Siarylated human factor H protein and its therapeutic use

Abstract

summary The present invention relates to in vitro sialylated human factor H protein, or to biologically active sialylated fragments or biologically active sialylated variants thereof. The present invention also relates to methods for producing such proteins in vitro, and to methods for using such proteins in the treatment of complement-mediated diseases such as C3 glomerulosis, atypical hemolytic uremic syndrome, or age-related macular degeneration.

Description

[Technical Field]

[0001] The present invention relates to in vitro sialylated human factor H protein, or to a biologically active sialylated fragment or biologically active sialylated variant thereof. The present invention also relates to a method for producing such protein in vitro, and to the use of such protein in methods for treating complement-mediated diseases such as C3 glomerulosis (C3G), atypical hemolytic uremic syndrome (aHUS), or age-related macular degeneration (AMD). [Background technology]

[0002] The glycoprotein complement factor H (FH) plays a central role in regulating alternative pathways (APs) for complement activation, and mutations or antibodies affecting FH function can lead to serious renal diseases such as atypical hemolytic uremic syndrome and C3 glomerulopathy. FH polymorphisms are also associated with age-related macular degeneration, a common form of visual impairment. In aHUS, FH dysfunction leads to uncontrolled complement activation, ultimately resulting in the formation of C5b-9 terminal attack complexes on glomerular endothelial cells. This is accompanied by cytotoxicity, glomerular thrombotic microangiopathy, and acute renal failure (ARF), and historically, more than 60% of genetically diagnosed patients have died or progressed to end-stage renal failure. In C3G, more than 50% of patients progress to end-stage renal failure within 10 years, and characteristic complement deposition is observed in the glomerular basement membrane. Uncontrolled AP activation in C3G is caused by mutations in complement genes (particularly FH) or autoantibodies that affect C3 convertase activity.

[0003] Treatment options for aHUS and C3G are limited, including plasma exchange therapy or plasma replacement with fresh frozen plasma (FFP), immunosuppressive therapy, and kidney transplantation, but these carry a high risk of relapse of the underlying disease. Among the complement-targeted therapies currently under investigation, eculizumab, a humanized monoclonal anti-C5 antibody, inhibits C5b-9 formation and is approved as a treatment for aHUS. However, due to the mode of inhibition by eculizumab, the bactericidal activity inhibitory ability of the complement system in patients is also impaired. For C3G, there is still no established treatment. The application of eculizumab to C3G patients has resulted in partial responses in only some patients.

[0004] FH regulates the activation of C3 convertase in serum and on the cell surface. FH consists of 1213 amino acids, comprising 20 repeating spherical short-chain consensus repeat (SCR) domains, 40 disulfide bonds, and 9 putative sites of asparagine (Asn,N)-linked glycans. In mature proteins, Asn511, -700, -784, -804, -864, -893, -1011, and 1077 mainly contain unfucosylated, branched diciallylated glycans with a molecular weight of 155 kD (see UniProtKB-P08603-1 for sequence reference). SCR1-4 contain the protein's complement regulatory region. This region binds to C3b and, by competing with factor B (FB), inhibits the formation of C3 convertase (C3bBb) and further promotes the dissociation of already formed C3 convertase (dissociation-promoting activity). Furthermore, these domains are crucial for FH to act as a cofactor for the irreversible cleavage and inactivation of C3b to iC3b in a factor I (FI)-dependent manner. The C-terminus of FH (SCR18-20) is a surface-binding region that interacts with C3b, C3d, and the cell surface. Binding of FH to the cell surface or biological membrane occurs via polyanionic structures such as proteoglycans and sialic acid, or glycosaminoglycans (e.g., heparin), regulating local complement activation in endogenous cells such as glomerular endothelial cells. The majority of FH mutations seen in aHUS patients directly affect FH's ability to interact with C3b or host cells, leading to alterations in the activity of this important complement regulator (http: / / www.fh-hus.org / ).

[0005] Since administering FFP instead of FH can restore normal complement activity in aHUS or C3G, purified plasma-derived FH and recombinant FH are suggested to be valuable treatment options for these patients. In H factor-deficient (FH- / -) mice exhibiting a C3G-like phenotype, administration of purified plasma-derived human H factor (FHplasma) rapidly normalized plasma C3 concentration and eliminated glomerular C3 deposition. Furthermore, FHplasma supplementation normalized serum-induced in vitro hemolysis in the serum of aHUS patients. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The biopharmaceutical production of FH is complex and requires considerable effort and cost. Recombinant FH has been expressed in mammalian cells, insect cells, yeast, and mosses. However, these approaches have not yet demonstrated therapeutic efficacy, and there is still a need for a means to provide recombinant FH usable for the treatment of FH-related diseases on an industrial scale. Therefore, the object of this invention was to provide recombinant FH usable for therapeutic purposes. [Means for solving the problem]

[0007] This issue is resolved by the attached claims and the subject matter described below.

[0008] As shown below, the inventors have surprisingly found that FH protein produced in the moss Physcomitrium patens and subsequently sialylated in vitro can be effectively used as a substitute for serum-derived FH protein. Although the glycosylation profile of the thus produced FH protein is not identical to that of human serum-derived FH protein, it surprisingly exhibits essentially the same functional properties as serum-derived human FH protein. Even more surprisingly, such FH protein has a higher targeting rate to the kidneys compared to serum-derived FH, and recombinant sialylated FH protein is particularly suitable for the acute treatment of factor H-related kidney disease. A further advantage of moss-produced sialylated FH protein is, for example, that it surprisingly inhibits the growth of Neisseria fungus more effectively than serum-derived FH protein or unsialylated moss-derived FH protein.

[0009] Accordingly, in a first embodiment, the present invention relates to a sialylated human factor H protein, or a biologically active sialylated fragment or biologically active sialylated variant thereof, wherein the protein, fragment or variant does not contain a trisialylated N-glycan of structure A3G3S3(NaNaNa) and / or contains a monosialylated N-glycan of structure A1G1S1(NaM). This aspect of the present invention also includes a pharmaceutical composition comprising a sialylated human factor H protein, or a biologically active sialylated fragment or biologically active sialylated variant thereof, and a pharmaceutically acceptable diluent, excipient or carrier.

[0010] In a second embodiment, the present invention relates to a method for sializing a human FH protein (or a fragment or variant thereof), the method comprising the step of sializing a galactosylated but unsialized human FH protein (or a fragment or variant thereof) in vitro.

[0011] In a third aspect, the present invention relates to sialylated human factor H (or a fragment or variant thereof) produced by a method according to the present invention (second aspect of the present invention). This aspect of the present invention also includes pharmaceutical compositions comprising a sialylated human factor H protein or a fragment or variant thereof and a pharmaceutically acceptable diluent, excipient, or carrier.

[0012] In a fourth aspect, the present invention relates to a sialylated human factor H protein according to a first or third aspect of the present invention, or a pharmaceutical composition containing the same, for use in treating the human body, particularly complement-mediated diseases and disorders such as C3 glomerulosis, atypical hemolytic uremic syndrome, and age-related macular degeneration, especially dry AMD.

[0013] In a fifth aspect, the present invention relates to a method for treating complement-mediated disorders or conditions in a subject, the method comprising administering a therapeutically effective amount of factor H protein or a pharmaceutical composition containing the same as described in a first or third aspect of the present invention to a subject in need thereof. Complement-mediated disorders or conditions may be selected from, but are not limited to, C3 glomerulopathy, atypical hemolytic uremic syndrome, and age-related macular degeneration, particularly dry AMD.

