Sialylated human factor h protein for the treatment of paroxysmal nocturnal hemoglobinuria

Abstract

The present invention relates to the use of an in vitro sialylated human Factor H protein or a biologically active sialylated fragment or biologically active sialylated variant thereof for the treatment of paroxysmal nocturnal hemoglobinuria, for the treatment of thromboinflammation, for the treatment of pathological platelet aggregation formation, for the treatment of microangiopathy and / or for the treatment of long COVID. A combination with a C5 inhibitor, such as eculizumab, is also contemplated.

Description

Technical Field

[0001] This invention relates to the in vitro sialylation of human factor H protein or its biologically active sialylated fragments or biologically active sialylated variants for the treatment of paroxysmal nocturnal hemoglobinuria, thrombotic inflammation, pathological platelet aggregation, microangiopathy, and / or long-term COVID. The invention also relates to a method for treating paroxysmal nocturnal hemoglobinuria in a subject in need, comprising administering eculizumab to the subject along with said human factor H or its biologically active fragments or biologically active variants. Background Technology

[0002] The complement system is a major humoral component of the innate immune response, characterized by a complex network of plasma and membrane-associated proteins. Its primary functions are to recognize, destroy, and clear invading pathogens, apoptotic cells, immune complexes, and damaged host cells. In addition to its classical, systemic effects, the complement system also has important non-classical functions, such as synaptic pruning, T cell differentiation, B cell antibody production, and the control of fundamental cellular processes through intracellular complement (also known as complement bodies). Depletion or dysfunction of complement components can lead to uncontrolled complement activation, manifesting in various human diseases, such as paroxysmal nocturnal hemoglobinuria (PNH).

[0003] Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia. In addition to hemolysis, it can also lead to thrombosis, dystonia, chronic kidney disease, and bone marrow failure. PNH is caused by somatic mutations in the class A gene (PIGA) of phosphatidylinositol glycan anchoring biosynthesis in one or more long-lived hematopoietic stem cell (HSC) clones. PIGA encodes a glycosyltransferase, an enzyme essential for the biosynthetic pathway of glycosylphosphatidylinositol (GPI). Mutations in PIGA result in a deficiency of GPI-anchoring proteins, including the complement inhibitory proteins CD55 and CD59. The lack of these complement regulators is crucial for the erythrocytes in PNH to be vulnerable to complement-mediated attack. Current treatments for PNH involve the humanized monoclonal antibodies targeting C5, eculizumab and ravulizumab, and the C3 inhibitor pegcetacoplan. Importantly, most current complement therapies cause complete blockade of the complement system or its activation and effector pathways, thereby preventing the opsonization of C5 and blocking the membrane attack complex (MAC). Therefore, patients treated with eculizumab (a humanized monoclonal anti-C5 antibody) have a significantly higher incidence of Neisseria meningitidis (… Neisseriameningitidis ) and other pathogenic bacteria (including Haemophilus influenzae) Haemophilus influenza ) and Streptococcus pneumoniae ( Streptococcus pneumoniae The risk of life-threatening infections caused by this condition. Therefore, the object of this invention is to provide a novel method for treating paroxysmal nocturnal hemoglobinuria and other complement-related conditions such as thrombotic inflammation, pathological platelet aggregation, microangiopathy, and chronic COVID.

[0004] This problem is addressed by the subject matter set forth in the appended claims and the following description. Summary of the Invention

[0005] As will be shown below, the inventors of this invention unexpectedly discovered that in the moss *Moss styrax* (… Physcomitrium patens FH protein produced in this manner and subsequently sialylated in vitro can be effectively used to treat paroxysmal nocturnal hemoglobinuria and conditions such as thrombotic inflammation, pathological platelet aggregation, microangiopathy, or chronic COVID. The glycosylation profile of the FH protein produced in this way is different from that of human serum-derived FH protein, but surprisingly, it exhibits essentially the same functional properties. Even more surprisingly, this FH protein shows increased targeting to the kidneys compared to serum-derived FH. Furthermore, it unexpectedly inhibits the growth of Neisseria meningitidis more effectively than serum-derived FH protein or non-sialylated lichen-derived FH protein.

[0006] Therefore, in a first aspect, the present invention relates to a method for treating paroxysmal nocturnal hemoglobinuria (PNH), thrombotic inflammation, platelet aggregation, microangiopathy, and / or chronic COVID in subjects in need of sialylated human factor H protein or its biologically active sialylated fragments or variants thereof, wherein the protein, fragment, or variant does not contain a trisialylated N-glycan with the structure A3G3S3 (NaNaNa) and / or a monosialylated N-glycan with the structure A1G1S1 (NaM). Preferably, the method for treating paroxysmal nocturnal hemoglobinuria (PNH) is described in the sialylated human factor H protein or its biologically active sialylated fragments or variants thereof. This aspect of the invention also includes methods for using pharmaceutical compositions in subjects in need to treat paroxysmal nocturnal hemoglobinuria (PNH), to treat thrombotic inflammation, to treat platelet aggregation, to treat microangiopathy, or to treat chronic COVID, wherein the composition comprises the 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. Preferably, the pharmaceutical composition is used in methods for treating paroxysmal nocturnal hemoglobinuria (PNH).

[0007] In a second aspect, the present invention relates to a method for treating paroxysmal nocturnal hemoglobinuria (PNH), thrombotic inflammation, platelet aggregation, microangiopathy, and / or chronic COVID in subjects of need, the method comprising administering to the subject a therapeutically effective amount of sialylation factor H protein or a biologically active sialylation fragment or biologically active sialylation variant thereof, wherein the protein, fragment, or variant does not contain a trisialylated N-glycan with the structure A3G3S3 (NaNaNa) and / or a monosialylated N-glycan containing the structure A1G1S1 (NaM).

[0008] In a third aspect, the present invention relates to a method for treating paroxysmal nocturnal hemoglobinuria in a subject of need using human factor H protein or its biologically active fragment or biologically active variant, wherein the method comprises administering eculizumab and the human factor H or its biologically active fragment or biologically active variant to the subject.

[0009] In a fourth aspect, the present invention relates to a method for treating paroxysmal nocturnal hemoglobinuria (PNH) in a subject of need, the method comprising administering to the subject a therapeutically effective amount of factor H protein or a biologically active fragment or variant thereof, and eculizumab.

[0010] As described above, this invention relates to human factor H protein, particularly its sialylated form, for use in methods of treating PNH, thrombotic inflammation, pathological platelet aggregation, microangiopathy, and / or chronic COVID. Numerous polymorphisms and sequence variants of human factor H are known in the art, all of which are included in the term "human factor H protein" herein. For example, database entry UniProtKB-P08603 discloses numerous natural variants of human factor H protein, and the invention is not limited to any particular polymorphism or isotype of human factor H protein. Preferably, the human factor H protein is a "mature" factor H protein, i.e., lacking the N-terminal signal peptide. A particularly preferred form of "mature" human factor H comprises 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-natural sequence variants of factor H protein. Furthermore, the term "human factor H protein" defines only the amino acid sequence of factor H protein but does not, in itself, impose any limitation on post-translational modifications that may or may not be present on the protein. Specifically, the terminology does not require the factor H protein to precisely exhibit the post-translational modifications commonly found on factor H proteins derived from human serum. The term "sialylated human factor H protein" (or its respective sialylated fragments or sialylated variants) requires the factor H protein to exhibit sialylated N-glycans. Preferably, the sialic acid is N-acetylneuraminic acid (Neu5Ac). Furthermore, if referred to herein as "canonical FH," it refers to the sequence provided in UniProt Knowledge Database entry P08603-1 (entry version 245, sequence version 4), including the signal peptide. Any reference to canonical FH herein is for illustrative purposes only, such as explaining the location of certain glycosylation sites. While the specific amino acid sequence of P08603-1 is covered by this invention, the invention is not specifically limited to this single embodiment of the human factor H amino acid sequence.

