Complement inhibiting factors and uses thereof

Abstract

The present application relates to multi-module polypeptides comprising (i) an Fc receptor binding module; (ii) a first complement regulatory protein repeat (CCP) module; and (iii) a second CCP module that binds to at least one host cell surface marker, binds to a complement factor C3b, binds to a complement factor C4b, binds to a degradation product of a complement factor C3b, and / or binds to a degradation product of a complement factor C4b; wherein the second CCP module is the C-terminus of the Fc receptor binding module and the first CCP module. The present application also relates to polynucleotides encoding said multi-module polypeptides, to the use of said multi-module polypeptides for the treatment and / or prevention of inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom, and to in vitro methods for preventing or reducing the extent of complement activation.

Description

[0001] This application is a divisional application of the patent application for invention with the application date of June 21, 2019, application number 201980055246.X, and invention title "Complement Inhibitory Factor and Its Use". Technical Field

[0002] The present invention relates to a multimodular polypeptide comprising (i) an Fc receptor binding module; (ii) a first complement control protein repeat (CCP) module; and (iii) a second CCP module that binds to at least one host cell surface marker, binds to complement factor C3b, binds to complement factor C4b, binds to a degradation product of complement factor C3b, and / or binds to a degradation product of complement factor C4b; wherein the second CCP module is the C-terminus of the Fc receptor binding module and the first CCP module. The present invention also relates to a polynucleotide encoding the multimodular polypeptide, and to the use of the multimodular polypeptide for medical treatment, particularly for treating and / or preventing inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom. Moreover, the present invention relates to an in vitro method for preventing or reducing the degree of complement activation, which comprises applying the multimodular polypeptide to a reaction mixture, tissue, and / or organ containing complement factors, thereby preventing or reducing the degree of complement activation in the reaction mixture, tissue, and / or organ. Background Art

[0003] The immune system can be divided into two branches: phylogenetically earlier innate immunity and adaptive immune responses. Immune responses generated by the adaptive or acquired immune system are generally more specific than innate immune responses. Other characteristics of the adaptive immune system are the development of immune memory and the delay usually observed between antigen exposure and the maximum immune response.

[0004] Even in primitive organisms, the innate immune system is highly conserved. The cellular effectors of this branch mainly include neutrophils, monocytes, and macrophages, while the soluble innate immune effectors mainly consist of the complement system and other effectors such as acute phase proteins or pore-forming peptides (Parkin and Cohen (2001) The Lancet 357:1777 - 89). The complement system consists of heat-labile components in serum, which Paul Ehrlich described as being able to "complement" the antibody response against bacteria. Other functions of the complement system are the opsonization of invading microorganisms, immune complexes, debris, apoptotic, and necrotic cells to support efficient clearance by phagocytosis (Ricklin et al. (2010) Nature Immunology 11:785 - 797). The complement system is divided into three activation pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP).

[0005] Activation of the CP is usually achieved in an antibody - dependent manner by the complement component C1q, which acts as a pattern recognition molecule (PRM). After a series of proteolytic activation events, the CP C3 convertase (C4bC2a) cleaves the complement component C3 into C3a, an anaphylatoxin, and C3b (opsonin), and C3 is central to all three complement activation pathways. Due to this cleavage, a conformational change occurs and the previously internal thioester bond protrudes to the protein surface of C3b. This active and short - lived thioester bond once exposed can covalently bind to the hydroxyl and amino groups of molecules located on the cell surface or can be cleaved by water ("quenched"). Thus, without down - regulation of the C3 convertase, cell opsonization can occur through many C3b molecules. The production of a large number of C3b molecules promotes the activation of C5 by the C5 convertase. The C5 convertase cleaves C5 into C5a (the most potent anaphylatoxin) and C5b, which recruits complement factors C6 - 9 to form the membrane attack complex (MAC), which assembles pores in the cell membrane to lyse and kill the cell.

[0006] The lectin pathway (LP) is organized similarly to the CP. Activation occurs by recognition of pathogen - associated molecular patterns (PAMPs) or danger - associated molecular patterns (DAMPs). In the LP, PAMPs or DAMPs can be detected by several pattern recognition molecules homologous to C1q (the pattern recognition molecule of the CP): mannose - binding lectin (MBL) and various types of collagen lectins and ficolins. After PAMP or DAMP binding, the bound MBL undergoes a conformational change and then binds to MBL - associated serine proteases (MASP). MBL is homologous to C1q in structure and function. Similar to the CP, MASP2 proteolytically activates C2 into C2a and C2b and C4 into C4a and C4b. The activated components can build the LP C3 convertase C4bC2a, which is the same as that of the CP, and cleave C3 into C3a and C3b. Similar to the CP, without strict regulation of the C3 convertase, the production of more C3b molecules promotes the activation of C5 by the C5 convertase. The proteolytic activation of C5 is the starting point of the terminal and lytic complement pathway where C5b initiates MAC formation.

[0007] The alternative pathway is activated by a low - level self - activation process. This process is called the "slow" activation of C3. C3 molecules have an inherent metastable conformation. At any time, a small fraction of C3 molecules undergo spontaneous conformational changes (activation), thus exposing the previously internal thioester module. The thioester can be quenched by water or can attach indiscriminately (to self or foreign) to nucleophiles on the cell surface. This "auto - activated" C3 is called C3(H 2 O), which has a structure similar to the C3b molecule. C3b or C3(H2 O) exposes new protein surfaces hidden in C3. These new surfaces bind factor B, another complement factor that acts as an AP. When factor B binds to C3b or C3(H 2 O), it can be cleaved by protease D into Ba and Bb. Bb remains bound to C3(H 2 O) (or C3b) and forms the C3 convertase of the AP, C3bBb. Similar to the CP and LP C3 convertases, C3bBb can generate C3b and C3a molecules by cleaving C3. The protein properdin is a positive regulator of the AP and plays an important role by stabilizing the protein–protein interactions of the AP C3 convertase. Without regulation, any C3b generated by the alternative pathway, classical pathway or lectin pathway can build more AP C3 convertases and also increase the number of C3b molecules generated in the positive feedback loop. This step is called the “amplification loop” of the AP. Thus, the three activated pathways converge at the level of C3 activation and, without regulation, accumulate in the formation of the MAC.

[0008] The classical pathway and lectin pathway are inactivated until they are specifically activated by sensing pathogens or endogenous danger molecules. In contrast, the AP is always activated at a low level and generates C3b (or the initial C3(H 2 O)) molecules indiscriminately. More than ten different regulatory proteins are known within the complement system. Some regulatory proteins inhibit at the level of initiation of the CP and LP, but the most tightly controlled part of the cascade is the convertase (which acts as an amplifier of the activation signal), and C3b forms the platform for the formation of C3 convertases and inflammatory C5 convertases. There are also some regulatory proteins that specifically control the lytic MAC.

[0009] Regulatory proteins can be divided into decay-accelerating factors, which destabilize C3 convertases and cause faster degradation of the convertases. Another group involves proteins that degrade C3b or / and C4b, such as factor H and factor I; for the inactivation of C3b or C4b, the presence of a cofactor protein that binds to the target and recruits factor I (such as FH or CR1) is required to prevent non-specific degradation of the soluble protease factor I. Another group of regulatory proteins inhibits the formation of the MAC.

[0010] Many diseases, particularly genetic diseases, are associated with abnormal complement function, in particular complement overactivation. Thus, to provide artificial regulatory proteins of the complement system, the monoclonal antibody eculizumab that specifically binds to complement protein C5 and inhibits terminal activation was developed (Hillmen et al. (2006), NEJM 355(12):1233). In a similar development route, the C5 inhibitory protein rEV576 (coversin) was developed (Romay-Penabad et al. (2014), Lupus 23(12):1324). In addition, a protein called "mini-FH" was obtained that links the complement control protein repeats (CCP domains) 1 to 4 and 19 to 20 of complement factor H via a linker (WO 2013 / 142362 A1). In several in vitro and ex vivo assays, miniFH had a complement regulatory activity that was enhanced tenfold in terms of regulatory activity against the alternative complement pathway and was superior to FH.

[0011] Nevertheless, there is still a need in the art for improved complement inhibitory factors to avoid the disadvantages of the prior art. This problem is solved by the devices and methods disclosed herein. Summary of the Invention

[0012] Accordingly, the present invention relates to a multimodular polypeptide comprising (i) an Fc receptor binding module; (ii) a first complement control protein repeat (CCP) module; and (iii) a second CCP module that binds to at least one host cell surface marker, binds to complement factor C3b, binds to complement factor C4b, binds to a degradation product of complement factor C3b, and / or binds to a degradation product of complement factor C4b; wherein the second CCP module is the C-terminus of the Fc receptor binding module and the first CCP module.

[0013] As used hereinafter, the terms "having", "comprising", or "including" are used in a non-exclusive manner. Thus, these terms can refer both to the case where there are no other features in the entity described herein other than the features introduced by these terms, and to the case where there is one or more other features. For example, the expressions "A has B", "A comprises B", and "A includes B" can all refer to the case where there are no other elements in A other than B (i.e., the case where A consists solely and exclusively of B), and to the case where there is one or more other elements in entity A other than B, such as element C, elements C and D, or even other elements.

[0014] In addition, as used hereinafter, the terms "preferred", "more preferred", "most preferred", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features without restricting further possibilities. Thus, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. Those skilled in the art to which the present invention pertains will recognize that the present invention can be practiced by using the optional features. Similarly, features introduced by "in an embodiment of the present invention" or similar expressions are intended to be optional features without imposing any limitation on other embodiments of the present invention, without imposing any limitation on the scope of the present invention, and without imposing any limitation on the possibility of combining the features introduced in this way with other optional or non-optional features of the present invention.

[0015] Unless otherwise stated, as used herein, the term "standard conditions" refers to IUPAC standard ambient temperature and pressure (SATP) conditions, namely a temperature of preferably 25 °C and an absolute pressure of 100 kPa. It is also preferred that the standard conditions include a pH of 7. In addition, unless otherwise stated, the term "about" refers to the value indicated by the generally accepted technical precision in the relevant field, preferably to the indicated value ±20%, more preferably ±10%, most preferably ±5%. In addition, the term "substantially" means that there is no deviation that affects the indicated result or use, i.e., the potential deviation does not cause the indicated result to deviate by more than ±20%, more preferably ±10%, most preferably ±5%. Thus, "substantially consisting of" or "consisting essentially of" means including the specified components, but not including other components except for materials present as impurities, inevitable materials due to the processes used to provide the components, and components added for purposes other than the technical purpose of the present invention. For example, a composition defined using the phrases "substantially consisting of" or "consisting essentially of" encompasses any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition substantially consisting of a group of components contains less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of non-specified components. In the context of nucleic acid sequences, the term "substantially identical" means having an % identity value of at least 80%, preferably at least 90%, more preferably at least 98%, most preferably at least 99%. It will be understood that the term substantially identical includes 100% identity. The term "substantially complementary" applies mutatis mutandis to the foregoing.

