Factor H enhancing antibodies and uses thereof

Abstract

Provided herein are novel isolated, synthetic, or recombinant antibodies and fragments thereof, or nucleic acids or vectors encoding such antibodies and fragments, specific for Factor H. Further provided herein are uses of such antibodies, fragments, nucleic acids, or vectors for inhibiting complement activation and treating disorders associated with complement activation.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 875,309, filed July 17, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes. This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on August 4, 2020, is named GEM-013WO_SL.txt and is 18,037 bytes in size. [Background technology]

[0002] The complement system is a key component of innate immunity that contributes to protecting many organisms, including mammals, from invading pathogens. It consists of more than 30 different components that are primarily synthesized in the liver. Activation of the complement system occurs by three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. The three pathways converge with the formation of C3 convertase, which has a different but similar activity in each pathway.

[0003] In the classical complement pathway, binding to an antibody-antigen complex leads to activation of the complement component (C)1 complex, which consists of C1q, C1r, and C1s. The C1 complex cleaves C4 and C2, leading to the formation of the C3 convertase, which consists of C4bC2a. The C3 convertase cleaves C3 into the active components C3a and C3b. In the lectin pathway, mannose-binding lectins bind to mannose residues on the surface of pathogens and activate the serine proteases MASP-1 and MASP-2, which can cleave C4 and C2. As in the classical pathway, this leads to the formation of the C4bC2a C3 convertase, which can bind to C3b to form the C5 convertase. Contrary to the classical and lectin pathways, the alternative pathway has a low level of sustained activity due to the spontaneous hydrolysis of C3 to C3(H2O) in plasma. This C3b-like C3(H2O) can form fluid-phase C3 convertase by binding to factor B (FB), which is then activated to Bb by factor D. Similarly, when C3b binds to a surface, it can bind to FB to form C3bB. This complex is cleaved by factor D to form the alternative pathway C3 convertase, C3bBb, which can be stabilized to C3bBbP by properdin (factor P). This C3 convertase can cleave C3 to C3a and C3b. In addition to this process, the alternative pathway acts as an amplification loop for the classical pathway and the lectin activation pathway, because C3b generated in these pathways can serve as the starting point for the alternative pathway. The amplification loop thereby results in the reinforcement of the classical pathway and the lectin pathway. The C3 convertase formed in one of the three activation pathways can bind to C3b to form C5 convertase. The C5 convertases of all three complement pathways activate C5 to C5a and C5b, which initiate the terminal pathway of the complement system. C5b binds to C6, C7, C8, and C9 to form the membrane attack complex (MAC), which forms a transmembrane channel and causes cell lysis.

[0004] In addition to forming pores in the membrane of pathogens, complement helps to eliminate pathogens or altered self-cells by opsonization with C3b molecules and production of proinflammatory peptides such as C3a and C5a that attract and activate immune cells to the site of infection. Due to the strong proinflammatory nature of complement, host cells are well protected by several membrane and soluble complement regulatory proteins.

[0005] The alternative pathway contributes 80-90% of the total complement activity. Regulation of this pathway is therefore crucial. C3(H2O), formed by spontaneous hydrolysis of C3, and C3b are normally rapidly inactivated by factor H (FH), factor I (FI), and host cell surface molecules in the absence of pathogen binding, thereby inhibiting the formation of C3 convertase. CD55 (also known as decay accelerating factor or DAF) binds to C3b on the host cell surface. FI cleaves C3b to an inactive form, which depends on either cofactors expressed on the cell surface (CD46, MCP) or cofactors circulating in the plasma (FH).

[0006] FH is a plasma glycoprotein essential for controlling the alternative pathway of complement both in lytic conditions and on the cell surface. FH binds C3b at the same position as FB, thereby preventing the formation of C3 convertase. FH also has decay-accelerating activity, i.e., promoting the dissociation of alternative pathway C3 convertases once they are formed. Whether FH binds to C3b depends on the carbohydrates present on the cell surface. Sialic acid, glycosaminoglycans, and heparin present on the host cell surface promote FH binding to C3b, whereas binding of C3b to molecules expressed on the surface of pathogens results in binding of FB. Thus, FH exerts its complement inhibitory activity on host cells but not on the surface of pathogenic cells, because the cell surface molecules that bind FH are expressed on host cells but not usually on pathogenic cells. FH contains 20 complement control protein (CCP) domains, numbered 1-20 starting from the N-terminus of FH. CCP domains are also called short consensus repeats (SCRs) or sushi domains. CCP1-4 are domains involved in regulation, CCP19-20 are involved in binding C3b, and CCP6, 7, 8, 19, and 20 bind to GAG and sialic acid expressed on the surface of cells. Antibodies that bind to CCP19 and / or CCP20 inhibit the activity of FH.

[0007] Factor H-related proteins (CFHR) are plasma glycoproteins related in structure and antigenicity to FH. FHR proteins are also composed of CCP domains, which have various degrees of homology to the CCP domains found in FH. For example, FHR1 contains domains corresponding to CCP6, CCP7, CCP18, CCP19 and CCP20. In contrast to FH, CFHRs do not have strong complement inhibitory activity. A common feature of CFHRs is that they bind to the C3b component of complement, thereby competing with FH for binding to C3b, and are therefore considered positive regulators of the alternative pathway of complement.

[0008] FH deficiency or impaired recognition of host surfaces due to mutations is associated with complement-mediated tissue damage and disease. In addition to controlling complement activation during normal hemostasis, FH also plays an important role in limiting complement-mediated damage to diseased cells and tissues. Multiple mutations in the FH gene that can lead to loss of function of the FH protein have been reported. The C-terminal region of FH is a hotspot for disease mutations. This is the critical region for FH binding to host cells. Most disease-associated mutations in this region interfere with FH binding. Most patients with mutant FH genes have heterozygous mutations, meaning that roughly half of the circulating FH has normal function. However, this does not appear to be sufficient to protect self-surfaces under certain conditions where complement is activated. FH deficiency can lead to kidney diseases such as membranoproliferative glomerulonephritis (MPGN) and atypical hemolytic uremic syndrome (aHUS). More recently, a relationship between FH mutations and age-related macular disease (AMD) has been reported.

[0009] Currently, the standard treatment for FH deficiency, such as in aHUS, is plasma replacement or plasma exchange. In such therapy, the missing complement regulator is replaced. Plasma exchange further removes mutant complement factors and / or autoantibodies against complement factors. However, plasma therapy also has some limitations. No prospective clinical trials to date have shown that plasma exchange is safe or effective for the treatment of aHUS, and that the efficiency of plasma therapy may depend on the underlying mutation. Some patients may develop anaphylactic reactions to fresh frozen plasma, necessitating the cessation of all forms of plasma therapy. Furthermore, plasma exchange may worsen the clinical picture of aHUS due to the administration of active pathogenic complement components derived from the plasma.

[0010] Recently, the therapeutic monoclonal antibody eculizumab has been approved for the treatment of aHUS and paroxysmal nocturnal hemoglobinuria (PNH) in several countries, including the United States and European countries. Eculizumab is a humanized murine monoclonal antibody specific for C5 that prevents C5 from being cleaved to C5a and C5b, thus preventing activation of the terminal pathway and reducing the influx of immune cells. However, the use of eculizumab is associated with unwanted side effects. Because it blocks C5, a crucial component for the initiation of the terminal pathway, patients treated with eculizumab are more susceptible to infection with encapsulated bacteria (such as Neisseria meningitidis) whose clearance is highly dependent on MAC formation. Therefore, vaccination against Neisseria meningitidis is required for patients before undergoing treatment with eculizumab. Furthermore, because eculizumab acts downstream of C3, C3 deposition is maintained, which is detrimental in several disorders involving unnecessary or excessive complement activation. Furthermore, eculizumab treatment is associated with high costs and availability of the antibody is limited.

[0011] A mouse monoclonal antibody that binds to CCP18 has been reported by Cheng et al. (Clinical Chemistry, 2005). This antibody, called X52.1, has been reported to increase the binding of FH to C3b and C3d, which is believed to be caused by dimerization of FH. The increased binding of FH to C3b and C3d induced by X52.1 leads to increased complement-mediated lysis of cells, including RBCs and several types of cancer cells, as shown by Corey et al. (J Biol Chem. 2000). This demonstrates that the antibody X52.1 inhibits the complement inhibitory activity of FH. Indeed, Corey et al. suggest that this antibody could be used to treat cancer by enhancing complement-mediated lysis of cancer cells.

[0012] There remains a need for new and improved therapeutic agents that bind to FH, such as agents useful in the treatment of disorders associated with unwanted or excessive complement activation. Summary of the Invention

[0013] The present invention provides antibodies or antigen-binding fragments thereof, e.g., isolated, synthetic, or recombinant antibodies or antigen-binding fragments thereof, that specifically bind to complement control protein domain 18 (CCP18) of factor H (FH). Such antibodies and antigen-binding fragments are useful for enhancing the activity of FH, e.g., increasing the binding affinity of FH for C3b, increasing the ability of FH to inhibit C3 deposition, and / or increasing the ability of FH to inhibit hemolytic activity. In some embodiments, the antibody or fragment comprises a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO:1) or the sequence TSVTY (SEQ ID NO:13), a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO:2) or the sequence ASS (SEQ ID NO:14), a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO:3), a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO:6) or the sequence IWSGGTT (SEQ ID NO:10), and a heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDY (SEQ ID NO:7) or the sequence ARNFGNYAMDF (SEQ ID NO:11).

[0014] In some embodiments, the antibody or fragment comprises a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO:1), a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO:2), a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO:3), a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence IWSGGTT (SEQ ID NO:10), and a heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDF (SEQ ID NO:11). In some embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4 or 16, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8 or 12. In some embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:12.

[0015] In some embodiments, the antibody or fragment comprises a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO:1), a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO:2), a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO:3), a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO:6), and a heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDY (SEQ ID NO:7). In some embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8.

[0016] In some embodiments, the antibody or fragment comprises a light chain CDR1 sequence comprising the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence comprising the sequence ASS (SEQ ID NO: 14), a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO: 3), a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO: 5), a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO: 6), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7). In some embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:16, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8.

[0017] In some embodiments, the antibody or fragment comprises a light chain CDR1 sequence comprising the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence comprising the sequence ASS (SEQ ID NO: 14), a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO: 3), a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO: 5), a heavy chain CDR2 sequence comprising the sequence IWSGGTT (SEQ ID NO: 10), and a heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDF (SEQ ID NO: 11). In some embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:16, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:12.

[0018] In some embodiments, the antibody or fragment has a titer of 2.5×10 to FH. -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D or the antibody or fragment has a binding affinity for FH of 1.25×10 -8 K below M D and / or 0.04 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D In some embodiments, the antibody or fragment has a binding affinity for FH of 2.5×10 -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below MD or the antibody or fragment has a binding affinity of 0.6×10 to FH. -8 K below M D and / or 0.6 × 10 for the FH fragment consisting of CCP18-20 -11 K below M D In some embodiments, the antibodies or fragments of the disclosure inhibit C3 deposition in lipopolysaccharide (LPS) in vitro in the presence of 10% (v / v) serum with an IC of 38 nM or less. 50 value, e.g., IC below 30 nM 50 value and / or in vitro hemolytic activity in the presence of 10% (v / v) serum with an IC value of 150 nM or less 50 value, e.g., IC below 130 nM 50 value and / or the K of FH against C3b in vitro in the presence of 10% (v / v) serum. D and / or increase the binding affinity of FH to C3b in vitro in the presence of 10% (v / v) serum by at least 3-fold. In certain embodiments, the serum is normal human serum.

[0019] In some embodiments, the antibody or fragment enhances FH activity, and optionally the FH activity is inhibition of alternative complement activation, and further optionally the inhibition of alternative complement activation includes inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition, and / or increased binding of FH to C3b, iC3b, and / or C3d. In some embodiments, the antibody or fragment comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, and the antibody or fragment further comprises an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region. In some embodiments, the antibody or fragment comprises an IgG constant region. In some embodiments, the IgG constant region is IgG1, IgG2, IgG3, or IgG4 (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibody or fragment comprises an IgG4 (e.g., human IgG4) constant region. In some embodiments, the IgG4 constant region comprises a mutation at position 297. In certain embodiments, the IgG4 constant region comprises amino acid residue Q at position 297. In certain embodiments, the IgG4 constant region comprises amino acid residue A at position 297.

[0020] In some embodiments, the fragment comprises at least a Fab fragment. In some embodiments, the antibody or fragment is a monoclonal antibody or a fragment thereof. In some embodiments, the antibody or fragment is a chimeric or humanized antibody comprising human light and heavy chain constant regions or a fragment thereof. In some embodiments, the antibody or fragment is PEGylated. In some embodiments, the antibody or fragment is a PEGylated Fab fragment.

[0021] In some embodiments, the present disclosure provides an isolated, synthetic, or recombinant nucleic acid comprising a nucleic acid sequence encoding any of the antibodies or fragments disclosed herein. In some embodiments, the present disclosure provides a vector comprising any of the nucleic acids disclosed herein. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0022] In some embodiments, the disclosure provides a recombinant cell comprising any of the nucleic acids disclosed herein, or any of the vectors disclosed herein.

[0023] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any antibody or fragment disclosed herein, any nucleic acid disclosed herein, any vector disclosed herein, or any recombinant cell disclosed herein, and a pharma- ceutically acceptable carrier, diluent, and / or excipient.

[0024] In some embodiments, the disclosure provides any antibody or fragment disclosed herein, any nucleic acid disclosed herein, any vector disclosed herein for use in therapy. In some embodiments, the disclosure provides any antibody or fragment disclosed herein, any nucleic acid disclosed herein, or any vector disclosed herein for use in inhibiting alternative complement activation. In some embodiments, the disclosure provides any antibody or fragment disclosed herein, any nucleic acid disclosed herein, or any vector disclosed herein for use in treating, alleviating, or preventing a disorder associated with alternative pathway complement activation. In some embodiments, the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy.

[0025] In some embodiments, the disclosure provides a use of any antibody or fragment disclosed herein, any nucleic acid disclosed herein, or any vector disclosed herein for the preparation of a medicament for the treatment, alleviation, or prevention of a disorder associated with alternative pathway complement activation. In some embodiments, the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy.

[0026] In some embodiments, the disclosure provides a method of treating, alleviating, or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof a therapeutically effective amount of any antibody or fragment disclosed herein, any nucleic acid disclosed herein, any vector disclosed herein, or any pharmaceutical composition disclosed herein. In some embodiments, the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy.

[0027] In some embodiments, the present disclosure provides a method of inhibiting alternative complement activation comprising administering to a subject any antibody or fragment disclosed herein, any nucleic acid disclosed herein, any vector disclosed herein, or any pharmaceutical composition disclosed herein.

