Factor H enhancing antibodies and their uses

Abstract

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

Description

[0001] Related Applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 875,309, filed on July 17, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] 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 was created on August 4, 2020, named GEM-013WO_SL.txt, and is 18,037 bytes in size. Background of the Invention

[0006] The complement system is an important part of innate immunity, which helps protect many organisms (such as mammals) from invasive pathogens. The complement system consists of more than 30 different components, which are mainly synthesized in the liver. Activation of the complement system occurs through three different pathways, namely the classical pathway, the lectin pathway, and the alternative pathway. The three pathways converge when the C3 convertase is formed, and each of the pathways is different but has similar activities.

[0007] In the classical complement pathway, activation of the complement component (C) 1 complex, composed of C1q, C1r, and C1s, occurs upon binding to an antibody-antigen complex. The C1 complex cleaves C4 and C2, thereby forming a C3 convertase composed of C4bC2a. The C3 convertase cleaves C3 into active components C3a and C3b. In the lectin pathway, mannose-binding lectins bind to mannose residues on the surface of pathogens, thereby activating serine proteases MASP-1 and MASP-2 that can cleave C4 and C2. As in the classical pathway, this leads to the formation of C4bC2a C3 convertase. This C3 convertase can bind to C3b to form C5 convertase. In contrast to the classical and lectin pathways, the alternative pathway has a low level of continuous activity due to the spontaneous hydrolysis of C3 to C3 (H2O) in plasma. This C3b-like C3 (H2O) can form a fluid-phase C3 convertase by binding to factor B (FB), which in turn is activated to Bb by factor D. Similarly, when C3b is bound to the surface, it can bind FB to form C3bB. This complex is cut into C3bBb by factor D, which is an alternative pathway C3 convertase that can be stabilized as C3bBbP by properdin (factor P). This C3 convertase can cut C3 into C3a and C3b. In addition to this process, the alternative pathway also serves as an amplification loop of the classical pathway and the lectin activation pathway, because the C3b produced in these pathways can serve as the starting point of the alternative pathway. Thus, the amplification loop enhances the classical pathway and the lectin pathway. The C3 convertase formed in one of the three activation pathways can bind C3b to form a C5 convertase. The C5 convertases of all three complement pathways activate C5 into C5a and C5b, thereby initiating the terminal pathway of the complement system. C5b binds C6, C7, C8 and C9 to form a membrane attack complex (MAC), thereby forming a transmembrane channel and causing cell lysis.

[0008] Following the formation of pores in the pathogen's membrane, complement aids in the clearance of pathogens or altered autologous cells by aiding phagocytosis with the C3b molecule and producing pro-inflammatory peptides (such as C3a and C5a) that attract and activate immune cells to the site of infection. Due to the strong pro-inflammatory properties of complement, host cells are well protected by several membrane and soluble complement regulatory proteins.

[0009] The alternative pathway accounts for 80% to 90% of total complement activity. Therefore, regulation of this pathway is critical. C3(H2O) and C3b, formed by spontaneous hydrolysis of C3, are generally rapidly inactivated by factor H (FH), factor I (FI), and host cell surface molecules when not bound by pathogens, thereby inhibiting C3 convertase formation. CD55 (also known as decay-accelerating factor or DAF) binds to C3b on the surface of host cells. FI cleaves C3b into an inactive form, but is dependent on cofactors expressed on the cell surface (CD46, MCP) or circulating in plasma (FH).

[0010] FH is a plasma glycoprotein that is essential for controlling the alternative complement pathway in solution and on the cell surface. FH binds C3b at the same position as FB, thereby preventing C3 convertase from forming. FH also has decay-accelerating activity, that is, it promotes their dissociation after the alternative pathway C3 convertase has been formed. Whether FH binds to C3b is determined by the carbohydrates present on the cell surface. Sialic acid, glycosaminoglycans and heparin present on the host cell surface promote FH to bind to C3b, and C3b binds to molecules expressed on the surface of pathogens and causes FB to bind. FH therefore exerts its complement inhibition activity on host cells rather than on the surface of pathogenic cells, because the cell surface molecules that bind FH are expressed on host cells but are generally not expressed on pathogenic cells. FH contains 20 complement control protein (CCP) domains, numbered 1 to 20 from the N-terminus of FH. CCP domains are also called short common repeats (SCR) or sushi domains. CCP 1-4 are domains involved in regulation, CCP 19-20 are involved in binding C3b, and CCP 6, 7, 8, 19 and 20 bind to GAG and sialic acid expressed on the cell surface. Antibodies that bind to CCP19 and / or CCP20 inhibit the activity of FH.

[0011] Factor H related protein (CFHR) is a plasma glycoprotein related to the structure and antigenicity of FH. The FHR protein also includes CCP domains, and these domains have varying degrees of homology with the CCP domains found in FH. For example, FHR1 includes domains corresponding to CCP6, CCP7, CCP18, CCP19 and CCP20. Compared with FH, CFHR does not have strong complement inhibitory activity. A common feature of CFHR is that they bind to the C3b component of complement, thereby competing with FH for binding to C3b, and are therefore considered to be positive regulators of the alternative complement pathway.

[0012] FH deficiency or impaired host surface recognition due to mutations is associated with complement-mediated tissue damage and disease. Following the control of complement activation in 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 may cause the FH protein to lose function have been described. The C-terminal region of FH is a mutation hotspot for the disease. This is the key region for FH to bind to host cells. Most disease-related mutations in this region interfere with FH binding. Most patients with mutant FH genes have heterozygous mutations, meaning that about half of the circulating FH has normal function. However, under certain conditions where complement is activated, this is apparently not enough to protect the self-surface. FH deficiency may lead to kidney disease, such as membranoproliferative glomerulonephritis (MPGN) and atypical hemolytic uremic syndrome (aHUS). Recently, the relationship between FH mutations and age-related macular disease (AMD) has been described.

[0013] Currently, the standard treatment for FH deficiency (such as in aHUS) is plasma supplementation or plasma exchange therapy. With this type of therapy, the missing complement regulators are replenished. Plasma exchange therapy also removes mutant complement factors and / or autoantibodies to complement factors. However, plasma therapy also has some limitations. There are no prospective clinical studies that have demonstrated that plasma exchange therapy is safe or effective in treating aHUS, and the efficiency of plasma therapy may depend on the underlying mutation. Some patients have developed allergic reactions to fresh frozen plasma, which may require discontinuation of any form of plasma therapy. In addition, plasma exchange may worsen the clinical expression of aHUS due to the administration of active pathogenic complement components derived from plasma.

[0014] Recently, the therapeutic monoclonal antibody eculizumab has been approved in several countries, including the United States and European countries, for the treatment of aHUS and paroxysmal nocturnal hemoglobinuria (PNH). Eculizumab is a C5-specific humanized mouse monoclonal antibody that can prevent C5 from being cleaved into C5a and C5b. It therefore prevents activation of the terminal pathway and reduces the influx of immune cells. However, the use of eculizumab is associated with adverse side effects. Since it blocks C5, a key component that triggers the terminal pathway, patients treated with eculizumab become susceptible to infection with encapsulated bacteria (such as Neisseria meningitidis), the clearance of which is highly dependent on MAC formation. Therefore, patients are required to be vaccinated with a meningococcal vaccine before receiving eculizumab treatment. In addition, since eculizumab works downstream of C3, C3 deposition is maintained, which is disadvantageous in several conditions involving poor or excessive complement activation. In addition, eculizumab treatment involves high costs and the availability of antibodies is limited.

[0015] Cheng et al. (Clinical Chemistry, 2005) described a mouse monoclonal antibody that binds CCP18. It is described that this antibody (called X52.1) increases the binding of FH to C3b and C3d, which is believed to be caused by FH dimerization. X52.1 induces FH to bind to C3b and C3d and increases the dissolution of complement-mediated cells (including RBCs and several types of cancer cells as shown in Corey et al. (J Biol Chem. 2000)). This proves that antibody X52.1 inhibits the complement inhibition activity of FH. In fact, Corey et al. show that the antibody can be used to treat cancer by enhancing complement-mediated cancer cell dissolution.

[0016] There continues to be a need for new and improved therapeutic agents that bind FH, such as agents that can be used to treat disorders associated with inappropriate or excessive complement activation. SUMMARY OF THE INVENTION

[0017] The present invention provides antibodies or antigen-binding fragments thereof, such as 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 can be used to enhance the activity of FH, for example, to increase the binding affinity of FH to C3b, to increase the ability of FH to inhibit C3 deposition, and / or to increase 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 (SEQID 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).

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In some embodiments, the binding affinity of the antibody or fragment to FH is measured as K. D 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 M or less, or wherein the binding affinity of the antibody or fragment to FH is expressed as K D 1.25×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.04×10 -9 In some embodiments, the binding affinity of the antibody or fragment to FH is expressed as K D 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 M or less, or wherein the binding affinity of the antibody or fragment to FH is expressed as K D 0.6×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.6×10-11 In some embodiments, the antibodies or fragments of the invention inhibit C3 deposition on lipopolysaccharide (LPS) in vitro in the presence of 10% (v / v) serum, wherein IC 50 Values ​​of 38 nM or lower, such as IC 50 value of 30 nM or less; and / or inhibiting in vitro hemolytic activity in the presence of 10% (v / v) serum, where IC 50 Values ​​of 150 nM or less, such as IC 50 and / or in the presence of 10% (v / v) serum, the K of FH for C3b in vitro is 130 nM or less; D value (increase binding affinity) to 2 μM or less; and / or increase the in vitro binding affinity of FH to C3b by at least 3-fold in the presence of 10% (v / v) serum. In certain embodiments, the serum is normal human serum.

[0023] In some embodiments, the antibody or fragment enhances FH activity, optionally, wherein the FH activity is to inhibit alternative complement activation, further optionally, wherein inhibiting alternative complement activation includes: inhibiting hemolytic activity; inhibiting complement component 3 (C3) deposition; and / or increasing the 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 wherein the antibody or fragment also comprises an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region. In some embodiments, the antibody or fragment comprises a constant region of IgG. In some embodiments, the constant region of IgG is IgG1, IgG2, IgG3 or IgG4 (eg, human IgG1, IgG2, IgG3 or IgG4). In some embodiments, the antibody or fragment comprises a constant region of IgG4 (eg, human IgG4). In some embodiments, the constant region of IgG4 comprises a mutation at position 297. In certain embodiments, the constant region of IgG4 comprises amino acid residue Q at position 297. In certain embodiments, the constant region of IgG4 comprises amino acid residue A at position 297.

[0024] 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 or a fragment thereof comprising a human light chain and a heavy chain constant region. In some embodiments, the antibody or fragment is PEGylated. In some embodiments, the antibody or fragment is a PEGylated Fab fragment.

[0025] In some embodiments, the present invention 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 invention 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.

[0026] In some embodiments, the invention provides a recombinant cell comprising any of the nucleic acids disclosed herein or any of the vectors disclosed herein.

[0027] In some embodiments, the present invention provides a pharmaceutical composition comprising any of the antibodies or fragments disclosed herein, any of the nucleic acids disclosed herein, any of the vectors disclosed herein, or any of the recombinant cells disclosed herein, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0028] In some embodiments, the present invention provides any one of the antibodies or fragments disclosed herein, any one of the nucleic acids disclosed herein, and any one of the vectors disclosed herein for use in treatment. In some embodiments, the present invention provides any one of the antibodies or fragments disclosed herein, any one of the nucleic acids disclosed herein, or any one of the vectors disclosed herein for use in inhibiting alternative complement activation. In some embodiments, the present invention provides any one of the antibodies or fragments disclosed herein, any one of the nucleic acids disclosed herein, or any one of the vectors disclosed herein for use in treating, alleviating or preventing disorders associated with complement activation of alternative pathways. 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), membranous proliferative glomerulonephritis (MPGN) and IgA nephropathy.

[0029] In some embodiments, the present invention provides a use of any of the antibodies or fragments disclosed herein, any of the nucleic acids disclosed herein, or any of the vectors disclosed herein for the preparation of 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 uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN) and IgA nephropathy.

[0030] In some embodiments, the present invention provides a method of treating, alleviating or preventing a disorder associated with alternative pathway complement activation, the method comprising administering to an individual in need thereof a therapeutically effective amount of any of the antibodies or fragments disclosed herein, any of the nucleic acids disclosed herein, any of the vectors disclosed herein, or any of the pharmaceutical compositions 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), membranous proliferative glomerulonephritis (MPGN) and IgA nephropathy.

[0031] In some embodiments, the present invention provides a method of inhibiting alternative complement activation, the method comprising administering to a subject any of the antibodies or fragments disclosed herein, any of the nucleic acids disclosed herein, any of the vectors disclosed herein, or any of the pharmaceutical compositions disclosed herein.

