Antagonists of complement system for use in methods of treating paraproteinemic neuropathies

Abstract

To provide a therapeutic agent targeting a complement cascade.SOLUTION: The present invention relates to a method of treating a paraproteinemic neuropathy in a subject by administering to the subject an antagonist of a complement system. The antagonist inhibits the complement system upstream of complement factor C5. More specifically, the antagonist may be an antibody or an antigen-binding fragment thereof that blocks or inhibits the complement system by inhibiting a C2b domain of complement factor C2. The paraproteinemic neuropathies that may be treated include, in particular, multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre syndrome (GBS).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods of treating paraproteinemic neuropathies using antagonists of the complement system that block or inhibit the complement system upstream of complement factor C5. Paraproteinemic neuropathies that may be treated include, inter alia, multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre syndrome (GBS). [Background technology]

[0002] BACKGROUND OF THEINVENTION The complement system is an important aspect of the innate immune system that enhances (complements) the ability of antibodies and phagocytes to eliminate microbial invaders and damaged cells from the organism. Complement therefore forms an essential line of defense against infection.

[0003] The response to infection must be rapid and comprehensive enough to prevent risk to the host, yet selective enough to avoid damage to healthy cells. Complement normally achieves this delicate balance by utilizing a stepwise and tightly regulated cascade system of more than 30 soluble and cell surface expressed proteins. Complement factors circulate in the blood as inactive precursor proteins. Activation of this system results in an activation cascade in which one factor activates subsequent factors by specific proteolysis of complement proteins further downstream in the cascade. This cascade can ultimately result in the production of anaphylatoxins that attract and activate macrophages and leukocytes; formation of a lytic membrane attack complex (MAC); and opsonization of targets for phagocytosis and destruction.

[0004] Activation of the complement system can occur via three pathways: the classical pathway; the lectin pathway; and the alternative pathway (see FIG. 1). Each pathway activates the central complement component C3, which leads to activation of the common terminal pathway and the formation of MAC (Muller-Eberhrd, Annu Rev Biochem 1988, 57:321). The classical pathway is often referred to as antibody-dependent because it is strongly triggered by IgM or IgG clusters. This pathway is usually activated when hexameric C1q binds to the Fc region of IgG or IgM molecules present in antibody / antigen complexes. Upon binding, C1s cleaves C4 to produce C4a and C4b. C2 then binds to surface-bound C4b (Mg 2+ C4bC2 complexes bind to C1s (in the presence of C1s) to form C4bC2 complexes, which are then cleaved by activated C1s into two fragments: a smaller 30 kDa fragment, C2b, and a larger 70 kDa fragment, C2a. The larger 70 kDa fragment remains bound to C4b to form the C4bC2a classical pathway C3 convertase. The C3 convertase cleaves C3 into C3a (an anaphylatoxin; potentiates inflammation) and C3b, which can trigger amplification and downstream effector functions.

[0005] Activation of the lectin pathway is mediated by binding of mannose-binding lectin (MBL) or ficolins to bacterial carbohydrate motifs expressed on the surface of pathogens or microorganisms. MBL binding subsequently stimulates activation of MBL-associated serine proteinase-1 (MASP-1) and MASP-2, leading to cleavage of C4 and C2 and the generation of the C4bC2a lectin pathway C3 convertase.

[0006] The alternative pathway can be thought of as an amplification loop that is engaged regardless of the initial trigger. C3b binds directly to targets on the cell surface of microorganisms, foreign bodies, or damaged tissue. Surface-bound C3b can then bind to factor B to form C3bB. This complex is cleaved into Ba and Bb in the presence of factor D. Bb remains associated with C3b to form C3bBb, which is the alternative pathway C3 convertase.

[0007] These three pathways therefore converge at the central C3 convertase. The C3 convertases C4bC2a or C3bBb form multimeric complexes with additional C3b molecules to give rise to the C5 convertases C4bC2aC3b and C3bBbC3b, respectively. These enzymes preferentially cleave the complement factor C5, liberating C5a (an anaphylatoxin; potentiates inflammation) and the fragment C5b. C5b recruits and associates with C6 and C7; this complex becomes inserted into the cell membrane and interacts with C8; this induces the binding of multiple units of C9 molecules to form the C5b-9 membrane attack complex (MAC) or soluble terminal complement complex (TCC). The MAC inserts itself into the cell membrane to form pores, primarily resulting in cytolysis of anuclear cells (e.g., old red blood cells and certain gram-negative bacteria). However, in nucleated cells, MAC formation is tightly regulated and its lytic effect can be counteracted by ion pumps. Furthermore, sublytic levels of MAC can induce host cell damage or activation and act as proinflammatory mediators. In addition to the MAC-mediated effects of the cascade, the anaphylatoxins C3a and C5a act as potent immune modulators, recruiting immune cells to sites of activation. The opsonins C3b and C4b also bind to various complement receptors and can mediate immune complex removal, phagocytosis, or stimulation of B cell responses.

[0008] The complement system is further controlled by several complement regulatory proteins, including C1-inhibitor, which inhibits the initiation steps of the classical and lectin pathways, factor H, which dissociates C3 convertase, factor I, which degrades C4b and C3b, and the plasma proteins vitronectin and clusterin and membrane protein CD59, which inhibit MAC formation (Sahu et al., Immunol Res 1998, 17:109; Campbell et al., Annu Rev Immunol 1988, 6:161).

[0009] Complement forms an essential line of defense against pathogenic organisms, but if not properly controlled, these defenses can be detrimental to host cells and induce or exacerbate immune, inflammatory, and degenerative conditions. When complement is overactivated, as occurs in autoimmune diseases or in individuals with dysfunctional regulatory proteins, it promotes severe inflammatory responses in many organs (Noris and Remuzzi, Semin Nephrol 2013, 33(6): 479-492). Given the ubiquitous expression of complement proteins throughout the body, it is believed that the complement system may play a role in many diseases with an immune component, such as inflammatory diseases, degenerative diseases, cancer, and transplant rejection. The complement system is also increasingly implicated in diseases of the central nervous system, such as Alzheimer's disease (Carpanini et al., Front Immunol 2019, 10:362).

[0010] The unique position of complement as both a first detector of foreign or damaging material and a downstream orchestrator of the immune response makes it an attractive therapeutic target. Indeed, clinical development programs have recently been reported for inhibitors against over a dozen different complement targets.

[0011] Several soluble complement inhibitors are in production. C1-INH (various manufacturers) is currently approved for the treatment of hereditary angioedema and is being evaluated in other disorders such as sepsis and ischemia-reperfusion injury. However, C1-INH is a broad serine protease inhibitor that blocks proteases of both the classical and lectin pathways as well as initiation of noncomplement proteases of the coagulation and contact systems. In contrast, stimulimab (also known as BIVV009, formerly TNT009) is a humanized monoclonal antibody designed to selectively inhibit the classical complement pathway by targeting C1s and has shown promise in the treatment of hemolytic anemia and cold agglutinin disease. Other antibodies that inhibit key proteins of this cascade are also in development. Annexon is developing a monoclonal antibody (ANX005) acting at the level of C1q for neurodegenerative and autoimmune disorders, while Omeros has developed a monoclonal antibody (OMS721) against MASP-2 as a clinical candidate for the treatment of atypical hemolytic uremic syndrome (aHUS).

[0012] Acting on the other side of the complement cascade is the anti-C5 antibody eculizumab (Soliris®), currently approved for use in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and aHUS. This antibody binds to a site on C5 that prevents its activation by the C5 convertase, thereby impairing the release of C5a and the formation of MAC. An anti-C5a antibody (IFX-1) is also being evaluated, as well as several small molecule C5aR1 antagonists (PMX53; PMX205; CCX186) that have shown promise as potential treatments for a variety of disorders.

[0013] Antibodies have also been developed to target the C2 protein of the complement cascade. International Patent Application WO2014 / 189378 describes binding molecules, such as antibodies, with specific C2 activity inhibitory properties. Such binding molecules are described as being useful in treating various human diseases, such as inflammatory diseases or symptoms of ischemia-reperfusion injury.

[0014] Despite the significant efforts to develop therapeutic agents that target the complement cascade, the clinical success of complement inhibitors remains limited.This is likely due to the complex nature of the complement cascade.There is still a need to better understand the role of overactive complement activity in various disorders in order to develop effective therapeutics based on complement inhibition. Summary of the Invention

[0015] SUMMARY OF THE PRESENTINVENT: The present inventors have discovered that targeting the complement cascade upstream of complement factor C5 is likely to be an effective strategy in the treatment of paraproteinemic neuropathies, particularly neuropathies such as multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre syndrome (GBS). Paraproteinemic neuropathies are peripheral neuropathies characterized by the presence of "paraproteins" in the serum. Paraproteins are specific types of monoclonal antibodies or immunoglobulins that are produced in relative excess by abnormal clonal proliferation of B-lymphocytes or plasma cells. Unlike normal immunoglobulin antibodies, paraproteins are not usually able to fight infections.

[0016] As reported and exemplified herein, the complement system plays an important role in the pathology of paraproteinemic neuropathies. In particular, sera from patients with MMN, CIDP, and GBS show in vivo It was found that opsonization of both Schwann cells and motor neurons in vitro activates complement. Importantly, the results presented herein further show that Schwann cells and motor neurons express high levels of the complement regulatory protein CD59, a factor that prevents terminal polymerization of the membrane attack complex (MAC). These results also show that Schwann cells are resistant to complement-mediated lysis. Taken together, this indicates that in paraproteinemic neuropathies there is protection from complement-mediated lysis and that the pathology observed is likely MAC-independent, i.e., caused by complement proteins upstream of the MAC. Thus, the present invention is directed to the treatment of paraproteinemic neuropathies by targeting the complement system upstream of complement factor C5.

[0017] In a first aspect, the invention provides a method of treating a paraproteinaceous neuropathy in a subject, the method comprising administering to the subject an antagonist of the complement system, wherein the antagonist inhibits a pathway upstream of complement factor C5. The invention also provides an antagonist of the complement system for use in treating a paraproteinaceous neuropathy in a subject, wherein the antagonist inhibits a pathway upstream of complement factor C5.

[0018] In certain embodiments, the antagonist inhibits the classical complement pathway and / or the lectin complement pathway.

[0019] In certain embodiments, the paraproteinaceous neuropathy is a demyelinating neuropathy.

[0020] In certain embodiments, the paraproteinaceous neuropathy is characterized by the presence of IgM, IgA, or IgG immunoglobulins.

[0021] In certain embodiments, the paraproteinaceous neuropathy is characterized by the presence of autoantibodies, which may be immunoglobulins of the IgM, IgA, or IgG classes.

[0022] In some embodiments, the paraproteinaceous neuropathy is characterized by the presence of autoantibodies against a neural antigen. The neural antigen may be a ganglioside, or the neural antigen may be myelin-associated glycoprotein (MAG). For embodiments in which the neural antigen is a ganglioside, the ganglioside may be selected from GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GT3, and GQ1b. In some preferred embodiments, the ganglioside is GM1.

[0023] In some embodiments, the paraproteinaceous neuropathy is selected from: multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), Miller Fisher syndrome, acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), chronic ataxic neuropathy-ophthalmoplegia-IgM paraprotein-cold agglutinin-disialosyl antibody (CANOMAD) syndrome, distal acquired demyelinating symmetric (DADS) neuropathy, monoclonal gammopathy-associated peripheral neuropathy, anti-MAG peripheral neuropathy, and POEMS syndrome. In some preferred embodiments, the paraproteinaceous neuropathy is multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), or Guillain-Barre syndrome (GBS). The paraproteinaceous neuropathy is preferably multifocal motor neuropathy (MMN).

[0024] In some embodiments, the antagonist inhibits the complement system upstream of complement factor C3. In some embodiments, the antagonist inhibits C1, C1q, C1r, or C1s. In some embodiments, the antagonist inhibits complement factor C2, C2a, or C2b. In some embodiments, the antagonist inhibits complement factor C3, C3a, or C3b. In some embodiments, the antagonist inhibits complement factor C4, C4a, or C4b.

[0025] In certain embodiments, the antagonist is selected from: an inhibitory RNA species, e.g., siRNA or shRNA; a small molecule inhibitor; a biological antagonist, e.g., an inhibitory peptide or an antibody mimetic, e.g., an affibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, fynomer, versabody, or duocalin; or an antibody or antigen-binding fragment thereof.

[0026] In certain embodiments, the antagonist is selected from: compstatin Cp40 (Amyndas); PEG-Cp40 (Amyndas); AMY-101 (Amyndas); AMY-201 (Amyndas); APL-1 and APL-2 (Apellis); Cinryze (Shire); CDX-1135 (Celldex); APT070 myrococept (MRC); HC3-1496 (InCode); nafamostat (Torii Pharmaceutical), and vaccinia virus complement control protein (VCP).

[0027] In a preferred embodiment, the antagonist is an antibody or antigen-binding fragment thereof, preferably an IgG antibody or antigen-binding fragment thereof.

[0028] In certain embodiments, the antigen-binding fragment is selected from: an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), an F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a one-arm (monovalent) antibody, a diabody, a triabody, a tetrabody, a unibody, a domain antibody, and a nanobody.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof binds to complement factors C1, C1q, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b.

[0030] In certain embodiments, the antibody or antigen-binding fragment is selected from: stimulimab (Bioverativ); ANX005 (Annexon); mAb H17 (Elusys Therapeutics); and TNT003 (True North).

[0031] In certain embodiments, the antibody or antigen-binding fragment binds to complement factor C2. In certain embodiments, the antibody or antigen-binding fragment binds to the C2b domain of complement factor C2.

[0032] In certain embodiments, the antibody or antigen-binding fragment comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains have the CDR sequences: [ka] HCDR3 comprising or consisting of: [ka] HCDR2 comprising or consisting of: [ka] HCDR1 comprising or consisting of: [ka] LCDR3 comprising or consisting of: [ka] LCDR2 comprising or consisting of: [ka] The LCDR1 comprises or consists of:

[0033] In certain embodiments, the antibody or antigen-binding fragment comprises a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 70% identity thereto. In certain embodiments, the antibody or antigen-binding fragment comprises a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 9.

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG heavy chain constant domain.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:16 and a light chain comprising the amino acid sequence of SEQ ID NO:20.

[0036] In certain embodiments, the method further comprises administering IVIg to the subject.