[0028] In some embodiments, the disclosure provides a method of producing any of the antibodies or fragments disclosed herein, comprising providing a cell with any of the nucleic acids disclosed herein or any of the vectors disclosed herein and causing the cell to translate the nucleic acid sequence contained in the nucleic acid or vector, thereby producing the antibody or fragment. In some embodiments, the method further comprises harvesting, purifying, and / or isolating the antibody or fragment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] A. General Techniques and Definitions Unless otherwise defined herein, scientific and technical terms described herein shall have the meanings commonly understood by those skilled in the art. Generally, the nomenclature related to pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein, and the techniques thereof, are well known and commonly used in the art. In case of discrepancy, the present specification, including definitions, shall prevail.

[0030] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of a person of ordinary skill in the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press, Animal Cell Culture (RIFreshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-1998) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Gene Transfer Vectors for Mammalian Cells(JMMiller and Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001), Ausubel et al. al.This is explained in detail in publications such as Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998), Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003), and Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0031] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly practiced in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0032] Throughout this specification and the embodiments, the term "comprise" or variations such as "comprises" or "comprising" should be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0033] Whenever an embodiment is described herein using the term "comprising," it is understood that similar embodiments are also provided except that they are described using the terms "consisting of" and / or "consisting essentially of."

[0034] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0035] Any example or examples following the term "eg" or "for example" are not intended to be exhaustive or limiting.

[0036] Unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0037] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0038] "Treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. With respect to a disease or condition (e.g., an eye disease), treatment refers to reducing or reversing the progression, severity, and / or duration of an infection (e.g., an eye disease or symptoms associated therewith), or ameliorating one or more symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents).

[0039] As used herein, the terms "specific" and "specifically binds" or "capable of specifically binding" refer to a non-covalent interaction between an antibody and its epitope. This indicates that the antibody or fragment binds preferentially to its epitope over other binding sites or other antigens. Thus, although the antibody or fragment may bind non-specifically to other binding sites or antigens, the binding affinity of the antibody or fragment to its epitope is significantly higher than the binding affinity of the antibody or fragment to any other binding site or antigen.

[0040] The percentage of identity of an amino acid or nucleic acid sequence, or the term "% sequence identity", is defined herein as the percentage of residues in a full-length amino acid or nucleic acid sequence that are identical to the residues in a reference amino acid or nucleic acid sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art, such as "Align 2".

[0041] As used herein, "FH.07" refers to an antibody comprising the variable light chain amino acid sequence of SEQ ID NO:32 and the variable heavy chain amino acid sequence of SEQ ID NO:36.

[0042] As used herein, "FHR-1.3B4" refers to an antibody comprising the variable light chain amino acid sequence of SEQ ID NO:48 and the variable heavy chain amino acid sequence of SEQ ID NO:52.

[0043] In the amino acid sequences presented herein, amino acids are represented by single letter codes. These one-letter and three-letter symbols are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine.

[0044] The term "enhancing FH activity" means that the activity of FH is increased when the antibody or fragment of the present disclosure binds to FH. In some embodiments, the activity of FH enhanced by the antibodies and fragments of the present disclosure is the inhibition of alternative complement activation, for example in an individual. As used herein, the term "alternative complement activation" refers to activation of the complement system via the alternative pathway, i.e., involving at least the formation of the alternative pathway C3 convertase, i.e., C3bBb / C3bBbP, or involving an increase in the formation of this C3 convertase. Alternative complement activation may further involve cleavage of C3 to C3a and C3b by the alternative pathway C3 convertase, formation of the alternative pathway C5 convertase, i.e., C3bBbC3b / C3bBbC3bP, and / or cleavage of C5 and subsequent binding of C6, C7, C8, and C9 to form MAC. Alternative complement activation may further include an increase in the alternative pathway amplification loop.

[0045] In some embodiments, any of the antibodies or fragments disclosed herein are capable of inhibiting any one or more of the alternative complement activation processes disclosed herein. In some embodiments, alternative complement activation is inhibited in an individual (e.g., in an individual's bodily fluids, such as blood, interstitial fluid, or cerebrospinal fluid). As used herein, the term "individual" is used synonymously with "subject" or "patient" and refers to an animal, such as a human or a mammal (e.g., rodent, monkey, horse, cow, pig, dog, cat), that includes a complement system as part of its immune system. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a human.

[0046] As used herein, "inhibition of alternative complement activation" includes any change in the amount or activity of a component, factor, or activity of the alternative complement system that causes or is a result of its inhibition. Inhibition of alternative complement activation can include, for example, inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition in the cells of a subject, increased binding of FH to C3b, iC3b, and / or C3d, inhibition of formation of the alternative complement pathway C3 convertase C3bBb / C3bBbP, inhibition of binding of factor B to C3b and / or inhibition of the interaction between C3b and factor B, inhibition of cleavage of C3 into C3a and C3b by the alternative pathway C3 convertase, inhibition of the induction of C3 by the alternative pathway C3 convertase in host cells, particularly those expressed in host cells. These include increasing the binding of FH to sialic acid, glycosaminoglycans and / or heparin, inhibiting the amplification loop of the alternative complement pathway, and inhibiting the formation of the alternative complement pathway C5 convertase C3bBbC3bP / C3bBbC3bP, inhibiting the cleavage of C5 into C5a and C5b by the alternative pathway C5 convertase, increasing the decay acceleration activity of FH, i.e., promoting the dissociation of the alternative pathway C3 convertase when it is formed, and / or increasing the FI cofactor activity resulting in the degradation of C3b. In certain embodiments, inhibition of alternative complement activation by FH enhanced by the anti-FH antibodies and fragments of the present disclosure includes inhibiting hemolytic activity, inhibiting C3 deposition in the subject's cells, and / or increasing the binding of FH to C3b, iC3b and / or C3d.

[0047] As used herein, "inhibition" preferably means that the indicated activity is reduced by at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Thus, "inhibition of alternative complement activation" means that the activity of the alternative complement pathway is reduced by at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Similarly, "inhibition of hemolytic activity" means that the hemolytic activity is reduced by at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0048] As used herein, "increase" preferably means that the indicated activity is increased by at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Thus, "increase in binding of FH to C3b" means that the binding of FH to C3b is increased by at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Similarly, "increasing binding of FH to host cells" means that binding of FH to host cells is increased by at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%.

[0049] As used herein, "hemolytic activity" refers to the rupture of red blood cells and the subsequent release of the cell's contents, e.g., into the circulation, induced by the activation of the complement system, preferably as a result of the formation of MAC on the cell surface. Hemolytic activity is measured, for example, by using the hemolysis assay reported by Sanchez-Corral et al. (2004) and Wouters et al. (2008), optionally with some modifications, as described in the Examples herein. In this representative and non-limiting assay, red blood cells, such as sheep red blood cells (SRBCs), are incubated with serum (e.g., human serum) for 1.25 hours, e.g., at 37°C, with shaking. Serum containing low levels of FH or dysfunctional FH results in the lysis of SRBCs. Lysis can be stopped by the addition of veronal buffer containing 20 mM EDTA, followed by centrifugation for 2.5 minutes in a pre-cooled centrifuge (e.g., at 7°C). The percentage of red blood cell lysis is determined by measuring the absorbance of the supernatant at 412 nm. The serum may be, for example, from a healthy human individual or from a human individual suffering from a disorder associated with unnecessary or excessive alternative pathway complement activation, such as aHUS. The ability of the antibody or fragment to inhibit hemolytic activity may be determined by incubating red blood cells with serum in the presence of the antibody or fragment.

[0050] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of an antibody or functional portion or functional equivalent and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (in this application, an antibody and an antigen). As used herein, binding affinity is defined as the k a vs. k d The equilibrium dissociation constant (K D) See, for example, Chen, Y., et al., 1999. Affinity can be measured by common methods known in the art, such as, for example, surface plasmon resonance (SPR) assays, such as the BiaCore (GE Healthcare Life Sciences GE Healthcare Life Sciences) or IBIS-iSPR instruments from IBIS Technologies BV (Hengelo, the Netherlands), or solution phase assays, such as Kinexa.

[0051] B. Anti-FH Antibodies and Fragments Thereof WO 2016 / 028150 describes a murine agonist anti-FH antibody called FH.07 that inhibits alternative pathway activation as shown by increased binding of FH to C3b, inhibition of mediated C3 deposition, and inhibition of hemolytic activity. Fab' and F(ab')2 fragments of the antibody were shown to have the same FH enhancing effect. WO 2019 / 139481 describes another murine agonist anti-FH antibody called FHR-1.3B4 that has a higher binding affinity for FH than FH.07 and an increased ability to enhance FH activity.

[0052] In one embodiment, the present disclosure provides agonistic anti-FH antibodies (e.g., humanized antibodies) and fragments thereof, i.e., antibodies and fragments that enhance FH activity. In one embodiment, these antibodies compete with antibody FH.07 or antibody FHR-1.3B4 for binding to the same epitope in domain CCP18 of FH. Enhanced anti-FH antibodies are potent inhibitors of alternative complement pathway activation and are therefore useful in the treatment of disorders associated with unnecessary or excessive activation of the alternative pathway of the complement system. FH specifically inhibits the amplification loop of the alternative pathway, in which C3 is cleaved to C3b, which then binds to FB at the cell surface, forming C3 convertase and promoting further cleavage of the C3 molecule to C3b. The main advantage of the fact that the antibodies and fragments of the present disclosure interfere with complement activation at the level of C3 is that the accumulation of C3b on the surface and the release of C3a are avoided. Conversely, when complement activation is inhibited at the C5 level, as with eculizumab, the accumulation of C3b and the release of C3a are not inhibited. C3b acts as an opsonin and C3a is an anaphylatoxin. It is therefore preferable to prevent the accumulation of C3b and the formation of C3a, since these processes result in the attraction of immune cells and opsonophagocytosis of targets. This means that, for example, PNH patients receiving eculizumab still require transfusions because red blood cells are opsonized by the accumulation of C3b and then cleared by the liver and spleen. Furthermore, treatment with anti-C5 antibodies leads to the formation of C3b and the accumulation of C3a in cells that would otherwise be lysed by MAC. An important drawback of anti-C5 antibodies is that they also interfere with pathogen-induced complement activation, making patients more susceptible to infection. This is circumvented by targeting regulators of the complement system that protect host cells.

[0053] The term "antibody" as used herein refers to an immunoglobulin protein comprising at least a heavy chain variable region (VH) paired with a light chain variable region (VL) specific for a target epitope. The term includes both polyclonal and monoclonal antibodies. It refers to any form of antibody, including full-length immunoglobulins, that specifically binds to CCP18 of FH. An antibody or fragment thereof according to the present disclosure comprises at least one antigen-binding site. The term "antigen-binding site" as used herein refers to a site of an antibody or fragment thereof that comprises at least one CDR sequence, preferably at least two CDR sequences, more preferably at least three CDR sequences. For example, an antigen-binding site comprises light chain CDR1-3 or heavy chain CDR1-3. In some embodiments, an antigen-binding site comprises light chain CDR1-3 and heavy chain CDR1-3.

[0054] As is well known to those skilled in the art, an antibody contains two heavy chains and two light chains. The heavy chain of an antibody is the larger of the two types of chains that make up an immunoglobulin molecule. The heavy chain contains a constant domain and a variable domain, and the variable domain is involved in antigen binding. The light chain of an antibody is the smaller of the two types of chains that make up an immunoglobulin molecule. The light chain contains a constant domain and a variable domain. The variable domain of the light chain is often, but not always, combined with the variable domain of the heavy chain that is involved in antigen binding. Complementarity determining regions (CDRs) are hypervariable regions present in the heavy chain variable domain and the light chain variable domain. In the case of a full-length antibody, CDRs 1-3 of the heavy chain and CDRs 1-3 of the connected light chain combine to form an antigen binding site.

[0055] CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. There are three CDRs in each of the heavy and light chain variable domains, which are referred to as CDR1, CDR2, and CDR3 in each of the variable domains. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single heavy or light chain variable domain that can bind to a target antigen. The exact boundaries of these CDRs are defined differently in different systems. The three heavy chain CDRs are sometimes referred to as CDRH1, CDRH2, and CDRH3, and the three light chain CDRs are sometimes referred to as CDRL1, CDRL2, and CDRL3.

[0056] An "antigen-binding fragment of an antibody" is defined herein as a portion of an antibody that can specifically bind to the same antigen as the antibody (e.g., CCP18 of FH), although not necessarily to the same extent. A fragment of an FH activity enhancing antibody also enhances FH activity, although not necessarily to the same extent. A fragment of an FH activity inhibiting antibody also inhibits FH activity, although not necessarily to the same extent. In some embodiments, an antibody fragment according to the present disclosure comprises the heavy chain CDR1, CDR2, and CDR3 sequences of an antibody (e.g., any antibody disclosed herein), and in some embodiments, further comprises the light chain CDR1, CDR2, and CDR3 sequences of an antibody. Non-limiting examples of antibody fragments are single domain antibodies, single chain antibodies, nanobodies, unibodies, single chain variable fragments (scFv), Fab fragments, Fab' fragments, F(ab')2 fragments, and F(ab)2 fragments. In some embodiments, an antibody fragment comprises at least the heavy chain variable domain (VH) and / or the light chain variable domain (VL). In some embodiments, the fragment comprises at least a Fab fragment. In some embodiments, a Fab' fragment of the enhancing anti-FH antibody FH.07 or antibody FHR-1.3B4, which competes with the antibodies of the present disclosure for binding to the same epitope, has been demonstrated to retain the ability to enhance the function of FH. Thus, in some embodiments, a fragment of an antibody of the present disclosure is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a F(ab)2 fragment of an antibody of the present disclosure. In another embodiment, a fragment of an antibody of the present disclosure comprises an immunoglobulin heavy chain variable region, an immunoglobulin heavy chain constant region, an immunoglobulin light chain variable region, and an immunoglobulin light chain constant region.

[0057] In some embodiments, the antibodies and fragments of the present disclosure are capable of enhancing the activity of FH, such as human FH.

[0058] In some embodiments, the inhibition of C3 deposition is measured using a C3 deposition assay, such as the C3 deposition assay described in the Examples herein.This representative and non-limiting assay includes coating a microtiter plate with LPS.The plate is then incubated with serum from a healthy individual or an individual suffering from a disorder associated with unnecessary or excessive alternative complement activation, for example, as described above, in the presence or absence of antibody or fragment.C3 deposition in LPS can be detected with anti-C3 antibody.