[0032] In some embodiments, the present invention provides a method for producing any of the antibodies or fragments disclosed herein, the method comprising providing any of the nucleic acids disclosed herein or any of the vectors disclosed herein to a cell, and allowing the cell to translate the nucleic acid or the nucleic acid sequence contained in the vector, thereby producing the antibody or fragment. In some embodiments, the method further comprises collecting, purifying and / or isolating the antibody or fragment. DETAILED DESCRIPTION

[0033] A. General Techniques and Definitions

[0034] Unless otherwise defined herein, the scientific and technical terms described herein should have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature and techniques used in conjunction with 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 are well-known and commonly used in the art. In the event of a conflict, this specification (including definitions) shall prevail.

[0035] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. Such techniques are fully described in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd 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 (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Thayer and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (Academic Press, Inc.); and Methods in Enzymology (Academic Press, Inc.). Cells (edited by JMMiller and MPCalos, 1987); Current Protocols in Molecular Biology (FMAusubel et al., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., 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); Short Protocols in Molecular Biology (Wiley and Sons, 1999). .

[0036] Enzymatic reactions and purification techniques are as commonly practiced in the art or as described herein according to manufacturer's specifications. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Chemical syntheses and chemical analyses are performed using standard techniques.

[0037] Throughout the present specification and embodiments, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or groups of integers.

[0038] It should be understood that where embodiments are described herein with the wording "comprising," other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided.

[0039] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0040] Any examples following the term "eg or for example" are not intended to be exhaustive or limiting.

[0041] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0042] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0043] "Treating" a condition or patient means taking steps to obtain beneficial or desired results, including clinical results. With respect to a disease or condition (e.g., an eye disease), treatment means reducing or ameliorating the progression, severity, and / or duration of an infection (e.g., an eye disease or symptoms associated therewith), or ameliorating one or more symptoms by administering one or more therapies (including, but not limited to, one or more prophylactic or therapeutic agents).

[0044] As used herein, the terms "specific for" and "specifically binds to" or "capable of specifically binding" refer to the non-covalent interaction between an antibody and its epitope. It indicates that the antibody or fragment preferentially binds to the epitope rather than other binding sites or other antigens. Thus, although the antibody or fragment may non-specifically bind 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.

[0045] The percent identity of an amino acid sequence or nucleic acid sequence or the term "% sequence identity" is defined herein as the percentage of residues that are identical to the residues in a reference amino acid sequence or nucleic acid sequence over the entire length of the amino acid sequence 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".

[0046] As used herein, “FH.07” refers to an antibody comprising a variable light chain amino acid sequence of SEQ ID NO:32 and a variable heavy chain amino acid sequence of SEQ ID NO:36.

[0047] 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.

[0048] In the amino acid sequences as shown herein, amino acids are represented by single letter symbols. These single letter symbols 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.

[0049] The term "enhancing FH activity" means that the activity of FH increases when the antibody or fragment according to the present invention is bound to FH. In some embodiments, the FH activity enhanced by the antibody and fragment of the present invention is to inhibit alternative complement activation, such as in an individual. As used herein, the term "alternative complement activation" refers to activating the complement system via an alternative pathway, that is, at least involving the formation of the C3 convertase (i.e., C3bBb / C3bBbP) of the alternative pathway, or involving increasing the formation of such a C3 convertase. Alternative complement activation may also involve cutting C3 into C3a and C3b, forming an alternative pathway C5 convertase (i.e., C3bBbC3b / C3bBbC3bP) and / or cutting C5 by an alternative pathway C3 convertase and subsequently combining C6, C7, C8 and C9 to form a MAC. Alternative complement activation may also include an increase in the alternative pathway amplification loop.

[0050] 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 a body fluid of the individual, such as blood, interstitial fluid, or cerebrospinal fluid). As used herein, the term "individual" is used interchangeably with "subject" or "patient" and refers to a person or animal, such as a mammal (e.g., rodent, ape, horse, cow, pig, dog, cat), that contains 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.

[0051] As used herein, "inhibiting alternative complement activation" includes any change in the amount or activity of an alternative complement system component, factor, or activity that results from or results in its inhibition. For example, inhibition of alternative complement activation includes inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition on cells of a subject, increasing 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 interaction between C3b and factor B, inhibition of cleavage of C3 into C3a and C3b by the alternative pathway C3 convertase, increasing binding of FH to host cells (specifically, to sialic acid, glycosaminoglycans and / or heparin expressed on host cells), inhibition of the alternative complement pathway amplification loop and inhibition of formation of the alternative complement pathway C5 convertase C3bBbC3bP / C3bBbC3bP, inhibition of cleavage of C5 into C5a and C5b by the alternative pathway C5 convertase, increasing the decay accelerating activity of FH (i.e., promoting the dissociation of the alternative pathway C3 convertase after its formation), and / or increasing FI cofactor activity thereby causing degradation of C3b. In certain embodiments, the enhanced inhibition of alternative complement activation by FH antibodies and fragments of the invention comprises inhibition of hemolytic activity, inhibition of C3 deposition on cells of a subject, and / or increased binding of FH to C3b, iC3b, and / or C3d.

[0052] As used herein, "inhibit" preferably means that a specified 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, "inhibiting 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, "inhibiting 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%.

[0053] As used herein, "increase" preferably means that the specified 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, "increases the 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 the binding of FH to host cells" means that the 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%.

[0054] As used herein, "hemolytic activity" refers to the rupture of red blood cells and the subsequent release of cellular contents into the circulation, preferably due to the formation of MAC on the cell surface, and then, for example, by the activation of the complement system. For example, as described in the embodiments herein, hemolytic activity is measured by using a hemolytic assay as described in Sanchez-Corral et al., (2004) and Wouters et al., (2008), optionally with some modifications. In this representative non-limiting assay, red blood cells such as sheep red blood cells (SRBC) are incubated with serum (e.g., human serum) for 1.25 hours at 37 ° C while shaking. Serum with low levels of FH or dysfunctional FH causes SRBC to dissolve. Dissolution can be terminated by adding a veronal buffer containing 20mM EDTA, followed by centrifugation in a precooled centrifuge (e.g., 7 ° C) for 2.5 minutes. The percentage of red blood cell lysis is determined by measuring the absorbance of the supernatant at 412nm. The serum may be, for example, from a healthy human individual or a human individual suffering from a disorder associated with unwanted or excessive alternative pathway complement activation, such as aHUS. The ability of an antibody or fragment to inhibit hemolytic activity may be determined by incubating erythrocytes with the serum in the presence of the antibody or fragment.

[0055] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of an antibody or functional part or functional equivalent and its binding partner (e.g., antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (antibody and antigen in this application). Binding affinity is herein represented by the equilibrium dissociation constant (K D ) means that it is calculated as ka With k d Ratio, see, e.g., Chen, Y. et al., 1999. Affinity can be measured by common methods known in the art, such as surface plasmon resonance (SPR) assays, such as BiaCore (GE Healthcare Life Sciences GE Healthcare LifeSciences) or IBIS Technologies BV (Henglo, The Netherlands) IBIS-iSPR instruments, or solution phase assays, such as Kinexa.

[0056] B. Anti-FH antibodies and fragments thereof

[0057] WO 2016 / 028150 describes a murine agonist anti-FH antibody, called FH.07, which inhibits alternative pathway activation as shown by increased FH binding 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 enhancement effect. WO 2019 / 139481 describes another murine agonist anti-FH antibody, called FHR-1.3B4, which has a higher binding affinity for FH than FH.07 and an increased ability to enhance FH activity.

[0058] In one aspect, the present invention provides agonistic anti-FH antibodies (e.g., humanized antibodies) and fragments thereof, i.e., antibodies and fragments that enhance FH activity. In one aspect, 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 effective inhibitors of activation of the alternative complement pathway, and are therefore useful for treating disorders associated with undesired or excessive activation of the alternative pathway of the complement system. FH specifically inhibits the alternative pathway amplification loop, in which C3 is cut into C3b, which is then bound to FB on the cell surface, and the formation of C3 convertase promotes the cutting of other C3 molecules into C3b. The main advantage of the fact that the antibodies and fragments of the present invention interfere with complement activation at the C3 level is to avoid the accumulation of C3b on the surface and the release of C3a. In contrast, if complement activation is inhibited at the C5 level, such as with eculizumab, C3b accumulation and C3a release will not be inhibited. C3b acts as an opsonin, while C3a is an anaphylatoxin. It is therefore preferred to prevent C3b accumulation and C3a formation, since these processes lead to the attraction of immune cells and opsonophagocytosis of the target. This means, for example, that PNH patients receiving eculizumab still require blood transfusions, since C3b accumulation leads to opsonization of red blood cells, which are then cleared in the liver and spleen. In addition, treatment with anti-C5 antibodies leads to C3b accumulation and C3a formation on cells that would otherwise be lysed by MAC. An important disadvantage of anti-C5 antibodies is that patients become susceptible to infection, since the antibodies also interfere with complement activation induced by pathogens. This can be avoided by targeting regulators of the complement system that protect host cells.

[0059] As used herein, the term "antibody" refers to an immunoglobulin that at least comprises a heavy chain variable region (VH) paired with a light chain variable region (VL) and is specific for a target epitope. The term encompasses polyclonal and monoclonal antibodies. It refers to any form of antibody that specifically binds to CCP18 of FH, including full-length immunoglobulins. The antibody or fragment thereof according to the present invention comprises at least one antigen binding site. As used herein, the term "antigen binding site" refers to a site comprising at least one CDR sequence, preferably at least two CDR sequences, more preferably at least three CDR sequences in an antibody or its fragment. For example, an antigen binding site comprises light chain CDR 1-3 or heavy chain CDR 1-3. In some embodiments, the antigen binding site comprises light chain CDR 1-3 and heavy chain CDR 1-3.

[0060] As is well known to those skilled in the art, antibodies contain 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, which participate 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 usually, but not always, participates in antigen binding together with the variable domain of the heavy chain. The complementary determining region (CDR) is a hypervariable region present in the heavy chain variable domain and the light chain variable domain. In the case of a full-length antibody, the heavy chain CDR 1-3 and the connected light chain CDR 1-3 form an antigen binding site together.

[0061] CDR participates in antigen binding and confers antigen specificity and binding affinity to the antibody. There are three CDRs in each variable domain of the heavy chain and light chain, which are named CDR1, CDR2 and CDR3 of each variable domain. As used herein, the term "CDR set" refers to a group of three CDRs present in a single heavy chain or light chain variable domain that can bind to a target antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The three heavy chain CDRs can be referred to as CDRH1, CDRH2 and CDRH3, and the three light chain CDRs can be referred to as CDRL1, CDRL2 and CDRL3.

[0062] "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 antigen bound by the antibody (e.g., CCP18 of FH), but not necessarily to the same extent. In addition, fragments of FH activity-enhancing antibodies also enhance FH activity, but not necessarily to the same extent. In addition, fragments of FH activity-inhibiting antibodies also inhibit FH activity, but not necessarily to the same extent. In some embodiments, an antibody fragment according to the present invention comprises a heavy chain CDR1, CDR2, and CDR3 sequence of an antibody (e.g., any of the antibodies disclosed herein), and in some embodiments also comprises a light chain CDR1, CDR2, and CDR3 sequence of an antibody. Non-limiting examples of antibody fragments include single-domain antibodies, single-chain antibodies, nanobodies, monofunctional antibodies, 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 a heavy chain variable domain (VH) and / or a light chain variable domain (VL). In some embodiments, a fragment comprises at least a Fab fragment. In some embodiments, the Fab' fragments of the enhanced anti-FH antibody FH.07 or antibody FHR-1.3B4 that compete with the antibodies of the present invention for binding to the same epitope have been shown to retain the ability to enhance FH function. In some embodiments, the fragment of the antibody of the present invention is therefore a Fab fragment, a Fab' fragment, a F(ab')2 fragment or a F(ab)2 fragment of an antibody according to the present invention. In another embodiment, the fragment of the antibody according to the present invention 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.

[0063] In some embodiments, the antibodies and fragments according to the invention are capable of enhancing the activity of FH, such as human FH.

[0064] In some embodiments, inhibition of C3 deposition is measured using a C3 deposition assay, such as the C3 deposition assay described herein in the Examples. This representative, non-limiting assay comprises coating a microtiter plate with LPS. The plate is then incubated with, for example, serum from a healthy individual or an individual suffering from a condition associated with unwanted or excessive alternative complement activation as noted above, in the presence or absence of an antibody or fragment. C3 deposition on LPS can be detected with an anti-C3 antibody.