[0014] In a sixth aspect, the present invention relates to a method for galactosylating human FH protein (or a fragment or variant thereof). This method includes the step of galactosylating an ungalactosylated human FH protein in vitro. In this context, the present invention also relates to galactosylated but unsialylated human H factor (or a fragment or variant thereof), in particular to galactosylated but unsialylated human FH protein (or a fragment or variant thereof) produced by the aforementioned method for galactosylating human FH protein (or a fragment or variant thereof). [Brief explanation of the drawing]

[0015] The attached drawings are briefly described below. These drawings are intended to illustrate aspects of the present invention in more detail, but are not intended to limit the overall scope of the invention. [Figure 1] Figure 1 schematically illustrates the function of factor H as a central regulator of the complement system, particularly the alternative pathway to the complement pathway (figure quoted from Kopp et al, Biomolecules. 2012;2(1):46-75). [Figure 2] Figure 2A) shows the structure of the human FH protein including the glycosylation site used (quoted from Schmidt et al., Protein Expr Purif. 2011;76(2):254-263); B) schematicly shows a two-step sialylation process for one type of N-glycan that can be used to sialylate FH proteins produced in vitro in plants such as the moss Physcomitrella patens. [Figure 3A] Figure 3 shows A) recombinant FH protein produced by the moss P. patent (double knockout of α-1,3-fucosyltransferase (FT3) and β-1,2-xylosyltransferase (XT)) (hereinafter referred to as "moss-FH"), [Figure 3B] B) The in vitro sialylated recombinant FH protein (hereinafter referred to as "sial-moss-FH") first produced in the moss P. patent (double knockout of FT3 and XT), and [Figure 3C] C) Exemplary HILIC-HPLC elution and glycosylation profiles of human serum-derived (sd)FH protein (hereinafter referred to as "sd-FH") are shown. [Figure 4] Figure 4 shows the major glycan morphologies determined by HILIC-FLD-HPLC analysis of three FH variants. The average values ​​from nine different production batches are shown for moss-FH, four batches for sial-moss-FH, and one representative batch for sd-FH. "Unassigned, Other*" refers to N-glycan structures not specifically mentioned in the figure. Clear differences in glycan composition between moss-FH and sial-moss-FH are evident. In sial-moss-FH, the major glycan morphology of moss-FH is modified by the addition of a (Neu5Ac-Gal-GlcNAc) structure. In contrast to sd-FH, sial-moss-FH lacks trisialylated NaNaNa glycans. [Figure 5] Figure 5 shows a comparison of the in vitro properties of the sialylated FH protein of the present invention (right) and serum-derived FH protein (left). Specifically, in an SDS-PAGE assay using Coomassie staining, dose-dependent cleavage of the C3bα chain showed equivalent activity in sd-FH and the sialylated FH protein of the present invention. [Figure 6]Figure 6 shows the in vitro characterization of the sialylated FH protein of the present invention. A) Hemolysis assay, B) Characterization in the MAC formation assay (TCC ELISA). The FH protein of the present invention showed in vitro activity similar to serum-derived FH. When the amount of the sialylated FH protein of the present invention was increased, complement-induced hemolysis and MAC (terminal attack complex) formation decreased. [Figure 7A] Figure 7 shows that, despite the short plasma half-life, sial-moss-FH shows superior / equivalent efficacy compared to sd-FH in FH(- / -) mice, which are an animal model of C3G. A) PK profiles of three 125I-SIB-labeled FH variants in CD-1 mice (n = 3). The plots are the FH concentrations in plasma. Calculated half-lives from the initial time points (2 minutes to 6 hours) considering a one-compartment model system: moss-FH: 35 minutes, sial-moss-FH: 2.66 hours, sd-FH: 5.35 hours. [Figure 7B] B) PK profiles of three FH variants in FH(- / -) mice. The plots are the FH concentrations in serum. Correlating with the PK profiles obtained in CD-1 mice, moss-FH has a short half-life and the concentration in serum is below the detection limit 4 hours after injection. In contrast, the concentration of sial-moss-FH is almost the same as that of sd-FH immediately after injection, and sd-FH remains slightly longer in serum for more than 24 hours after injection. [Figure 7C] C) Mice injected with sial-moss-FH showed a significant increase in serum C3 concentration, exceeding the effect achieved with sd-FH. The plotted values are the serum C3 concentrations. [Figure 7D] D) Complement C3 kidney deposits 4 days after injection of FH variants into knockout mice are expressed as a ratio to the control group (PBS-treated group). There was no significant difference in C3 deposits in mice injected with moss-FH compared to mice injected with PBS. In contrast, in mice injected with sial-moss-FH, a significant decrease in glomerular C3 deposits was observed, comparable to the level of decrease achieved in animals administered sd-FH. [Figure 8]Figure 8 shows that sial-moss-FH does not inhibit the bactericidal activity of the complement system. When heat-inactivated normal human serum (HI NHS) is added to the test system, the survival rate of N. meningitidis increases significantly (the inventors observed a similar level after adding eculizumab to the test system; data not shown). In contrast, the addition of sial-moss-FH has little effect (even less than sd-FH or moss-FH) on the ability of complement to inactivate N. meningitidis. The concentration range tested corresponds to twice the normal FH concentration (assay in 500 mg / L of normal FH concentration, 50% normal human serum, NHS). N. meningitidis was added to 50% NHS and incubated in the presence of different concentrations of sd-FH or sial-moss-FH. The survival rate of N. meningitidis was evaluated by counting colony-forming units (cfu). Since there are various serotypes of N. meningitidis, the two most common serotypes (serotype B and serotype W) were used in the experiment. Heat-inactivated NHS and NHS were used as controls. A) Serotype W, B) Serotype B. [Figure 9] Figure 9 shows the in vivo distribution of 125I-SIB-labeled FH proteins (sial-moss-FH and sd-FH) 30 minutes after intravenous injection in selected organs of CD-1 mice. The two different figures show that 125I-SIB-labeled sial-moss-FH has better targeting ability (about twice) to the kidney than sd-FH. A) Injected dose per gram of organ (%ID), B) Organ-to-blood concentration ratio: in the case of the kidney (sial-moss-FH: 0.59 vs sd-FH: 0.33). Due to the improved targeting ability of sial-moss-FH to the kidney, this compound becomes a particularly useful candidate for the treatment of complement-related kidney diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). [Figure 10] Figure 10 shows the experimental setup for evaluating the effect of sial-moss-FH (5 μg) on photo-induced retinal degeneration in a mouse model of dry-type AMD compared to a PBS vehicle control. [Figure 11]Figure 11 shows that sial-moss-FH reduces the degree of degeneration and neuroinflammation induced by photo-induced retinal injury in the eyes of BALB / cJ mice (n=9, 18 readings from the start of analysis for both eyes). A) Retinal thickness (circle diameter 6 mm) measured by SD-OCT: Animals administered with sial-moss-FH showed significantly higher retinal thickness compared to individuals administered with PBS on days 3 and 4 after photoinjury. B) TUNEL staining of the outer granular layer (ONL). TUNEL (terminal deoxynucleotidyltransferase dUTP nick end labeling) is used to visualize apoptotic cells. Treatment with sial-moss-FH significantly reduced the number of apoptotic cells in the ONL from day 1 to after photoinjury compared to PBS. C) Number of microglia per 15 mm2 in the subretinal space (SRS) (visualized by immunohistochemistry using anti-lba1 antibody). Microglia are the primary immune cells of the central nervous system, playing a role in directing and resolving the immune response. The migration of microglia to the subretinal space is one of the characteristics of AMD. Pretreatment with sial-moss-FH significantly reduced the number of microglial cells in controls treated with PBS compared to controls treated with SRS. D) The formation of membrane attack complexes (MAC:C5b-9) was significantly reduced from day 1 after treatment with sial-moss-FH compared to the PBS-treated group. Significant differences between means were determined by one-way ANOVA and Tukey's multiple comparison test. Data are presented mean ± SEM, *P<0.05, **P<0.01, ***P≦0.001. [Figure 12]Figure 12 shows that in the eyes of BALB / cJ mice, sial-moss-FH treatment reduced levels of pro-inflammatory cytokines (IL-6) and complement C3 activation 4 days after photo-induced retinal injury (n=9, 18 readouts for bilateral analysis). The following transcription levels were observed: A) Measured levels of C3 in the retina; B) IL-6 in the retina analyzed by quantitative real-time PCR; C) Protein levels of IL-6 in the retina measured by ELISA. For all parameters, a significant decrease in levels was observed after sial-moss-FH pretreatment compared to the PBS-treated group. Significant differences between means were determined by one-way ANOVA and Tukey's multiple comparison test. Data are presented mean ± SEM, *P<0.05, **P<0.01, ***P≦0.001. [Figure 13] Figure 13 shows experiments conducted to define appropriate clinical dose ranges: A) Inhibition of hemolysis by adding increased amounts of moss-FH to the serum of various aHUS patients; Patient 1: C-terminal FH deletion; Patient 2: FH mutation combined with C3 mutation; Patient 3: FH mutation R1215Q; Carrier 1: Healthy sibling of Patient 3, carrier of FH mutation R1215Q; B) Replacement of C3NEF antibody by increasing the amount of moss-FH; MAC concentrations were measured by ELISA, and the plotted values ​​are normalized, 100% = starting value for normal human serum (NHS), Patients 1-3 possess C3NEF antibody. [Modes for carrying out the invention]

[0016] As described above, the present invention relates to the human factor H protein, and more particularly to its sialylated form. Numerous polymorphisms and sequence variations of human factor H are known, and in this specification, all of these are included in the term “human factor H protein.” For example, database entry UniProtKB-P08603 publishes numerous native variants of human factor H protein, and the present invention is not limited to any particular polymorphism or isoform of human factor H protein. Preferably, the human factor H protein of the present invention is a “mature” factor H protein, i.e., lacking the N-terminal signal peptide. A particularly preferred form of the “mature” human factor H according to the present invention includes the amino acid sequence of SEQ ID NO: 1. The term “human factor H protein” refers to the full-length sequence of (mature) human factor H and does not include fragments or non-native sequence variants of the factor H protein. Furthermore, the term “human factor H protein” defines only the amino acid sequence of the factor H protein and does not impose any restrictions on post-translational modifications, whether present or not, in the protein. In particular, this term does not require that the factor H protein of the present invention be exactly the same as the post-translational modifications typically found in human serum-derived factor H protein. The term “sially

[0017] The present invention also relates to sialylated fragments and variants of human factor H.