[0059] In some embodiments, the increase in binding of FH to C3b is measured using ELISA, as described in the Examples herein. This representative and non-limiting assay involves coating a microtiter ELISA plate with C3b and incubating the plate with serum from a healthy individual or an individual suffering from a disorder associated with unnecessary or excessive alternative complement activation, for example, as described above. Bound FH can be detected with an anti-FH antibody, such as peroxidase-labeled polyclonal anti-FH. The ability of an antibody or fragment to enhance FH binding to C3b can be determined by pre-incubating serum in the presence of the antibody or fragment prior to incubation with coated C3b. As another example, binding of FH to C3b can be determined using surface plasmon resonance (SPR), as described in the Examples herein, for example. SPR is a technique that measures biomolecular interactions in real time in a label-free environment. One of the interactors, e.g., C3b, is immobilized on a sensor surface, and the other, e.g., FH, is free in solution and passed over the surface, e.g., in the presence or absence of an antibody or fragment of the present disclosure (at different concentrations). In some embodiments, an antibody or fragment thereof according to the present disclosure increases the binding affinity of FH for C3b in vitro by up to 2 μM, up to 1.95 μM, up to 1.8 μM, up to 1.7 μM (K DIn certain embodiments, the antibody or fragment thereof of the present disclosure reduces the binding affinity of FH to C3b in vitro (K value) and / or increases the binding affinity of FH to C3b in vitro by at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. D In certain embodiments, the antibody or fragment thereof of the present disclosure increases the in vitro binding affinity of FH to C3b (K D ) to 250 nM to 1 μM. In certain embodiments, the antibody or fragment thereof of the present disclosure increases the binding affinity of FH to C3b in vitro by at least 3-5 fold.

[0060] In some embodiments, the antibodies or fragments thereof provided by the present disclosure have a low in vitro IC 50 values, such as, in some embodiments, the in vitro IC of antibody FH.07 or antibody FHR-1.3B4 in the same functional assay. 50 Lower in vitro IC value 50 "IC 50 " is a term well known in the art and refers to the concentration of an antibody or fragment required to inhibit or reduce a particular functional activity by 50%. The IC of an antibody or fragment 50The lower the value, the stronger the inhibitory activity of the antibody or fragment and the greater its potential as a therapeutic agent. In some embodiments, the functional assay is a C3b deposition assay and / or a hemolysis assay as described herein above. In some embodiments, an antibody or fragment thereof of the present disclosure inhibits C3 deposition in LPS in vitro in the presence of 10% (v / v) normal human serum with an IC of 38 nM or less, 35 nM or less, 32 nM or less, 30 nM or less, 28 nM or less, 27 nM or less, 25 nM or less, 23 nM or less, 20 nM or less, 18 nM or less, 15 nM or less, or 10 nM or less. 50 In some embodiments, the antibodies or fragments thereof of the present disclosure inhibit C3 deposition in vitro in LPS in the presence of 10% (v / v) normal human serum with an IC value of 15 nM to 30 nM. 50 In some embodiments, the antibodies or fragments thereof of the present disclosure inhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC value of 150 nM or less, 130 nM or less, 120 nM or less, 115 nM or less, 110 nM or less, 105 nM or less, 100 nM or less, 95 nM or less, 90 nM or less, 85 nM or less, 80 nM or less, 75 nM or less, 70 nM or less, 65 nM or less, or 60 nM or less. 50 In some embodiments, the antibodies or fragments thereof of the present disclosure inhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC value of 60 nM to 80 nM. 50 Inhibit by value.

[0061] In some embodiments, the antibodies or fragments of the present disclosure inhibit C3 deposition in vitro in LPS in the presence of 10% (v / v) normal human serum with a low IC as described herein above. 50 The in vitro hemolytic activity was inhibited at IC values ​​in the presence of 10% (v / v) normal human serum with a low IC value as described above. 50 In some embodiments, the antibodies or fragments of the present disclosure inhibit C3 deposition in vitro in LPS in the presence of 10% (v / v) normal human serum with an IC value of 38 nM or less. 50value and in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC 50 value or inhibited C3 deposition in vitro in LPS in the presence of 10% (v / v) normal human serum with an IC of 30 nM or less. 50 value and in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC 50 value or in vitro LPS-induced C3 deposition in the presence of 10% (v / v) normal human serum with an IC of less than 27 nM. 50 value and inhibited in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC 50 In some embodiments, the IC value of C3 deposition in LPS in vitro in the presence of 10% (v / v) normal human serum is 50 The IC value is determined in a C3 deposition assay as described herein above. In some embodiments, the IC value of hemolytic activity is 50 The value is determined in a hemolytic activity assay as described herein above.

[0062] In some embodiments, the antibodies or fragments thereof provided by the present disclosure have a high binding affinity for FH and / or FH fragments comprising domains CCP18-20, e.g., in some embodiments, higher than the binding affinity of antibody FH.07 or antibody FHR-1.3B4. In some embodiments, the in vivo therapeutic activity of the antibody or fragment typically requires a high binding affinity, e.g., to minimize the binding of the antibody or fragment to binding sites and / or antigens other than the epitope or antigen to which it is specific, and to minimize the amount of antibody or fragment that needs to be administered in vivo. In some embodiments, antibodies with high binding affinity are preferred. In some embodiments, the antibody or fragment has a binding affinity of 2.5×10 to FH. -8 The dissociation constant (K D) and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D Thus, in one embodiment, the disclosure provides an isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to factor H (FH) and enhances FH activity, the antibody having a binding affinity of 2.5×10 to FH. -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D In some embodiments, the antibody or fragment of the present disclosure has a K for FH of 22.5 nM or less, 20 nM or less, 17.5 nM or less, 15 nM or less, 12.5 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, or 0.6 nM or less. D In some embodiments, the antibody or fragment according to the present disclosure has a binding affinity of 0.09 × 10 to an FH fragment consisting of CCP18-20. -9 M or less, 0.08×10 -9 M or less, 0.07×10 -9 M or less, 0.06×10 -9 M or less, 0.05×10 -9 M or less, 0.04×10 -9 M or less, 0.03×10 -9 M or less, 0.02×10 -9 M or less, 1×10 -11 M or less, 0.9×10 -11 M or less, 0.8×10 -11 M or less, 0.7×10 -11 M or less, or 0.6 x 10 -11 K below M D In some embodiments, the antibody or fragment thereof has a binding affinity for FH of 1.25×10 -8 K below M Dand / or 0.04 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D In some embodiments, the antibody or fragment thereof has a binding affinity for FH of 0.6×10 -8 K below M D and / or 0.6 × 10 for the FH fragment consisting of CCP18-20 -11 K below M D In some embodiments, the binding affinity is determined using surface plasmon resonance (SPR), for example in an assay described herein, i.e., in an assay in which binding affinity is determined by SPR on a ProtA chip where the antibody or fragment is captured before flowing over the surface either full-length FH (for binding affinity of FH) or a fragment of FH consisting of domains 18-20 (for binding affinity of CCP18-20).

[0063] In some embodiments, the antibody or fragment thereof provided by the present disclosure increases the interaction between FH and C3b. In some embodiments, the antibody or fragment thereof has a dissociation constant (K D In some embodiments, the antibodies or fragments of the present disclosure have a K of 2 μM or less, 1.5 μM or less, 1 μM or less, 500 nM or less, 400 nM or less, or 300 nM or less. D In some embodiments, the antibody or fragment of the present disclosure increases the binding affinity of FH to C3b in vitro by at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold (K D (reduces noise).

[0064] In some embodiments, the antibodies or fragments of the present disclosure have high binding affinity and inhibit C3 deposition in LPS in vitro with low IC 50 Inhibits in vitro hemolytic activity at low IC levels and / or50 In some embodiments, the antibody or fragment of the present disclosure inhibits FH at a level of 2.5×10 -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D and inhibited C3 deposition in vitro in LPS with an IC of 38 nM or less in the presence of 10% (v / v) normal human serum. 50 value and / or inhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC of 150 nM or less 50 In some embodiments, the antibody or fragment inhibits FH at a level of 1.25×10 -8 K of 10 nM or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) D and / or 0.04 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D and inhibited C3 deposition in vitro in LPS with an IC of 30 nM or less. 50 value and inhibited in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC 50 In some embodiments, the antibody or fragment inhibits FH at a level of 1.25×10 -8 K of 10 nM or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) D and / or 0.04 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D and inhibited C3 deposition in vitro in LPS with an IC of 27 nM or less in the presence of 10% (v / v) normal human serum. 50 It inhibited in vitro hemolytic activity at IC values ​​below 100 nM in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the antibody or fragment inhibits FH at a level of 1.25×10 -8K of 10 nM or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) D and / or 0.04 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D and inhibited C3 deposition in vitro in LPS with an IC of less than 25 nM in the presence of 10% (v / v) normal human serum. 50 It inhibited in vitro hemolytic activity at IC values ​​below 80 nM in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the antibody or fragment inhibits FH at a level of 1.25×10 -8 K of 10 nM or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) D and / or 0.04 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D and inhibited C3 deposition in vitro in LPS with IC of 15 nM to 25 nM in the presence of 10% (v / v) normal human serum. 50 It inhibited in vitro hemolytic activity at IC values ​​between 15 nM and 25 nM in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the binding affinity is determined by SPR as described herein. In some embodiments, the IC value of C3 deposition in LPS in vitro is determined. 50 The IC value is determined in a C3 deposition assay as described herein above. In some embodiments, the IC value of hemolytic activity is 50 The value is determined in a hemolytic activity assay as described herein above.

[0065] In some embodiments, any antibody or antigen-binding fragment disclosed herein has a binding affinity for an FH fragment comprising FH and / or CCP18 that is higher than the binding affinity of antibody FH.07. In some embodiments, any antibody or antigen-binding fragment disclosed herein has a binding affinity for an FH fragment comprising FH and / or CCP18-20 that is higher than the binding affinity of antibody FH.07. In some embodiments, any antibody or antigen-binding fragment disclosed herein has a binding affinity for an FH fragment comprising FH and / or CCP18-20 that is higher than the binding affinity of antibody FH.07. 50 IC value lower than 50 In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein exhibit greater inhibition of C3 deposition in LPS than antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein exhibit greater inhibition of hemolytic activity than antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein compete with antibody FH.07 for binding to the same epitope on CCP18 of FH. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high storage capacity, particularly improved storage stability compared to antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high stability, particularly improved stability compared to antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high selectivity, particularly increased selectivity compared to antibody FH.07.

[0066] In some embodiments, any antibody or antigen-binding fragment disclosed herein has a binding affinity for an FH fragment comprising FH and / or CCP18 that is higher than the binding affinity of antibody FHR-1.3B4. In some embodiments, any antibody or antigen-binding fragment disclosed herein has a binding affinity for an FH fragment comprising FH and / or CCP18-20 that is higher than the binding affinity of antibody FHR-1.3B4. In some embodiments, any antibody or antigen-binding fragment disclosed herein has a binding affinity for an FH fragment comprising FH and / or CCP18-20 that is higher than the binding affinity of antibody FHR-1.3B4. 50 IC value lower than 50 In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein inhibit C3 deposition in LPS better than antibody FHR-1.3B4. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein exhibit greater inhibition of hemolytic activity than antibody FHR-1.3B4. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein compete with antibody FHR-1.3B4 for binding to the same epitope on CCP18 of FH. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high storage capacity, particularly improved storage stability compared to antibody FHR-1.3B4. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high stability, particularly improved stability compared to antibody FHR-1.3B4. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high selectivity, particularly increased selectivity compared to antibody FHR-1.3B4.

[0067] In some embodiments, an antibody or fragment thereof provided by the present disclosure competes with an anti-FH.07 antibody for binding to FH, particularly the CCP18 domain of FH.

[0068] Exemplary antibodies of the disclosure are listed in Table 1. CDR sequences are numbered according to the IMGT numbering system (Lefranc 1997, Lefranc 1999, and Lefranc et al. 2003). LC is the light chain, HC is the heavy chain, CDR is the complementarity determining region, VH is the heavy chain variable region, and VL is the light chain variable region. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0069] In some embodiments, the disclosure provides isolated, synthetic, or recombinant antibodies or antigen-binding fragments thereof that specifically bind to complement control protein domain 18 (CCP18) of factor H (FH), including antibodies having VH and VL sequences disclosed in Table 1, similar to those described by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196:901-917), MacCallum (MacCallum RM et al. The present invention provides an antibody or fragment thereof comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, as determined by the methods described herein (see, for example, U.S. Pat. No. 6,313,163, or U.S. Pat. No. 6,313,163, or any other CDR determination method known in the art).

[0070] In some embodiments, the antibody of the present disclosure is Antibody 1. Table 1 provides a summary of the variable heavy and light chain sequences and individual CDR sequences of Antibody 1. In some embodiments, the antibody or fragment comprises at least one, two, or three of the heavy chain CDR sequences of Antibody 1. In some embodiments, the antibody or fragment comprises at least one, two, or three of the light chain CDR sequences of Antibody 1. As used herein, the term "Antibody 1" encompasses all antibodies and fragments that comprise at least the heavy and light chain CDR1, CDR2, and CDR3 regions shown in Table 1, e.g., isolated and / or purified antibodies or recombinantly produced antibodies. In some embodiments, Antibody 1 competes with antibody FH.07 or antibody FHR-1.3B4 for binding to the same epitope on CCP18 of FH.

[0071] In some embodiments, the antibody of the present disclosure is antibody 2. Table 1 provides a summary of the variable heavy and light chain sequences and individual CDR sequences of antibody 2. In some embodiments, the antibody or fragment comprises at least one, two, or three of the heavy chain CDR sequences of antibody 2. In some embodiments, the antibody or fragment comprises at least one, two, or three of the light chain CDR sequences of antibody 2. As used herein, the term "antibody 2" encompasses all antibodies and fragments that comprise at least the heavy and light chain CDR1, CDR2, and CDR3 regions shown in Table 1, e.g., isolated and / or purified antibodies or recombinantly produced antibodies. In some embodiments, antibody 2 competes with antibody FH.07 or antibody FHR-1.3B4 for binding to the same epitope on CCP18 of FH.

[0072] In some embodiments, the antibody of the present disclosure is antibody 3. Table 1 provides a summary of the variable heavy and light chain sequences and individual CDR sequences of antibody 3. In some embodiments, the antibody or fragment comprises at least one, two, or three of the heavy chain CDR sequences of antibody 3. In some embodiments, the antibody or fragment comprises at least one, two, or three of the light chain CDR sequences of antibody 3. As used herein, the term "antibody 3" encompasses all antibodies and fragments that comprise at least the heavy and light chain CDR1, CDR2, and CDR3 regions shown in Table 1, e.g., isolated and / or purified antibodies or recombinantly produced antibodies. In some embodiments, antibody 3 competes with antibody FH.07 or antibody FHR-1.3B4 for binding to the same epitope on CCP18 of FH.