[0037] In certain embodiments, the method further comprises administering rituximab to the subject. [Brief description of the drawings]

[0038] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 is a schematic diagram showing the cell signaling cascades involved in the classical, lectin, and alternative pathways of the complement system. [Diagram 2] Figure 2 shows the expression profile of various membrane proteins in live Schwann cells (sNF02.2). Schwann cells were stained for various membrane proteins, particularly those associated with the complement cascade, using flow cytometry analysis. The seven graphs show: (A) CD46, (B) CD55, (C) CD59, (D) CD64, (E) CD88, (F) GM1, and (G) MAG. [Diagram 3]Figure 3 shows the expression profile of CRP in sNF02.2 Schwann cells with or without treatment with phospholipase-C (PL-C). sNF02.2 Schwann cells were treated with various concentrations of PL-C (1-0.5-0.25 U / mL) and incubated at 37°C for 1 h, and then stained with antibodies detecting CD46 (A), CD55 (B), and CD59 (C), respectively. [Figure 4] FIG. 4 shows the resistance of Schwann cells (sNF96.2) to complement-mediated lysis. Each graph in FIG. 4 shows "no PL-C treatment" on the left half of the graph and "PL-C treatment" on the right half of the graph. (A) PL-C treatment reduced both CD59 and CD55 expression by cleavage of the GPI anchor. Expression of the transmembrane protein CD46 was unaffected (right y-axis). PL-C also had no effect on GM1 expression (left y-axis, panel A). (B) Cells are essentially protected from C3 fixation. C3 fixation was observed in the absence of opsonization with MMN patient serum, and C3 fixation was slightly increased by opsonization with MMN patient serum. PL-C-treated cells were more susceptible to C3 fixation, especially after opsonization. ARGX-117 inhibited C3 fixation, but only a small inhibition was seen after opsonization and PL-C treatment. This is likely due to a combination of opsonization density of anti-GM1 antibodies and serum antibodies against other epitopes. (C) Similar results to those seen in panel B were observed when MAC fixation was quantified. (D) Schwann cells were protected from complement-mediated cytolysis - this is due to high level CD59 expression. Cells not treated with PL-C remained viable after opsonization and incubation with complement-active serum. After PL-C treatment, cytolysis was observed when Schwann cells were opsonized with patient-derived anti-GM1 antibodies, which was inhibited to baseline by ARGX-117. [Diagram 5]Figure 5 shows complement C3 fixation in sNF02.2 Schwann cells by anti-HLA mAb. Cells were opsonized with increasing concentrations of W6 / 32 (anti-HLA antibody) and then HPS (pooled human serum) was added to activate the complement pathway. Both EDTA and TNT009 (480 μg / mL) were added to assess complement specificity. [Figure 6] Figure 6 shows GM1 expression and IgM binding on Schwann cells. (A) sNF02.2 Schwann cells were stained with CTb and GM1 expression was detected using flow cytometry. (B) Flow cytometric detection of IgM binding to cultured sNF02.2 Schwann cells after opsonization with MMN patient serum (shaded bars) or without complement activation (white bars). [Figure 7] Figure 7 shows binding of IgM in MMN patient sera to sNF02.2 Schwann cells. Schwann cells were opsonized with various patient samples containing various GM1 titers, and all showed IgM binding upon incubation with sNF02.2 cells. (A) MFI of IgM staining. (B) Percentage of IgM positive Schwann cells. [Figure 8]Figure 8 shows optimization of C3 fixation on sNF02.2 Schwann cells after opsonization with MMN patient serum. 50,000 Schwann cells were seeded in a 96-well plate and opsonized with MMN patient serum (1 h, RT). Each of the graphs in Figure 8 shows "without opsonization" on the left half of the graph and "with opsonization" on the right half of the graph. (A) Complement was activated using various percentages of complement-active serum: 10% (left graph), 5% (middle graph), or 2.5% (right graph). Complement-active serum (black bars) and stripped serum (=complement-active serum previously incubated with Schwann cells) (gray bars) were examined. (B) Summary of the results shown in (A) comparing serum (black bars) and stripped serum (light gray bars). (C) C3 fixation on Schwann cells using different C3 detection antibodies: C3 FITC (LSBio, clone 6C9) (left graph), C3-BIO (LSBio, clone 6C9) + streptavidin APC (middle graph), or C3-BIO (polyclonal sheep anti-human C3) + streptavidin APC (right graph). Cells were activated with either 10% serum (black bars) or 5% serum (gray bars). White bars represent EDTA controls, all as expected. [Figure 9] Figure 9 shows the C2-dependence of complement activation in Schwann cells. Schwann cells were seeded in 96-well plates, opsonized (1 h, RT) with MMN patient serum (MMN-005), and then incubated with C2-depleted serum supplemented with increasing concentrations of rhC2 from 1.11 μg / mL up to 30 μg / mL (physiological concentration). After 1 h (37°C), C3 fixation was measured using flow cytometry after staining with C3-BIO (LSBio, clone 6C9). [Figure 10]FIG. 10 shows dose-dependent inhibition of C3 fixation by ARGX-117 on sNF02.2 Schwann cells opsonized with MMN patient serum. Schwann cells were transferred to 96-well plates (50,000 cells / well), opsonized with MMN patient serum (1 h, RT), and then incubated with 5% complement-active serum preincubated with complement blocking antibodies or EDTA (20 min, RT). Detection of C3 fixation was performed by staining Schwann cells with C3-BIO (LSBio, clone 6C9) and streptavidin-APC. (A) C3 fixation on Schwann cells by MFI value of APC. (B) Percentage inhibition of C3 fixation on Schwann cells calculated by setting 5% serum as 0% inhibition and EDTA 10 mM as 100% inhibition. [Figure 11] FIG. 11 shows cytokine secretion by sNF02.2 Schwann cells after complement activation induced by MMN serum. Schwann cells were seeded in 24-well plates and opsonized with MMN patient serum (1 hr, RT), and complement-activating serum was added in the presence or absence of complement-blocking antibodies. After 48 hr, supernatants were collected and cytokine secretion was measured using a Luminex platform. Three graphs show: (A) IL-6, (B) IL-8, and (C) MCP-1. Each of the graphs in FIG. 11 shows the following bars in order (from left to right): no stimulation, IL-1b 10 ng / mL, IL-1b 5 ng / mL, IL-1b 2.5 ng / mL, TNF-a 50 ng / mL, TNF-a 25 ... 12.5 ng / mL, serum only, MMN-05 only, MMN-05 + serum, MMN-05 + serum MgEGTA, MMN-05 + serum ARGX-117, MMN-05 + serum TNT009, MMN-05 + serum eculizumab, MMN-05 + serum HI, MMN-73 only, MMN-73 + serum, MMN-73 + serum MgEGTA, MMN-73 + serum ARGX-117, MMN-73 + serum + TNT009, MMN-73 + serum eculizumab, MMN-73 + serum HI. [Figure 12] FIG. 12 shows a proposed mechanism of complement events triggered by anti-GM1 autoantibodies present in MMN patients. [Figure 13] Figure 13 shows the expression of membrane complement proteins, including complement regulatory protein (CRP), in fixed iPSC-MNs. Induced pluripotent stem cell-derived motor neurons (iPSC-MNs) were cultured and fixed on glass coverslips with 4% PFA and then stained for expression markers. MGV = mean grey value. [Figure 14] Figure 14 shows the C2-dependence of C3 fixation. iPSC-derived motor neurons were opsonized with C2-depleted serum and reconstituted with increasing concentrations of purified human C2 (hC2) to assess complement activation by measuring C3 fixation. iPSC-MNs were cultured for 3 days before being fixed and then stained to detect GM1 expression and C3 deposition. Images were analyzed at 40x magnification and mean grey values ​​(MGV) were calculated for GM1 alone, C3 alone, or the ratio between the two. [Figure 15] FIG. 15 shows that ARGX-117 blocked complement in other immune-mediated neuropathies. Motor neurons were opsonized with GBS or CIPD patient serum in the presence or absence of ARGX-117 (200 μg / mL). iPSC-derived motor neurons were cultured for 12-14 days and then fixed and stained to detect GM1 expression and C3 deposition. Images were analyzed at 40x magnification and mean gray values ​​(MGV) were calculated for GM1 alone, C3 alone, or the ratio between the two (C3 / GM1). Each graph in FIG. 15 compares "serum only" (left bar), "serum + EDTA" (middle bar), and "ARGX-117 200 μg / mL" (right bar). [Figure 16]FIG. 16 shows the effect of IVIg on C3 fixation using iPSC-MNs opsonized with MMN patient samples. iPSC-derived motor neurons were cultured for 12-14 days, then fixed and subsequently stained for GM-1 expression and C3 deposition. Two different IVIg batches were tested: GammaQuin and Nanogam (both used at 50 mg / mL). Images were analyzed at 40x magnification and mean grey values ​​(MGV) were calculated for GM1 only, C3 only, or the ratio between the two (C3 / GM1). Each of the graphs in FIG. 16 shows the following ordered bars (from left to right): serum only, serum+EDTA, GammaQuin ops, GammaQuin ops+comp, GammaQuin comp, Nanogram ops, Nanogram ops+comp, Nanogram comp. [Figure 17] Figure 17 shows the anti-idiotypic effect of IVIg on GM1 binding by MMN patient sera. ELISA was performed with MMN patient sera MMN005 and MMN073. GM1 was coated onto 96-well plates and incubated with MMN patient sera with or without the addition of 50 μg / mL IVIg (2 batches: GammaQuin and Nanogam), after which IgM was detected using an anti-human IgM antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] DETAILED DESCRIPTION A. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Without limiting any term, further clarification of some of the terms used herein is provided below.

[0040] "Paraproteinaceous Neuropathies" - As used herein, the term "paraproteinaceous neuropathies" or "PPN" describes a series of peripheral neuropathies characterized by the presence of homogeneous immunoglobulins in serum. Homogeneous immunoglobulins are known as "paraproteins." Abnormal clonal proliferation of B-lymphocytes or plasma cells, which may or may not occur in association with hematological malignancies, produces immunoglobulins in excess. Several disorders of the peripheral nervous system are closely associated with the presence of excessive amounts of abnormal immunoglobulins in the blood. PPN may result from the interaction of antibodies with specific antigenic targets or from the deposition of immunoglobulins in peripheral nerves. Exemplary paraproteinaceous neuropathies that may be treated by the present invention include MMN, CIDP, and GBS.

[0041] "Multifocal Motor Neuropathy" - Multifocal motor neuropathy or MMN is a rare disorder with a prevalence of approximately 0.6 per 100,000 individuals, with men more frequently affected than women (ratio of 2.7:1) (Harschnitz et al., J Clin Immunol 2014, 34:112-119). MMN is a chronic immune-mediated neuropathy characterized by asymmetric distal-predominant muscle weakness of the limbs. The hallmark of the disease is the presence of multifocal motor conduction block, and patients often show high serum levels of IgM antibodies against the glycosphingolipid GM1, which is abundantly expressed in the perinodal regions of peripheral nerves. These autologous GM1 IgM antibodies have complement-activating properties and are a determinant of disease severity (Vlam et al., Neurol Neuroimmunol Neuroinflamm 2015, 25;2(4)). GM1 is abundantly expressed in peripheral motor nerves and is localized in both the axon sheath and myelin of peripheral nerves. GM1 has several important functions necessary for action potential propagation and maintaining conduction velocity. Maximal GM1 expression is found at the nodes of Ranvier and adjacent paranodes, where GM1 anchors potassium channels and clusters sodium channels, maintaining tight junctions through stabilization of the paranodes. In addition, GM1 functions both as a receptor modulator for neurotrophic factors that control neuritogenesis and apoptosis, and as part of multimolecular assemblies in lipid rafts in membrane signaling and trafficking. Disruption of these functions results in failure of conduction across the paranodal region. Anti-GM1 IgM antibodies present in MMN patients are produced by activated B cells (plasma cells); however, the mechanism of this B cell activation has yet to be established.

[0042] "Guillain-Barré syndrome" - Guillain-Barré syndrome or GBS has an incidence of 0.81-1.89 cases per 100,000 individuals, with men more frequently affected than women (ratio 3:2) (Kieseier et al., Nature Reviews, 2018, 4:31). In 60-70% of cases, the first symptoms of GBS appear 1-3 weeks after an acute infection, usually an upper respiratory tract or gastrointestinal infection. The initial symptoms of GBS are sensory changes or pain accompanied by muscle weakness, usually beginning in the hands and feet. This often spreads to the arms and upper body. Autoantibodies against various gangliosides found in axons are used to aid in the diagnosis of certain subtypes of GBS. For example, anti-GM1 and anti-GD1a IgG antibodies are found in the serum of patients suffering from acute motor axonal neuropathy (AMAN) and acute motor and sensory axonal neuropathy (AMSAN). These antibodies bind to the nodes of Ranvier where they disrupt the ultrastructural arrays involved in sodium channel clustering, resulting in slowed axonal conduction and loss of function, or they bind to motor nerve terminals and cause degeneration of presynaptic nerve terminals.

[0043] "Chronic Inflammatory Demyelinating Polyneuropathy" - Chronic inflammatory demyelinating polyneuropathy or CIDP is the most common immune-mediated neuropathy, with a reported prevalence ranging from 0.8 to 8.9 cases per 100,000 individuals (Kieseier et al., Nature Reviews, 2018, 4:31). Men are more frequently affected than women (2:1 ratio). CIDP is closely related to GBS, which is considered the chronic counterpart of that acute disease. The most common symptoms of CIDP are weakness, numbness, and tingling in the legs, arms, fingers, and hands. Other symptoms include fatigue, pain, balance problems, and impaired walking ability. Some variants of CIDP exhibit autoimmunity against proteins of the nodes of Ranvier. These variants include a subgroup of inflammatory neuropathies with IgG4 autoantibodies against the paranodal proteins neurophasin-186, neurophasin-155, contactin-1, and caspr-1 (Querol et al., Nat Rev Neurol. 2017, 13(9):533-547). These proteins play a crucial role in compartmentalization of myelinated axons into nodes, paranodes, and internodes. Compartmentalization is required for saltatory conduction, as it maintains the separation of voltage-gated sodium and voltage-gated potassium channels involved in the transmission of action potentials. Disruption of these regions can result in slowed or blocked nerve conduction.

[0044] "Antagonist of the Complement System" - As used herein, the phrase "antagonist of the complement system" or "complement antagonist" refers to any agent capable of blocking or inhibiting the function of a complement factor or component of the complement cascade, thereby inhibiting or reducing complement activity. Complement system antagonists can block or inhibit the classical complement pathway, the lectin complement pathway, the alternative complement pathway, or any combination thereof. Preferably, complement system antagonists inhibit the classical and lectin complement pathways by targeting complement factors common to both of these pathways. Complement antagonists for use with the methods described herein inhibit the complement system upstream of complement factor C5. This means that the antagonist inhibits or reduces complement activity by inhibiting any component or factor of the complement pathway prior to C5 in the complement cascade, i.e., the antagonist does not directly inhibit C5 or inhibit any complement factor downstream of C5. For example, an antagonist can inhibit the function of complement factors C1, C2, C3, or C4, or any combination thereof. Inhibiting the function of a complement factor means that the complement factor cannot play its role in the overall cascade of complement activation, even in the presence of its upstream activation signal.

[0045] Antagonists for use in the present invention can take the form of any suitable agent and can directly or indirectly block or inhibit the function of a complement factor or component. Antagonists can inhibit the function of a target by downregulating the expression of the target, for example, by siRNA technology. In this regard, suitable antagonists include inhibitory RNA species, such as siRNA or shRNA. Antagonists can inhibit the function of a target by directly binding to the target; for example, antagonists can directly bind to the target and inhibit the target from activating the next complement factor in the cascade. In a preferred embodiment, the antagonist is specific to its target. For example, an antagonist of C2 preferentially inhibits the function of C2 compared to other molecular targets. The antagonist usually achieves the required level of specificity by directly interacting with its target, for example by selectively binding to complement proteins. Suitable agents that can serve as antagonists for use in the methods described herein include, but are not limited to: small molecule inhibitors; and biological antagonists including inhibitory peptides, antibody mimetics, such as affibodies, affilins, affitins, adnectins, atrimers, evasins, DARPins, anticalins, avimers, finomers, versabodies, and duocalins. In a preferred embodiment, the antagonist for use in the present invention is an antibody or an antigen-binding fragment thereof.

[0046] "Antibody" or "Immunoglobulin" - As used herein, the term "immunoglobulin" includes a polypeptide having a combination of two heavy chains and two light chains, regardless of whether it possesses any relevant specific immune reactivity. "Antibody" refers to such an aggregate having a significant known specific immune reactive activity against an antigen of interest. Antibodies and immunoglobulins comprise light and heavy chains with or without interchain covalent bonds between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0047] The general term "immunoglobulin" includes five different classes of antibodies that can be biochemically distinguished. For IgG, immunoglobulins contain two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chains with a molecular weight of 53,000-70,000. These four chains are linked in a "Y" configuration by disulfide bonds. In this "Y" configuration, the light chains support the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. The light chains of antibodies are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either kappa or lambda light chains. In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulins are produced by either hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom end of each chain. Those skilled in the art will recognize that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization. Modified forms of each of these classes and isotypes are readily discernible to those skilled in the art in view of the present disclosure and are therefore within the scope of the present invention.