[0018] The sialylated FH protein (or its biologically active sialylated fragment or biologically active sialylated variant) according to the first aspect of the present invention is preferably characterized by not having a trisialylated N-glycan of structure A3G3S3(NaNaNa). In general, those skilled in the art will readily understand the types of N-glycan structures that may be present on glycoproteins and the corresponding nomenclature. Table 1 below shows typical abbreviations used herein, Oxford notes (where possible), summation formulas, and exemplary structures of N-glycans related to the present invention.

[0019] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0020] In Oxford nomenclature, N-acetylglucosamine is represented by A, galactose by G, fucose by F, mannose by M, N-acetylgalactosamine by GN, and sialic acid by S. The glycans are read from the backbone towards the reducing end, and there is no specific mention of the core, which consists of two GlcNAcs and three mannoses common to all N-glycans.

[0021] The sialylated factor H protein (or fragment or mutant) according to a first aspect of the present invention is preferably characterized by not containing a trisialylated N-glycan of the NaNaNa (A3G3S3 according to the established Oxford annotation, see Table 1 above) structure. Trisialylated NaNaNa glycan is typically found in human serum-derived factor H (sd FH), but is not found in human factor H, which was first recombinantly produced in the moss Physcomitrella patens and subsequently sialylated in vitro. Where it is stated herein that a human factor H protein does not contain a particular type of N-glycan, e.g., NaNaNa N-glycan, this means that the respective N-glycan species cannot be detected in a sample of total N-glycan using standard means known in the art, i.e., its level is below the detection threshold level of the method. Appropriate methods for determining the presence or absence of N-glycans such as NaNaNa N-glycan are described in the Examples section. Human factor H, recombinantly produced in Physcomitrium and subsequently sialylated in vitro, typically does not exhibit NaNaNa N-glycans and can therefore be easily distinguished from human serum-derived FH, which exhibits a NaNaNa structure (see, for example, Figure 4). Most preferably, the sialylated human factor H protein of the present invention contains no trisialylated N-glycans at all.

[0022] The sialylated human factor H protein (or its fragments or variants) according to the present invention may preferably also include NaM-type N-glycan structures. Preferably, less than 20%, more preferably less than 15%, of the total N-glycans of the FH protein (or its fragments or variants) according to the present invention are NaM-type N-glycan structures. In this specification, "total N-glycans" refers to N-glycans that can be cleaved from glycosylated factor H protein by PNGase F treatment. PNGase F is an amidase that acts on N-linked glycoproteins and glycopeptides by cleaving between the innermost N-acetylglucosamine (GlcNAc) residue and asparagine residue of high-mannose, hybrid, and complex oligosaccharides. As a result, deaminated proteins or peptides and free glycans are obtained. These free glycans (representing the total N-glycans of the glycoprotein) can then be analyzed quantitatively and qualitatively in more detail. Exemplary methods for determining the total N-glycans of factor H, as well as the presence and amount of different types of N-glycans, are described in the Examples section. The typical range of NaM N-glycan structures on sialylated human factor H protein (or its fragments or variants) is, for example, about 5% to about 15% of the total N-glycans. Most preferably, the sialylated human factor H protein (or its fragments or variants) of the present invention does not contain trisialylated N-glycans with a NaNaNa structure, but contains monosialylated N-glycans with a NaM structure.

[0023] Preferably, the sialylated human factor H protein (or its fragment or variant) of the present invention contains a NaNa N-glycan structure (A2G2S2, see Table 1 above). More preferably, at least 40%, at least 45%, or at least 50% of the total N-glycans of the FH protein (or its fragment or variant) are NaNa N-glycan structures. Most preferably, the sialylated human factor H protein (or its fragment or variant) of the present invention contains NaNa N-glycan structures in the range of about 45% to about 60% of the total N-glycans of the FH protein (or its fragment or variant). These glycan structures are abundant, for example, in human serum-derived FH protein, and in human factor H, which was first recombinantly produced in the moss Physcomitrella patens and subsequently sialylated in vitro (see Figure 4).

[0024] Similarly, the sialylated human factor H protein (or its fragments or variants) of the present invention preferably contains NaA(A2G2S1, see Table 1 above) N-glycan structures. More preferably, at least about 3.0% of the total N-glycans of the protein (or its fragments or variants) are NaA N-glycan structures. For example, about 5% to about 15% of the total N-glycans may be NaA N-glycan structures. Preferably, the total N-glycans of the sialylated human FH protein of the present invention contain 15% or less of NaA N-glycan structures.

[0025] The N-glycans of the H factor protein of the present invention may also include methylated N-glycans (e.g., NaM*1Me, see Table 1). For clarity, it should be noted that in this specification, when a specific glycan structure is referred to without the suffix "*xMe" (where x indicates the number of methylations), it refers to an unmethylated glycan structure. There is no clear Oxford annotation for methylated N-glycans, but the NaM*1Me structure is, for example, a monomethylated A1G1S1 structure, and the NaM*2Me structure is a dimethylated A1G1S1 structure, etc. Preferably, less than 20%, more preferably less than 15%, of the total N-glycans of the FH protein (or fragment or variant) of the present invention are methylated N-glycans such as NaM*1Me and NaM*2Me. For example, the sialylated human FH protein (or fragment or variant) of the present invention may contain less than 10% NaM*1Me N-glycan structures and / or less than 5% NaM*2Me N-glycan structures. The typical range for the NaM*1Me N-glycan structure is, for example, 5-10% of the total N-glycan. The typical range for the NaM*2Me N-glycan structure in the FH protein (or fragment or mutant) of the present invention is, for example, 2.0-4.0% of the total N-glycan.

[0026] The sialylated human factor H protein (or its fragments or mutants) of the present invention may have methylated N-glycan structures, such as the NaM*1Me or NaM*2Me type N-glycan structures described above. Methyl groups on sugar residues have rarely been reported and are not found in mammals in particular, but they are present in bacteria, fungi, algae, and some plant species. For example, the human factor H protein produced in the moss Physcomitrium patens and subsequently sialylated in vitro may exhibit such methylation modifications.

[0027] Another post-translational modification not found in humans but found in plants is the β1,2-xylose or α1,3-fucose-containing N-glycan structure. However, it is preferable that the sialylated human factor H protein (or its fragments or mutants) of the present invention does not contain the β1,2-xylose or α1,3-fucose-containing N-glycan structure. This can be achieved, for example, by knocking out the genes for α-1,3-fucosyltransferase (FT3) and β-1,2-xylosetransferase (XT), which are enzymes that lead to the β1,2-xylose or α1,3-fucose-containing N-glycan structure, even when the human FH protein of the present invention is produced in plants such as Physcomitrella patens.

[0028] The sialylated human factor H protein (or its fragments or variants) of the present invention may contain a Na(FA)-type α1,4-fucose-containing N-glycan structure. Again, although a clear Oxford annotation is not available, the Na(FA) structure is a fucosylated A2G2S1 N-glycan (see Table 1). Preferably, the Na(FA)-type α1,4-fucose-containing N-glycan structure accounts for less than 20% of the total N-glycans in the FH protein (or its fragments or variants) of the present invention. A typical range for the Na(FA) N-glycan structure is, for example, 10-15% of the total N-glycans, more preferably 11-14%.

[0029] In a particularly preferred embodiment, the sialylated human factor H protein (or a fragment or variant thereof) of the present invention is characterized by exhibiting one, two, or more of the following: a NaNa N-glycan structure in the range of about 45% to about 60% of the total N-glycan, a NaA glycan structure in the range of about 5% to about 15% of the total N-glycan, a Na(FA) glycan structure in the range of about 10% to about 15% of the total N-glycan, and a NaM glycan structure in the range of about 5% to about 15% of the total N-glycan. Most preferably, the sialylated human factor H protein (or a fragment or variant thereof) of the present invention includes a NaNa N-glycan structure in the range of about 45% to about 60%, a NaA glycan structure in the range of about 5% to about 15%, a Na(FA) glycan structure in the range of about 10% to about 15% of the total N-glycan, and a NaM glycan structure in the range of about 5% to about 15%.