[0011] The present invention also considers the use of sialylated fragments and variants of human factor H for the treatment of PNH, thrombotic inflammation, pathological platelet aggregation, microangiopathy and / or chronic COVID.

[0012] Sialinated FH proteins (or their biologically active sialylated fragments or biologically active sialylated variants), particularly in the context of the first and second aspects of the invention, are preferably characterized by not having any trisialylated N-glycans with the structure A3G3S3(NaNaNa). In the context of the third and fourth aspects of the invention, the FH protein can be any FH protein (or its fragments or variants) known to those skilled in the art, including non-sialylated FH proteins and sialylated FH proteins having trisialylated N-glycans with the structure A3G3S3(NaNaNa), such as serum-derived FH proteins. Generally, those skilled in the art are very familiar with the types of N-glycan structures that may be present on glycoproteins and the corresponding nomenclature. Table 1 below provides the general abbreviations, Oxford notes (where possible), general formulas, and exemplary structures of N-glycans related to the invention.

[0013] Table 1: Summary of relevant N-glycans. The table lists abbreviations, general formulas, and exemplary structures.

[0014] In the Oxford nomenclature, A represents N-acetylglucosamine, G represents galactose, F represents fucose, M represents mannose, GN represents N-acetylglucosamine, and S represents sialic acid. Among these, the glycans are read from the backbone to the reducing end, while the core, which consists of two GlcNAcs and three galactoses (a common feature of all N-glycans), is not specifically mentioned.

[0015] The preferred feature of the sialylated factor H protein (or a fragment or variant thereof) is that it does not contain any trisialylated N-glycans with the structure NaNaNa (A3G3S3 according to the established Oxford notes; see Table 1 above). Trisialylated NaNaNa glycans are commonly found in factor H (sd FH) derived from human serum, but not in human factor H originally recombined in the sphagnum moss *Sphagnum spp.* and subsequently sialylated in vitro. If the human factor H protein mentioned herein does not contain a certain type of N-glycan, such as NaNaNa N-glycan herein, it means that the corresponding N-glycan species cannot be detected in the total N-glycan sample by standard methods known in the art, i.e., its level is below the detection threshold level of the method. Suitable methods for determining the presence or absence of N-glycans (such as NaNaNa N-glycans) are provided in the Examples section. Originally in the sphagnum moss genus *Sphagnum* (… PhyscomitriumHuman factor H, recombinantly generated and subsequently sialylated in vitro, typically does not contain any NaNaNa N-glycans and can therefore be easily distinguished from human serum-derived FH, which has a NaNaNa structure (see example). Figure 4 Most preferably, the sialylated human factor H protein does not contain any trisialylated N-glycans.

[0016] Sialized human factor H protein (or fragments or variants thereof) may also preferably contain NaM-type N-glycan structures. Preferably, less than 20%, more preferably less than 15%, of the total N-glycans in the FH protein (or fragments or variants thereof) are NaM-type N-glycan structures. As used herein, "total N-glycans" refers to those N-glycans that can be cleaved from glycosylated factor H protein by treatment with PNGase F. PNGase F is an amidase that functions by cleaving the innermost N-acetylglucosamine (GlcNAc) and asparagine residues of N-linked glycoproteins and glycopeptides in high-mannose, hybrid, and complex oligosaccharides. This results in the production of deaminated proteins or peptides and free glycans. These free glycans (representing the total N-glycans of the glycoprotein) can then be subjected to more detailed quantitative and qualitative analysis. The Examples section provides exemplary methods for determining the total N-glycans of factor H and the presence and content of different types of N-glycans. The typical range of NaMN-glycan structures on sialylated human factor H protein (or fragments or variants thereof) is, for example, about 5% to about 15% of total N-glycans. Most preferably, sialylated human factor H protein (or fragments or variants thereof) does not contain trisialylated N-glycans with the structure NaNaNa, but contains monosialylated N-glycans with the structure NaM.

[0017] Preferably, the sialylated human factor H protein (or a fragment or variant thereof) comprises a NaNa N-glycan structure (A2G2S2, see Table 1 above). Even more preferably, at least 40%, at least 45%, or even at least 50% of the total N-glycans of the FH protein (or a fragment or variant thereof) are NaNa N-glycan structures. Most preferably, the sialylated human factor H protein (or a fragment or variant thereof) comprises NaNa N-glycan structures ranging from about 45% to about 60% of the total N-glycans of the FH protein (or a fragment or variant thereof). These glycan structures are abundant, for example, in FH proteins derived from human serum, and in human factor H proteins originally recombined in the bryophyte *Phyllostachys nigra* and subsequently sialylated in vitro (see Table 1 above). Figure 4 ).

[0018] Similarly, sialylated human factor H protein (or a fragment or variant thereof) preferably comprises a NaA (A2G2S1, see Table 1 above) N-glycan structure. Even more preferably, at least about 3.0% of the total N-glycans of the protein (or a fragment or variant thereof) 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 sialylated human FH protein comprise equal to or less than 15% NaA N-glycan structures.

[0019] The N-glycan of factor H protein used according to the first and second aspects of the present invention may further comprise methylated N-glycans (e.g., NaM). 1Me (see Table 1). For clarity, it is hereby noted that any reference to a given glycan structure in this article without the suffix "-me" indicates that the glycan structure is not included. Where xMe" (where x represents the number of methylations) refers to unmethylated glycan structures. Although there is no explicit Oxford annotation for methylated N-glycans, NaM... The 1Me structure is, for example, a monomethylated A1G1S1 structure, NaM The 2Me structure is a dimethylated A1G1S1 structure, and so on. Preferably, less than 20%, and even more preferably less than 15%, of the total N-glycans in the FH protein (or its fragments or variants) are methylated N-glycans, such as NaM. 1Me and NaM 2Me. For example, sialylated human FH protein (or fragments or variants thereof) may contain less than 10% NaM. 1Me N-glycan structure and / or less than 5% NaM 2Me N-glycan structure. NaM The typical range for 1Me N-glycan structures is, for example, 5% to 10% of total N-glycans. NaM on FH proteins (or fragments or variants thereof) The typical range for 2Me N-glycan structures is, for example, 2.0% to 4.0% of total N-glycans.

[0020] Sialidized human factor H protein (or fragments or variants thereof) can carry methylated N-glycan structures, such as the aforementioned NaM. 1Me type or NaM 2Me type N-glycan structure. Although methylation of sugar residues appears to be a rarely reported event, especially not found in mammals, it exists in certain species of bacteria, fungi, algae, and plants. For example, in the moss *Sphaerocarpus septemlobus* (… Physcomitrium patens Human factor H protein, which is produced in vivo and subsequently sialylated in vitro, can exhibit this type of methylation modification.

[0021] Another post-translational modification is the inclusion of an N-glycan structure containing β1,2-xylose or α1,3-fucose, which is not found in humans but can be found in plants. However, preferably, the sialylated human factor H protein (or a fragment or variant thereof) does not contain an N-glycan structure containing β1,2-xylose or α1,3-fucose. This can be achieved even when the human FH protein is produced in plants such as *Sphagnum moss*, for example by knocking out the corresponding α-1,3-fucosyltransferase (FT3) and β-1,2-xyloseyltransferase (XT) genes, which result in an N-glycan structure containing β1,2-xylose or α1,3-fucose.

[0022] Sialidized human factor H protein (or fragments or variants thereof) may contain a Na(FA)-type N-glycan structure containing α1,4-fucose. Similarly, although not explicitly noted in the Oxford notes, the Na(FA) structure is a fucosylated A2G2S1 N-glycan (see Table 1). Preferably, less than 20% of the total N-glycans in the FH protein (or fragments or variants thereof) are Na(FA)-type N-glycan structures containing α1,4-fucose. A typical range for the Na(FA) N-glycan structure is, for example, 10% to 15% of the total N-glycans, more preferably 11% to 14%.