[0048] As shown above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and the VH domain of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This four-chain antibody structure forms an antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three complementarity determining regions (CDRs) on each of the VH and VL chains.

[0049] The term "antibody" as used herein is also intended to encompass "VHH antibodies" or "heavy chain only antibodies".

[0050] "VHH antibody" - As used herein, the term "VHH antibody" or "heavy chain only antibody" refers to a type of antibody produced by species of the Camelidae family, which includes camels, llamas, and alpacas. Heavy chain only antibodies or VHH antibodies are composed of two heavy chains and lack light chains. Each heavy chain has a variable domain at the N-terminus, and these variable domains are referred to as "VHH" domains to distinguish them from the variable domains of the heavy chains of conventional heterotetrameric antibodies, i.e., VH domains, described above.

[0051] "Variable Region" or "Variable Domain" - The terms "variable region" and "variable domain" are used interchangeably herein and are intended to have equivalent meanings. The term "variable" refers to the fact that certain portions of the variable domains, VH and VL, differ extensively in sequence among antibodies and is used with respect to the binding and specificity of each particular antibody for its target antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains that form part of the antigen-binding site. The first, second, and third hypervariable loops of a Vlamda light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and can be defined as comprising residues 24-33 (L1(λ) of 9, 10, or 11 amino acid residues), 49-53 (L2(λ) of 3 residues), and 90-96 (L3(λ) of 5 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of the V kappa light chain domain are referred to herein as L1(κ), L2(κ), and L3(κ), and can be defined as comprising residues 25-33 (L1(κ) of 6, 7, 8, 11, 12, or 13 residues), 49-53 (L2(κ) of 3 residues), and 90-97 (L3(κ) of 6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of a VH domain are referred to herein as H1, H2, and H3, and can be defined as comprising residues 25-33 (H1 consisting of 7, 8, or 9 residues), 52-56 (H2 consisting of 3 or 4 residues), and 91-105 (H3, which varies greatly in length) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

[0052] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops, respectively, of the VL domain and include hypervariable loops from both the Vkappa and Vlambda isotypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops, respectively, of the VH domain and include hypervariable loops from any of the known heavy chain isotypes, including gamma, epsilon, delta, alpha, or mu.

[0053] Hypervariable loops L1, L2, L3, H1, H2, and H3 may each comprise a portion of a "complementarity determining region" or "CDR" as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous, since hypervariable loops (HV) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined on the basis of sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983), and the boundaries of HV and CDR differ in some VH and VL domains.

[0054] The CDRs of the VL and VH domains can generally be defined as comprising the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Thus, HVs can be contained within the corresponding CDRs, and unless otherwise indicated, references herein to the "hypervariable loops" of the VH and VL domains should be construed to encompass the corresponding CDRs, and vice versa.

[0055] The more highly conserved parts of the variable domains are called framework regions (FRs), as defined below. Natural heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4, respectively) that are connected by three hypervariable loops and are primarily in a β-sheet configuration. The hypervariable loops of each chain are held in close proximity by the FRs and, together with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of the antibody. Structural analysis of antibodies has revealed a relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high degree of sequence variability, five of these six loops adopt only a small repertoire of main-chain conformations, called "canonical structures". These conformations are determined firstly by the length of the loop and secondly by the presence of key residues at specific positions in the loop and in the framework regions that determine the conformation by their packing, hydrogen bonding, or ability to adopt unusual main-chain conformations.

[0056] "CDR" - As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Proteins of Immunological Interest (1991), as well as Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where these definitions include overlapping or subsets of amino acid residues when compared with each other. The amino acid residues encompassing the CDRs defined by each of the above cited references are provided for comparison. Preferably, the term "CDR" refers to the CDRs defined by Kabat based on sequence comparisons. Table 1: CDR definitions [Table 1] 1 Residue numbering follows the nomenclature of Kabat et al. (supra). 2 Residue numbering follows the nomenclature of Chothia et al. (supra). 3 Residue numbering follows the nomenclature of MacCallum et al. (supra).

[0057] "Framework Region" - The term "framework region" or "FR region" as used herein includes amino acid residues that are part of the variable region (e.g., using the Kabat definition of CDR) but are not part of the CDRs. Thus, the variable region framework is about 100-120 amino acids in length, but includes only amino acids outside the CDRs. For the specific example of a heavy chain variable domain, and for the CDRs defined by Kabat et al., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1-30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acid 103 to the end of the variable region. The framework regions of the light chain are similarly separated by each of the light chain variable region CDRs. Similarly, using the definitions for CDRs by Chothia et al. or McCallum et al., the framework region boundaries are separated by the respective CDR ends as described above. In a preferred embodiment, the CDRs are as defined by Kabat.

[0058] In natural antibodies, the six CDRs present on each monomeric antibody are short non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding site when the antibody assumes its three-dimensional shape in an aqueous environment. The remainder of the heavy and light variable domains show less intermolecular variability in terms of amino acid sequence and are called framework regions. The framework regions largely adopt a β-sheet conformation. The CDRs form loops that connect to, and in some cases form part of, the β-sheet structure. These framework regions thus act to form a scaffold that orients the six CDRs by non-covalent interactions between the chains. The antigen-binding site formed by the positioned CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The position of the CDRs can be easily identified by one skilled in the art.

[0059] "Constant Region" - As used herein, the term "constant region" refers to the portion of an antibody outside the variable domain or regions. Immunoglobulin light chains have a single domain, the "constant region", usually called the "CL" or "CL1 domain". This domain is C-terminal to the VL domain. Immunoglobulin heavy chains differ in their constant regions depending on the immunoglobulin class (gamma, mu, alpha, delta, epsilon). Heavy chains gamma, alpha, and delta have a constant region consisting of three immunoglobulin domains (called CH1, CH2, and CH3) with a flexible hinge region separating the CH1 and CH2 domains. Heavy chains mu and epsilon have a constant region consisting of four domains (CH1-CH4). The heavy chain constant domain is located C-terminal to the VH domain.

[0060] The numbering of amino acids in heavy and light immunoglobulin chains runs from the N-terminus at the forked end of the Y to the C-terminus at the bottom end of each chain. Various numbering systems are used to define the constant domains of immunoglobulin heavy and light chains. According to the EU numbering system, the heavy chain constant domain of an IgG molecule is identified as follows: CH1-amino acid residues 118-215; CH2-amino acid residues 231-340; CH3-amino acid residues 341-446. According to the Kabat numbering system, the heavy chain constant domain of an IgG molecule is identified as follows: CH1-amino acid residues 114-223; CH2-amino acid residues 244-360; CH3-amino acid residues 361-477. The term "Fc domain" or "Fc region" defines the portion of the constant region of a heavy chain that typically includes the CH2 and CH3 domains. The Fc region may also include some residues from the hinge region. The "hinge region" comprises the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux KH et al., J. Immunol. 161:4083-90 1998). Antibodies of the invention that comprise a "fully human" hinge region can contain one of the hinge region sequences shown in Table 2 below. Table 2: Human hinge sequences [Table 2]

[0061] "Fragment" - The term "fragment" or "antigen-binding fragment" refers to a part or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which they were derived) for antigen binding. As used herein, the term "fragment" of an antibody molecule includes antigen-binding fragments of an antibody, such as an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, a Fd fragment, a Fv fragment, a one-arm (monovalent) antibody, a diabody, a triabody, a tetrabody, or any antigen-binding molecule formed by combination, assembly, or conjugation of such antigen-binding fragments. The term "antigen-binding fragment" as used herein is further intended to encompass an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody. Fragments can be obtained, for example, by chemical or enzymatic treatment of an intact or complete antibody or antibody chain, or by recombinant means.

[0062] "Specificity" and "multispecific antibodies" -Antibodies for use in the methods described herein bind to a target antigen within the complement system. The antibody preferably "specifically binds" to its target antigen, where the term "specifically binds" refers to the ability of any antibody to preferentially immunoreact with a given target, e.g., C1, C2, C3, or C4. An antibody can be monospecific and contain one or more binding sites that specifically bind to a particular target. An antibody can be incorporated into a "multispecific antibody" format, e.g., a bispecific antibody, where the multispecific antibody binds to more than one target antigen. To achieve multispecificity, "multispecific antibodies" are usually engineered to contain different combinations or pairings of heavy and light chain polypeptides with different VH-VL pairs. Multispecific, and particularly bispecific, antibodies can be engineered to adopt the overall conformation of a native antibody, e.g., a Y-shaped antibody with Fab arms of different specificities conjugated to an Fc region. Alternatively, multispecific antibodies, e.g., bispecific antibodies, can be engineered to adopt a non-native conformation, e.g., where variable domains or variable domain pairs with different specificities are positioned at opposite ends of the Fc region.

[0063] "Modified antibodies" - As used herein, the term "modified antibodies" includes synthetic forms of antibodies that have been modified to be non-natural, such as antibodies that contain at least two heavy chain portions but do not contain two complete heavy chains (e.g., domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been modified to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules connected to scFv molecules, etc. scFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. Additionally, the term "modified antibodies" includes, but is not limited to, multivalent forms of antibodies (e.g., trivalent, tetravalent, etc. antibodies that bind to three or more copies of the same antigen). In another embodiment, the modified antibodies of the invention are fusion proteins that include at least one heavy chain portion that lacks a CH2 domain and a binding domain of a polypeptide that includes the binding portion of one member of a receptor-ligand pair.

[0064] "Humanizing substitution" - As used herein, the term "humanizing substitution" refers to an amino acid substitution in which an amino acid residue present at a particular position in a VH or VL domain of an antibody is replaced with an amino acid residue occurring at an equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be a VH or VL domain encoded by human germline. The humanizing substitution may be made in the framework regions and / or CDRs of an antibody defined herein.

[0065] "Humanized variant" - As used herein, the term "humanized variant" or "humanized antibody" refers to a variant antibody that contains one or more "humanizing substitutions" compared to a reference antibody, where portions of the reference antibody (e.g., the VH domain and / or VL domain or portions thereof containing at least one CDR) have amino acids derived from a non-human species, and where the "humanizing substitutions" occur within the amino acid sequence derived from the non-human species.

[0066] "Germlined variant" - The term "germlined variant" or "germlined antibody" is used herein to specifically refer to a "humanized variant" in which "humanizing substitutions" result in the replacement of one or more amino acid residues present at a particular position in the VH or VL domain of an antibody with an amino acid residue occurring at an equivalent position in a reference human VH or VL domain encoded by human germline. For any given "germlined variant", the substituted amino acid residues substituted into the germlined variant are generally taken exclusively or preferentially from a single VH or VL domain encoded by human germline. The terms "humanized variant" and "germlined variant" are often used interchangeably. The introduction of one or more "humanizing substitutions" into a camelid-derived (e.g., llama-derived) VH or VL domain results in the production of a "humanized variant" of the camelid (llama)-derived VH or VL domain. Where the substituted amino acid residues are derived from a single VH or VL domain sequence that is preferentially or exclusively encoded by the human germline, this can result in a "human germlined variant" of a camelid (llama)-derived VH or VL domain.

[0067] "Affinity variant" - As used herein, the term "affinity variant" refers to a variant antibody that exhibits one or more changes in amino acid sequence compared to a reference antibody, where the affinity variant exhibits modified affinity to a target antigen compared to the reference antibody. For example, the affinity variant exhibits altered affinity to the target when compared to the reference antibody. Preferably, the affinity variant exhibits improved affinity to the target antigen when compared to the reference antibody. Affinity variants usually exhibit one or more changes in amino acid sequence in the CDRs when compared to the reference antibody. Such substitutions can result in the replacement of the original amino acid present at a given position in the CDR with a different amino acid residue, which can be a natural amino acid residue or a non-natural amino acid residue. The amino acid substitutions can be conservative or non-conservative.

[0068] "Subject" - As used herein, the term "subject" refers to a mammal, preferably a human. A subject can be either male or female. A subject can exhibit one or more symptoms consistent with paraproteinemic neuropathy. In certain embodiments, a subject can be a patient, where a patient is an individual who is receiving medical care for the treatment of paraproteinemic neuropathy and / or is actively seeking medical care for the treatment of paraproteinemic neuropathy.

[0069] B. Methods of Treatment The present invention provides a method of treating paraproteinemic neuropathy. The method involves administration of an antagonist of the complement system to a subject, where the antagonist inhibits the complement system upstream of complement factor C5. The present invention also provides an antagonist of the complement system for use in treating paraproteinemic neuropathy, where the antagonist inhibits the complement system upstream of complement factor C5.

[0070] Paraproteinaceous neuropathies are defined elsewhere herein and include a class of peripheral neuropathies characterized by the presence of homogeneous immunoglobulins or "paraproteins" in serum. Peripheral neuropathies are diseases or degenerative conditions of peripheral nerves in which motor, sensory, or vasomotor nerve fibers are affected. Of particular interest are immune-mediated neuropathies, which represent a class of peripheral neuropathies caused by immune-mediated damage to peripheral nerves. In general, immune-mediated peripheral neuropathies are characterized by progressive muscle weakness, often accompanied by sensory disturbances such as pain and numbness. These disorders are presumed to be caused by autoreactive antibodies in serum that bind to components of myelin or proteins at the nodes of Ranvier. A common feature of these disorders is that the blood-nerve barrier (BNB) is compromised, allowing autoantibodies, complement components, and inflammatory cells access to the inner membrane of the nerve. This breakdown of BNB is thought to be triggered by circulating cytokines such as VEGF, TNFα, and IL1-β, as well as metalloproteases secreted by T cells. Upon entry into the nerve, autoantibodies can bind to neuroantigens such as myelin-associated glycoprotein (MAG) or gangliosides, present on myelin or at the junctions between axons, respectively. As explained elsewhere herein, the formation of antibody / antigen immune complexes can trigger the classical pathway of the complement system by recruitment of C1q.

[0071] Paraproteinemic neuropathies are a group of immune-mediated neuropathies characterized by excess immunoglobulins in the serum. PPN is often associated with the presence of autoantibodies. Existing treatment strategies for this class of neuropathies currently include intravenous immunoglobulin (IVIg), plasmapheresis, corticosteroids, and azathioprine. These include rituximab, chlorambucil, fludarabine, and melphalan (see Rison and Beydoun, BMC Neurology. (2016) 16:13).

[0072] As reported herein, immunoglobulins present in the serum of patients with various paraproteinaceous neuropathies can activate complement, supporting a role for complement-mediated tissue damage in the pathology seen in PPN patients. However, previous clinical studies testing eculizumab (Soliris™-anti-C5 antibody) in the treatment of multifocal motor neuropathy (MMN) have shown no efficacy (Fitzpatrick et al., J Peripher Nerv Syst, 2011, 16(2):84-91).

[0073] Importantly, we have shown that both Schwann cells and motor neurons upregulate the complement regulatory protein CD59, a protein that protects cells from MAC-mediated lysis. This observation indicates that elements of the complement cascade upstream of C5 play a more important role in PPN pathology. This may well explain the lack of efficacy previously seen with eculizumab. Without wishing to be bound by theory, it is believed that complement activation in paraproteinemic neuropathies induces the release of cytokines and / or chemokines through activation of C3aR, which has been shown to be expressed on Schwann cells and motor neurons. Chemokines such as MCP-1 may play a role in attracting inflammatory cells and thereby promoting neurological damage.

[0074] The present invention seeks to improve the treatment of PPN by targeting complement activity upstream of complement factor C5.