[0016] As used herein, the term "Fc receptor" refers to a receptor that has an affinity for the Fc portion of an antibody, preferably IgG, on the cell surface or in endosomes; thus, preferably, the Fc receptor is the neonatal Fc receptor (FcRn) (or Fc-gamma receptor), and more preferably, the Fc receptor is selected from CD64, CD32, CD16a, and CD16b. Preferably, the Fc receptor is a mammalian Fc receptor, and more preferably a human Fc receptor. Thus, as used herein, the term "Fc receptor binding module" refers to a module of a multimodular polypeptide that has an affinity for an Fc receptor as described above, preferably a polypeptide domain. Preferably, the dissociation constant K D is at most 10 -6 M, more preferably at most 10 -7 M, and even more preferably at most 10 -8 M. Preferably, the Fc receptor is FcRn as described herein, and the dissociation constant of the FcRn / Fc receptor binding module complex at pH 6 is less than 10 -6 M, preferably less than 10 -7 M. Also preferably, the Fc receptor is FcRn as described above herein, and the dissociation constant of the FcRn / Fc receptor binding module complex at pH 7 is at least 10 -6 M, preferably at least 10 -5 M. Thus, preferably, the Fc receptor is FcRn as described above herein, and the dissociation constant of the FcRn / Fc receptor binding module complex at pH 6 is less than 10 -6 M, preferably less than 10 -7 M, and at least 10 -6 M at pH 7, preferably at least 10 -5M. Also preferably, compared to pH 6, the dissociation constant of the FcRn / Fc receptor binding module complex is at least 5-fold, more preferably at least 10-fold, and even more preferably at least 20-fold at pH 7. Preferably, the Fc receptor binding module is the Fc module of an immunoglobulin (Ig), more preferably the Fc module of IgG, and even more preferably the Fc module of IgG1. In a preferred embodiment, the Fc receptor binding module contains at most one cysteine residue that forms a disulfide bond with a second molecule of the Fc receptor binding module, and in a more preferred embodiment, the Fc receptor binding module does not contain cysteine residues that form disulfide bonds. Thus, in a preferred embodiment, the multimodule polypeptide forms a non-covalent homodimer. Most preferably, the Fc receptor binding module contains a peptide having the amino acid sequence SEQ ID NO: 3 or a sequence having at least 70% identity thereto, preferably containing a peptide having the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 12. In a preferred embodiment, the Fc receptor binding module contains albumin, preferably the Fc-receptor binding sub-sequence of human albumin (Genbank accession number AAA98797.1) or mouse albumin (Genbank accession number AAH49971.1).

[0017] In principle, the term "complement control protein repeat domain" is known in the art and is also referred to herein as "complement control protein repeat", "CCP domain" or "CCP", and is also referred to in the art as "short complement-like repeat", "short consensus repeat" or "SCR". The CCP domain is reviewed, for example, in Schmidt et al. (2008), Clin Exp Immunol. 151(1):14-24. The CCP domain is a peptide sequence containing approximately 60 to 70 amino acids, including a conserved tryptophan and four conserved cysteine residues that form two disulfide bonds, and the sequence of the remaining amino acids varies widely. In addition to binding to complement proteins C3b and / or C4b, the CCP domain has been found to mediate other activities, including the decay-accelerating activity and factor I cofactor activity described below.

[0018] As used herein, the term "first CCP module" refers to a module of a multimodular polypeptide that comprises at least one CCP domain and is (i) an enzymatic decay module for the convertases of the classical pathway and / or alternative pathway of complement activation; and / or is (ii) a binding module for complement factor C3b and / or C4b, preferably also having factor I cofactor activity. Thus, preferably, the first CCP module comprises at least one CCP domain having decay accelerating activity against the C3 convertase of the alternative pathway and / or classical pathway of complement activation. The term "decay accelerating activity" as used herein refers to the property of a CCP domain or CCP module to mediate the decay, preferably inactivation, of the C3 convertase of the alternative pathway of complement activation, i.e., C3bBb, and / or the C3 convertase of the classical pathway of complement activation, i.e., C4bC2a. Preferably, the decay accelerating activity of the CCP domain or CCP module is determined by surface plasmon resonance (SPR). Preferably, the first CCP module comprises two to ten, more preferably two to five, still more preferably three to four CCP domains having or contributing to the aforementioned activity. Preferably, the first CCP module comprises CCP domains 1 to 4 of factor H, preferably CCP domains 1 to 4 of human factor H; comprises CCP domains 1 to 3 of complement receptor type 1 (CR1), preferably CCP domains 1 to 3 of human CR1, as described below; comprises CCP domains 1 to 4 of decay accelerating factor (DAF), preferably CCP domains 1 to 4 of human DAF, as described below, and / or comprises CCP domains 1 to 3 of C4 binding protein (C4BP), preferably CCP domains 1 to 3 of human C4BP. Preferably, the module comprising the first CCP comprises CCP domains 1 to 3 of complement receptor type 1 (CR1) as in the naturally occurring sequence; and / or comprises CCP domains 1 to 4 of decay accelerating factor (DAF) as in the naturally occurring sequence. Preferably, the first CCP module comprises CCP domains 1 to 4 of human factor H. More preferably, the first CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity to the amino acid sequence shown in SEQ ID NO:1, and has the activity of an enzymatic decay module for the convertases of the classical pathway and / or alternative pathway of complement activation. More preferably, the first CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO:1.

[0019] Also preferably, the first CCP module comprises at least one, preferably at least two, more preferably at least three CCP domains having binding activity for complement factor C3b and / or C4b, and preferably also has factor I cofactor activity. As will be understood by those skilled in the art, one or more CCP domains having binding activity for complement factor C3b and / or C4b may be CCP domains different from the CCP domains having decay-accelerating activity as described above. Preferably, at least one, more preferably at least two, still more preferably at least three CCP domains having binding activity for complement factor C3b and / or C4b are also CCP domains having decay-accelerating activity. More preferably, the CCP having binding activity for complement factor C3b and / or C4b is the CCP having decay-accelerating activity as described above. The term "having binding activity for complement factor C3b and / or C4b" is understood by those skilled in the art. Preferably, this term relates to the property that the first CCP module and / or at least one of its CCP domains binds at least one of the complement proteins C3b and C4b with a measurable affinity, and the measurable affinity is more preferably K D of at most 5 x 10 -5 M, more preferably at most 1 x 10 -5 M, even more preferably at most 10 -6 M. Preferably, the binding affinity of the CCP or CCP module for C3b or C4b is determined by surface plasmon resonance (SPR). Preferably, at least one CCP having binding activity for complement factor C3b and / or C4b is selected from CCP domains 1 to 4 of factor H, CR1, preferably CCP domains 8 to 10 and 15 to 17 of human CR1, and C4BP, preferably CCP domains 1 to 3 of human C4BP. Thus, preferably, the first CCP module comprises or further comprises CR1, preferably CCP domains 8 to 10 and / or 15 to 17 of human CR1, as described elsewhere herein. More preferably, the module comprising the first CCP comprises or further comprises CR1, preferably CCP domains 15 to 17 of human CR1.

[0020] In principle, the term "complement receptor type 1" or "CR1" is known to those skilled in the art as a member of the regulatory proteins of the complement activation (RCA) family of proteins, also known as the C3b / C4b receptor or cluster of differentiation 35 protein (CD35). Preferably, CR1 is mammalian CR1, more preferably, CR1 is human CR1. Most preferably, CR1 is human CR1 having the amino acid sequence shown in Genbank accession number P17927.3 GI:290457678.

[0021] In principle, the term "decay-accelerating factor" or "DAF" is also known to those skilled in the art, referring to a cell surface-bound regulatory protein of the complement system, which is also called cluster of differentiation 55 protein (CD55). Preferably, DAF is mammalian DAF, more preferably human DAF. Most preferably, DAF is human DAF having the amino acid sequence shown in Genbank accession number P08174.4 GI:60416353.

[0022] The term "factor H" is also recognized by those skilled in the art as a cofactor for inactivating C3b by factor I and is also used to increase the dissociation rate of C3bBb complex (C3 convertase) and C3bBb3b complex (C5 convertase) in the alternative complement pathway. Preferably, factor H is mammalian factor H, more preferably human factor H. Most preferably, factor H is human factor H having the amino acid sequence shown in Genbank accession number NP_000177.2.

[0023] The term "C4BP" is also known to those skilled in the art as a control molecule of the classical pathway of complement activation, which binds as a cofactor to C3b / C4b for proteolytic inactivation of complement protein C4b by serum protease factor I. C4BP also increases the dissociation rate of C4b2a complex (C3 convertase) and C4b2a3b complex (C5 convertase) in the classical complement pathway. Preferably, C4BP is mammalian C4BP, more preferably human C4BP. Most preferably, C4BP is human C4BP having the amino acid sequence shown in Genbank accession number NP_000706.1.

[0024] As used herein, the term "second CCP module" refers to a module of a multimodular polypeptide that has activity of binding to at least one host cell surface marker, to complement factor C3b, to complement factor C4b, to degradation products of complement factor C3b, and / or to degradation products of complement factor C4b. Preferably, the second CCP module has activity of binding to polyanionic carbohydrates containing sialic acid and / or glycosaminoglycan, and / or activity of binding to complement factor C3b or C4b and / or their degradation products, preferably activity of binding to iC3b, C3dg, C3d, iC4b, C4dg, and / or C4d. Preferably, the second CCP module has activity of binding to at least one host cell surface marker. More preferably, the second CCP module has activity of binding to at least one host cell surface marker and at least C3b. Preferably, the second CCP module does not have detectable convertase decay activity and / or does not have detectable factor I cofactor activity. Thus, more preferably, the second CCP module only has binding activity to at least one of the above-mentioned molecules, and most preferably, it does not have complement regulatory activity. Preferably, the second CCP module comprises at least one, preferably at least two CCP domains, which have activity of binding to a host cell surface marker, preferably polyanionic carbohydrates containing sialic acid and / or glycosaminoglycan, and / or activity of binding to degradation products of complement factor C3b, preferably iC3b, C3dg, C3d, iC4b, C4dg, and / or C4d. Preferably, the second CCP module comprises the complement factor H as described above, preferably CCP domains 6 to 8 and / or 19 to 20 of human complement factor H, or any CCP domains 1 to 8 of the α-chain of C4BP. More preferably, the second CCP module comprises the complement factor H as described above, preferably CCP domains 6 to 8 and / or 19 to 20 of human complement factor H. Even more preferably, the second CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity to SEQ ID NO: 2, preferably having activity of binding to a host cell surface marker, preferably polyanionic carbohydrates containing sialic acid and / or glycosaminoglycan, and / or activity of binding to degradation products of complement factor C3b, preferably binding to iC3b and / or C3dg. More preferably, the second CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 2. Preferably, surface plasmon resonance (SPR) is used to determine the activity of binding to a host cell surface marker and the activity of binding to degradation products of complement factor C3b of the CCP or host cell recognition module.