[0030] The amino acid residues that are normally glycosylated in the human factor H protein and are preferably glycosylated in the factor H protein (or its fragments or variants) of the present invention are the asparagine residues at positions 529, 718, 802, 822, 882, 911, 1029, and 1095 of the standard FH (see UniProtKB-P08603-1). Importantly, these amino acid positions are provided for the P08603-1 sequence in the UniProt knowledge database, which includes an 18-amino acid signal peptide, and these positions may differ slightly in other human FH proteins, such as proteins lacking the signal peptide. For example, in Sequence ID No. 1, which is a preferred amino acid sequence for the factor H protein according to the present invention, the corresponding positions are amino acid residues 511, 700, 784, 804, 864, 893, 1011, and 1077 (due to the absence of the N-terminal signal peptide sequence). Although an asparagine residue is also present at position 217 (199 in SEQ ID NO: 1), it is not normally glycosylated in human FH protein, and it is preferable that it is not glycosylated in the H factor protein of the present invention.

[0031] As described above, multiple polymorphisms have been disclosed in the human factor H protein, and the present invention is not limited to any particular form of the human factor H sequence. However, the inventors have investigated several specific native variants that are particularly useful for the purposes of the present invention. For example, the FH protein (or fragment or variant thereof) preferably has valine or isoleucine (corresponding to the 44th amino acid residue of SEQ ID NO: 1) corresponding to the 62nd amino acid residue of standard FH (UniProtKB-P08603-1), with isoleucine being preferred over valine. Similarly, the FH protein (or fragment or variant thereof) preferably shows histidine or tyrosine (corresponding to the 384th amino acid residue of SEQ ID NO: 1) at the amino acid residue corresponding to the 402nd position of standard FH (UniProtKB-P08603-1), with tyrosine being more preferred than histidine. In one embodiment, the FH protein (or its fragment or variant) exhibits either I62, Y402, or I62 and Y402 (corresponding to I44, Y384, or I44 and Y384 in SEQ ID NO: 1) at amino acid residues corresponding to positions 62 and / or 402 of standard FH (UniProtKB-P08603-1). Most preferably, the sialylated human factor H protein of the present invention contains the amino acid sequence of SEQ ID NO: 1. As described above, the sialylated human factor H protein of the present invention preferably represents a mature FH protein, i.e., does not contain the FH signal peptide (see amino acids 1-18 of standard FH).

[0032] The FH protein according to the present invention is biologically active, i.e., it has at least one activity common to human serum-derived H factor. For example, the FH protein according to the present invention (or a fragment or variant thereof) is preferably able to bind to human C3b protein. The corresponding test is described in the Examples section of this application. Similarly, the FH protein according to the present invention (or a fragment or variant thereof) can preferably proteolytically cleave the α chain of C3b. The corresponding test is also described in the Examples section of this application. Similarly, the FH protein (or a fragment or variant thereof) can preferably protect sheep red blood cells from lysis. The corresponding test (hemolysis test) is described in the Examples section of this application. In this regard, the inventors note that protection from lysis does not mean that lysis does not occur, but rather that in the presence of the FH protein of the present invention, lysis is statistically significantly less than in the absence of the FH protein of the present invention. Most preferably, the FH protein of the present invention protects sheep red blood cells within a range (±15-20%) equivalent to that of human serum-derived FH protein, for example at 100 nM. Furthermore, the FH protein of the present invention (or its fragments or variants) can preferably interact with glycosaminoglycans on the cell surface. Most preferably, the FH protein of the present invention has two or more, or all, of these properties (binding to human C3b protein, proteolytic cleavage of the α-chain of C3b protein, protection of sheep red blood cells from lysis, and interaction with glycosaminoglycans on the cell surface).

[0033] Preferably, the sialylated FH protein (or fragments or variants thereof) of the present invention can reduce the number of glomerular C3 deposits in a C3G mouse model. Glomerular C3 deposition and decreased serum C3 concentration are characteristic pathological abnormalities in C3G patients. For example, FH- / - mice also exhibit abnormal glomerular C3 accumulation and decreased serum C3 concentration due to AP hyperactivation, but these can be restored by supplementation with human FH, making them a useful C3G model for testing the therapeutic effects of recombinant FH. Corresponding assays are described in the examples of this application. Most preferably, the FH protein of the present invention reduces the number of glomerular C3 deposits to a range comparable to that of human serum-derived FH protein.

[0034] Preferably, the sialylated FH protein according to the present invention is detectable in the serum of FH- / - mice 24 hours after intravenous injection of 1 mg of FH protein into the tail vein of FH- / - mice. A corresponding readout assay for testing this requirement is described in the examples of this application. Preferably, the sialylated FH protein according to the present invention has a half-life in mouse plasma ranging from 1.5 to 3 hours, preferably about 2.0 to 3.0 hours, and most preferably about 2.6 to 2.7 hours.

[0035] In a second aspect, the present invention relates to an in vitro method for producing sialylated human FH protein (or fragments or variants thereof). The method comprises the step of sialylating a galactosylated but unsialylated human FH protein in vitro. As used in the context of the method of the present invention, “in vitro” is intended to be limited to ex vivo methods that do not involve living cells. Therefore, sialylating processes occurring within living cells are not included in the term “in vitro” as used herein, regardless of whether the cells are from a multicellular organism (e.g., a mammal), cells isolated from a multicellular organism (e.g., an immortalized cell line), or single-celled microorganisms (e.g., Escherichia coli, yeast). One method to achieve sialylation is to contact a galactosylated but sialylated human FH protein (or its fragment or variant) with α-2,6-sialyltransferase and CMP-N-acetylneuraminic acid in vitro (i.e., in a suitable container) under conditions that allow for sialylation of the galactosylated N-glycan of the FH protein (or its fragment or variant).

[0036] The human factor H protein (or fragment or derivative thereof) to be sialylated in the method of the second aspect of the present invention may be any galactosylated but not sialylated human FH protein (or fragment or derivative thereof) known to those skilled in the art. The FH protein (or fragment or derivative thereof) is a galactosylated but not sialylated human FH protein (or fragment or derivative thereof), that is, it has galactose-terminated N-glycans but does not have sialic acid-terminated N-glycans. The method is not limited to specific polymorphisms of human FH protein or naturally occurring or unnaturally occurring variants. However, as with the FH protein according to the first aspect of the present invention, the inventors also consider specific natural variants that are particularly useful in carrying out the method of the second aspect of the present invention. In particular, all embodiments described above for the sialylated human factor H protein (or fragment or derivative thereof) according to the first aspect of the present invention (especially the glycan composition and glycan content described above) are equally suitable embodiments for human FH protein (or fragment or derivative thereof) to be sialylated using the method of the second aspect of the present invention. However, each FH protein (or its fragment or variant) is, naturally, only galactosylated and not yet sialylated. For example, as described above, the FH protein according to the first aspect of the present invention may include a monosialylated N-glycan of structure A1G1S1(NaM). Therefore, the corresponding galactosylated but not sialylated human FH protein (or its fragment or derivative) of the present invention includes a non-sialylated N-glycan of structure A1G1. Thus, all embodiments described above for sialylated N-glycans also apply to galactosylated but not sialylated human FH proteins, except for the presence of a sialic acid moiety that is necessarily absent. Galactosylated but not sialylated human FH proteins (or its fragment or derivative) are included within the scope of the present invention, as will be described later in the context of the sixth aspect of the present invention.As described above, the galactosylated FH protein (or its fragment or mutant) preferably exhibits valine or isoleucine (corresponding to the 44th amino acid residue of SEQ ID NO: 1), with isoleucine being preferred over valine. Similarly, the galactosylated FH protein (or its fragment or mutant) preferably exhibits histidine or tyrosine (corresponding to the 384th amino acid residue of SEQ ID NO: 1) at the amino acid residue corresponding to position 402 of the standard FH (UniProtKB-P08603-1), with tyrosine being more preferred than histidine. In one embodiment, the galactosylated FH protein (or its fragment or mutant) exhibits I62, Y402, or I62 and Y402 at the amino acid residues corresponding to positions 62 and / or 402 of the standard FH (UniProtKB-P08603-1). Most preferably, the galactosylated human factor H protein of the present invention contains the amino acid sequence of SEQ ID NO: 1. As described above, the galactosylated human factor H protein of the present invention preferably does not contain mature FH protein, i.e., FH signal peptide (see amino acids 1-18 of standard FH). Preferably, the galactosylated human FH protein sialylated in the method of the second aspect of the present invention is a galactosylated mature human FH protein. Most preferably, the galactosylated human FH protein sialylated in this method contains the amino acid sequence of SEQ ID NO: 1. Preferably, the asparagine residues corresponding to positions 529, 718, 802, 822, 882, 911, 1029, and 1095 of standard FH (see UniProtKB-P08603-1) are galactosylated (glycosylated and terminally). Preferably, in the human FH protein (or fragment or mutant thereof) sialylated by the method of the second aspect of the present invention, the 217th asparagine residue of standard FH (199 in SEQ ID NO: 1) is not glycosylated (and therefore not galactosylated).Human factor H protein (or its fragments or derivatives) is produced and glycosylated in plant host cells, preferably in Physcomitrella patens, and more preferably in double knockout strains of α-1,3-fucosyltransferase (FT3) and β-1,2-xylosyltransferase (XT) of Physcomitrella patens, and subsequently galactosylated in vitro (see below).