[0073] In some embodiments, the antibody of the present disclosure is antibody 4. Table 1 provides a summary of the variable heavy and light chain sequences and individual CDR sequences of antibody 4. In some embodiments, the antibody or fragment comprises at least one, two, or three of the heavy chain CDR sequences of antibody 4. In some embodiments, the antibody or fragment comprises at least one, two, or three of the light chain CDR sequences of antibody 4. As used herein, the term "antibody 4" encompasses all antibodies and fragments that comprise at least the heavy and light chain CDR1, CDR2, and CDR3 regions shown in Table 1, e.g., isolated and / or purified antibodies or recombinantly produced antibodies. In some embodiments, antibody 4 competes with antibody FH.07 or antibody FHR-1.3B4 for binding to the same epitope on CCP18 of FH.

[0074] In some embodiments, the present disclosure provides an isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to complement control protein domain 18 (CCP18) of Factor H (FH), comprising: - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1) or the sequence TSVTY (SEQ ID NO: 13), - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 2) or the sequence ASS (SEQ ID NO: 14), - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6) or IWSGGTT (SEQ ID NO: 10), and / or - a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7) or the sequence ARNFGNYAMDF (SEQ ID NO: 11), and / or - Any combination of the above The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0075] In certain embodiments, the antibody or fragment comprises: - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1), - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO:2), - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6), and - a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7).

[0076] In certain embodiments, the antibody or fragment comprises: - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1), - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO:2), - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 10), and - a heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO: 11).

[0077] In certain embodiments, the antibody or fragment comprises: - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13), - a light chain CDR2 sequence having the sequence ASS (SEQ ID NO: 14), - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6), and - a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7).

[0078] In certain embodiments, the antibody or fragment comprises: - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13), - a light chain CDR2 sequence having the sequence ASS (SEQ ID NO: 14), - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 10), and - a heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO: 11).

[0079] In some embodiments, the antibody or fragment enhances FH activity, e.g., inhibition of alternative complement activation, e.g., inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition, and / or increased binding of FH to C3b, iC3b, and / or C3d. In some embodiments, the fragment comprises at least a heavy chain variable domain (VH) and / or a light chain variable domain (VL). In some embodiments, the fragment comprises at least a Fab fragment.

[0080] In some embodiments, the antibody or fragment has a titer of 2.5×10 to FH. -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D For example, 1.25×10 -8 K below M D and / or 0.04 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D The antibody or fragment has a binding affinity for

[0081] In a further embodiment, the disclosure provides an isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO:1), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO:2), and a light chain CDR3 having the sequence QHRSSSNPLT (SEQ ID NO:3), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO:6), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO:7). In one embodiment, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the antibody comprises a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:8. In certain embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence of SEQ ID NO:4 and a variable heavy chain sequence comprising a sequence of SEQ ID NO:8. In some embodiments, the antibody or fragment enhances FH activity, e.g., inhibition of alternative complement activation, e.g., inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition, and / or increased binding of FH to C3b, iC3b, and / or C3d. In some embodiments, the fragment comprises at least a heavy chain variable domain (VH) and / or a light chain variable domain (VL). In some embodiments, the fragment comprises at least a Fab fragment.

[0082] In a further embodiment, the disclosure provides an isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO:1), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO:2), and a light chain CDR3 having the sequence QHRSSSNPLT (SEQ ID NO:3), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO:10), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO:11). In one embodiment, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:4. In some embodiments, the antibody or fragment comprises a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 12. In certain embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence of SEQ ID NO: 4, and a variable heavy chain sequence comprising a sequence of SEQ ID NO: 12. In some embodiments, the antibody or fragment enhances FH activity, inhibition of alternative complement activation, such as inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition, and / or increased binding of FH to C3b, iC3b, and / or C3d. In some embodiments, the fragment comprises at least a heavy chain variable domain (VH) and / or a light chain variable domain (VL). In some embodiments, the fragment comprises at least a Fab fragment.

[0083] In a further embodiment, the disclosure provides an isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence having the sequence ASS (SEQ ID NO: 14), and a light chain CDR3 having the sequence QHRSSSNPLT (SEQ ID NO: 3), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5), a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7). In one embodiment, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments, the antibody or fragment comprises a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:8. In certain embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence of SEQ ID NO:16, and a variable heavy chain sequence comprising a sequence of SEQ ID NO:8. In some embodiments, the antibody or fragment enhances FH activity, such as inhibition of alternative complement activation, such as inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition, and / or increased binding of FH to C3b, iC3b, and / or C3d. In some embodiments, the fragment comprises at least a heavy chain variable domain (VH) and / or a light chain variable domain (VL). In some embodiments, the fragment comprises at least a Fab fragment.

[0084] In a further embodiment, the disclosure provides an isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence having the sequence ASS (SEQ ID NO: 14), and a light chain CDR3 having the sequence QHRSSSNPLT (SEQ ID NO: 3), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 10), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO: 11). In one embodiment, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments, the antibody or fragment comprises a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 12. In certain embodiments, the antibody or fragment comprises a variable light chain sequence comprising a sequence of SEQ ID NO: 16, and a variable heavy chain sequence comprising a sequence of SEQ ID NO: 12. In some embodiments, the antibody or fragment enhances FH activity, such as inhibition of alternative complement activation, such as inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition, and / or increased binding of FH to C3b, iC3b, and / or C3d. In some embodiments, the fragment comprises at least a heavy chain variable domain (VH) and / or a light chain variable domain (VL). In some embodiments, the fragment comprises at least a Fab fragment.

[0085] In some embodiments, any of the antibodies or fragments disclosed herein has an activity against FH of 2.5×10 -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D In some embodiments, the antibody or fragment has a K for FH of 22.5 nM or less, 20 nM or less, 17.5 nM or less, 15 nM or less, 12.5 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, or 0.6 nM or less. D and / or 0.09 × 10 for the FH fragment consisting of CCP18-20 -9 M or less, 0.08×10 -9 M or less, 0.07×10 -9 M or less, 0.06×10 -9 M or less, 0.05×10 -9 M or less, 0.04×10 -9 M or less, 0.03×10 -9 M or less, 0.02×10 -9 M or less, 1×10 -11 M or less, 0.9×10 -11 M or less, 0.8×10 -11 M or less, 0.7×10 -11 M or less, or 0.6 x 10 -11 K below M D has a binding affinity of

[0086] In some embodiments, any of the antibodies or fragments disclosed herein inhibit C3 deposition in vitro in LPS in the presence of 10% (v / v) normal human serum with an IC of 38 nM or less. 50In some embodiments, the antibody or fragment inhibits C3 deposition in vitro in LPS in the presence of 10% (v / v) normal human serum with an IC value of 35 nM or less, 32 nM or less, 30 nM or less, 28 nM or less, 25 nM or less, 23 nM or less, 20 nM or less, 18 nM or less, 15 nM or less, or 10 nM or less. 50 In some embodiments, the antibodies or fragments thereof of the present disclosure inhibit C3 deposition in vitro in LPS in the presence of 10% (v / v) normal human serum with an IC value of 15 nM to 30 nM. 50 Inhibit by value.

[0087] In some embodiments, any of the antibodies or fragments disclosed herein exhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC of 150 nM or less. 50 In some embodiments, the antibody or fragment inhibits in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC value of 10 nM or less. 50 In some embodiments, the antibodies or fragments thereof of the present disclosure inhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC value of 150 nM or less, 130 nM or less, 115 nM or less, 105 nM or less, 100 nM or less, 95 nM or less, 75 nM or less, 70 nM or less, 65 nM or less, or 60 nM or less. 50 In some embodiments, the antibodies or fragments thereof of the present disclosure inhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum with an IC value of 60 nM to 80 nM. 50 Inhibit by value.

[0088] In some embodiments, any of the antibodies or fragments disclosed herein may be used to measure the in vitro binding affinity of FH to C3b (K D) to 2 μM or less, 1.5 μM or less, 1 μM or less, 500 nM or less, 400 nM or less, or 300 nM or less, and / or increase the in vitro binding affinity of FH to C3b by at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold. In some embodiments, any antibody or fragment disclosed herein increases the in vitro binding affinity of FH to C3b (K D In some embodiments, any of the antibodies or fragments disclosed herein increases the in vitro binding affinity of FH to C3b (K D In some embodiments, any of the antibodies or fragments disclosed herein increases the in vitro binding affinity of FH to C3b (K D In some embodiments, any of the antibodies or fragments disclosed herein increases the in vitro binding affinity of FH to C3b (K D ) to 250-300 nM.

[0089] In some cases, the sequence of at least one of any one or more of the CDRs disclosed herein is optimized to generate a variant antibody or fragment, e.g., to (further) improve binding affinity, selectivity, FH-enhancing ability, and / or in vivo or storage stability. In some embodiments, the antibody or fragment according to the present disclosure has high storage stability, particularly improved storage stability compared to antibody FH.07 or antibody FHR-1.3B4. In some embodiments, the antibody or fragment according to the present disclosure has high in vivo stability, particularly improved in vivo stability compared to antibody FH.07 or antibody FHR-1.3B4. In some embodiments, the antibody or fragment according to the present disclosure has high selectivity, particularly increased selectivity compared to antibody FH.07 or antibody FHR-1.3B4.

[0090] In addition, in some cases, at least one sequence in at least one of the framework regions of the antibody or fragment of the present disclosure is optimized, for example, to improve the binding efficacy or stability of the antibody or fragment, or to reduce its side effects after administration of the non-human sequence to humans. This is done, for example, by mutagenesis procedures. It is possible for a person skilled in the art to generate antibody variants that contain at least one altered CDR or framework sequence. The CDR and / or framework sequence is optimized, for example, by mutating the nucleic acid encoding such framework sequence. For example, conservative amino acid substitutions are applied. In some embodiments, any of the CDR, framework, variable heavy chain, variable light chain sequences disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions (e.g., conservative substitutions) compared to any one or more of the amino acid sequences disclosed herein. In some embodiments, any of the CDR, framework, variable heavy, variable light sequences disclosed herein comprise at least 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions (e.g., conservative substitutions) compared to any one or more of the amino acid sequences disclosed herein. In some embodiments, any of the CDR, framework, variable heavy, variable light sequences disclosed herein comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions (e.g., conservative substitutions) compared to any one or more of the amino acid sequences disclosed herein. In some embodiments, any of the CDR, framework, variable heavy, variable light sequences disclosed herein comprise no more than 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or insertions (e.g., conservative substitutions) compared to any one or more of the amino acid sequences disclosed herein.Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another, and the substitution of one polar residue for another, e.g., arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0091] To select improved antibodies or fragments, the resulting variant antibodies or fragments may be tested for binding affinity, FH enhancing ability, and / or stability, for example using the tests described herein. Once an antibody or fragment specific for FH, particularly CCP18 of FH, is obtained, its desired biological activity, i.e. its ability to enhance the activity of FH, can be tested by several methods known to those skilled in the art. As described hereinbefore, enhancing the activity of FH may include inhibiting hemolytic activity, inhibiting C3 deposition in cells, and / or increasing the binding of FH to C3b. Functional assays for testing these activities are described hereinbefore and in detail in the Examples. Usually, up to three amino acid residues of the CDR sequence may be changed while maintaining the same specificity, depending on the number of amino acids that make up the CDR. Thus, in some embodiments, an antibody or fragment according to the present disclosure may contain heavy and light chain CDR1, CDR2, and CDR3 sequences in which up to three, up to two, or up to one amino acid of each CDR has been altered compared to the heavy and light chain CDR1, CDR2, and CDR3 sequences of Table 1. In some embodiments, an antibody or fragment comprises the same antibody light chain CDR3 and heavy chain CDR3 as shown in Table 1, an antibody light chain CDR2 with up to one amino acid alteration, and a light chain CDR1, heavy chain CDR1, and heavy chain CDR2 with up to three amino acid alterations. In some embodiments, an antibody or fragment comprises the same antibody light chain CDR2 and CDR3 and heavy chain CDR3 as shown in Table 1, and a light chain CDR1, heavy chain CDR1, and heavy chain CDR2 with up to two amino acids or up to one amino acid alteration.

[0092] Thus, the present disclosure further provides an isolated, synthetic, or recombinant antibody or fragment thereof that specifically binds to complement control protein domain 18 (CCP18) of factor H (FH), comprising: - a light chain CDR1 sequence having at least 60% or at least 80% identity with the sequence SSVTY (SEQ ID NO:1), a light chain CDR2 sequence having at least 60% identity with the sequence ATS (SEQ ID NO:2) and a light chain CDR3 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence QHRSSSNPLT (SEQ ID NO:3), a heavy chain CDR1 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence VWSGGTT (SEQ ID NO:6) and a heavy chain CDR3 sequence having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence ARNFGNYAMDY (SEQ ID NO:7), - a light chain CDR1 sequence having at least 60% or at least 80% identity with the sequence SSVTY (SEQ ID NO: 1), a light chain CDR2 sequence having at least 60% identity with the sequence ATS (SEQ ID NO: 2) and a light chain CDR3 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence QHRSSSNPLT (SEQ ID NO: 3), a heavy chain CDR1 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence GFSLTNYG (SEQ ID NO: 5), a heavy chain CDR2 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence IWSGGTT (SEQ ID NO: 10) and a heavy chain CDR3 sequence having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence ARNFGNYAMDF (SEQ ID NO: 11), - a light chain CDR1 sequence having at least 60% or at least 80% identity with the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence having at least 60% identity with ASS (SEQ ID NO: 14) and a light chain CDR3 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence QHRSSSNPLT (SEQ ID NO: 3), a heavy chain CDR1 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence GFSLTNYG (SEQ ID NO: 5), a heavy chain CDR2 having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence VWSGGTT (SEQ ID NO: 6) and a heavy chain CDR3 sequence having at least 60%, at least 70%, at least 80% or at least 90% identity with the sequence ARNFGNYAMDY (SEQ ID NO: 7), a light chain CDR1 sequence having at least 60% or at least 80% identity with the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence having at least 60% identity with ASS (SEQ ID NO: 14) and a light chain CDR3 having at least 60%, at least 70%, at least 80%, or at least 90% identity with the sequence QHRSSSNPLT (SEQ ID NO: 3), a heavy chain CDR1 having at least 60%, at least 70%, at least 80%, or at least 90% identity with the sequence GFSLTNYG (SEQ ID NO: 5), a heavy chain CDR2 having at least 60%, at least 70%, at least 80%, or at least 90% identity with the sequence IWSGGTT (SEQ ID NO: 10) and a heavy chain CDR3 sequence having at least 60%, at least 70%, at least 80%, or at least 90% identity with the sequence ARNFGNYAMDF (SEQ ID NO: 11). The present invention provides an antibody or fragment thereof comprising:

[0093] In some embodiments, the antibody or fragment enhances FH activity. In some embodiments, the antibody or fragment comprises a heavy chain CDR1, CDR2 and / or CDR3 sequence, and / or a light chain CDR1, CDR2 and / or CDR3 sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences shown.

