Factor H enhancing antibodies and their uses

Abstract

The present invention relates to novel isolated, synthetic or recombinant antibodies and fragments thereof that are specific for Factor H. The present invention also relates to the use of such antibodies and fragments for inhibiting complement activation and treating complement activation-related disorders.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and medicine. In particular, the present invention relates to factor H specific antibodies and uses thereof. Background Art

[0002] The complement system is an important element of innate immunity that helps protect many organisms, such as mammals, from invading pathogens. The complement system is composed of more than 30 different components that are synthesized primarily in the liver. Activation of the complement system occurs through three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. These three pathways converge in the formation of the C3 convertase, which is different for each pathway but has similar activities.

[0003] In the classical complement pathway, activation of the complement component (C) 1 complex, composed of C1q, C1r, and C1s, occurs upon binding of an antibody-antigen complex. The C1 complex cleaves C4 and C2, resulting in the formation of 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 pathogenic surfaces, thereby activating serine proteases MASP-1 and MASP-2 that are capable of cleaving C4 and C2. As with the classical pathway, this results in the formation of the C4bC2a C3 convertase. The C3 convertase can bind to C3b to form the C5 convertase. In contrast to the classical and lectin pathways, the alternative pathway has a lower level of continuous activity due to the spontaneous hydrolysis of C3 in plasma to C3(H2O). This C3b-like C3 (H2O) can form a liquid-phase C3 convertase by binding to factor B (FB), which in turn can be activated to Bb by factor D. Similarly, when C3b is bound to a surface, it may bind to FB to form C3bB. This complex is cleaved by factor D to C3bBb, which is the C3 convertase of the alternative pathway and can be stabilized as C3bBbP by properdin (factor P). This C3 convertase is able to cleave C3 into C3a and C3b. In addition to this process, the alternative pathway also serves as an amplification loop for the classical pathway and the lectin activation pathway, because the C3b generated in these pathways can serve as the starting point of the alternative pathway. Therefore, the amplification loop leads to the enhancement of the classical and lectin pathways. The C3 convertase formed in one of the three activation pathways can bind to 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 to C6, C7, C8, and C9 to form the membrane attack complex (MAC), which forms a transmembrane channel and causes cell lysis.

[0004] Immediately following the formation of pores in the pathogen's membrane, complement helps to clear the pathogen or altered self-cells by opsonizing 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. Because complement has strong pro-inflammatory properties, host cells are well protected by a variety of membrane and soluble complement regulatory proteins.

[0005] The alternative pathway accounts for 80-90% of the total complement activity. Therefore, it is crucial to regulate this pathway. C3 (H2O) and C3b (if not bound to a pathogen) formed by the spontaneous hydrolysis of C3 are usually rapidly inactivated by factor H (FH), factor I (FI) and host cell surface molecules, thereby inhibiting the formation of the C3 convertase. CD55 (also known as decay accelerating factor or DAF) binds to C3b on the host cell surface. FI cuts C3b into an inactive form, but depends on cofactors (CD46, MCP) expressed on the cell surface or cofactors (FH) circulating in the plasma. FH is a plasma glycoprotein that is essential for controlling the alternative complement pathway in solution and on the cell surface. FH binds to C3b at the same position as FB, thereby preventing the formation of C3 convertase. FH also has decay accelerating activity, that is, once formed, it promotes the dissociation of the alternative pathway C3 convertase. Whether FH binds to C3b depends on the carbohydrates present on the cell surface. Sialic acid, glycosaminoglycans, and heparin present on the surface of host cells promote the binding of FH to C3b, while the binding of C3b to molecules expressed on the surface of pathogens leads to the binding of FB. Therefore, FH exerts complement inhibitory activity on the surface of host cells rather than pathogenic cells because the cell surface molecules bound to FH are expressed on host cells but are not usually expressed on pathogenic cells. FH contains 20 complement control protein (CCP) domains, numbered 1-20 from the N-terminus of FH. CCP domains are also called short common repeats (SCR) or sushi domains. CCP 1-4 is a domain involved in regulation, CCP 19-20 is involved in binding C3b, and CCP 6, 7, 8, 19, and 20 bind to GAGs and sialic acid expressed on the cell surface. Antibodies that bind to CCP19 and / or CCP20 inhibit the activity of FH.

[0006] Factor H related proteins (CFHR) are plasma glycoproteins that are structurally and antigenically related to FH. FHR proteins are also composed of CCP domains and have varying degrees of homology to the CCP domains found in FH. For example, FHR1 contains domains corresponding to CCP6, CCP7, CCP18, CCP19, and CCP20. In contrast to FH, CFHR do not have potent 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 positive regulators of the alternative complement pathway.

[0007] FH deficiency or impaired host surface recognition due to mutations is associated with complement-mediated tissue damage and disease. After controlling complement activation in normal hemostasis, FH also plays an important role in limiting the damage of complement-mediated diseased cells and tissues. Multiple mutations in the FH gene have been described, which may lead to loss of FH protein function. The C-terminal region of FH is a hot spot for mutations in diseases. 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, which means that about half of the circulating FH has normal function. However, this is obviously not enough to protect the self surface under certain conditions where complement is activated. FH deficiency may lead to kidney diseases, 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.

[0008] Currently, the standard treatment for FH deficiency such as aHUS is plasma replacement or plasma exchange therapy. With this therapy, deficient complement regulators are replaced. Plasma exchange therapy can also remove mutant complement factors and / or autoantibodies against complement factors. However, plasma therapy also has some limitations. No prospective clinical studies have shown that plasma exchange therapy is safe or effective for the treatment of aHUS, and the effectiveness of plasma therapy may depend on the underlying mutation. Some patients have allergic reactions to fresh frozen plasma, which may require discontinuation of any form of plasma therapy. In addition, plasma exchange may worsen the clinical condition of aHUS due to the administration of active pathogenic complement components derived from plasma.

[0009] Recently, the therapeutic monoclonal antibody eculizumab has been approved for the treatment of aHUS and paroxysmal nocturnal hemoglobinuria (PNH) in several countries, including the United States and European countries. Eculizumab is a humanized mouse monoclonal antibody specific for C5 that prevents C5 from being cleaved into C5a and C5b. Therefore, it blocks the activation of the terminal pathway and reduces the influx of immune cells. However, the use of eculizumab is associated with adverse side effects. Because it blocks C5, which is a key component for initiating the terminal pathway, patients treated with eculizumab become susceptible to infection with enveloped bacteria (such as Neisseria meningitidis), whose clearance is very dependent on the formation of MAC. Therefore, before receiving eculizumab treatment, patients need to be vaccinated against meningococci. In addition, because eculizumab acts downstream of C3, the deposition of C3 is maintained, which is harmful in a variety of diseases involving undesirable or excessive complement activation. In addition, eculizumab treatment involves high costs, and the availability of antibodies is limited.

[0010] Cheng et al. (Clinical Chemistry, 2005) described a mouse monoclonal antibody that binds to CCP18. The antibody, called X52.1, was described to enhance the binding of FH to C3b and C3d, which is thought to be caused by the dimerization of FH. The increased binding of FH to C3b and C3d induced by X52.1 also resulted in increased complement-mediated cell lysis (including RBCs and a variety of cancer cells), as described by Corey et al. (J Biol Chem. 2000). This suggests that antibody X52.1 inhibits the complement inhibitory activity of FH. In fact, Corey et al. suggested that the antibody could be used to treat cancer by enhancing complement-mediated cancer cell lysis.

[0011] WO 2016 / 028150 describes an agonistic anti-FH antibody called FH.07 that inhibits alternative pathway activation as shown by increased binding of FH to C3b, inhibition of mediated C3 deposition, and inhibition of hemolytic activity. The Fab' and F(ab')2 fragments of this antibody were shown to have the same FH enhancing effect.

[0012] There is a continuing need for new and improved therapeutic agents that bind FH, such as agents that can be used to treat diseases associated with undesirable or excessive complement activation. Summary of the invention

[0013] The object of the present invention is to provide novel antibodies that bind to FH. Another object is to provide antibodies that specifically recognize epitopes in FH, the binding of which leads to enhanced FH activity, particularly epitopes located in the domain CCP18 of FH. Another object of the present invention is to provide antibodies with high binding affinity to FH.

[0014] Thus, the present invention 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 or fragment for FH is D 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 M or less, preferably wherein the K of the binding affinity of the antibody or fragment to FH is D 1.25×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.04×10 -9 M or lower, more preferably wherein the K of the binding affinity of the antibody or fragment to FH is D 1.25×10 -8M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.04×10 -9 M or less or 0.1×10 -9 M or less. Preferred antibodies or fragments preferably have a binding affinity for FH of K D 0.6×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.6×10 -11 M or lower. The antibody or fragment preferably has an IC of 38nM or lower. 50 values ​​inhibited C3 deposition on LPS in vitro and / or had an IC of 150 nM or less in vitro 50 The antibody or fragment preferably increases the binding affinity (K) of FH to C3b in vitro. D ) to a maximum of 2 μM and / or increase the binding affinity of FH for C3b at least 3-fold in vitro. Binding affinity was determined using surface plasmon resonance (SPR).

[0015] In a further aspect, the present invention 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 antibody or fragment has an IC of 38 nM or less in vitro. 50 The value inhibited the deposition of C3 on LPS.

[0016] In a further aspect, the present invention 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 antibody or fragment increases the binding affinity (K) of FH for C3b in vitro. D ) to a maximum of 2 μM and / or increase the binding affinity of FH for C3b at least 3-fold in vitro.

[0017] In a further aspect, the present invention 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 antibody or fragment has an IC of 150 nM or less in vitro. 50 The IC 50 The value is preferably 130 nM or lower, more preferably 115 nM or lower, more preferably 105 nM or lower, more preferably 100 nM or lower.

[0018] The antibody or fragment according to the present invention preferably binds to the complement control protein domain 18 (CCP18) of factor H (FH). The FH activity is preferably to inhibit alternative complement activation. Inhibition of alternative complement activation includes: inhibiting hemolytic activity, inhibiting the deposition of complement component 3 (C3) on cells, and / or increasing the binding of FH to C3b, iC3b and / or C3d.

[0019] In a further aspect, 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), comprising:

[0020] - a light chain CDR1 sequence having the sequence SSVXY, wherein X is R, T or N (SEQ ID NO: 91) or the sequence QSLVHSNGNTY (SEQ ID NO: 49),

[0021] - a light chain CDR2 sequence having the sequence X1X2S, wherein X1=A, K or Y and X2=T or L (SEQ ID NO: 92),

[0022] - a light chain CDR3 having a sequence selected from QQWGTKPPT (SEQ ID NO: 19), QQRSSSNPLT (SEQ ID NO: 35), SQSTHVPFT (SEQ ID NO: 51) and QQFTSSPLT (SEQ ID NO: 67),

[0023] - a heavy chain CDR1 having the sequence X1FSLTX2X3G, wherein X1 = D or G, X2 = N or S and X3 = S or Y (SEQ ID NO: 93),

[0024] - a heavy chain CDR2 having the sequence IWSGGXT, wherein x = T, N or S (SEQ ID NO: 94), and

[0025] - A heavy chain CDR3 sequence having the sequence ARNX1GNYX2X3DY, wherein X1 = F or G, X2 = A or Y and X3 = V, M or F (SEQ ID NO: 95) or AKNGDYGYTMDY (SEQ ID NO: 55).

[0026] In a further aspect, 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), comprising:

[0027] - a light chain CDR1 sequence having the sequence SSVXY, wherein X is R or T (SEQ ID NO: 96),

[0028] - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 97);

[0029] - a light chain CDR3 having a sequence selected from QQWGTKPPT (SEQ ID NO: 19) and QQRSSSNPLT (SEQ ID NO: 35);

[0030] - a heavy chain CDR1 having the sequence X1FSLTNX2G, wherein X1 = D or G and X2 = S or Y (SEQ ID NO: 98),

[0031] - a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 99), and

[0032] - A heavy chain CDR3 sequence having the sequence ARNFGNYAXDY, wherein X=V or M (SEQ ID NO: 100).

[0033] In a further aspect, 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), comprising a light chain CDR1 sequence having the sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 18), and a light chain CDR3 having the sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 having the sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 22), and a heavy chain CDR3 having the sequence ARNFGNYAVDY (SEQ ID NO: 23). The antibody or fragment preferably has a binding affinity for FH of K D 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 M or less, preferably wherein the K of the binding affinity of the antibody or fragment to FH is D 1.25×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.04×10 -9 M or lower. The antibody or fragment preferably has an IC of 38nM or lower in vitro. 50 The value inhibits the deposition of C3 on LPS, preferably C 50 The value is 30 nM or lower, more preferably C 50The antibody or fragment preferably has an IC of 150 nM or less in vitro. 50 The value of inhibition of hemolytic activity, preferably IC 50 The value is 130 nM or lower, and more preferably IC 50 The antibody or fragment preferably has a binding affinity (K) of FH to C3b in vitro. D ) to a maximum of 2 μM, more preferably 1.8 μM and / or increase the binding affinity of FH for C3b in vitro by at least 3-fold, more preferably 3.5-fold.

[0034] In a further aspect, the present invention provides an isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds to factor H (FH), comprising a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 33), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 34), and a light chain CDR3 having the sequence QQRSSSNPLT (SEQ ID NO: 35), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 37), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 38), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 39). The antibody or fragment preferably has a binding affinity for FH of D 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 M or less, preferably wherein the K of the binding affinity of the antibody or fragment to FH is D 0.6×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.6×10 -11 M or less. The antibody or fragment preferably has a binding affinity (K of FH to C3b) of D ) to a maximum of 2 μM, more preferably 1.95 μM and / or increase the binding affinity of FH for C3b in vitro by at least 3-fold, more preferably 3.1-fold.