[0037] Siarylation of galactosylated human factor H protein (or its fragments or derivatives) can be achieved, for example, by enzymatic means. In this case, the method of the second aspect of the present invention includes the step of contacting a galactosylated but not sialylated human FH protein or its fragments or variants in vitro with, for example, α-2,6-sialyltransferase and CMP-N-acetylneuraminic acid under conditions that enable sialylation of the galactosylated N-glycan of the galactosylated FH protein or its fragments or variants. The α-2,6-sialyltransferase adds N-acetylneuraminic acid (Neu5Ac), which is a sialic acid, to the terminal galactosylated portion of the N-linked glycan of the FH protein (or its fragments or derivatives). Preferably, the α-2,6-sialyltransferase sializes all of the galactosylated N-linked glycans of the FH protein (or its fragments or derivatives), for example, asparagine residues at positions 529, 718, 802, 822, 882, 911, 1029, and 1095 of standard FH (see UniProtKB-P08603-1). Neu5Ac is generally preferred as the sialic acid used for sialization.

[0038] In a particularly preferred embodiment of the method according to a second aspect of the present invention, the method comprises, in a first step, galactosylating a glycosylated but galactosylated and sialylated human factor H protein or a fragment or variant thereof in vitro, for example, as a first step, contacting the glycosylated but galactosylated and sialylated human factor H protein or a fragment or variant thereof in vitro with an enzyme such as β-1,4-galactosyltransferase and UDP-galactose under conditions that enable galactosylation of the N-glycan of the factor H protein or a fragment or variant thereof, and then, as a further subsequent step, contacting the galactosylated human factor H protein or a fragment or variant thereof with an enzyme such as α-2,6-sialyltransferase and CMP-N-acetylneuraminic acid under conditions that enable sialylation of the already galactosylated N-glycan of the factor H protein or a fragment or variant thereof in vitro By contacting them in vitro, the same considerations disclosed above for the H factor protein (or its fragment or derivative) of the first aspect of the present invention apply to non-galactosylated and non-sialylated human factor H protein or its fragment or variant. For example, the FH protein according to the first aspect of the present invention may contain a monosialylated N-glycan of structure A1G1S1(NaM). Thus, the corresponding non-galactosylated and non-sialylated human FH protein (or its fragment or derivative) contains a nonsialylated and non-galactosylated N-glycan of structure A1. Therefore, all embodiments described above for galactosylated and sialylated N-glycans also apply to non-galactosylated and non-sialylated human FH protein, except for the presence of galactose and sialic acid moieties (which are necessarily absent).The two-step method, which involves first galactosylating the factor H protein, or its fragment or mutant, and then sialylating the galactosylated factor H protein, or its fragment or mutant, is particularly useful in embodiments in which the human factor H protein (or its fragment or mutant) is produced and glycosylated in double knockout strains of α-1,3-fucosyltransferase (FT3) and β-1,2-xylosyltransferase (XT) from plant host cells, preferably Physcomitrium, and more preferably Physcomitrium.

[0039] As described above, the present invention relates to sialylated human factor H (or a fragment or variant thereof) sialylated by a method according to a second aspect of the present invention. Such compounds are also included herein when referring to the FH protein of the present invention or the FH protein according to the present invention. This aspect of the present invention also includes pharmaceutical compositions comprising the human factor H protein of the present invention or a fragment or variant thereof sialylated by the method of the present invention, and further comprising pharmaceutically acceptable diluents, excipients or carriers.

[0040] A fourth aspect of the present invention relates to the FH protein (or fragments or variants thereof) of the present invention, i.e., sialylated human FH protein (or fragments or variants thereof) according to the first or third aspect of the present invention, or to a pharmaceutical composition containing the same for use in a method of treating the human body. As demonstrated in the examples, the FH protein (or fragments or variants thereof) of the present invention is a suitable and fully functional recombinant substitute for serum-derived human FH protein, and can, for example, bind to human C3b protein, proteolytically cleave the α chain of C3b, and protect sheep erythrocytes from lysis. The FH protein of the present invention has a reasonable serum half-life and can reduce the number of glomerular C3 depositions in the C3G mouse model. Therefore, the FH protein (or fragments or variants thereof) of the present invention can be used as a pharmaceutical in H factor-related disease conditions and disorders and can be used to restore normal complement activity. Such disease conditions and disorders may be selected from the group consisting of, for example, C3 glomerulopathy, atypical hemolytic uremic syndrome, and age-related macular degeneration, particularly dry AMD. Particularly preferred disease conditions to be treated with the H factor protein (or its fragments or variants) of the present invention are C3 glomerulosis and dry AMD. Methods of treating the human body typically involve administering an effective dose of the FH protein (or its fragments or variants) of the present invention. Those skilled in the art can determine such a therapeutically effective dose by conventional means. Those skilled in the art can also determine the most appropriate route of administration for a given disease condition or disorder. Particularly preferred clinical dose ranges may be, for example, 1.5 to 25 mg of the full-length FH protein of the present invention (or the corresponding amount of FH protein fragments or variants corresponding to 1.5 to 25 mg of the full-length protein) / kg body weight. More preferably, the clinical dose range is 3 to 15 mg per kg of body weight.

[0041] Preferably, the FH protein of the present invention used for the treatment of humans, particularly the treatment of the aforementioned disease conditions and disorders, is a sialylated, preferably mature, FH protein comprising SEQ ID NO: 1 and exhibiting the glycan composition and proportions described above for the first aspect of the present invention. Most preferably, such an FH protein is produced in Physcomitrella patens and then sialylated by the method according to the second aspect of the present invention.

[0042] Similar to the fourth aspect of the present invention, the present invention, in a fifth aspect, directly relates to a method for treating complement-mediated disorders in a subject, the method comprising administering a therapeutically effective amount of factor H protein described in the first or third aspect of the present invention to a subject in need thereof, or administering a pharmaceutical composition containing thereof. Complement-mediated disorders may be selected from, but are not limited to, C3 glomerulosis, atypical hemolytic uremic syndrome (aHUS), and age-related macular degeneration (AMD).

[0043] Finally, in a sixth aspect of the present invention, the present invention relates to a second method, namely, a method for galactosylating a human FH protein (or a fragment or variant thereof). This method comprises the step of galactosylating a glycated human FH protein (or a fragment or derivative thereof) in vitro. The glycated FH protein is initially glycated but not galactosylated; that is, it initially has an N-glycan but does not contain a galactose moiety (or sialic acid moiety, of course). The FH protein (or a fragment or derivative thereof) may be, for example, the same as those defined in any other aspect of the present invention, but conditionally not galactosylated (not yet galactosylated) and not yet sialylated (not yet sialylated). Galactosylation can be achieved, for example, using an enzyme such as β-1,4-galactosyltransferase and UDP-galactose. The present invention also relates to a galactosylated but not sialylated human H factor (or a fragment or variant thereof), in particular to a galactosylated human FH protein produced by a method according to the sixth aspect of the present invention. The galactosylated but non-sialylated human FH protein (or its fragment or variant) of the present invention may contain the sequence of SEQ ID NO: 1. The galactosylated but non-sialylated human FH protein (or its fragment or variant) preferably does not contain the N-glycan of structure A3G3. Preferably, the galactosylated but non-sialylated human FH protein (or its fragment or variant) contains the N-glycan of structure A1G1 and / or A2G2. The galactosylated but non-sialylated human FH protein (or its fragment or variant) of the present invention may contain methylated N-glycan. The galactosylated but non-sialylated human FH protein (or its fragment or variant) of the present invention can be used, for example, as a starting material for a method according to a second aspect of the present invention.

[0044] As used herein, the term “contains” should not be construed to mean “consisting of” (i.e., excluding the presence of any additional other substances). Rather, “contains” means that additional substances may optionally be present. The term “contains” encompasses, in particularly assumed embodiments within its scope, “consisting of” (i.e., excluding the presence of any additional other substances) and “contains but not composed of” (i.e., requiring the presence of any additional other substances), with the former being preferred. [Examples]

[0045] The following are specific examples illustrating embodiments and aspects of the present invention. However, the scope of the present invention is not limited by the specific examples described herein. In fact, various modifications of the present invention, in addition to those described herein, will be readily apparent to those skilled in the art from the foregoing description and the following examples. All such modifications are included in the appended claims.