[0094] The present disclosure further provides an isolated, synthetic, or recombinant antibody or fragment thereof that specifically binds to complement control protein domain 18 (CCP18) of factor H (FH), comprising: - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1), optionally with one amino acid substitution, a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 2), and a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3), optionally with one or two amino acid substitutions, a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5), optionally with one or two amino acid substitutions, a heavy chain CDR2 sequence having the sequence VWSGGTT (SEQ ID NO: 6), optionally with one or two amino acid substitutions, and a heavy chain CDR3 sequence ARNFGNYAMDY (SEQ ID NO: 7), optionally with one or two amino acid substitutions; - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1), optionally with one amino acid substitution, a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 2), and a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3), optionally with one or two amino acid substitutions, a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5), optionally with one or two amino acid substitutions, a heavy chain CDR2 sequence having the sequence IWSGGTT (SEQ ID NO: 10), optionally with one or two amino acid substitutions, and a heavy chain CDR3 sequence ARNFGNYAMDF (SEQ ID NO: 11), optionally with one or two amino acid substitutions; - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13), optionally with one or two amino acid substitutions, a light chain CDR2 sequence ASS (SEQ ID NO: 14), and a light chain CDR3 having the sequence QHRSSSNPLT (SEQ ID NO: 3), optionally with one or two amino acid substitutions, a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5), optionally with one or two amino acid substitutions, a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6), optionally with one or two amino acid substitutions, and a heavy chain CDR3 ARNFGNYAMDY (SEQ ID NO: 7), optionally with one or two amino acid substitutions, or - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13) optionally with one amino acid substitution, a light chain CDR2 sequence having the sequence ASS (SEQ ID NO: 14) and a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3) optionally with one or two amino acid substitutions, a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5) optionally with one or two amino acid substitutions, a heavy chain CDR2 sequence having the sequence IWSGGTT (SEQ ID NO: 10) optionally with one or two amino acid substitutions and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO: 11) optionally with one or two amino acid substitutions The present invention provides an antibody or fragment thereof comprising:

[0095] In some embodiments, any antibody or antigen-binding fragment disclosed herein comprises at least one or more CDR(s) from any of the heavy or light chain sequences disclosed herein. In some embodiments, any antibody or antigen-binding fragment disclosed herein comprises at least one or more CDR(s) from any of the variable heavy or variable light chain sequences of SEQ ID NO: 4, 8, 12, or 16. In some embodiments, any antibody or antigen-binding fragment disclosed herein comprises at least one or more CDR(s) from any of the heavy or light chain sequences disclosed herein, the CDRs being numbered using the Kabat system. In some embodiments, any antibody or antigen-binding fragment disclosed herein comprises at least one or more CDR(s) from any of the heavy or light chain sequences disclosed herein, the CDRs being numbered using the Chothia system. In some embodiments, any antibody or antigen-binding fragment disclosed herein comprises at least one or more CDR(s) from any of the heavy or light chain sequences disclosed herein, the CDRs being numbered using the MacCallum system. In some embodiments, any antibody or antigen-binding fragment disclosed herein comprises at least one or more CDRs derived from any of the heavy or light chain sequences disclosed herein, where the CDRs are numbered using the AbM system. In some embodiments, any antibody or antigen-binding fragment disclosed herein comprises at least one or more CDRs derived from any of the heavy or light chain sequences disclosed herein, where the CDRs are numbered using the IMGT system.

[0096] The system described by Kabat, also referred to as "numbered according to Kabat," "Kabat numbering," "Kabat definition," and "Kabat labeling," provides an unambiguous residue numbering system applicable to any variable domain of an antibody and provides precise residue boundaries defining the three CDRs of each chain (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991), the contents of which are incorporated herein by reference in their entirety). These CDRs are referred to as Kabat CDRs and include residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain and residues 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain. When the CDRs are defined according to Kabat, the light chain FR residues are located at approximately residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located at approximately residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) of the heavy chain. "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies.

[0097] Other CDR numbering systems are also used in the art. Chothia et al. found that certain sub-portions of Kabat CDRs adopt nearly identical peptide backbone conformations despite the large diversity at the amino acid sequence level. (Chothia et al. (1987) J. Mol. Biol. 196:901-917, and Chothia et al. (1989) Nature 342:877-883). These sub-portions are designated L1, L2, and L3, or H1, H2, and H3, with "L" and "H" representing the light and heavy chain regions, respectively. These CDRs are sometimes referred to as "Chothia CDRs," "Chothia numbering," or "numbered according to Chothia," and include residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and residues 26-32 (CDR1), 52-56 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain. Mol. Biol. 196:901-917 (1987).

[0098] The system described by MacCallum, also referred to as "numbered according to MacCallum" or "MacCallum numbering", includes residues 30-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) in the light chain variable domain, and residues 30-35 (CDR1), 47-58 (CDR2), and 93-101 (CDR3) in the heavy chain variable domain. MacCallum et al. ((1996) J. Mol. Biol. 262(5):732-745).

[0099] The system described by AbM, also known as "numbering according to AbM" or "AbM numbering", includes residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and residues 26-35 (CDR1), 50-58 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain.

[0100] The variable region numbering according to IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) can also be used, which is the numbering of immunoglobulin variable heavy or light chain residues according to the method of IIMGT described in Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell receptors and Ig-like domains", The Immunologist, 7, 132-136 (1999), which is expressly incorporated herein by reference in its entirety. As used herein, "IMGT sequence numbering" or "numbering according to IMGT" refers to the numbering of sequences encoding variable regions according to IMGT. For heavy chain variable domains, when numbered according to IMGT, the hypervariable region spans amino acids 27-38 for CDR1, amino acids 56-65 for CDR2, and amino acids 105-117 for CDR3. For the light chain variable domain, the hypervariable region spans amino acids 27-38 for CDR1, 56-65 for CDR2, and 105-117 for CDR3, numbered according to IMGT. In some embodiments of the constructs and antigen binding arms described herein, the CDRs described herein include residues 24-34 (CDR1), 49-56 (CDR2), and 89-97 (CDR3) of the light chain variable domain, and residues 27-35 (CDR1), 49-60 (CDR2), and 93-102 (CDR3) of the heavy chain variable domain, numbered according to Chothia numbering. In some embodiments, CDR2 of the light chain variable domain may include amino acids 49-56, numbered according to Chothia numbering.

[0101] The antibody or fragment thereof according to the present disclosure is, in some embodiments, a monoclonal antibody or fragment. A monoclonal antibody is an antibody that is substantially composed of a single molecular species. Monoclonal antibodies are obtained from a homogeneous population of antibodies that have the same sequence and bind to the same epitope, with the exception of possible variant antibodies or fragments that have one or more mutations that naturally occurred, e.g., during production. Monoclonal antibodies can advantageously be produced recombinantly, so that amounts of the antibody can be obtained that are significantly higher than the amount of polyclonal antibodies present in antisera. However, polyclonal antibodies and fragments are also encompassed by the present disclosure. In some embodiments, the antibody or fragment according to the present disclosure is a chimeric or humanized antibody. In some embodiments, the antibody or fragment thus comprises at least human light and heavy chain constant regions. In some embodiments, the antibody or fragment also comprises human framework regions within the heavy and light chain variable regions. In some embodiments, a human antibody or fragment is provided that is composed entirely of human sequences. The use of non-human antibodies or fragments for the treatment of human diseases is hindered by several factors, so in some embodiments, the use of chimeric, humanized, or human antibodies is more desirable than the use of non-human antibodies. The human body may recognize non-human antibodies as foreign, resulting in an immune response against the non-human antibody or fragment, leading to adverse side effects and / or rapid clearance of the antibody or fragment from the circulation. The potential for side effects is reduced when chimeric, humanized, or human antibodies are administered to humans. Furthermore, longer circulating half-lives are generally achieved with chimeric, humanized, or human antibodies due to reduced clearance compared to non-human antibodies. In some embodiments, human germline sequences are used in the framework regions of the antibodies or fragments according to the present disclosure. The use of human germline sequences minimizes the risk of immunogenicity of the antibodies or fragments, because these sequences are unique to the individual from which the framework regions are derived and are less likely to contain somatic mutations that may cause an immunogenic response when applied to another human individual.

[0102] Means for humanizing antibodies or providing chimeric antibodies are well known in the art. Various recombinant DNA-based approaches have been established that aim to increase the content of amino acid residues in an antibody occurring at the same or similar positions of a human antibody while maintaining the specificity and affinity of the parent non-human antibody. For example, the framework regions of the variable regions of a murine antibody are replaced by the corresponding human framework regions with the highest degree of homology, leaving the non-human CDRs intact. Further methods suitable for humanizing antibodies according to the present disclosure include, but are not limited to, CDR grafting (Queen, C et.al. 1989, Carter, P et.al. 1992), resurfacing (Padlan, EA, et.al. 1991), superhumanization (Tan, PDA, et.al. 2002), human string content optimization (Lazar, GA et.al. 2007), and humaneering (Almagro, JC, et.al. 2008).

[0103] In one embodiment, the antibody or fragment of the present disclosure is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different antigens and / or epitopes. In one embodiment, a bispecific antibody has binding specificity for FH, for example, comprises one variable light chain and one variable heavy chain that specifically binds to CCP18 of FH as described herein, and has binding specificity for another antigen. In another embodiment, a bispecific antibody can bind to two different epitopes of FH.

[0104] The antibody or fragment according to the present disclosure can be of any class. The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody according to the present disclosure comprises a constant region (e.g., Fc domain) of an IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the antibody according to the present disclosure comprises a constant region (e.g., Fc domain) of a human IgG (e.g., human IgG1, IgG2, IgG3, or IgG4). The Fc domain mediates the effector functions of the antibody, including the ability to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In certain embodiments, the antibody disclosed herein comprises an Fc domain with one or more mutations that reduce the effector functions of the Fc. In some embodiments, the antibodies disclosed herein comprise an Fc domain comprising a mutation (e.g., a substitution relative to wild-type human IgG1) at one or more positions selected from 234, 235, 236, 237, 270, 297, 318, 320, 322, 329, and 331. Such mutations are known in the art and include, but are not limited to, L234A, L235A, G237A, P329A or P329G, A330S, P331S, N297Q, N297A, and combinations thereof. The Fc domain also binds to the neonatal Fc receptor FcRn, thereby facilitating antibody recycling and thereby extending the half-life of the antibody. In certain embodiments, the antibodies disclosed herein comprise an Fc domain having one or more mutations that increase the binding affinity of Fc to FcRn. Such mutations are known in the art and include, but are not limited to, M252Y / T256D, T256D / T307Q, and T256D / T307W.

[0105] In certain embodiments, any antibody or fragment disclosed herein may further comprise post-translational modifications in addition to those naturally occurring in the native antibody or fragment. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, PEGylation (polyethylene glycol), and acylation. As a result, modified polypeptides may contain non-amino acid elements such as polyethylene glycol, lipids, mono- or polysaccharides, and phosphates. In certain embodiments, the antibody or fragment is PEGylated. In further particular embodiments, the antibody or fragment is a PEGylated Fab molecule.

[0106] Antibodies specific for a particular antigen, such as FH according to the present disclosure, can be prepared by various methods known in the art. For example, human FH can be used as an immunogen to elicit antibodies. As another example, the CCP18 domain of the FH-related protein human FH can be used as an immunogen. One such method is by immunization and hybridoma generation as described in the Examples. Mouse monoclonal antibodies against FH can be generated, for example, by immunizing mice (e.g., BALB / c mice) with intraperitoneal administration of human factor H or an FH-related protein, such as FHR-1, at intervals of, for example, four weeks, optionally in the presence of an adjuvant such as Montanide. A few days after the fourth immunization, spleen cells can be fused, for example, with the myeloma cell line SP2 / 0. The presence of factor H-specific antibodies in the hybridoma supernatant can be tested by ELISA. For example, microtiter plates can be coated with moAb (e.g., rat anti-mouse kappa moAb RM19) to capture mouse IgG antibodies. The specificity of the antibodies can be determined by biotinylated factor H. Another example of the method of producing FH-specific antibodies is by screening phage display library expressing recombinant nucleic acid sequence encoding immunoglobulin chain.The method of antibody phage display has been used in the art and has been widely reported.The screening of antibody library can be carried out with the same antigen as that used for immunization, for example, human FH, FH-related protein, or CCP18 domain of human FH.

[0107] C. Nucleic Acids and Vectors The present disclosure further provides one or more isolated, synthetic, or recombinant nucleic acids comprising a nucleic acid sequence encoding any of the antibodies or fragments thereof disclosed herein. In some embodiments, the nucleic acid encodes at least the heavy chain CDR1, CDR2, and CDR3, and / or the light chain CDR1, CDR2, and CDR3, of antibody 1 shown in Table 1. In some embodiments, the nucleic acid encodes at least the heavy chain CDR1, CDR2, and CDR3, and / or the light chain CDR1, CDR2, and CDR3, of antibody 2 shown in Table 1. In some embodiments, the nucleic acid encodes at least the heavy chain CDR1, CDR2, and CDR3, and / or the light chain CDR1, CDR2, and CDR3, of antibody 3 shown in Table 1. In some embodiments, the nucleic acid encodes at least the heavy chain CDR1, CDR2, and CDR3, and / or the light chain CDR1, CDR2, and CDR3, of antibody 4 shown in Table 1.

[0108] In some embodiments, the nucleic acid of the present disclosure has a length of at least 30 nucleotides, at least 50 nucleotides, or at least 75 nucleotides. In some embodiments, the nucleic acid of the present disclosure encodes a chimeric or humanized monoclonal antibody or fragment thereof comprising the heavy and light chain CDR sequences and / or the heavy and light chain variable regions of antibody 1. In some embodiments, the nucleic acid of the present disclosure encodes a chimeric or humanized monoclonal antibody or fragment thereof comprising the heavy and light chain CDR sequences and / or the heavy and light chain variable regions of antibody 2. In some embodiments, the nucleic acid of the present disclosure encodes a chimeric or humanized monoclonal antibody or fragment thereof comprising the heavy and light chain CDR sequences and / or the heavy and light chain variable regions of antibody 3. In some embodiments, the nucleic acid of the present disclosure encodes a chimeric or humanized monoclonal antibody or fragment thereof comprising the heavy and light chain CDR sequences and / or the heavy and light chain variable regions of antibody 4.

[0109] Nucleic acid sequences encoding the heavy and light chains of antibodies 5 to 7 are shown in Table 1. However, nucleic acids encoding the heavy or light chain CDRs of the antibodies of the present disclosure, which differ from the nucleic acid sequences shown in Table 1 but include nucleic acid sequences that include nucleic acid codons that encode the amino acid sequences of the heavy chain, light chain, heavy chain CDR, or light chain CDR sequences shown in Table 1, are also encompassed by the present disclosure.