[0065] In some embodiments, the increase in FH binding to C3b is measured using an ELISA as described herein in the Examples. This representative non-limiting assay includes coating a micro ELISA plate with C3b and incubating the plate with, for example, serum from a healthy individual or an individual suffering from a disorder associated with undesired or excessive replacement complement activation as noted above. The bound FH can be detected with an anti-FH antibody (such as a peroxidase-labeled polyclonal anti-FH). The ability of the antibody or fragment to enhance the binding of FH to C3b can be determined by pre-incubating the serum in the presence of the antibody or fragment before incubating with the coated C3b. As another example, surface plasmon resonance (SPR) can be used to determine the binding of FH to C3b, for example, as described herein in the Examples. SPR is a technique for measuring biomolecular interactions in real time in a label-free environment. One of the interactants (e.g., C3b) is fixed to the sensor surface, and another interactant (e.g., FH) is freed in solution and passed through the surface, for example, in the presence or absence of (different concentrations of) antibodies or fragments of the present invention. In some embodiments, the antibodies or fragments thereof according to the present invention increase the binding affinity of FH to C3b in vitro (increase K D value) to at most 2 μM, at most 1.95 μM, at most 1.8 μM, at most 1.7 μM and / or increase the in vitro binding affinity of FH to C3b 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. In certain embodiments, the antibodies or fragments thereof according to the invention increase the in vitro binding affinity (K) of FH to C3b by at least 2 μM, at most 1.95 μM, at most 1.8 μM, at most 1.7 μM and / or increase the in vitro binding affinity of FH to C3b 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 ) to less than 1 μM, less than 750 nM, less than 500 nM, less than 300 nM or less than 250 nM. In certain embodiments, the antibodies or fragments thereof according to the invention increase the in vitro binding affinity (K) of FH to C3b to less than 1 μM, less than 750 nM, less than 500 nM, less than 300 nM or less than 250 nM. D ) to between 250 nM and 1 μM. In a specific embodiment, the antibodies or fragments thereof according to the invention increase the binding affinity of FH for C3b in vitro by at least 3 to 5 fold.

[0066] In some embodiments, the antibodies or fragments thereof provided herein have low in vitro IC in one or more functional assays. 50 values, such as, in some embodiments, its in vitro IC 50 The value is lower than the in vitro IC of antibody FH.07 or antibody FHR-1.3B4 50 Value. 50 " is a term well known in the art and refers to the concentration of an antibody or fragment necessary to inhibit or reduce a functional activity by 50%. The IC50 The 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 according to the present invention inhibits C3 deposition on LPS in vitro in the presence of 10% (v / v) normal human serum, IC 50 The value is 38nM or less, 35nM or less, 32nM or less, 30nM or less, 28nM or less, 27nM or less, 25nM or less, 23nM or less, 20nM or less, 18nM or less, 15nM or less, or 10nM or less. In some embodiments, the antibodies or fragments thereof according to the present invention inhibit C3 deposition on LPS in vitro in the presence of 10% (v / v) normal human serum, IC 50 The value is between 15 nM and 30 nM. In some embodiments, the antibody or fragment thereof according to the present invention inhibits in vitro hemolytic activity in the presence of 10% (v / v) normal human serum, IC 50 The value is 150nM or less, 130nM or less, 120nM or less, 115nM or less, 110nM or less, 105nM or less, 100nM or less, 95nM or less, 90nM or less, 85nM or less, 80nM or less, 75nM or less, 70nM or less, 65nM or less, or 60nM or less. In some embodiments, the antibodies or fragments thereof according to the present invention inhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum, IC 50 Values ​​ranged between 60 nM and 80 nM.

[0067] In some embodiments, the antibodies or fragments of the invention are expressed in the presence of 10% (v / v) normal human serum at a low IC value as described herein. 50 The inhibitory effect of 10% (v / v) normal human serum on C3 deposition on LPS in vitro was as low as described above. 50 In some embodiments, the antibodies or fragments according to the present invention inhibit hemolytic activity in vitro with an IC value of 38 nM or less in the presence of 10% (v / v) normal human serum. 50 The results showed that the C3 deposition on LPS in vitro was inhibited with an IC value of 150 nM or less in the presence of 10% (v / v) normal human serum. 50 The inhibitory effect was shown to inhibit in vitro hemolytic activity with an IC value of 30 nM or lower in the presence of 10% (v / v) normal human serum. 50 The results showed that the C3 deposition on LPS in vitro was inhibited with an IC value of 150 nM or less in the presence of 10% (v / v) normal human serum. 50In the presence of 10% (v / v) normal human serum, the IC value was 27 nM or lower. 50 The results showed that the C3 deposition on LPS in vitro was inhibited with an IC value of 100 nM or less in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the IC of C3 deposition on LPS in vitro in the presence of 10% (v / v) normal human serum is determined in a C3 deposition assay as described herein above. 50 In some embodiments, the IC value of hemolytic activity is determined in a hemolytic activity assay as described herein above. 50 value.

[0068] In some embodiments, the antibodies or fragments thereof provided herein have high binding affinity to FH and / or a fragment of FH comprising the domain CCP18-20, such as, in some embodiments, a binding affinity higher than the binding affinity of antibody FH.07 or antibody FHR-1.3B4. In some embodiments, the in vivo therapeutic activity of an antibody or fragment typically requires a high binding affinity, for example, in order to minimize the binding of the antibody or fragment to a binding site and / or an antigen other than its specific epitope or antigen, and to minimize the amount of the antibody or fragment that needs to be administered in vivo. In some embodiments, antibodies with high binding affinity are preferred. In some embodiments, the binding affinity of an antibody or fragment to FH is expressed as a dissociation constant (K D ) is 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 In one embodiment, the present invention therefore provides an isolated synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds to factor H (FH) and enhances FH activity, wherein the binding affinity of the antibody to FH is expressed as K D 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 In some embodiments, the binding affinity of the antibody or fragment according to the present invention to FH is expressed as K DIn some embodiments, the binding affinity of an antibody or fragment according to the present invention to a FH fragment comprising CCP18-20 is expressed as K. D 0.09×10 -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 0.6×10 -11 In some embodiments, the binding affinity of the antibody or fragment thereof to FH is expressed as K D 1.25×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.04×10 -9 In some embodiments, the binding affinity of the antibody or fragment thereof to FH is expressed as K D 0.6×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.6×10 -11 In some embodiments, the binding affinity is determined using surface plasmon resonance (SPR), such as in an assay as described herein, i.e., in an assay where the binding affinity is determined by capturing the antibody or fragment on a ProtA chip and then flowing the full-length FH (for binding affinity to FH) or a FH fragment comprising domains 18-20 (for binding affinity to CCP18-20) over the surface to perform SPR.

[0069] In some embodiments, the antibodies or fragments thereof provided herein increase the interaction between FH and C3b. In some embodiments, the antibodies or fragments thereof increase the binding affinity of FH to C3b, wherein the dissociation constant (K D ) is 2 μM or less. In some embodiments, the antibodies or fragments according to the present invention increase the binding affinity for C3b, wherein K D 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. In some embodiments, the antibodies or fragments according to the invention increase the binding affinity of FH to C3b in vitro (increase K D or a factor of 5.

[0070] In some embodiments, the antibodies or fragments according to the invention have high binding affinity and low IC 50 values ​​inhibit C3 deposition on LPS in vitro and / or with low IC 50 In some embodiments, the binding affinity of the antibody or fragment according to the present invention to FH is expressed as K D 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 M or lower, and with an IC of 38 nM or lower in the presence of 10% (v / v) normal human serum 50 The results showed that the C3 deposition on LPS in vitro was inhibited with an IC value of 150 nM or less in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the binding affinity of the antibody or fragment to FH is expressed as K D 1.25×10 -8 M or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) and / or the binding affinity to the FH fragment comprising CCP18-20 is expressed as K D 0.04×10 -9 M or lower, and with an IC of 30 nM or lower 50 The results showed that the C3 deposition on LPS in vitro was inhibited with an IC value of 115 nM or lower in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the binding affinity of the antibody or fragment to FH is expressed as K D 1.25×10 -8M or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) and / or the binding affinity to the FH fragment comprising CCP18-20 is expressed as K D 0.04×10 -9 M or lower, and with an IC of 27 nM or lower in the presence of 10% (v / v) normal human serum 50 The results showed that the C3 deposition on LPS in vitro was inhibited with an IC value of 100 nM or less in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the binding affinity of the antibody or fragment to FH is expressed as K D 1.25×10 -8 M or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) and / or the binding affinity to the FH fragment comprising CCP18-20 is expressed as K D 0.04×10 -9 M or lower, and with an IC of 25 nM or lower in the presence of 10% (v / v) normal human serum 50 The results showed that the C3 deposition on LPS in vitro was inhibited with an IC value of 80 nM or less in the presence of 10% (v / v) normal human serum. 50 In some embodiments, the binding affinity of the antibody or fragment to FH is expressed as K D 1.25×10 -8 M or less (e.g., 10 nM or less, 5 nM or less, or 1 nM or less) and / or the binding affinity to the FH fragment comprising CCP18-20 is expressed as K D 0.04×10 -9 M or lower, and with an IC between 15 nM and 25 nM in the presence of 10% (v / v) normal human serum 50 The values ​​inhibited C3 deposition on LPS in vitro and in the presence of 10% (v / v) normal human serum with an IC between 15 nM and 25 nM. 50 In some embodiments, the binding affinity is determined by SPR as described herein. In some embodiments, the IC of C3 deposition on LPS in vitro is determined in a C3 deposition assay as described herein above. 50 In some embodiments, the IC value of hemolytic activity is determined in a hemolytic activity assay as described herein above. 50 value.

[0071] In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have a higher binding affinity for FH and / or a fragment of FH comprising CCP18 than the binding affinity of antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have a higher binding affinity for FH and / or a fragment of FH comprising CCP18-20 than the binding affinity of antibody FH.07. In some embodiments, the IC 50 The values ​​are lower than the IC of antibody FH.07 50 Value. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein show greater inhibition of C3 deposition on LPS than antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein show 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 in CCP18 of FH. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high storage capacity, specifically, improved storage stability compared to antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high stability, specifically, improved stability compared to antibody FH.07. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high selectivity, specifically, increased selectivity compared to antibody FH.07.

[0072] In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have a higher binding affinity for FH and / or a fragment of FH comprising CCP18 than the binding affinity of antibody FHR-1.3B4. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have a higher binding affinity for FH and / or a fragment of FH comprising CCP18-20 than the binding affinity of antibody FHR-1.3B4. In some embodiments, the IC 50 The values ​​were lower than the IC of antibody FHR-1.3B4 50Value. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein inhibits C3 deposition on LPS to a greater extent than antibody FHR-1.3B4. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein show 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 in CCP18 of FH. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high storage capacity, specifically, 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, specifically, improved stability compared to antibody FHR-1.3B4. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein have high selectivity, specifically, increased selectivity compared to antibody FHR-1.3B4.

[0073] In some embodiments, the antibodies or fragments thereof provided by the present invention compete with the antibody anti-FH.07 for binding to FH, specifically, binding to the CCP18 domain of FH.

[0074] Table 1 lists exemplary antibodies of the invention. CDR sequences are numbered according to the IMGT numbering system (Lefranc 1997; Lefranc 1999; and Lefranc et al., 2003). LC = light chain, HC = heavy chain, CDR = complementarity determining region, VH = heavy chain variable region, VL = light chain variable region.

[0075] Table 1: Amino acid and nucleotide sequences of Antibodies 1 to 4, FH.07 and FHR-1.3B4

[0076]

[0077]

[0078]

[0079]

[0080] In some embodiments, the present invention 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), the antibody or fragment comprising the heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of the antibodies disclosed in Table 1 determined according to Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk AM, (1987), J. Mol. Biol. 196:901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262:732-745), or any other CDR determination method known in the art.

[0081] In some embodiments, the antibody according to the present invention is antibody 1. Table 1 provides an overview 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. The term "antibody 1" as used herein encompasses all antibodies and fragments including at least the heavy and light chain CDR1, CDR2 and CDR3 regions as shown in Table 1, such as 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 in CCP18 of FH.

[0082] In some embodiments, the antibody according to the present invention is antibody 2. Table 1 provides an overview 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. The term "antibody 2" as used herein encompasses all antibodies and fragments including at least the heavy and light chain CDR1, CDR2 and CDR3 regions as shown in Table 1, such as 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 in CCP18 of FH.

[0083] In some embodiments, the antibody according to the present invention is antibody 3. Table 1 provides an overview of the variable heavy chain 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. The term "antibody 3" as used herein encompasses all antibodies and fragments including at least the heavy and light chain CDR1, CDR2 and CDR3 regions as shown in Table 1, such as 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 in CCP18 of FH.

[0084] In some embodiments, the antibody according to the present invention is antibody 4. Table 1 provides an overview 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. The term "antibody 4" as used herein encompasses all antibodies and fragments including at least the heavy and light chain CDR1, CDR2 and CDR3 regions as shown in Table 1, such as 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 in CCP18 of FH.