[0046] Example 1: Production of recombinant H factor protein in Physcomitrium patens Recombinant human factor H with the amino acid sequence of Sequence ID No. 1 was produced in Physcomitrella patens, as previously described (Michelfelder et al.; J Am Soc Nephrol.; 2017; 28(5): 1462-1474). In short, the moss Physcomitrella patens can be used for the recombinant production of human FH on an industrial scale. However, post-translational modifications, particularly N-glycosylation which differs to some extent between plants and humans, must be considered. Both human and plant glycoproteins have a branched complex N-glycan with the same core structure GnGn (A2 according to Oxford annotations, see Table 1), but this core has two plant-specific sugar residues that may be immunogenic in mammals. These sugars, xylose and fucose, were completely removed by genome engineering via homologous recombination, modifying the glycosylation pattern of the moss and enabling the production of human factor H. In this study, we used double knockout strains of Physcomitrella patens to replace the genes for α-1,3-fucosyltransferase (FT3) and β-1,2-xylosyltransferase (XT) with knockout constructs. Both constructs contain shortened versions of the respective enzymes and additional stop codons. This causes translation to terminate prematurely, producing shortened, non-functional enzymes. Consequently, all proteins in these strains, including the recombinant human FH protein, lack the plant-specific glycan structure containing β1,2-xylose or α1,3-fucose. Furthermore, the recombinant H factor lacks the terminal sialic acid of the glycan, which is not normally found in plants.

[0047] Production was carried out by suspension culture of moss cells in a 500L disposable stirred tank reactor with illumination (Sartorius Biostat STR500), followed by purification using standard chromatography and filtration procedures.

[0048] Example 2: Siarylation of recombinant factor H protein Next, the recombinant FH protein produced in Example 1 was subjected to a two-step in vitro sialylation, as schematically shown in Figure 2B. The initial reaction buffer was prepared as 80 mM citrate, 4% propylene glycol, 80 mM arginine, 200 mM Tris + 0.0025% Tween® 20, pH 6.5. The FH concentration was adjusted to 10-15 mg / ml. In the first enzymatic reaction, galactosyltransferase (final concentration 5 mg / L), its substrate UDP-galactose (final concentration 8 mM), and the cofactor manganese chloride (MnCl2) (final concentration 4 mM) were added. Specifically, the substrate and cofactor were added as 10-fold concentrated solutions. The reaction solution was incubated at 37°C for 7 hours. Subsequently, a second enzymatic reaction was performed using sialyltransferase (final concentration 28.5 mg / L) and CMP-N-acetylneuraminic acid (final concentration 2.2 mM). CMP-N-acetylneuraminic acid was concentrated 10-fold. Simultaneously, alkaline phosphatase (final concentration 11 mg / L) and its coenzyme, zinc chloride (ZnCl2) (final concentration 50 μM), were added. The phosphatase inhibits the reverse reaction of sialyltransferase by cleaving CMP. This second enzymatic reaction was incubated at 37°C for 24 hours.

[0049] Example 3: Structural analysis of FH protein N-glycan analysis by HILIC-HPLC-MS: To identify and evaluate the amount of N-glycans bound to proteins, glycans were released from proteins and labeled with procainamide. First, two 50 μg aliquots were taken from the provided glycoprotein solution and reduced in 100 mM ammonium bicarbonate (pH 8.2) with DTT (final concentration 5 mM) at 56°C for 45 minutes. Iodoacetamide was added to a final concentration of 25 mM, and S-alkylation was carried out in the dark for 30 minutes. Next, the sample was precipitated in 4 times the volume of -20°C acetone at -20°C for 2 hours. After washing the pellet with 80% -20°C acetone, the sample was briefly dried under vacuum and subjected to PNGase F digestion overnight. The released N-glycans were purified by passing the reaction solution through a 25 mg C18 Hypersep centrifuge cartridge (Thermo Scientific) and collecting the effluent while retaining the protein. The samples were vacuum-dried and labeled with procainamide in the presence of hydrocyanoborate (65°C, 3 hours, dark). After derivatization, HILIC SPE cleanup was performed using a Discovery glycan cartridge (50 mg, Supelco). The bound derivatized glycans were eluted with 500 μL of 20% acetonitrile aqueous solution. The glycans were vacuum-dried and analyzed after dissolving in 20 μL of water. Separation was performed using a Nexera X2 HPLC system (RF-20Axs fluorescence detector, semi-microflow cell (Shimadzu, Korneuburg, Austria)) with an Acquity UPLC Glycan BEH amide column (2.1 × 150 mm, 1.7 μm, Waters) fitted with a Security Guard Ultra pre-column (Phenomenex). Solvent A consisted of 80 mM formic acid buffered to pH 4.4 with ammonia, and solvent B consisted of 80% acetonitrile in solvent A. The applied gradient was initially maintained at 99% solvent B for 8 minutes, then reduced to 57% B over 60 minutes, and finally reduced to 25% B over 2 minutes, with a flow rate of 0.4 ml min -1The column oven was set to 45°C and the flow cell thermostat to 40°C. Fluorescence was measured at wavelengths of 308nm and 359nm (Ex / Em). The injection volume was 2.5 μL. Peak identification was performed using the same HPLC system connected to a Bruker amaZon speed ETD ion trap mass spectrometer equipped with a standard ESI ion source. Spectra were recorded in positive ion mode.

[0050] Example 4: In vitro functional analysis of sialylated FH protein a)C3b cutting Complement factor H functions as a cofactor of complement factor I and cleaves C3b through its proteolytic activity. More precisely, factor I cleaves the α chain of C3b, while the β chain remains intact. This reaction can be measured with a simple in vitro assay. Briefly, gradually increasing amounts (0.5-10 ng) of sialylated human factor H obtained in Example 2 were mixed with 0.5 μg of factor I and 2 μg of C3b and incubated at 37°C for 30 minutes. Subsequently, the degradation of C3b, more precisely the degradation of the α chain of C3b, was visualized by Coomassie-stained SDS-PAGE as shown in Figure 5. C3b consists of an α chain of approximately 100 kDa and a β chain of approximately 65 kDa, which can be separated by reduced SDS-PAGE. Increased levels of sialylated recombinant H factor lead to a dose-dependent decrease in the complete C3b α chain and an increase in various degradation fragments of sizes 43, 46, and 68 kDa.

[0051] b) Protection of sheep red blood cells Sheep erythrocytes (sE) are used as a model for studying complement activation pathway II (AP) activity. Similar to human erythrocytes, sE express glycosaminoglycans on their surface and are normally protected from complement-mediated hemolysis by plasma-derived FH. Incubation of sE with FH-depleted serum induces AP-mediated cell surface complement activation, C5b-9 formation, and subsequent hemolysis.

[0052] c) Hemolysis test The hemolysis assay was performed according to a previously described method (Sanchez-Corral et al.; Mol Immunol 2004;41:81-84). Briefly, 5 × 10⁻¹⁴ 7 Individual fresh sE GVB / Mg 2+ The solution was diluted to a final volume of 25 μL with EGTA buffer. Various amounts of factor H or control were added to the GVB / Mg solution. 2+ The mixture was diluted to a final volume of 15 μL with EGTA buffer, and 10 μL of serum was added. The reaction mixture was then incubated at 37°C for 30 minutes, and the reaction was stopped by adding 200 μL of GVB / EDTA buffer. After centrifugation, the OD of the supernatant was measured at 414 nm using a microplate reader, and the corresponding blank value (without serum) was subtracted from each value.

[0053] d) Measurement of MAC formation using TCC-ELISA Sial-moss-FH or sd-FH were diluted with NHS serum and incubated in LPS-precoated wells in the presence of AP-specific buffer. After washing, active C5b-9 formation was detected using an alkaline phosphatase-labeled mAb. This mAb recognized the C9 neoantigen formed during C5b-9 formation, and was then incubated with alkaline phosphatase substrate solution for 30 minutes. Readings were performed at 405 nm using a microplate reader.

[0054] e) C3b binding The binding behavior of sialylated human factor H and sd-FH was analyzed using a Biacore T200 analyzer with a C1 sensor chip. C3b was first diluted to 0.05 mg / mL in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15% NaCl, 0.005% surfactant P20), and then diluted to 1 μg / mL in acetate buffer (10 mM, pH 5.0). All four channels of the chip were first analyzed using EDC(N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide). The channels were activated with a solution of 60 μL of hydrochloride and 60 μL of NHS (N-hydroxysuccinimide). The solution was flowed at 10 μL / min for 420 seconds until the RU (Response Unit) of all channels reached at least 70. One channel was used as a reference and was not immobilized. C3b solution was flowed through the channels at 5 μL / min until the RU reached approximately 150. After immobilization, ethanolamine solution was flowed through all four channels at a flow rate of 10 μL / min for 420 seconds. Serial dilutions of sd-FH, moss-FH, and sial-moss-FH (250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2 pM) were prepared in HBS-P buffer and the system was flowed at 30 μL / min with a contact time of 60 seconds and a dissociation time of 300 seconds. The analysis temperature and sample room temperature were set to 10°C. Reaction kinetic parameters were calculated using Biacore T200 evaluation software.