[0025] As used herein, the term "multimodular polypeptide" refers to any chemical molecule that contains at least the polypeptide modules described below. It should be understood that the chemical bonds between the modules do not necessarily have to be peptide bonds. The present invention also contemplates that the chemical bonds between the modules are ester bonds, disulfide bonds or any other suitable covalent chemical bonds known to those skilled in the art. Non-covalent bonds are also contemplated, which have such a low dissociation constant that the degree of dissociation of a module from other modules is only negligible. Preferably, the dissociation constant of the non-covalent bond is less than 10 -5 M (as in the case of Strep-Tag:Strep-Tactin binding), less than 10 -6 M (as in the case of Strep-TagII:Strep-Tactin binding), less than 10 -8 M, less than 10 -10 M or less than 10- 12 M (as in the case of streptavidin:biotin binding). Methods for determining the dissociation constant are well known to those skilled in the art and include, for example, spectroscopic titration, surface plasmon resonance measurements, equilibrium dialysis, etc. In addition, it is also contemplated that the binding between the modules of the multimodular polypeptide is indirect, for example, the modules contain a tag that has an affinity for biotin and binds to another molecule or particle that contains a biotin moiety. Preferably, the chemical bond between the modules is a peptide bond, i.e., preferably, the multimodular polypeptide is a fusion polypeptide that contains or consists of the modules of the present invention. In a preferred embodiment, the polypeptide consists of the components described herein.

[0026] Preferably, mention is made of polypeptides, especially multimodular polypeptides, and / or modules, especially CCP modules, including variants of the specific polypeptides and modules described herein. As used herein, the terms "polypeptide variant" and "module variant" refer to any chemical molecule that contains at least one or more modules as specified herein, but has a structure different from that of the polypeptide or module. Preferably, the polypeptide variant or module variant contains a peptide having an amino acid sequence corresponding to an amino acid sequence of 25 to 500 amino acids, more preferably 30 to 300 amino acids, most preferably 35 to 150 consecutive amino acids included in the polypeptides or modules described herein. In addition, it should be understood that the polypeptide variant or module variant according to the present invention should have an amino acid sequence that is different due to at least one amino acid substitution, deletion, and / or addition, wherein the amino acid sequence of the variant is still preferably at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identical to the amino acid sequence of the specific polypeptide or module. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences in a comparison window, wherein the fragment of the amino acid sequence in the comparison window can include additions or deletions (e.g., gaps or overhangs) compared to the sequence being compared to obtain the best alignment. By preferably determining the number of positions at which identical amino acid residues occur in the two sequences over the full length of the peptide to produce the 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, the percentage of sequence identity is obtained. The optimal alignment of the comparison sequences can be carried out by the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the method of similarity search of Pearson and Lipman (1988), by the computerized implementation of these algorithms (GAP, BESTFIT, BLAST, PASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by visual inspection. Assuming that the two sequences to be compared have been determined, it is preferred to use GAP and BESTFIT to determine their optimal alignment, thereby determining the degree of identity. Preferably, the default value of 5.00 is used for the gap weight and the default value of 0.30 is used for the gap weight length. The polypeptide variants or module variants mentioned above can be derived from allelic variants or any other species-specific homologs, paralogs or orthologs. In addition, the polypeptide variants referred to herein include fragments of the specific polypeptides or variants of the above type, provided that these fragments and / or variants contain the above modules.Such fragments can be derived from or be derivatives of, for example, degradation products or splice variants of polypeptides. Also included are variants that differ due to post-translational modifications such as phosphorylation, glycosylation, ubiquitination, SUMOylation or myristoylation, by including non-natural amino acids, and / or by being peptidomimetics.

[0027] Preferably, at least two modules of the multimodular polypeptide are linked by a "linker" peptide. Suitable linker peptides are in principle known in the art. Preferred linker peptides comprise or preferably consist of glycine, alanine and / or proline residues. More preferably, the linker peptide is a polyglycine linker peptide. Most preferably, the linker peptide, in particular the linker peptide connecting the first CCP module and the second CCP module as described elsewhere herein, is a linker consisting of, preferably consisting of 14 or 15 glycine residues. Other preferred linkers are linkers consisting of 5 or 8 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 a G residue.

[0028] As used herein, the term "module comprising an amino acid sequence having at least 70% identity to X" relates to a module comprising a variant of said module having an amino acid sequence having at least 70% identity to said module. Preferably, the module comprising an amino acid sequence having at least 70% identity to X is a variant of X having the activity of X, more preferably having the activity as described herein.

[0029] Thus, preferably, the multimodular polypeptides and variants thereof of the present invention have activity as a complement activation inhibitor, i.e., have the activity of inhibiting the complement reaction, preferably in vitro and / or in vivo. Preferably, the multimodular polypeptides and variants thereof have the activity of inhibiting at least two, more preferably all three, activation pathways of the complement system. More preferably, the multimodular polypeptides and variants thereof have the activity of inhibiting at least the alternative pathway and the classical pathway of complement activation, preferably having the activity of inhibiting at least the alternative pathway, the classical pathway and the lectin pathway of complement activation.

[0030] Preferably, the multimodular polypeptide comprises at least two of its modules, preferably all three of its modules as a contiguous polypeptide sequence, i.e., the multimodular polypeptide is preferably a fusion polypeptide comprising said three modules. Preferably, in principle, as long as the second CCP module is at the C-terminus of the Fc receptor-binding module and the first CCP module, the three modules can be included in such a fusion polypeptide in any order considered appropriate by those skilled in the art. Preferably, the modules comprised by the multimodular polypeptide are, in N-terminal to C-terminal order, an Fc receptor-binding module, a first CCP module, and a second CCP module. It should be understood that the multimodular polypeptide may preferably comprise other domains and structural elements in addition to the domains and structural elements referred to herein. More preferably, the multimodular polypeptide consists of the elements mentioned herein. However, preferably, one or more CCP domains having activity of binding to at least one host cell surface marker, to complement factor C3b, to complement factor C4b, to degradation products of complement factor C3b, and / or to degradation products of complement factor C4b are at the C-terminus of the other CCP domains and the Fc receptor-binding module, and more preferably, the CCP domain having activity of binding to at least one host cell surface marker, to complement factor C3b, to complement factor C4b, to degradation products of complement factor C3b, and / or to degradation products of complement factor C4b is the C-terminal element of the multimodular polypeptide. Even more preferably, the multimodular polypeptide comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10, or is a variant thereof as described above, wherein, preferably, the variant still has the activity of a complement activation inhibitor as described above. Most preferably, the multimodular polypeptide comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10.

[0031] Advantageously, in the work underlying the present invention, it has been found that it is important to construct a multimodular polypeptide comprising an Fc receptor-binding module and a cell-binding CCP domain such that the cell-binding CCP domain is located at or near the C-terminus of the resulting molecule. Unexpectedly, such a position has no significant effect on the plasma half-life of the multimodular polypeptide, but has a significant effect on the effective concentration / dose.

[0032] The definitions described above apply to the further modifications described below. The additional definitions and explanations described further below also apply to the necessary modifications of all embodiments described in this specification.

[0033] The present invention also relates to polynucleotides encoding the multimodular polypeptides of the present invention.

[0034] The term "polynucleotide" as used according to the present invention relates to a polynucleotide comprising a nucleic acid sequence encoding a multimodular polypeptide comprising the modules as specified above. According to the present invention, polynucleotides encoding multimodular polypeptides comprising the above-mentioned modules have been obtained by synthesizing polynucleotides encoding the relevant modules using well-known techniques.

[0035] Thus, the polynucleotide preferably comprises the nucleic acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, which encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10. It should be understood that due to the degenerate genetic code, polypeptides having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10 can also be encoded by other polynucleotides.

[0036] In addition, the term "polynucleotide" as used according to the present invention also includes variants of the above-mentioned specific polynucleotides. Polynucleotide variants preferably comprise a nucleic acid sequence characterized in that the sequence can be derived from the above-mentioned specific nucleic acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11 by at least one nucleotide substitution, addition and / or deletion, wherein the variant nucleic acid sequence should still encode a polypeptide comprising the above activities. Variants include the following polynucleotides: the polynucleotides comprise nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with at least one of the nucleic acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11. In addition, the following polynucleotides are also included: the polynucleotides comprise nucleic acid sequences encoding amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with the amino acid sequences shown in SEQ ID NO: 8 or SEQ ID NO: 10. Preferably, the percentage identity values are calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on various algorithms are available to those skilled in the art to compare different sequences. In this case, the Needleman and Wunsch algorithm or the Smith and Waterman algorithm give particularly reliable results. For sequence alignment, the PileUp program (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the Gap and Best Fit programs (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))), which are part of the GCG software package (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)) are used. The sequence identity values expressed as percentages (%) listed above are preferably determined using the GAP program over the entire sequence region with the following settings: gap weight: 50, length weight: 3, average match: 10.000 and average mismatch: 0.000, which should always be used as the standard settings for sequence alignment unless otherwise stated. Variants also cover polynucleotides comprising nucleic acid sequences that preferably are capable of hybridizing with the above-mentioned specific nucleic acid sequences under stringent hybridization conditions.These stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 - 6.3.6. A preferred example of stringent hybridization conditions is hybridization in 6× sodium chloride / sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2× SSC, 0.1% SDS at 50°C to 65°C. Those skilled in the art know that these hybridization conditions vary depending on the type of nucleic acid and, for example, the temperature and concentration of the buffer when organic solvents are present. For example, under "standard hybridization conditions", in an aqueous buffer with a concentration of 0.1 to 5× SSC (pH 7.2), the temperature varies from 42°C to 58°C depending on the type of nucleic acid. If an organic solvent such as 50% formamide is present in the above buffer, the temperature under standard conditions is approximately 42°C. The hybridization conditions for DNA:DNA hybrids are preferably, for example, 0.1× SSC and 20°C to 45°C, preferably 30°C to 45°C. The hybridization conditions for DNA:RNA hybrids are preferably, for example, 0.1× SSC and 30°C to 55°C, preferably 45°C to 55°C. For example, in the absence of formamide, for a nucleic acid with a length of approximately 100 bp (= base pairs) and a G + C content of 50%, the above hybridization temperature is determined. Those skilled in the art know how to determine the required hybridization conditions by referring to textbooks such as the above textbook or the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants can be obtained by PCR-based techniques, such as oligonucleotide primer-based DNA amplification based on mixtures, i.e., using degenerate primers directed against conserved motifs of the polypeptides of the invention. The conserved motifs of the polypeptides of the invention can be determined by comparing the nucleic acid sequence of the polynucleotides of the invention or the amino acid sequence of the polypeptides with the sequences of other CCP domains. As a template, DNA or cDNA from animals, preferably mammals, more preferably humans can be used.