[0085] In some embodiments, the present invention 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) and comprises:

[0086] - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1) or the sequence TSVTY (SEQ ID NO: 13);

[0087] - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 2) or the sequence ASS (SEQ ID NO: 14);

[0088] - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3);

[0089] - a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5);

[0090] - a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6) or the sequence IWSGGTT (SEQ ID NO: 10); and / or

[0091] - a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7) or the sequence ARNFGNYAMDF (SEQ ID NO: 11); and / or

[0092] - Any combination of the above.

[0093] In a specific embodiment, the antibody or fragment comprises:

[0094] - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1);

[0095] - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 2);

[0096] - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3);

[0097] - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5);

[0098] - a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6); and

[0099] - A heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7).

[0100] In a specific embodiment, the antibody or fragment comprises:

[0101] - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1);

[0102] - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 2);

[0103] - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3);

[0104] - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5);

[0105] - a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 10); and

[0106] - A heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO: 11).

[0107] In a specific embodiment, the antibody or fragment comprises:

[0108] - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13);

[0109] - a light chain CDR2 sequence having the sequence ASS (SEQ ID NO: 14);

[0110] - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3);

[0111] - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5);

[0112] - a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6); and

[0113] - A heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 7).

[0114] In a specific embodiment, the antibody or fragment comprises:

[0115] - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13);

[0116] - a light chain CDR2 sequence having the sequence ASS (SEQ ID NO: 14);

[0117] - a light chain CDR3 sequence having the sequence QHRSSSNPLT (SEQ ID NO: 3);

[0118] - a heavy chain CDR1 sequence having the sequence GFSLTNYG (SEQ ID NO: 5);

[0119] - a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 10); and

[0120] - A heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO: 11).

[0121] In some embodiments, the antibody or fragment enhances FH activity, such as inhibiting alternative complement activation, such as inhibiting hemolytic activity, inhibiting complement component 3 (C3) deposition, and / or increasing the 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.

[0122] In some embodiments, the binding affinity of the antibody or fragment to FH is measured as K. D 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 M or lower, such as the binding affinity for FH is expressed as K D 1.25×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.04×10 -9 M or lower antibodies or fragments.

[0123] In another embodiment, the present invention provides an isolated synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH and 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); 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 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 SEQ ID NO: 8. In a specific embodiment, the antibody or fragment comprises 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. In some embodiments, the antibody or fragment enhances FH activity, such as inhibiting alternative complement activation, such as inhibiting hemolytic activity, inhibiting complement component 3 (C3) deposition, and / or increasing the 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.

[0124] In another embodiment, the present invention provides an isolated synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH and 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); 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 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 SEQ ID NO: 12. In a specific embodiment, the antibody or fragment comprises 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. In some embodiments, the antibody or fragment enhances FH activity, inhibits alternative complement activation, such as inhibiting hemolytic activity, inhibiting complement component 3 (C3) deposition, and / or increasing the 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.

[0125] In another embodiment, the present invention provides an isolated synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH and 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); 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 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 SEQ ID NO: 8. In a specific embodiment, the antibody or fragment comprises 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. In some embodiments, the antibody or fragment enhances FH activity, such as inhibiting alternative complement activation, such as inhibiting hemolytic activity, inhibiting complement component 3 (C3) deposition, and / or increasing the 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.

[0126] In another embodiment, the present invention provides an isolated synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH and 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); 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 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 SEQ ID NO: 12. In a specific embodiment, the antibody or fragment comprises 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. In some embodiments, the antibody or fragment enhances FH activity, such as inhibiting alternative complement activation, such as inhibiting hemolytic activity, inhibiting complement component 3 (C3) deposition, and / or increasing the 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.

[0127] In some embodiments, the binding affinity of any of the antibodies or fragments disclosed herein for FH is measured as K. D 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 In some embodiments, the binding affinity of the antibody or fragment to FH is expressed as K DThe binding affinity of the present invention is 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, and / or the binding affinity for the FH fragment comprising CCP18-20 is measured as K D 0.09×10 -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 0.6×10 -11 M or lower.

[0128] In some embodiments, any of the antibodies or fragments disclosed herein inhibit C3 deposition on LPS in vitro in the presence of 10% (v / v) normal human serum, IC 50 In some embodiments, the antibody or fragment inhibits C3 deposition on LPS in vitro in the presence of 10% (v / v) normal human serum, IC 50 In some embodiments, the antibodies or fragments thereof according to the present invention inhibit C3 deposition on LPS in vitro in the presence of 10% (v / v) normal human serum, IC 50 Values ​​ranged between 15 nM and 30 nM.

[0129] In some embodiments, any of the antibodies or fragments disclosed herein inhibits in vitro hemolytic activity in the presence of 10% (v / v) normal human serum, IC 50 In some embodiments, the antibody or fragment inhibits in vitro hemolytic activity in the presence of 10% (v / v) normal human serum, IC50 In some embodiments, the antibody or fragment thereof according to the present invention inhibits in vitro hemolytic activity in the presence of 10% (v / v) normal human serum, IC 50 The value is 150nM or less, 130nM or less, 115nM or less, 105nM or less, 100nM or less, 95nM or less, 75nM or less, 70nM or less, 65nM or less, or 60nM or less. In some embodiments, the antibodies or fragments thereof according to the present invention inhibit in vitro hemolytic activity in the presence of 10% (v / v) normal human serum, IC 50 The values ​​were between 60 nM and 80 nM.

[0130] In some embodiments, any of the antibodies or fragments disclosed herein increases the binding affinity (K) of FH to C3b in vitro. 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 of the antibodies or fragments disclosed herein increase the in vitro binding affinity (K) of FH to C3b by at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold. D ) to between 50 and 500 nM. In some embodiments, any of the antibodies or fragments disclosed herein increases the in vitro binding affinity (K) of FH to C3b by D ) to between 50 and 300 nM. In some embodiments, any of the antibodies or fragments disclosed herein increases the in vitro binding affinity (K) of FH to C3b by D ) to between 100 and 300 nM. In some embodiments, any of the antibodies or fragments disclosed herein increases the in vitro binding affinity (K) of FH to C3b by D ) increased to between 250 and 300 nM.

[0131] Optionally, the sequence of at least one of any one or more of the CDRs disclosed herein is optimized to produce variant antibodies or fragments, for example to (further) improve binding affinity, selectivity, FH enhancing ability and / or in vivo or storage stability. In some embodiments, the antibodies or fragments according to the present invention have high storage stability, in particular, improved storage stability compared to antibody FH.07 or antibody FHR-1.3B4. In some embodiments, the antibodies or fragments according to the present invention have high in vivo stability, in particular, improved in vivo stability compared to antibody FH.07 or antibody FHR-1.3B4. In some embodiments, the antibodies or fragments according to the present invention have high selectivity, in particular, increased selectivity compared to antibody FH.07 or antibody FHR-1.3B4.

[0132] In addition, at least one sequence in at least one of the framework regions of the antibody or fragment of the present invention is optionally optimized, for example, to improve the binding efficacy or stability of the antibody or fragment or to reduce the side effects of the non-human sequence after it is administered to humans. This is, for example, performed by an induction procedure. Those skilled in the art can produce antibody variants comprising at least one altered CDR or framework sequence. CDR and / or framework sequences are optimized, for example, by mutating nucleic acids encoding such framework sequences. For example, conservative amino acid substitutions are applied. In some embodiments, any one of the CDR, framework, variable heavy chain, and variable light chain sequences disclosed herein comprises 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 CDRs, frameworks, variable heavy chains, and variable light chain sequences disclosed herein include 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 CDRs, frameworks, variable heavy chains, and variable light chain sequences disclosed herein include 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 CDRs, frameworks, variable heavy chains, and variable light chain sequences disclosed herein include 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 substitution of one hydrophobic residue (such as isoleucine, valine, leucine or methionine) for another hydrophobic residue, and substitution of one polar residue for another polar residue, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0133] In order to select improved antibodies or fragments, the binding affinity, FH enhancing ability and / or stability of the obtained variant antibodies or fragments can be tested, for example, using the tests described herein. Once an antibody or fragment specific to FH (specifically, CCP18 to FH) has been obtained, its desired biological activity, i.e., the ability to enhance FH activity, can be tested by several methods known to those skilled in the art. As previously described herein, enhancing FH activity can encompass inhibiting hemolytic activity, inhibiting C3 deposition on cells and / or increasing FH binding to C3b. Functional assays for testing these activities have been previously described herein and described in detail in the embodiments. Typically, up to three amino acid residues in a CDR sequence can vary while retaining the same specificity, depending on the number of amino acids that the CDR comprises. Therefore, in some embodiments, an antibody or fragment according to the present invention contains heavy and light chain CDR1, CDR2 and CDR3 sequences, wherein at most 3, at most 2 or at most 1 amino acid in each CDR is mutated compared to the heavy and light chain CDR1, CDR2 and CDR3 sequences of Table 1. In some embodiments, the antibody or fragment comprises a light chain CDR3 and a heavy chain CDR3 of the same antibody as shown in Table 1, a light chain CDR2 of the same antibody with at most 1 amino acid variation, and a light chain CDR1, a heavy chain CDR1, and a heavy chain CDR2 with at most 3 amino acid variations. In some embodiments, the antibody or fragment comprises a light chain CDR2 and CDR3 and a heavy chain CDR3 of the same antibody as shown in Table 1, and a light chain CDR1, a heavy chain CDR1, and a heavy chain CDR2 with at most 2 amino acids or at most 1 amino acid variation.

[0134] The present invention therefore also provides an isolated synthetic or recombinant antibody or fragment thereof which specifically binds to the complement control protein domain 18 (CCP18) of factor H (FH) and comprises:

[0135] - a light chain CDR1 sequence having a sequence at least 60% or at least 80% identical to the sequence SSVTY (SEQ ID NO: 1); a light chain CDR2 sequence having a sequence at least 60% identical to the sequence ATS (SEQ ID NO: 2); and a light chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence QHRSSSNPLT (SEQ ID NO: 3); a heavy chain CDR1 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence GFSLTNYG (SEQ ID NO: 5); a heavy chain CDR2 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence VWSGGTT (SEQ ID NO: 6); and a heavy chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence ARNFGNYAMDY (SEQ ID NO: 7); ID NO:7) a sequence that is at least 60%, at least 70%, at least 80% or at least 90% identical;

[0136] - a light chain CDR1 sequence having a sequence at least 60% or at least 80% identical to the sequence SSVTY (SEQ ID NO: 1); a light chain CDR2 sequence having a sequence at least 60% identical to the sequence ATS (SEQ ID NO: 2); and a light chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence QHRSSSNPLT (SEQ ID NO: 3); a heavy chain CDR1 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence GFSLTNYG (SEQ ID NO: 5); a heavy chain CDR2 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence IWSGGTT (SEQ ID NO: 10); and a heavy chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence ARNFGNYAMDF (SEQ ID NO: 11). ID NO: 11) a sequence that is at least 60%, at least 70%, at least 80% or at least 90% identical;

[0137] a light chain CDR1 sequence having a sequence at least 60% or at least 80% identical to the sequence TSVTY (SEQ ID NO: 13); a light chain CDR2 sequence having a sequence at least 60% identical to the sequence ASS (SEQ ID NO: 14); and a light chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence QHRSSSNPLT (SEQ ID NO: 3); a heavy chain CDR1 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence GFSLTNYG (SEQ ID NO: 5); a heavy chain CDR2 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence VWSGGTT (SEQ ID NO: 6); and a heavy chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence ARNFGNYAMDY (SEQ ID NO: 7); ID NO:7) a sequence that is at least 60%, at least 70%, at least 80% or at least 90% identical;

[0138] - a light chain CDR1 sequence having a sequence at least 60% or at least 80% identical to the sequence TSVTY (SEQ ID NO: 13); a light chain CDR2 sequence having a sequence at least 60% identical to the sequence ASS (SEQ ID NO: 14); and a light chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence QHRSSSNPLT (SEQ ID NO: 3); a heavy chain CDR1 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence GFSLTNYG (SEQ ID NO: 5); a heavy chain CDR2 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence IWSGGTT (SEQ ID NO: 10); and a heavy chain CDR3 having a sequence at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence ARNFGNYAMDF (SEQ ID NO: 11). ID NO: 11) is at least 60%, at least 70%, at least 80% or at least 90% identical to the sequence.

[0139] 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 a specified sequence.