[0075] Neuropathies to be treated according to the methods described herein include all peripheral neuropathies classified as "paraproteinemic neuropathies". This includes both acute and chronic disorders. Paraproteinemic neuropathies can be classified as demyelinating or axonal, or a combination thereof, depending on whether myelin and / or axons are damaged. In some embodiments, the neuropathy to be treated is an axonal neuropathy. In some embodiments, the neuropathy to be treated is a demyelinating neuropathy, e.g., a chronic demyelinating neuropathy.

[0076] Paraproteinaceous neuropathies are often characterized by the presence of autoantibodies. Thus, in certain embodiments, the neuropathy to be treated according to the methods described herein is characterized by the presence of autoantibodies. High titers of serum autoantibodies that recognize neural antigens occur in some forms of peripheral sensory, motor, and sensorimotor neuropathies. The antibodies often react with glycosylated cell surface molecules, including glycolipids, glycoproteins, and glycosaminoglycans, but antibodies against intracellular proteins have also been described. There are some correlations between antibody specificity and clinical symptoms, suggesting that the neuropathy may be caused by the antibodies. Typically, autoantibodies of IgM or IgG isotypes can activate the classical pathway of the peripheral nerve complement system, as described elsewhere herein. Access of autoantibodies and complement to peripheral nerves becomes possible after the breakdown of the blood-nerve barrier (BNB). In certain embodiments, paraproteinaceous neuropathies are characterized by high titers of autoantibodies. In certain embodiments, the paraproteinaceous neuropathy is characterized by the presence of IgG, IgM, or IgA autoantibodies.

[0077] In one embodiment, the paraproteinaceous neuropathy is characterized by the presence of autoantibodies against neural antigens. In one embodiment, the neural antigen is a protein at the node of Ranvier. In another embodiment, the neural antigen is a protein at the myelin sheath. Myelin-associated glycoprotein (MAG) is a component of peripheral and central nervous system myelin. High titer IgM antibodies against MAG are associated with sensorimotor demyelinating peripheral neuropathy. MAG antibodies are usually associated with the presence of IgM monoclonal protein. In one embodiment, the neural antigen is myelin-associated glycoprotein (MAG).

[0078] Gangliosides are a group of glycosphingolipids that are widely distributed in membrane components of the nervous system. In some embodiments, the neuroantigen is a ganglioside. The gangliosides most commonly recognized by neuropathy-associated autoantibodies are GM1, GD1a, GD1b, and GQ1b. In some embodiments, the ganglioside is selected from GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GT3, and GQ1b. In a preferred embodiment, the ganglioside is GM1. Individual patients may carry antibodies against a single ganglioside or against multiple gangliosides. Thus, in some embodiments, paraproteinaceous neuropathy is characterized by the presence of autoantibodies against one or more neuroantigens. In some embodiments, peripheral neuropathy is characterized by the presence of autoantibodies against one or more gangliosides. In some embodiments, the neuroantigen is a paranodal protein. In certain embodiments, the neural antigen may be a paranodal protein selected from contactin 1, NF155, NF186, and NF140. Autoantibodies known to be associated with various peripheral neuropathies are listed in Table 3 below. For example, IgG4 autoantibodies against paranodal proteins have been identified in patients with CIDP. Detection of GM1 antibodies, usually of the IgM isotype, is associated with multifocal motor neuropathy and lower motor neuropathy, characterized by muscle weakness and atrophy. GM1 IgM may be present as a monoclonal IgM paraprotein or as a polyclonal IgM. Table 3. Autoantibodies in peripheral neuropathies [Table 3]

[0079] Particular paraproteinemic neuropathies to be treated according to the methods described herein may be selected from multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), Miller Fisher syndrome, acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), chronic ataxic neuropathy-ophthalmoplegia-IgM paraprotein-cold agglutinin-disialosyl antibody (CANOMAD) syndrome, distal acquired demyelinating symmetric (DADS) neuropathy, monoclonal gammopathy-associated peripheral neuropathy, anti-MAG peripheral neuropathy, and POEMS syndrome. In certain preferred embodiments, the paraproteinemic neuropathy is selected from multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre syndrome (GBS). The methods described herein are particularly preferred for the treatment of multifocal motor neuropathy (MMN).

[0080] The method described herein is for treating paraproteinaceous neuropathy in a subject in need thereof. The subject is preferably a human subject. The subject may be a patient, where the patient is an individual who is receiving medical care for the treatment of paraproteinaceous neuropathy and / or actively seeking medical care for the treatment of paraproteinaceous neuropathy. The subject to be treated typically exhibits one or more symptoms consistent with paraproteinaceous neuropathy, including, but not limited to, gradual onset of numbness; tingling or burning pain in the feet or hands that may spread up the legs and arms; sharp, stabbing, throbbing, freezing, or burning pain; extreme sensitivity to touch; lack of coordination and falls; twitching and muscle spasms; thinning (atrophy) of muscles; muscle weakness or paralysis. The subject to be treated may have previously been diagnosed as having paraproteinaceous neuropathy by any standard evaluation criteria. The subject may have been identified or previously diagnosed based on excess immunoglobulins in serum. Alternatively or additionally, the subject may possess autoantibodies against one or more neural antigens.

[0081] The subject to be treated may already be undergoing treatment for a paraproteinemic neuropathy or may have previously undergone treatment for a paraproteinemic neuropathy. In some embodiments, the subject has previously undergone IVIg or is undergoing IVIg. In other embodiments, the subject has previously undergone rituximab or is undergoing rituximab. In some embodiments, the subject has undergone plasma exchange or is undergoing plasma exchange. The subject may have failed to respond to previous treatments or may have developed resistance to previous treatments such that their symptoms have worsened.

[0082] The methods of treatment described herein may result in complete or partial amelioration of one or more symptoms associated with PPN. Improved performance may be measured using any standard criterion suitable for evaluating PPN treatments.

[0083] The method of the present invention can include the administration of one or more additional therapeutic agents for the treatment of paraproteinemic neuropathy. The one or more additional therapeutic agents can be administered simultaneously with the complement antagonist as a combination therapy. Alternatively, the one or more additional agents can be administered before or after the administration of the complement antagonist, i.e., the agents are administered sequentially. Additional therapeutic agents that can be administered according to the method of the present invention include, but are not limited to, IVIg, rituximab, corticosteroids, azathioprine, chlorambucil, fludarabine, melphalan, cyclophosphamide / prednisone, melphalan, gabapentin, pregabalin, valproate, dextromethorphan, tramadol, duloxetine, amitriptyline, and venlafaxine.

[0084] C. Complement Antagonists The results presented herein indicate that the final step of the complement cascade, in particular MAC-mediated lysis, may not play a significant role in the pathology of PPN because Schwann cells and motor neurons express high levels of the complement regulatory protein CD59, which prevents MAC-mediated lysis. It therefore follows that the complement-related pathology associated with paraproteinaceous neuropathies described herein may be mediated by factors upstream of C5, for example, by C3aR. Therefore, the present invention provides a method of treating paraproteinaceous neuropathies by inhibiting the complement pathway upstream of C5.

[0085] The method includes administering to a subject an antagonist of the complement system, wherein the antagonist inhibits the complement system upstream of complement factor C5. The antagonist inhibiting the complement system upstream of complement factor C5 does not directly inhibit either complement factor C5 itself or the downstream factors (C6, C7, C8, C9) that combine to form the membrane attack complex. In fact, the antagonist inhibiting the complement system upstream of complement factor C5 targets factors or components of the complement cascade before complement factor C5. The complement factors upstream of C5 include C1, C2, C4, and C3. Thus, in some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C2, C4, and C3. In certain embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b.

[0086] In some embodiments, the antagonist inhibits the complement system upstream of complement factor C3. The upstream complement factors of C3 include C1, C4, and C2. Thus, in some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C2, and C4. In some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, or C4b.

[0087] Initiation factors of the classical complement pathway is complement factor C1. Thus, in some embodiments, the antagonist inhibits complement factor C1. In other embodiments, the antagonist inhibits complement factors C1q, C1r, or C1s. Inhibiting the complement cascade with complement factor C1 makes it possible to prevent or reduce the cleavage of C1-specific targets C4 and C2, as well as to specifically inhibit complement activation through the classical pathway of the complement system. Inhibiting C1 can reduce the deposition of opsonin C4b, thereby reducing the targeting of cells for phagocytosis and destruction. In addition, inhibiting C1 can prevent or reduce the formation of the C3 convertase C4bC2a. C4 and C2 can also be cleaved by MASP enzymes via the lectin pathway; however, the total level of C3 convertase can also be significantly inhibited by targeting complement factor C1.

[0088] In some embodiments, the antagonist inhibits complement factor C4. In other embodiments, the antagonist inhibits complement factors C4a or C4b. Targeting complement factor C4 makes it possible to prevent the formation of the C3 convertase C4bC2a and inhibit complement activation through both the classical and lectin pathways of the complement system.

[0089] In some embodiments, the antagonist inhibits complement factor C2. In other embodiments, the antagonist inhibits complement factor C2a or C2b. Inhibition of C2 can also prevent or reduce the formation of C3 convertase, thereby reducing the deposition of anaphylatoxin C3a and opsonin C3b, as well as other complement activation products downstream of C2. Antagonists of C2 can directly inhibit C2a, preventing the formation of C3 convertase. Alternatively, antagonists can inhibit C2b, preventing the initial binding of C2 to surface-bound C4b. Antagonists of C2b can leave the binding of C2a to C4b intact. Nevertheless, C2 activity can be significantly inhibited by antagonists of C2b.

[0090] In some embodiments, the antagonist inhibits complement factor C3. In other embodiments, the antagonist inhibits complement factors C3a or C3b. Inhibition of C3 can prevent or reduce the formation of the C5 convertase C3bBbC3b, thereby reducing deposition of the anaphylatoxin C5a and the MAC generating factor C5b.

[0091] Depending on the point in the complement cascade where the antagonist inhibits the complement system, the antagonist can inhibit the classical complement pathway, the lectin complement pathway, the alternative complement pathway, or a combination of these pathways.In some embodiments, the antagonist inhibits the classical and lectin complement pathways, but does not affect the alternative complement pathway.Antagonists that target C2 and C4 can inhibit the classical and lectin complement pathways while leaving the alternative pathway intact.In some cases, it may be beneficial to leave one of the complement pathways intact so that this important weapon of the innate immune system is not completely impaired.Alternatively or in addition, the antagonist may only need to cause partial inhibition of complement activity to achieve a therapeutic effect.

[0092] Antagonists for use in the therapeutic methods described herein inhibit factors or components of the complement system upstream of complement factor C5 to inhibit or reduce complement activity. As reported herein, it is beneficial to target the complement cascade upstream of C5 to inhibit the activity of complement factors that precede the formation of the MAC complex. Antagonists for use according to the methods described herein can reduce or inhibit complement activity by inhibiting the generation of biologically active complement-derived peptides, such as C4a, C4b, C3a, C3b, and C5a. Antagonists can prevent, at least in part, the damaging effects of complement-derived peptides on cells and tissues. Complement system antagonists can inhibit or reduce complement activity by reducing deposition of anaphylatoxins, reducing deposition of opsonins, reducing production or secretion of cytokines and / or chemokines, reducing phagocytosis, reducing immune cell recruitment, and any combination thereof. In some embodiments, the antagonist of the complement system inhibits deposition of anaphylatoxins. In some embodiments, the antagonist inhibits deposition of C3a or C5a. In some embodiments, the antagonist inhibits deposition of opsonins. In some embodiments, the antagonist inhibits deposition of C3b or C4b. In some embodiments, the antagonist inhibits production and / or secretion of inflammatory cytokines and / or chemokines. In some embodiments, the antagonist inhibits production and / or secretion of MCP-1.

[0093] Antagonists for use in the present invention can be any agent capable of inhibiting the function of a complement factor, thereby inhibiting or reducing complement activity. As described elsewhere herein, antagonists for use in the present methods preferably exhibit specificity for their target. This specificity is usually achieved by antagonists that directly bind to and inhibit the function of their target.

[0094] In some embodiments, the antagonist is selected from: inhibitory RNA species, e.g., siRNA or shRNA; small molecule inhibitors; inhibitory peptides, antibody mimetics, e.g., affibodies, affilins, affitins, adnectins, atrimers, evasins, DARPins, anticalins, avimers, finomers, versabodies, and duocalins; biological antagonists, including antibodies and antigen-binding fragments thereof.

[0095] In a preferred embodiment, the antagonist is an antibody or an antigen-binding fragment thereof. In an embodiment, the antibody or an antigen-binding fragment thereof binds to complement factors C1, C1q, C1s, C1r, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b. In an embodiment, the antibody or an antigen-binding fragment thereof binds to complement factors C1, C1q, C1s, C1r, C2, C2a, C2b, C4, C4a, or C4b. In a preferred embodiment, the antibody or an antigen-binding fragment thereof binds to C2, preferably C2b.

[0096] The antibodies and antigen-binding fragments for use in the methods described herein are intended for human therapeutic use and therefore are usually of the IgA, IgD, IgE, IgG, IgM type, mostly of the IgG type, which may belong to any of the four subclasses IgG1, IgG2a and b, IgG3, or IgG4. In a preferred embodiment, the antibody is an IgG antibody, optionally an IgG1 antibody. The antibody may be monoclonal, polyclonal, or multispecific (e.g., bispecific) provided that they exhibit the appropriate immunological specificity for their target. Monoclonal antibodies are preferred, as they are highly specific and directed against a single antigenic site.

[0097] Antigen-binding fragments as described herein typically comprise a portion of a full-length antibody, usually the antigen-binding or variable domain thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, bispecific Fab', and Fv fragments, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments (see Holliger and Hudson (2005) Nature Biotechnol. 23:1126-36, incorporated herein by reference).

[0098] The antibody or antigen-binding fragment for use according to the methods described herein can exhibit high human homology. Such antibody molecules with high human homology can include antibodies that contain VH and VL domains of native non-human antibodies that exhibit sufficiently high % sequence identity to human germline sequences. In some embodiments, the antibody molecule is a humanized or germline variant of a non-human antibody.

[0099] In non-limiting embodiments, the antibody may comprise a CH1 domain and / or a CL domain (from the heavy and light chain, respectively), the amino acid sequence of which is fully or substantially human. For antibody molecules intended for therapeutic use in humans, it is typical that the entire constant region of the antibody or at least a part thereof has a fully or substantially human amino acid sequence. Thus, one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may be fully or substantially human in terms of its amino acid sequence. The CH1 domain, hinge region, CH2 domain, CH3 domain, and / or CL domain (and / or CH4 domain, if present) may be derived from a human antibody, preferably a human IgG antibody, more preferably a human IgG1 antibody of subtype IgG1, IgG2, IgG3, or IgG4.

[0100] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may all have fully or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% amino acid sequence identity with a human constant region. The term "human amino acid sequence" in this context refers to an amino acid sequence encoded by a human immunoglobulin gene, including rearranged and somatically mutated germline genes.

[0101] In one embodiment, the antibody or antibody fragment comprises a human Fc domain that contains one or more mutations designed to increase the serum half-life of the antibody or antibody molecule. Such optimization efforts aim to improve antibody circulation in vivo. Examples of mutations in the human IgG Fc domain that affect half-life include His433Lys+Asn434Phe(NHance); Arg435His; Asn434Ala; Met252Tyr+Ser254Thr+Thr256Glu(YTE); Met428Leu +Asn434Ser(LS); Thr252Leu+Thr253Ser+Thr254Phe(LSF); Glu294delta+Thr307Pro+Asn434Tyr(C6A-66); Thr256Asn+Ala378Val+Ser383Asn+Asn434Tyr(C6A-78); and Glu294delta(del). Depending on the serum half-life of the complement inhibitor, it may be administered as a single dose or it may be administered as multiple doses with intervals between subsequent doses ranging from one day to one month.

