently, shRBCs were sensitized by incubating with rabbit anti-sheep RBC antibody (hemolysin) diluted 1:40 in 5 mM PBSE (Rockland Immunochemicals, Inc., USA). These sensitized cells were incubated at 37°C for 20 min. After the hemolysin incubation time, the cells were washed twice in DPBS and resuspended in PBS++ to the same initial concentration (value determined at A405) and held on ice before use for assay. Human serum from different healthy donors was prepared for assay via a pre-absorption step; this meant incubating the serum with a similar volume of washed (shRBCs) (1:1) on ice for 30 min with intermittent shaking to ensure all antibodies were pre-absorbed from the serum. The incubated serum was then washed twice at 2500 g for 2 min each time. The supernatant (pre-absorbed serum) was transferred and mixed with magnesium and calcium ions at concentrations of 0.5 mM and 0.15 mM, respectively. This is done to ensure the effective function of CP, as calcium ions are important for the function of the C1 complex, while magnesium ions are essential for invertase formation. Test proteins were prepared using DPBS in serial dilutions (1:2).

[0147] After preparing each component for assay (diluted serum, test protein, and shRBCs), all components were mixed in a 96-well plate (Corning Inc., USA) such that each well contained 80 µl of diluted serum (i.e., +PBS + 0.15 mM CaCl2 + 0.5 mM MgCl2; 5% final serum concentration), 10 µl of protein inhibitor solution, and 10 µl of A405-adjusted, sensitized cells. First, the diluted serum was transferred, followed by serially diluted test proteins. Both were thoroughly mixed and allowed to incubate for 30 minutes (on ice) before adding the last added sensitized shRBCs. To terminate the reaction, 50 µl of ice-cold PBS / EDTA (50 mM) was added to each well. Afterward, the plates were centrifuged at 1500g for 3 minutes, and 100µl of supernatant from each well was carefully transferred to a new flat-bottomed 96-well plate (BRANDplates®, 96-well, BRAND GmbH, Germany) for absorbance measurement (405nm). For quality control of each experiment, a series of blank and control groups were prepared. A serum blank was prepared by mixing 80µl of diluted serum with 20µl of DPBS, and a positive control or complete lysis was performed by transferring 90µl of deionized water (osmotic lysis) with 10µl of cells. Another positive control, called the PBS control, was performed by incubating 80µl of the serum mixture with 10µl of PBS and 10µl of sensitized cells without any inhibitors (expected complete lysis). In addition, a PBS / EDTA 10mM control (referred to as the EDTA 10mM control) was prepared by mixing 80 µl of serum mixture with 10 µl of 100 mM EDTA plus 10 µl of sensitized shRBCs. The EDTA was intended to chelate all calcium and magnesium ions, thereby inhibiting cell lysis. A final control was the PBS / EGTA 10mM control (referred to as the EGTA 10mM control), prepared by transferring 80 µl of serum mixture with 10 µl of 100 mM EGTA plus 10 µl of sensitized shRBCs. In all hemolysis assays, each analyte was repeated, and each assay was performed at least three times.

[0148] Example 5: Combination of FH (19 to 20) with C3d (Control Example)

[0149] Factor H (FH) is a regulator of the alternative pathway of the complement system (=AP), and therefore binds to proteins that bind to AP as expected and frequently described in the literature. Figure 1Experimental results of binding affinity measurements by surface plasmon resonance spectroscopy (SPR) are presented. For this purpose, the cleavage product C3d was immobilized, and the recombinant FH fragment (=FH(19 to 20)) consisting of the last two protein domains was injected into the immobilized C3d, and the binding signal was measured. Furthermore, a mutant variant of this protein fragment was observed, in which the aspartic acid at position 1119 of FH was replaced with glycine. This mutation has been described as significantly reducing binding to either C3b or C3d (C3d being a fragment of C3b).

[0150] Example 6: Combination of FH (19 to 20) with C4d

[0151] Repeating Example 5 with C4d (the cleavage product of the classical activation pathway (CP)) instead of C3d as the decoy unexpectedly revealed a concentration-dependent binding signal (see Example 5). Figure 2 ).

[0152] In both cases, the dissociation constant cannot be precisely determined due to the relatively weak interaction. The given affinity values ​​are only approximations, as it is impossible to study sufficiently high concentrations to achieve a precise determination of the affinity.

[0153] FH(19–20) or the mutant FH(19–20)1119G was fused with the regulatory active regions of different regulators of the complement system, and the affinity of the fused constructs or regulatory active regions was re-determined (see [link to original text]). Figure 3 This experiment clearly demonstrates that C4BP (1 to 4) or its two copies (2XC4BP) do not bind to C4d. In contrast, mini-C4BP (= fusion of C4BP (1 to 4) with FH (19 to 20)1119G) binds to C4d in a concentration-dependent manner.

[0154] Example 7: Comparative Example

[0155] Example 6 was repeated using other control proteins, which are also protein segments of complement regulators, and their concentration-dependent binding to C4d is not shown (see [link to example 6]). Figure 4 ).