[0110] Thus, there is provided an isolated, synthetic or recombinant nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 1, 2, 3, 5, 10, and 11, or any combination or fragment thereof. Further, there is provided an isolated, synthetic or recombinant nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 1, 2, 3, 5, 10, and 11, or any combination or fragment thereof. Further provided are isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 3, 5, 10, 11, 13, and 14, or any combination or fragment thereof. Further provided are isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 3, 5, 10, 11, 13, and 14, or any combination or fragment thereof.

[0111] Further provided are isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 4 and 8, or any combination or fragment thereof. Further provided are isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 4 and 12, or any combination or fragment thereof. Further provided are isolated, synthetic or recombinant nucleic acids comprising nucleic acid sequences encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 8 and 16, or any combination or fragment thereof. Further provided are isolated, synthetic or recombinant nucleic acids comprising nucleic acid sequences encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs: 12 and 16, or any combination or fragment thereof. Nucleic acids encoding heavy and / or light chain CDRs or antibodies modified, for example, by conservative amino acid substitutions, are also encompassed by the present disclosure.

[0112] In some embodiments, the nucleic acid or nucleic acid sequence of the present disclosure comprises a strand of nucleotides (i.e., DNA and / or RNA). However, the nucleic acid or nucleic acid sequence of the present disclosure may comprise other types of nucleic acid structures, such as, for example, DNA / RNA helices, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and / or ribozymes. Such other nucleic acid structures are referred to as functional equivalents of the nucleic acid sequence and are encompassed by the present disclosure. The term "functional equivalent of a nucleic acid sequence" also encompasses strands that include non-natural nucleotides, modified nucleotides and / or non-nucleotide components that exhibit the same function as natural nucleotides.

[0113] The present disclosure further provides a vector comprising one or more nucleic acids according to the present disclosure. In some embodiments, the vector is a plasmid. A plasmid is defined herein as circular. In some embodiments, the plasmid is a double-stranded DNA molecule. Methods for preparing vectors comprising nucleic acids according to the present disclosure are well known in the art. Non-limiting examples of vectors suitable for generating vectors according to the present disclosure are retroviral vectors and lentiviral vectors. Vectors according to the present disclosure can be used for various applications. Vectors according to the present disclosure are used in some embodiments for in vitro expression of nucleic acids according to the present disclosure in cells, for example for the generation of antibodies or fragments according to the present disclosure. Furthermore, vectors according to the present disclosure comprising nucleic acids according to the present disclosure can be used for therapy. When such vectors are administered to an individual, such as a human in need thereof, antibodies or fragments according to the present disclosure are expressed in vivo.

[0114] In some embodiments, the disclosure provides a vector encoding any of the antibodies or fragments disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. The recombinant AAV (rAAV) vectors of the disclosure can be generated from various adeno-associated viruses. For example, the ITRs of any AAV serotype are expected to have similar structures and functions with respect to replication, integration, excision, and transcription mechanisms. Examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, the rAAV vector is generated from serotype AAV1, AAV2, AAV4, AAV5, or AAV8. These serotypes are known to target photoreceptor cells or retinal pigment epithelium. In certain embodiments, the rAAV vector is generated from serotype AAV2. In certain embodiments, the AAV serotype comprises AAVrh8, AAVrh8R or AAVrh10. It will also be appreciated that the rAAV vector may be a chimera of two or more serotypes selected from serotypes AAV1-AAV12. The tropism of the vector may be altered by packaging a recombinant genome of one serotype into a capsid derived from another AAV serotype. In some embodiments, the ITRs of the rAAV virus may be based on any one of the ITRs of AAV1-12, and may be combined with an AAV capsid selected from any one of AAV1-12, AAV-DJ, AAV-DJ8, AAV-DJ9, or other modified serotypes. In certain embodiments, any AAV capsid serotype may be used with the vectors of the present disclosure. Examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, or AAVrh10. In certain embodiments, the AAV capsid serotype is AAV2.

[0115] Further provided is a recombinant cell comprising a nucleic acid or vector according to the disclosure. In some embodiments, the nucleic acid or vector is introduced into a cell such that the nucleic acid translation machinery of the cell produces the encoded antibody or fragment. In some embodiments, the nucleic acid or vector according to the disclosure is expressed in so-called producer cells, such as, for example, cells of the Chinese Hamster Ovary (CHO), NSO (mouse myeloma), or 293(T) cell line, some of which are adapted for commercial antibody production. Propagation of such producer cells results in a producer cell line capable of producing the antibody or fragment according to the disclosure. In some embodiments, the producer cell line is suitable for producing antibodies for use in humans. In some embodiments, the producer cell line is free of pathogenic agents, such as pathogenic microorganisms.

[0116] The disclosure further provides a method of producing an antibody or fragment according to the disclosure, comprising providing a nucleic acid or vector according to the disclosure to a cell and causing the cell to translate a nucleic acid sequence contained in the nucleic acid or vector, thereby producing an antibody or fragment according to the disclosure. In some embodiments, the method according to the disclosure further comprises harvesting, purifying, and / or isolating the antibody or fragment. Antibodies or fragments obtained using the method of producing an antibody or fragment according to the disclosure are also provided.

[0117] D. Pharmaceutical Compositions In some embodiments, the antibody or fragment of the present disclosure can be advantageously used in therapeutic applications. Thus, a pharmaceutical composition is provided that includes an antibody or fragment of the present disclosure and a pharma- ceutically acceptable carrier, diluent, and / or excipient. Also provided is a pharmaceutical composition that includes a nucleic acid or vector (e.g., an AAV vector) of the present disclosure and at least one pharma- ceutically acceptable carrier, diluent, and / or excipient. Non-limiting examples of suitable carriers are, for example, keyhole limpet hemocyanin (KLH), serum albumin (e.g., BSA or RSA), and ovalbumin. In some embodiments, the carrier is a solution, such as an aqueous solution, for example, a saline solution, or an oily solution. Non-limiting examples of excipients that can be incorporated into tablets, capsules, and the like are binders such as gum tragacanth, acacia, corn starch, or gelatin, excipients such as microcrystalline cellulose, disintegrants such as corn starch, pregelatinized starch, and alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, or saccharin, and flavoring agents such as oil of peppermint, wintergreen, or cherry. In some embodiments, the unit dosage form is a capsule. In some embodiments, the unit dosage form contains a liquid carrier, such as a fatty oil, in addition to one or more of the above excipients. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac and / or sugar, or both. In some embodiments, the pharmaceutical compositions according to the present disclosure are suitable for use in humans.

[0118] The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering a pharmaceutical composition comprising an antibody according to the present disclosure and a pharma- ceutically acceptable carrier by intraocular, intravitreal, subretinal, oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. In certain embodiments, any of the antibodies, fragments, vectors, or pharmaceutical compositions disclosed herein can be administered intravitreally to a subject. In certain embodiments, any of the antibodies, fragments, vectors, or pharmaceutical compositions disclosed herein can be administered subretinal to a subject. For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice by dissolving or suspending the antibody or fragment of the present disclosure in an injectable vehicle such as water or natural oils such as sesame oil, coconut oil, peanut oil, cottonseed oil, or synthetic fatty vehicles such as ethyl oleate, and may also incorporate buffers, preservatives, and / or antioxidants.

[0119] E. Therapeutic / Prophylactic Uses The present disclosure further provides an antibody or fragment according to the present disclosure for use in therapy. Further, a nucleic acid according to the present disclosure for use in therapy is provided. The therapy can be therapeutic or prophylactic. The antibody or fragment according to the present disclosure is particularly suitable for treating, alleviating, or preventing disorders associated with alternative pathway complement activation. Thus, an antibody or fragment according to the present disclosure is provided for use in treating, alleviating, or preventing disorders associated with alternative pathway complement activation. Also, a nucleic acid according to the present disclosure is provided for use in treating, alleviating, or preventing disorders associated with alternative pathway complement activation. As used herein, "alternative complement activation-associated disorder" is defined herein as a disorder in which unnecessary and / or excessive alternative pathway complement activation leads to cell, tissue, or extracellular matrix damage. Cells that may be damaged by unnecessary and / or excessive alternative pathway activation are any cells in contact with blood, such as red blood cells, epithelial cells, particularly liver and / or kidney epithelial cells, platelets, white blood cells, endothelial cells. In some embodiments, the disorder is a disorder associated with FH dysfunction or FH deficiency. In some embodiments, the disorder is associated with FH dysfunction or FH deficiency, but not with the absence of FH. Because the antibodies and fragments of the present disclosure enhance the function of FH, the antibodies and fragments are particularly suitable for blocking or reducing the effects of FH dysfunction or FH deficiency. However, the enhancing anti-FH antibodies of the present disclosure can also inhibit the lysis of red blood cells incubated with serum from healthy individuals in which FH has been artificially blocked. Thus, the antibodies and fragments can also be used to block or reduce unnecessary and / or excessive alternative pathway complement activation caused by factors other than FH dysfunction or FH deficiency. Non-limiting examples of such disorders that can be treated are atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), and membranoproliferative glomerulonephritis (MPGN).

[0120] Atypical hemolytic uremic syndrome (aHUS), also called complement-mediated HUS, is characterized by hemolytic anemia, thrombocytopenia, systemic thrombotic microangiopathy (TMA), and renal failure. Onset of aHUS is typically in childhood, and episodes of the disease are associated with, for example, infection, pregnancy, other illnesses, surgery, or trauma. More than 60% of aHUS patients die or develop end-stage renal disease (ESRD) despite plasma exchange or plasma replacement. Several mutations in components or factors of the complement system have been identified in aHUS patients. Mutations in FH, FI FB, membrane cofactor protein (MCP), thrombomodulin (THBD), or C3 constitute approximately 50% of known mutations in aHUS patients (approximately 20–30% of aHUS patients), with mutations in FH being the most frequent. The majority of patients are heterozygous for the mutation, but pathological FH deficiency nevertheless occurs. Furthermore, in about 10% of patients, aHUS is due to autoantibodies against FH, which also results in a reduction of functional FH. Currently, the standard treatment for aHUS is plasma replacement or plasma exchange therapy. In addition, eculizumab is used to treat aHUS patients. Kidney transplantation is associated with a high risk of recurrence depending on the mutations underlying aHUS. In children with mutations in FH, FB, FI, C3 or THBD, transplantation is contraindicated due to the increased risk of recurrence. The antibodies or fragments, such as antibodies or fragments comprising at least a Fab fragment, according to the present disclosure are particularly suitable for treating, mitigating, or preventing aHUS due to mutations in FH or the presence of anti-FH autoantibodies. The enhancing effect on FH is independent of the CCP domain of FH that has the mutation. For example, an enhancing FH antibody or fragment thereof can inhibit alternative complement activation in aHUS patients with mutations in CCP1, CCP6, CCP7, CCP14, CCP17, CCP18, CCP19 and CCP20 of FH. However, because the antibodies and fragments of the present disclosure can enhance the activity of FH even in the absence of FH dysfunction or FH deficiency, all forms of complement-dependent aHUS can be advantageously treated, alleviated, or prevented by the antibodies or fragments of the present disclosure.

[0121] Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a genetic mutation on the X chromosome of totipotent hematopoietic stem cells. This mutation results in a deficiency of phosphatidylinositol glycan class A proteins, which are important for the synthesis of glycosylphosphatidylinositol membrane-anchored proteins (GPI-APs). The complement system inhibitor CD55 is an example of such a protein, which prevents the formation of C3 convertase by binding to C3b on the host cell surface. Thus, deficiencies of these proteins result in unnecessary or excessive complement activation. One of the main consequences of PNH is the lysis of red blood cells as a result of excessive activation of the complement system. Recently, eculizumab has been approved for the treatment of PNH in several countries. Other therapies include blood transfusions, erythrocyte-stimulating agent therapy, and treatment with corticosteroids and anabolic steroids. The antibodies and fragments of the present disclosure may also be advantageously used in PNH patients, since they may enhance the activity of FH independent of FH levels or function, thereby inhibiting activation of the alternative pathway of the complement system. Furthermore, the antibodies and fragments of the present disclosure act at the level of C3 deposition, unlike eculizumab, which acts further downstream in the activation pathway, resulting in less cells being opsonized by C3b, thus reducing cellular attrition in the liver.

[0122] Age-related macular degeneration (AMD) is damage to the retina that usually affects older individuals and results in loss of vision in the macula, the center of vision. Recently, mutations and SNPs (single nucleotide polymorphisms) in FH have been implicated in approximately 35% of AMD patients. This SNP is located in CCP7 of FH and has been demonstrated to affect the binding of FH to heparin, thereby impairing the ability of FH to bind to host cell surfaces and extracellular matrix. The antibodies or fragments, such as antibodies or fragments comprising at least a Fab fragment, according to the present disclosure are particularly suitable for treating, mitigating, or preventing AMD characterized by reduced FH function (i.e., SNPs in the gene encoding FH).

[0123] Membranoproliferative glomerulonephritis (MPGN) is a rare cause of chronic nephritis that occurs mainly in children and young adults. It causes glomerular injury as a result of mesangial and endothelial cell proliferation and mesangial matrix expansion, thickening of peripheral capillary walls with subendothelial immune deposits and / or intramembranous dense deposits, and inclusion of mesangial stroma in capillary walls. MPGN is often associated with a complete lack of FH. In some embodiments, MPGN that can be treated with the antibody and / or fragment of the present disclosure is associated with FH dysfunction or FH deficiency, but not with the absence of FH.

[0124] Thus, the present disclosure provides an antibody or fragment or nucleic acid according to the present disclosure for use in treating, alleviating, or preventing a disorder associated with alternative pathway complement activation. In some embodiments, the disorder is selected from the group consisting of atypical hemolytic uraemic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN). Also provided is the use of an antibody or fragment, nucleic acid, or vector according to the present disclosure for preparing a medicament for treating, alleviating, or preventing a disorder associated with alternative pathway complement activation. In some embodiments, the disorder is selected from the group consisting of atypical hemolytic uraemic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy. In certain embodiments, the disorder is IgA nephropathy. In some embodiments, the antibody or fragment used as a pharmaceutical or prophylactic according to the present disclosure is an antibody or fragment thereof (i.e., a Fab, Fab' or F(ab)2 or F(ab')2 fragment) that comprises the heavy and light chain CDR1, CDR2, and CDR3 of an antibody shown in Table 1. In some embodiments, the antibody or fragment is a humanized or chimeric monoclonal antibody or fragment.