[0035] In a further aspect, the present invention provides an isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds factor H (FH), comprising a light chain CDR1 sequence having the sequence QSLVHSNGNTY (SEQ ID NO:49), a light chain CDR2 sequence having the sequence KLS (SEQ ID NO:50), and a light chain CDR3 having the sequence SQSTHVPFT (SEQ ID NO:51), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO:53), a heavy chain CDR2 having the sequence IWSGGNT (SEQ ID NO:54), and a heavy chain CDR3 sequence having the sequence AKNGDYGYTMDY (SEQ ID NO:55).

[0036] In a further aspect, the present invention provides an isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds to factor H (FH), comprising a light chain CDR1 sequence having the sequence SSVNY (SEQ ID NO:65), a light chain CDR2 sequence having the sequence YTS (SEQ ID NO:66) and a light chain CDR3 having the sequence QQFTSSPLT (SEQ ID NO:67), a heavy chain CDR1 having the sequence GFSLTSYG (SEQ ID NO:69), a heavy chain CDR2 having the sequence IWSGGST (SEQ ID NO:70) and a heavy chain CDR3 sequence having the sequence ARNGGNYYFDY (SEQ ID NO:71).

[0037] In a further aspect, the present invention provides an isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds Factor H (FH), comprising: a variable light chain sequence comprising a sequence having at least 80% sequence identity to a sequence selected from the group consisting of QIVLSQSPAILSASPGEKVTMTCRASSSVRYMHWYQQKAGSSPTAWIFATSNLASGVPPRFSGSGSGTSYSLTISRVEAEDAATYYCQQWGTKPPTFGAGTKLELK (SEQ ID NO:20), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLKQSGPGLVQPSQSLSITCTVSDFSLTNSGVHWVRQSPGKGLEWLGVIWSGGTTEYNAAFMSRLTITKDNSKSQVFFKMNSLLVDDTGIYYCARNFGNYAVDYWGQGTSVTVSS (SEQ ID NO:24).

[0038] In a further aspect, the present invention provides an isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds Factor H (FH), comprising: a variable light chain sequence comprising a sequence having at least 80% sequence identity to the sequence QIVLSQSPTILSASPGEKVTMTCRASSSVTYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQRSSSNPLTFGAGTKLELK (SEQ ID NO:36), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLRQSGPGLVQPSQSLSITCTVSGFSLTNYGVYWVRQSPGKGLEWLGVIWSGGTTDYSAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARNFGNYAMDYWGQGTSVTVSS (SEQ ID NO:40).

[0039] In a further aspect, the present invention provides an isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds Factor H (FH), comprising: a variable light chain sequence comprising a sequence having at least 80% sequence identity to the sequence DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIK (SEQ ID NO:52), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQPPGKGLEWLGVIWSGGNTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCAKNGDYGYTMDYWGQGTSVTVSS (SEQ ID NO:56).

[0040] In a further aspect, the present invention provides an isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds factor H (FH), comprising: a variable light chain sequence comprising a sequence having at least 80% sequence identity to the sequence ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASKLSWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGAGTKLELK (SEQ ID NO:68), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARNGGNYYFDYWGQGTTLTVSS (SEQ ID NO:72).

[0041] The antibody or fragment according to the present invention comprising the light chain CDR1-3 sequence and the heavy chain CDR1-3 sequence specified herein or the variable light chain sequence and the variable heavy chain sequence specified herein preferably enhances FH activity. The FH activity is preferably to inhibit alternative complement activation. The inhibition of 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.

[0042] In a further aspect, the present invention provides an isolated, synthetic or recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or fragment according to the present invention.

[0043] In a further aspect, the present invention provides a vector comprising the nucleic acid molecule according to the present invention.

[0044] In a further aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule or vector according to the present invention.

[0045] In a further aspect, the present invention provides a pharmaceutical composition comprising the antibody or fragment according to the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0046] In a further aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid molecule according to the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0047] In a further aspect, the present invention provides a pharmaceutical composition comprising the carrier according to the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0048] In a further aspect, the invention provides an antibody or fragment according to the invention for use in therapy.

[0049] In a further aspect, the present invention provides a nucleic acid molecule according to the present invention for use in therapy.

[0050] In a further aspect, the invention provides a vector according to the invention for use in therapy.

[0051] In a further aspect, the invention provides an antibody or fragment according to the invention for use in inhibiting alternative complement activation.

[0052] In a further aspect, the present invention provides a nucleic acid molecule according to the present invention for use in inhibiting alternative complement activation.

[0053] In a further aspect, the invention provides a vector according to the invention for use in inhibiting alternative complement activation.

[0054] In a further aspect, the invention provides an antibody or fragment according to the invention for use in the treatment, alleviation or prevention of a disorder associated with alternative pathway complement activation.

[0055] In a further aspect, the present invention provides a nucleic acid molecule according to the present invention for use in the treatment, alleviation or prevention of a disorder associated with alternative pathway complement activation.

[0056] In a further aspect, the present invention provides a vector according to the present invention for use in the treatment, alleviation or prevention of a disorder associated with alternative pathway complement activation.

[0057] In a further aspect, the present invention provides use of the nucleic acid molecule according to the present invention for the preparation of a medicament for treating, alleviating or preventing a disorder associated with alternative complement activation.

[0058] In a further aspect, the present invention provides use of the vector according to the present invention for preparing a medicament for treating, alleviating or preventing a disorder associated with alternative complement activation.

[0059] In a further aspect, the present invention provides use of the antibody or fragment according to the present invention for the preparation of a medicament for treating, alleviating or preventing a disorder associated with alternative complement activation.

[0060] In a further aspect, the present invention provides a method for treating, ameliorating or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody or fragment according to the present invention.

[0061] In a further aspect, the present invention provides a method of treating, ameliorating or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof a therapeutically effective amount of a nucleic acid molecule according to the present invention.

[0062] In a further aspect, the present invention provides a method for treating, ameliorating or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof a therapeutically effective amount of a vector according to the present invention.

[0063] In a further aspect, the present invention provides a method for treating, ameliorating or preventing a disorder associated with alternative pathway complement activation, comprising administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical composition according to the present invention.

[0064] In a further aspect, the invention provides a method of inhibiting alternative complement activation comprising administering to a subject an antibody or fragment according to the invention.

[0065] In a further aspect, the invention provides a method of inhibiting alternative complement activation comprising administering to a subject a nucleic acid molecule according to the invention.

[0066] In a further aspect, the present invention provides a method of inhibiting alternative complement activation, comprising administering to a subject a vector according to the present invention.

[0067] In a further aspect, the present invention provides a method for producing the antibody or fragment according to the present invention, the method comprising providing a cell having a nucleic acid molecule or vector according to the present invention, and allowing the cell to translate the nucleic acid sequence contained by the nucleic acid molecule or vector, thereby producing the antibody or fragment according to the present invention. DETAILED DESCRIPTION

[0068] In one aspect, the present invention provides agonistic anti-FH antibodies and fragments thereof, i.e., antibodies and fragments that enhance FH activity. In one aspect, these antibodies compete with antibody FH.07 for binding to the same epitope in domain CCP18 of FH. Enhanced anti-FH antibodies are effective inhibitors of activation of alternative complement pathways and can therefore be used to treat disorders associated with undesirable or excessive activation of alternative pathways of the complement system. FH specifically inhibits the amplification loop of the alternative pathway, in which C3 is cleaved into C3b, which is then combined with FB on the cell surface and forms a C3 convertase, which promotes the cleavage 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 that the accumulation of C3b on the surface and the release of C3a are avoided. In contrast, if complement activation is inhibited at the C5 level, such as with eculizumab, the accumulation of C3b and the release of C3a will not be inhibited. C3b acts as an opsonin and C3a is an anaphylatoxin. Thus, accumulation of C3b and formation of C3a are preferably prevented, since these processes lead to attraction of immune cells and opsonophagocytosis of the target. This means, for example, that PNH patients receiving eculizumab still require blood transfusions, since accumulation of C3b leads to opsonization of erythrocytes, which are subsequently cleared in the liver and spleen. In addition, treatment with anti-C5 antibodies leads to accumulation of C3b and formation of C3a on cells, which need to be cleaved by MAC in addition. An important disadvantage of anti-C5 antibodies is that patients become susceptible to infections, 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.

[0069] As used herein, the term "antibody" refers to an immunoglobulin protein specific for a target epitope, which comprises at least a heavy chain variable region (VH) and a light chain variable region (VL) paired therewith. The term encompasses polyclonal and monoclonal antibodies. It refers to any form of antibody that specifically binds to CCP18 (preferably CCP18) of FH, including full-length immunoglobulins. The antibody according to the present invention, or its fragment, comprises at least one antigen binding site. As used herein, the term "antigen binding site" refers to a site of an antibody or its fragment comprising at least one CDR sequence (preferably at least two CDR sequences, more preferably at least three CDR sequences). For example, the antigen binding site comprises light chain CDR 1-3 or heavy chain CDR 1-3. Particularly preferred antigen binding sites comprise light chain CDR 1-3 and heavy chain CDR 1-3.

[0070] As is well known to those skilled in the art, antibodies comprise two heavy chains and two light chains. The heavy chain of an antibody is the larger of the two chains that make up the immunoglobulin molecule. The heavy chain includes a constant domain and a variable domain, which participates in antigen binding. The light chain of an antibody is the smaller of the two chains that make up the immunoglobulin molecule. The light chain includes a constant domain and a variable domain. The variable domain of the light chain often, 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 in the heavy chain variable domain and the light chain variable domain. If it is a full-length antibody, the CDR 1-3 of the heavy chain and the CDR 1-3 of the connected light chain together form an antigen binding site.

[0071] Herein, "antigen-binding fragment of an antibody" is defined as a part of an antibody that can specifically bind to the same antigen as the antibody (i.e., CCP18 of FH), although not necessarily to the same extent. Fragments of FH activity-enhancing antibodies can also enhance FH activity, although not necessarily to the same extent. Fragments of FH activity-inhibiting antibodies can also inhibit FH activity, although not necessarily to the same extent. The antibody fragment according to the present invention preferably comprises the heavy chain CDR1, CDR2 and CDR3 sequences of the antibody, and the light chain CDR1, CDR2 and CDR3 sequences of the antibody. Non-limiting examples of antibody fragments are single-domain antibodies, single-chain antibodies, nanobodies, monomers, single-chain variable fragments (scFv), Fab fragments, Fab' fragments, F(ab')2 fragments and F(ab)2 fragments. Preferred fragments of antibodies contain at least a heavy chain variable domain (VH) and / or a light chain variable domain (VL). More preferred fragments contain at least a Fab fragment. The Fab' fragment of the enhanced anti-FH antibody FH.07 competes with the antibody of the present invention for binding to the same epitope, and has been shown to retain the ability to enhance FH function. Therefore, particularly preferred fragments of the antibodies of the present invention are Fab fragments, Fab' fragments, F(ab')2 fragments and F(ab)2 fragments of the antibodies of the present invention. In another embodiment, the fragments of the antibodies of the present invention include immunoglobulin heavy chain variable regions, immunoglobulin heavy chain constant regions, immunoglobulin light chain variable regions and immunoglobulin light chain constant regions.

[0072] As used herein, the terms "specific for" and "specifically binds" or "capable of specifically binding" refer to the non-covalent interaction between an antibody and its epitope. This indicates that the antibody or fragment preferentially binds to the epitope relative to 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.

[0073] 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 along the entire length of the amino acid sequence or nucleic acid sequence that are identical to the residues in a reference 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, for example "Align 2".

[0074] In the amino acid sequences described 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.

[0075] Preferred antibodies and fragments according to the present invention are capable of enhancing the activity of FH, preferably the activity of human FH. The term "enhancing FH activity" means that if the antibody or fragment according to the present invention binds to FH, the activity of FH increases. The activity of FH enhanced by the antibodies and fragments of the present invention is preferably to inhibit alternative complement activation, preferably in an individual. As used herein, the term "alternative complement activation" refers to the activation of the complement system via the alternative pathway, i.e., at least involving the formation of the C3 convertase of the alternative pathway, i.e., C3bBb / C3bBbP, or involving an increase in the formation of such a C3 convertase. Alternative complement activation may further involve the cleavage of C3 into C3a and C3b by the alternative pathway C3 convertase, the formation of the alternative pathway C5 convertase, i.e., C3bBbC3b / C3bBbC3b, and / or the cleavage of C5 and the subsequent combination of C6, C7, C8 and C9 to form a MAC. Alternative complement activation may further include an increase in the alternative pathway amplification loop. The alternative complement activation is preferably inhibited in the individual, preferably in a body fluid of the individual, preferably in the blood, interstitial fluid or cerebrospinal fluid, more preferably in the blood. As used herein, a "subject" is a human or animal that comprises a complement system as part of its immune system. Preferably, the individual is a mammal, more preferably a human.