[0055] The obtained affinity constants are summarized in the table below. The KD ratio between sd-FH and the sialylated recombinant FH of Example 2 was 2.3 to 2.8 in all channels (Fc with different immobilized C3b concentrations), indicating that the sd-FH concentration required for complex formation in equilibrium is approximately twice the concentration required for FH according to the present invention.

[0056] [Table 2]

[0057] Example 5: Functional characteristics of sialylated FH protein in mice FH(- / -) knockout mice are characterized by persistently activated AP, resulting in decreased serum C3 concentration and marked C3 deposition in the kidneys. FH(- / -) mice aged 8–12 weeks were used for functional analysis of FH protein mutants (n=3 at each time point). Animals were intravenously injected via the tail vein at a dose of 40 mg / kg (equivalent to 100–150 μL). Control animals were injected with PBS. Approximately 60–100 μL of blood was collected from the tail vein at predetermined time points (0, 0.5, 1, 2, 4, 6, 24, 48, 72, and 96 hours) and separated into plasma and serum (blood was allowed to coagulate for 30 minutes, then centrifuged at 2000 g for 10 minutes). Serum samples were analyzed for FH and C3 concentrations using appropriate ELISA-based assays.

[0058] After 96 hours (4 days), the animals were slaughtered, and the kidneys were collected in PBS and rapidly frozen at -80°C. Glomerular C3 deposits were analyzed by fluorescence staining as follows: 10 μm frozen sections were prepared from mouse kidneys using a Leica CM 3050S cryostat, mounted on SuperFrost® plus glass slides, and stored at -80°C. After fixation with 4% paraformaldehyde solution, the sections were permeabilized with PBS 0.5% Tween® 20, and C3 was detected using goat anti-mouse C3 antibody, followed by rabbit anti-goat Alexa Fluor 488 labeled antibody. After washing, the slides were mounted on mounting medium and covered with coverslips. The sections were observed under a microscope using appropriate software. For quantitative immunofluorescence staining, the average fluorescence intensity of three glomeruli per section was measured using ImageJ software (National Institutes of Health, USA), and expressed in arbitrary fluorescence units (AFU). The changes in C3 deposition in each treatment group were compared as a percentage, with the control group (PBS treatment group) set at 100%.

[0059] Example 6: Evaluation of Neisseria growth inhibition N. meningitidis serotype B and N. meningitidis serotype W were cultured overnight and adjusted to an OD of 0.1 with 2x THY broth. The bacteria were then treated with complement-activated human pooled serum (NHS) (50%) or heat-inactivated (1 hour at 56°C) human serum (HI-NHS). The effects of sial-moss-FH or sd-FH on cell proliferation were investigated for bacteria treated with complement-activated NHS. For this purpose, factor H was serially diluted using PBS at concentrations of 0.004, 0.008, 0.015, 0.03, 0.06, 0.125, and 0.25 mg / mL.

[0060] N. meningitidis serotype B and N. meningitidis serotype W were cultured overnight and adjusted to an OD of 0.1 with 2x THY broth. The bacteria were then treated with complement-activated human pooled serum (NHS) (50%) or heat-inactivated (1 hour at 56°C) human serum (HI-NHS). The effects of sial-moss-FH or sd-FH on cell proliferation were investigated for bacteria treated with complement-activated NHS. For this purpose, factor H was serially diluted using PBS at concentrations of 0.004, 0.008, 0.015, 0.03, 0.06, 0.125, and 0.25 mg / mL.

[0061] As a control, eculizumab and BSA were added at the same concentrations. Bacteria were incubated at 37°C for 60 minutes in the presence of 5% CO2. After serum loading, the samples were placed on ice to inhibit complement action. Subsequently, the bacteria were serially diluted with THY broth, and the diluted samples were transferred to blood agar. After incubation at 37°C for 24 hours in the presence of 5% CO2, colony-forming units (CFUs) were measured. Each assay was repeated at least three times for each serotype, starting with an independent culture.

[0062] Example 7: 125 In vivo distribution of I-SIB-labeled FH protein To evaluate the systemic exposure, pharmacokinetic profiles, tissue distribution, and excretion of sial-moss-FH and sd-FH, various FH variants were radiolabeled and administered intravenously to healthy CD-1 mice.

[0063] Details: 1.65 mCi (61 MBq, 0.75 nmol) of iodinated NHS ester ligand 125 I-SIB was transferred to a 1.5 mL tube and evaporated to dryness under a nitrogen stream. 125 To the tube containing I-SIB, 400 μg of FH protein and approximately 130 μL of 1X PBS pH 7.4 were added. 0.2 M borate buffer pH 8.3 was added to adjust the pH of the solution to 8 and the final volume was made 0.2 mL. The mixture was gently stirred at room temperature for 30 minutes. 20 mM Tris, 0.15 M NaCl pH 8.5 containing 0.02% PS20 was added to make the volume up to 0.5 mL. The fraction of associated radioactivity was measured by instant thin layer chromatography (ITLC) using 10% TCA as the eluent. After the incubation time, unreacted 125 I-SIB was removed by a PD-10 desalting column containing Sephadex G-25 resin in 20 mM Tris, 0.15 M NaCl pH 8.5 containing 0.02% Tween® 20. The radiochemical purity (ITLC / 10% TCA precipitation) and total activity in solution were determined from the final labeled protein solution. The quality of the radiolabeled protein was evaluated by radioactive SE-HPLC and SDS-PAGE. Each 125 The radiochromatogram of the I-SIB-H factor mutant solution showed the presence of one major peak (>98%) corresponding to the H factor mutant as a monomer. The electrophoretic profile obtained after Coomassie blue staining was identical to that of the unlabeled and 125 I-SIB-H factor mutant. Thus, no protein degradation due to the radiolabeling process was observed compared to the unlabeled mutant. The dosing solutions were prepared by diluting the labeled sial-moss-FH and sd-FH with their respective unlabeled proteins to a specific activity of approximately 0.025 mCi / mg (0.925 MBq / mg) and an FH concentration of 6.7 mg / mL.

[0064] For in vivo administration, CD-1 mice weighing approximately 25-32g (7 weeks old) (n=3 at each time point) were used. Anesthetized mice (isoflurane gas) were intravenously injected with a solution at a dose of 40 mg / kg (volume 148-190 μL, equivalent to 0.93-1.19 MBq of radioactivity). This intravenous injection was performed via the posterior orbital plexus using a 0.3 mL syringe fitted with a 29 gauge needle. At intermediate time points, approximately 60-100 μL of blood was collected from the posterior orbital plexus of anesthetized mice. At the final time point, anesthetized mice were intraperitoneally injected with a ketamine / xylazine mixture, and blood samples were collected from blood loss via cardiac puncture. The blood samples were collected in pre-weighed Microvette® tubes supplemented with lithium heparin (Sarstedt), and their radioactivity was measured using a gamma counter. Next, plasma was separated from the blood sample (centrifuged at 2000g for 5 minutes), and the plasma sample was also analyzed for radioactivity using a gamma counter. Radioactivity in the plasma sample was expressed as the percentage of injected volume per gram (%ID / g). In these calculations, the total blood volume of the mouse was estimated to be 7.5% of the mouse body weight, and the hematocrit value was assumed to be 0.53% (Janvier Labs-Hematology data, 10-week-old female CD-1 mice). Furthermore, a blood density of 1.06 and a plasma density of 1 were assumed. The half-life of each compound was calculated from an early time point (2 minutes to 6 hours), considering a one-compartment model system.

[0065] At the final point (30 minutes after injection), mice were euthanized by intraperitoneal overdose of a mixture of ketamine hydrochloride and xylazine hydrochloride, followed by rapid euthanasia by intracardiac paracentesis. The target organs were extracted, washed with 0.9% NaCl, placed in vials, weighed, and their radioactivity was measured using a gamma counter. The selected tissues / organs were the bladder (empty), liver, pancreas, spleen, kidneys, lungs, heart, digestive tract (stomach, small intestine, and colon with contents), brain, skeletal muscle, thyroid gland, head, and tail. Both kidneys were counted separately. The liver was counted as a whole by cutting it in half and counting each fragment individually. Radioactivity concentration was expressed as the percentage of injected volume per gram of organ (%ID / g).