[0037] Polynucleotides comprising fragments of any of the above nucleic acid sequences are also included in the polynucleotides of the present invention. The fragment should encode a polypeptide comprising the above-specified modules and preferably still have the activity specified above. Thus, the polypeptide may comprise or consist of the modules of the present invention that confer the said biological activity. The fragments described herein preferably comprise at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of the above nucleic acid sequence or an amino acid sequence encoding at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids comprising the above amino acid sequence.

[0038] The polynucleotides of the present invention consist of or comprise the above nucleic acid sequences. Thus, they may also comprise other nucleic acid sequences. Specifically, the polynucleotides of the present invention may encode a fusion protein, wherein one partner of the fusion protein is a multimodular polypeptide encoded by the above nucleic acid sequence. Such a fusion protein may comprise, as an additional part, other polypeptides for monitoring expression (e.g., green, yellow, blue or red fluorescent proteins, alkaline phosphatase, etc.) or so-called "tags", which may be used as detectable markers or aids for purification purposes. Tags for different purposes are well known in the art and include FLAG tags, 6-histidine tags, MYC tags, etc.

[0039] The polynucleotides of the present invention should preferably be provided in isolated form (i.e., isolated from their natural environment) or in a genetically modified form. The polynucleotide is preferably DNA, including cDNA, or RNA. The term encompasses single-stranded and double-stranded polynucleotides. In addition, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides.

[0040] Thus, preferably, the polynucleotides of the present invention a) are polynucleotides having at least 70% sequence identity with SEQ ID NO: 9, b) encode polypeptides having at least 70% sequence identity with SEQ ID NO: 8, and / or c) are polynucleotides capable of hybridizing with SEQ ID NO: 9 under stringent conditions. More preferably, the polynucleotide a) is a polynucleotide comprising, preferably consisting of, the nucleic acid sequence SEQ ID NO: 9 or SEQ ID NO: 11, and / or b) encodes a polypeptide comprising, preferably consisting of, the amino acid sequence SEQ ID NO: 8 or SEQ ID NO: 10. Preferably, the polynucleotides of the present invention encode multimodular polypeptides having the activity as described above.

[0041] The present invention also relates to vectors comprising the polynucleotides of the present invention.

[0042] The term "vector" preferably encompasses phage, plasmid, viral or retroviral vectors and artificial chromosomes, such as bacterial or yeast artificial chromosomes. In addition, the term also relates to targeting constructs which allow random or site-directed integration of the targeting construct into genomic DNA. Such target constructs preferably contain DNA of sufficient length for homologous or heterologous recombination, as described in detail below. Vectors containing the polynucleotides of the invention preferably also contain a selectable marker for propagation and / or selection in a host. The vector can be incorporated into the host cell by a variety of techniques well known in the art. For example, plasmid vectors can be introduced into precipitates, such as calcium phosphate precipitates or rubidium chloride precipitates, or into complexes with charged lipids, or into carbon-based clusters, such as fullerenes. Alternatively, plasmid vectors can be introduced by heat shock or electroporation techniques. If the vector is viral, it can be packaged in vitro using an appropriate packaging cell line prior to application to the host cell. Retroviral vectors can be replication-competent or replication-defective. In the latter case, viral propagation generally only occurs in complementary host / cells.

[0043] More preferably, in the vector of the present invention, the polynucleotide is operably linked to an expression control sequence, thereby allowing expression in prokaryotic and / or eukaryotic cells or their isolated fractions. Expression of the polynucleotide includes transcription of the polynucleotide, preferably into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They preferably comprise regulatory sequences ensuring transcription initiation, and optionally a poly-A signal ensuring transcription termination and transcript stability. Other regulatory elements may include transcriptional enhancers and translational enhancers. Regulatory elements that may be expressed in prokaryotic host cells include, for example, the lac, trp or tac promoters in Escherichia coli, while examples of regulatory elements allowing expression in eukaryotic host cells are the AOX1 or GAL1 promoters in yeast or the CMV-promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells. In addition, inducible expression control sequences may be used in the expression vectors covered by the present invention. Such inducible vectors may contain tet or lac operator sequences or sequences that can be induced by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to the elements responsible for transcription initiation, such regulatory elements may also contain a transcription termination signal downstream of the polynucleotide, such as the SV40-poly-A site or the tk-poly-A site. In this regard, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (InVitrogene) or pSPORT1 (GIBCO BRL). Preferably, the vector is an expression vector and a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses or bovine papillomaviruses can be used to deliver the polynucleotide or vector of the present invention to a target cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).

[0044] Preferably, the vector is a vector that mediates the expression of the polynucleotide of the present invention in a host cell. Those skilled in the art know how to select a combination of a vector and a host cell for the propagation of the vector and / or for the expression of a protein encoded by the vector.

[0045] Furthermore, the present invention relates to a host cell comprising the polynucleotide or vector of the present invention.

[0046] As used herein, "host cell" refers to a bacterial, archaeal or eukaryotic cell that has the ability to propagate the vector of the present invention and / or produce a multimodular polypeptide encoded on the vector or the polynucleotide of the present invention. Preferably, the host cell is a bacterial cell from the Escherichia coli species, a lepidopteran insect, a mouse, a rat or a human cell; preferably, the host cell is Escherichia coli. More preferably, the cell is a yeast cell, preferably a yeast cell of the genus Pichia, more preferably a Pichia pastoris cell. Preferably, the host cell is a cell cultured in vitro. In another preferred embodiment, the host cell is an in vivo cell, preferably a retinal pigment epithelial cell, an endothelial cell within the choroidal vasculature and / or another cell within the retina or choroid.

[0047] The present invention also relates to the multimodular polypeptide according to the present invention, the polynucleotide according to the present invention, the vector according to the present invention and / or the host cell according to the present invention for medical use. Furthermore, the present invention also relates to the multimodular polypeptide according to the present invention, the polynucleotide according to the present invention, the vector according to the present invention and / or the host cell according to the present invention for the treatment and / or prevention of inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom.

[0048] As used herein, the term "inappropriate complement activation" refers to complement activation that exceeds the normal level of complement activation in terms of time and / or magnitude in a given situation. Thus, preferably, inappropriate complement activation is, in a given situation, complement activation that exceeds, preferably significantly exceeds, the complement activation level of a healthy reference, preferably an apparently healthy subject. Preferably, inappropriate complement activation is complement activation that causes symptoms of the patient's disease. Symptoms of inappropriate complement activation are known in the art and include hemolysis, macular degeneration, episodic swelling, such as hereditary angioedema, etc. Preferably, inappropriate complement activation is determined by determining the activity of complement factor C3 and / or C4 in a sample.

[0049] As is known to those skilled in the art, various diseases are associated with and / or caused by inappropriate complement activation. Thus, preferably, the present invention also relates to a multimodular polypeptide according to the present invention, a polynucleotide according to the present invention, or a vector according to the present invention for treating and / or preventing diseases with inappropriate complement activation as a symptom. In a preferred embodiment, the diseases with inappropriate complement activation as a symptom are selected from the diseases disclosed in Ricklin et al. (2017), Mol Immunol. 89:10-21. Preferably, the diseases with inappropriate complement activation as a symptom are selected from ischemic reperfusion injury, antibody-mediated graft rejection, post-transplant thrombotic microangiopathy, autoimmune hemolytic anemia, acute and delayed hemolytic transfusion reactions, cold agglutinin disease, rheumatoid arthritis, aquaporin-4-antibody-positive neuromyelitis optica, CD59 deficiency, C3 glomerulopathy, atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, and age-related macular degeneration.

[0050] As used herein, the term "treatment" refers to alleviating the disease or disorder or symptoms associated therewith mentioned herein, preferably to a great extent. The treatment as used herein also includes the overall health restoration with respect to the disease or disorder mentioned herein. It should be understood that the treatment used according to the present invention may not be effective in all subjects to be treated. However, the term should preferably require the ability to successfully treat a statistically significant proportion of the subjects suffering from the disease or disorder mentioned herein. Those skilled in the art can readily use various well-known statistical assessment tools to determine whether the proportion is statistically significant, such as determination of confidence intervals, p-value determination, 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%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment is effective for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population.

[0051] As used herein, the term "prophylaxis" refers to maintaining health in an object for a period of time with respect to the diseases or disorders mentioned herein. It should be understood that the period of time depends on the amount of the pharmaceutical compound administered and the individual factors of the object discussed elsewhere in this specification. It should also be understood that prophylaxis may not be effective in all objects treated with the compounds of the present invention. However, the term preferably requires effectively preventing a statistically significant portion of a cohort or population of objects from suffering from the diseases or disorders or their concomitant symptoms described herein. Preferably, in such cases, a cohort or population of objects is envisioned which would, typically, i.e., in the absence of prophylactic measures according to the present invention, develop the diseases or disorders referred to herein. Those skilled in the art can use various well-known statistical assessment tools discussed elsewhere in this specification to determine whether a portion is statistically significant.

[0052] The present invention also relates to a multimodular polypeptide according to the present invention, a polynucleotide according to the present invention, or a vector according to the present invention, which is combined with a complement protein C5 inhibitory polypeptide, preferably eculizumab, for treating and / or preventing inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom.

[0053] The present invention also relates to a combination of a complement protein C5 inhibitory polypeptide, preferably eculizumab or rEV576 (coversin), with a multimodular polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and / or a host cell according to the present invention, for treating and / or preventing inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom.

[0054] The term "complement protein C5" is understood by those skilled in the art to relate to the protein that is cleaved after activation of the complement pathway to produce complement protein C5a and C5b. Accordingly, a "complement protein C5 inhibitory polypeptide" is a polypeptide that specifically recognizes and inhibits complement protein C5, preferably an antibody, more preferably a monoclonal antibody. Preferably, the complement protein C5 inhibitory polypeptide is an antibody that specifically binds to C5 and inhibits terminal activation. More preferably, the complement protein C5 inhibitory polypeptide is eculizumab (CAS No.: 219685-50-4). Still more preferably, the complement protein C5 inhibitory polypeptide is rEV576 (coversin).

[0055] The present invention also relates to a combined preparation for simultaneous, separate or sequential use, which comprises (i) a multimodular polypeptide according to the present invention and (ii) a complement protein C5 inhibitory polypeptide, preferably eculizumab or rEV576 (coversin).