[0125] The present disclosure further provides a method of inhibiting alternative complement activation, comprising administering to an individual an antibody or fragment according to the present disclosure, or a nucleic acid or vector according to the present disclosure. The present disclosure further provides a method of treating, alleviating, or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof an antibody or fragment according to the present disclosure in a therapeutically effective amount. Also provided is a method of treating, alleviating, or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof a nucleic acid or vector according to the present disclosure in a therapeutically effective amount. Also provided is a method of treating, alleviating, or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof a pharmaceutical composition according to the present disclosure in a therapeutically effective amount. In some embodiments, the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), and membranoproliferative glomerulonephritis (MPGN). As used herein, an "individual" is a human or an animal (e.g., a mammal) having a complement system as part of its immune system. In certain embodiments, the individual is a human. In some embodiments, the antibody used in the disclosed methods is an antibody or a fragment thereof, such as a Fab, Fab', F(ab)2 or F(ab')2 fragment, that comprises the heavy and light chain CDR1, CDR2, and CDR3 of antibody 1, the heavy and light chain CDR1, CDR2, and CDR3 of antibody 2, the heavy and light chain CDR1, CDR2, and CDR3 of antibody 3, or the heavy and light chain CDR1, CDR2, and CDR3 of antibody 4. In some embodiments, the antibody or fragment is a humanized or chimeric monoclonal antibody or fragment.

[0126] Compositions containing the antibodies, fragments, nucleic acids of the present disclosure may be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the antibodies, fragments, nucleic acids, or compositions of the present disclosure are administered to an individual (e.g., a human) already suffering from and / or already exhibiting symptoms of the disease in an amount sufficient to alleviate the symptoms of the disease and / or its complications. In prophylactic applications, the antibodies, fragments, nucleic acids, or compositions of the present disclosure are administered to an individual before the individual exhibits symptoms of the disorder to prevent the development of these symptoms or complications. For example, individuals with genetic mutations that may cause or cause disorders associated with alternative complement activation may be treated prophylactically with the antibodies, fragments, nucleic acids, or compositions of the present disclosure. The antibodies, fragments, nucleic acids, or vector molecules are typically present in pharmaceutical compositions of the present disclosure in a therapeutically effective amount, which is an amount sufficient to address disorders associated with unnecessary or excessive activation of the alternative pathway of the complement system.

[0127] For purposes of clarity and concise description, multiple features may be described herein as part of the same or separate aspects or embodiments of the present disclosure. Those skilled in the art will appreciate that the scope of the present disclosure may include embodiments having all or some combination of the features described herein as part of the same or separate embodiments. EXAMPLES

[0128] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0129] reagent Human purified factor H was obtained from CompTech (Tyler, Texas USA). Rat anti-mouse kappa (RM19) was obtained from Sanquin (Business Unit reagents, Sanquin, Amsterdam, the Netherlands). High performance ELISA buffer (HPE) was obtained from Sanquin. Recombinant FH CCPs (CCP15–18, CCP15–19, CCP18–20, or CCP19–10) were generated as previously described (Schmidt et al. 2008). Mouse monoclonal antibodies (mAbs) against human FH were generated as previously described. Anti-IL-6.8 was used as an isotype control and was obtained from Sanquin. Anti-C3.19 may react with an epitope on the C3d fragment of the molecule (Wolbink et al. 1993).

[0130] Expression of rhFHR protein Recombinant human factor H-related (rhFHR) protein containing a C-terminal six histidine (6xHis) tag was generated and purified as previously reported (Pouw et al. 2015). Briefly, the protein was expressed by transient transfection of the pcDNA3.1 expression vector in HEK293F cells, followed by Ni-cleavage using HisTrap™ High Performance 1 ml columns (GE Healthcare Life Sciences, Freiburg, Germany). 2+ Proteins were purified from the supernatant by affinity chromatography. rhFHR was filtered and concentrated using Amicon® Ultra Centrifugal Filter Devices (Merck Millipore, Darmstadt, Germany).

[0131] antibody generation Monoclonal antibody FHR-1.3B4 was generated by immunizing BALB / c mice intraperitoneally at 4-week intervals with 25 μg recombinant human factor H-related protein 1 (FHR-1) and 25 μg rhFHR-1 in Montanide as adjuvant. Three days after the fourth booster immunization, spleen cells were fused with myeloma cell line SP2 / 0. The presence of FHR-1-specific antibodies in the hybridoma supernatants was tested by ELISA. Briefly, microtiter plates were coated with rat anti-mouse kappa moAb (RM19) to capture mouse IgG antibodies. Antibody specificity was determined by biotinylated rhFHR-1. Briefly, binding of biotinylated rhFHR-1 was determined by incubation with 0.1% (v / v) streptavidin-HRP conjugate in HPE for 30 min. The ELISA was further developed using 100 μg / mL 3,5,3',5'-tetramethylbenzidine (TMB) in 0.1 M sodium acetate, pH 5.5, containing 0.003% (v / v) H2O2. Substrate conversion was stopped by adding 100 μL H2SO4, and absorbance was measured at 450 nm and corrected to absorbance at 540 nm using a Synergy 2 Multi-Mode plate reader (BioTek Instruments, Winooski, VT, USA). All ELISA steps were performed in a final volume of 100 μL per well.

[0132] FHR-1.3B4 antibody was humanized by grafting the CDR sequences into the framework regions of human germline antibody. As a result of humanization, antibody 5 was generated. Then, specific amino acids of antibody 5 were mutated to improve the binding affinity to FH. Antibody 1, antibody 2, antibody 3, and antibody 4 were the antibodies that exhibited the highest affinity to FH, as disclosed in Table 1.

[0133] Binding affinity of antibodies to factor H To determine the binding affinity of antibodies 1-4, surface plasmon resonance (SPR) experiments were performed using a BiaCore T200 (GE Healthcare) and a CM5 sensor chip (GE Healthcare) according to the manufacturer's instructions. Briefly, antibodies 1-9 or anti-FHR-1.3B4 were captured on a chip conjugated with protein A or protein G, and decreasing concentrations of full-length FH were flowed over the captured moAB. The binding kinetics obtained from the SPR experiments are presented in Table 2. [Table 2]

[0134] As shown in Table 2, Antibody 1, Antibody 2, Antibody 3, and Antibody 4 bound to human FH with nanomolar or subnanomolar affinity. Furthermore, Antibodies 1-4 had higher binding affinity for full-length FH than that of FHR-1.3B4.

[0135] Epitope mapping of mAbs and competition assays The binding sites of certain monoclonal antibodies (mAbs) were mapped. Specifically, the reactivity of the mAbs was tested against recombinant FH fragments containing various CCP domains (CCP15-18, CCP15-19, CCP18-20, and CCP19-20) and full-length human FH. Recombinant human FH fragments composed of multiple CCP domains (15-18, 15-19, 18-20, or 19-20) were used to determine the location of the epitopes of the mAbs, such as those disclosed in Table 1. Briefly, FH.07 and FHR-1.3B4 were captured on microtiter plates coated with RM-19 to ensure optimal binding conformation. Biotinylated FH mixed with 100-fold higher concentrations of the indicated unlabeled recombinant FH fragments was then incubated on the plates for 1 h. Binding of biotinylated FH was determined by incubation with 0.01% (v / v) streptavidin-polyHRP conjugate in HPE for 30 min. The ELISA was further developed as previously reported. FH.07 and FHR-1.3B4 were found to bind to CCP18 of human FH.

[0136] A competition assay using the antibodies or fragments disclosed in Table 1 was performed with the agonist anti-FH antibody FH.07. A similar setup as above was used to determine whether the humanized anti-FHR-1 mAbs disclosed in Table 1 compete with FH.07 for FH binding. Briefly, mAbs were directly coated onto plates and binding of biotinylated FH (FH-bt) in the absence or presence of 10-fold higher concentrations of the indicated mAbs was assessed by ELISA as previously reported. FHR-1.3B4 was found to compete with FH.07.

[0137] C3 Deposition in LPS To investigate whether antibodies 1–4 affect alternative pathway inhibition by FH, a C3 deposition assay in LPS was performed. Briefly, 96-well microtiter plates (Nunc) were coated with PBS containing Salmonella typhosa LPS (40 μg / mL, L-6386 Sigma-Aldrich) overnight at room temperature. LPS was used to activate the alternative pathway of complement. Plates were washed with PBS + 0.1% (w / v) Tween-20, 10% (v / v). Normal human serum (NHS, final concentration 10% (v / v)) was incubated in veronal buffer (VB; 3 mM barbital, 1.8 mM sodium barbital, 145 mM NaCl, pH 7.4) containing 0.05% (w / v) gelatin, 5 mM MgCl2, 10 mM EGTA, and 0.1% (w / v) Tween-20 in the presence or absence of the indicated concentrations of anti-FH / anti-FHR-1 mAbs (antibodies 1-4 or FHR-1.3B4), isotype controls, or aIL6-8 AB as a negative control. C3b deposition was detected with biotinylated mAb anti-C3.19 (0.55 μg / mL in HPE), followed by incubation with 0.01% (v / v) streptavidin-polyHRP conjugate for 30 min in HPE. ELISA was developed as described above. Table 3 shows the IC of antibodies 1 to 4 and FHR-1.3B4. 50 The IC values ​​for antibodies 1 to 4 are shown in Table 4. 50 Values ​​(2 replicates) are reported. [Table 3] [Table 4]

[0138] As shown in Tables 3 and 4, antibodies 1 to 4 inhibited C3b deposition in LPS. This indicated that the inhibitory function of FH against alternative pathway activation was enhanced by the addition of antibodies 1, 2, 3, or 4. Furthermore, as shown in Table 3, antibodies 1 to 4 showed lower IC 50As shown by the values, it was more effective than FHR-1.3B4.

[0139] SRBC hemolysis assay To investigate the effect of antibodies 1–4 on factor H function, we performed a hemolysis assay as previously outlined by Sanchez-Corral et al. (2004) and Wouters et al. (2008). Briefly, pre-diluted human serum (20%, v / v) containing 20 μg / ml anti-FH.09 (a blocking antibody against FH) was pre-incubated with the indicated mAb (antibodies 1–4 or FHR-1.3B4) and mixed with sheep red blood cells (SRBCs) in a 1:1 ratio and incubated at 37°C for 1 h in VB containing 5 mM MgCl2 and 10 mM EGTA, or in VB containing 10 mM EDTA as blank, with 10% (v / v) serum and 1.05*10 8 Cells were incubated at a final concentration of 100 μl of ice-cold VB containing 20 mM EDTA for 75 min at 37° C. with shaking. Lysis was stopped by the addition of 100 μl of ice-cold VB containing 20 mM EDTA, followed by centrifugation (2.5 min, 1,800 RPM / 471 RCF, 7° C.). Hemolysis was measured as the absorbance of the supernatant at 412 nm, corrected for background absorbance measured at 690 nm, and expressed as a percentage of the 100% lysis control (SRBCs incubated with 0.6% (w / v) saponin). As a negative control, SRBCs were incubated with serum diluted in VB supplemented with 10 mM EDTA to prevent complement activation.

[0140] Using Graphpad prism 7.04, IC of hemolysis was calculated from multiple experiments using various antibodies as disclosed in Table 1. 50 The IC values ​​of antibodies 1 to 4 and FHR-1.3B4 were calculated and statistically compared using standard one-way ANOVA and Dunnett's multiple comparison test. 50 The IC values ​​for antibodies 1 to 4 are shown in Table 6. 50 Values ​​(2 replicates) are reported. [Table 5] [Table 6]

[0141] As shown in Tables 5 and 6, antibodies 1-4 inhibited SRBC lysis. Furthermore, as shown in Table 6, antibodies 1-3 had lower IC 50 As shown by the values, it was more effective than FHR-1.3B4.

[0142] Binding affinity of factor H to C3b in the presence of anti-FH antibodies Binding of FH to C3b in the presence of antibodies 1-4 as disclosed in Table 1 was determined by surface plasmon resonance using a Biacore T200 instrument (GE Healthcare, Little Chalfont, UK). Specifically, purified C3b (Complement Technologies) was immobilized on the flow cell of a CM5 Biacore Sensor Chip (GE Healthcare) by using standard amine coupling. The remaining flow cell was used as a reference surface and was prepared by performing the coupling reaction without adding protein. After coupling with C3b, a response of 2000 response units (RU) was obtained. SPR experiments were performed at 37 °C in phosphate buffered saline (PBS, Orphi Farma) at pH 7.4 (PBS-T) supplemented with 0.01% (w / v) Tween-20 (Merck) using a flow rate of 10 μl / min.

[0143] To determine the effect of antibodies without possible interference from cross-linking by moAbs, recombinantly produced Fab' fragments of mAbs (antibodies 1-4) were used. Fab' fragments were mixed with plasma purified FH (pdFH). Different concentrations of FH (10-0,01953 μM for FH alone and 5-0,01953 μM for FH complexed with moAb Fab' fragments) were injected for 60 s over the chip in the absence or presence of 10 μM (at least 2-fold molar excess) of anti-FHR-1 3B4 Fab' fragment or Fab' fragments of antibodies 1-4. Each Fab' fragment was also injected without the addition of FH to determine the presence or absence of interaction of the Fab' fragment with the surface. Injection of pdFH was followed by a 60 s dissociation and the surface was regenerated with one injection of 1 M NaCl (Merck) between each cycle.