[0102] In another embodiment, the Fc domain may contain one or more mutations designed to impair the effector function of the Fc domain. Such mutations are well known to those skilled in the art. In the case of human IgG1, the antibody may contain a human Fc domain that has been modified to disable or impair its effector function. Such Fc domain mutations usually involve changing at least one amino acid from the heavy chain constant region at positions 234, 235, 236, 237, 297, 318, 320, or 322, thereby causing alteration of effector function while retaining binding to the antigen. Examples of effector-impairing mutations in the human IgG Fc domain include Leu234Ala+Leu235Ala (termed LALA); Leu234Ala+Leu235Ala+Pro329Gly (termed LALA-PG); Ser228Pro+Leu235Glu in IgG4; Pro331Ser+Leu234Glu+Leu235Phe; and Pro331Ser+Leu234Ala+Leu235Ala.

[0103] Examples of antagonists suitable for use in the present invention that inhibit complement factor C1 include Cinryze (Shire), stimulimab - also known as TNT009 and BIV009 (Bioverativ), TNT003 (True North), ANX005 (Annexon), and nafamostat (Torii Pharmaceutical).

[0104] Examples of antagonists suitable for use in the present invention that inhibit complement factor C3 include compstatin Cp40 (Amyndas), PEG-Cp40 (Amyndas), AMY-101 (Amyndas), AMY-201 (Amyndas), APL-1 and APL-2 (Apellis), CDX-1135 (Celldex), APT070 myrococept (MRC), HC3-1496 (InCode), humanized monoclonal antibody H17 (Elusys Therapeutics), or vaccinia virus complement control protein (VCP).

[0105] (D. Anti-C2 Antibodies and antigen-binding fragments) Particularly preferred complement antagonists for use in the therapeutic methods described herein are those that inhibit complement factor C2. In a preferred embodiment, the complement antagonist for use in the therapeutic methods is an antibody or antigen-binding fragment thereof that binds to C2. Suitable anti-C2 antibodies and antigen-binding fragments for use in the methods described herein include the antibodies and antigen-binding fragments identified in International Patent Application WO2014 / 189378, the contents of which are incorporated herein in their entirety.

[0106] C2 is a 90-100 kDa glycoprotein involved in the classical and lectin pathways of complement activation. As mentioned above, C2 can be activated by C1s in the classical pathway or by activated MASP2 in the lectin pathway. C2 binds to surface-bound C4b (Mg 2+ C4bC2 complexes bind to C4bC2 (in the presence of C1s or MASP2) to form complexes that are subsequently cleaved by activated C1s or MASP2 into two fragments: a larger 70 kDa fragment C2a that remains bound to C4b to form the C3-convertase C4bC2a, and a smaller 30 kDa N-terminal fragment C2b that is released into the fluid phase. Once activated and bound to C4b, C2a constitutes the catalytic subunit of the C3 and C5 convertases that can cleave C3 and C5, respectively.

[0107] The amino acid sequence of human C2 is known (GenBank Accession No. NM_000063) and is shown below in SEQ ID NO: 1. Amino acid sequence of human C2 (SEQ ID NO: 1): [ka]

[0108] In certain embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH and VL domains have the CDR sequences: [ka] HCDR3 comprising or consisting of: [ka] HCDR2 comprising or consisting of: [ka] HCDR1 comprising or consisting of: [ka] LCDR3 comprising or consisting of: [ka] LCDR2 comprising or consisting of: [ka] The LCDR1 comprises or consists of:

[0109] In certain embodiments, the antibody or antigen-binding fragment binds C2b and comprises a variable heavy chain (VH) domain that comprises or consists of a sequence at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 8 and a variable light chain (VL) domain that comprises or consists of a sequence at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 9. In certain embodiments, the antibody or antigen-binding fragment that binds C2b comprises a variable heavy chain domain (VH domain) that comprises or consists of SEQ ID NO: 8 and a variable light chain domain (VL domain) that comprises or consists of SEQ ID NO: 9.

[0110] In certain embodiments, the antibody or antigen-binding fragment binds C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises or consists of a sequence at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:8, and the VL domain comprises the CDR sequence: [ka] LCDR3 comprising or consisting of: [ka] LCDR2 comprising or consisting of: [ka] The LCDR1 comprises or consists of:

[0111] In certain embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises or consists of the amino acid sequence of SEQ ID NO:8 and the VL domain comprises the CDR sequences: [ka] LCDR3 comprising or consisting of: [ka] LCDR2 comprising or consisting of: [ka] The LCDR1 comprises or consists of:

[0112] In certain embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain has the CDR sequences: [ka] HCDR3 comprising or consisting of: [ka] HCDR2 comprising or consisting of: [ka] and the VL domain comprises or consists of a sequence at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:9.

[0113] In certain embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain has the CDR sequences: [ka] HCDR3 comprising or consisting of: [ka] HCDR2 comprising or consisting of: [ka] and the VL domain comprises or consists of the amino acid sequence of SEQ ID NO:9.

[0114] For embodiments in which antibody or antigen-binding fragment domains are defined by a particular percentage sequence identity to a reference sequence, the VH and / or VL domains may retain CDR sequences identical to those present in the reference sequence, such that mutations are present only in the framework regions. Table 4. VH and VL Domain Sequences [Table 4]

[0115] In certain embodiments, the anti-C2b antibody comprises the CH1, hinge, CH2, and / or CH3 domains of a human antibody, in particular human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the anti-C2b antibody comprises the CH1, hinge, CH2, and CH3 domains of a human IgG1, and comprises substitutions L234A and L235A in the CH2 domain, where the positions are defined according to the EU numbering. Alternatively or additionally, the anti-C2b antibody comprises the CH1, hinge, CH2, and CH3 domains of a human IgG1, and comprises substitutions H433K and N434F in the CH3 domain, where the positions are defined according to the EU numbering. EU numbering refers to the convention for Fc regions described in Edelman, GM et al., Proc. Natl. Acad. Sci. USA, 63: 78-85 (1969); and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th ed., 1991.

[0116] In one embodiment, the anti-C2b antibody comprises the CH1, hinge, CH2, and CH3 domains of human IgG4. In one embodiment, the antibody comprises the CH1, hinge, CH2, and CH3 domains of human IgG4 and contains the substitution S228P in the hinge domain.

[0117] In certain embodiments, the anti-C2b antibody comprises the CH1, hinge, CH2, and CH3 domains of human IgG4, and contains the substitution L445P in the CH3 domain.

[0118] In certain embodiments, the anti-C2b antibody comprises the CH1, hinge, CH2, and CH3 domains of human IgG4, and contains both the S228P substitution in the hinge domain and the L445P substitution in the CH3 domain.

[0119] In certain embodiments, the anti-C2b antibody comprises the CH1, hinge, CH2, and CH3 domains of human IgG4, and contains substitutions H433K and N434F in the CH3 domain.

[0120] In certain embodiments, the anti-C2b antibody comprises the CH1, hinge, CH2, and CH3 domains of human IgG4, and contains the substitution S228P in the hinge domain and the substitutions H433K and N434F in the CH3 domain.

[0121] In certain embodiments, the anti-C2b antibody comprises a human IgG heavy chain constant domain. In certain embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain. In certain embodiments, the heavy chain constant domain consists of a human IgG1 heavy chain constant domain. In certain embodiments, the heavy chain constant domain comprises a human IgG1 heavy chain constant domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10 or 11.

[0122] In certain embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain. In certain embodiments, the heavy chain constant domain consists of a human IgG4 heavy chain constant domain. In certain embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 12, 13, or 14.

[0123] The heavy chain constant domains are shown in Table 5 below. Table 5 Heavy Chain Constant Domains [Table 5]

[0124] In certain embodiments, the anti-C2b antibody has a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, and one of the following: (i) a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; (ii) a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16; (i) a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17; (iv) a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18; and (v) a heavy chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:19.

[0125] In certain embodiments, the anti-C2b antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 20 and a heavy chain having an amino acid sequence selected from SEQ ID NOs: 15-19.

[0126] In a preferred embodiment, the anti-C2b antibody comprises a light chain having the amino acid sequence of SEQ ID NO:20 and a heavy chain having the amino acid sequence of SEQ ID NO:16.

[0127] The heavy and light chain sequences are shown in Table 6 below. Table 6 Heavy and Light Chains [Table 6] TIFF2025038109000033.tif168170

[0128] In a preferred embodiment, the anti-C2b antibody is a monoclonal IgG antibody.

[0129] For embodiments in which the heavy and / or light chain of an antibody is defined by a particular percentage sequence identity to a reference sequence, the heavy and / or light chain may retain identical CDR sequences as present in the reference sequence, such that mutations are only present outside of the CDR regions.

[0130] Unless otherwise specified in the present application, the % sequence identity between two amino acid sequences can be determined by comparing these two sequences aligned in an optimal manner, where the amino acid sequence to be compared can contain additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions where the amino acid residues are identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences. For example, it is possible to use the BLAST program "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), the parameters used being those given by default (in particular the parameters "Open Gap Penalty" and "Open Gap Penalty"). The selected matrix is, for example, the matrix "BLOSUM 62" proposed by this program), and the percentage of identity between the two sequences to be compared is calculated directly by this program.

[0131] Anti-C2 antibodies can be modified in the Fc region to increase their binding affinity to the neonatal receptor FcRn, preferably human FcRn. The increased binding affinity can be measurable at acidic pH (e.g., about pH 5.5 to about pH 6.0). The increased binding affinity can also be measurable at neutral pH (e.g., about pH 6.9 to about pH 7.4). By "increased binding affinity" is meant increased binding affinity to FcRn compared to the binding affinity of the unmodified Fc region. Typically, the unmodified Fc region possesses the wild-type amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. In such embodiments, the increased binding affinity to FcRn of an antibody molecule having a modified Fc region is measured compared to the binding affinity of wild-type IgG1, IgG2, IgG3, or IgG4 to FcRn, preferably human FcRn.

[0132] E. Pharmaceutical Compositions The antagonists, particularly antibodies and antigen-binding fragments, for use in the therapeutic methods described herein can be formulated as pharmaceutical compositions for administration to a subject.

[0133] Pharmaceutical compositions can be formulated with a pharma- ceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques, such as those disclosed in (Remington: The Science and Practice of Pharmacy, 19th ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1995). The term "pharma-ceutically acceptable carrier" refers to a carrier or excipient that is essentially non-toxic. Examples of such excipients include, but are not limited to, saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous excipients, such as fixed oils and ethyl oleate, can also be used.

[0134] Pharmaceutical compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions include water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0135] Pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonicity agents in the compositions, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol, or sodium chloride. Pharmaceutically acceptable antioxidants may also be included, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0136] The pharmaceutical composition can be administered by any suitable mode of administration. For example, administration can be parenteral, preferably by intravenous (iv) or subcutaneous (sc) injection or infusion. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0137] INCORPORATION BY REFERENCE Various publications are cited in the foregoing description and throughout the examples which follow, each of which is incorporated herein by reference in its entirety. EXAMPLES

[0138] EXAMPLES The invention will be further understood with reference to the following non-limiting examples.

[0139] Example 1 Complement Inhibition in an In Vitro Model of Multifocal Motor Neuropathy (MMN) Using Live Schwann Cells Schwann cells are myelin-secreting glial cells that spirally wrap around axons in the peripheral nervous system to form the myelin sheath. These cells are attached to the axons by the protein GM1. Their most important function is the myelination of axons to increase neuronal saltatory conduction, but they also aid in neuronal survival and signaling. Dysfunction of these cells causes demyelination resulting in reduced signaling. Thus, Schwann cells are associated with several demyelinating disorders such as MMN. A human Schwann cell line - sNF02.2 - exists, which was derived from a lung metastasis of a patient diagnosed as a malignant peripheral nerve sheath tumor. The cells were established by numerous passagings in culture of primary tumor material until they formed a homogenous Schwann cell-like population that showed a clonal morphology positive for Schwann cell markers S100 and p75. This cell line, purchased from ATCC®, was used in the experiments described herein.

[0140] A. Methods 1.1 Protocol for culturing Schwann cells sNF02.2 (ATCC® CRL-2885™) or sNF96.2 (ATCC® CRL-2884) Schwann cells were cultured at 37° C., 5% CO2 in DMEM medium supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% FCS. Twice a week, cells were passaged or used in experiments when they reached >80% confluency. The medium was discarded and cells were washed with 10 ml PBS. To dissociate cells, 3 mL (T75) or 5 mL (T175) of Accutase cell detachment solution (eBioscience™, Thermo Fischer Scientific; Cat. No. 00-455-56) was added and cells were incubated at 37° C. for 5 min or until cells were completely detached. Culture medium (7 mL in T75 and 10 mL in T175) was then added and the cells were transferred to a 15 mL tube and then centrifuged (125×g, 10 min). The pellet was resuspended in 5 ml of culture medium and counted using trypan blue to distinguish between live and dead cells. Cells were then adjusted to the desired concentration and either seeded into culture flasks (10 mL in T75, 20 mL in T175) or used in FACS experiments.

[0141] 1.2 Protocol for staining Schwann cells Cells were cultured as described above and, after counting, 50,000 cells were transferred to a v-bottom plate. Cells were washed once in FACS buffer (PBS 1% BSA 0.01% sodium azide) and stained with the respective antibodies diluted in FACS buffer for 45 minutes on ice in the dark (see Table 7 for FACS staining antibodies). If necessary, cells were washed once by adding 100 μL of FACS buffer and then centrifuged at 125×g for 5 minutes. Cells were then incubated with secondary antibodies for 45 minutes on ice in the dark. After incubation, 100 μL of FACS buffer was added and cells were centrifuged (125×g for 5 minutes). Finally, cell pellets were resuspended in 100 μL of FACS buffer and analyzed using a FACS Canto II and associated software. Table 7 FACS staining antibodies [Table 7]

[0142] 1.3 Complement activation assay on live Schwann cells When assessing complement activation, 50,000 cells were transferred to a 96-well v-bottom plate and then opsonized with 20 μL of each opsonizing agent diluted in VB++ (1 h, RT). Then, 100 μL of VB++ was added and the samples were centrifuged (125×g for 5 min). The supernatant was then discarded and the cells were incubated with 100 μL of complement-activated serum (preincubated with EDTA, MgEDTA, or Ab for 15 min at RT) for 1 h at 37° C. The cells were then centrifuged at 125×g for 5 min and the supernatant was discarded. Staining for complement activation was then performed according to the staining protocol described above.

[0143] 1.4 Cytokine Secretion Assay sNF02.2 cells were cultured as described above. After Accutase treatment and counting, cells were transferred to 24-well plates at a density of 10,000 cells / well in 1 mL of sNF02.2 culture medium. After 2 days, culture medium was discarded and cells were opsonized with 100 μL of heat-inactivated MMN patient serum diluted 1:50 in VB++ for 1 h at RT. Then, 100 μL of 15% complement-activated serum (pre-incubated with MgEGTA or antibody for 15 min at RT) was added to the opsonized cells, followed by incubation at 37° C. for 1 h. Then, 300 μL of culture medium was added to the cells, resulting in a final volume / well of 500 μL. After 24 and 48 hours, 200 μL of supernatant was collected for in-house analysis by multiple core centers at the University Medical Centre in Utrecht (MC Utrecht).