[0066] Example 8: AMD Experiment The Institute of Experimental Immunology of the Eye has established a white light damage paradigm in a mouse model to mimic dry AMD, and this established white light damage paradigm can mimic phenomena similar to retinal degeneration (Department of Ophthalmology, University of Cologne, Germany, Experimental Immunology of the Eye). In this model, 10,000 lux of white light containing a blue peak in the spectrum is irradiated for 30 minutes, inducing severe retinal thinning over 4 days. The analysis pipeline uses non-invasive in vivo longitudinal spectral optical coherence tomography (SD-OCT) and histology of DAPI-stained frozen retinal sections to grade retinal thickness and degeneration. In addition, gene expression and protein analysis are performed, as well as morphometric analysis of retinal sections and flat mounts to record the number, migration / localization of microglia and assess the degree of neuroinflammation.

[0067] In this mouse model, experiments were conducted to evaluate the effects of sial-moss-FH on photo-induced retinal degeneration compared to a PBS solvent control.

[0068] The day before light-induced damage induction, 5 μg of sial-moss-FH was injected intravitreously into one eye of wild-type mice. The control group was injected with PBS. An untreated control group was also included. On day 0, all mice were exposed to 30 minutes of white light at 10,000 lux, including a blue peak in the spectrum. The following analyses were performed on days 1, 3, and 4 after light exposure, and the results were obtained:

[0069] Measurement of retinal thickness Animals treated with Sial-moss FH showed significantly higher retinal thickness on days 3 and 4 compared to animals treated with PBS, demonstrating recovery of retinal thickness from photo-induced damage.

[0070] Accumulation of autofluorescent substances The measured degree of autofluorescence correlated with the number of "dead" cells. Sial-moss FH-treated animals showed a significantly reduced autofluorescence area on days 3 and 4 compared to PBS-treated animals, indicating recovery of the retinal condition after photodamage.

[0071] TUNEL (terminal deoxyribonucleotide transferase dUTP nick-end labeling) is used to visualize apoptotic cells. Sial-moss-FH treatment significantly reduced the amount of apoptotic cells after photodamage, starting from day 1.

[0072] Measurement of microglia reactivity in the subretinal space (SRS) and outer plexiform layer (OPL) Microglia are the primary immune cells of the central nervous system, playing a crucial role in inducing and resolving immune responses. The migration of microglia to the subretinal space is a characteristic feature of age-related macular degeneration (AMD). Pretreatment with sial-moss-FH significantly reduced the number of microglial cells during SRS and the number of "dead" microglial cells compared to the PBS-treated control group.

[0073] Formation of membrane attack complex (MAC:C5b-9): Treatment with sial-moss-FH significantly reduced MAC formation from day 1 compared to the PBS-treated group.

[0074] Müller cell gliosis The primary role of Müller cells is to maintain the structural and functional stability of retinal cells. There are indications that treatment with sial-moss-FH may show potential efficacy.

[0075] Based on these results, we can conclude that sial-moss-FH significantly reduces the degree of neuroinflammation induced by photo-induced retinal damage.

[0076] Example 9: Clinical Dose Range To establish the clinical dose range of sial-moss-FH, in vitro studies were conducted using serum from aHUS and C3G patients. Moss-FH (which has the same amino acid sequence but different glycans) was used as an alternative test substance in these studies. This approach is considered valid because moss-FH, sial-moss-FH, and sd-FH have been demonstrated to show comparable reactions in the hemolysis tests that underpin the studies performed. Therefore, the calculated dose range for moss-FH is considered valid for sial-moss-FH as well.

[0077] Serum from aHUS and C3G patients is characterized by excessive complement system activation, leading to increased serum C3 depletion and persistent formation of terminal membrane invasion complexes (MACs). Since complement factor H modulates complement activity, it is expected that adding moss-FH to patient serum will decrease complement activity.

[0078] Complement activity was evaluated using a sheep erythrocyte hemolysis assay (see above). In this assay, sheep erythrocytes are mixed with human serum. Since erythrocytes are susceptible to complement-mediated hemolysis, the erythrocyte hemolysis level (measured by absorbance at 414 nm) serves as an indicator of complement activity level. Normal human serum was used as a control (baseline).

[0079] Addition of 100-500 nM moss-FH reduced complement activity to the level of a healthy control group. Based on these results, a clinical dose range of 3-15 mg of moss-FH / kg body weight was calculated (Table 3).

[0080] [Table 3]

[0081] Approximately 80% of C3G patients possess C3NEF antibodies that stabilize C3 convertase. This leads to increased C3 cleavage and hyperactivation of the complement cascade (higher concentration of terminal membrane invasion complex (MAC)). Moss-FH concentrations were gradually increased in the serum of different C3G patients with C3NEF antibodies. Terminal membrane MAC formation was then evaluated by ELISA.

[0082] Increasing the dose of moss-FH reduced the terminal membrane attack complex. This suggests that moss-FH can displace C3NEF antibodies and induce the dissociation of C3 convertases, which can lead to a balance adjustment of the complement system. Furthermore, at doses of 100-500 nM (equivalent to 3-15 mg / kg body weight of moss-FH), the complex was reduced to control levels.

[0083] Based on the above data, it can be concluded that the appropriate dosage range for sial-moss FH is preferably in the range of 3 to 15 mg / kg body weight.

Claims

1. A sialylated human H factor (FH) protein, wherein the protein does not contain a trisialylated N-glycan of structure A3G3S3(NaNaNa).

2. A sialylated FH protein according to claim 1, wherein at least 40%, preferably at least 45%, and more preferably at least 50% of the total N-glycans of the protein are A2G2S2(NaNa)N-glycan structures.

3. A sialylated FH protein according to claim 1 or 2, wherein at least about 3.0% of the total N-glycans of the protein are A2G2S1(NaA)N-glycan structures, and / or about 5% to about 15% of the total N-glycans of the protein are A1G1S1(NaM)N-glycan structures, and / or about 10% to about 15% of the total N-glycans of the protein are α1,4-fucose-containing N-glycan structures (Na(FA)).

4. A sialylated FH protein according to any one of claims 1 to 3, wherein the sialylated FH protein does not contain a β1,2-xylose or α1,3-fucose-containing N-glycan structure.

5. A sialylated FH protein according to any one of claims 1 to 4, wherein the amino acid residues corresponding to positions 529, 718, 802, 822, 882, 911, 1029, and 1095 of standard FH (UniProtKB-P08603-1; entry version 245, sequence version 4) are glycosylated in the FH protein, fragment, or variant.

6. A sialylated FH protein according to any one of claims 1 to 5, wherein the protein exhibits either I62, Y402, or I62 and Y402 at each amino acid residue corresponding to positions 62 and / or 402 of standard FH (UniProtKB-P08603-1, entry version 245, sequence version 4).

7. A sialylated FH protein according to any one of claims 1 to 6, wherein the protein contains the amino acid sequence described in SEQ ID NO:

1.

8. A sialylated FH protein according to any one of claims 1 to 7, wherein the protein binds to a human C3b protein, proteolytically cleaves the α chain of C3b, and / or protects sheep red blood cells from lysis.

9. A method for producing sialylated human FH protein in vitro, comprising the step of sialylating a galactosylated but not sialylated human FH protein in vitro.

10. A method according to claim 9, comprising the step of contacting a galactosylated but not sialylated human FH protein in vitro with α-2,6-sialyltransferase and CMP-N-acetylneuraminic acid, thereby sialylating the N-glycan of the galactosylated FH protein.

11. A method according to claim 9, comprising the following steps: a) A step of galactosylating the N-glycan of the factor H protein by contacting a glycosylated but not galactosylated or sialylated human factor H protein in vitro with β-1,4-galactosyltransferase and UDP-galactose. b) A step of contacting galactosylated human factor H protein with α-2,6-sialyltransferase and CMP-N-acetylneuraminic acid in vitro, thereby sializing the N-glycan of the galactosylated factor H protein.

12. The method according to claim 11, wherein the human factor H protein is produced and glycosylated in plant host cells, preferably in Physcomitrium patentes, and more preferably in a double knockout strain of α-1,3-fucosyltransferase (FT3) and β-1,2-xylosyltransferase (XT) of Physcomitrium patentes.

13. A sialylated factor H protein produced by the method described in any one of claims 9 to 12.

14. A pharmaceutical composition comprising a sialylated human factor H protein according to any one of claims 1 to 8 or claim 13, or a sialylated human factor H protein for use in a method of treating the human body.

15. A sialylated human factor H protein or composition for use according to claim 14, wherein the method is a method for treating complement disorders or disorders such as C3 glomerulosis, atypical hemolytic uremic syndrome, or age-related macular degeneration, particularly dry AMD.

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