[0056] The term "combination preparation" as referred to in the present application relates to a preparation comprising the pharmaceutically active compound of the present invention in one preparation. Preferably, the combination preparation is contained in a container, i.e., preferably, the container contains all the pharmaceutically active compounds of the present invention. Preferably, the container contains the pharmaceutically active compounds of the present invention as separate preparations, i.e., preferably, one preparation of the multimodular polypeptide and one preparation of the complement protein C5 inhibitory polypeptide. As will be understood by those skilled in the art, the term "preparation" relates to a mixture of preferably pharmaceutically acceptable compounds which comprises or consists of at least one pharmaceutically active compound of the present invention. Preferably, the combination preparation comprises the complement protein C5 inhibitory polypeptide and the multimodular polypeptide in a single solid pharmaceutical form, such as a tablet, wherein, more preferably, one compound of the present invention is comprised in an immediate-release or rapid-release preparation, while the second compound of the present invention is comprised in a sustained-release or delayed-release preparation; more preferably, the compounds of the present invention are comprised in two separate, preferably liquid, preparations; the separate liquid preparations are preferably for injection, preferably into different parts of the body of the subject.

[0057] Preferably, the combination preparation is for administration alone or in combination. As used herein, "administration alone" relates to an administration in which at least two pharmaceutically active compounds of the present invention are administered by different routes and / or to different parts of the subject. For example, one compound can be administered by enteral administration (e.g., orally), while the second compound is administered by parenteral administration (e.g., intravenously). Preferably, the combination preparation for administration alone comprises at least two physically separate preparations for administration alone, each of which comprises at least one pharmaceutically active compound; this alternative is preferred, for example, in cases where the pharmaceutically active compounds of the combination preparation must be administered by different routes, such as parenterally and orally, due to their chemical or physiological properties. In contrast, "administration in combination" relates to an administration in which the pharmaceutically active compounds of the present invention are administered by the same route, such as orally or preferably intravenously.

[0058] Also preferably, the combination preparation is for simultaneous or sequential administration. As used herein, "simultaneous administration" relates to an administration in which the pharmaceutically active compounds of the present invention are administered simultaneously, i.e., preferably, the administration of the pharmaceutically active compounds starts within a time interval of less than 15 minutes, more preferably within a time interval of less than 5 minutes. Most preferably, the administration of the pharmaceutically active compounds starts simultaneously, for example, by swallowing a tablet containing the pharmaceutically active compounds, or swallowing a tablet containing one of the pharmaceutically active compounds and injecting the second compound simultaneously, or by intravenous injection of a solution containing one pharmaceutically active compound and injecting the second compound in a different manner to different parts of the body. In contrast, as used herein, "sequential administration" relates to an administration that enables the plasma concentration of the pharmaceutically active compounds in the subject to achieve the synergistic effect of the present invention, but preferably, it is not the simultaneous administration as described above. Preferably, sequential administration is an administration in which the pharmaceutically active compounds, preferably all pharmaceutically active compounds, start within a time interval of 1 day or 2 days, more preferably within a time interval of 12 hours, still more preferably within a time interval of 4 hours, even more preferably within a time interval of 1 hour, and most preferably within a time interval of 5 minutes.

[0059] Preferably, the combination preparation is a pharmaceutically compatible combination preparation. As used herein, the terms "pharmaceutically compatible preparation" and "pharmaceutical composition" relate to a composition comprising the compounds of the present invention and optionally one or more pharmaceutically acceptable carriers. The compounds of the present invention can be formulated into pharmaceutically acceptable salts. Preferred acceptable salts are acetate, methyl ester salt, HCl salt, sulfate, chloride, etc. The pharmaceutical composition is preferably administered topically, or more preferably systemically. Suitable routes of administration commonly used for drug administration are oral, intravenous, subcutaneous or parenteral administration and inhalation. However, depending on the nature and mode of action of the compound, the pharmaceutical composition can also be administered by other routes. In addition, the compound can be administered in combination with other drugs in the form of a common pharmaceutical composition or in the form of a separate pharmaceutical composition as specified elsewhere herein, where the separate pharmaceutical composition can be provided in the form of a kit set. Preferably, the combination preparation is a sustained-release preparation for one or more compounds.

[0060] The compound is preferably administered in a conventional dosage form, which is prepared by combining the drug with a standard pharmaceutical carrier according to a conventional method. These procedures can include mixing the ingredients, granulating, compressing or dissolving to suit the required preparation. It should be understood that the form and properties of the pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient combined therewith, the route of administration and other well-known variables.

[0061] Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and harmless to the subject. The pharmaceutical carriers used can be, for example, solids, gels or liquids. Examples of solid carriers are lactose, gypsum powder, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, etc. Exemplary liquid carriers are phosphate buffered saline solutions, syrups, oils such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions, etc. Similarly, the carrier or diluent can include retardation materials well known in the art, such as glyceryl monostearate or glyceryl distearate alone or together with waxes. The suitable carriers include those mentioned above and other carriers well known in the art, see, for example, Remington′s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0062] A diluent is selected that does not affect the biological activity of one or more compounds. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution and Hank's solution. Additionally, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, reactive oxygen scavengers, etc.

[0063] A therapeutically effective dose refers to the amount of the compound in the pharmaceutical composition of the present invention that is used to prevent, ameliorate or treat the symptoms associated with the diseases or conditions mentioned in this specification. The therapeutic efficacy and toxicity of such compounds can be determined in cell culture or experimental animals by standard pharmaceutical methods, such as ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, and can be expressed as the ratio of LD50 / ED50.

[0064] The dosing regimen will be determined by the attending physician and other clinical factors; preferably according to any of the methods described above. As is well known in the medical art, the dosage for any given patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic evaluation. A typical dosage can be, for example, from 1 mg to 1500 mg; however, dosages below 1 mg or above 1500 mg can be envisioned, particularly in view of the above factors. Generally, a conventional dosing regimen as a pharmaceutical composition should be in the range of 100 μg to 100 mg units per day. If the regimen is by continuous infusion, it should also be in the range of 100 μg to 100 mg units per kilogram of body weight per minute. Preferably, a sustained-release formulation of each drug is injected at intervals from once every week to once every two months or even longer. Progress can be monitored by periodic evaluation. The preferred dosages and concentrations of the compounds of the present invention are specified elsewhere herein.

[0065] For example, the plasma concentration of the multimodular polypeptide is preferably not less than 25 nM, more preferably not less than 50 nM. Also preferably, the plasma concentration of the multimodular polypeptide is from 20 nM to 20 μM, more preferably from 50 nM to 5 μM. The effective concentration of the complement protein C5 inhibitory polypeptide, particularly eculizumab, is known in the art. Due to the synergistic effect of the multimodular polypeptides of the present invention, the effective concentration of the complement protein C5 inhibitory polypeptide in combination therapy may be lower.

[0066] The pharmaceutical compositions and formulations referred to herein are preferably administered at least once, for example, in the case of a sustained-release formulation, for the treatment or amelioration or prevention of the diseases or conditions described in this specification. However, the pharmaceutical compositions can be administered multiple times, for example, from once a day to four times a day, for an unlimited number of days. Similarly, some compounds with short clearance times can also be used by infusion to provide an effective dose systemically over a longer treatment period.

[0067] The specific pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art and contain at least one of the active compounds mentioned above herein, which is mixed or otherwise associated with a pharmaceutically acceptable carrier or diluent. To prepare those specific pharmaceutical compositions, one or more active compounds are generally mixed with a carrier or diluent, or enclosed or encapsulated in a capsule, sachet, cachet, paper, or other suitable container or carrier. The resulting formulation should be suitable for the mode of administration, i.e., in the form of tablets, capsules, suppositories, solutions, suspensions, etc. Dosage recommendations should be indicated in the prescriber's or user's instructions in order to adjust the dosage according to the recipient considered.

[0068] The present invention also relates to a medicament which comprises (i) a multimodular polypeptide, (ii) a complement protein C5 inhibitory polypeptide, and (iii) at least one pharmaceutically acceptable carrier; and to said medicament for use in the above-mentioned treatment and / or prophylaxis.

[0069] The term "medicament" is understood by the person skilled in the art. It should be understood that the definition of the term "combination preparation" given above herein preferably applies, mutatis mutandis, to the term medicament of the present invention.

[0070] Furthermore, the present invention relates to a method for treating and / or preventing inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom in a subject, the method comprising administering an effective dose of the multimodular polypeptide of the present invention, the polynucleotide according to the present invention, the vector according to the present invention and / or the host cell according to the present invention, so as to treat and / or prevent inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom in the subject.

[0071] The treatment and / or prophylaxis method of the present invention is preferably an in vivo method. Furthermore, in addition to those explicitly mentioned above, a plurality of other steps may be included. For example, the other steps may involve, for example, diagnosing inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom, or administering additional compounds, such as a complement protein C5 inhibitory polypeptide. Furthermore, one or more than one of said steps may be performed by an automated device.

[0072] As used herein, the term "subject" relates to an animal having a complement system, preferably to a mammal. More preferably, the subject is a cow, pig, sheep, horse, cat, dog, mouse or rat, and most preferably a human.

[0073] Furthermore, the present invention relates to an in vitro method for preventing or reducing the degree of complement activation, which comprises applying the multimodular polypeptide according to the present invention, the polynucleotide according to the present invention, the vector according to the present invention and / or the host cell according to the present invention to a reaction mixture, tissue and / or organ containing complement factors, so as to prevent or reduce the degree of complement activation in said reaction mixture, tissue and / or organ.

[0074] The in vitro method of the present invention for preventing or reducing the degree of complement activation may include steps in addition to those explicitly mentioned above. For example, the other steps may involve, for example, introducing the polynucleotide or vector of the present invention into a host cell, or determining the degree of complement activation in said reaction mixture, tissue or organ. Furthermore, one or more than one of said steps may be performed by an automated device. Preferably, the method is performed on a reaction mixture in vitro.

[0075] Furthermore, the present invention relates to the use of a multimodular polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and / or a host cell according to the present invention in the treatment and / or prevention of inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom; and to the use of a multimodular polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, and / or a host cell according to the present invention in the preparation of a medicament for the treatment and / or prevention of inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom.

[0076] In view of the above, the following embodiments are preferred:

[0077] 1. A multimodular polypeptide comprising

[0078] (i) an Fc receptor binding module;

[0079] (ii) a first complement control protein repeat (CCP) module; and

[0080] (iii) a second CCP module that binds to at least one host cell surface marker, binds to complement factor C3b, binds to complement factor C4b, binds to a degradation product of complement factor C3b, and / or binds to a degradation product of complement factor C4b;

[0081] wherein the second CCP module is the C-terminus of the Fc receptor binding module and the first CCP module.

[0082] 2. The multimodular polypeptide of embodiment 1, wherein the modules comprised by the multimodular polypeptide are, in the N-terminus to C-terminus order, an Fc receptor binding module, a first CCP module, and a second CCP module.

[0083] 3. The multimodular polypeptide of embodiment 1 or 2, wherein the first CCP module and / or the second CCP module comprises a plurality of CCP domains.