[0144] B. Results 1.5 Expression of Complement Receptors by Live Schwann Cells To investigate MMN biology using Schwann cells, expression of complement regulatory factors was evaluated. Therefore, sNF02.2 Schwann cells were cultured and FACS staining of expression markers was performed. The results are shown in FIG. 2 and summarized in Table 8 below. Table 8: Expression of Complement Regulatory Proteins on sNF02.2 Schwann Cells [Table 8]

[0145] These results reveal that complement regulatory proteins (CD46, CD55, and CD59) are expressed in Schwann cells, with high expression of CD59, a key regulator of the terminal pathway. High CD59 expression in Schwann cells suggests that complement-mediated lysis is limited in these cells, as the formation of MAC is inhibited. Indeed, inflammation is induced in the neural compartment through cell signaling. Schwann cells also showed expression of Fcγ receptor 1 (CD64), which is involved in phagocyte clearance. As CR1 expression in Schwann cells occurs with the onset of myelination and these cultured sNF02.2 Schwann cells lack myelin, no detection of CR1 (CD35) was observed (Terenghi F et al., Neurology, 2004, 62: 666-668). Only C3aR showed low expression, and C5aR was absent. In conclusion, Schwann cells highly express CD59, suggesting sublytic MAC formation that induces inflammation within the neural compartment.

[0146] To examine the sensitivity of complement regulatory proteins (CRPs) on Schwann cells to complement-mediated lysis, sNF02.2 cells were treated with various concentrations of phospholipase-C (PL-C), which cleaves glycosylphosphatidylinositol-anchored proteins, including CD59 (Fitzpatrick A, Mann C, et al., J Peripher Nerv Syst, 2011, 16(2): 84-91). The results are shown in Figure 3 and indicate that both CD55 and CD59 are sensitive to complement-mediated lysis, as the expression levels of both CRPs were reduced by PL-C treatment.

[0147] 1.6 Resistance of Schwann cells to complement-mediated lysis Previous experiments showed high expression of CD59 and CD55 on Schwann cells (sNF92.2 and sNF02.2). To investigate the functional implications of this expression, anti-GM1-mediated C3 and MAC fixation on sNF96.2 Schwann cells with or without CD59 and CD55 was evaluated. The effect of complement activation on cell survival was also examined. For this purpose, Schwann cells were cultured and transferred to v-bottom plates for further experimental analysis. Treatment with phospholipase C (PL-C) is commonly used to remove GPI-linked proteins such as CD59 and CD55 from the cell surface. Indeed, PL-C treatment led to the removal of CD59 and CD55 while leaving the surface expression of CD46 and GM1 unaffected (Figure 4A). To evaluate the effect of PL-C treatment on the susceptibility to C3 and MAC fixation, Schwann cells were incubated with 100 mM NaCl, 100 mM NaCl, and 100 mM NaCl. The cells were opsonized (1 h at RT) with MMN patient serum containing 5% complement activity or with veronal buffer (VB) as a control. The cells were then incubated with 5% complement activity serum or 5% complement activity serum preincubated with 480 μg / ml ARGX-117 for 1 h at 37° C. Finally, C3 and MAC fixation was detected using biotin-labeled antibodies against C3 and MAC, respectively, and stained with APC-conjugated streptavidin. The results are shown in FIG. 4A, which reveal that C3 fixation was slightly increased after opsonization with MMN-05 serum compared to opsonization with VB alone in non-PL-C treated Schwann cells. However, ARGX-117 completely inhibited C3 fixation in both conditions. Classical pathway-mediated complement activation in the VB control is probably caused by antibodies (e.g., anti-HLA antibodies) present in the complement activity serum. An increase in C3 fixation was observed with PL-C treatment, and ARGX-117 certainly, but not completely, inhibited C3 fixation induced by MMN-005 serum on PL-C-treated cells (Figure 4B). In this condition, a high opsonization density was expected and reached by IgM antibodies against GM1 and IgM / IgG antibodies against non-GM1 targets, modulating in strong complement activation due to the lack of mCRP. A similar observation was seen for MAC fixation, where untreated cells showed a low level of MAC fixation, which was also slightly increased after opsonization with MMN-005 serum. PL-C treatment led to an increase in MAC fixation after opsonization with MMN-005 serum, which was inhibited by ARGX-117 (Figure 4C). Finally, to assess the induction of complement-mediated cytotoxicity (CDC), viability was measured by staining with 7AAD in combination with Annexin-V, and double-positive cells were considered as late apoptosis / death. Using cells expressing normal levels of complement regulatory proteins, CDC was not detectable even after opsonization with MMN-005 serum, however, PL-C treatment increased apoptosis in MMN-05-opsonized cells, and addition of ARGX-117 provided complete protection from complement-mediated lysis (Figure 4D).In conclusion, Schwann cells are intrinsically protected from complement-mediated lysis due to their high expression levels of CD59 and CD55. In the absence of mCRP, CDC was detected that could be (partially) prevented by ARGX-117.

[0148] 1.7 Detection of Complement Activation Using Schwann Cells To examine complement activation in live Schwann cells, cells were transferred to 96-well v-bottom plates (50,000 cells / well) and opsonized with anti-HLA antibody (W6 / 32 - BioLegend; Cat. No. 311402) for 30 min at RT. Complement-activating serum (preincubated with either EDTA, MgEGTA, or TNT009 for 15 min at RT) was then added to the cells at a final concentration of 5% serum. After 1 h of incubation (37° C.), cells were transferred to 96-well V-bottom plates and stained (45 min, ice) before measuring C3 fixation. The results are shown in FIG. 5.

[0149] These results indicate that C3 fixation occurs after activation of complement with 5% HPS (pooled human serum). Moreover, C3 fixation was complement specific, since it decreased to basal levels after the addition of EDTA (10 mM) or TNT009 (480 mg / mL). Furthermore, C3 was blocked in the presence of MgEDTA, negating that it occurs primarily via the alternative complement pathway. Of note, C3 fixation was independent of opsonization with W6 / 32.

[0150] 1.8 Binding of MMN Patient-Derived Autoantibodies to Cultured Schwann Cells Before determining the pathogenicity of IgM anti-GM1 antibodies, we examined the binding of these antibodies to human Schwann cells in vitro. For this, sNF02.2 Schwann cells were cultured and transferred to 50 μL of sNF02.2 culture medium in 96-well round-bottom plates (50,000 cells / well). The cells were then stained with cholera toxin B-AF488 to detect GM1 expression. The results shown in Figure 6A show that in vitro cultured human sNF0.2.2 Schwann cells express GM1. Furthermore, incubation with MMN patient serum (containing autoantibodies against GM1) resulted in higher anti-GM1 expression, and more importantly, IgM binding to Schwann cells was detected after opsonization with MMN patient serum (Figure 6B). Various sera from MMN patients with a wide range of IgM anti-GM1 antibody titers were examined, and all showed IgM binding to Schwann cells. Furthermore, a titer effect of IgM binding on Schwann cells was detected for all patient sera examined (Fig. 7 ).

[0151] Next, we evaluated the complement activation ability of patient IgM anti-GM1 antibodies on Schwann cells. For this, human sNF02.2 Schwann cells were transferred to 96-well v-bottom plates (50,000 cells / well) and opsonized with MMN patient serum (1 h, RT). C3 was then detected using FITC-conjugated antibodies after addition of complement-activating serum (1 h, 37°C).

[0152] Initially, only limited C3 fixation was observed due to poor C3 detection by the antibodies used. Therefore, different approaches were used to enhance C3 detection and to reduce background complement activation. First, different percentages of complement-active serum were tested (10%, 5%, and 2.5%), preincubated with 200 μg / mL of anti-C5 mAb (1 h, 37° C.) to prevent terminal complement pathway activation and thus lysis of sNF02.2 Schwann cells. To reduce complement background, conditions using stripped serum (=complement-active serum that was incubated on Schwann cells for 4×10 min at 4° C. to remove antibodies in the serum) were also tested.

[0153] The results shown in Figures 8A and 8B show no difference between complement-activated serum (black bars) and depleted serum (gray bars), suggesting that antibodies still present in the serum did not contribute to complement activation and therefore to C3 fixation. EDTA (white bars) was used as a control. However, the use of 5% complement-activated serum seemed optimal, since the window between no opsonization and opsonization was the largest. Next, different anti-C3 antibodies were tested to increase the detection window. The results are shown in Figure 8C. This showed an improvement of the window by 10% to 25% using biotinylated anti-C3 (LSBio, clone 6C9) to detect C3 fixation on Schwann cells.

[0154] Thus, optimal detection of C3 fixation on Schwann cells was observed when complement-activated serum was used at a final concentration of 5% and together with a biotinylated anti-C3 detection antibody (clone 6C9) from LSBio. In conclusion, C3 fixation is detected on Schwann cells after opsonization with MMN patient serum, demonstrating that IgM anti-GM1 antibodies present in MMN patient serum are able to activate the classical complement pathway.

[0155] (1.9 C2 Dependence) To confirm the importance of complement factor C2 in the pathogenesis of MMN, C2 dependency was evaluated. sNF02.2 Schwann cells were opsonized with MMN patient serum and subjected to C2-depleted serum (Complement Technology; Catalog No. A312) reconstituted with increasing concentrations of recombinant human C2, rhC2 (U-protein express; Catalog No.: C001, 1987) to evaluate C3 fixation. The results are shown in Figure 9. This revealed that C2-depleted serum alone yielded relatively high mean fluorescence intensity (MFI) values ​​- a higher MFI signal compared to the EDTA control and a smaller experimental window. Nevertheless, serum reconstituted with 30 μg / mL rhC2 restored C3 fixation in Schwann cells, back to normal levels. Moreover, C3 fixation was restored with approximately 5-10 μg / mL rhC2, which corresponds to 20-40% of physiological C2 levels. These results indicate that low levels of C2 do not result in C3 fixation or complement activation.

[0156] 1.10 Efficacy of ARGX-117 in inhibiting C3 fixation in sNF02.2 Schwann cells A variety of inhibitory monoclonal antibodies against complement factors have been generated, some of which are approved for clinical use. For example, TNT009 (BIVV009) is an anti-C1s humanized antibody that specifically blocks the classical complement pathway (Jager U, D'Sa et al., Blood, 2019, 133(9): 893-901), while OMS646 targets MASP-2, thereby blocking the lectin pathway. Eculizumab inhibits C5, thereby blocking MAC deposition induced by all three pathways (Brodsky R, Young N et al., Blood, 2018, 111: 1840-1847).

[0157] ARGX-117 is a monoclonal antibody that targets C2 and is unique because it inhibits the classical and lectin pathways while leaving the alternative pathway intact. The above results show that IgM anti-GM1 autoantibodies in serum from MMN patients can specifically bind to sNF02.2 Schwann cells. Furthermore, this autoantibody can activate the complement cascade, leading to C3 fixation on Schwann cells. To evaluate the therapeutic potential of this antibody, the effect of ARGX-117 on C3 fixation mediated by MMN serum was examined. For this, Schwann cells were cultured, transferred to v-bottom plates (50,000 cells / well), opsonized with MMN patient serum (1 h, RT), and then incubated with 5% complement-active serum preincubated with complement-blocking antibodies or EDTA (20 min, RT). Detection of C3 fixation was performed by staining Schwann cells with C3-BIO (LSBio, clone 6C9) and streptavidin-APC. The results are shown in FIG.

[0158] These results revealed that C3 fixation was inhibited by both ARGX-117 and TNT009 in a dose-dependent manner. However, the inhibition of both antibodies did not reach the EDTA control. TNT009 was able to block C3 fixation up to 53 μg / mL, but a higher concentration of ARGX-117 was required to block to the same extent (160 μg / mL). Importantly, anti-C5 was not added to the cells, which was previously done to block the terminal complement pathway so that C3 fixation could be detected. Surprisingly, the cells were not lysed, meaning that the cells are essentially protected from complement-mediated lysis, probably due to the high expression of CD59 on Schwann cells. In conclusion, ARGX-117 is able to block C3 fixation on Schwann cells in a dose-dependent manner, and these cells are essentially protected from complement-mediated lysis.

[0159] (1.11 Cytokine Secretion Assay) To elucidate the pathophysiology of the origin of neuronal damage in MMN patients, complement-mediated cytokine production was measured. Schwann cells were seeded in 24-well plates (10,000 cells / well) and opsonized with MMN patient serum (1 h, RT), and complement-active serum was added in the presence or absence of complement-blocking antibodies. After 48 h, supernatants were collected and cytokine secretion was measured using the Luminex platform. The results are shown in FIG. 11 and show that stimulation of Schwann cells with IL1-β or TNF-α leads to increased secretion of IL-6, IL-8, and MCP-1. No increase in IL-6 or IL-8 was observed with MMN patient serum. Nevertheless, MCP-1 levels in cells incubated with MMN patient serum were increased two-fold compared to controls (FIG. 11C). This increase could be blocked by the addition of ARGX-117 or TNT009, whereas eculizumab was only able to partially block MCP-1 secretion.

[0160] MCP-1 plays a role in the recruitment of inflammatory immune cells, monocytes, and macrophages to the site of infection and is also involved in the pathogenesis of several diseases, including neuroinflammatory processes characterized by neurodegeneration. For example, circulating levels of MCP-1 increase during GBS progression (Orlikowski et al., J of neuroimmunol, 2003, 134(118-27)). In conclusion, MCP-1 is produced by sNF02.2 Schwann cells after opsonization with MMN patient serum and subsequent complement activation. ARGX-117 was able to block MCP-1 production.

[0161] C. Conclusions: sNF02.2 Schwann cells were used to evaluate the regulation of the complement system. Data show that Schwann cells express high levels of the complement regulatory proteins CD46, CD55, and CD59. Furthermore, we show that IgM anti-GM1 antibodies from MMN patients activate the classical complement pathway and that C3 fixation is dependent on the presence of C2 in the MMN model system examined. ARGX-117 efficiently blocked complement activation in sNF02.2 cells sensitized with anti-GM1 antibodies.

[0162] These results suggest a novel mechanism by which complement events induced by anti-GM1 autoantibodies contribute to pathology in MMN patients, which is shown diagrammatically in Figure 12. It has been reported that IgM anti-GM1 autoantibodies activate the classical complement pathway, but lytic MAC is not formed due to the high expression of CD59 on Schwann cells. Therefore, direct complement-dependent cytotoxicity (CDC) is an unlikely mechanism contributing to neuronal damage under this condition. However, sublytic MAC complex formation has been described to cause cell activation that can induce the production and secretion of inflammatory mediators. In the present study, complement-dependent production of the chemokine MCP-1 by sNF02.2 cells was observed. Eculizumab, an anti-C5 antibody, failed to inhibit MCP-1 secretion in one of the two MMN sera tested. This suggests that MCP-1 secretion by sNF02.2 cells is triggered by a mechanism upstream of sublytic MAC formation.

[0163] Complement activation by anti-GM1 antibodies likely induces the release of cytokines and / or chemokines via C3aR, which were found to be expressed in these cells. These chemokines, such as MCP-1, may play a role in attracting inflammatory cells, leading to further neuronal dysfunction and damage. Here, complement activation by anti-GM1 antibodies in Schwann cells leads to MCP-1 secretion, which is inhibited by ARGX-117, suggesting a possible therapeutic mechanism of this antibody. In conclusion, the pathophysiology of neuronal destruction and demyelination in MMN patients appears to occur upstream of MAC formation, making components of the complement cascade upstream of MAC an interesting therapeutic target for treating this disease. ARGX-117 targets the C2 complement protein and thus represents an example of a suitable molecule for treating this indication.