[0156] Example 8: Combination of FH (19 to 20) with C4b

[0157] FH (19 to 20) also binds to C4b (an activation product of the classical pathway (CP) of complement cascade, which produces C4d upon further degradation), further confirming the binding to C4d. Figure 5The results show that FH(19 to 20)1119G binds unusually weakly to C4b on its own, but the affinity increases from about 2.9 µM for individual C4BP(1 to 4) to 0.6 µM for the combination of C4BP(1 to 4) and FH(19 to 20)1119G (= mini-C4BP), which demonstrates that FH(19 to 20)1119G increases the initial affinity of C4BP(1 to 4) through protein fusion.

[0158] This is a general effect because the fusion of FH (19 to 20) with regulators other than C4BP also increases the regulator's affinity for C4b (see [link to product]). Figure 6 This varies depending on the different modifiers and also on the joint (or its length) in the fusion between the modifier and FH (19 to 20).

[0159] Example 9: Complement Inhibition

[0160] This positive effect can be used to target, for example, mini-C4BP fusion constructs to surfaces that must be protected from CP activation, or surfaces that have recently suffered CP activation damage and are therefore occupied by C4d. In functional cytology assays using erythrocytes, we demonstrated that the protective effect of the CP regulator C4BP (1 to 4) was enhanced by fusion with FH (19 to 20)1119G (see [link to cytology]). Figure 7 In this experiment, C4BP alone (1 to 4) achieved an IC50 of 0.59 µM, while the fusion with FH(19 to 20)1119G (= mini-C4BP) achieved an IC50 of 0.06 µM. These results demonstrate and impressively prove that despite the lack of high affinity of FH(19 to 20)1119G for the CP cleavage product C4d, the cytoprotective effect is enhanced (10-fold).

[0161] References

[0162]

Claims

1. A modulator protein comprising (i) a modulator module that regulates the classical pathway of complement activation and / or the lectin pathway of complement activation, and (ii) a binding module comprising, preferably, a CCP domain 19 and a CCP domain 20 of a factor H; for the prevention and / or treatment of diseases caused and / or aggravated by activation of the classical pathway of complement activation and / or the lectin pathway.

2. The modulator protein for the use of claim 1, wherein the binding modulator comprises the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 70% identity with it.

3. The modulated protein for use according to claim 1 or 2, wherein in the amino acid sequence of the binding modulator, aspartic acid at position 1119 is replaced with glycine.

4. The modulator protein for use according to any one of claims 1 to 3, wherein the modulator module comprises a module of an effector protein of the classical pathway of complement activation and / or the lectin pathway, preferably an invertase decay acceleration module and / or a specific module having cofactor activity of proteolytic cleavage of C4b, or C4b and C3b, via factor I.

5. The modulator protein for use according to any one of claims 1 to 4, wherein the modulator module comprises, preferably, an effector module consisting of a C4b binding protein, a decay-accelerating protein, a membrane cofactor protein, and / or a complement receptor (CR).

6. The modulator protein for use according to any one of claims 1 to 5, wherein the C4b-binding protein is a C4b-binding protein, wherein the decay-accelerating protein is CD55, wherein the membrane cofactor protein is CD46, and / or wherein the CR is CR1.

7. The modulator protein according to any one of claims 1 to 6, wherein the modulator module comprises an amino acid sequence of at least one of SEQ ID NO: 3 and SEQ ID NO: 8 to SEQ ID NO: 12 or a sequence having at least 70% identity with it.

8. The modulator protein for use according to any one of claims 1 to 7, wherein the modulator module comprises the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 70% identity with it.

9. The modulator protein for use according to any one of claims 1 to 8, wherein the modulator protein is a polypeptide.

10. The modulator protein for use according to any one of claims 1 to 9, wherein the modulator protein comprises the amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 7, or a sequence having at least 70% identity with it.

11. The modulated protein according to any one of claims 1 to 10, wherein the disease is a pathogenic microbial infection, atypical hemolytic uremic syndrome, autoimmune hemolytic anemia, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, antiphospholipid syndrome, catastrophic antiphospholipid syndrome, platelet ineffective transfusion, IgA nephropathy, transplant-associated microangiopathy, hematopoietic stem cell transplant-associated microangiopathy, antibody-mediated rejection, ischemia-reperfusion injury, secondary membranous nephropathy, xenotransplantation, acute kidney injury, or Guillain-Barré syndrome.

12. A modulator protein comprising (i) a modulator module that regulates the classical pathway of complement activation and / or the lectin pathway of complement activation, and (ii) a binding module comprising, preferably, a CCP domain 19 and a CCP domain 20 of an H factor.

13. The modulator protein of claim 12, wherein the modulator protein is the modulator protein as defined in any one of claims 1 to 11.

14. A kit comprising a modulator protein according to claim 12 or 13, wherein the modulator protein is contained in a shell.

15. A method for preventing damage to host cells and / or surfaces caused by components of the complement-activated classical pathway and / or lectin pathway, preferably an in vitro method, said method comprising: (A) Contacting the host cell and / or surface with the modulated protein according to claim 12 or 13; and (B) thereby preventing damage to the host cells and / or surfaces.

Images