[0084] 4. The multimodular polypeptide of any one of embodiments 1 to 3, wherein the first and / or second CCP module is composed of 2 to 10, preferably 2 to 5, more preferably 3 to 4 CCP domains.

[0085] 5. The multimodular polypeptide of any one of embodiments 1 to 4, wherein the first CCP module comprises CCP domains 1 to 4 of factor H, preferably CCP domains 1 to 4 of human factor H; comprises CCP domains 1 to 3 of complement receptor type 1 (CR1), preferably CCP domains 1 to 3 of human CR1; comprises CCP domains 1 to 4 of decay-accelerating factor (DAF), preferably CCP domains 1 to 4 of human DAF, and / or comprises CCP domains 1 to 3 of C4-binding protein (C4BP), preferably CCP domains 1 to 3 of human C4BP.

[0086] 6. The multimodular polypeptide of any one of embodiments 1 to 5, wherein the first CCP module (i) is a convertase decay module for the convertases of the classical pathway and / or alternative pathway of complement activation, and / or (ii) is a binding module for complement factor C3b and / or C4b, and the first CCP module preferably also has factor I cofactor activity.

[0087] 7. The multimodular polypeptide of any one of embodiments 1 to 6, wherein the first CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 70% identity thereto.

[0088] 8. The multimodular polypeptide of any one of embodiments 1 to 7, wherein the first CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 1.

[0089] 9. The multimodular polypeptide of any one of embodiments 1 to 8, wherein the second CCP module binds to polyanionic carbohydrates including sialic acid, glycosaminoglycan and / or complement factor C3b or its degradation products.

[0090] 10. The multimodular polypeptide of any one of embodiments 1 to 9, wherein the second CCP module comprises CCP domains 19 to 20 of factor H, preferably CCP domains 19 to 20 of human factor H.

[0091] 11. The multimodular polypeptide of any one of embodiments 1 to 10, wherein the second CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 70% identity thereto.

[0092] 12. The multimodular polypeptide of any one of embodiments 1 to 11, wherein the second CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 2.

[0093] 13. The multimodular polypeptide of any one of embodiments 1 to 12, wherein the Fc receptor binding module is the Fc module of an immunoglobulin (Ig).

[0094] 14. The multimodular polypeptide of any one of embodiments 1 to 13, wherein the Fc receptor binding module is an Fc module of IgG, preferably IgG1.

[0095] 15. The multimodular polypeptide of any one of embodiments 1 to 14, wherein the Fc receptor binding module comprises a peptide having the amino acid sequence SEQ ID NO: 3 or a sequence having at least 70% identity therewith, preferably comprises a peptide having the amino acid sequence SEQ ID NO: 3.

[0096] 16. The multimodular polypeptide of any one of embodiments 1 to 15, wherein the multimodular polypeptide comprises at least two of said modules as a continuous polypeptide sequence, preferably comprises all three of said modules as a continuous polypeptide sequence.

[0097] 17. The multimodular polypeptide of any one of embodiments 1 to 16, wherein the first CCP module and / or the second CCP module comprises CCP domains 1 to 4 of factor H, preferably CCP domains 1 to 4 of human factor H.

[0098] 18. The multimodular polypeptide of any one of embodiments 1 to 17, wherein the first CCP module and the second CCP module are together as mini-FH, and the mini-FH comprises the amino acid sequence of one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 or a sequence having at least 70% identity with at least one of said sequences, preferably comprises the amino acid sequence of one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0099] 19. The multimodular polypeptide of any one of embodiments 1 to 18, wherein at least two of said modules are connected by a linker peptide, preferably a poly-AG or poly-G linker peptide.

[0100] 20. The multimodular polypeptide of any one of embodiments 1 to 19, wherein the first CCP module and the second CCP module are connected by a linker, and the linker comprises, preferably consists of 1 to 15, preferably 1, 5, 8, 14 or 15 glycine residues.

[0101] 21. The multimodular polypeptide of any one of embodiments 1 to 20, wherein the multimodular polypeptide comprises, preferably consists of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 70% identity therewith.

[0102] 22. The multimodular polypeptide of any one of embodiments 1 to 21, wherein the multimodular polypeptide comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 8.

[0103] 23. The multimodular polypeptide of any one of embodiments 1 to 22, wherein the first CCP module comprises at least one, preferably at least two, more preferably at least three CCPs having binding activity to complement factor C3b and / or C4b and having factor I cofactor activity.

[0104] 24. The multimodular polypeptide of embodiment 23, wherein the CCP having binding activity to complement factor C3b and / or C4b has factor I cofactor activity.

[0105] 25. The multimodular polypeptide of any one of embodiments 1 to 24, wherein the multimodular polypeptide has the activity of inhibiting the complement reaction.

[0106] 26. The multimodular polypeptide of any one of embodiments 1 to 25, wherein the multimodular polypeptide has the activity of inhibiting at least one, preferably two, more preferably all three activation pathways of the complement system.

[0107] 27. The multimodular polypeptide of any one of embodiments 1 to 26, wherein the multimodular polypeptide has the activity of inhibiting at least the alternative pathway and the classical pathway of complement activation, preferably has the activity of inhibiting at least the alternative pathway, the classical pathway and the lectin pathway of complement activation.

[0108] 28. A polynucleotide encoding the multimodular polypeptide of any one of embodiments 1 to 27.

[0109] 29. The polynucleotide of embodiment 28, wherein the polynucleotide

[0110] a) is a polynucleotide having at least 70% sequence identity with SEQ ID NO: 9,

[0111] b) encodes a polypeptide having at least 70% sequence identity with SEQ ID NO: 8, and / or

[0112] c) is a polynucleotide capable of hybridizing with SEQ ID NO: 9 under stringent conditions.

[0113] 30. The polynucleotide of embodiment 28 or 29, wherein the polynucleotide

[0114] a) is a polynucleotide comprising, preferably consisting of, the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11, and / or

[0115] b) encoding a polypeptide comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10.

[0116] 31. A vector comprising the polynucleotide of any one of embodiments 28 to 30.

[0117] 32. A host cell comprising the polynucleotide of any one of embodiments 28 to 30 and / or the vector of embodiment 31.

[0118] 33. The multimodular polypeptide according to any one of embodiments 1 to 27, the polynucleotide according to any one of embodiments 28 to 30, the vector according to embodiment 31 and / or the host cell according to embodiment 32, for medical use.

[0119] 34. The multimodular polypeptide according to any one of embodiments 1 to 27, the polynucleotide according to any one of embodiments 28 to 30, the vector according to embodiment 31 and / or the host cell according to embodiment 32, for the treatment and / or prevention of inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom.

[0120] 35. The multimodular polypeptide according to any one of embodiments 1 to 27, the polynucleotide according to any one of embodiments 28 to 30, the vector according to embodiment 31 or the host cell according to embodiment 32, for the treatment and / or prevention of ischemia-reperfusion injury, antibody-mediated graft rejection, post-transplant thrombotic microangiopathy, autoimmune hemolytic anemia, acute and delayed hemolytic transfusion reactions, cold agglutinin disease, rheumatoid arthritis, aquaporin-4-antibody-positive neuromyelitis optica, CD59 deficiency, C3 glomerulopathy, atypical or typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and age-related macular degeneration.

[0121] 36. The multimodular polypeptide according to any one of embodiments 1 to 27, the polynucleotide according to any one of embodiments 28 to 30, the vector according to embodiment 31 or the host cell according to embodiment 32, in combination with a complement protein C5 inhibitory polypeptide, preferably eculizumab or rEV576 (coversin), for the treatment and / or prevention of inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom.

[0122] 37. A complement protein C5 inhibitory polypeptide, preferably eculizumab, in combination with the multimodular polypeptide according to any one of embodiments 1 to 27, the polynucleotide according to any one of embodiments 28 to 30, the vector according to embodiment 31 or the host cell according to embodiment 32, for the treatment and / or prevention of inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom.

[0123] 38. A combination preparation for simultaneous, separate or sequential use, comprising (i) a multimodular polypeptide according to any one of embodiments 1 to 27, a polynucleotide according to any one of embodiments 28 to 30, a vector according to embodiment 31 or a host cell according to embodiment 32 and (ii) a complement protein C5 inhibitory polypeptide, preferably eculizumab or rEV576 (coversin).

[0124] 39. A method for treating and / or preventing inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom in a subject, comprising administering an effective dose of a multimodular polypeptide according to any one of embodiments 1 to 27, a polynucleotide according to any one of embodiments 28 to 30, a vector according to embodiment 31 and / or a host cell according to embodiment 32, thereby treating and / or preventing inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom in the subject.

[0125] 40. An in vitro method for preventing or reducing the degree of complement activation, comprising applying a multimodular polypeptide according to any one of embodiments 1 to 28 to a reaction mixture, tissue and / or organ containing complement factors, thereby preventing or reducing the degree of complement activation in the reaction mixture, tissue and / or organ.

[0126] 41. Use of a multimodular polypeptide according to any one of embodiments 1 to 27, a polynucleotide according to any one of embodiments 28 to 30, a vector according to embodiment 31 and / or a host cell according to embodiment 32 for treating and / or preventing inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom.

[0127] 42. Use of a multimodular polypeptide according to any one of embodiments 1 to 27, a polynucleotide according to any one of embodiments 28 to 30, a vector according to embodiment 31 and / or a host cell according to embodiment 32 in the preparation of a medicament for treating and / or preventing inappropriate complement activation and / or a disease having inappropriate complement activation as a symptom.

[0128] 43. A multimodular polypeptide according to any one of embodiments 1 to 27, wherein the Fc receptor-binding module comprises at most one cysteine residue that forms a disulfide bridge with a second molecule of the Fc receptor-binding module, preferably does not comprise a cysteine residue that forms a disulfide bridge.

[0129] 44. A multimodular polypeptide according to any one of embodiments 1 to 27 or 43, wherein the multimodular polypeptide forms a non-covalent homodimer.

[0130] All references cited in this specification are incorporated by reference in their entireties, and the disclosures specifically mentioned in this specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] In Figure 1 Figure 1A , Figure 1B and Figure 1C : Natural, engineered, and Fc-fused constructs. (A) Schematic representation of the native complement regulator factor H (FH) and the previously engineered FH variant miniFH domain. Amino acid numbering is based on the FH sequence (UniProt accession number: P08603) encoding the signal sequence. Each oval represents a CCP domain (labeled with the domain number). The native N-terminal and C-terminal residues are represented by a single letter code; non-native linker sequences are boxed. Key functional attributes of the CCP domains are highlighted in the upper panel. (B) Schematic representation of the domain structure of an IgG molecule and three miniFH Fc-fusion molecules. Note that only the inter-polypeptide chain disulfide bonds (S-S bridges) are depicted and highlighted with arrows. Inter-chain disulfide bridges are present between the constant domains of the IgG heavy and light chains, and between the heavy chains of IgG, in miniFH-Fc and Fc-miniFH. Intra-chain disulfide bonds, i.e., disulfide bonds within the polypeptide chain, are not shown or highlighted. (C) SDS-PAGE gel analysis of three different Fc-fusion variants of miniFH and miniFH. 2 μg of each protein was loaded onto a Novex NuPAGE 4-12% Bis-Tris SDS-PAGE gel under reducing and non-reducing conditions and visualized by Coomassie staining.