[0164] Example 2 Complement Inhibition in an In Vitro Model of Multifocal Motor Neuropathy (MMN) Using Fixed Schwann Cells A. Methods 2.1 Protocol for Cultivating and Fixing Schwann Cells on Glass Coverslips Schwann cells were cultured at 37°C and 5% CO2 in DMEM medium supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% FCS. Twice a week, when the confluency reached >80%, the cells were passaged or used in the experiments. The medium was discarded and the cells were washed with 10 ml of PBS. To dissociate the cells, 3 mL (T75) or 5 mL (T175) of Accutase cell detachment solution was added and the cells were incubated at 37°C for 5 min or until the cells were completely detached. Then, culture medium (7 mL for T75 and 10 mL for T175) was added and the cells were transferred to a 15 mL tube and then centrifuged (125×g, 10 min). The pellet was resuspended in 5 ml of culture medium and counted using trypan blue to distinguish between live and dead cells. Cells were then adjusted to the desired concentration and seeded into culture flasks (10 mL in T75, 20 mL in T175) or onto glass coverslips placed in 24-well plates.

[0165] 2.2 Protocol for in vitro evaluation of ARGX-117 sNF02.2 Schwann cells were cultured on glass coverslips at 37°C and 5% CO2 for 3 days. Cells were fixed with 4% PFA (10 min at 4°C), washed once with 500 μL PBS, and coverslips were removed from the 24-well plate. To minimize nonspecific staining, cells were quenched with 100 μL NH4Cl for 5 min at RT, washed once with 100 μL PBS, and then blocked with 100 μL PBS + 2% BSA for 2 h at RT. After washing with 100 μL PBS, cells were incubated face down with heat-inactivated MMN patient serum diluted 1:50 in PBS + 2% BSA for 60 min. The cells were then washed once (500 μL PBS+2% BSA), incubated face down with 15% complement-active serum (with or without preincubation with complement-blocking antibody) for 30 min at RT, and washed once (100 μL PBS+2% BSA). The cells were then stained with 100 μL primary antibody (diluted in PBS+2% BSA), incubated face down (1 h, RT, in the dark), and washed in 100 μL PBS+2% BSA. The cells were then incubated face down with 100 μL streptavidin-APC (diluted 1:100 in PBS+2% BSA) for 1 h at RT in the dark, and then washed once with 100 μL PBS and once with 100 μL MilliQ water. After drying the coverslips on the tissue, 7 μL of ProLong™ Diamond Antifade Mountant with DAPI was pipetted onto the coverslips and dried overnight at 4° C. before being fixed with nail polish. Cells were analyzed using a Zeiss Z1 microscope with Colibri LED at the following settings: 40x magnification, 25% LED, Alexa Fluor™. 400ms for the 488 channel, 100ms for the APC channel, and 50ms for the DAPI channel. The antibodies used for staining are shown in Table 9 below. Table 9 Staining Antibodies [Table 9] (1) It reacts with both human C3a and C3b. (2) The anti-C5b-9 clone aE11 is directed against a neoepitope exposed on C9 when incorporated into TCC.

[0166] B. Results For experiments performed with fixed Schwann cells, very similar results to those reported in Example 1 above with live Schwann cells were observed.

[0167] 2.3 Expression of Complement Receptors sNF02.2 Schwann cells were cultured and fixed on glass coverslips and then stained for expression markers. The results are shown in Table 10 below, which reveal that all complement regulatory proteins are expressed on Schwann cells. CD59 is highly expressed, suggesting that CD59 is a key regulator of the terminal pathway and therefore protects motor neurons from MAC-mediated lysis. CD46, CD55, and C3aR showed moderate expression on Schwann cells, while C5aR was highly expressed on the cell body of Schwann cells. CD35, CD11b, and CD11c were all absent from Schwann cells. Table 10 Expression of Complement Regulatory Proteins on Fixed Schwann Cells [Table 10]

[0168] 2.4 Binding of MMN patient-derived autoantibodies to fixed Schwann cells Binding of MMN patient-derived autoantibodies to fixed Schwann cells was examined and similar results were observed as reported in Example 1.8 above.

[0169] sNF02.2 Schwann cells were cultured on coverslips in 1 mL of sNF02.2 culture medium for 3 days (50,000 cells / coverslip) and then fixed with 4% PFA. Coverslips were then washed in PBS, quenched with NH4Cl4 (5 min, RT), and then blocked with PBS-2% BSA for 2 h. Staining with cholera toxin B-Alexa488 was performed to detect GM1 expression. Results showed that not only in vitro cultured human sNF0.2.2 Schwann cells, but also neuroblastoma-derived mouse N2a cells expressed GM1.

[0170] Incubation with MMN patient sera produced prominent anti-GM1 staining that colocalized with the IgM staining. Various sera from MMN patients with a wide range of IgM anti-GM1 antibody titers were examined, and all showed IgM binding to Schwann cells. Furthermore, the binding of IgM antibodies to human Schwann cells was GM-1 specific, since excess amounts of soluble unlabeled cholera toxin prevented the binding of IgM antibodies from MMN sera. Preincubation with 100 μg / mL unlabeled cholera toxin efficiently prevented anti-GM1 antibody binding to Schwann cells due to competition for binding between cholera toxin and anti-GM1 antibodies for GM1 ganglioside.

[0171] The complement activation capacity of patient IgM anti-GM1 antibodies against Schwann cells was evaluated. For this, human Schwann cells were incubated with heat-inactivated serum from MMN patients (containing autoreactive IgM anti-GM1 antibodies) and HPS (pooled human serum) serving as an exogenous complement source. Using specific antibodies, the deposition of complement factors, e.g., C4 and C3 formation, was determined. The results showed that C4 fixation could be detected on Schwann cells, which correlated with anti-GM1 titers. However, high IgM anti-GM1 titers were not necessarily associated with high C4 deposition.

[0172] Detection of C3 was assessed by staining coverslips with various anti-C3 antibodies. Schwann cells subsequently opsonized with various MMN patient sera showed C3 fixation on these cells. However, high anti-IgM GM1 titers did not necessarily correlate with high C3 deposition. All EDTA controls were negative.

[0173] Table 11 below summarizes the expression levels of various complement factors in various MMN patient samples. Table 11: C3 and C4 fixation in various MMN patient samples [Table 11] * Determined based on GM1 ELISA

[0174] In conclusion, C4 and C3 fixation were detected on fixed Schwann cells after opsonization with MMN patient sera, indicating that IgM anti-GM1 antibodies from MMN patients are capable of activating the classical complement pathway.

[0175] (2.5 C2 Dependence) To confirm the importance of complement factor C2 in the pathogenesis of MMN, C2 dependency was assessed. Therefore, Schwann cells were opsonized with C2-depleted serum and reconstituted with increasing concentrations of purified human C2 (hC2) and C3 formation was assessed. Results revealed an absence of C3 fixation with C2-depleted serum. However, addition of C2 restored C3 fixation in a concentration-dependent manner. Interestingly, complete blockade of C2 was not required to block C3 fixation; 4.58 μg / mL of hC2 (approximately 20% of physiological levels) showed almost no C3 formation.

[0176] In summary, C3 fixation was dependent on the presence of C2, and complete inhibition of C2 was not required to block the classical complement pathway and thereby prevent C3 formation in Schwann cells.

[0177] 2.6 Efficacy of ARGX-117 on fixed Schwann cells Schwann cells were cultured on coverslips for 3 days and then fixed. Cells were then washed, quenched, blocked, and then opsonized with patient serum. Cells were then incubated for 20 min at RT with complement-activating serum in the presence or absence of various complement-blocking antibodies or IVIg. Finally, cells were stained and imaged using 40x magnification.

[0178] C4 fixation in Schwann cells was inhibited by TNT009 (200 μg / mL) and 12.5 mg / mL IVIg treatment.

[0179] While C3 inhibition was observed with ARGX-117 and TNT009, both used at 200 μg / mL, as expected, no effect was seen with eculizumab or OMS646. Only IVIg at a concentration of 12.5 mg / mL showed partial inhibition of C3 fixation in Schwann cells. Thus, a high concentration (200 μg / mL) of a complement inhibitory antibody was able to block C4 and C3 fixation in this in vitro disease model system of MMN.

[0180] To determine the differential effect of anti-complement antibodies, titrations were performed to analyze downstream complement events. Fixed Schwann cells were opsonized with MMN patient serum and complement was activated in the presence of complement blocking antibodies (TNT009, eculizumab, and ARGX-117) at increasing concentrations starting at 15 μg / mL up to 480 μg / mL. Because eculizumab inhibits downstream of C3, the results showed no inhibitory effect on C3 fixation by eculizumab. ARGX-117 showed complete inhibition of C3 fixation up to a concentration of approximately 30 μg / mL.

[0181] C. Conclusions The complement inhibitory effect of ARGX-117, which targets human C2b, was evaluated in an in vitro model using fixed Schwann cells, thereby mimicking the pathophysiology of MMN. Data show that Schwann cells expressed high levels of the complement regulatory proteins CD46, CD55, and CD59. Furthermore, it was shown that IgM anti-GM1 antibodies from MMN patients activate the classical complement pathway and that C3 fixation is dependent on the presence of C2 in the MMN model system. ARGX-117 efficiently blocked complement activation and was superior to both eculizumab and TNT009.

[0182] Example 3 Complement Inhibition in an In Vitro Model of Multifocal Motor Neuropathy (MMN) Using Induced Pluripotent Stem Cells (iPSCs) A. Methods 3.1 Protocol for Differentiation of Spinal Motor Neurons from iPSCs Induced pluripotent stem cell (iPSC) derived motor neuron-like cells (MN) were prepared as described (Harschnitz O et al., J Clin Immunol. 2014, Jul;34 Suppl 1:S112-9) and in collaboration with Dr. L van der Pol and co-workers at the Department of Neurology, UMCU, The Netherlands. Briefly, human fibroblasts were obtained from skin biopsies from healthy individuals under an Institutional Review Board approved protocol. These cells were cultured in mouse embryonic fibroblast medium containing DMEM GlutaMAX supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were cultured in MEF medium at 37°C and 5% CO2. The cells were reprogrammed within the first 5 passages according to the following protocol: Human fibroblasts were plated in 6-well dishes at a density of 10,000 cells per well and cultured in MEF medium for 24 hours. Viral transduction was then performed in a mixture containing MEF medium, 4 mg / mL hexadimethrine bromide, and lentiviral vectors expressing Oct4, Klf4, Sox2, and c-Myc. After 24 hours of incubation, the cells were washed three times with PBS, pH 7.4, and cultured in MEF medium for an additional 5 days. The cells were then incubated with trypsin-EDTA and transferred to a 10 cm dish pre-coated with 0.1% gelatin and containing a confluent layer of irradiated MEFs in the MEF medium. The culture medium was replaced with human embryonic stem cell (huES) medium containing DMEM-F12, knockout serum replacement, 1% penicillin / streptomycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, and 20 ng / mL recombinant human fibroblast growth factor-basic. After 3–6 weeks, iPSC colonies were manually picked for further expansion and characterization. iPSCs were maintained in huES medium and cryopreserved after 4–6 passages and stored in liquid nitrogen. iPSCs were cultured on irradiated MEFs in huES medium and passaged manually. Feeder culture of iPSCs was performed on Geltrex and maintained in mTeSR1 medium. Feeder-free cultured iPSCs were enzymatically passaged using Accutase.

[0183] (3.2 Protocol for in vitro evaluation of ARGX-117) iPSCs were cultured on coverslips at 37°C, 5% CO2 for 12-14 days in MN orientation. Next, iPSC-MNs were fixed with 4% PFA (10 min at 4°C), washed three times with 500 μL PBS, and coverslips were removed from the 24-well plate. To minimize nonspecific staining and to neutralize the fixative, cells were quenched with 100 μL 50 mM NH4Cl for 5 min at RT, washed once with PBS (all washing steps were by immersing the coverslip), and then blocked with 100 μL PBS + 2% BSA for 2 h at RT. After washing with PBS, cells were incubated face down for 60 min with heat-inactivated MMN patient serum diluted 1:50 in PBS + 2% BSA. The cells were then washed once in PBS, incubated face down with 15% complement-active serum (with or without preincubation with complement-blocking antibody) for 30 min at RT, and washed once in PBS. The cells were then stained with 100 μL of primary antibody (diluted in PBS+2% BSA), incubated face down (1 h, RT, in the dark), and then washed in PBS. The cells were then incubated face down with 100 μL of streptavidin-APC (diluted 1:100 in PBS+2% BSA) for 1 h at RT in the dark, and then washed once with PBS and once with MilliQ water (also with the coverslip immersed). After drying the coverslip on tissue, 7 μL of ProLong™ Diamond Antifade Mountant with DAPI was pipetted onto the objective glass, the coverslip was placed face down on the drop, and dried overnight at RT before being fixed with nail polish. Cells were analyzed using a Zeiss Z1 microscope with Colibri LED at the following settings: 40x or 20x magnification (as appropriate for the experiment), 25% LED, Alexa Fluor™ (unless otherwise indicated). 400ms for the 488 channel, 100ms for the APC channel, and 50ms for the DAPI channel. Four pictures were taken per condition across the entire coverslip. All pictures were normalized to positive and negative controls and exported in uncompressed 8-bit TIFF format in single and combined channels using ZEN 2012 software. The mean grey value of each single channel was calculated with ImageJ (Fiji). Ratios were calculated in Microsoft Excel 2010 and visualized using GraphPad Prism 7. The antibodies used for staining are shown in Table 12 below. Table 12 Staining Antibodies [Table 12] (1) It reacts with both human C3a and C3b. (2) The anti-C5b-9 clone aE11 is directed against a neoepitope exposed on C9 when incorporated into TCC.

[0184] 3.3 Protocol for determining GM1 using ELISA NUNC maxisorp plates were coated with GM1 (5 μg / mL) in methanol. Methanol was allowed to evaporate for + / - 2,5 hours in a laminar flow cabinet. The wells were then blocked with 200 μL of 1% BSA-PBS for 2 hours at RT. MMN patient sera were diluted in 1% BSA-PBS (both preincubated with IVIg) and 100 μL was added to each well followed by O / N incubation at 4°C. Next, the plates were washed 6 times with PBS and then incubated with a primary antibody detecting human IgM in 1% BSA-PBS (1 hour at RT). After 6 washing steps with PBS, the wells were incubated with 100 μL of HRP-conjugated secondary antibody for 1 hour at RT. After 6 washing steps with PBS, TMB was added and the reaction was stopped with hydrochloric acid. The plates were analyzed at 415 nm using a BioRad ELISA reader.

[0185] (B. Results) Currently, an animal model of MMN is not available, so we developed an in vitro model of MMN to investigate the pathogenicity of anti-GM1 IgM antibodies in MMN patients. Briefly, human induced pluripotent stem cells were generated from fibroblasts and differentiated into motor neurons as described above. These motor neurons were then fixed on glass coverslips using paraformaldehyde (PFA) and quenched with NH4Cl. The cells were then opsonized for 1 h with MMN patient serum containing anti-GM1 IgM autoantibodies, allowing binding of these autoantibodies to GM1. After washing, complement was activated for 30 min using human pooled serum (HPS) in the presence or absence of complement blocking antibodies. Finally, cells were stained with antibodies against complement factors to assess complement activity. The results of these studies are described below.

[0186] 3.4 Expression of Complement Receptors in iPSC-Derived Motor Neurons To better understand the role of complement in the pathophysiology of MMN, the expression of complement regulatory proteins and complement receptors was assessed in iPSC-MN. Therefore, iPSC-MN were cultured and fixed on glass coverslips with 4% PFA and then stained for expression markers. The results are shown in Table 13 and Figure 13. Table 13 Expression of Complement Regulatory Proteins on iPSC-MN [Table 13]

[0187] These results demonstrate that complement regulatory proteins (CD46, CD55, and CD59) are all expressed in fixed motor neurons. CD59 was highly expressed, suggesting that motor neurons are protected from MAC-mediated lysis. CD46 and CD55 showed moderate expression localized to the cell body. Both C3R and C5R were expressed in motor neurons, and C5aR was expressed only in the cell body and not in the neurites. CD35 expression was low, while both CD11b and CD11c were absent.