[0076] As used herein, "inhibition of alternative complement activation" includes any change in the amount or activity of a component, factor or activity of the alternative complement system that causes or results from its inhibition. Inhibition of alternative complement activation includes, for example, inhibition of hemolytic activity, inhibition of complement component 3 (C3) deposition on cells of the individual, increased binding of FH to C3b, iC3b and / or C3d, inhibition of formation of 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 alternative pathway C3 convertase, increased binding of fH to host cells ( In particular, it binds to sialic acid, glycosaminoglycans and / or heparin expressed on host cells, inhibits the amplification loop of the alternative complement pathway, and inhibits the formation of the alternative complement pathway C5 convertase C3bBbC3bP / C3bBbC3bP, inhibits the cleavage of C5 into C5a and C5b by the alternative pathway C5 convertase, increases the decay accelerating activity of FH (i.e., once the alternative pathway C3 convertase is formed, it promotes its dissociation), and / or increases the activity of the FI cofactor, resulting in the degradation of C3b. The inhibition of alternative complement activation by FH enhanced by the anti-FH antibodies and fragments of the present invention preferably includes inhibiting hemolytic activity, inhibiting the deposition of C3 on cells of the individual, and / or increasing the binding of FH to C3b, iC3b and / or C3d.

[0077] As used herein, "inhibit" preferably means that the indicated activity is reduced by at least about 25%, more preferably at least about 50%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95%. Thus, "inhibiting alternative complement activation" preferably means that the activity of the alternative complement pathway is reduced by at least about 25%, more preferably at least about 50%, 75%, 80%, 85%, 90% or 95%. Similarly, "inhibiting hemolytic activity" preferably means that the hemolytic activity is reduced by at least about 25%, more preferably at least about 50%, 75%, 80%, 85%, 90% or 95%.

[0078] As used herein, "increase" preferably means that the indicated activity is increased by at least about 25%, more preferably at least about 50%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95%. Thus, "increase the binding of FH to C3b" preferably means that the binding of FH to C3b is increased by at least about 25%, more preferably at least about 50%, 75%, 80%, 85%, 90% or 95%. Similarly, "increase the binding of FH to host cells" preferably means that the binding of FH to host cells is increased by at least about 25%, more preferably at least about 50%, 75%, 80%, 85%, 90% or 95%.

[0079] As used herein, "hemolytic activity" refers to the rupture of red blood cells and the subsequent release of red blood cell contents into, for example, the circulation, induced by the activation of the body system, preferably due to the formation of MAC on the cell surface. For example, as described in the Examples herein, hemolytic activity is measured using a hemolytic assay as described by Sanchez-Corral et al. (2004) and Wouters et al. (2008), optionally with some modifications. In this assay, red blood cells such as sheep red blood cells (SRBC) are incubated with serum (such as human serum), for example, at 37°C with shaking for 1.25 hours. Serum in which low FH levels or FH dysfunction lead to SRBC lysis can be stopped by adding veronal buffer containing 20mM EDTA and then centrifuged 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 can be, for example, from a healthy human individual or a human individual suffering from a disorder associated with undesirable or excessive activation of the alternative complement pathway (e.g., aHUS). The ability of an antibody or fragment to inhibit hemolytic activity can be determined by incubating erythrocytes with serum in the presence of the antibody or fragment.

[0080] For example, inhibition of C3 deposition is measured using a C3 deposition assay as described herein in the Examples. The assay involves coating a microtiter plate with LPS. The plate is then incubated with serum (e.g., serum from healthy individuals, or serum from individuals suffering from a disorder associated with undesirable or excessive surrogate entity activation) as described above in the presence or absence of an antibody or fragment. C3 deposition on LPS can be detected using an anti-C3 antibody.

[0081] For example, an ELISA as described in the Examples herein is used to measure the increase in the binding of FH to C3b. The assay comprises coating a microtiter ELISA plate with C3b and incubating the plate with serum (e.g., serum from a healthy individual or from an individual suffering from a condition associated with the above-mentioned undesirable or excessive alternative complement activation). The bound FH can be detected with an anti-FH antibody, such as a peroxidase-labeled polyclonal anti-FH. The ability of an 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 prior to incubation with the coated C3b. As another example, the binding of FH to C3b is determined using surface plasmon resonance (SPR), such 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 immobilized on the sensor surface, while the other (e.g., FH) is free in solution and, for example, passes over the surface in the presence or absence (of varying concentrations) of an antibody or fragment of the invention. Preferred antibodies or fragments thereof according to the present invention can increase the binding affinity (K) of FH to C3b in vitro. D ) to a maximum of 2 μM, more preferably to a maximum of 1.95 μM, more preferably to a maximum of 1.8 μM, more preferably to a maximum of 1.7 μM and / or the binding affinity of FH for C3b in vitro is increased by at least 3-fold, more preferably at least 3.1-fold, more preferably at least 3.2-fold, more preferably at least 3.3-fold, more preferably at least 3.5-fold, more preferably at least 3.6-fold.

[0082] The preferred antibodies or fragments thereof provided by the present invention have low in vitro IC in one or more functional assays. 50 value, preferably, for the same functional assay, its in vitro IC 50 The value is lower than the in vitro IC of antibody FH.07 50 Value. 50 " is a term well known in the art and refers to the concentration of an antibody or fragment required to inhibit or reduce a certain functional activity by 50%. The IC 50 The lower the value, the stronger the inhibitory activity of the antibody or fragment, and the greater its potential as a therapeutic agent. The functional assay is preferably a C3b deposition assay and / or a hemolysis assay as described above. In a preferred aspect, the antibody or fragment thereof according to the present invention has an IC of 38 nM or less, more preferably 35 nM or less, more preferably 32 nM or less, more preferably 30 nM or less, more preferably 28 nM or less, more preferably 27 nM or less in vitro. 50In a further preferred embodiment, the antibody or fragment thereof according to the present invention inhibits the deposition of C3 on LPS with an IC value of 150 nM or less, more preferably 130 nM or less, more preferably 115 nM or less, more preferably 105 nM or less, more preferably 100 nM or less, more preferably 95 nM or less in vitro. 50 Inhibitory hemolytic activity.

[0083] Further preferred antibodies or fragments of the present invention are capable of performing in vitro with a low IC 50 The values ​​inhibited the deposition of C3 on LPS and in vitro with low IC 50 Therefore, the preferred antibodies or fragments according to the present invention inhibit hemolytic activity in vitro with an IC value of 38 nM or lower. 50 The values ​​inhibited the deposition of C3 on LPS and had an IC of 150 nM or lower in vitro 50 The hemolytic activity is inhibited by an IC value of 30 nM or less in vitro. 50 The values ​​inhibited the deposition of C3 on LPS and had an IC of 150 nM or lower in vitro 50 The hemolytic activity is inhibited by an IC value of 27 nM or less in vitro. 50 The values ​​inhibited the deposition of C3 on LPS and had an IC of 100 nM or lower in vitro 50 The IC of C3 deposition on LPS in vitro is preferably determined in a C3 deposition assay as described above. 50 The IC value of hemolytic activity is preferably determined in a hemolytic activity assay as described above. 50 value.

[0084] "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 intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (antibody and antigen in this application). Binding affinity is herein expressed by the equilibrium dissociation constant (K D ) indicates that k a With k dSee, 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 the IBIS-iSPR instrument at IBIS Technologies BV (Hengelo, the Netherlands), or solution phase assays, such as Kinexa.

[0085] The preferred antibodies or fragments thereof provided by the present invention have high binding affinity for FH and / or FH fragments comprising the domain CCP18-20, and more preferably the binding affinity is higher than the binding affinity of antibody FH.07. The in vivo therapeutic activity of antibodies or fragments generally requires high binding affinity, for example, minimizing the binding of the antibody or fragment to binding sites and / or antigens other than its specific epitope or antigen, and minimizing the amount of antibody or fragment that needs to be administered in vivo. Therefore, antibodies with high binding affinity are preferred. The dissociation constant (K) of the binding affinity of the antibody or fragment to FH is preferably D ) is 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 M or less. Therefore, in one embodiment, the present invention 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 K D 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 M or lower. Preferably, the K of the binding affinity of the antibody or fragment according to the present invention to FH is D 2.25×10 -8 M or less, more preferably 2×10 -8 M or less, more preferably 1.75×10 -8 M or less, more preferably 1.5×10 -8 M or less, more preferably 1.25×10 -8 M or less, more preferably 1×10 -8 M or less, more preferably 0.9×10 -8 M or less, more preferably 0.8×10 -8 M or less, more preferably 0.7×10 -8M or less, more preferably 0.6×10 -8 M or lower, and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.09×10 -9 M or less, more preferably 0.08×10 -9 M or less, more preferably 0.07×10 -9 M or less, more preferably 0.06×10 -9 M or less, more preferably 0.05×10 -9 M or less, more preferably 0.04×10 -9 M or less, more preferably 0.03×10 -9 M or less, more preferably 0.02×10 -9 M or less, more preferably 1×10 -11 M or less, more preferably 0.9×10 - 11 M or less, more preferably 0.8×10 -11 M or less, more preferably 0.7×10 -11 M or less, more preferably 0.6×10 -11 In a preferred aspect, the antibody or fragment thereof preferably has a K of 1.5 or less for its binding affinity to FH. D 1.25×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.04×10 -9 In a further preferred aspect, the antibody or fragment thereof preferably has a K of 1.5 or less for its binding affinity to FH. D 0.6×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.6×10 -11 The binding affinity is preferably determined using surface plasmon resonance (SPR), more preferably using SPR in an assay as described herein, i.e. in an assay, the binding affinity is determined by SPR on a ProtA chip, the antibody or fragment being captured by the ProtA chip before full-length FH (for binding affinity to FH) or a FH fragment consisting of domains 18-20 (for binding affinity to CCP18-20) is passed over the surface.

[0086] Further preferred antibodies or fragments according to the present invention have high binding affinity and low IC 50 values ​​inhibited C3 deposition on LPS and / or in vitro with low IC 50Therefore, the K value of the binding affinity of the preferred antibodies or fragments according to the present invention to FH is D 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 M or lower, and in vitro with an IC of 38 nM or lower 50 values ​​inhibited C3 deposition on LPS and / or in vitro with an IC of 150 nM or less 50 More preferably, the K value of the antibody or fragment for FH binding affinity is D 1.25×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.04×10 -9 M or lower, and in vitro with an IC of 30 nM or lower 50 The values ​​inhibited the deposition of C3 on LPS and in vitro with an IC of 115 nM or lower 50 Even more preferably, the K of the binding affinity of the antibody or fragment to FH is D 1.25×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.04×10 -9 M or lower, and in vitro with an IC of 27 nM or lower 50 The values ​​inhibited the deposition of C3 on LPS and in vitro with an IC of 100 nM or lower 50 The binding affinity is preferably determined by SPR as described herein. The IC of C3 deposition on LPS in vitro 50 The value is preferably determined in a C3 deposition assay as described above. IC of hemolytic activity 50 The values ​​are preferably determined in a hemolytic activity assay as described above.

[0087] A particularly preferred antibody according to the present invention is the antibody anti-FHR-1.8E4, the preparation and identification of which is described in the examples. Table 1 provides an overview of the variable heavy and light chain sequences and the individual CDR sequences of the antibody anti-FHR-1.8E4. Even more preferred are monoclonal chimeric or humanized antibodies or fragments thereof comprising the heavy chain CDR sequences and light chain CDR sequences of the antibody anti-FHR-1.8E4 or the heavy chain variable regions and light chain variable regions of the antibody anti-FHR-1.8E4. The term "anti-FHR-1.8E4" as used herein encompasses all antibodies and fragments having at least the heavy and light chain CDR1, CDR2 and CDR3 regions shown in Table 1, such as, for example, isolated and / or purified antibodies or recombinantly produced antibodies. The antibody anti-FHR-1.8E4 competes with the antibody FH.07 for binding to the same epitope in CCP18 of FH. The antibody anti-FHR-1.8E4 has a low IC of 26.3 nM for inhibiting C3 deposition on LPS in vitro. 50 value and a low IC value of 94.0 nM for in vitro hemolytic inhibition 50 The antibody anti-FHR-1.8E4 further has a high binding affinity for full-length FH, K D 1.04×10 -8 , and has high binding affinity to the FH fragment composed of CCP18-20, K D 3.13×10 -11 The antibody anti-FHR-1.8E4 further confirmed the in vitro binding affinity of FH for C3b (K D ) increased from 6 μM to 1.66 μM, a 3.6-fold increase.

[0088] Another particularly preferred antibody according to the present invention is the antibody anti-FHR-1.3B4, the preparation and identification of which are described in the examples. Table 1 provides an overview of the variable heavy and light chain sequences and the individual CDR sequences of the antibody anti-FHR-1.3B4. Even more preferred are monoclonal chimeric or humanized antibodies or fragments thereof comprising the heavy chain CDR sequences and light chain CDR sequences of the antibody anti-FHR-1.3B4 or the heavy chain variable regions and light chain variable regions of the antibody anti-FHR-1.3B4. The term "anti-FHR-1.3B4" as used herein encompasses all antibodies and fragments having at least the heavy and light chain CDR1, CDR2 and CDR3 regions as shown in Table 1, such as, for example, isolated and / or purified antibodies or recombinantly produced antibodies. The antibody anti-FHR-1.3B4 competes with the antibody FH.07 for binding to the same epitope in CCP18 of FH. The antibody anti-FHR-1.3B4 has a low IC of 38.41 nM for inhibiting C3 deposition on LPS in vitro. 50 value and a low IC value of 127.5 nM for in vitro hemolytic inhibition 50The antibody anti-FHR-1.3B4 further has a high binding affinity for full-length FH, K D 0.58×10 -8 , and has high binding affinity to the FH fragment composed of CCP18-20, K D 5.44×10 -12 The antibody anti-FHR-1.3B4 further confirmed the in vitro binding affinity of FH to C3b (K D ) increased from 6 μM to 1.9 μM, i.e. 3.2 times.