[0023] In a particularly preferred embodiment, the sialylated human factor H protein (or a fragment or variant thereof) is characterized by exhibiting one, two, or more of the following: a NaNa N-glycan structure comprising about 45% to about 60% of the total N-glycans, a NaA glycan structure comprising about 5% to about 15% of the total N-glycans, a Na(FA) glycan structure comprising about 10% to about 15% of the total N-glycans, and a NaM glycan structure comprising about 5% to about 15% of the total N-glycans. Most preferably, the sialylated human factor H protein (or a fragment or variant thereof) comprises a NaNa N-glycan structure comprising about 45% to about 60% of the total N-glycans, a NaA glycan structure comprising about 5% to about 15% of the total N-glycans, a Na(FA) glycan structure comprising about 10% to about 15% of the total N-glycans, and a NaM glycan structure comprising about 5% to about 15% of the total N-glycans.

[0024] The amino acid residues that are typically glycosylated in human factor H proteins, and preferably also in factor H proteins (or fragments or variants thereof), are asparagine residues corresponding to positions 529, 718, 802, 822, 882, 911, 1029, and 1095 of the canonical FH sequence (see UniProtKB-P08603-1). Importantly, these amino acid positions are for the P08603-1 sequence in the UniProt knowledge base, which contains an 18-amino acid signal peptide, and may be located at slightly different positions in other human FH proteins (e.g., those lacking a signal peptide). For example, in SEQ ID NO:1 (the preferred amino acid sequence of the factor H protein according to the invention), the corresponding positions are amino acid residues 511, 700, 784, 804, 864, 893, 1011, and 1077 (due to the lack of a signal peptide sequence at the N-terminus). There is another asparagine residue at position 217 (position 199 in SEQ ID NO:1), which is not normally glycosylated in human FH protein and preferably is not glycosylated in factor H protein used according to the invention.

[0025] As previously described, various polymorphisms of human factor H protein are disclosed in the art, and the present invention is not limited to a specific form of human factor H sequence. However, the inventors of the present invention have considered certain specific natural variations to be particularly useful for the purposes of the present invention. For example, preferably, the FH protein (or a fragment or variant thereof) exhibits valine or isoleucine (corresponding to position 44 in SEQ ID NO:1) at the corresponding amino acid residue corresponding to position 62 of the canonical FH (UniProtKB-P08603-1), wherein isoleucine is preferred over valine. Similarly, it is preferred if the FH protein (or a fragment or variant thereof) exhibits histidine or tyrosine (corresponding to position 384 in SEQ ID NO:1) at the corresponding amino acid residue corresponding to position 402 of the canonical FH (UniProtKB-P08603-1), wherein tyrosine is preferred over histidine. In one embodiment, the FH protein (or a fragment or variant thereof) exhibits one of the following at the corresponding amino acid residues corresponding to positions 62 and / or 402 of the canonical FH (UniProtKB-P08603-1): I62, Y402, or I62 and Y402 (corresponding to I44, Y384, or I44 and Y384 in SEQ ID NO:1). Most preferably, the sialylated human factor H protein comprises the amino acid sequence of SEQ ID NO:1. As described above, the sialylated human factor H protein preferably represents the mature FH protein, i.e., does not contain the FH signal peptide (see amino acids 1 to 18 of the canonical FH).

[0026] The FH protein used according to the present invention has biological activity, namely, at least one activity shared with human serum-derived factor H. For example, the FH protein (or a fragment or variant thereof) according to the present invention is preferably capable of binding human C3b protein. The Examples section of this application provides corresponding tests. Similarly, the FH protein (or a fragment or variant thereof) according to the present invention is preferably capable of cleaving the α chain of C3b via proteolytic action (via complement factor I). The Examples section of this application also provides corresponding tests. Likewise, the FH protein (or a fragment or variant thereof) is preferably capable of protecting sheep erythrocytes from lysis. The Examples section of this application provides corresponding tests (hemolysis test). In this case, the inventors note that protection from lysis does not mean the absence of lysis, but rather that the lysis occurring in the presence of the FH protein of the present invention is statistically significantly less than in the absence of the FH protein of the present invention. Most preferably, the protection of sheep erythrocytes by the FH protein of the present invention is the same as that protected by human serum-derived FH protein (+ / - 15%-20%), for example at 100 nM. Furthermore, the FH protein (or a fragment or variant thereof) according to the present invention is preferably capable of interacting with glycosaminoglycans on the cell surface. Most preferably, the FH protein of the present invention has two or more or even all of these properties (binding to human C3b protein, cleaving the α chain of C3b protein by proteolysis, protecting sheep red blood cells from lysis, and interacting with glycosaminoglycans on the cell surface).

[0027] Preferably, sialylated FH protein (or a fragment or variant thereof) is able to reduce the amount of glomerular C3 deposits in a C3G mouse model. Glomerular C3 deposition and low serum C3 levels are characteristic pathological abnormalities in patients with C3G. For example, FH- / - mice also develop abnormal glomerular C3 accumulation and low serum C3 levels due to AP overactivation, which can be reversed by human FH supplementation, thus providing a useful C3G model for testing the therapeutic efficacy of recombinant FH. The embodiments section of this application provides corresponding detection methods. Most preferably, the FH protein of the present invention reduces the amount of glomerular C3 deposits to the same extent as human serum-derived FH protein.

[0028] Preferably, the sialylated FH protein used according to the present invention remains detectable in the serum of FH- / - mice 24 h after intravenous injection of 1 mg of FH protein into the tail of FH- / - mice. The Examples section of this application provides a corresponding reading assay for testing this requirement. Preferably, the half-life of the sialylated FH protein in mouse plasma is in the range of 1.5 h to 3 h, more preferably about 2.0 h to 3.0 h, and most preferably about 2.6 h to 2.7 h.

[0029] The sialylated human FH protein (or a fragment or variant thereof) used according to the present invention can be produced by sialylating galactosylated but non-sialylated human FH protein in vitro. "In vitro," as used in the context of this invention, means limited to in vitro methods not involving living cells. Therefore, sialylation processes occurring in living cells are not covered by the term "in vitro" as used herein, whether the cells are cells in multicellular organisms (e.g., in mammals), cells isolated from multicellular organisms (e.g., immortalized cell lines), or cells themselves being single-celled microorganisms (e.g., *Escherichia coli*, yeast). One way to achieve sialylation is to contact galactosylated but non-sialylated human FH protein (or a fragment or variant thereof) in vitro (i.e., in a suitable container) with sialyltransferases (α-2,6) and CMP-N-acetylneuraminic acid under conditions that allow sialylation of the galactosylated N-glycans of said FH protein (or a fragment or variant thereof).

[0030] The human factor H protein (or a fragment or derivative thereof) to be sialylated can be any galactosylated but non-sialylated human FH protein (or a fragment or derivative thereof) known to those skilled in the art. The FH protein (or a fragment or derivative thereof) is a galactosylated but non-sialylated human FH protein (or a fragment or derivative thereof), i.e., it carries an N-glycan terminally modified with galactose but not a terminally modified with sialic acid. This method is not limited to specific polymorphisms or naturally occurring or non-natural variants of the human FH protein. The human factor H protein (or a fragment or derivative thereof) may have been produced and glycosylated in plant host cells, preferably in *Sphaerocarpus spp.* (a plant host cell). Physcomitrium patens More preferably, in a double knockout of α-1,3-fucosyltransferase (FT3) and β-1,2-xylosyltransferase (XT) line of *Sclerotium spp.*, followed by in vitro galactosylation (see below).

[0031] Sialization of galactosylated human factor H protein (or fragments or derivatives thereof) can be achieved, for example, by an enzymatic method. In this case, galactosylated but not sialylated human FH protein, or fragments or variants thereof, is contacted in vitro with, for example, sialyltransferases (α-2,6) and CMP-N-acetylneuraminic acid under conditions that allow sialylation of the galactosylated N-glycans of the galactosylated FH protein, or fragments or variants thereof. Sialyltransferases (α-2,6) add sialic acid N-acetylneuraminic acid (Neu5Ac) to the terminal galactosylated portion of the N-linked glycan chain of the FH protein (or fragments or derivatives thereof). Preferably, sialyltransferases (α-2,6) sialylate all galactosylated N-glycans of the FH protein (or fragments or derivatives thereof), for example, corresponding to asparagine residues at positions 529, 718, 802, 822, 882, 911, 1029, and 1095 of the canonical FH (see UniProtKB-P08603-1). Neu5Ac is generally the preferred sialic acid used for sialylation.