[0140] The present invention also provides an isolated synthetic or recombinant antibody or fragment thereof which specifically binds to complement control protein domain 18 (CCP18) of factor H (FH) and comprises:

[0141] - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1) optionally with 1 amino acid substitution; 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) optionally with 1 or 2 amino acid substitutions; a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5) optionally with 1 or 2 amino acid substitutions; a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6) optionally with 1 or 2 amino acid substitutions; and a heavy chain CDR3 sequence ARNFGNYAMDY (SEQ ID NO: 7) optionally with 1 or 2 amino acid substitutions;

[0142] - a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 1) optionally with 1 amino acid substitution; 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) optionally with 1 or 2 amino acid substitutions; a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5) optionally with 1 or 2 amino acid substitutions; a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 10) optionally with 1 or 2 amino acid substitutions; and a heavy chain CDR3 sequence ARNFGNYAMDF (SEQ ID NO: 11) optionally with 1 or 2 amino acid substitutions;

[0143] - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13) optionally having 1 or 2 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 having 1 or 2 amino acid substitutions; a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5) optionally having 1 or 2 amino acid substitutions; a heavy chain CDR2 having the sequence VWSGGTT (SEQ ID NO: 6) optionally having 1 or 2 amino acid substitutions; and a heavy chain CDR3ARNFGNYAMDY (SEQ ID NO: 7) optionally having 1 or 2 amino acid substitutions; or

[0144] - a light chain CDR1 sequence having the sequence TSVTY (SEQ ID NO: 13) optionally having 1 amino acid substitution; 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) optionally having 1 or 2 amino acid substitutions; a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 5) optionally having 1 or 2 amino acid substitutions; a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 10) optionally having 1 or 2 amino acid substitutions; and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDF (SEQ ID NO: 11) optionally having 1 or 2 amino acid substitutions.

[0145] In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein include at least one or more CDRs from any of the heavy or light chain sequences disclosed herein. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein include at least one or more CDRs from any of the variable heavy or light chain sequences of SEQ ID NO: 4, 8, 12 or 16. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein include at least one or more CDRs from any of the heavy or light chain sequences disclosed herein, wherein the CDRs are determined using the Kabat system. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein include at least one or more CDRs from any of the heavy or light chain sequences disclosed herein, wherein the CDRs are determined using the Chothia system. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein include at least one or more CDRs from any of the heavy or light chain sequences disclosed herein, wherein the CDRs are determined using the MacCallum system. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein comprise at least one or more CDRs from any of the heavy chain or light chain sequences disclosed herein, wherein the CDRs are determined using the AbM system. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein comprise at least one or more CDRs from any of the heavy chain or light chain sequences disclosed herein, wherein the CDRs are determined using the IMGT system.

[0146] The system described by Kabat, also referred to as "numbering according to Kabat," "Kabat numbering," "Kabat definition," and "Kabat notation," provides an unambiguous residue numbering system applicable to any variable domain of an antibody and provides the exact 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 by reference in their entirety. These CDRs are referred to as Kabat The CDRs are approximately located at residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain and 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 approximately located at residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are approximately located at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0147] Other CDR numbering systems are also used in the art. Chothia and colleagues found that certain subsections within the Kabat CDRs adopted nearly identical peptide backbone conformations despite the vast differences in amino acid sequence. (Chothia et al., (1987) J. Mol. Biol. 196:901-917; and Chothia et al., (1989) Nature 342:877-883). These subsections are named L1, L2, and L3 or H1, H2, and H3, where "L" and "H" represent the light chain and heavy chain regions, respectively. These CDRs may be referred to as "Chothia CDRs," "Chothia numbering," or "numbered according to Chothia," and comprise approximately residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain and 26-32 (CDR1), 52-56 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain. Mol. Biol. 196:901-917 (1987).

[0148] The system described by MacCallum, also referred to as "according to MacCallum numbering" or "MacCallum numbering," encompasses approximately residues 30-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) in the light chain variable domain and 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).

[0149] The system described by AbM, also referred to as "by AbM numbering" or "AbM numbering," encompasses approximately residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain and 26-35 (CDR1), 50-58 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain.

[0150] The IMGT (International Immunogenetics Information System) numbering of the variable region may also be used, which is the numbering of residues in the immunoglobulin variable heavy or light chain according to the IMGT method as described in Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains", The Immunologist, 7, 132-136 (1999), and is expressly incorporated herein in its entirety by introduction. As used herein, "IMGT sequence numbering" or "numbering according to IMTG" refers to the numbering of sequences encoding variable regions according to IMGT. For the heavy chain variable domain, when numbered according to IMGT, the hypervariable region is in the range of amino acid positions 27 to 38 for CDR1, in the range of amino acid positions 56 to 65 for CDR2, and in the range of amino acid positions 105 to 117 for CDR3. For the light chain variable domain, when numbered according to IMGT, the hypervariable region is in the range of amino acid positions 27 to 38 for CDR1, in the range of amino acid positions 56 to 65 for CDR2, and in the range of amino acid positions 105 to 117 for CDR3. In some embodiments of the constructs and antigen binding arms described herein, when numbered according to Chothia numbering, the CDRs described herein approximately comprise residues 24-34 (CDR1), 49-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and 27-35 (CDR1), 49-60 (CDR2), and 93-102 (CDR3) in the heavy chain variable domain. In some embodiments, when numbered according to Chothia numbering, CDR2 in the light chain variable domain may comprise amino acids 49-56.

[0151] The antibody or fragment thereof according to the present invention is a monoclonal antibody or fragment in some embodiments. A monoclonal antibody is an antibody consisting essentially of a single molecule species. Monoclonal antibodies are obtained from a homogeneous antibody population having the same sequence and binding to the same epitope, except for possible variant antibodies or fragments having one or more mutations that occur spontaneously, for example, during the production process. Advantageously, monoclonal antibodies can be recombinantly produced so that the amount of antibodies that can be obtained is significantly higher than the amount of polyclonal antibodies present in the antiserum. However, the present invention also encompasses polyclonal antibodies and fragments. In some embodiments, the antibody or fragment according to the present invention is a chimeric or humanized antibody. In some embodiments, the antibody or fragment therefore at least comprises a human light chain and a heavy chain constant region. In some embodiments, the antibody or fragment also comprises a human framework region in the heavy and light chain variable regions. In some embodiments, a human antibody or fragment consisting entirely of human sequences is provided. In some embodiments, it is more desirable to use chimeric, humanized or human antibodies than to use non-human antibodies, because many factors hinder the use of non-human antibodies or fragments to treat human diseases. In some embodiments, human germline sequences are used for the framework regions in antibodies or fragments according to the present invention. The immunogenicity risk of antibodies or fragments can be minimized by using human germline sequences, because these sequences are not likely to contain framework regions that are peculiar to the individuals from which they are derived and may cause somatic variation of immunogenic responses when applied to another human individual.

[0152] The procedure of antibody humanization or providing chimeric antibodies is well known in the art. Various recombinant DNA-based methods have been established, the goal of which is to increase the content of amino acid residues that also appear in the same or similar positions in human antibodies in antibodies, while retaining the specificity and affinity of the parent non-human antibody. For example, the framework region of the mouse antibody variable region is replaced by the corresponding human framework region with the highest degree of homology, thereby keeping the non-human CDR intact. Other methods suitable for humanizing antibodies according to the present invention include but are not limited to CDR transplantation (Queen, C et al., 1989; Carter, P et al., 1992); surface reforming (Padlan, EA et al., 1991), superhumanization (Tan, PDA et al., 2002), human string content optimization (Lazar, GA et al., 2007) and human engineering (Almagro, JC et al., 2008).

[0153] In one embodiment, the antibody or fragment according to the present invention is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different antigens and / or epitopes. In one embodiment, the bispecific antibody has binding specificity to FH, for example, it comprises a variable light chain and a variable heavy chain of CCP18 that specifically binds to FH as described herein, and has binding specificity to another antigen. In another embodiment, the bispecific antibody can be bound to two different epitopes of FH.

[0154] The antibody or fragment according to the present invention may belong to any category. The "category" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five major antibody categories: 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 invention comprises a constant region (e.g., Fc domain) of IgG (e.g., IgG1, IgG2, IgG3 or IgG4). In certain embodiments, the antibody according to the present invention comprises a constant region (e.g., Fc domain) of human IgG (e.g., human IgG1, IgG2, IgG3 or IgG4). The Fc domain mediates the effector functions of antibodies, including the ability to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In certain embodiments, antibodies disclosed herein comprise Fc domains with one or more mutations that reduce Fc effector functions. In some embodiments, the antibodies disclosed herein are included in an Fc domain comprising a mutation (e.g., relative to a substitution of 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 promoting the recycling of the antibody and thereby increasing the half-life of the antibody. In certain embodiments, the antibodies disclosed herein include 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.

[0155] In certain embodiments, any of the antibodies or fragments disclosed herein may also include post-translational modifications, in addition to any modifications naturally present in the native antibody or fragment. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, pegylation (polyethylene glycol) and acylation. Therefore, the modified polypeptide may contain non-amino acid elements, such as polyethylene glycol, lipids, monosaccharides or polysaccharides and phosphates. In specific embodiments, the antibody or fragment is PEGylated. In other specific embodiments, the antibody or fragment is a PEGylated Fab molecule.

[0156] Antibodies specific to specific antigens (such as FH according to the present invention) can be prepared by various methods known in the art. For example, human FH can be used as an immunogen to trigger antibodies. As another example, the CCP18 domain of human FH of FH-related proteins can be used as an immunogen. An example of such a method is produced by immunization and hybridoma as described in the embodiments. For example, mouse monoclonal antibodies against FH can be produced by, for example, immunizing mice such as BALB / c mice with human factor H or FH-related proteins such as FHR-1 at four-week intervals, optionally in the presence of adjuvants such as montanide. Several days after the fourth immunization, splenocytes can be fused with, for example, myeloma cell lines SP2 / 0. The presence of factor H-specific antibodies in hybridoma supernatants can be tested by ELISA. For example, microtiter plates are coated with moAb (e.g., rat anti-mouse κ moAb RM19) to capture mouse IgG antibodies. The specificity of the antibody can be determined by biotinylated factor H. Another example of a method for providing FH-specific antibodies is by screening a phage display library expressing a recombinant nucleic acid sequence encoding an immunoglobulin chain. Methods for antibody phage display have been widely used and described in the art. The antibody library can be screened with an antigen identical to that used for immunization, such as the CCP18 domain of human FH, a FH-related protein, or human FH.

[0157] C. Nucleic Acids and Vectors

[0158] The present invention also 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 as 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 as 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 as 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 as shown in Table 1.

[0159] In some embodiments, the nucleic acid according to the present invention has a length of at least 30 nucleotides, at least 50 nucleotides, or at least 75 nucleotides. In some embodiments, the nucleic acid encoding according to the present invention comprises a monoclonal chimeric or humanized antibody or a fragment thereof comprising the heavy chain CDR sequence and light chain CDR sequence and / or the heavy chain variable region and the light chain variable region of antibody 1. In some embodiments, the nucleic acid encoding according to the present invention comprises a monoclonal chimeric or humanized antibody or a fragment thereof comprising the heavy chain CDR sequence and light chain CDR sequence and / or the heavy chain variable region and the light chain variable region of antibody 2. In some embodiments, the nucleic acid encoding according to the present invention comprises a monoclonal chimeric or humanized antibody or a fragment thereof comprising the heavy chain CDR sequence and light chain CDR sequence and / or the heavy chain variable region and the light chain variable region of antibody 3. In some embodiments, the nucleic acid encoding according to the present invention comprises a monoclonal chimeric or humanized antibody or a fragment thereof comprising the heavy chain CDR sequence and light chain CDR sequence and / or the heavy chain variable region and the light chain variable region of antibody 4.

[0160] The nucleic acid sequences encoding the heavy and light chains of antibodies 5-7 are shown in Table 1. However, the present invention also encompasses nucleic acids encoding the heavy or light chain CDRs of the antibodies according to the present invention, which comprise nucleic acid sequences different from the nucleic acid sequences shown in Table 1, but comprise nucleic acid codons encoding the amino acid sequences of the heavy chains, light chains, heavy chain CDRs or light chain CDR sequences shown in Table 1.

[0161] Thus, an isolated synthetic or recombinant nucleic acid is provided, 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 one of the sequences of SEQ ID NO: 1, 2, 3, 5, 6 and 7, or any combination or fragment thereof. Also provided is 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 one of the sequences of SEQ ID NO: 1, 2, 3, 5, 10 and 11, or any combination or fragment thereof. Also provided is 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 one of the sequences of SEQ ID NOs: 3, 5, 6, 7, 13 and 14, or any combination or fragment thereof. Also provided is 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 one of the sequences of SEQ ID NOs: 3, 5, 10, 11, 13 and 14, or any combination or fragment thereof.

[0162] Also provided is 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 one of the sequences of SEQ ID NOs: 4 and 8, or any combination or fragment thereof. Also provided is 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 one of the sequences of SEQ ID NOs: 4 and 12, or any combination or fragment thereof. Also provided is 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 one of the sequences of SEQ ID NOs: 8 and 16, or any combination or fragment thereof. Also provided is 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 one of the sequences of SEQ ID NOs: 12 and 16, or any combination or fragment thereof. The present invention also encompasses nucleic acids encoding heavy and / or light chain CDRs or antibodies that are modified, for example, by conservative amino acid substitutions.