[0132] Figure 2 : Determination of the plasma half-life of miniFH and three Fc-fusion variants in mice. Mean plasma circulating levels of miniFH or three miniFH Fc-fusion variants. Plasma levels of the protein (0.1 mg) in plasma prepared from blood collected at the indicated times after injection were measured by ELISA. Mean ± SD; miniFH n = 3, Fc-miniFH-short n = 5, all others n = 4.

[0133] Figure 3 : C3b-binding activity of miniFH and Fc-fusion variants of miniFH. Surface plasmon resonance (SPR) sensorgrams of C3b binding for miniFH (A), miniFH-Fc (C), Fc-miniFH (E), and Fc-miniFH-short (G) (measured over the indicated concentration range; 4030 RU of C3b was deposited by amine). (B) Individual concentration-response plots showing the fitted affinity (1:1 steady-state affinity fit) of miniFH (B), miniFH-Fc (D), Fc-miniFH (F), and Fc-miniFH-short (H) for C3b binding. The affinity equilibrium constants are shown in the figure.

[0134] Figure 4: To protect rabbit red blood cells from lysis mediated by the alternative complement pathway. Rabbit red blood cells were incubated in serum (from healthy donors) for 30 minutes, which had been mixed with one of the indicated complement inhibitory factors. The final serum concentration in the assay was 25%. Lysis of rabbit red blood cells was measured by the release of hemoglobin and normalized to the lysis observed in water (mean and standard deviation of 3 independent assays are shown).

[0135] Figure 5 : Decay-accelerating factor (DAF or CD55) construct fused to Fc. Schematic diagram of the domain structure of an IgG molecule and two DAF Fc fusion molecules.

[0136] Figure 6 : Bb binding activity of Fc-DAF and DAF-Fc fusion variants. SPR sensorgrams of Bb binding for Fc-DAF (A) and DAF-Fc (B).

[0137] Figure 7 : C3b binding activity of Fc-DAF and DAF-Fc fusion variants. C3b binding of Fc-DAF (A) and DAF-Fc (B) was measured; (C) SPR sensorgram of C3b binding for Fc-DAF and DAF-Fc (single injection, each at a concentration of 1.0 μM).

[0138] Figure 8 : To protect rabbit red blood cells from lysis mediated by the alternative complement pathway. Rabbit red blood cells were incubated in serum (from a pool of healthy donors) for 30 minutes, which had been mixed with one of the indicated complement inhibitory factors. The final serum concentration in the assay was 25%. Lysis of rabbit red blood cells was measured by hemoglobin release and normalized to the lysis observed in water. Detailed Description of the Invention

[0139] The following examples are only for illustrative purposes of the present invention. In any case, they should not be construed as limiting the scope of the present invention.

[0140] Example 1: Recombinant Proteins and FH

[0141] The naturally occurring (full-length) plasma protein factor H (FH) was purified from plasma and purchased from the commercial source CompTech (Tyler, USA). The production and purification processes of the recombinant proteins miniFH, and three different Fc fusions of miniFH (SEQ ID NO: 4), miniFH-Fc (SEQ ID NO: 13), Fc-miniFH (SEQ ID NO: 10), and Fc-miniFH-short (SEQ ID NO: 8) were as previously described (Schmidt et al. (2013), J. Immunol. 190(11):5712 - 5721). To construct DNA constructs for different Fc:miniFH fusion proteins, codon-optimized (for the host Pichia pastoris) DNA encoding miniFH and codon-optimized (for the host Pichia pastoris) DNA encoding the Fc portion were cloned into the Pichia pastoris expression vector pPICZαB (Invitrogen). The codon-optimized DNA was obtained from GeneArt. The DNA encoding the Fc portion and miniFH was digested with specifically introduced restriction enzyme sites (PstI, XmaI, and XbaI) and ligated into the pPICZαB Pichia pastoris expression vector in a manner that produced the desired orientation (an overview of the constructs is shown in Figure 1). The expression cassette was transformed into the Pichia pastoris strains KM71H or GS115 (Invitrogen) according to the manufacturer's instructions and expressed in Pichia pastoris using a fermentor following a described procedure (with minor variations) (Schmidt et al. (2011), Protein Expr. Purif.; 76(2):254 - 263). The proteins were purified by successive cation exchange and / or anion exchange chromatography steps, followed by size exclusion chromatography. The native amino acid sequences of all recombinant constructs were expected to start with the non-native sequences EAEAAG (SEQ ID NO: 14), EAAG (SEQ ID NO: 15), or AG (SEQ ID NO: 16: Ala Gly), where the EA sequence is a residue of yeast secretion signal peptide processing (Cereghino et al. (2000), FEMS Microbiol. Rev. 24(1):45 - 66), and AG is a cloned artificial amino acid.

[0142] Example 3: Alternative pathway rabbit erythrocyte hemolysis assay

[0143] Assays were performed as previously published (Schmidt et al. (2016), Immunobiology. 221(4):503-511), with minor variations. Briefly, 20 μl of complement inhibitor in PBS was mixed with 10 μl of NHS (CompTech) containing Mg-EGTA (final assay concentration 5 mM). 10 μl of suspended rabbit red blood cells (rRBC) was added and the mixture was incubated at 37 °C for 30 minutes. To terminate the reaction, 120 μl of ice-cold PBS / EDTA (5 mM) was added. rRBC lysis was quantified by measuring the A405 of 100 μl of supernatant.

[0144] Example 4: Monitoring the binding of complement Fc fusion variants to C3b by surface plasmon resonance (SPR)

[0145] Surface plasmon resonance experiments were performed on a Reichert SR7500DC SPR instrument at 25 °C with a flow rate of 25 μl / min in PBS containing 0.005% Tween 20. 4030 response units (RU) of C3b were immobilized on a carboxymethyl dextran (CMD) 500 sensor chip (Xantec), and binding to amine-coupled C3b was assayed. Analytes were injected at the specified concentrations. To probe binding, a series of concentrations of miniFH or miniFH Fc fusions (for 2.5 min at 25 μl / min) were injected, followed by 300 s of buffer flow and a regeneration step that included injection of 1 M NaCl for 30 s. The highest concentration of each series was injected twice to assess reproducibility. Where appropriate, affinity constants were extracted by plotting the response at steady state against molar concentration and then fitting the affinity using a 1:1 steady-state affinity model with TRACEDRAWER software. Sensorgrams with reference subtracted were always shown.

[0146] Example 5: Determination of mouse plasma half-life

[0147] A solution of 0.1 mg of analyte protein in sterile PBS was injected intravenously into BALB / c mice. If necessary, the protein analyte under study should be cleared of endotoxin prior to administration, such that the dose contains no more than 5 EU endotoxin / kg body weight. Typically, blood was taken from the tail at 1 h, 2 h, 4 h, 8 h, 24 h, 30 h, 48 h, 54 h, and 72 h after injection of the protein analyte and mixed with an equal volume of PBS containing 5 mM EDTA to terminate the clotting reaction. Plasma was prepared by spinning the EDTA-blood mixture at 2000 g to 3000 g for 3 minutes. Plasma was snap-frozen in liquid nitrogen and stored at -80 °C before being used in a sandwich ELISA to determine the level of analyte protein in mouse plasma at each time point.

[0148] To determine miniFH or one of the Fc fusion forms of miniFH in mouse plasma, microtiter plates (MaxiSorp; Nunc) were coated with 2 μg / ml capture antibody (anti-human complement factor H (clone: C18 / 3), stock solution: 1 mg / ml) in 50 μl / well in PBS at room temperature for 2 h, or overnight at 4°C. After washing twice with 200 μl / well PBST (PBS + 0.05% Tween), the wells were blocked with 200 μl / well of 1% BSA in PBS solution (=BSA-PBS) at room temperature for 1 h. Then, the wells were exposed to dilutions of mouse plasma samples or standard curve samples of the corresponding analyte, which were prepared by mixing the analyte at a set concentration with untreated mouse plasma. The samples were incubated in the wells at room temperature for 30 minutes and then washed 3 times with PBST (200 μl). Then, 50 μl of goat anti-human factor H polyclonal antibody (unconjugated, stock solution: 1.0 mg / ml, cat# A237, CompTech) was added in a 1:1000 PBS-BSA (PBS containing 1% BSA) solution and incubated at room temperature for 30 min. Then the wells were washed 3 times with PBST (200 μl). Then, 50 μl of donkey anti-goat IgG HRP (santa cruz biotechnology) diluted 1:1000 in PBS-BSA was added and incubated at room temperature for 30 minutes. After washing 3 times with PBST (200 μl), the plates were developed by adding a mixture of 50 μl of freshly prepared 10 ml of 0.1 M citric acid buffer at pH 4.3, 5 mg of ABTS (Roche), and 10 μl of 30% H 2 O 2 The absorbance was read at 405 nm.

[0149] Example 6: Production of different Fc fusion versions of miniFH

[0150] To increase the plasma half-life of miniFH, the polypeptide of miniFH was fused to the Fc portion of an IgG antibody. Three different ways of linking miniFH to the Fc portion were achieved ( Figure 1B)。First, the C-terminus of miniFH was fused to the N-terminus of the Fc portion, i.e., miniFH-Fc. In the second construct, the N-terminus of miniFH was fused to the C-terminus of the Fc portion, i.e., Fc-miniFH. And in the third construct, the N-terminus of miniFH was also fused to the C-terminus of the Fc portion, but this time the Fc portion consisted of a shorter N-terminal sequence and thus lacked the disulfide dimerization domain of the heavy chain of IgG, i.e., Fc-miniFH-short. However, due to the very high-affinity, non-covalent interaction between the two identical CH3 domains, this construct remained a stable dimer under physiological conditions (e.g., as could be established by size exclusion chromatography). This is consistent with published reports (Ridgway et al. (1996), Protein Eng. 9(7):617-621; McAuley et al. (2008), Protein Sci. Publ. Protein Soc. 17(1):95-106). All three Fc fusion variants of miniFH could be successfully recombinantly produced and purified to high purity in the host Pichia pastoris ( Figure 1C )。

[0151] Example 6: Fusion of miniFH to the N-terminus or C-terminus of the Fc portion resulted in a similar prolongation of plasma half-life

[0152] To evaluate how the Fc fusions affected plasma half-life, approximately 0.1 mg of miniFH or one of the three Fc fusion variants of miniFH was injected intravenously into mice, and the plasma half-life was determined by collecting plasma samples at several time points and analyzing the amount of protein present in these samples. Figure 2 The β-phase plasma half-life of miniFH was shown to be approximately 2.5 h, while the β-phase plasma half-life (T(1 / 2)) of any of the Fc fusion variants of miniFH was significantly increased by approximately 20 h (miniFH-Fc T(1 / 2) = 23.1 h; Fc-miniFH T(1 / 2) = 21.1 h; Fc-miniFH-short (T(1 / 2) = 16.5 h). It could be determined that there was no substantial difference in plasma half-life among the three different Fc fusion forms of miniFH.