[0032] The method may further include, in a first step, galactosylating a glycosylated but non-galactosylated and non-sialylated human factor H protein or a fragment or variant thereof in vitro, for example by contacting the glycosylated but non-galactosylated and non-sialylated human factor H protein or a fragment or variant thereof in vitro with an enzyme (such as galactosyltransferase (β-1,4)) and UDP-galactose under conditions that allow galactosylation of the N-glycan of the factor H protein or a fragment or variant thereof, and then in a further subsequent step, contacting the galactosylated human factor H protein or a fragment or variant thereof with sialyltransferase (α-2,6) and CMP-N-acetylneuraminic acid in vitro under conditions that allow sialylation of the previously galactosylated N-glycan of the factor H protein or a fragment or variant thereof.

[0033] As shown in the Examples section, sialylated recombinant FH protein (or a fragment or variant thereof) is a suitable and fully functional alternative to serum-derived human FH protein, for example, capable of binding to human C3b protein, resulting in proteolytic cleavage of the α-chain of C3b (via complement factor I), and protecting sheep erythrocytes from lysis. It has a reasonable serum half-life and is able to reduce the amount of glomerular C3 deposits in the C3G mouse model. Therefore, sialylated FH protein (or a fragment or variant thereof) can be used as a medicine in factor H-related disease states and conditions, and can be used to restore normal complement activity, including in the treatment of PNH. Such treatments typically involve administering an effective amount of sialylated FH protein (or a fragment or variant thereof). Those skilled in the art will be able to determine the effective amount of such treatment using conventional methods. Those skilled in the art will also be able to determine the most suitable route of administration. Particularly preferred clinical dose ranges include, for example, 1.5 mg / kg to 25 mg / kg body weight of sialylated full-length FH protein (or a matching amount of an FH protein fragment or variant corresponding to 1.5 mg to 25 mg of full-length protein). More preferably, the clinical dose range is from 3 mg / kg to 15 mg / kg body weight.

[0034] Preferably, the FH protein used in subjects seeking treatment for paroxysmal nocturnal hemoglobinuria (PNH), thromboinflammatory disease, platelet aggregation, microangiopathy, or chronic COVID is a sialylated, preferably mature FH protein, comprising SEQ ID NO:1 and exhibiting the glycan composition and percentages described above with respect to the first aspect of the invention. Most preferably, such FH proteins are produced in *Pseudomonas aeruginosa* and have been sialylated using the methods described above. The FH protein may also be bound to a second therapeutic agent for treating, for example, PNH. The second therapeutic agent may be, for example, a C5 inhibitor. The C5 inhibitor may be an anti-C5 antibody. In particular, the second therapeutic agent may be eculizumab. The second therapeutic agent may be administered to the patient before, simultaneously with, or after the administration of the FH protein. In embodiments where the FH protein is used to treat thromboinflammatory disease, platelet aggregation, or microangiopathy in subjects, the subjects are preferably subjects suffering from complement disorders or conditions (such as atypical hemolytic uremic syndrome (aHUS)). An example of a disease involving complement dysregulation (which the inventors have specifically considered for treatment) includes long-term COVID, characterized by increased complement activation and thrombotic inflammation, including activated platelets and erythrocyte lysis markers (Cervia-Hasler et al. Science 2024383 eadg7942).

[0035] As described above, the present invention relates in a third aspect to a method for treating paroxysmal nocturnal hemoglobinuria (PNH) in a subject requiring treatment using human factor H protein or a biologically active fragment or variant thereof, wherein the method involves administering eculizumab and the human factor H protein or its biologically active fragment or variant to the subject. In the context of the third aspect of the invention (i.e., combined treatment of PNH with eculizumab and factor H protein), the factor H protein (or a fragment or derivative thereof) can be any factor H protein (or a fragment or derivative thereof) known to those skilled in the art, i.e., it can be non-sialylated or trisialylated N-glycan having an A3G3S3 (NaNaNa) structure. In some embodiments, the factor H protein of the third aspect of the invention is a serum-derived human factor H protein. However, preferably, the factor H protein (or a fragment or derivative thereof) is the same as defined above with respect to the first and second aspects of the invention. The human factor H protein (or a fragment or derivative thereof) can be administered to the subject before, simultaneously with, or after eculizumab has been administered. Preferably, the human factor H protein (or a fragment or derivative thereof) is administered to the subject concurrently with or after eculizumab has been administered.

[0036] In a fourth aspect, the present invention relates to a method for treating paroxysmal nocturnal hemoglobinuria (PNH) in a subject of need, the method comprising administering to the subject a therapeutically effective amount of factor H protein or a biologically active fragment or variant thereof, and eculizumab. The method of the fourth aspect of the invention is applicable to the same content as described above with respect to the third aspect of the invention. In particular, the FH protein (or a fragment or derivative thereof) may be any factor H protein (or a fragment or derivative thereof) known to those skilled in the art, including sd-FH, but is preferably the factor H protein (or a fragment or derivative thereof) as defined above with respect to the first and second aspects of the invention.

[0037] The term "comprising" as used herein should not be construed as limited to the meaning of "consisting of" (i.e., excluding the presence of additional substances). Rather, "comprising" implies that additional substances may optionally be present. The term "comprising" covers both "consisting of" (i.e., excluding the presence of additional substances) and "comprising but not constituting of" (i.e., requiring the presence of additional substances) of a particular contemplated embodiment falling within its scope, with the former being preferred. Attached Figure Description

[0038] A brief description of the accompanying drawings is given below. These drawings are intended to illustrate various aspects of the invention in more detail. However, they are not intended to limit the overall scope of the invention.

[0039] Figure 1 The function of the complement system, particularly factor H, as a central regulator of the complement pathway bypass is illustrated (Figure taken from Kopp et al, Biomolecules. 2012;2(1):46-75).

[0040] Figure 2 A) shows the structure of the human FH protein (Figure taken from Schmidt et al., Protein Expr Purif. 2011;76(2):254-263), including the glycosylation sites used; B) schematically shows a two-step sialylation process of a type of N-glycan that can be used to sialylate the FH protein in vitro, which is found in plants such as the moss *Bryum simonii*. Physcomitrium patens It was generated in ).

[0041] Figure 3 Exemplary HILIC-HPLC elution and glycosylation profiles are provided: A) recombinant FH protein produced in *Mossula spp.* (double knockout of α-1,3-fucosyltransferase (FT3) and β-1,2-xylosyltransferase (XT), hereinafter referred to as "moss-FH"); B) recombinant FH protein originally produced in *Mossula spp.* (FT3 and XT double knockout), hereinafter referred to as "sial-moss-FH"; and C) (sd) FH protein derived from human serum, hereinafter referred to as "sd-FH".

[0042] Figure 4 The results represent the major glycan forms of the three FH variants as determined by HILIC-FLD-HPLC: the average results for moss-FH were from nine different production batches, the average results for sialylated-moss-FH were from four batches, and the average results for sd-FH were from one representative batch. "Unspecified, other" "Refers to the N-glycan structure not otherwise mentioned in the figure. It demonstrates a clear difference in glycan composition between sd-FH and sialylated-sd-FH. In sialylated-sd-FH, the dominant glycan form of sd-FH is modified by the addition of a (Neu5Ac-Gal-GlcNAc) structure. Unlike sd-FH, sialylated-sd-FH does not carry trisialylated NaNaNa glycans."

[0043] Figure 5A comparison of in vitro characterization of sialylated FH protein (right) and serum-derived FH (left) is provided. Specifically, the figure shows dose-dependent cleavage of the C3b α chain as detected by Coomassie staining SDS-PAGE, comparable to the activity of sd-FH and sialylated FH protein.

