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

Abstract

To provide a method of treating a paraproteinemic neuropathy in a subject by administering to the subject an antagonist of the complement system.SOLUTION: An antagonist of the complement system 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 the 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 uses antagonists of the complement system to treat paraproteinaceous neuropathies. The antagonist blocks or inhibits the complement pathway upstream of complement factor C5. The treatable protein-mediated neuropathies include multifocal motor neuropathy (MMN). ), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barré syndrome (GBS). can be done. [Background technology]

[0002] BACKGROUND OF THEINVENTION The complement system increases the ability of antibodies and phagocytes to eliminate microbial invaders and damaged cells from the organism. Complement is an important aspect of the innate immune system, which strengthens the immune system and therefore plays an essential role in preventing infection. Forming an essential line of defense.

[0003] The response to infection is rapid and comprehensive enough to prevent risk to the host, but It must be selective enough to avoid damage to healthy cells. , a stepwise and tightly regulated cascade of more than 30 soluble and cell surface expressed proteins. This delicate balance is achieved by utilizing the complement system. It circulates in the blood as a catabolic protein. Activation of this system occurs when a factor initiates a cascade It activates subsequent factors by specific proteolysis of downstream complement proteins. This cascade ultimately leads to the activation of macrophages and leukocytes. Production of anaphylatoxins that attract and activate blood cells; formation of the lytic membrane attack complex (MAC); and opsonization of targets for phagocytosis and destruction.

[0004] Activation of the complement system occurs via three pathways: the classical pathway; the lectin pathway; and the alternative pathway. Each pathway activates the central complement component C3, which then acts as a cofactor for the This leads to activation of the terminal pathway, resulting in the formation of the MAC (Muller-Eberhrd, Annu Rev. Biochem 1988, 57:321). The classical pathway is strongly triggered by IgM or IgG clusters. This pathway is often referred to as antibody-dependent. It is normally initiated by the hexameric C1q binding to the antibody / antigen complex. It is activated when it binds to the Fc region of IgG or IgM molecules present in the blood. C4 is cleaved to produce C4a and C4b. C2 then binds to the surface-bound C4b (Mg 2+ The existence of (below) bind to form the C4bC2 complex, which is then split into two by activated C1s. Fragments: C2b, a smaller 30 kDa fragment, and C2a, a larger 70 kDa fragment. The larger 70 kDa fragment remains bound to C4b to form the C4bC2a classical pathway C3 convertase. Convertase cleaves C3 into C3a (an anaphylatoxin that enhances inflammation) and C3b, which This can trigger amplification and downstream effector functions.

[0005] Activation of the lectin pathway is mediated by bacterial carbohydrate motifs expressed on the surface of pathogens or microorganisms. MBL binding is mediated by the binding of mannose-binding lectin (MBL) or ficolin to the nucleosomes. This then stimulates the activation of MBL-associated serine proteinase-1 (MASP-1) and MASP-2, leading to the upregulation of C4 and and C2, resulting in 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 They bind directly to targets on the cell surface of microorganisms, foreign bodies, or damaged tissue. Combined C3b can bind to factor B to form C3bB. This complex is involved in the synthesis of factor D. In the presence of C3b, it is cleaved into Ba and Bb. Bb remains associated with C3b to form C3bBb, which Alternative pathway C3 convertase.