[0089] Another particularly preferred antibody according to the present invention is the antibody anti-FHR-1.11E1, the preparation and identification of which are described in the examples. Table 1 provides an overview of the variable heavy and light chain sequences and individual CDR sequences of the antibody anti-FHR-1.11E1. Even more preferred are monoclonal chimeric or humanized antibodies or fragments thereof comprising the heavy chain CDR sequences and light chain CDR sequences of the antibody anti-FHR-1.11E1 or the heavy chain variable regions and light chain variable regions of the antibody anti-FHR-1.11E1. The term "anti-FHR-1.11E1" as used herein encompasses all antibodies and fragments having at least the heavy and light chain CDR1, CDR2 and CDR3 regions as shown in Table 1, such as, for example, isolated and / or purified antibodies or recombinantly produced antibodies. The antibody anti-FHR-1.11E1 competes with the antibody FH.07 for binding to the same epitope in CCP18 of FH. The antibody anti-FHR-1.11E1 has a low IC of 25.04 nM for inhibiting C3 deposition on LPS in vitro 50 value.

[0090] Another particularly preferred antibody according to the present invention is the antibody anti-FHR-1.12F4, the preparation and identification of which are described in the examples. Table 1 provides an overview of the variable heavy and light chain sequences and individual CDR sequences of the antibody anti-FHR-1.12F4. Even more preferred are monoclonal chimeric or humanized antibodies or fragments thereof comprising the heavy chain CDR sequences and light chain CDR sequences of the antibody anti-FHR-1.12F4 or the heavy chain variable regions and light chain variable regions of the antibody anti-FHR-1.12F4. The term "anti-FHR-1.12F4" as used herein encompasses all antibodies and fragments having at least the heavy and light chain CDR1, CDR2 and CDR3 regions as shown in Table 1, such as, for example, isolated and / or purified antibodies or recombinantly produced antibodies. The antibody anti-FHR-1.12F4 competes with the antibody FH.07 for binding to the same epitope in CCP18 of FH. The antibody anti-FHR-1.12F4 has a low IC of 345.2 nM for inhibiting C3 deposition on LPS in vitro 50 value.

[0091] Thus, there is provided 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:

[0092] - a light chain CDR1 sequence having the sequence SSVXY, wherein X is R, T or N (SEQ ID NO: 91) or the sequence QSLVHSNGNTY (SEQ ID NO: 49),

[0093] - a light chain CDR2 sequence having the sequence X1X2S, wherein X1=A, K or Y and X2=T or L (SEQ ID NO: 92),

[0094] - a light chain CDR3 having a sequence selected from QQWGTKPPT (SEQ ID NO: 19), QQRSSSNPLT (SEQ ID NO: 35), SQSTHVPFT (SEQ ID NO: 51) and QQFTSSPLT (SEQ ID NO: 67),

[0095] - a heavy chain CDR1 having the sequence X1FSLTX2X3G, wherein X1 = D or G, X2 = N or S and X3 = S or Y (SEQ ID NO: 93),

[0096] - a heavy chain CDR2 having the sequence IWSGGXT, wherein x = T, N or S (SEQ ID NO: 94), and

[0097] - A heavy chain CDR3 sequence having the sequence ARNX1GNYX2X3DY, wherein X1 = F or G, X2 = A or Y and X3 = V, M or F (SEQ ID NO: 95) or AKNGDYGYTMDY (SEQ ID NO: 55).

[0098] In a preferred embodiment, the antibody comprises:

[0099] - a light chain CDR1 sequence having the sequence SSVXY, wherein X is R or T (SEQ ID NO: 96),

[0100] - a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 97),

[0101] - a light chain CDR3 having a sequence selected from QQWGTKPPT (SEQ ID NO: 19) and QQRSSSNPLT (SEQ ID NO: 35),

[0102] - a heavy chain CDR1 having the sequence X1FSLTNX2G, wherein X1 = D or G and X2 = S or Y (SEQ ID NO: 98),

[0103] - a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 99), and

[0104] - A heavy chain CDR3 sequence having the sequence ARNFGNYAXDY, wherein X=V or M (SEQ ID NO: 100).

[0105] The antibody or fragment preferably enhances FH activity, preferably 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. The fragment preferably comprises at least a heavy chain variable domain (VH) and / or a light chain variable domain (VL). A more preferred fragment comprises at least a Fab fragment.

[0106] The antibody or fragment preferably has a binding affinity for FH of D 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 M or less, preferably wherein the K of the binding affinity of the antibody or fragment to FH is D 1.25×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.04×10 -9 M or lower.

[0107] In a further preferred embodiment, the present invention provides an isolated, synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 18) and a light chain CDR3 having the sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 having the sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 22) and a heavy chain CDR3 sequence having the sequence ARNFGNYAVDY (SEQ ID NO: 23). In one embodiment, the antibody comprises: a variable light chain sequence comprising a sequence having at least 80% sequence identity to the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVRYMHWYQQKAGSSPTAWIFATSNLASGVPPRFSGSGSGTSYSLTISRVEAEDAATYYCQQWGTKPPTFGAGTKLELK (SEQ ID NO:20), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLKQSGPGLVQPSQSLSITCTVSDFSLTNSGVHWVRQSPGKGLEWLGVIWSGGTTEYNAAFMSRLTITKDNSKSQVFFKMNSLLVDDTGIYYCARNFGNYAVDYWGQGTSVTVSS (SEQ ID NO:24). In a further embodiment, the antibody or fragment comprises: a variable light chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 20, and a variable heavy chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 24. In a specific embodiment, the antibody or fragment comprises a variable light chain sequence comprising the sequence SEQ ID NO: 20 and a variable light chain sequence comprising the sequence SEQ ID NO: 24. The antibody or fragment preferably enhances FH activity, preferably 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.The fragment preferably comprises at least the heavy chain variable domain (VH) and / or the light chain variable domain (VL). More preferably, the fragment comprises at least a Fab fragment.

[0108] The antibody or fragment comprising a light chain CDR1 sequence having the sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 18), and a light chain CDR3 having the sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 having the sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 22), and a heavy chain CDR3 sequence having the sequence ARNFGNYAVDY (SEQ ID NO: 23), preferably with a K of binding affinity to FH. D 2.5×10 -8 M or lower and / or a binding affinity K for the FH fragment consisting of CCP18-20 D 0.1×10 -9 Preferably, the K of the binding affinity of the antibody or fragment to FH is D 2.25×10 -8 M or less, more preferably 2×10 - 8 M or less, more preferably 1.75×10 -8 M or less, more preferably 1.5×10 -8 M or less, more preferably 1.25×10 -8 M or lower, and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.09×10 -9 M or less, more preferably 0.08×10 -9 M or less, more preferably 0.07×10 -9 M or less, more preferably 0.06×10 -9 M or less, more preferably 0.05×10 -9 M or less, more preferably 0.04×10 -9 M or lower.

[0109] The antibody or fragment comprising a light chain CDR1 sequence having the sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 18), and a light chain CDR3 having the sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 having the sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 22), and a heavy chain CDR3 sequence having the sequence ARNFGNYAVDY (SEQ ID NO: 23), preferably in vitro with an IC of 38 nM or less 50 Preferably, the antibody or fragment inhibits the deposition of C3 on LPS with an IC value of 35 nM or less, more preferably 32 nM or less, more preferably 30 nM or less, more preferably 28 nM or less, more preferably 27 nM or less in vitro. 50 The value inhibited the deposition of C3 on LPS.

[0110] The antibody or fragment comprising a light chain CDR1 sequence having the sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 18), and a light chain CDR3 having the sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 having the sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 22), and a heavy chain CDR3 sequence having the sequence ARNFGNYAVDY (SEQ ID NO: 23), preferably in vitro with an IC of 150 nM or less 50 Preferably, the antibody or fragment inhibits hemolytic activity in vitro with an IC value of 115 nM or lower, more preferably 105 nM or lower. 50 Inhibitory hemolytic activity.

[0111] The antibody or fragment comprising a light chain CDR1 sequence having the sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 18), and a light chain CDR3 having the sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 having the sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 22), and a heavy chain CDR3 sequence having the sequence ARNFGNYAVDY (SEQ ID NO: 23), preferably in vitro to enhance the binding affinity (K) of FH to C3b D) to a maximum of 2 μM, more preferably 1.8 μM and / or increase the binding affinity of FH for C3b in vitro by at least 3-fold, more preferably 3.5-fold.

[0112] In a further preferred embodiment, the present invention provides an isolated, synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 33), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 34) and a light chain CDR3 having the sequence QQRSSSNPLT (SEQ ID NO: 35), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 37), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 38) and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 39). In one embodiment, the antibody comprises: a variable light chain sequence comprising a sequence having at least 80% sequence identity to the sequence QIVLSQSPTILSASPGEKVTMTCRASSSVTYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQRSSSNPLTFGAGTKLELK (SEQ ID NO:36), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLRQSGPGLVQPSQSLSITCTVSGFSLTNYGVYWVRQSPGKGLEWLGVIWSGGTTDYSAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARNFGNYAMDYWGQGTSVTVSS (SEQ ID NO:40). In a further embodiment, the antibody or fragment comprises: a variable light chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 36, and a variable heavy chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 40. In a specific embodiment, the antibody or fragment comprises a variable light chain sequence comprising the sequence SEQ ID NO: 36 and a variable light chain sequence comprising the sequence SEQ ID NO: 40. The antibody or fragment preferably enhances FH activity, preferably 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.The fragment preferably comprises at least the heavy chain variable domain (VH) and / or the light chain variable domain (VL). More preferably, the fragment comprises at least a Fab fragment.

[0113] The antibody or fragment comprising a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 33), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 34), a light chain CDR3 having the sequence QQRSSSNPLT (SEQ ID NO: 35), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 37), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 38), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 39), preferably with a K of 1.5 for binding affinity to FH. D 2.5×10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1×10 -9 Preferably, the K of the binding affinity of the antibody or fragment to FH is D 2.25×10 -8 M or less, more preferably 2×10 -8 M or less, more preferably 1.75×10 -8 M or less, more preferably 1.5×10 -8 M or less, more preferably 1.25×10 - 8 M or less, more preferably 1×10 -8 M or less, more preferably 0.9×10 -8 M or less, more preferably 0.8×10 -8 M or less, more preferably 0.7×10 -8 M or less, more preferably 0.6×10 -8 M or lower, and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.09×10 -9 M or less, more preferably 0.08×10 -9 M or less, more preferably 0.07×10 -9 M or less, more preferably 0.06×10 -9 M or less, more preferably 0.05×10 -9 M or less, more preferably 0.04×10 -9 M or less, more preferably 0.03×10 -9 M or less, more preferably 0.02×10 -9 M or less, more preferably 1×10-11 M or less, more preferably 0.9×10 -11 M or less, more preferably 0.8×10 -11 M or less, more preferably 0.7×10 -11 M or less, more preferably 0.6×10 -11 M or lower.

[0114] The antibody or fragment comprising a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 33), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 34), a light chain CDR3 having the sequence QQRSSSNPLT (SEQ ID NO: 35), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 37), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 38), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 39), preferably in vitro with an IC of 150 nM or less 50 Preferably, the antibody or fragment inhibits hemolytic activity in vitro with an IC value of 105 nM or lower. 50 Inhibitory hemolytic activity.

[0115] The antibody or fragment comprising a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO: 33), a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 34), a light chain CDR3 having the sequence QQRSSSNPLT (SEQ ID NO: 35), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 37), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 38), and a heavy chain CDR3 sequence having the sequence ARNFGNYAMDY (SEQ ID NO: 39), preferably increases the binding affinity (K) of FH to C3b in vitro. D ) to a maximum of 2 μM, more preferably 1.95 μM, and / or increase the binding affinity of FH for C3b in vitro by at least 3-fold, more preferably 3.1-fold.

[0116] In a further preferred embodiment, the present invention provides an isolated, synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence QSLVHSNGNTY (SEQ ID NO: 49), a light chain CDR2 sequence having the sequence KLS (SEQ ID NO: 50) and a light chain CDR3 having the sequence SQSTHVPFT (SEQ ID NO: 51), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 53), a heavy chain CDR2 having the sequence IWSGGNT (SEQ ID NO: 54) and a heavy chain CDR3 sequence having the sequence AKNGDYGYTMDY (SEQ ID NO: 55). In one embodiment, the antibody comprises: a variable light chain sequence comprising a sequence having at least 80% sequence identity to the sequence DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIK (SEQ ID NO:52), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQPPGKGLEWLGVIWSGGNTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCAKNGDYGYTMDYWGQGTSVTVSS (SEQ ID NO:56). In a further embodiment, the antibody or fragment comprises: a variable light chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 52, and a variable heavy chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 56. In a specific embodiment, the antibody or fragment comprises a variable light chain sequence comprising the sequence SEQ ID NO: 52 and a variable light chain sequence comprising the sequence SEQ ID NO: 56. The antibody or fragment preferably enhances FH activity, preferably 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.The fragment preferably comprises at least the heavy chain variable domain (VH) and / or the light chain variable domain (VL). More preferably, the fragment comprises at least a Fab fragment.