[0044] Figure 6 This invention provides in vitro characterization of the sialylated FH protein in A) hemolysis detection and B) MAC formation detection (TCC ELISA). The FH protein of this invention exhibits the same in vitro activity as serum-derived FH. Increasing the amount of the sialylated FH protein of this invention reduces complement-induced hemolysis and MAC (terminal attack complex) formation.

[0045] Figure 7 It was demonstrated that, despite a shorter half-life in the blood, sialylated-lichenified-FH showed better / comparable efficacy compared to sd-FH in FH(- / -) mice (C3G animal model): A) three 125 PK curves of I-SIB-labeled FH variants in CD-1 mice (n=3); the figure shows plasma FH concentrations. Considering the single-compartment model system: half-lives calculated from early time points (2 minutes to 6 hours): *Moss-FH: 35 min; *Sialoyl-Moss-FH: 2.66 h; *sd-FH: 5.35 h. B) PK curves of the three FH variants in FH(- / -) mice; the figure shows serum FH concentrations. Corresponding to the PK curves obtained in CD-1 mice, *Moss-FH showed a short half-life, with undetectable levels in serum 4 h post-injection. In contrast, the concentrations of *Sialoyl-Moss-FH* were comparable to those of directly injected *sd-FH*, with *sd-FH* showing a slightly longer retention time in serum more than 24 hours post-injection. C) Serum C3 levels were significantly elevated in mice injected with *Sialoyl-Moss-FH*, exceeding the effect achieved with *sd-FH*; the figure shows serum C3 concentrations. D) Complement C3 renal deposits 4 days after injection of the FH variant into gene knockout mice, expressed as a percentage of the control (PBS-treated) group: There was no significant difference in C3 deposits between mice injected with lichen-FH and those injected with PBS. In contrast, mice injected with sialylated-lichen-FH showed a significant reduction in glomerular C3 deposits, comparable to the reduction achieved in sd-FH-treated animals.

[0046] Figure 8 This demonstrates that sialylated-lichen-FH does not inhibit the bactericidal activity of the complement system. Adding heat-inactivated normal human serum (HI NHS) to the test system induced Neisseria meningitidis (… N. meningitidisThe survival rate of *Neisseria meningitidis* was significantly improved (similar levels were observed by the inventors after adding eculizumab to the test system; data not shown). In contrast, the addition of sialylated-moss-FH had only a slight effect on the ability of *Neisseria meningitidis* to inactivate complement (even less than sd-FH or moss-FH). The test concentration range was twice the normal FH concentration (normal FH concentration was 500 mg / L, detected in 50% normal human serum (NHS)). *Neisseria meningitidis* was added to 50% NHS and incubated in the presence of different concentrations of sd-FH or sialylated-moss-FH. The survival rate of *Neisseria meningitidis* was scored by counting colony-forming units (CFU). Since there are different serotypes of *Neisseria meningitidis*, the two most common serotypes (type B and type W) were used in the experiment. NHS and heat-inactivated NHS were used as controls. A) Serotype W; B) Serotype B.

[0047] Figure 9 Showing 125 Biodistribution of I-SIB-labeled FH proteins (sialylated-moss-FH and sd-FH) in selected organs of CD-1 mice 30 minutes after intravenous injection. Two different graphs show... 125 Compared with sd-FH, I-SIB radioisotope-labeled sialyl-moss-FH showed better renal targeting: A) percentage of injected dose per gram of organ (ID%); B) organ to blood concentration ratio: kidney (sialyl-moss-FH: 0.59 and sd-FH: 0.33).

[0048] Figure 10 This indicates that pre-exposure to untreated aHUS serum (n=3) resulted in the formation of large platelet aggregates on HMEC-1 cells upon normal whole blood perfusion. When added to the serum of aHUS patients, sialylation-lichen-FH inhibited platelet aggregation on endothelial cells.

[0049] Figure 11This demonstrates how sialylation-lichen-FH protects PNH erythrocytes from complement-mediated opsonization. A) Left panel: PNH erythrocytes are characterized by the absence of the cell surface complement regulator CD59, making them susceptible to complement-mediated lysis upon exposure to ABO-matched acidified serum. Eculizumab prevents PNH erythrocyte lysis but leaves C3 deposits on the surface of CD59- erythrocytes, making them targets for extravascular hemolysis. Right panel: Summary and analysis of flow cytometry data from three PNH patients. Complement activation via aNHS leads to significant lysis of CD59-negative cells. While eculizumab treatment prevented lysis of CD59-negative erythrocytes, significant amounts of C3 deposits (CD59-C3c+) were detected. Conversely, treatment of PNH erythrocytes with sialylation-lichen-FH or pegcetacoplan prevented both erythrocyte lysis and pathological complement deposition. The data were analyzed using ANOVA and Tukey's HSD test for CD59- and CD59-C3c+ against aNHS. P<0.05; P<0.01; ###P<0.001). aNHS—acidified normal human serum. B) Red blood cells from PNH patients were incubated alone with acidified serum, eculizumab, or a combination of eculizumab and the C3 inhibitor peggac or sialylated-lichen-FH for 24 h. Red blood cells were then stained for CD59 and C3c, and the percentage of positive and negative staining cells was analyzed by flow cytometry. Left panel: Treatment of red blood cells with a combination of eculizumab and peggac or sialylated-lichen-FH effectively prevented pathological C3 deposition on PNH red blood cells. Right panel: Using the same experimental setup, red blood cells from PNH patients were treated with eculizumab in combination with increasing concentrations of sialylated-lichen-FH, and the number of C3c-positive PNH red blood cells was analyzed by flow cytometry. At concentrations above 1000 nM, sialylated-lichen-FH completely prevented pathological C3 deposition on PNH red blood cells.

[0050] Figure 12 Experiments conducted to determine the appropriate clinical dosage range are shown: A) Adding increased amounts of lichen-FH to the serum of different aHUS patients inhibited hemolysis; Patient 1: C-terminal FH deletion; Patient 2: FH mutation combined with C3 mutation; Patient 3: FH mutation R1215Q; Carrier 1: Patient 3's healthy sibling, a carrier of the FH mutation R1215Q; B) Increased amounts of lichen-FH caused C3NEF antibody shift; MAC concentration was measured by ELISA, and the figure shows the normalized value, 100% = starting value of normal human serum (NHS), and patients 1-3 had C3NEF antibodies.

[0051] Example Specific embodiments illustrating various aspects of the present invention will be presented below. However, the scope of the invention should not be limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described herein, will become readily apparent to those skilled in the art from the foregoing description and the following embodiments. All such modifications fall within the scope of the appended claims.

[0052] Example 1: In small bowl moss ( Physcomitrium patens Production of recombinant factor H protein in ) Recombinant human factor H with the amino acid sequence SEQ ID NO:1 is produced in *Sphagnum moss*, as previously described (Michelfelder et al.; J Am Soc Nephrol.; 2017;28(5):1462-1474). In short, the moss *Sphagnum moss* ( Physcomitrium patens This can be used for the industrial-scale recombinant production of human factor H. However, post-translational modifications, particularly N-glycosylation, must be considered, as there are some differences between plants and humans. While both human and plant glycoproteins contain a biantennary complex of N-glycans with the same core structure GnGn (A2 according to the Oxford notes, see Table 1), this core is linked to two plant-specific sugar residues that may be immunogenic to mammals. Through genome engineering via homologous recombination, these sugars (i.e., xylose and fucose) are completely removed, thereby altering the glycosylation pattern of mosses to produce human factor H. In this case, a double-knockout *Phyllostachys nigra* strain was used, in which the corresponding genes for α-1,3-fucosyltransferase (FT3) and β-1,2-xyloseyltransferase (XT) were replaced by knockout constructs, respectively. Both constructs contain truncated versions of the corresponding enzymes and additional stop codons. This results in premature termination of translation and the production of non-functional truncated enzymes. Therefore, none of the proteins in the plant, including the recombinant human FH protein, possess the plant-specific glycan structures containing β1,2-xylose or α1,3-fucose. Furthermore, recombinant factor H lacks the terminal sialic acid, which is typically absent in plants, in its glycans.