[0144] Data was analyzed using Scrubber 2 (Biologic Software) and affinities were determined by equilibrium analysis. Table 7 lists the binding kinetics of antibodies 1-4. [Table 7]

[0145] As shown in Table 7, the addition of Fab' fragments of antibodies 1 to 4 increased the response on the C3b-coated surface, indicating that antibodies 1 to 4 enhanced the binding of pdFH to C3b. EMBODIMENTS OF THE PRESENT DISCLOSURE Embodiments of the present invention are further described in the following sections: [Section 1] 1. An isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to complement control protein domain 18 (CCP18) of factor H (FH), - a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO: 1) or the sequence TSVTY (SEQ ID NO: 13), - a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO: 2) or the sequence ASS (SEQ ID NO: 14), - a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO: 6) or IWSGGTT (SEQ ID NO: 10), and - a heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDY (SEQ ID NO: 7) or the sequence ARNFGNYAMDF (SEQ ID NO: 11) The antibody or antigen-binding fragment thereof. [Section 2] - a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO: 1), - a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO: 2), - a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 sequence comprising the sequence IWSGGTT (SEQ ID NO: 10), and - the heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDF (SEQ ID NO: 11) The antibody or fragment according to claim 1, [Section 3] - a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO: 1), - a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO: 2), - a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO: 6), and - the heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDY (SEQ ID NO: 7) The antibody or fragment according to claim 1, [Section 4] - a light chain CDR1 sequence comprising the sequence TSVTY (SEQ ID NO: 13), - a light chain CDR2 sequence comprising the sequence ASS (SEQ ID NO: 14), - a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO: 6), and - the heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7) The antibody or fragment according to claim 1, [Section 5] - a light chain CDR1 sequence comprising the sequence TSVTY (SEQ ID NO: 13), - a light chain CDR2 sequence comprising the sequence ASS (SEQ ID NO: 14), - a light chain CDR3 sequence comprising the sequence QHRSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO: 5), - a heavy chain CDR2 sequence comprising the sequence IWSGGTT (SEQ ID NO: 10), and - the heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDF (SEQ ID NO: 11) The antibody or fragment according to claim 1, [Section 6] The antibody or fragment has a titer of 2.5×10 -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D or the antibody or fragment has a binding affinity for FH of 1.25×10 -8 K below M D and / or 0.04 × 10 for the FH fragment consisting of CCP18-20-9 K below M D 6. The antibody or fragment according to any one of items 1 to 5, having a binding affinity of [Section 7] The antibody or fragment has a titer of 2.5×10 -8 K below M D and / or 0.1 × 10 for the FH fragment consisting of CCP18-20 -9 K below M D or the antibody or fragment has a binding affinity for FH of 0.6×10 -8 K below M D and / or 0.6 × 10 for the FH fragment consisting of CCP18-20 -11 K below M D 6. The antibody or fragment according to any one of items 1 to 5, having a binding affinity of [Section 8] The antibody or fragment thereof - In the presence of 10% (v / v) serum, C3 deposition in lipopolysaccharide in vitro was inhibited with an IC of 38 nM or less. 50 value, e.g., IC below 30 nM 50 Inhibit at the value, and / or - In vitro hemolytic activity in the presence of 10% (v / v) serum with an IC of 150 nM or less 50 value, e.g., IC below 130 nM 50 Inhibit at the value, and / or - K of FH against C3b in vitro D 8. The antibody or fragment according to any one of claims 1 to 7, which reduces the binding affinity of FH to C3b in vitro by at least 3-fold (increasing the binding affinity) to a maximum of 2 μM. [Section 9] - a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or 16; - a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 12; The antibody or fragment according to any one of items 1 to 8, comprising: [Section 10] 10. The antibody or fragment according to any one of items 1 to 9, comprising a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4, and a variable heavy chain sequence comprising a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8. [Section 11] The antibody or fragment according to any one of items 1 to 9, comprising a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:12. [Section 12] The antibody or fragment according to any one of items 1 to 9, comprising a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. [Section 13] The antibody or fragment according to any one of items 1 to 9, comprising a variable light chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16, and a variable heavy chain sequence comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. [Section 14] The antibody or fragment enhances FH activity, optionally wherein the FH activity is inhibition of alternative complement activation, and further optionally wherein the inhibition of alternative complement activation is - Inhibition of hemolytic activity, - inhibition of complement component 3 (C3) deposition, and / or - Increased binding of FH to C3b, iC3b and / or C3d 14. The antibody or fragment according to any one of items 1 to 13, comprising: [Section 15] 15. The antibody or fragment according to any one of items 1 to 14, wherein the antibody or fragment comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, and further comprises an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region. [Section 16] 16. The antibody or fragment thereof according to any one of items 1 to 15, wherein the antibody or fragment thereof comprises a Fab fragment. [Section 17] 17. The antibody or fragment according to any one of items 1 to 16, wherein the antibody or fragment is monoclonal. [Section 18] 18. The antibody or fragment thereof according to any one of items 1 to 17, which comprises human light chain and heavy chain constant regions. [Section 19] 19. The antibody or fragment thereof according to any one of items 1 to 18, wherein the antibody or fragment is PEGylated. [Section 20] 20. The antibody or fragment thereof according to item 19, wherein the antibody or fragment is a PEGylated Fab fragment. [Section 21] 21. An isolated, synthetic, or recombinant nucleic acid comprising a nucleic acid sequence encoding the antibody or fragment thereof according to any one of items 1 to 20 above. [Section 22] 22. A vector comprising the nucleic acid according to 21 above. [Section 23] 23. The vector according to claim 22, wherein the vector is an AAV vector. [Section 24] 24. The vector according to claim 23, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. [Section 25] A recombinant cell comprising the nucleic acid according to item 21 above, or the vector according to any one of items 22 to 24 above. [Section 26] A pharmaceutical composition comprising the antibody or fragment thereof according to any one of items 1 to 20, the nucleic acid according to item 21, the vector according to any one of items 22 to 24, or the recombinant cell according to item 25, and a pharma- ceutically acceptable carrier, diluent, and / or excipient. [Section 27] The antibody or fragment thereof according to any one of items 1 to 20, the nucleic acid according to item 21, the vector according to any one of items 22 to 24, the recombinant cell according to item 25, or the pharmaceutical composition according to item 26, for use in a therapeutic agent. [Section 28] Item 27. The antibody or fragment thereof according to any one of items 1 to 20, the nucleic acid according to item 21, the vector according to any one of items 22 to 24, the recombinant cell according to item 25, or the pharmaceutical composition according to item 26, for use in inhibiting alternative complement activation. [Section 29] The antibody or fragment according to any one of items 1 to 20, the nucleic acid according to item 21, the vector according to any one of items 22 to 24, the recombinant cell according to item 25, or the pharmaceutical composition according to item 26, for use in the treatment, alleviation, or prevention of a disorder associated with alternative pathway complement activation. [Section 30] The antibody or fragment, nucleic acid, recombinant cell, or pharmaceutical composition for use according to item 29 above, wherein the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy. [Section 31] Use of the antibody or fragment according to any one of items 1 to 20, the nucleic acid according to item 21, the vector according to any one of items 22 to 24, the recombinant cell according to item 25, or the pharmaceutical composition according to item 26, for the preparation of a medicament for the treatment, alleviation, or prevention of a disorder associated with alternative pathway complement activation. [Section 32] The use according to item 31 above, wherein the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy. [Section 33] A method for treating, alleviating, or preventing a disorder associated with alternative pathway complement activation, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or fragment according to any one of items 1 to 20, the nucleic acid according to item 21, the vector according to any one of items 22 to 24, the recombinant cell according to item 25, or the pharmaceutical composition according to item 26. [Section 34] 34. The method according to claim 33, wherein the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy. [Section 35] A method for inhibiting alternative complement activation, comprising administering to a subject the antibody or fragment thereof according to any one of items 1 to 20, the nucleic acid according to item 21, the vector according to any one of items 22 to 24, the recombinant cell according to item 25, or the pharmaceutical composition according to item 26. [Section 36] A method for producing an antibody or a fragment thereof, comprising: supplying a cell with the nucleic acid according to Item 21 or the vector according to any one of Items 22 to 24; and causing the cell to translate the nucleic acid or the nucleic acid sequence contained in the vector, thereby producing the antibody or the fragment thereof according to any one of Items 1 to 20. [Section 37] 37. The method of claim 36, further comprising harvesting, purifying and / or isolating the antibody or fragment. References · Almagro JC, Fransson J. 2008. Humanization of antibodies. Frontiers in Bioscience 13: 1619-33. · Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, Shepard HM. 1992. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. U.S.A. 89(10):4285-9. · Chen Y, Wiesmann C, Fuh G, Li B, Christinger HW, McKay P, de Vos AM, Lowman HB. 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[0146] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0147] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. An isolated, synthetic, or recombinant antibody or antigen-binding fragment thereof that specifically binds to complement control protein domain 18 (CCP18) of factor H (FH), a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO: 1) or the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO: 2) or the sequence ASS (SEQ ID NO: 14), a light chain CDR3 sequence comprising the sequence QHRSSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence IWSGGTT (SEQ ID NO: 10) or the sequence VWSGGTT (SEQ ID NO: 6), and - a heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDF (SEQ ID NO: 11) or the sequence ARNFGNYAMDY (SEQ ID NO: 7); The antibody or antigen-binding fragment thereof.

2. a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO: 1), a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO:2), a light chain CDR3 sequence comprising the sequence QHRSSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence IWSGGTT (SEQ ID NO: 10), and - heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDF (SEQ ID NO: 11) The antibody or fragment of claim 1 , comprising:

3. a light chain CDR1 sequence comprising the sequence SSVTY (SEQ ID NO: 1), a light chain CDR2 sequence comprising the sequence ATS (SEQ ID NO:2), a light chain CDR3 sequence comprising the sequence QHRSSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO: 6), and - heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDY (SEQ ID NO: 7) The antibody or fragment of claim 1 , comprising:

4. a light chain CDR1 sequence comprising the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence comprising the sequence ASS (SEQ ID NO: 14), a light chain CDR3 sequence comprising the sequence QHRSSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence VWSGGTT (SEQ ID NO: 6), and - heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7) The antibody or fragment of claim 1 , comprising:

5. a light chain CDR1 sequence comprising the sequence TSVTY (SEQ ID NO: 13), a light chain CDR2 sequence comprising the sequence ASS (SEQ ID NO: 14), a light chain CDR3 sequence comprising the sequence QHRSSSSNPLT (SEQ ID NO: 3), - a heavy chain CDR1 sequence comprising the sequence GFSLTNYG (SEQ ID NO:5), a heavy chain CDR2 sequence comprising the sequence IWSGGTT (SEQ ID NO: 10), and - heavy chain CDR3 sequence comprising the sequence ARNFGNYAMDF (SEQ ID NO: 11) The antibody or fragment of claim 1 , comprising:

6. The antibody or fragment has a titer of 2.5×10 －８ K below M Ｄ and / or a binding affinity of 0.1×10 to the FH fragment consisting of CCP18-20 －９ K below M Ｄ or the antibody or fragment has a binding affinity for FH of 1.25×10 －８ K below M Ｄ and / or 0.04×10 to the FH fragment consisting of CCP18-20 －９ K below M Ｄ The antibody or fragment according to any one of claims 1 to 5, having a binding affinity of

7. The antibody or fragment thereof - In the presence of 10% (v / v) serum, C3b deposition in lipopolysaccharide in vitro was inhibited with an IC of 38 nM or less. ５０ Inhibit at the value, and / or - In vitro hemolytic activity in the presence of 10% (v / v) serum with an IC of 150 nM or less ５０ Inhibit at the value, and / or - K of FH against C3b in vitro Ｄ 7. The antibody or fragment of claim 1, which reduces the binding affinity of FH to C3b in vitro by at least 3-fold (increasing the binding affinity) to a maximum of 2 μM.

8. - a variable light chain sequence comprising the sequence of SEQ ID NO: 4 or SEQ ID NO: 16, and - a variable heavy chain sequence comprising the sequence of SEQ ID NO: 12 or SEQ ID NO: 8 The antibody or fragment according to any one of claims 1 to 7, comprising:

9. The antibody or fragment according to any one of claims 1 to 8, comprising a variable light chain sequence comprising the sequence of SEQ ID NO:4 and a variable heavy chain sequence comprising the sequence of SEQ ID NO:

12.

10. The antibody or fragment according to any one of claims 1 to 8, comprising a variable light chain sequence comprising the sequence of SEQ ID NO:4 and a variable heavy chain sequence comprising the sequence of SEQ ID NO:

8.

11. The antibody or fragment according to any one of claims 1 to 8, comprising a variable light chain sequence comprising the sequence of SEQ ID NO: 16 and a variable heavy chain sequence comprising the sequence of SEQ ID NO:

8.

12. The antibody or fragment according to any one of claims 1 to 8, comprising a variable light chain sequence comprising the sequence of SEQ ID NO: 16 and a variable heavy chain sequence comprising the sequence of SEQ ID NO:

12.

13. The antibody or fragment enhances FH activity, optionally wherein the FH activity is inhibition of alternative complement activation, and further optionally wherein the inhibition of alternative complement activation is - inhibition of hemolytic activity, - inhibition of C3b deposition, and / or - increased binding of FH to C3b, iC3b and / or C3d The antibody or fragment according to any one of claims 1 to 12, comprising:

14. The antibody or fragment according to any one of claims 1 to 13, wherein the antibody or fragment comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, and further comprises an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region.

15. The antibody or fragment of any one of claims 1 to 14, wherein the antibody or fragment comprises a Fab fragment.

16. The antibody or fragment of any one of claims 1 to 15, wherein the antibody or fragment is monoclonal.

17. The antibody or fragment of any one of claims 1 to 16, wherein the antibody or fragment comprises human light and heavy chain constant regions.

18. The antibody or fragment according to claim 17, wherein the constant region comprises a human IgG4 Fc domain.

19. 19. The antibody or fragment according to claim 18, wherein the Fc domain of human IgG4 comprises the amino acid residue Ala or Gln at position 297.

20. The antibody or fragment of any one of claims 1 to 19, wherein the antibody or fragment is PEGylated.

21. The antibody or fragment of claim 20, wherein the antibody or fragment is a PEGylated Fab fragment.

22. 22. An isolated, synthetic or recombinant nucleic acid comprising a nucleic acid sequence encoding the antibody or fragment of any one of claims 1 to 21.

23. A vector comprising the nucleic acid of claim 22.

24. The vector of claim 23, wherein the vector is an AAV vector.

25. 25. The vector of claim 24, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

26. A recombinant cell comprising the nucleic acid according to claim 22 or the vector according to any one of claims 23 to 25.

27. A pharmaceutical composition comprising an antibody or fragment according to any one of claims 1 to 21, a nucleic acid according to claim 22, a vector according to any one of claims 23 to 25, or a recombinant cell according to claim 26, and a pharma- ceutically acceptable carrier, diluent, and / or excipient.

28. An antibody or fragment according to any one of claims 1 to 21, a nucleic acid according to claim 22, a vector according to any one of claims 23 to 25, a recombinant cell according to claim 26, or a pharmaceutical composition according to claim 27 for use in therapy.

29. An antibody or fragment according to any one of claims 1 to 21, a nucleic acid according to claim 22, a vector according to any one of claims 23 to 25, a recombinant cell according to claim 26, or a pharmaceutical composition according to claim 27, for use in inhibiting alternative complement activation.

30. The antibody or fragment of any one of claims 1 to 21, the nucleic acid of claim 22, the vector of any one of claims 23 to 25, the recombinant cell of claim 26, or the pharmaceutical composition of claim 27, for use in the treatment, alleviation, or prevention of a disorder associated with alternative pathway complement activation.

31. 31. The antibody or fragment, nucleic acid, recombinant cell, or pharmaceutical composition for use according to claim 30, wherein the disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), and IgA nephropathy.

32. A method for producing an antibody or fragment thereof, comprising providing a cell with a nucleic acid according to claim 22 or a vector according to any one of claims 23 to 25, and causing the cell to translate the nucleic acid sequence contained in the nucleic acid or vector, thereby producing the antibody or fragment according to any one of claims 1 to 21.

33. 33. The method of claim 32, further comprising harvesting, purifying, and / or isolating the antibody or fragment.

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