[0117] In a further preferred embodiment, the present invention provides an isolated, synthetic or recombinant antibody or an antigen-binding fragment thereof, which specifically binds to CCP18 of FH, comprising a light chain CDR1 sequence having the sequence SSVNY (SEQ ID NO: 65), a light chain CDR2 sequence having the sequence YTS (SEQ ID NO: 66) and a light chain CDR3 having the sequence of QQFTSSPLT (SEQ ID NO: 67), a heavy chain CDR1 having the sequence GFSLTSYG (SEQ ID NO: 69), a heavy chain CDR2 having the sequence IWSGGST (SEQID NO: 70) and a heavy chain CDR3 sequence having the sequence ARNGGNYYFDY (SEQ ID NO: 71). In one embodiment, the antibody comprises: a variable light chain sequence comprising a sequence having at least 80% sequence identity to the sequence ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASKLSWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGAGTKLELK (SEQ ID NO:68), and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to the sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARNGGNYYFDYWGQGTTLTVSS (SEQ ID NO:72). In a further embodiment, the antibody or fragment comprises a variable light chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 68, and a variable heavy chain sequence comprising a sequence having at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity with the sequence SEQ ID NO: 72. In a specific embodiment, the antibody or fragment comprises a variable light chain sequence comprising the sequence SEQ ID NO: 68 and a variable light chain sequence comprising the sequence SEQ ID NO: 72. The antibody or fragment preferably enhances FH activity, preferably 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.The fragment preferably comprises at least the heavy chain variable domain (VH) and / or the light chain variable domain (VL). More preferably, the fragment comprises at least a Fab fragment.

[0118] Optionally, the sequence of at least one of the CDRs is optimized, thereby generating a variant antibody or fragment, for example to (further) improve binding affinity, selectivity, FH enhancing ability and / or in vivo or storage stability. In a preferred embodiment, the antibody or fragment according to the invention has high storage stability, in particular improved storage stability compared to antibody FH.07. In a further preferred embodiment, the antibody or fragment according to the invention has high in vivo stability, in particular improved in vivo stability compared to antibody FH.07. In a further preferred embodiment, the antibody or fragment according to the invention has high selectivity, in particular improved selectivity compared to antibody FH.07.

[0119] In addition, at least one sequence in at least one framework region of the antibody or fragment of the present invention is optionally preferred, for example, to improve the binding efficacy or stability of the antibody or fragment, or to reduce the side effects after the non-human sequence is applied to the human body. For example, this is accomplished by a mutagenesis procedure. Those skilled in the art can produce antibody variants comprising at least one altered CDR or framework sequence. For example, the CDR and / or framework sequence are optimized by mutations of nucleic acid molecules encoding such framework sequences. For example, conservative amino acid substitutions are applied. Examples of conservative amino acid substitutions include a hydrophobic residue such as isoleucine, valine, leucine or methionine replacing another hydrophobic residue, and a polar residue replacing another polar residue, such as arginine replacing lysine, glutamic acid replacing aspartic acid or glutamine replacing asparagine. In order to select improved antibodies or fragments, it is preferred to test the binding affinity, FH enhancing ability and / or stability of the obtained variant antibodies or fragments, for example, using tests described herein. Once an antibody or fragment specific for FH (particularly CCP18 specific for FH) is obtained, its desired biological activity, i.e., their ability to enhance FH activity, can be tested by a variety of methods known to those skilled in the art. As previously described herein, the activity of enhancing FH preferably encompasses inhibition of hemolytic activity, inhibition of deposition of C3 on cells and / or increased binding of FH to C3b. Functional assays for testing these activities have been described previously herein and are described in detail in the Examples. Typically, up to three amino acid residues of a CDR sequence may vary, depending on the number of amino acids that make up the CDR, while maintaining the same specificity. Therefore, the antibody or fragment according to the present invention preferably contains heavy and light chain CDR1, CDR2 and CDR3 sequences, wherein at most 3, preferably at most 2, more preferably at most 1 amino acid of each CDR is changed compared to the heavy and light chain CDR1, CDR2 and CDR3 sequences of Table 1. Further preferably, the antibody or fragment comprises the light chain CDR3 and heavy chain CDR3 of the same antibody as shown in Table 1, the light chain CDR2 of the antibody in which at most 1 amino acid is changed, and the light chain CDR1, heavy chain CDR1 and heavy chain CDR2 in which at most 3 amino acids are changed. More preferably, the antibody or fragment comprises the light chain CDR2 and CDR3 and the heavy chain CDR3 of the same antibody as shown in Table 1, and the light chain CDR1, heavy chain CDR1 and heavy chain CDR2 in which at most 2 amino acids (more preferably at most 1 amino acid) are changed.

[0120] Therefore, the present invention further 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), comprising:

[0121] - a light chain CDR1 sequence having a sequence at least 80% identical to the sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence having a sequence ATS (SEQ ID NO: 18) and a light chain CDR3 having a sequence at least 80% identical to the sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 having a sequence at least 80% identical to the sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 having a sequence at least 80% identical to the sequence IWSGGTT (SEQ ID NO: 22), and a heavy chain CDR3 sequence having a sequence at least 80% identical to the sequence ARNFGNYAVDY (SEQ ID NO: 23),

[0122] - a light chain CDR1 sequence having a sequence at least 80% identical to the sequence SSVTY (SEQ ID NO:33), a light chain CDR2 sequence having a sequence ATS (SEQ ID NO:34) and a light chain CDR3 having a sequence at least 80% identical to the sequence QQRSSSNPLT (SEQ ID NO:35), a heavy chain CDR1 having a sequence at least 80% identical to the sequence GFSLTNYG (SEQ ID NO:37), a heavy chain CDR2 having a sequence at least 80% identical to the sequence IWSGGTT (SEQ ID NO:38), and a heavy chain CDR3 sequence having a sequence at least 80% identical to the sequence ARNFGNYAMDY (SEQ ID NO:39),

[0123] - a light chain CDR1 sequence having a sequence at least 80% identical to the sequence QSLVHSNGNTY (SEQ ID NO:49), a light chain CDR2 sequence having the sequence KLS (SEQ ID NO:50) and a light chain CDR3 having a sequence at least 80% identical to the sequence SQSTHVPFT (SEQ ID NO:51), a heavy chain CDR1 having a sequence at least 80% identical to the sequence GFSLTNYG (SEQ ID NO:53), a heavy chain CDR2 having a sequence at least 80% identical to the sequence IWSGGNT (SEQ ID NO:54), and a heavy chain CDR3 sequence having a sequence at least 80% identical to the sequence AKNGDYGYTMDY (SEQ ID NO:55),

[0124] - A light chain CDR1 sequence having a sequence at least 80% identical to the sequence SSVNY (SEQ ID NO:65), a light chain CDR2 sequence having a sequence YTS (SEQ ID NO:66) and a light chain CDR3 having a sequence at least 80% identical to the sequence QQFTSSPLT (SEQ ID NO:67), a heavy chain CDR1 having a sequence at least 80% identical to the sequence GFSLTSYG (SEQ ID NO:69), a heavy chain CDR2 having a sequence at least 80% identical to the sequence IWSGGST (SEQ ID NO:70), and a heavy chain CDR3 sequence having a sequence at least 80% identical to the sequence ARNGGNYYFDY (SEQ ID NO:71).

[0125] The antibody preferably enhances FH activity. Preferably, 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, which has at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity with the indicated sequence.

[0126] The present invention further 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), comprising:

[0127] a light chain CDR1 sequence having the sequence SSVRY (SEQ ID NO: 17), optionally with 1 amino acid substitution, a light chain CDR2 sequence having the sequence ATS (SEQ ID NO: 18), and a light chain CDR3 having the sequence QQWGTKPPT (SEQ ID NO: 19), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR1 having the sequence DFSLTNSG (SEQ ID NO: 21), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO: 22), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), and a heavy chain CDR3 sequence ARNFGNYAVDY (SEQ ID NO: 23), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution),

[0128] a light chain CDR1 sequence having the sequence SSVTY (SEQ ID NO:33) optionally with 1 amino acid substitution, a light chain CDR2 sequence having the sequence ATS (SEQ ID NO:34) and a light chain CDR3 having the sequence QQRSSSNPLT (SEQ ID NO:35) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO:37) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR2 having the sequence IWSGGTT (SEQ ID NO:38) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), and a heavy chain CDR3 sequence ARNFGNYAMDY (SEQ ID NO:39) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution),

[0129] a light chain CDR1 sequence having the sequence QSLVHSNGNTY (SEQ ID NO: 49), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a light chain CDR2 sequence KLS (SEQ ID NO: 50), and a light chain CDR3 having the sequence SQSTHVPFT (SEQ ID NO: 51), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR1 having the sequence GFSLTNYG (SEQ ID NO: 53), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR2 having the sequence IWSGGNT (SEQ ID NO: 54), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), and a heavy chain CDR3 sequence AKNGDYGYTMDY (SEQ ID NO: 55), optionally with 2 amino acid substitutions (preferably 1 amino acid substitution),

[0130] - a light chain CDR1 sequence having the sequence SSVNY (SEQ ID NO:65) optionally with 1 amino acid substitution, a light chain CDR2 sequence having the sequence YTS (SEQ ID NO:66) and a light chain CDR3 having the sequence QQFTSSPLT (SEQ ID NO:67) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR1 having the sequence GFSLTSYG (SEQ ID NO:69) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), a heavy chain CDR2 having the sequence IWSGGST (SEQ ID NO:70) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution), and a heavy chain CDR3 sequence having the sequence ARNGGNYYFDY (SEQ ID NO:71) optionally with 2 amino acid substitutions (preferably 1 amino acid substitution).

[0131] The antibody or fragment thereof according to the present invention is preferably a monoclonal antibody or fragment. A monoclonal antibody is an antibody consisting essentially of a single molecular species. A monoclonal antibody is 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. Monoclonal antibodies can advantageously be recombinantly produced so that the amount of antibody 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. The antibody or fragment according to the present invention is further preferably a chimeric or humanized antibody. Therefore, the antibody or fragment preferably comprises at least a human light chain and a heavy chain constant region. More preferably, the antibody or fragment also comprises a human framework region in the heavy chain and light chain variable regions. Further preferred are human antibodies or fragments consisting entirely of human sequences. The use of chimeric, humanized or human antibodies is superior to the use of non-human antibodies because many factors hinder the use of non-human antibodies or fragments to treat human diseases. The human body may recognize non-human antibodies as foreign matter, which will produce an immune response to the non-human antibodies or fragments, thereby causing adverse side effects and / or the antibodies or fragments to be quickly removed from the circulation. When chimeric, humanized or human antibodies are applied to humans, the chance of side effects is reduced. In addition, when chimeric, humanized or human antibodies are used, a longer half-life is usually obtained in the circulation due to the reduced clearance rate compared to non-human antibodies. Preferably, human germline sequences are used for the framework regions in the antibodies or fragments according to the present invention. The use of human germline sequences minimizes the immunogenicity risk of the antibodies or fragments, because these sequences are unlikely to contain somatic alterations, which are peculiar to the individuals from which the framework regions are derived, and may cause immunogenic reactions when applied to another human individual.

[0132] The procedure of antibody humanization or providing chimeric antibodies is well known in the art. Various recombinant DNA-based methods have been established, which are intended to increase the content of amino acid residues in antibodies that are also present in similar positions in human 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 with the corresponding human framework region with the highest homology, while keeping the non-human CDR intact. Other methods suitable for humanization of 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 resurfacing (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).

[0133] In one embodiment, the antibody or fragment according to the present invention is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody with binding specificity to at least two different antigens and / or epitopes. In one embodiment, a bispecific antibody has binding specificity to FH (preferably comprising a variable light chain and a variable heavy chain of CCP18 that binds to FH as described herein) and has binding specificity to another antigen. In another embodiment, a bispecific antibody can bind to two different epitopes of FH.

[0134] The antibody or fragment according to the present invention can be of any class. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3 and IgG4. In a preferred embodiment, the antibody or fragment according to the present invention is of the IgG class, preferably IgG1 or IgG3.

[0135] 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 this method is the production of immunization and hybridomas, as described in the embodiments. For example, it is possible to immunize mice (such as BALB / c mice) to produce mouse monoclonal antibodies against FH by intraperitoneal injection of human factor H or FH-related proteins (such as FHR-1), optionally in the presence of adjuvants (such as montanide), for example every four weeks. A few days after the fourth immunization, spleen cells 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 (such as rat anti-mouse κ moAb RM19) to capture mouse IgG antibodies. The specificity of the antibody is 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 used and widely described in the art. Antibodies can be screened in a library using the same antigen used for immunization (e.g., the CCP18 domain of human FH, FH-associated proteins, or human FH).