[0053] Moss cells were cultured in suspension in a 500L illuminated disposable stirred tank reactor (Sartorius Biostat STR500) and then purified using standard chromatographic and filtration procedures for production.

[0054] Example 2: Sialization of recombinant factor H protein Then, as Figure 2 As shown in Figure B, the FH protein produced by recombinant synthesis in Example 1 underwent a two-step in vitro sialylation process. The initial reaction buffer was prepared with the following conditions: 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. Then, in the first enzymatic reaction, galactosyltransferase (final concentration 5 mg / L) and its substrate UDP-galactose (final concentration 8 mM) and cofactor manganese chloride (MnCl2) (final concentration 4 mM) were added. Specifically, the substrate and cofactor were added in 10× concentrate form. The reaction was then incubated at 37°C for 7 hours. Subsequently, a second enzymatic reaction was carried out using sialyltransferase (final concentration 28.5 mg / L) and CMP-N-acetylneuraminic acid (final concentration 2.2 mM), the latter being a 10× concentrate. Simultaneously, alkaline phosphatase (final concentration 11 mg / L) and its cofactor zinc chloride (ZnCl2) (final concentration 50 μM) were added. The phosphatase cleaves CMP, thus preventing the sialyltransferase from reversing the reaction. The second enzymatic reaction was incubated at 37°C for 24 hours.

[0055] Example 3: Structural Analysis of FH Protein N-Glycans were analyzed by HILIC-HPLC-MS: To assess the characteristics and quantity of protein-linked N-glycans, glycans were released from the protein and labeled with procainamide. First, 50 μg aliquots were repeatedly taken from the provided glycoprotein solution and reduced with DTT (final concentration 5 mM) in 100 mM ammonium bicarbonate at pH 8.2 at 56 °C for 45 min. Iodoacetamide was added to a final concentration of 25 mM, and S-alkylation was performed in the dark for 30 min. The sample was then precipitated with 4 volumes of -20 °C acetone at -20 °C for 2 h. After washing the precipitate with 80% -20 °C acetone, the sample was rapidly dried under vacuum and digested overnight with PNGase F. The reaction solution was passed through a 25 mg C18 Hypersep centrifuge cartridge (ThermoScientific), collecting the flowing solution while retaining the protein, thus purifying the released N-glycans. The sample was then vacuum dried and labeled with procainamide in the presence of cyanoborohydride (65°C; 3 h; dark). After derivatization, it was purified by HILIC SPE on a Discovery glycan kit (50 mg; Supelco). The bound derivatized glycans were eluted with 500 µL of 20% acetonitrile aqueous solution. Before analysis, the glycans were dried under vacuum and dissolved in 20 μL of water. Separation was performed on a Nexera X2 HPLC system with an Acquity UPLC Glycan BEH amide column (2.1 × 150 mm, 1.7 μm; Waters) equipped with a Security Guard Ultra pre-packed column (Phenomenex) and an RF-20Axs fluorescence detector with a semi-microfluidic cell (Shimadzu, Korneuburg, Austria). Solvent A consisted of 80 mM formic acid adjusted to pH 4.4 with ammonia, and solvent B was 80% acetonitrile dissolved in solvent A. The gradient application started with an initial concentration of solvent B of 99%, maintained for 8 minutes, then decreased to 57% B over 60 minutes, followed by a decrease to 25% B over 2 minutes, at a flow rate of 0.4 ml / min. -1 The column temperature was set to 45°C, and the flow cell thermostat was set to 40°C. Fluorescence measurement wavelengths were Ex / Em 308 nm and 359 nm. The injection volume was 2.5 μl. Peak characteristics were evaluated by coupling the same HPLC system with a Bruker amaZon speed ETD ion trap mass spectrometer equipped with a standard ESI source. Spectra were recorded in positive ion mode.

[0056] Example 4: In vitro functional characterization of sialylated FH protein a) C3b cutting Complement factor H acts as a cofactor of complement factor I, whose proteolytic activity cleaves C3b. More specifically, factor I cleaves the α-chain of C3b while the β-chain remains intact. This reaction can be detected by a simple in vitro assay. Briefly, an increased amount of sialylated human factor H (0.5-10 ng) obtained from Example 2 is mixed with 0.5 μg of factor I and 2 μg of C3b and incubated at 37°C for 30 minutes. Subsequently, as... Figure 5 As shown, the degradation of C3b, more precisely, the degradation of the C3b α chain, was observed by Coomassie staining SDS-PAGE. C3b consists of an α chain of approximately 100 kDa and a β chain of approximately 65 kDa, which can be separated on reducing SDS-PAGE. Increased amounts of sialylated recombinant factor H resulted in a dose-dependent reduction of the intact C3b α chain and an increase in various degradation fragments of sizes 43, 46, and 68 kDa.

[0057] b) Protect sheep red blood cells Sheep erythrocytes (sE) serve as a model for studying complement activity via the alternative pathway (AP). Like human erythrocytes, sE expresses glycosaminoglycans on its surface and is normally protected from complement-mediated lysis by plasma-derived ferrous sulfate (FH). Incubation of sE with FH-depleted serum leads to AP-mediated activation of cell surface complement, C5b-9 formation, and subsequent hemolysis.

[0058] c) Hemolysis detection Perform hemolysis testing as described previously (Sanchez-Corral et al.; Mol Immunol 2004;41:81-84). In short, use 5x10... 7 Freshly prepared sE in GVB / Mg 2+ Dilute to a final volume of 25 µl in EGTA buffer. Use GVB / Mg 2+ Different amounts of factor H or controls were diluted to a final volume of 15 µl using GVB / EDTA buffer, and then 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 value of the supernatant was measured at 414 nm using a microplate reader, and the corresponding blank value (without serum) was subtracted from each value.

[0059] d) TCC-ELISA, measuring MAC formation Sialidized-moss-FH or sd-FH was diluted with NHS serum and incubated in wells pre-coated with LPS in the presence of AP-specific buffer. After washing, the formation of active C5b-9 was detected using an alkaline phosphatase-conjugated mAb that recognizes the C9 neoantigen formed during C5b-9 assembly. The wells were then incubated with alkaline phosphatase substrate solution for 30 minutes. Readings were taken at 405 nm using a microplate reader.

[0060] e) C3b combination The binding behavior of sialylated human factor H and sd-FH was analyzed using the C1 sensor chip in the Biacore T200 instrument used for the measurements. 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 activated with a solution of 60 µL EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) mixed with 60 µL NHS (N-hydroxysuccinimide). The solution was run at 10 µL / min for 420 seconds until the RU (response unit) of all channels reached at least 70. A single channel was used as a reference and was not fixed. The C3b solution was then run through the channels at 5 µL / min until an RU of approximately 150 was reached. After fixation, the solution was run through all four channels for 420 seconds at a flow rate of 10 µl / min using ethanolamine solution. A series of dilutions (250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2 pM) of sd-FH, moss-FH, and sialylated-moss-FH were prepared in HBS-P buffer and run through the system at 30 µl / min with a contact time of 60 seconds and a dissociation time of 300 seconds. The analytical and sample chamber temperatures were set to 10 °C. Kinetic parameters were calculated using Biacore T200 evaluation software.

[0061] The table below summarizes the obtained affinity constants. For all channels (with different C3b concentrations of Fc), the KD ratio between sd-FH and sialylated recombinant FH in Example 2 was 2.3-2.8, indicating that the concentration of sd-FH required to reach equilibrium and form the complex is more than twice the concentration of FH required in this invention.