[0163] In some embodiments, nucleic acid or nucleic acid sequence according to the present invention comprises nucleotide chain (that is, DNA and / or RNA). However, nucleic acid or nucleic acid sequence of the present invention may comprise other types of nucleic acid structures, such as DNA / RNA helices, peptide nucleic acids (PNA), locked nucleic acids (LNA) and / or ribozymes. Such other nucleic acid structures are referred to as functional equivalents of nucleic acid sequences and are encompassed by the present invention. The term "functional equivalent of nucleic acid sequence" also encompasses chains comprising non-natural nucleotides, modified nucleotides and / or non-nucleotide building blocks that exhibit the same function as natural nucleotides.

[0164] The present invention also provides a kind of vector, it comprises one or more nucleic acids according to the present invention.In some embodiments, the vector is a plasmid.Plasmid is defined as circular herein.In some embodiments, plasmid is a double-stranded DNA molecule.The method for preparing the vector comprising nucleic acid according to the present invention is well known in the art.Non-limiting examples of vectors suitable for producing vectors of the present invention include retrovirus and lentiviral vectors.The vector according to the present invention can be used for a variety of applications.In some embodiments, the vector according to the present invention is used for expressing nucleic acid according to the present invention in vitro in cells, such as for producing antibodies or fragments according to the present invention.In addition, the vector according to the present invention comprising nucleic acid according to the present invention can be used for treatment.Administering such a vector to an individual (such as a person) in need causes the antibody or fragment according to the present invention to be expressed in vivo.

[0165] In some embodiments, the present invention 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) vector of the present invention can be produced by a variety of adeno-associated viruses. For example, in terms of replication, integration, cutting and transcription mechanisms, it is expected that ITRs from any AAV serotype have similar structures and functions. 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 produced by serotype AAV1, AAV2, AAV4, AAV5 or AAV8. These serotypes are known to target photoreceptor cells or retinal pigment epithelial cells. In specific embodiments, the rAAV vector is produced by serotype AAV2. In certain embodiments, AAV serotypes include AAVrh8, AAVrh8R or AAVrh10. It should also be understood that the rAAV vector can be a chimera of two or more serotypes selected from serotypes AAV1 to AAV12. The tropism of the vector can be changed by packaging the recombinant genome of a serotype into a capsid derived from another AAV serotype. In some embodiments, the ITR of the rAAV virus can be based on the ITR of any one of AAV1-12, and can 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 can be used with the vector of the present invention. 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.

[0166] A recombinant cell is also provided, which comprises a nucleic acid or a vector according to the present invention. In some embodiments, nucleic acid or a vector is introduced into a cell so that the nucleic acid translation mechanism of the cell will produce an encoded antibody or fragment. In some embodiments, nucleic acid or a vector according to the present invention is expressed in so-called production cells, such as cells of Chinese hamster ovary (CHO), NSO (mouse myeloma) or 293 (T) cell lines, some of which are suitable for commercial antibody production. The propagation of such production cells produces a production cell line that can produce an antibody or a fragment according to the present invention. In some embodiments, the production cell line is suitable for producing antibodies for human use. In some embodiments, the production cell line does not contain pathogens, such as pathogenic microorganisms.

[0167] The present invention also provides a method for producing an antibody or fragment according to the present invention, the method comprising providing a nucleic acid or vector according to the present invention to a cell, and allowing the cell to translate the nucleic acid sequence contained in the nucleic acid or vector, thereby producing an antibody or fragment according to the present invention. In some embodiments, the method according to the present invention also includes collecting, purifying and / or separating the antibody or fragment. Also provided is an antibody or fragment obtained by the method for producing an antibody or fragment according to the present invention.

[0168] D. Pharmaceutical Composition

[0169] In some embodiments, antibodies or fragments according to the present invention can be advantageously used for therapeutic applications. Therefore, a pharmaceutical composition comprising an antibody or fragment according to the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient is provided. A pharmaceutical composition comprising a nucleic acid or a vector (e.g., an AAV vector) according to the present invention and at least one pharmaceutically acceptable carrier, diluent and / or excipient is also provided. Non-limiting examples of suitable carriers include, 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, such as saline, or an oil-based solution. Non-limiting examples of excipients that can be incorporated into tablets, capsules, and the like include adhesives, such as gum tragacanth, gum arabic, 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 flavorings, such as peppermint oil, wintergreen oil, or cherry oil. In some embodiments, the dosage unit form is a capsule. In some embodiments, in addition to one or more of the excipients noted above, the dosage unit form also contains a liquid carrier, such as a fatty oil. Various other materials may exist as coating agents or be used to adjust the physical form of the dosage unit. For example, tablets may be coated with shellac and / or sugar or both. In some embodiments, pharmaceutical compositions according to the present invention are suitable for human use.

[0170] The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering pharmaceutical compositions comprising antibodies according to the present invention and containing pharmaceutically acceptable carriers via intraocular, intravitreal, subretinal, oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal and intracranial methods. In a specific embodiment, any of the antibodies, fragments, carriers or pharmaceutical compositions disclosed herein can be administered to a subject via intravitreal administration. In a specific embodiment, any of the antibodies, fragments, carriers or pharmaceutical compositions disclosed herein can be administered to a subject via subretinal administration. For oral administration, the active ingredient can be in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. According to conventional pharmaceutical practices, sterile compositions for injection can be prepared by dissolving or suspending the antibodies or fragments of the present invention in injection vehicles, such as water or naturally occurring oils such as sesame oil, coconut oil, peanut oil, cottonseed oil or synthetic fat vehicles such as ethyl oleate. Buffers, preservatives and / or antioxidants can also be incorporated.

[0171] E. Therapeutic / preventive applications

[0172] The present invention also provides antibodies or fragments according to the present invention for use in therapy. Nucleic acids according to the present invention are also provided for use in therapy. The therapy may be therapeutic or prophylactic. Antibodies or fragments according to the present invention are particularly suitable for treating, alleviating or preventing disorders associated with alternative pathway complement activation. Therefore, an antibody or fragment according to the present invention is provided for use in treating, alleviating or preventing disorders associated with alternative pathway complement activation. A nucleic acid according to the present invention is also provided for use in treating, alleviating or preventing disorders associated with alternative pathway complement activation. As used herein, "disorders associated with alternative complement activation" are defined herein as disorders in which unwanted and / or excessive alternative pathway complement activation causes damage to cells, tissues or extracellular matrix. Cells that may be damaged by unwanted and / or excessive alternative pathway activation are any cells in contact with blood, such as red blood cells, epithelial cells (specifically, liver and / or kidney epithelial cells), platelets, leukocytes, endothelial cells. In some embodiments, the disorder is a disorder associated with impaired FH function or FH deficiency. In some embodiments, the disorder is a disorder associated with impaired FH function or FH deficiency but not associated with the absence of FH. Because the antibodies and fragments of the present invention enhance the function of FH, the antibodies and fragments are particularly suitable for blocking or reducing the effects of impaired FH function or FH deficiency. However, the enhanced anti-FH antibodies of the present invention can also inhibit the lysis of red blood cells incubated with serum from healthy individuals in which FH has been artificially blocked. Therefore, the antibodies and fragments can also be used to block or reduce undesirable and / or excessive alternative pathway complement activation caused by factors other than impaired FH function or FH deficiency. Non-limiting examples of such conditions that can be treated are atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), and membranous proliferative glomerulonephritis (MPGN).

[0173] Atypical hemolytic uremic syndrome (aHUS), also known as complement-mediated HUS, is characterized by hemolytic anemia, thrombocytopenia, systemic thrombotic microangiopathy (TMA) and renal failure. The onset of aHUS is usually in childhood, and the acute onset of the disease is associated with, for example, infection, pregnancy, other diseases, surgery or trauma. Despite plasma exchange or plasma supplementation, more than 60% of aHUS patients die or develop end-stage renal disease (ESRD). Several mutations in the components or factors of the complement system have been identified in aHUS patients. Mutations in FH, FIFB, membrane cofactor protein (MCP), thrombomodulin (THBD) or C3 account for about 50% of the known mutations in aHUS patients, of which FH mutations are the most frequent (accounting for about 20% to 30% of aHUS patients). Most patients' mutations are heterozygous, but still lead to pathological FH deficiency. In addition, in about 10% of patients, aHUS is caused by autoantibodies against FH, which also leads to functional FH reduction. Currently, the standard treatment for aHUS is plasma supplementation or plasma exchange therapy. In addition, eculizumab is also used to treat aHUS patients. Kidney transplantation is associated with a high risk of relapse, depending on the underlying mutation of aHUS. Children with mutations in FH, FB, FI, C3 or THBD are contraindicated for transplantation due to increased risk of relapse. The antibodies or fragments according to the present invention, such as antibodies or fragments comprising at least a Fab fragment, are particularly suitable for treating, alleviating or preventing aHUS caused by mutations in FH or due to the presence of anti-FH autoantibodies. The enhancement of FH is independent of the CCP domain of FH carrying mutations. For example, enhanced FH antibodies or fragments thereof can inhibit alternative complement activation in aHUS patients carrying mutations in CCP1, CCP6, CCP7, CCP14, CCP17, CCP18, CCP19 and CCP20 of FH. However, because the antibodies and fragments of the present invention can also enhance FH activity in the absence of impaired FH function or FH deficiency, the antibodies or fragments of the present invention can be advantageously used to treat, alleviate or prevent any form of complement-dependent aHUS.

[0174] Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a gene mutation in the X chromosome of a totipotent hematopoietic stem cell. The mutation results in a lack of phosphatidylinositol glycan class A proteins that are essential for the synthesis of glycosylphosphatidylinositol membrane anchored proteins (GPI-APs). Inhibitors of the complement system CD55 are examples of such proteins, which bind to C3b on the surface of host cells, thereby preventing the formation of C3 convertase. Therefore, the lack of these proteins leads to unnecessary or excessive complement activation. One of the main consequences of PNH is that red blood cells are dissolved due to the excessive activity of the complement system. Recently, several countries have approved eculizumab for the treatment of PNH. Other therapies include blood transfusions, erythrocyte stimulator therapy, treatment with corticosteroids and anabolic steroids. Because the antibodies and fragments of the present invention can also enhance the activity of FH regardless of FH or FH functional levels, thereby inhibiting activation of the complement system alternative pathway, the antibodies and fragments can be advantageously used in PNH patients. Additionally, because the antibodies and fragments of the invention act at the level of C3 deposition, in contrast to eculizumab which acts further downstream in the activation pathway, cellular depletion in the liver is reduced because fewer cells are opsonized by C3b.

[0175] Age-related macular degeneration (AMD) is retinal damage, usually affects elderly individuals, causes macula (center of visual field) vision loss.Recently, mutation and SNP (single nucleotide polymorphism) in FH have been involved in about 35% of AMD patients.SNP is located in the CCP7 of FH, and is proved to affect FH to be combined with heparin, thereby impairing the ability of FH in conjunction with host cell surface and extracellular matrix.Antibody according to the present invention or fragment, such as the antibody or fragment comprising at least Fab fragment are particularly suitable for treating, alleviating or preventing AMD characterized by FH hypofunction (that is, with the SNP in the gene encoding FH).

[0176] Membrane proliferative glomerulonephritis (MPGN) is a rare cause of chronic nephritis that occurs primarily in children and adolescents. It causes glomerular damage due to proliferation of mesangial and endothelial cells and expansion of mesangial matrix, thickening of peripheral capillary walls by subendothelial immune deposits and / or dense intramembranous deposits, and insertion of mesangial cells into the capillary walls. MPGN is often associated with the complete absence of FH. In some embodiments, MPGN that can be treated with the antibodies and / or fragments of the invention is associated with impaired FH function or FH deficiency, but is not associated with the absence of FH.

[0177] The present invention therefore provides an antibody or fragment or nucleic acid according to the present invention 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), membranous proliferative glomerulonephritis (MPGN). Also provided is the use of an antibody or fragment, nucleic acid or vector according to the present invention for the preparation of a drug 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 uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranous proliferative glomerulonephritis (MPGN) and IgA nephropathy. In a specific embodiment, the disorder is IgA nephropathy. In some embodiments, the antibody or fragment for use as a medicament or prophylactic agent according to the present invention is an antibody or fragment thereof (i.e., Fab, Fab' or F(ab)2 or F(ab')2 fragment) comprising the antibody heavy and light chain CDR1, CDR2 and CDR3 as shown in Table 1. In some embodiments, the antibody or fragment is a monoclonal humanized or chimeric antibody or fragment.