[0007] Therefore, these three pathways converge at a central C3 convertase. The enzymes C4bC2a and C3bBb form multimeric complexes with additional C3b molecules to form C5 These enzymes give rise to the convertases C4bC2aC3b and C3bBbC3b, which preferentially convert complement factor C5. C5a selectively cleaves C5a to release C5a (an anaphylatoxin that enhances inflammation) and the fragment C5b. This complex then recruits and associates with C6 and C7; this complex then becomes inserted into the cell membrane. This leads to the binding of multiple C9 molecules, resulting in the C5b-9 membrane attack. The complex (MAC) or soluble terminal complement complex (TCC) is formed. The MAC attaches itself to the cell membrane. By inserting the cells, they form pores and mainly secrete non-nucleated cells (e.g., old red blood cells and some types of granular cells). However, in nucleated cells, MAC formation is tightly regulated. The dissolution effect can be countered by ion pumps. MAC induces host cell damage or activation and acts as a pro-inflammatory mediator In addition to the MAC-mediated effects of the cascade, the anaphylatoxins C3a and C5a It acts as a potent immune modulator, recruiting immune cells to sites of activation. The complement receptors C3b and C4b also bind to various complement receptors and mediate immune complex clearance, phagocytosis, or B cell responses. It can mediate stimulation of.

[0008] The complement system is further regulated by several complement regulatory proteins. These include ancient C1-inhibitor inhibits the initiation step of the canonical and lectin pathways; dissociates C3 convertase factor H, which degrades C4b and C3b; factor I, which degrades C4b and C3b; and the plasma protein vitreous, which inhibits MAC formation. These include ronectin and clusterin and the membrane protein CD59 (Sahu et al., Immunol. es 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 regulated, These defenses may then be adversely affected by the host cells, inducing immune, inflammatory, and degenerative conditions. Complement may be compromised in autoimmune diseases or with dysfunctional regulatory proteins. When overactivated, as occurs in individuals, 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, the complement system plays an important role in the pathogenesis of inflammatory and degenerative diseases, cancer, and transplantation. It is believed that it may play a role in many diseases that have an immune component, such as rejection. The system is increasingly being implicated in diseases of the central nervous system, such as Alzheimer's. (Carpanini et al., Front Immunol 2019, 10:362).

[0010] As both a first detector of foreign or damaging material and a downstream orchestrator of the immune response The unique location of complement makes it an attractive therapeutic target. Recently, inhibitors against more than a dozen different complement targets have been reported. There are.

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

[0012] On the other side of the complement cascade are currently the causes of paroxysmal nocturnal hemoglobinuria (PNH) and and eculizumab (Soliris®), an anti-C5 antibody approved for use in treating aHUS. This antibody binds to a site on C5 that prevents its activation by the C5 convertase, This impairs the release of C5a and the formation of MAC. Several small molecule C5aR1 antagonists (PMX53; P MX205; CCX186) are also being evaluated.

[0013] Antibodies have also been developed to target the C2 protein of the complement cascade. WO2014 / 189378 describes binding molecules, such as antibodies, that have specific C2 activity inhibitory properties. Such binding molecules are useful in treating a variety of human diseases, such as inflammatory diseases or ischemia- It is stated to be useful in treating symptoms of reperfusion injury.

[0014] Despite significant efforts to develop therapeutic agents that target the complement cascade, The clinical success of inhibitors remains limited. In order to develop effective therapies based on complement inhibition, A better understanding of the role of overactive complement activity in various disorders remains to be achieved. It is needed. Summary of the Invention

[0015] (Summary of the invention) The present inventors have demonstrated that targeting the complement cascade upstream of complement factor C5 inhibits the synthesis of abnormal proteins. Neuropathies, especially multifocal motor neuropathy (MMN) and chronic inflammatory demyelinating multiple neuropathy (CDMA) In the treatment of neuropathies such as chronic obstructive pulmonary disease (CIDP) and Guillain-Barré syndrome (GBS) We found that this is likely to be an effective strategy to treat paraprotein-mediated neuropathy. It is a peripheral neuropathy characterized by the presence of "paraproteins" in the serum. Paraproteins are relatively abundant and are produced by abnormal clonal proliferation of B-lymphocytes or plasma cells. It is a specific type of monoclonal antibody or immunoglobulin produced in the body during normal immune response. Unlike globulin antibodies, paraproteins are not normally able to fight infection.