[0136] The present invention further provides an isolated, synthetic or recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or fragment thereof according to the present invention. A preferred nucleic acid molecule encodes at least the heavy chain CDR1, CDR2 and CDR3 and / or the light chain CDR1, CDR2 and CDR3 of the antibody FHR-1.8E4 as shown in Table 1. A further preferred nucleic acid molecule encodes at least the heavy chain CDR1, CDR2 and CDR3 and / or the light chain CDR1, CDR2 and CDR3 of the antibody FHR-1.3B4 as shown in Table 1. A further preferred nucleic acid molecule encodes at least the heavy chain CDR1, CDR2 and CDR3 and / or the light chain CDR1, CDR2 and CDR3 of the antibody FHR-1.11E1 as shown in Table 1. A further preferred nucleic acid molecule encodes at least the heavy chain CDR1, CDR2 and CDR3 and / or the light chain CDR1, CDR2 and CDR3 of the antibody FHR-1.12F4 as shown in Table 1. Preferably, the length of the nucleic acid molecule according to the present invention is at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 75 nucleotides. Preferably, the nucleic acid molecule according to the present invention encodes 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 the antibody FHR-1.8E4 as shown in Table 1. Further preferred nucleic acid molecules according to the present invention encode 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 the antibody FHR-1.3B4 as shown in Table 1. Further preferred nucleic acid molecules according to the present invention encode 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 the antibody FHR-1.11E1 as shown in Table 1. Further preferred nucleic acid molecules according to the present invention encode monoclonal chimeric or humanized antibodies or fragments thereof comprising the heavy chain CDR sequence and light chain CDR sequence and / or the heavy chain variable region and light chain variable region of the antibody FHR-1.12F4 as shown in Table 1. Table 1 shows the nucleic acid sequences encoding the heavy chain and light chain CDRs of the antibodies FHR-1.8E4, FHR-1.3B4, FHR-1.11E1 and FHR-1.12F4. However, the present invention also encompasses nucleic acid molecules encoding the heavy chain or light chain CDRs of the antibodies according to the present invention comprising nucleic acid sequences different from the CDR nucleic acid sequences shown in Table 1, but comprising nucleic acid codons encoding the amino acid sequences of the heavy chain or light chain CDR sequences shown in Table 1.

[0137] Therefore, an isolated, synthetic or recombinant nucleic acid molecule is provided, comprising a nucleic acid sequence encoding at least the sequence of SEQ ID NOs 17, 18, 19, 21, 22 and 23. Further provided is an isolated, synthetic or recombinant nucleic acid molecule, comprising a nucleic acid sequence encoding at least the sequence of SEQ ID NOs 33, 34, 35, 37, 38 and 39. Further provided is an isolated, synthetic or recombinant nucleic acid molecule, comprising a nucleic acid sequence encoding at least the sequence of SEQ ID NOs 49, 50, 51, 53, 54 and 55. Further provided is an isolated, synthetic or recombinant nucleic acid molecule, comprising a nucleic acid sequence encoding at least the sequence of SEQ ID NOs 65, 66, 67, 69, 70 and 71. Further provided is an isolated, synthetic or recombinant nucleic acid molecule, comprising a nucleic acid sequence encoding at least the sequence of SEQ ID NOs 20 and 24. Further provided is an isolated, synthetic or recombinant nucleic acid molecule, comprising a nucleic acid sequence encoding at least the sequence of SEQ ID NOs 36 and 40. Further provided is an isolated, synthetic or recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least the sequences of SEQ ID NOs 52 and 56. Further provided is an isolated, synthetic or recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least the sequences of SEQ ID NOs 68 and 72. The present invention also encompasses nucleic acid molecules encoding heavy and / or light chain CDRs or antibodies modified, for example, by conservative amino acid substitutions.

[0138] Nucleic acid molecules or nucleic acid sequences according to the present invention preferably comprise nucleotide chains, more preferably DNA and / or RNA. However, nucleic acid molecules or nucleic acid sequences of the present invention can comprise nucleic acid structures of other types, such as, for example, DNA / RNA spirals, peptide nucleic acids (PNA), locked nucleic acids (LNA) and / or ribozymes. Such other nucleic acid structures are referred to as the functional equivalents of nucleic acid sequences, and are encompassed by the present invention. The term "functional equivalents of nucleic acid sequences" also encompasses chains comprising non-natural nucleotides, modified nucleotides and / or non-nucleotide structural units that show the same functions as natural nucleotides.

[0139] The present invention further provides a vector comprising a nucleic acid molecule according to the present invention. A preferred vector is a plasmid. A plasmid is defined herein as a circular, preferably double-stranded, DNA molecule. Methods for preparing vectors comprising nucleic acid molecules according to the present invention are well known in the art. Non-limiting examples of vectors suitable for producing vectors of the present invention are retroviral and lentiviral vectors. The vector according to the present invention can be used for various applications. The vector according to the present invention is preferably used for expressing nucleic acid molecules according to the present invention in cells in vitro, preferably for producing antibodies or fragments according to the present invention. Further, the vector according to the present invention comprising nucleic acid molecules according to the present invention can be used for treatment. Administration of such a vector to an individual (preferably a human) in need results in the expression of antibodies or fragments according to the present invention in vivo.

[0140] A recombinant cell is further provided, comprising a nucleic acid molecule or vector according to the present invention. Such a nucleic acid molecule or vector is preferably used for being introduced into the cell so that the nucleic acid translation mechanism of the cell will produce the encoded antibody or fragment. Preferably, the nucleic acid molecule or vector according to the present invention is expressed in so-called producer cells (such as, for example, Chinese hamster ovary cells (CHO), NSO (mouse myeloma) or 293 (T) cell lines, some of which are suitable for commercial antibody production). The proliferation of such producer cells produces a producer cell line capable of producing antibodies or fragments according to the present invention. Preferably, the producer cell line is suitable for producing antibodies for use in humans. Therefore, the producer cell line is preferably free of pathogens, such as pathogenic microorganisms.

[0141] The present invention further provides a method for producing an antibody or fragment according to the present invention, comprising providing a cell having a nucleic acid molecule or vector according to the present invention, and causing the cell to translate a nucleic acid sequence contained by the nucleic acid molecule or vector, thereby producing an antibody or fragment according to the present invention. The method according to the present invention preferably further comprises harvesting, purifying and / or isolating the antibody or fragment. Antibodies or fragments obtained using the method for producing an antibody or fragment according to the present invention are also provided.

[0142] The antibodies or fragments according to the present invention can be advantageously used for therapeutic applications. Therefore, a pharmaceutical composition is provided, comprising an antibody or fragment according to the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient. A pharmaceutical composition is also provided, comprising a nucleic acid molecule or vector according to the present invention and at least one pharmaceutically acceptable carrier, diluent and / or excipient. Non-limiting examples of suitable carriers are, for example, keyhole limpet hemocyanin (KLH), serum albumin (e.g., BSA or RSA) and ovalbumin. Preferred carriers are solutions, such as aqueous solutions, such as saline or oil-based solutions. Non-limiting examples of excipients that can be incorporated into tablets, capsules, etc. are adhesives, such as 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, wintergreen oil or cherry oil. When the dosage unit form is a capsule, in addition to one or more of the above-mentioned excipients, it preferably also contains a liquid carrier, such as a fatty oil. Various other materials may exist as coatings or be used to change the physical form of the dosage unit. For example, tablets may be coated with shellac and / or sugar or both. The pharmaceutical composition according to the present invention is preferably suitable for human use.

[0143] Pharmaceutical compositions as 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 oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, percutaneous, intrathecal and intracranial methods. For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. According to conventional pharmaceutical practices, sterile compositions for injection can be prepared by dissolving or suspending the antibody or fragment of the present invention in an injection carrier (such as water or natural oil, such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc.) or a synthetic fat carrier (such as ethyl oleate). Buffers, preservatives and / or antioxidants can also be incorporated.

[0144] The present invention further provides an antibody or fragment according to the present invention for use in treatment. A nucleic acid molecule according to the present invention for use in treatment is further provided. The treatment may be therapeutic or prophylactic. The antibody or fragment according to the present invention is particularly suitable for treating, alleviating or preventing a condition associated with alternative pathway complement activation. Therefore, an antibody or fragment according to the present invention for use in the treatment, alleviating or preventing a condition associated with alternative pathway complement activation is provided. A nucleic acid molecule according to the present invention for use in the treatment, alleviating or preventing a condition associated with alternative pathway complement activation is also provided. As used herein, "a condition associated with alternative complement activation" is defined herein as a condition in which cells, tissues or extracellular matrix are damaged by undesirable and / or excessive alternative pathway complement activation. Cells that may be damaged by undesirable and / or excessive alternative pathway activation are any cells in contact with blood, such as red blood cells, epithelial cells (particularly liver and / or kidney epithelial cells), platelets, leukocytes, endothelial cells. The condition is preferably a condition associated with impaired FH function or FH deficiency. More preferably, the disease is a condition associated with impaired FH function or FH deficiency but not associated with FH deficiency. 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 the serum of healthy individuals in which FH is 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 membranoproliferative glomerulonephritis (MPGN).

[0145] 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 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). Multiple mutations in complement system components or factors have been identified in aHUS patients. Mutations in FH, FI, FB, membrane cofactor protein (MCP), thrombomodulin (THBD) or C3 account for about 50% of known mutations in aHUS patients, of which FH mutations are the most common (about 20-30% of aHUS patients). Most patients have heterozygous mutations, but still lead to pathological FH deficiency. In addition, in approximately 10% of patients, aHUS is caused by autoantibodies against FH, which also leads to reduced functional FH. Currently, the standard treatment for aHUS is plasma supplementation or plasma exchange therapy. In addition, eculizumab is used in the treatment of aHUS patients. Kidney transplantation is associated with a high risk of recurrence, depending on the underlying mutation of aHUS. Children with mutations in FH, FB, FI, C3 or THBD are contraindicated for transplantation because of their increased risk of recurrence. The antibody or fragment according to the present invention (preferably at least comprising a Fab fragment) is particularly suitable for treating, alleviating or preventing aHUS caused by FH mutations or by the presence of anti-FH autoantibodies. The enhancement of FH is independent of the CCP domain of FH with mutations. For example, an enhanced FH antibody or fragment thereof can inhibit the activation of alternative entities in aHUS patients carrying mutations in CCP1, CCP6, CCP7, CCP14, CCP17, CCP18, CCP19 and CCP20 of FH. However, since the antibodies and fragments of the present invention can also enhance the activity of FH without impaired FH function or FH deficiency, any form of complement-dependent aHUS can be advantageously treated, alleviated or prevented with the antibodies or fragments of the present invention.

[0146] Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a gene mutation on the X chromosome of a totipotent hematopoietic stem cell. The mutation results in a deficiency of phosphatidylinositol glycan class A proteins, which are essential for the synthesis of glycosylphosphatidylinositol membrane anchored proteins (GPI-APs). An example of such a protein is an inhibitor of the complement system CD55, which can bind C3b on the surface of host cells, thereby preventing the formation of C3 convertase. Therefore, the lack of these proteins can lead to undesirable or excessive entity activation. One of the main consequences of PNH is the lysis of red blood cells due to excessive activity of the complement system. Recently, eculizumab has been approved for the treatment of PNH in many countries. Other therapies include blood transfusions, erythrocyte stimulator therapy, treatment with corticosteroids and anabolic steroids. Since the antibodies and fragments of the present invention can also enhance the activity of FH independently of the level of FH or FH function, thereby inhibiting the activation of the alternative pathway of the complement system, the antibodies and fragments can be advantageously used in PNH patients. Furthermore, 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, depletion of cells in the liver is reduced since fewer cells are opsonized by C3b.

[0147] Age-related macular degeneration (AMD) is the damage to the retina, usually affects the elderly, causes the macula (center of visual field) vision loss. Recently, about 35% of AMD patients have been related to the mutation and SNP (single nucleotide polymorphism) in FH. SNP is located in the CCP7 of FH, and is proved to affect the combination of FH and heparin, thereby damaging the ability of FH in conjunction with host cell surface and extracellular matrix. Antibody according to the present invention or fragment (preferably comprising Fab fragment at least) are particularly suitable for treating, alleviating or preventing AMD characterized by (preferably characterized by the SNP in the gene encoding FH) with FH function reduction.

[0148] Membranoproliferative glomerulonephritis (MPGN) is a rare cause of chronic nephritis, occurring primarily in children and young adults. It causes glomerular damage due to proliferation of mesangial and endothelial cells and expansion of the mesangial matrix, thickening of the peripheral capillary walls by subendothelial immune deposits and / or dense intramembrane deposits, and insertion of the mesangium into the capillary walls. MPGN is usually associated with a complete lack of FH. MPGN that can be treated with the antibodies and / or fragments of the invention is preferably associated with impaired FH function or FH deficiency, and is not associated with FH deficiency.

[0149] Therefore, the present invention provides an antibody or fragment or nucleic acid molecule according to the present invention for use in the treatment, alleviation or prevention of a disorder associated with alternative pathway complement activation, wherein the disorder is selected from atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN). Also provided is the use of an antibody or fragment, nucleic acid molecule or vector according to the present invention for the preparation of a medicament for treating, alleviating or preventing a disorder associated with alternative pathway complement activation. The disorder is preferably selected from atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN). The preferred antibody used as a medicament or preventive agent according to the present invention is an antibody or fragment thereof comprising heavy and light chain CDR1, CDR2 and CDR3 of an antibody as shown in Table 1, preferably Fab, Fab' or F(ab)2 or F(ab')2 fragment. The antibody or fragment is preferably a monoclonal humanized or chimeric antibody or fragment.

[0150] The present invention further provides a method of inhibiting alternative complement activation, comprising administering to an individual the antibody or fragment according to the present invention, or the nucleic acid molecule or vector according to the present invention.