[0178] The present invention also provides a method for inhibiting alternative complement activation, the method comprising administering an antibody or fragment according to the present invention, or a nucleic acid or vector according to the present invention to an individual. The present invention also provides a method for treating, alleviating or preventing a condition associated with alternative complement activation, the method comprising administering a therapeutically effective amount of an antibody or fragment according to the present invention to an individual in need. A method for treating, alleviating or preventing a condition associated with alternative complement activation is also provided, the method comprising administering a therapeutically effective amount of a nucleic acid or vector according to the present invention to an individual in need. A method for treating, alleviating or preventing a condition associated with alternative complement activation is also provided, the method comprising administering a therapeutically effective amount of a nucleic acid or vector according to the present invention to an individual in need. A pharmaceutical composition according to the present invention is also provided for treating, alleviating or preventing a condition associated with alternative complement activation, the method comprising administering a therapeutically effective amount of a pharmaceutical composition according to the present invention to an individual in need. In some embodiments, the condition is selected from the group consisting of: atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranous proliferative glomerulonephritis (MPGN). As used herein, an "individual" is a human or animal (e.g., a mammal) having a complement system as part of its immune system. In a specific embodiment, the individual is a human. In some embodiments, the antibody used in the methods of the invention is an antibody or fragment thereof, such as a Fab, Fab', F(ab)2, or F(ab')2 fragment, comprising 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 monoclonal humanized or chimeric antibody or fragment.

[0179] Compositions containing antibodies, fragments, nucleic acids of the present invention can be administered for preventive and / or therapeutic treatment. In therapeutic applications, antibodies, fragments, nucleic acids or compositions according to the present invention are administered to individuals (e.g., humans) who are already ill and / or have shown symptoms of the disease in an amount sufficient to offset the symptoms of the disease and / or its complications. In preventive applications, antibodies, fragments, nucleic acids or compositions according to the present invention are administered to individuals before the symptoms of the disease are displayed to prevent the development of these symptoms or its complications. For example, individuals carrying genetic mutations that may or will cause diseases associated with alternative complement activation can be prophylactically treated with antibodies, fragments, nucleic acids or compositions according to the present invention. Antibodies, fragments, nucleic acids or carrier molecules are typically present in a pharmaceutical composition according to the present invention in a therapeutically effective amount, which is an amount sufficient to treat diseases associated with undesirable or excessive activation of the alternative pathway of the complement system.

[0180] For the purpose of clarity and brief description, many features may be described herein as part of the same or separate aspects or embodiments of the present invention. It will be appreciated by those skilled in the art that the scope of the present invention may include embodiments having a combination of all or some features described herein as part of the same or separate embodiments.

[0181] Example

[0182] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0183] Reagents

[0184] 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, Netherlands). High performance ELISA buffer (HPE) was obtained from Sanquin. Recombinant FH CCPs (CCP 15-18, CCP 15-19, CCP 18-20 or CCP 19-10) were produced as previously described (Schmidt et al., 2008). Mouse monoclonal antibodies (mAbs) against human FH were made as previously described. Anti-IL-6.8 was used as an isotype control and was obtained from Sanquin. Anti-C3.19 can react with an epitope on the C3d fragment of the molecule (Wolbink et al., 1993).

[0185] Expression of rhFHR protein

[0186] Recombinant human factor H-related (rhFHR) protein containing a C-terminal 6x histidine (6xHis) tag was produced and purified as previously described (Pouw et al., 2015). Briefly, the protein was expressed by transiently transfecting pcDNA3.1 expression vector in HEK293F cells, followed by HisTrap TM High-efficiency 1 ml columns (GE Healthcare Life Sciences, Freiburg, Germany) were used to analyze the Ni 2+ Protein was purified from the supernatant by affinity chromatography. The rhFHR was filtered and concentrated using ultracentrifugal filter devices (Merck Millipore, Darmstadt, Germany).

[0187] Antibody production

[0188] The monoclonal antibody FHR-1.3B4 was generated by immunizing BALB / c mice intraperitoneally at four-week intervals with 25 μg of recombinant human factor H-related protein 1 (FHR-1) with 25 μg of rhFHR-1 in montanide as an adjuvant. Three days after the fourth booster immunization, splenocytes were fused with the myeloma cell line SP2 / 0. The presence of FHR-1-specific antibodies in hybridoma supernatants was tested by ELISA. Briefly, microtiter plates were coated with rat anti-mouse kappa moAb (RM19) to capture mouse IgG antibodies. The specificity of the antibody was determined by biotinylation of rhFHR-1. Briefly, binding of biotinylated rhFHR-1 was determined by incubation with 0.1% (v / v) streptavidin conjugated to HRP in HPE for 30 minutes. ELISA was further developed using 0.1M sodium acetate (containing 0.003% (v / v) H2O2, pH 5.5) containing 100 μg / mL 3,5,3',5'-tetramethylbenzidine (TMB). Substrate conversion was terminated by adding 100 μL H2SO4, and absorbance was measured at 450nm, and the absorbance at 540nm was corrected using Synergy 2 multi-mode plate reader (BioTek Instruments, Winooski, VT, USA). All ELISA steps were performed in a final volume of 100 μL / well.

[0189] The FHR-1.3B4 antibody was humanized by transplanting the CDR sequences into the framework region of a human germline antibody. As a result of humanization, antibody 5 was generated. Subsequently, certain amino acids in antibody 5 were mutated to improve the binding affinity to FH. Antibody 1, antibody 2, antibody 3, and antibody 4, as disclosed in Table 1, are antibodies that exhibit the highest affinity to FH.

[0190] Binding affinity of antibodies to factor H

[0191] To determine the binding affinity of Antibodies 1 to 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 to 9 or anti-FHR-1.3B4 were captured on a protein A or protein G coupled chip, and full-length FH was passed over the captured moAB at decreasing concentrations. The binding kinetics obtained from the SPR experiments are provided in Table 2.

[0192] Table 2: Binding kinetics of Antibodies 1 to 9.

[0193]

[0194]

[0195] As shown in Table 2, Antibody 1, Antibody 2, Antibody 3, and Antibody 4 bind to human FH with nanomolar or subnanomolar affinity. In addition, Antibody 1 to Antibody 4 have higher binding affinity to full-length FH than FHR-1.3B4.

[0196] Epitope mapping mAbs and competition assays

[0197] The binding sites of some monoclonal antibodies (mAbs) are located. Specifically, the reactivity of mAbs to recombinant FH fragments and full-length human FH comprising different CCP domains (CCP 15-18, CCP 15-19, CCP 18-20 and CCP 19-20) is tested. The position of the epitope of mAbs (such as those disclosed in Table 1) is determined using recombinant human FH fragments comprising multiple CCP domains (15-18, 15-19, 18-20 or 19-20). In short, FH.07 and FHR-1.3B4 are captured on the microtiter plate coated with RM-19 to ensure the best binding conformation. Next, the biotinylated FH mixed with 100 times high concentration of specified unlabeled recombinant FH fragments is incubated for 1 hour on the plate. Binding of biotinylated FH was determined by incubation with 0.01% (v / v) streptavidin conjugated to poly-HRP in HPE for 30 minutes. The ELISA was further developed as previously described. FH.07 and FHR-1.3B4 were found to bind CCP 18 of human FH.

[0198] Competition assays using antibodies or fragments disclosed in Table 1 were performed with the agonistic anti-FH antibody FH.07. To determine whether the humanized anti-FHR-1 mAbs disclosed in Table 1 would compete with FH.07 for binding to FH, a similar setup as described above was used. Briefly, mAbs were coated directly onto plates and binding of biotinylated FH (FH-bt) in the absence or presence of 10-fold higher concentrations of the indicated mAbs was evaluated by ELISA as previously described. FHR-1.3B4 was found to compete with FH.07.

[0199] C3 deposition on LPS

[0200] To investigate whether antibodies 1 to 4 affect alternative pathway inhibition of FH, a C3 deposition assay was performed on LPS. 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. The 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 indicated concentrations of anti-FH / anti-FHR-1 mAbs (Antibody 1 to Antibody 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 conjugated to poly-HRP in HPE for 30 minutes. ELISA was developed as described above. Table 3 describes the IC values ​​of Antibodies 1 to 4 and FHR-1.3B4 50 Table 4 describes the IC values ​​of Antibodies 1 to 4. 50 Value (2 repeated experiments).

[0201] Table 3: IC values ​​of Antibodies 1 to 4 and FHR-1.3B4 50 value.

[0202] Antibody μg / ml nM 1 2.26 15.07 2 2.82 18.79 3 3.47 23.12 4 3.51 23.4 FHR-1.3B4 5.7 38.01

[0203] Table 4: IC values ​​of Antibodies 1 to 4 50 value.

[0204]

[0205] As shown in Tables 3 and 4, antibodies 1 to 4 inhibited C3b deposition on LPS. This suggests that the addition of antibodies 1, 2, 3, or 4 enhances the inhibitory function of FH on alternative pathway activation. In addition, as shown in Table 3, antibodies 1 to 4 were more effective than FHR-1.3B4 in inhibiting C3b deposition on LPS, as shown by lower IC 50 Value indicated.

[0206] SRBC hemolysis assay

[0207] To investigate the effects of Antibody 1 to Antibody 4 on the function of Factor H, hemolytic assays were performed generally as previously described 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 (blocking antibody against FH) was pre-incubated with the indicated mAbs (Antibody 1 to Antibody 4 or FHR-1.3B4) and mixed with sheep red blood cells (SRBC) at a 1:1 ratio in VB containing 5 mM MgCl2 and 10 mM EGTA or VB containing 10 mM EDTA (as blank) to achieve 10% (v / v) serum and 1.05*10 8 The final concentration of cells / ml was then incubated at 37°C for 75 minutes while shaking. Dissolution was terminated by adding 100 μl of ice-cold VB containing 20 mM EDTA and then centrifuging (2.5 minutes, 1,800RPM / 471RCF, 7°C). Hemolysis was measured as the absorbance of the supernatant at 412 nm, corrected for the background absorbance measured at 690 nm, and expressed as a percentage of 100% dissolved control (SRBC incubated with 0.6% (w / v) saponin). As a negative control, SRBC was incubated with serum diluted in VB supplemented with 10 mM EDTA to prevent complement activation.

[0208] The IC of hemolysis was calculated using Graphpad prism 7.04 from multiple experiments using various antibodies such as those disclosed in Table 1. 50 , and statistical comparisons were performed by ordinary one-way ANOVA and Dunnett's multiple comparison test. Table 5 describes the IC values ​​of antibodies 1 to 4 and FHR-1.3B4 50 Table 6 describes the IC values ​​of Antibodies 1 to 4. 50 Value (2 repeated experiments).

[0209] Table 5: IC values ​​of Antibodies 1 to 4 and FHR-1.3B4 50 value.

[0210] ug / ml nM Antibody 1 10.6 70.7 2 10.1 67.3 3 9.4 62.7 4 11.5 76.7 FHR-1.3B4 11.4 76

[0211] Table 6: IC values ​​of Antibodies 1 to 4 50 value.

[0212] μg / ml nM μg / ml nM Antibody 1 10.7 71.33 10.9 72.67 2 11.45 76.33 10.4 69.33 3 11.1 74 9.1 60.67 4 11.47 76.47 11.78 78.53

[0213] As shown in Tables 5 and 6, Antibodies 1 to 4 inhibited SRBC lysis. In addition, as shown in Table 6, Antibodies 1 to 3 were more effective than FHR-1.3B4 in inhibiting SRBC lysis, as shown by lower IC 50 Value indicated.

[0214] Binding affinity of factor H for C3b in the presence of anti-FH antibodies

[0215] The binding of FH to C3b in the presence of antibodies 1 to 4 (such as those 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 fixed to the flow cell of a CM5 Biacore sensor chip (GE Healthcare) using standard amine coupling. The remaining flow cells were used as reference surfaces and were conditioned by coupling reactions without the addition of any protein. A response of 2000 response units (RU) was obtained after coupling with C3b. SPR experiments were performed at 37°C using a flow rate of 10 μl / min and in phosphate buffered saline pH 7.4 (PBS, Orphi Farma) (PBS-T) supplemented with 0.01% (w / v) Tween-20 (Merck).

[0216] In order to determine the role of the antibodies without interfering with possible cross-linking via moAbs, recombinantly produced mAb (Antibody 1 to Antibody 4) Fab' fragments were used. The Fab' fragments were mixed with plasma purified FH (pdFH). FH was injected onto the chip at different concentrations (10-0,01953 μM for FH alone; and 5-0,01953 μM for complexing with moAb Fab' fragments) in the absence or presence of 10 μM (at least 2-fold molar excess) of anti-FHR-1 3B4 Fab' fragments or Fab' fragments of Antibody 1 to Antibody 4 for 60 seconds. Each Fab' fragment was also injected without the addition of FH to determine any interaction of the Fab' fragment with the surface. pdFH dissociated for 60 seconds after injection, and the surface was regenerated by a single injection of 1M NaCl (Merck) between each cycle.