[0062] Table 2: Affinity constants of sialylated-moss-FH and sd-FH obtained Example 5: Functional characterization of sialylated FH protein in mice FH(- / -) knockout mice are characterized by persistently activated AP, which leads to low serum C3 levels and significant C3 deposition in the kidneys. Functional characterization of the FH protein variant was performed using 8–12-week-old FH(- / -) mice (n=3 at each time point). Animals were administered via tail vein injection at a dose of 40 mg / kg, equivalent to 100–150 µL. Control animals received PBS injections. At defined time points (0, 0.5, 1, 2, 4, 6, 24, 48, 72, and 96 hours), approximately 60–100 µL of blood was sampled via tail vein and separated into plasma and serum (blood was coagulated for 30 minutes and then centrifuged at 2000 g for 10 minutes). FH and C3 concentrations in serum samples were analyzed using appropriate ELISA-based assays.

[0063] After 96 hours (4 days), the animals were sacrificed, and the kidneys were collected in PBS and rapidly frozen at -80°C. Glomerular C3 deposits were analyzed by fluorescent staining as follows: 10µm frozen sections of mouse kidneys were cut using a Leica CM 3050S cryostat, mounted on SuperFrost® plus glass slides, and stored at -80°C. After fixation in 4% paraformaldehyde solution, the sections were frozen in PBS with 0.5% Tween. ® Twenty slides were permeabilized, and C3 was detected using goat anti-mouse C3 and second rabbit anti-goat Alexa Fluor 488 conjugated antibodies. After washing, slides were mounted in mounting medium and covered with coverslips. The slides were observed using a microscope with the appropriate software. For quantitative immunofluorescence staining, the mean fluorescence intensity of the three glomeruli on each slide was measured using ImageJ software (National Institutes of Health, USA) and expressed as arbitrary fluorescence units (AFU). The change (%) of C3 deposits in each treatment group was then compared with the control (PBS-treated) group (set as 100%).

[0064] Example 6: Inhibition of Neisseria spp. ( Neisseria Growth assessment It is expected that sialylation-lichen-FH can support or restore physiological complement regulation by controlling the overactivation of complement in host cells, while retaining protective bactericidal properties. To demonstrate this mode of action, bacteria supplemented with Neisseria meningitidis (… Neisseria meningitidis Growth experiments were conducted using human serum.

[0065] Overnight-grown Neisseria meningitidis sera type B and type W were adjusted to OD 0.1 in 2x THY broth. The bacteria were then challenged with a mixture of complement-active human serum (NHS) (50%) or heat-inactivated (1 h 56°C) human serum (HI-NHS). The effects of sialylated-moss-FH or sd-FH on cell growth were investigated for bacteria treated with complement-active NHS. For this purpose, factor H was serially diluted in PBS at concentrations ranging from 0.004, 0.008, 0.015, 0.03, 0.06, 0.125, and 0.25 mg / ml.

[0066] As a control, the same concentrations of eculizumab and BSA were added. The bacteria were then incubated at 37°C for 60 minutes in 5% CO2. After serum challenge, the samples were placed on ice to block complement action. Subsequently, the bacteria were serially diluted in THY broth, and the diluted samples were transferred to blood agar plates. After incubation at 37°C for 24 hours in 5% CO2, colony-forming units (CFU) were counted. At least three replicates were performed for each serotype, starting with independent cultures.

[0067] Example 7: 125 Biodistribution of I-SIB-labeled FH protein To evaluate the systemic exposure, pharmacokinetic characteristics, tissue distribution, and excretion of sialylated-moss-FH and sd-FH, different FH variants were radiolabeled and administered intravenously to healthy CD1 mice.

[0068] Detailed introduction: 1.65 mCi (61 MBq, 0.75 nmol) of iodinated NHS ester ligand 125 Transfer I-SIB to a 1.5 mL test tube and evaporate to dryness using a nitrogen stream. Add to the solution containing... 125 Add 400 μg of FH protein and approximately 130 µL of 1×PBS (pH 7.4) to an I-SIB tube. Add 0.2 M borate buffer (pH 8.3) to adjust the pH to 8, bringing the final volume to 0.2 mL. Gently stir the mixture at room temperature for 30 minutes. Adjust the volume to 0.5 mL with a solution of 20 mM Tris and 0.15 M NaCl (pH 8.5) containing 0.02% PS 20. Use 10% trichloroacetic acid as the eluent and determine the percentage of associated radioactivity by transient thin-layer chromatography (ITLC). After the incubation period, pass the solution through a PD-10 desalting column containing Sephadex G-25 resin and elute with 0.02% Tween... ® 20 mM Tris and 0.15 M NaCl solution at pH 8.5 were used to remove unreacted... 125I-SIB. Radiochemical purity (ITLC / 10% TCA precipitation) and total activity in solution were determined based on the final labeled protein solution. The quality of the radiolabeled proteins was evaluated by radioactive SE-HPLC and SDS-PAGE. Each 125 The radiochromatograms of the I-SIB-factor H variant solutions all showed a single main peak (>98%), corresponding to the factor H variant as a monomer. Unlabeled and 125 The I-SIB-factor H variant was consistent with the electrophoretic pattern obtained after Coomassie blue staining. Therefore, the radiolabeling process did not lead to protein degradation compared to the unlabeled variant. Dosing solutions were prepared by diluting the labeled sialylated-moss-FH and sd-FH with their respective unlabeled proteins, resulting in a specific activity of approximately 0.025 mCi / mg (0.925 MBq / mg) and an FH concentration of 6.7 mg / mL for each solution.

[0069] For in vivo administration: CD-1 mice weighing approximately 25–32 g (7 weeks old) were used (3 mice at each time point). Anesthetized mice were intravenously injected (using isoflurane gas) at a dose level of 40 mg / kg, corresponding to a volume of 148–190 µL, with an activity of 0.93–1.19 MBq. The injection was administered via the retro-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 sampled from the retro-orbital plexus of the anesthetized mice. At the final time point, blood samples were obtained by exsanguination via intracardiac puncture of mice anesthetized with a mixture of ketamine and toluene-thiazide. Blood samples were collected into pre-weighed Microvette® tubes containing lithium heparin (Sarstedt), and radioactivity was measured using a gamma counter. The blood samples were then processed to obtain plasma (centrifuged at 2000 g for 5 min), and the plasma samples were analyzed for radioactivity using a gamma counter. Radioactivity in plasma samples was expressed as a percentage of the injected dose per gram (ID / g%). For these calculations, the total blood volume of the mice was estimated to be 7.5% of the mouse body weight, and the hematocrit was assumed to be 0.53% (Janvier Laboratory—hematological data from 10-week-old female CD-1 mice). Furthermore, a blood density of 1.06 and a plasma density of 1 were assumed. Given the single-compartment model system, the half-life of each compound was calculated from early time points (2 min to 6 hours).

[0070] At the endpoint time (30 minutes post-injection), mice were intraperitoneally injected with an excess of a mixture of ketamine hydrochloride and toluidine hydrochloride, followed by rapid euthanasia via endocardial puncture. Target organs were harvested, rinsed with 0.9% NaCl, placed in vials, weighed, and their radioactivity measured using a gamma counter. Selected tissues / organs included the bladder (empty), liver, pancreas, spleen, kidneys, lungs, heart, gastrointestinal tract (stomach, small intestine, and colon and their contents), brain, skeletal muscle, thyroid gland, head, and tail. Both kidneys were counted separately. The liver was cut into two segments, each segment was counted separately, and the overall liver concentration was calculated. Radioactivity concentration was expressed as a percentage of the injected dose per gram of organ (ID / g%).

[0071] Example 8: Platelet aggregates on HMEC-1 under flow conditions In aHUS, complement activation leads to loss of endothelial antithrombotic properties, resulting in microvascular thrombosis. For this reason, the inventors investigated the ability of sialylation-lichen-FH to prevent platelet aggregate formation on microvascular endothelial cells.