[0153] Example 7: Fusion of miniFH to the C-terminus of the Fc portion resulted in a higher affinity for the complement activation product C3b

[0154] Since any Fc fusion variant introduces dimerization and thus has an affinity for the binding partner of miniFH, all Fc fusion variants are expected to bind better to the main target of miniFH (i.e., C3b) than miniFH itself. Since the driving factor for the expected higher affinity between miniFH and its target C3b is the introduction of affinity through dimerization, it is not expected that one Fc fusion strategy will result in better affinity than another Fc fusion strategy. However, Figure 3 showed that in surface plasmon resonance (SPR) experiments, two Fc-miniFH fusion variants (Fc-miniFH and Fc-miniFH-short) bound better to C3b than variants with different Fc orientations. Thus, the binding of Fc-miniFH and Fc-miniFH-short to the main complement target C3b was three times that of miniFH-Fc to the main complement target C3b. For miniFH used as a reference substance, the same affinity as previously measured was determined, thus cross-validating the SPR assay method (Harder et al. 2016, J. Immunol. Baltim. Md 1950196(2):866-876).

[0155] Example 8: The fusion of miniFH to the C-terminus of the Fc portion also resulted in higher complement regulatory activity in human serum, thus enhancing cytoprotection

[0156] It was then investigated whether the higher affinity for the main complement target C3b also resulted in enhanced complement regulatory activity. This was investigated in a hemolysis assay in which rabbit red blood cells were lysed via the alternative pathway of complement when human serum was mixed with rabbit red blood cells. MiniFH-Fc, Fc-miniFH, and Fc-miniFH-short were tested together with miniFH in this assay. Figure 4 showed that the higher affinity of Fc-miniFH and Fc-miniFH-short for C3b compared to miniFH-Fc did result in higher complement regulatory activity. Moreover, due to the avidity caused by dimerization, the affinity of miniFH-Fc for C3b was slightly higher (about two-fold) than that of miniFH. However, this slight increase did not result in a higher regulatory power over the alternative pathway activity of complement compared to miniFH. The activities of miniFH and miniFH-Fc in regulating the complement cascade were very similar. In contrast, compared to miniFH (or miniFH-Fc), the Fc fusion in the Fc-miniFH orientation resulted in a complement regulatory activity that was approximately four times higher. Thus, the atypical fusion of the N-terminus of miniFH to the C-terminus of the Fc portion not only resulted in a higher affinity for the main complement target C3b but also a significantly increased overall complement regulatory activity in human serum.

[0157] Example 9: To test the Bb binding activity of the Fc-DAF and DAF-Fc fusion variants ( Figure 5 ), each variant was analyzed twice by SPR at the same concentration of 0.5 μM to demonstrate reproducibility. 2600 RU of Bb was deposited onto a carboxymethyl dextran sensor chip by standard amine coupling. The running buffer was PBS supplemented with 1 mM MgCl 2 and 0.005% Tween 20. Figure 6 Only the sensorgrams minus reference are shown. Similarly, to test the C3b binding activity of the Fc-DAF and DAF-Fc fusion variants, each variant was assayed twice by SPR at the same concentration of 0.1 μM to demonstrate reproducibility. 5610 RU of C3b was deposited on a carboxymethyl dextran sensor chip by standard amine coupling. The running buffer was PBS supplemented with 1 mM MgCl 2 and 0.005% Tween 20. Figure 7 Only the sensorgrams minus reference are shown. In addition, as Figure 8 shown, the protection of rabbit red blood cells from complement alternative pathway-mediated lysis was tested.

[0158] Although there was little difference in the binding of the two fusion forms to Bb ( Figure 6 ), Fc-DAF bound substantially to C3b, while DAF-Fc failed to bind to C3b ( Figure 7 ), even though the two proteins were assayed at the same concentration in the same experiment. To further investigate whether this difference in binding to the isolated components also results in an overall regulatory difference in the serum complement cascade, a standard complement activation assay was used in which rabbit red blood cells were lysed by human serum complement-dependently, and the addition of complement inhibitory factors prevented this lysis from occurring. This assay demonstrated that the functional difference between Fc-DAF and DAF-Fc found in the binding assay ( Figure 7 ) was also reflected as a substantial difference in overall complement regulatory activity in the serum in the cell protection assay ( Figure 8 ). While Fc-DAF prevented the lysis of rabbit red blood cells at 2 μM, 2 μM of DAF-Fc resulted in almost complete lysis. As can be deduced from Example 9, fusing the Fc module to the N-terminus of a complement activation regulator produced higher regulatory activity than fusing the Fc module to the C-terminus.

[0159] Cited non-standard literature:

[0160] - Cereghino et al. (2000), FEMS Microbiol. Rev. 24(1); 45-66.

[0161] - Harder et al. (2016), J. Immunol. Baltim. Md 1950.196(2):866 - 876.

[0162] - Hillmen et al. (2006), NEJM 355(12):1233

[0163] - McAuley et al. (2008), Protein Sci. Publ. Protein Soc. 17(1):95 - 106.

[0164] - Parkin and Cohen (2001) The Lancet 357:1777 - 89.

[0165] - Ricklin et al. (2010) Nature Immunology 11:785 - 797

[0166] - Ricklin et al. (2017), Mol Immunol. 89:10 - 21

[0167] - Ridgway et al. (1996), Protein Eng. 9(7):617 - 621.

[0168] - Romay - Penabad et al. (2014), Lupus 23(12):1324

[0169] - Schmidt et al. (2008), Clin Exp Immunol. 151(1):14 - 24

[0170] - Schmidt et al. (2011), Protein Expr. Purif. 76(2):254 - 263.

[0171] - Schmidt et al. (2013), J. Immunol. 190(11):5712 - 5721.

[0172] - Schmidt et al. (2016). Immunobiology. 221(4):503 - 511.

[0173] - WO 2013 / 142362 A1

Claims

1. A multimodular polypeptide, comprising (i) an Fc receptor-binding module; (ii) a first complement control protein repeat (CCP) module; and (iii) a second CCP module that binds to at least one host cell surface marker, binds to complement factor C3b, binds to complement factor C4b, binds to a degradation product of complement factor C3b, and / or binds to a degradation product of complement factor C4b; wherein the second CCP module is the C-terminus of the Fc receptor-binding module and the first CCP module.

2. The multimodular polypeptide according to claim 1, wherein the first CCP module (i) is a convertase decay module for the convertase of the classical pathway and / or alternative pathway of complement activation, and / or (ii) is a binding module for complement factor C3b and / or C4b, and the first CCP module preferably further has factor I cofactor activity.

3. The multimodular polypeptide according to claim 1 or 2, wherein the first CCP module comprises CCP domains 1 to 4 of factor H, preferably CCP domains 1 to 4 of human factor H; comprises CCP domains 1 to 3 of complement receptor type 1 (CR1), preferably CCP domains 1 to 3 of human CR1; comprises CCP domains 1 to 4 of decay-accelerating factor (DAF), preferably CCP domains 1 to 4 of human DAF, and / or comprises CCP domains 1 to 3 of C4-binding protein (C4BP), preferably CCP domains 1 to 3 of human C4BP.

4. The multimodular polypeptide according to any one of claims 1 to 3, wherein the first CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 70% identity thereto.

5. The multimodular polypeptide according to any one of claims 1 to 4, wherein the second CCP module binds to polyanionic carbohydrates including sialic acid, glycosaminoglycan, and / or complement factor C3b or its degradation product.

6. The multimodular polypeptide according to any one of claims 1 to 5, wherein the second CCP module comprises CCP domains 19 to 20 of factor H, preferably CCP domains 19 to 20 of human factor H.

7. The multimodular polypeptide according to any one of claims 1 to 6, wherein the second CCP module comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 70% identity thereto.

8. The multimodular polypeptide according to any one of claims 1 to 7, wherein the Fc receptor-binding module is the Fc module of IgG, preferably the Fc module of IgG1.

9. The multimodular polypeptide according to any one of claims 1 to 8, wherein the Fc receptor-binding module comprises at most one cysteine residue that forms a disulfide bridge with a second molecule of the Fc receptor-binding module, preferably does not contain a cysteine residue that forms a disulfide bridge.

10. The multimodular polypeptide according to any one of claims 1 to 9, wherein the multimodular polypeptide forms a non-covalent homodimer.

11. The multimodular polypeptide according to any one of claims 1 to 10, wherein the Fc receptor binding module comprises a peptide having the amino acid sequence SEQ ID NO: 3 or a sequence having at least 70% identity therewith, preferably comprises a peptide having the amino acid sequence SEQ ID NO:

3.

12. The multimodular polypeptide according to any one of claims 1 to 11, wherein the first CCP module and the second CCP module are included together as mini-FH, and the mini-FH comprises the amino acid sequence of one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 or a sequence having at least 70% identity with at least one of said sequences, preferably comprises the amino acid sequence of one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:

7.

13. The multimodular polypeptide according to any one of claims 1 to 12, wherein the multimodular polypeptide comprises, preferably consists of, the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 70% identity therewith.

14. A polynucleotide encoding the multimodular polypeptide according to any one of claims 1 to 13.

15. The multimodular polypeptide according to any one of claims 1 to 13 and / or the polynucleotide according to claim 14, which is used in medicine, preferably for treating and / or preventing inappropriate complement activation and / or diseases with inappropriate complement activation as a symptom.

16. The multimodular polypeptide and / or polynucleotide for the use according to claim 15, wherein the disease with inappropriate complement activation as a symptom is ischemia-reperfusion injury, antibody-mediated graft rejection, post-transplant thrombotic microangiopathy, autoimmune hemolytic anemia, acute and delayed hemolytic transfusion reactions, cold agglutinin disease, rheumatoid arthritis, aquaporin-4-antibody-positive neuromyelitis optica, CD59 deficiency, C3 glomerulopathy, atypical or typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and / or age-related macular degeneration.

17. An in vitro method for preventing or reducing the degree of complement activation, which comprises applying the multimodular polypeptide according to any one of claims 1 to 13 to a reaction mixture, tissue and / or organ containing complement factors, thereby preventing or reducing the degree of complement activation in the reaction mixture, tissue and / or organ.

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