[0217] Data were analyzed using Scrubber 2 (Biologic Software) and affinities were determined by equilibrium analysis. Table 7 lists the binding kinetics of Antibodies 1 to 4.

[0218] Table 7: Binding kinetics of Antibody 1 to Antibody 4 (Fab' fragments).

[0219] name <![CDATA[K D (number of replicates used)]]> FWf 1.3μM(2) pdFH+FHR-1.3B4 (Fab' fragment) 299nM(9) pdFH+ Antibody 1 (Fab' fragment) 273nM(7) pdFH+ Antibody 2 (Fab' fragment) 284nM(7) pdFH+ Antibody 3 (Fab' fragment) 336nM(9) pdFH+ Antibody 4 (Fab' fragment) 292nM(8)

[0220] As shown in Table 7, the addition of Fab' fragments of Antibody 1 to Antibody 4 increased the response on the C3b-coated surface, indicating that Antibody 1 to Antibody 4 enhanced the binding of pdFH to C3b.

[0221] References

[0222] · Almagro JC, Fransson J. 2008. Humanization of antibodies. Frontiers in Bioscience 13: 1619-33.

[0223] 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.USA89(10):4285-9.

[0224] ·Chen Y, Wiesmann C, Fuh G, Li B, Christinger HW, McKay P, de Vos AM, Lowman HB. Selection and analysis of an optimized anti-VEGF antibody: crystalstructure of an affinity-matured Fab in complex with antigen. J Mol Biol. 1999; 293(4): 865-81.

[0225] Cheng ZZ, Corey MJ, M, Majno S, Hellwage J, Zipfel PF, Kinders RJ, Raitanen M, Meri S, Jokiranta TS. Complement factor H as a marker for detection of bladder cancer. Clin Chem. 2005;51(5):856-63.

[0226] ·Corey MJ,Kinders RJ,Poduje CM,Bruce CL,Rowley H,Brown LG,Hass GM,Vessella RL.Mechanistic studies of the effects of anti-factor H antibodies oncomplement-mediated lysis.J Biol Chem.2000;275(17):12917-25.

[0227] ·Lazar GA1,Desjarlais JR,Jacinto J,Karki S,Hammond PW.2007.Amolecular immunology approach to antibody humanization and functionaloptimization.Mol.Immunol.,44(8):1986-98.

[0228] ·Lefranc MP,“Unique database numbering system for immunogeneticanalysis”Immunology Today,18,509(1997).PMID:9386342.

[0229] ·Lefranc MP,″The IMGT unique numbering for immunoglobulins,T cellReceptors and Ig-like domains″,The Immunologist.1999;7,132-136.

[0230] ·Lefranc MP,Pommié C,Ruiz M,Giudicelli V,Foulquier E,Truong L,Thouvenin-Contet V,and Lefranc G,“IMGT unique numbering for immunoglobulinand T cell receptor variable domains and Ig superfamily V-like domains”Dev.Comp.Immunol.,27,55-77(2003).PMID 12477501.

[0231] ·Padlan,EA.1991.A possible procedure for reducing the immunogenicityof antibody variable domains while preserving their ligand-bindingproperties.Mol.hnmunol.,28(4-5):489-98.

[0232] ·Pouw,Richard B,Mieke C Brouwer,Judy Geissler,Laurens V van Herpen,Sacha S Zeerleder,Walter A Wuillemin,Diana Wouters,and Taco W Kuijpers.2016.“Complement Factor H-Related Protein3Serum Levels Are Low Compared to FactorH and Mainly Determined by Gene Copy Number Variation in CFHR3.”PLoS ONE 11(3):e0152164.

[0233] ·Queen C,Schneider WP,Selick HE,Payne PW,Landolfi NF,Duncan JF,Avdalovic NM,Levitt M,Junghans RP,Waldmann TA.1989.A humanized antibody thatbinds to the interleukin2receptor.Proc.Natl.Acad.Sci.U.S.A.86(24):10029-33.

[0234] ·Sánchez-Corral,Pilar,C González-Rubio,Santiago Rodríguez de Córdoba,and Margarita López-Trascasa.2004.“Functional Analysis in Serum fromAtypical Hemolytic Uremic Syndrome Patients Reveals Impaired Protection ofHost Cells Associated with Mutations in Factor H.”Molecular Immunology 41(1):81-84.

[0235] ·Schmidt,Christoph Q,Andrew P Herbert,Henry G Hocking,D Uhrín,andPaul N Barlow.2008.“Translational Mini-Review Series on Complement Factor H:Structural and Functional Correlations for Factor H.”Clinical andExperimental Immunology 151(1):14-24.

[0236] ·Tan P,Mitchell DA,Buss TN,Holmes MA,Anasetti C,Foote J.2002.″Superhumanized″antibodies:reduction of immunogenic potential bycomplementarity-determining region grafting with human germline sequences:application to an anti-CD28.J.Immunol.,169(2):1119-25.

[0237] Wolbink, GJ, J Bollen, JW Baars, RJ ten Berge, a J Swaak, J Paardekooper, and C Erik Hack. 1993. “Application of a Monoclonal Antibody against a Neoepitope on Activated C4 in an ELISA for the Quantification of Complement Activation via the Classical Pathway.” Journal of Immunological Methods 163(1):67-76.

[0238] ·Wouters, Diana, Mieke C Brouwer, Mohamed R Daha, and C Erik Hack. 2008. "Studies on the Haemolytic Activity of Circulating C1q-C3 / C4 Complexes." Molecular Immunology 45(7): 1893-99.

[0239] Incorporated by Reference

[0240] 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.

[0241] Equivalent solutions

[0242] The present invention may be implemented in other specific forms without departing from its spirit or essential features. The above embodiments are therefore considered in all respects to be illustrative, rather than limiting, the present invention described herein. The scope of the present invention is therefore specified by the appended claims rather than by the above invention description, and all changes within the meaning and scope of the equivalent scheme of the claims are intended to be included therein.

Claims

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), comprising: - the light chain CDR1 sequence as shown in the sequence SSVTY (SEQ ID NO: 1); - the light chain CDR2 sequence as shown in sequence ATS (SEQ ID NO: 2); - the light chain CDR3 sequence as shown in the sequence QHRSSSNPLT (SEQ ID NO: 3); - the heavy chain CDR1 sequence as shown in the sequence GFSLTNYG (SEQ ID NO: 5); - a heavy chain CDR2 sequence as shown in the sequence IWSGGTT (SEQ ID NO: 10); and - The heavy chain CDR3 sequence shown as sequence ARNFGNYAMDF (SEQ ID NO: 11).

2. 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: - the light chain CDR1 sequence as shown in the sequence SSVTY (SEQ ID NO: 1); - the light chain CDR2 sequence as shown in sequence ATS (SEQ ID NO: 2); - the light chain CDR3 sequence as shown in the sequence QHRSSSNPLT (SEQ ID NO: 3); - the heavy chain CDR1 sequence as shown in the sequence GFSLTNYG (SEQ ID NO: 5); - a heavy chain CDR2 sequence as shown in the sequence VWSGGTT (SEQ ID NO: 6); and - The heavy chain CDR3 sequence shown as sequence ARNFGNYAMDY (SEQ ID NO: 7).

3. 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: - the light chain CDR1 sequence as shown in the sequence TSVTY (SEQ ID NO: 13); - the light chain CDR2 sequence as shown in the sequence ASS (SEQ ID NO: 14); - the light chain CDR3 sequence as shown in the sequence QHRSSSNPLT (SEQ ID NO: 3); - the heavy chain CDR1 sequence as shown in the sequence GFSLTNYG (SEQ ID NO: 5); - a heavy chain CDR2 sequence as shown in the sequence VWSGGTT (SEQ ID NO: 6); and - The heavy chain CDR3 sequence shown as sequence ARNFGNYAMDY (SEQ ID NO: 7).

4. 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: - the light chain CDR1 sequence as shown in the sequence TSVTY (SEQ ID NO: 13); - the light chain CDR2 sequence as shown in the sequence ASS (SEQ ID NO: 14); - the light chain CDR3 sequence as shown in the sequence QHRSSSNPLT (SEQ ID NO: 3); - the heavy chain CDR1 sequence as shown in the sequence GFSLTNYG (SEQ ID NO: 5); - a heavy chain CDR2 sequence as shown in the sequence IWSGGTT (SEQ ID NO: 10); and - The heavy chain CDR3 sequence shown as sequence ARNFGNYAMDF (SEQ ID NO: 11).

5. The antibody or fragment according to any one of claims 1 to 4, wherein the binding affinity of the antibody or fragment to FH is expressed as K D 2.5×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.1×10 -9 M or lower.

6. The antibody or fragment according to any one of claims 1 to 4, wherein the binding affinity of the antibody or fragment to FH is expressed as K D 0.6×10 -8 M or lower and / or the binding affinity to the FH fragment containing CCP18-20 is expressed as K D 0.6×10 -11 M or lower.

7. The antibody or fragment according to any one of claims 1 to 4, wherein the antibody or fragment: - Inhibits C3 deposition on LPS in vitro in the presence of 10% v / v serum, with IC 50 A value of 38 nM or less; and / or -Inhibits in vitro hemolytic activity in the presence of 10% v / v serum, where IC 50 A value of 150 nM or less; and / or - In vitro FH K of C3b D to at most 2 μM and / or increase the in vitro binding affinity of FH for C3b by at least 3-fold.

8. The antibody or fragment according to claim 7, wherein the antibody or fragment: - Inhibits C3 deposition on LPS in vitro in the presence of 10% v / v serum, with IC 50 A value of 30 nM or less; and / or -Inhibits in vitro hemolytic activity in the presence of 10% v / v serum, where IC 50 The value was 130 nM or lower.

9. The antibody or fragment according to claim 2, comprising: a variable light chain sequence as shown in SEQ ID NO: 4; and a variable heavy chain sequence as shown in SEQ ID NO:

8. 10 . The antibody or fragment according to claim 1 , comprising: a variable light chain sequence as shown in SEQ ID NO: 4; and a variable heavy chain sequence as shown in SEQ ID NO:

12.

11. The antibody or fragment according to claim 3, comprising: a variable light chain sequence as shown in SEQ ID NO: 16; and a variable heavy chain sequence as shown in SEQ ID NO:

8.

12. The antibody or fragment according to claim 4, comprising: a variable light chain sequence as shown in SEQ ID NO: 16; and a variable heavy chain sequence as shown in SEQ ID NO:

12.

13. The antibody or fragment of any one of claims 1 to 4, wherein the antibody or fragment enhances FH activity, wherein the FH activity is inhibition of alternative complement activation.

14. The antibody or fragment of claim 13, wherein inhibiting alternative complement activation comprises: -Inhibit hemolytic activity; - Inhibits C3b deposition; and / or -Increases the binding of FH to C3b, iC3b and / or C3d.

15. The antibody or fragment according to any one of claims 1 to 4, 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.

16. The antibody or fragment according to any one of claims 1 to 4, wherein the antibody or fragment comprises a Fab fragment.

17. The antibody or fragment of any one of claims 1 to 4, wherein the antibody or fragment is monoclonal.

18. The antibody or fragment of any one of claims 1 to 4, wherein the antibody or fragment comprises human light and heavy chain constant regions.

19. The antibody or fragment of claim 18, wherein the constant region comprises the Fc domain of human IgG4.

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

21. The antibody or fragment of any one of claims 1 to 4, wherein the antibody or fragment is PEGylated.

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

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

24. A vector comprising the nucleic acid according to claim 23.

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

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

27. A recombinant cell comprising the nucleic acid of claim 23 or the vector of any one of claims 24 to 26.

28. A pharmaceutical composition comprising the antibody or fragment according to any one of claims 1 to 22, the nucleic acid according to claim 23, the vector according to any one of claims 24 to 26 or the recombinant cell according to claim 27, and a pharmaceutically acceptable carrier, diluent and / or excipient.

29. Use of an antibody or fragment according to any one of claims 1 to 22, a nucleic acid according to claim 23, a vector according to any one of claims 24 to 26, a recombinant cell according to claim 27, or a pharmaceutical composition according to claim 28 for the preparation of a medicament for treating, alleviating or preventing a disorder associated with alternative pathway complement activation, 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.

30. A method for producing an isolated synthetic or recombinant antibody or an antigen-binding fragment thereof, the method comprising providing a nucleic acid according to claim 23 or a vector according to any one of claims 24 to 26 to a cell, and allowing the cell to translate the nucleic acid or the nucleic acid sequence contained in the vector, thereby producing the antibody or fragment.

31. The method of claim 30, further comprising collecting, purifying and / or isolating the antibody or fragment.

Images