[0072] For platelet aggregate formation analysis, HMEC-1 cells were activated with 10 μM ADP and then incubated for 3 h with a pool of normal human serum (NHS) or primary aHUS serum (patient aHUS 6-8, 271, 1:2 diluted in HBSS + 0.5% BSA) in the presence or absence of sialylated-lichen-FH (500 μg / mL) 218 ​​or sd-FH (500 μg / mL) or peagtag (1 mg / mL). Subsequently, HMEC-1 cells were perfused in a flow chamber with heparinized whole blood from healthy subjects (laced with the fluorescent dye mipalin to label platelets), as reported (Aiello S et al., Blood Adv (2022) 6:866-881). After 3 minutes of perfusion, the endothelial cell monolayer was fixed in acetone. Fifteen images were acquired for each sample, and the area occupied by thrombi was assessed using ImageJ software. The highest and lowest values ​​were discarded, and the average value was calculated from the remaining 13 views.

[0073] ADP-activated HMEC-1 cells were pre-exposed to serum collected from patients with primary aHUS during the acute phase, followed by heparinized whole blood perfusion. The cell surface area covered by platelet aggregates increased by an average of 6-fold compared to cells exposed to a control serum pool. Figure 10 Compared to untreated serum, the addition of sialylated-lichen-FH, or peggac, or sd-FH to patient serum resulted in a significant reduction in cell surface area covered by platelet aggregates. Figure 10 Sialidized-lichen-FH inhibited serum-induced thrombosis of aHUS with comparable efficacy to sd-FH at the same concentration. Figure 10 ).

[0074] Example 9: Inhibition of C3c deposition on the surface of PNH erythrocytes Patients with proximal complement hypersensitivity (PNH) lack the surface complement regulators CD55 and CD59 on GPI-deficient stem cell progeny, leading to intravascular hemolysis in affected patients. Furthermore, terminal complement inhibition inevitably results in C3 deposition on erythrocytes, causing opsonization and extravascular destruction of macrophages in the liver and spleen. Therefore, many PNH patients treated with eculizumab still suffer from anemia. Proximal complement inhibitors and FH have the advantage of acting at AP-activated C3 levels, preventing erythrocyte C3-mediated opsonization and subsequent extravascular hemolysis.

[0075] The ability of sialylated-lichen-FH and sd-FH to prevent C3c deposition on the surface of PNH erythrocytes was tested, as previously described (Yuan X, et al. Haematologica (2017) 102:466-475). Briefly, EDTA-treated blood from PNH patients was washed three times in saline, and 2 μL aliquots of erythrocytes were mixed with either PBS alone or 0.5 μM eculizumab with increasing concentrations of sialylated-lichen-FH (0, 0.2, 0.5, 1, 2, 5 μM) or 12 μM PegTaq, for a total volume of 10 µL. The inventors then added 30 µL of ABO-matched, acid-activated (0.1 M HCl, 1:10 dilution) NHS, supplemented with 2 mM MgCl2, and incubated the cells at 37°C for 24 h. Cells were washed with PBS containing 2 mM EDTA and stained with antibodies against CD59 (anti-human CD59 PE; Biolegend, San Diego, USA) and C3c (anti-human C3c FITC; Dako / Agilent Technologies, Santa Clara, USA) prior to flow cytometry analysis.

[0076] While all tested inhibitors resulted in a significant increase in CD59-red blood cells, eculizumab treatment led to a significant increase in C3 deposition on erythrocytes. In contrast, treatment with sialylation-moss-FH and peggac almost completely prevented C3 deposition. Figure 11 ).

[0077] Example 10: Clinical Dosage Range To establish the clinical dose range for sialylated-lichen-FH, in vitro assays were performed using patient serum from aHUS and C3G patients. Lichen-FH (with the same amino acid sequence but different glycosylation) was used as an alternative assay for these procedures. This approach was considered valid because it demonstrated that lichen-FH, sialylated-lichen-FH, and sd-FH produced similar responses in hemolysis assays, which formed the basis for the experiments performed. Therefore, the calculated dose range for lichen-FH is also validly applicable to sialylated-lichen-FH.

[0078] The serum of patients with aHUS and C3G is characterized by overactivation of the complement system, leading to increased consumption of serum C3 and continuous formation of the terminal membrane attack complex (MAC). Since complement factor H can regulate complement activity, it is expected that the addition of lichen-FH to the patient's serum will result in a decrease in complement activity.

[0079] Complement activity was assessed using a sheep erythrocyte hemolysis assay (see above). In this assay, sheep erythrocytes were mixed with human serum. Because erythrocytes are susceptible to complement-mediated lysis, the level of erythrocyte lysis (measured by absorbance at 414 nm) is an indicator of complement activity. Normal human serum served as a control (baseline).

[0080] The addition of 100-500 nM lichen-FH resulted in a decrease in complement activity to levels seen in healthy controls. Based on these results, the calculated clinical dose range was 3-15 mg lichen-FH / kg body weight (Table 3).

[0081] Table 3: Calculation of Clinical Dosage Range; Calculated based on 40ml serum / kg body weight Approximately 80% of C3G patients possess C3NEF antibodies, which stabilize C3 convertase. This, in turn, leads to increased C3 cleavage and overactivation of the complement cascade (excessive terminal membrane attack complex, MAC). Increased levels of lichen-FH were supplemented in the serum of different C3G patients with C3NEF antibodies. The formation of terminal MAC was subsequently assessed by ELISA.

[0082] Adding increased amounts of moss-FH resulted in a reduction in the terminal membrane attack complex, indicating that moss-FH can displace the C3NEF antibody and induce the dissociation of C3 convertase. This, in turn, leads to a rebalancing of the supplemental system. Similarly, doses between 100-500 nM (equivalent to 3-15 mg moss-FH / kg body weight) resulted in a reduction to control levels.

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

Claims

1. A method for treating paroxysmal nocturnal hemoglobinuria (PNH), thrombotic inflammation, platelet aggregation, microangiopathy, or chronic COVID in subjects in need, wherein the sialylated FH protein does not contain a trisialylated N-glycan with the structure A3G3S3(NaNaNa).

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

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

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

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

6. The sialylated FH protein used according to any of the preceding claims, wherein the protein exhibits one of the following at the corresponding amino acid residues at position 62 and / or position 402 of the canonical FH (UniProtKB-P08603-1; entry version 245, sequence version 4): I62, Y402, or I62 and Y402.

7. The sialylated FH protein used according to any one of the preceding claims, wherein the protein comprises the amino acid sequence according to SEQ ID NO:

1.

8. The sialylated FH protein used according to any one of the preceding claims, wherein the protein binds to human C3b protein, cleaves the α chain of C3b by proteolytic means, and / or protects sheep red blood cells from lysis.

9. The sialylated FH protein used according to any one of the preceding claims, wherein the protein reduces platelet aggregation, thrombosis and / or C3 deposition.

10. The sialylated FH protein used according to any one of the preceding claims, wherein the sialylated FH protein is used to treat PNH.

11. The sialylated FH protein used according to claim 10, wherein the method further comprises administering a second therapeutic agent for treating PNH.

12. The sialylated FH protein used according to claim 11, wherein the second therapeutic agent for treating PNH is a C5 inhibitor.

13. The sialylated FH protein used according to claim 12, wherein the C5 inhibitor is eculizumab.

14. The sialylated FH protein used according to any one of claims 1 to 9, wherein the sialylated FH protein is used to treat platelet aggregation in a subject in need, particularly wherein the subject is a subject suffering from a complement disorder or condition.

15. The sialylated FH protein used according to any one of claims 1 to 9, wherein the sialylated FH protein is used to treat thrombotic inflammation in a subject in need, particularly wherein the subject is a subject suffering from complement disorder or condition.

16. The sialylated FH protein used according to any one of claims 1 to 9, wherein the sialylated FH protein is used to treat microvascular disease in a subject in need, particularly wherein the subject is a subject suffering from complement disease or condition.

17. The sialylated FH protein used according to any one of claims 1 to 9, wherein the sialylated FH protein is used to treat long-term COVID in a subject in need, particularly wherein the subject is a subject with a complement disorder or condition.

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