[0151] The present invention further provides a method for treating, alleviating or preventing a condition associated with alternative pathway complement activation, comprising administering a therapeutically effective amount of an antibody or fragment according to the present invention to an individual in need. Also provided is a method for treating, alleviating or preventing a condition associated with alternative pathway complement activation, comprising administering a therapeutically effective amount of a nucleic acid molecule or vector according to the present invention to an individual in need. Further provided is a method for treating, alleviating or preventing a condition associated with alternative pathway complement activation, comprising administering a therapeutically effective amount of a pharmaceutical composition according to the present invention to an individual in need. The condition is preferably selected from atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), and membranoproliferative glomerulonephritis (MPGN). As used herein, an "individual" is a human or animal having a complement system as part of its immune system, preferably a mammal. In a particularly preferred embodiment, the individual is a human. The antibody preferably used in the method of the present invention is an antibody or a fragment thereof, preferably a Fab, Fab', F(ab)2 or F(ab')2 fragment, which comprises the heavy chain and light chain CDR1, CDR2 and CDR3 of antibody FHR-1.8E4, the heavy chain and light chain CDR1, CDR2 and CDR3 of antibody FHR-1.3B4, the heavy chain and light chain CDR1, CDR2 and CDR3 of antibody FHR-1.11E1, or the heavy chain and light chain CDR1, CDR2 and CDR3 of antibody FHR-1.12F4 as shown in Table 1. The antibody or fragment is preferably a monoclonal humanized or chimeric antibody or fragment.

[0152] Compositions containing antibodies, fragments, and nucleic acid molecules of the present invention can be administered for preventive and / or therapeutic treatments. In therapeutic applications, antibodies, fragments, nucleic acid molecules, or compositions according to the present invention are administered to individuals, preferably humans, who have already suffered from 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 acid molecules, or compositions according to the present invention are administered to individuals before the individual exhibits symptoms of the disease to prevent the development of these symptoms or its complications. For example, individuals carrying genetic mutations that may or will cause disorders associated with alternative complement activation can be preventively treated using antibodies, fragments, nucleic acid molecules, or compositions according to the present invention. The antibody, fragment, or nucleic acid molecule is typically present in a pharmaceutical composition according to the present invention in a therapeutically effective amount, which is an amount sufficient to compensate for the disorder associated with the undesirable or excessive activation of the alternative pathway of the complement system.

[0153] For the sake of clear and concise description, features may be described herein as part of the same or different aspects or embodiments of the present invention. Those skilled in the art will recognize that the scope of the present invention may include all or some combinations of features described herein as part of the same or different embodiments.

[0154] The invention will be explained in more detail in the following non-limiting examples.

[0155] Table 1. Amino acid and nucleotide sequences of antibodies FH.07, FHR-1.8E3, FHR-1.3B4, FHR-1.11E1, FHR-1.12F4 and FHR-1.14C4 (CDRs 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.

[0156]

[0157]

[0158]

[0159]

[0160] BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 : Competition assay to map the epitope location of anti-FHR-1 antibodies.

[0162] A) Binding of each monoclonal antibody to biotinylated FH (FH-bt) in the presence or absence of unlabeled full-length FH or FH fragments was assessed by ELISA. Binding is expressed as a percentage of FH-bt binding observed in the absence of any added unlabeled competitor (set to 100%, dashed line). B) Competition ELISA with FH-bt using anti-FH and anti-FHR-1 mAbs as capture mAbs (indicated on the X-axis) and anti-FH.07 or anti-FH.16 (as controls) as competitors. C) Competition ELISA with FH-bt as in B, but only anti-FH.07 was used as capture mAb and anti-FHR-1 mAb (indicated on the X-axis) was used as competing mAb.

[0163] Figure 2: SPR analysis of anti-FHR-1 mAb to determine its affinity for FH. Affinity was determined by SPR on ProtA or ProtG chips.

[0164] (AB) Example sensorgrams of capture of the indicated mAbs prior to flowing over the surface full-length FH (A) or a fragment of FH consisting of domains 18-20 (B). Fits (black lines) and corresponding results indicated next to each plot were obtained following a 1:1 binding model.

[0165] (CD) Summary of all affinity analyses performed using ProtA or ProtG chips for antibodies FH.07, 3B4 and 8E4 full length (C) or CCP18-20 (D).

[0166] Figure 3 : Functional characterization of anti-FHR-1 mAbs.

[0167] A)

[0168] C3b deposition on LPS measured by ELISA using 10% (v / v) normal human serum with the indicated antibodies. Graphs are representative of multiple independent experiments, n=4.

[0169] Upper panel: IC50 for enhancement of C3b deposition.

[0170] Bottom: Representative graph of C3b deposition on LPS for increasing concentrations of FH.07, 3B4 and 8E4. C3b deposition in 10% (v / v) normal human serum without any added antibody was set to 100%. IgG1 ctrl is a negative control antibody.

[0171] B) Hemolysis assay using sheep red blood cells (SRBC) and 10% (v / v) normal human serum. Inhibition of FH using anti-FH.09 was used to induce hemolysis (serum + FH Inh.), resulting in 89% lysis. Monoclonal antibodies against FH and FHR-1 were added at the indicated hemolysis IC50 calculated from multiple experiments. n=2-4, statistical analysis was performed using ordinary one-way ANOVA, * indicates p≤0.05.

[0172] Figure 4 : A. SPR analysis sensorgrams of the interaction of FH with C3b with and without the addition of anti-FH.07, anti-FHR-1 3B4 or anti-FHR-1 8E4 Fab' fragments as shown. The addition of Fab' fragments increased the measured RU at least 2-fold on all surfaces, reflecting increased FH binding. B. Equilibrium analysis of binding and estimated KD of the interaction showed an increase in affinity of each Fab' by 2.7, 3.2 and 3.6 fold, respectively.

[0173] Example

[0174] Materials and methods

[0175] Reagents

[0176] Human purified factor H was obtained from CompTech. (Tyler, Texas USA). Rat anti-mouse kappa (RM19) was obtained from Sanquin (commercial unit reagents, Sanquin, Amsterdam, the Netherlands). High performance ELISA buffer (HPE) was obtained from Sanquin. Recombinant FH CCP (CCP 15-18, CCP 15-19, CCP 18-20 or CCP 19-10) was a gift from Dr. Christoph Schmidt and produced as previously described (Schmidt et al. 2008). Mouse monoclonal antibodies (mAbs) against human FH were previously made. Anti-FH.07 (murine IgG1) was directed against CCP 18, anti-FH.09 (murine IgG1) was directed against CCP 6 and anti-FH.16 (murine IgG1) was directed against CCP 16 / 17. Anti-IL-6.8 was used as an irrelevant isotype control and was obtained from Sanquin. Anti-C3.19 reacts with an epitope on the C3d fragment of the molecule and has been described previously (Wolbink et al. 1993).

[0177] Expression of rhFHR protein

[0178] 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 in HEK293F cells by transient transfection of the pcDNA3.1 expression vector, followed by HisTrap TM High-performance 1 ml chromatography columns (GE Healthcare LifeSciences, Freiburg, Germany) were used to analyze the 2+ Proteins were purified from the supernatant by affinity chromatography. The rFHRh was filtered and concentrated using ultracentrifugal filters (Merck Millipore, Darmstadt, Germany).

[0179] Immunization and hybridoma generation

[0180] BALB / c mice were immunized with 25 μg of rhFHR-1 in amantadine as adjuvant intraperitoneally every 4 weeks to generate mouse monoclonal antibodies against FHR-1. Three days after the fourth booster immunization, spleen cells were fused with the myeloma cell line SP2 / 0. The presence of FHR-1-specific antibodies in hybridoma cell 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 antibodies was determined by biotinylated rhFHR-1. Binding of biotinylated rhFHR-1 was determined in HPE by incubation for 30 minutes with 0.1% (v / v) streptavidin conjugated to HRP. The ELISA was further developed using 100 μg / mL 3,5,3',5'-tetramethylbenzidine (TMB) in 0.1 M sodium acetate (pH 5.5) containing 0.003% (v / v) H2O2. Substrate conversion was stopped by adding 100 μL H2SO4 and absorbance was measured at 450 nm and corrected for absorbance at 540 nm using a Synergy 2 multimode plate reader (BioTek Instruments, Winooski, VT, USA).All ELISA steps were performed in a final volume of 100 μL per well.

[0181] Epitope mapping mAbs and competition analysis

[0182] The location of the epitope of the anti-FHR-1 mAb was determined using a recombinant human FH fragment consisting of multiple CCP domains (15-18, 15-19, 18-20 or 19-20). In brief, the anti-FHR-1 mAb was captured on a RM-19 coated microtiter plate to ensure the best binding conformation. Subsequently, the biotinylated FH was mixed with a 100-fold higher concentration of the indicated unlabeled recombinant FH fragment and incubated on the plate for 1 hour. 0.01% (v / v) streptavidin conjugated with poly-HRP was incubated for 30 minutes in HPE to determine the binding of biotinylated FH. ELISA was further developed as described above. In order to determine whether the anti-FHR-1 mAb competes with anti-FH.07 for the binding of FH, a similar setup was used. In brief, the mAb was directly coated on the plate and the binding of biotinylated FH (FH-bt) in the absence or presence of 10-fold higher concentrations of the indicated mAb was evaluated by ELISA as described above.

[0183] Deposition of C3 on LPS

[0184] Polysorp 96-well microtiter plates (Nunc) were coated with Salmonella typhi LPS (40 μg / mL, L-6386 Sigma-Aldrich) in PBS, O / N at room temperature. LPS activates the alternative pathway of complement. After washing with PBS + 0.1% (w / v) Tween-20, 10% (v / v) NHS was incubated in Veronal buffer (VB; 3mM barbital, 1.8mM sodium barbital, 145mM NaCl, pH 7.4) containing 0.05% (w / v) gelatin, 5mM MgCl2, 10mM EGTA and 0.1% (w / v) Tween-20 in the presence or absence of indicated concentrations of anti-FH / anti-FHR-1 mAb, isotype control or aIL6-8AB as a negative control. C3b deposition was detected using biotinylated mAb anti-C3.19 (0.55 μg / mL in HPE) followed by incubation for 30 min with 0.01% (v / v) streptavidin conjugated with poly-HRP in HPE. The ELISA was further developed as described above.

[0185] SRBC hemolysis assay

[0186] FH function was determined using a hemolytic assay as previously described by Sanchez-Corral et al. (2004) and Wouters et al. (2008) with some modifications. Pre-diluted human serum (20%, v / v) containing 20 μg / ml anti-FH.09 was pre-incubated with the indicated mAbs and mixed with sheep red blood cells (SRBC) at a 1:1 ratio to achieve a final concentration of 10% (v / v) serum and was incubated at 1.05*10 μL in VB with 5 mM MgCl2 and 10 mM EGTA or in VB with 10 mM EDTA. 8 Cells / ml were used as blanks, followed by incubation at 37°C for 75 minutes under shaking. Lysis was stopped by adding 100 μl of ice-cold VB with 20 mM EDTA, followed by centrifugation (2.5 minutes, 1,800 RPM / 471 RCF, 7°C). Hemolysis was determined 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% lysis 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. Using graphpad prism 7.04, the EC50 of hemolysis was calculated from multiple experiments, and statistical comparisons were performed by ordinary one-way ANOVA and Dunnett's multiple comparison test.

[0187] Determine binding affinity (SPR)

[0188] Surface plasmon resonance (SPR) experiments were performed using a BiaCore T200 (GE Healthcare) and CM5 sensor chips (GE Healthcare) according to the manufacturer's instructions. Briefly, to determine their affinity, anti-FH.07, anti-FHR-1 3B4 or anti-FHR-1 8E4 were captured on ProtA or ProtG coupled chips, and full-length FH or FH fragments containing domains 18-20 were passed over the captured moAB at decreasing concentrations.

[0189] Binding affinity of factor H to C3b

[0190] The binding of FH to C3b in the presence of anti-FH moAb was determined by surface plasmon resonance using a Biacore T200 instrument (GE Healthcare, Little Chalfont, UK). Purified C3b (Complement Technologies) was immobilized on a flow cell of a CM5 Biacore sensor chip (GE Healthcare) using standard amine coupling. The remaining flow cell was used as a reference surface and prepared by a coupling reaction performed 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) supplemented with 0.01% (w / v) Tween-20 (Merck) (PBS-T).

[0191] To determine the effect of the antibody without interfering with possible cross-linking by the moAb, Fab' fragments of recombinantly produced moAbs were used. The Fab' fragments were mixed with plasma purified FH (pdFH). FH was injected on the chip at different concentrations for 60 seconds (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-FH.07, anti-FHR-1 3B4, or anti-FHR-1 8E4 Fab' fragments. Each Fab' fragment was also injected without the addition of FH to determine any interaction of the Fab' fragment with the surface. Dissociation for 60 seconds was followed by injection of pdFH and the surface was regenerated between each cycle by a single injection of 1 M NaCl (Merck).

[0192] Data were analyzed using Scrubber 2 (Biologic Software) and affinities were determined by equilibrium analysis.

[0193] result

[0194] Monoclonal antibodies

[0195] Monoclonal antibodies FHR-1.3B4, FHR-1.8E4, FHR-1.11E1, FHR-1.12F4, and FHR-1.14C4 were generated against recombinant human factor H related protein 1 (FHR-1).

[0196] Mapping binding sites

[0197] To map the binding site of the monoclonal antibody, the moAb was tested for reactivity against recombinant FH fragments containing different CCP domains (CCP 15-18, CCP 15-19, CCP 18-20, and CCP 19-20) as well as full-length human FH. Figure 1 As shown in A, FHR-1.3B4, FHR-1.8E4, FHR-1.11E1, FHR-1.12F4 and FHR-1.14C4 bind to recombinant fragments CCP 15-18, CCP 15-19 and CCP 18-20 with varying degrees, but not CCP 19-20. This indicates that all antibodies are specific for CCP 18 of FH.