[0188] (3.5 Binding of MMN patient-derived autoantibodies to iPSC-MN) Binding of IgM anti-GM1 antibodies to iPSC-MN was investigated in vitro. For this, iPSC-MN were cultured on coverslips in 1200 μL of culture hMN medium for 12–14 days (80,000–150,000 cells / 13 mm coverslip) and then fixed with 4% PFA. Coverslips were then washed in PBS, quenched with NH4Cl4 (5 min, RT), and then blocked with PBS-2% BSA for 2 h. To detect GM1 expression, staining with cholera toxin B-Alexa 488 was performed. Results showed that motor neurons derived from iPSCs express GM1 and that incubation with MMN patient serum resulted in prominent anti-GM1 staining that colocalized with the IgM staining.

[0189] The complement activation ability of patient IgM anti-GM1 antibodies in iPSC-derived motor neurons was also evaluated. For this, human iPSC-derived motor neurons were incubated with heat-inactivated serum from MMN patients (containing autoreactive IgM anti-GM1 antibodies) and HPS, which served as an exogenous complement source. Using specific antibodies, the deposition of complement factors such as C4 and C3 was determined. The results showed that C4 and C3 fixation could be detected in iPSC-MNs. However, high anti-IgM GM1 titers did not necessarily correlate with high C3 deposition. In conclusion, C4 and C3 fixation was detected in fixed iPSC-MNs after opsonization with MMN patient serum. This indicates that IgM anti-GM1 antibodies from MMN patients are able to activate the classical complement pathway.

[0190] 3.6 C2 Dependence To confirm the importance of complement factor C2 in the pathogenesis of MMN, C2 dependency was assessed. iPSC-derived motor neurons were opsonized with C2-depleted serum, reconstituted with increasing concentrations of purified human C2 (hC2), and complement activation was assessed by measuring C3 fixation. Results are shown in FIG. 14 and revealed an absence of C3 fixation in C2-depleted serum. However, addition of C2 restored C3 fixation in a concentration-dependent manner. Furthermore, ARGX-117 completely inhibited C3 fixation in C2-depleted serum reconstituted with physiological amounts of hC2.

[0191] In summary, C3 fixation is dependent on the presence of C2, as shown by the ability of ARGX-117 to inhibit C3 fixation in the presence of hC2. Complete inhibition of C2 does not appear to be necessary to block the classical complement pathway and prevent C3 formation.

[0192] 3.7 Efficacy of ARGX-117 to Prevent Complement Fixation in Fixed iPSC-MNs As described in section 1.9 above, various inhibitory monoclonal antibodies against complement factors have been generated, some of which have been approved for clinical applications. The effect of these inhibitory antibodies was examined in fixed iPSC-MNs. For this, iPSC-MNs were cultured on coverslips for 12–14 days and then fixed. Afterwards, cells were washed, quenched, blocked, and opsonized with patient serum. Afterwards, cells were incubated for 20 min at RT with complement-active serum in the presence or absence of various complement-blocking antibodies. Finally, cells were stained and imaged using 40x or 20x magnification.

[0193] As expected, C4 fixation on motor neurons was inhibited by TNT009 (200 μg / mL) but not by the other mAbs.

[0194] C3 inhibition was observed with ARGX-117 and TNT009, both used at 200 μg / mL, whereas eculizumab, OMS646, and rituximab used at the same concentrations had no effect. Thus, ARGX-117 at high concentrations (200 μg / mL), which blocks C2, was able to block downstream C3 fixation in this in vitro disease model of MMN.

[0195] To determine the differential effect of anti-complement antibodies, titrations were performed to analyze downstream complement events. Fixed iPSC-derived motor neurons were opsonized with MMN patient serum. They were preincubated with complement blocking antibodies (TNT009, OMS646, and ARGX-117) at increasing concentrations starting from 3 μg / mL up to 200 μg / mL, and then added to the iPSC-MNs. Since OMS646 inhibits the lectin pathway, the results showed no inhibitory effect on C3 fixation by OMS646. ARGX-117 showed complete inhibition of C3 fixation up to a concentration of about 12 μg / mL. The same results were obtained with TNT009, where C3 deposition was also blocked up to 12 μg / mL. Thus, ARGX-117 and TNT009 worked equally well in fixing C3 fixation in iPSC-derived motor neurons opsonized with MMN patient serum.

[0196] 3.8 Complement Inhibition by ARGX-117 in Other Immune-Mediated Neuropathies Autoimmune peripheral neuropathies represent a group of rare and disabling clinically heterogeneous disorders characterized by motor and / or sensory symptoms of disease severity. In MMN, these are direct evidence of autoimmune reactivity mediated by specific IgM antibodies targeting GM1. Other immune-mediated neuropathies have been identified, including chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) as the most common disorder and Guillain-Barre syndrome (GBS) as the most acute disorder. To evaluate the therapeutic effect of ARGX-117 on other immune-mediated neuropathies, the in vitro models described above were used. However, instead of opsonizing iPSC-derived motor neurons with MMN patient serum, serum from GBS and CIDP patients, respectively, was used. The results are shown in Figure 15. Thus, autoantibodies present in both patient sera were able to activate complement, measured as C3 fixation on motor neurons, which was reduced by the addition of 200 μg / mL ARGX-117. Thus, in addition to MMN, ARGX-117 also blocked complement in disease models of other immune-mediated neuropathies.

[0197] 3.9 Effect of IVIg on Complement Inhibition Using an In Vitro MMN Assay IVIg is a biological agent that is often used to treat various autoimmune diseases. In MMN, IVIg is the FDA approved first-line treatment. Several mechanisms are involved in immune modulation by IVIg, including direct neutralization of pathogenic immunoglobulins, FcR blockade, and modulation of several immune cells. IVIg also acts to inhibit complement activation, as IgG present in IVIg binds to C3, thereby removing it from the serum and preventing the terminal complement pathway (Terenghi F et al., Neurology, 2004, 62: 666-668; Fitzpatrick A, Mann C et al., J Peripher Nerv Syst, 2011, 16(2): 84-91). In MMN, IVIg has been shown to reduce the pathological deposition of anti-GM1-GM1 complexes (Bhatheja K, Field, J. Int J Biochem Cell Biol. 2006;38(12):1995). The standard treatment for MMN is IVIg; however, this treatment is not effective when administered over a long period of time. Therefore, an in vitro MMN model using iPSC-derived motor neurons was used to investigate the complement inhibitory effect of IVIg. For this, iPSC-derived motor neurons were cultured on coverslips, fixed, and opsonized with MMN patient serum, followed by the addition of complement-active serum to activate the complement cascade. Two types of IVIg (GammaQuin and Nanogam) were tested, and various approaches of IVIg treatment were evaluated as well. First, 50 mg / mL IVIg was added at the opsonization step, in which case IVIg was unable to block C3 fixation in motor neurons. Next, addition of IVIg during opsonization and complement activation, or only during complement activation, respectively, both resulted in a decrease in C3 fixation, with GammaQuin showing the strongest effect on complement inhibition compared to Nanogam (see Figure 16).

[0198] To evaluate the anti-idiotypic effect of IVIg, a competitive ELISA against GM1 was performed using MMN patient sera. The results are shown in Figure 17, which show that there was no competitive effect of IVIg on GM1 binding in this assay. In summary, IVIg treatment blocked C3 fixation when added at the complement activation step, confirming that IVIg removes C3 from serum. However, IVIg had no effect on idiotypic antibodies.

[0199] (C. Conclusion) The complement inhibitory effect of ARGX-117 targeting human C2b was evaluated in an in vitro model by using iPSC-derived motor neurons, thereby mimicking the possible pathophysiology of MMN. The data show that motor neurons in this system expressed complement regulatory proteins, with CD59 expression being the highest. Furthermore, it was shown that IgM anti-GM1 antibodies from MMN patients activated the classical complement pathway, and that C3 deposition was dependent on the presence of C2. ARGX-117 efficiently blocked complement activation in MMN patients, as well as in CIDP and GBS patient samples. Furthermore, IVIg only blocked complement at the complement activation step, and had no effect on anti-idiotypic antibodies. The present application provides the following aspects of the invention. (Aspect 1) A method of treating a paraproteinaceous neuropathy in a subject, comprising administering to the subject an antagonist of the complement system, wherein the antagonist inhibits the complement system upstream of complement factor C5. (Aspect 2) The method of Aspect 1, wherein the antagonist inhibits the classical complement pathway and / or the lectin complement pathway. (Aspect 3) The method of Aspect 1 or Aspect 2, wherein the paraproteinaceous neuropathy is a demyelinating neuropathy. (Aspect 4) The method of any one of Aspects 1 to 3, wherein the paraproteinaceous neuropathy is characterized by the presence of IgM, IgA, or IgG immunoglobulins. (Aspect 5) The method of any one of Aspects 1 to 4, wherein the paraproteinaceous neuropathy is characterized by the presence of autoantibodies. (Aspect 6) The method of any one of Aspects 1 to 5, wherein the paraproteinaceous neuropathy is characterized by the presence of autoantibodies against neural antigens. Aspect 7: The method of aspect 6, wherein the neural antigen is a ganglioside or myelin associated glycoprotein (MAG).Aspect 8: The method of aspect 7, wherein the ganglioside is selected from GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GT3, and GQ1b.Aspect 9: The method of aspect 8, wherein the ganglioside is GM1.(Aspect 10) The method of any one of Aspects 1 to 9, wherein the paraproteinaceous neuropathy is selected from: multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), Miller Fisher syndrome, acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), chronic ataxic neuropathy-ophthalmoplegia-IgM paraprotein-cold agglutinin-disialosyl antibody (CANOMAD) syndrome, distal acquired demyelinating symmetric (DADS) neuropathy, monoclonal gammopathy-associated peripheral neuropathy, anti-MAG peripheral neuropathy, and POEMS syndrome. (Aspect 11) The method of Aspect 10, wherein the paraproteinaceous neuropathy is MMN, CIDP, or GBS. (Aspect 12) The method of Aspect 10, wherein the paraproteinaceous neuropathy is MMN. (Aspect 13) The method according to any one of Aspects 1 to 12, wherein the antagonist inhibits the complement pathway upstream of complement factor C3. (Aspect 14) The method according to any one of Aspects 1 to 13, wherein the antagonist inhibits C1, C1q, C1r, or C1s. (Aspect 15) The method according to any one of Aspects 1 to 13, wherein the antagonist inhibits complement factor C2, C2a, or C2b. (Aspect 16) The method according to any one of Aspects 1 to 12, wherein the antagonist inhibits complement factor C3, C3a, or C3b. (Aspect 17) The method according to any one of Aspects 1 to 13, wherein the antagonist inhibits complement factor C4, C4a, or C4b. (Aspect 18) The method of any one of aspects 1 to 17, wherein the antagonist is selected from: an inhibitory RNA species, e.g., siRNA or shRNA; a small molecule inhibitor; a biological antagonist, e.g., an inhibitory peptide or an antibody mimetic, e.g., an affibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, fynomer, versabody, or duocalin; or an antibody or antigen-binding fragment thereof.Aspect 19: The method of any one of aspects 1 to 18, wherein the antagonist is selected from: compstatin Cp40 (Amyndas); PEG-Cp40 (Amyndas); AMY-101 (Amyndas); AMY-201 (Amyndas); APL-1 and APL-2 (Apellis); Cinryze (Shire); CDX-1135 (Celldex); APT070 mycocept (MRC); HC3-1496 (InCode); nafamostat (Torii Pharmaceutical); and vaccinia virus complement control protein (VCP). Aspect 20: The method of any one of aspects 1 to 17, wherein the antagonist is an antibody or an antigen-binding fragment thereof. Aspect 21: The method of aspect 20, wherein the antibody is an IgG antibody. Aspect 22: The method of aspect 20, wherein the antigen-binding fragment is selected from: an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a one-arm (monovalent) antibody, a diabody, a triabody, a tetrabody, a unibody, a domain antibody, and a nanobody. Aspect 23: The method of any one of aspects 20 to 22, wherein the antibody or antigen-binding fragment thereof binds to complement factors C1, C1q, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b. Aspect 24: The method of aspect 23, wherein the antibody or antigen-binding fragment thereof is selected from: stimulimab BIV009 / TNT009 (Bioverativ); ANX005 (Annexon); mAb H17 (Elusys Therapeutics); and TNT003 (True North). Aspect 25: The method of any one of aspects 20 to 22, wherein the antibody or antigen-binding fragment thereof binds to complement factor C2. Aspect 26: The method of aspect 25, wherein the antibody or antigen-binding fragment thereof binds to the C2b domain of complement factor C2.(Aspect 27) The method of aspect 26, wherein the antibody or antigen-binding fragment comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise the CDR sequences: - HCDR3 comprising or consisting of SEQ ID NO:2; - HCDR2 comprising or consisting of SEQ ID NO:3; - HCDR1 comprising or consisting of SEQ ID NO:4; - LCDR3 comprising or consisting of SEQ ID NO:5; - LCDR2 comprising or consisting of SEQ ID NO:6; and - LCDR1 comprising or consisting of SEQ ID NO:7. (Aspect 28) The method of aspect 27, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises or consists of the amino acid sequence of SEQ ID NO:8 and the VL domain comprises the CDR sequences: - LCDR3 comprising or consisting of SEQ ID NO:5; - LCDR2 comprising or consisting of SEQ ID NO:6; and - LCDR1 comprising or consisting of SEQ ID NO:7. (Aspect 29) The method of aspect 27 or aspect 28, wherein the antibody or antigen-binding fragment comprises a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 70% identity thereto. (Aspect 30) The method of aspect 29, wherein the antibody or antigen-binding fragment comprises a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 9. (Aspect 31) The method of any one of aspects 27 to 30, wherein the antibody or antigen-binding fragment comprises a human IgG heavy chain constant domain. (Aspect 32) The method of any one of aspects 25 to 27, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 20. (Aspect 33) The method of any one of aspects 1 to 32, further comprising administering IVIg to the subject. (Embodiment 34) The method of any one of embodiments 1 to 33, further comprising administering rituximab to the subject.(Embodiment 35) An antagonist of the complement system for use in treating a paraproteinaceous neuropathy in a subject, wherein the antagonist inhibits the complement system upstream of complement factor C5.

Claims

1. 1. A pharmaceutical composition comprising an anti-C2 antibody or antigen-binding fragment comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain for use in treating a paraproteinaceous neuropathy in a subject, wherein the VH domain comprises or consists of the amino acid sequence of SEQ ID NO: 8, and the VL domain comprises or consists of the amino acid sequence of SEQ ID NO: 9, the antibody or antigen-binding fragment binds to the C2b domain of C2, and the paraproteinaceous neuropathy is multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), or Guillain-Barré syndrome (GBS).

2. The pharmaceutical composition of claim 1 , wherein the anti-C2 antibody or antigen-binding fragment inhibits the classical complement pathway and / or the lectin complement pathway.

3. The pharmaceutical composition according to claim 1 or 2, wherein the paraproteinaceous neuropathy is characterized by the presence of IgM or IgG immunoglobulins.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the paraproteinaceous neuropathy is characterized by the presence of autoantibodies.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the paraproteinaceous neuropathy is characterized by the presence of autoantibodies against neural antigens.

6. The pharmaceutical composition according to claim 5, wherein the neuroantigen is a ganglioside or a myelin-associated glycoprotein (MAG).

7. 7. The pharmaceutical composition of claim 6, wherein the ganglioside is selected from GM1, GM1b, GD1a, GD1b, GD3, GT1a, GT1b, and GQ1b.

8. The pharmaceutical composition according to claim 7, wherein the ganglioside is GM1. composition.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the paraproteinaceous neuropathy is MMN.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment comprises a human IgG heavy chain constant domain.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

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