[0198] Epitope mapping

[0199] Competition assays were performed using the agonistic anti-FH antibody FH.07. Figure 1 B and Figure 1 As shown in C, the binding of FHbt to coated anti-FH.07, 11E1, 8E4, and 3B4 was competed by anti-FH.07, but not by 14C4 or isotype control anti-FH.16. This suggests that anti-FH.07, 11E1, 8E4, and 3B4 bind to the same or overlapping epitopes in CCP18, while 14C4 binds to another non-overlapping epitope in CCP 18. The binding of 12F4 was also competed by anti-FH.07, but to a lesser extent, indicating that this antibody binds to the same epitope, but perhaps with lower affinity.

[0200] Binding affinity

[0201] All antibodies FH.07, 8E4 and 3B4 bind human FH and CCP18-20 of FH with nanomolar or subnanomolar affinity. Figure 2 As shown in Table 2, antibodies 8E4 and 3B4 both have higher binding affinities than FH.07 for full-length FH and CCP18-20.

[0202] Table 2. Binding kinetics of antibodies FH.07, 3B4 and 8E4.

[0203]

[0204] Deposition of C3 on LPS

[0205] FH.07, 3B4, 8E4, 11E1, and 12F4 all reduced C3b deposition on LPS ( Figure 3 FH.07, 3B4, and 8E4 are shown in A). In contrast, 14C4 increased C3b deposition on LPS. Figure 3 A shows the deposition of C3 on LPS at increasing concentrations of FH.07, 3B4, and 8E4. It is shown that 8E4 inhibits C3 deposition to a greater extent than FH.07.

[0206] Thus, all mAbs that compete with anti-FH.07 also enhance FH function, resulting in decreased complement activation. Of note, the non-competitive mAb (14C4), despite also binding to CCP 18, had the opposite effect, inhibiting FH function, resulting in greater C3b deposition. As shown in Table 3, the IC 50 Lower than FH.07.

[0207] SRBC hemolytic activity

[0208] Anti-FH.07, 3B4, 8E4, and 11E1 reduced induced SRBC hemolysis. 12F4 showed the same effect, but to a lesser extent. Therefore, all mAbs that compete with anti-FH.07 also enhance the function of FH on the SRBC surface, resulting in a decrease in complement-mediated hemolysis. In this assay, 3B4, 8E4 both had a greater enhancement of FH at higher concentrations. As shown in Tables 3 and Figure 3 B shows the IC of 8E4 and 3B4 in the hemolytic assay. 50 Both are lower than FH.07.

[0209] Table 3: IC of anti-FHR-1 mAbs compared to anti-FH.07 in LPS C3b deposition assay and hemolysis assay 50 For FH.07, 3B4, and 8E4, the values ​​are based on multiple experiments.

[0210]

[0211] Binding affinity of factor H to C3b

[0212] Under normal conditions, FH was shown to interact with C3b with an estimated KD of 6.0 μM. However, the addition of Fab' fragments of anti-FH / 07, aFHR-1 3B4, or aFHR-1 8E4 increased the response to C3b-coated surfaces ( Figure 4A) and showed estimated increased affinities of 2.20 μM, 1.90 μM, and 1.66 μM, respectively ( Figure 4 B).

[0213] References

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[0216] · 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: crystal structure of an affinity - matured Fab in complex with antigen. J Mol Biol. 1999;293(4):865 - 81.

[0217] · 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.

[0218] ·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.

[0219] ·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.

[0220] ·Lefranc MP,“Unique database numbering system for immunogeneticanalysis”Immunology Today,18,509(1997).PMID:9386342.

[0221] ·Lefranc MP,"The IMGT unique numbering for immunoglobulins,T cellReceptors and Ig-like domains",The Immunologist.1999;7,132-136.

[0222] ·Lefranc MP,PommiéC,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.

[0223] ·Padlan,EA.1991.A possible procedure for reducing the immunogenicityof antibody variable domains while preserving their ligand-bindingproperties.Mol.Immunol.,28(4-5):489-98.

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[0225] ·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 interleukin 2 receptor.Proc.Natl.Acad.Sci.U.S.A.86(24):10029-33.

[0226] · -Corral,Pilar,C -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.

[0227] ·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.

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Claims

1. An isolated, synthetic or recombinant antibody or antigen-binding fragment thereof that specifically binds to the complement control protein domain 18 (CCP18) of factor H (FH), comprising: - light chain CDR1 sequence SSVRY (SEQ ID NO: 17), light chain CDR2 sequence ATS (SEQ ID NO: 18) and light chain CDR3 sequence QQWGTKPPT (SEQ ID NO: 19), heavy chain CDR1 sequence DFSLTNSG (SEQ ID NO: 21), heavy chain CDR2 sequence IWSGGTT (SEQ ID NO: 22) and heavy chain CDR3 sequence ARNFGNYAVDY (SEQ ID NO: 23), or - light chain CDR1 sequence SSVTY (SEQ ID NO: 33), light chain CDR2 sequence ATS (SEQ ID NO: 34) and light chain CDR3 sequence QQRSSSNPLT (SEQ ID NO: 35), heavy chain CDR1 sequence GFSLTNYG (SEQ ID NO: 37), heavy chain CDR2 sequence IWSGGTT (SEQ ID NO: 38) and heavy chain CDR3 sequence ARNFGNYAMDY (SEQ ID NO: 39), or - light chain CDR1 sequence QSLVHSNGNTY (SEQ ID NO: 49), light chain CDR2 sequence KLS (SEQ ID NO: 50), and light chain CDR3 sequence SQSTHVPFT (SEQ ID NO: 51), heavy chain CDR1 sequence GFSLTNYG (SEQ ID NO: 53), heavy chain CDR2 sequence IWSGGNT (SEQ ID NO: 54), and heavy chain CDR3 sequence AKNGDYGYTMDY (SEQ ID NO: 55), or -Light chain CDR1 sequence SSVNY (SEQ ID NO: 65), light chain CDR2 sequence YTS (SEQ ID NO: 66) and light chain CDR3 sequence QQFTSSPLT (SEQ ID NO: 67), heavy chain CDR1 sequence GFSLTSYG (SEQ ID NO: 69), heavy chain CDR2 sequence IWSGGST (SEQ ID NO: 70), and heavy chain CDR3 sequence ARNGGNYYFDY (SEQ ID NO: 71).

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain CDR1 sequence SSVRY (SEQ ID NO: 17), a light chain CDR2 sequence ATS (SEQ ID NO: 18) and a light chain CDR3 sequence QQWGTKPPT (SEQ ID NO: 19), a heavy chain CDR1 sequence DFSLTNSG (SEQ ID NO: 21), a heavy chain CDR2 sequence IWSGGTT (SEQ ID NO: 22) and a heavy chain CDR3 sequence ARNFGNYAVDY (SEQ ID NO: 23).

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the K of the binding affinity of the antibody or antigen-binding fragment thereof to FH is D 2.5 × 10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1 × 10 -9 M or lower.

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain CDR1 sequence SSVTY (SEQ ID NO: 33), a light chain CDR2 sequence ATS (SEQ ID NO: 34) and a light chain CDR3 sequence QQRSSSNPLT (SEQ ID NO: 35), a heavy chain CDR1 sequence GFSLTNYG (SEQ ID NO: 37), a heavy chain CDR2 sequence IWSGGTT (SEQ ID NO: 38) and a heavy chain CDR3 sequence ARNFGNYAMDY (SEQ ID NO: 39).

5. The antibody or antigen-binding fragment thereof according to claim 1 or 4, wherein the K of the binding affinity of the antibody or antigen-binding fragment thereof to FH is D 2.5 × 10 -8 M or lower and / or a K of the binding affinity to the FH fragment consisting of CCP18-20 D 0.1 × 10 -9 M or lower.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof: - In vitro with an IC of 38 nM or lower 50 value inhibits the deposition of C3 on LPS, and / or - In vitro with an IC of 150 nM or less 50 value to inhibit hemolytic activity, and / or - In vitro binding affinity of FH for C3b (K D ) up to 2 µM and / or increased the binding affinity of FH for C3b at least 3-fold in vitro.

7. The antibody or antigen-binding fragment thereof according to claim 1, comprising: - light chain CDR1 sequence QSLVHSNGNTY (SEQ ID NO:49), light chain CDR2 sequence KLS (SEQ ID NO:50) and light chain CDR3 sequence SQSTHVPFT (SEQ ID NO:51), heavy chain CDR1 sequence GFSLTNYG (SEQ ID NO:53), heavy chain CDR2 sequence IWSGGNT (SEQ ID NO:54) and heavy chain CDR3 sequence AKNGDYGYTMDY (SEQ ID NO:55), or -Light chain CDR1 sequence SSVNY (SEQ ID NO:65), light chain CDR2 sequence YTS (SEQ ID NO:66) and light chain CDR3 sequence QQFTSSPLT (SEQ ID NO:67), heavy chain CDR1 sequence GFSLTSYG (SEQ ID NO:69), heavy chain CDR2 sequence IWSGGST (SEQ ID NO:70) and heavy chain CDR3 sequence ARNGGNYYFDY (SEQ ID NO:71).

8. The antibody or antigen-binding fragment thereof according to claim 1, comprising: - a variable light chain sequence comprising a sequence having at least 80% sequence identity to QIVLSQSPAILSASPGEKVTMTCRASSSVRYMHWYQQKAGSSPTAWIFATSNLASGVPPRFSGSGSGTSYSLTISRVEAEDAATYYCQQWGTKPPTFGAGTKLELK (SEQ ID NO: 20); and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to QVQLKQSGPGLVQPSQSLITCTVSDFSLTNSGVHWVRQSPGKGLEWLGVIWSGGTTEYNAAFMSRLTITKDNSKSQVFFKMNSLLVDDDTGIYYCARNFGNYAVDYWGQGTSVTVSS (SEQ ID NO: 24), or - a variable light chain sequence comprising a sequence having at least 80% sequence identity to QIVLSQSPTILSASPGEKVTMTCRASSVSVTYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQRSSNPLTFGAGTKLELK (SEQ ID NO: 36); and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to QVQLRQSGPGLVQPSQSLITCTVSGFSLTNYGVYWVRQSPGKGLEWLGVIWSGGTTDYSAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARNFGNYAMDYWGQGTSVTVSS (SEQ ID NO: 40), or - a variable light chain sequence comprising a sequence having at least 80% sequence identity to DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKLSNFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYCSQSHVPFTFGSGTKLEIK (SEQ ID NO: 52); and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to QVQLKQSGPGLVQPSQSLITCTVSGFSLTNYGVHWVRQPPGKGLEWLGVIWSGGNTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCAKNGDYGYTMDYWGQGTSVTVSS (SEQ ID NO: 56), or - a variable light chain sequence comprising a sequence having at least 80% sequence identity to ENVLTQSPAIMSASLGEKVTMSCASSSVNYMYWYQQKSDASKLSWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGAGTKLELLK (SEQ ID NO: 68); and a variable heavy chain sequence comprising a sequence having at least 80% sequence identity to QVQLKQSGPGLVQPSQSLITCTVSGGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARNGGNYYFDYWGQGTTLTVSS (SEQ ID NO: 72).

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody enhances FH activity, wherein the FH activity is inhibition of alternative complement activation.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein inhibiting alternative complement activation comprises: - Inhibit hemolytic activity, - Inhibition of complement component 3 (C3) deposition, and / or - Increases the binding of FH to C3b, iC3b and / or C3d. 11 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antigen-binding fragment comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. 12 . The antibody or antigen-binding fragment thereof according to claim 11 , wherein the antigen-binding fragment further comprises an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment comprises at least a Fab fragment. The antibody or antigen-binding fragment thereof according to claim 1 , which is a monoclonal antibody or antigen-binding fragment thereof.

15. The antibody or antigen-binding fragment thereof according to claim 1, which is a chimeric or humanized antibody or antigen-binding fragment thereof, comprising human light chain and heavy chain constant regions.

16. An isolated, synthetic or recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to claim 1. A vector comprising the nucleic acid molecule according to claim 16 .

18. A recombinant cell comprising the nucleic acid molecule according to claim 16 or the vector according to claim 17.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid molecule according to claim 16, the vector according to claim 17 or the recombinant cell according to claim 18, and a pharmaceutically acceptable carrier and / or excipient.

20. The antibody or antigen-binding fragment thereof according to claim 1 or the nucleic acid molecule according to claim 16 for use in therapy.

21. The antibody or antigen-binding fragment thereof of claim 1 or the nucleic acid molecule of claim 16 for use in inhibiting alternative complement activation.

22. The antibody or antigen-binding fragment thereof according to claim 1 or the nucleic acid molecule according to claim 16, for use in treating, ameliorating or preventing a disorder associated with alternative pathway complement activation.

23. The antibody or antigen-binding fragment thereof according to claim 1 or the nucleic acid molecule according to claim 16, for use in treating, alleviating or preventing a disorder associated with alternative pathway complement activation, the disorder being selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), and membranoproliferative glomerulonephritis (MPGN).

24. Use of the antibody or antigen-binding fragment thereof according to claim 1, or the nucleic acid molecule according to claim 16, or the vector according to claim 17 in 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), and membranoproliferative glomerulonephritis (MPGN).

25. A method for producing an antibody or an antigen-binding fragment thereof according to claim 1, the method comprising providing a cell having a nucleic acid molecule according to claim 16 or a vector according to claim 17, and allowing the cell to translate a nucleic acid sequence contained by the nucleic acid molecule or the vector, thereby producing the antibody or an antigen-binding fragment thereof according to claim 1.

26. The method of claim 25, further comprising harvesting, purifying and / or isolating the antibody or antigen-binding fragment thereof.

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