[0016] As reported and exemplified herein, the complement system plays a key role in the treatment of paraproteinaceous neuropathies. In particular, serum from patients with MMN, CIDP, and GBS opsonization of both Schwann cells and motor neurons in vitro Importantly, the results presented here demonstrate that Schwarzenegger activates The researchers found that myocardium-binding proteins, which are essential for the synthesis of the membrane attack complex (MAC), are expressed in skeletal muscle cells and motor neurons. These results further demonstrate that Schwann cells express the complement regulatory protein CD59. It also shows that Wang cells are resistant to complement-mediated lysis. In proteinaceous neuropathies, there is protection from complement-mediated lysis, and The pathology observed is MAC-independent, i.e., triggered by complement proteins upstream of the MAC. Therefore, the present invention shows that the upstream of complement factor C5 is likely to be involved in the It is aimed at treating paraproteinaceous neuropathies by targeting the complement system.

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

[0018] In certain embodiments, the antagonist is an antagonist of the classical complement pathway and / or the lectin complement pathway. Inhibits.

[0019] In one embodiment, the paraproteinaceous neuropathy is a demyelinating neuropathy. do.

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

[0021] In one embodiment, the paraproteinaceous neuropathy is characterized by the presence of autoantibodies. The autoantibody may be an immunoglobulin of the IgM, IgA, or IgG class. .

[0022] In one embodiment, the paraproteinemic neuropathy is caused by autoantibodies to neural antigens. The neuroantigen may be a ganglioside or a neuro The antigen may be myelin associated glycoprotein (MAG). In certain embodiments, the gangliosides are GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD 3, GT1a, GT1b, GT3, and GQ1b. In a preferred embodiment, The ganglioside is GM1.

[0023] In one embodiment, the paraproteinaceous neuropathy is: multifocal motor neuropathy ( MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), - 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 a preferred embodiment, the paraproteinaceous neuropathy is selected from the group consisting of: Multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), or gi Lam-Barre syndrome (GBS). The paraprotein neuropathy is preferably multifocal. This is called sexual motor neuropathy (MMN).

[0024] In certain embodiments, the antagonist inhibits the complement pathway upstream of complement factor C3. In some embodiments, the antagonist inhibits C1, C1q, C1r, or C1s. In some embodiments, the antagonist inhibits complement factors C2, C2a, or C2b. In one embodiment, the antagonist inhibits complement factors C3, C3a, or C3b. In this case, the antagonist inhibits complement factors C4, C4a, or C4b.

[0025] In certain embodiments, the antagonist is: an inhibitory RNA species, e.g., an siRNA or shRNA small molecule inhibitors; biological antagonists, e.g., inhibitory peptides or antibody mimetics , e.g., affibody, affilin, affitin, adnectin, atrimer, ebashi DARPin, Anticalin, Avimer, Finomer, Versabody, or Duocalin or an antibody or antigen-binding fragment thereof.

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

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

[0028] In one embodiment, the antigen-binding fragment comprises: an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VL), Main (VH), single chain antibody (scFv), F(ab')2 fragment, Fab fragment, Fd fragment, Fv fragment, one-arm (single (valent) antibodies, diabodies, triabodies, tetrabodies, unibodies, domain antibodies, and and nanobodies.

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

[0030] In some embodiments, the antibody or antigen-binding fragment is: 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 one embodiment, 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). domain (VL), wherein the VH and VL domains comprise 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] LCDR1 comprising or consisting of Includes.

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

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG heavy chain constant domain. nothing.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof comprises 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.Definition) Unless otherwise defined, all technical and scientific terms used herein refer to the invention as defined by the appended claims. Any term shall have the same meaning as commonly understood by a person skilled in the relevant art. Without limitation, further clarification of some of the terms used herein is provided below. can be.

[0040] "Paraproteinaceous Neuropathy" - as used herein, The term "prostate neuropathy" or "prostate neuropathy" is based on the presence of homogeneous immunoglobulins in serum. They describe a spectrum of peripheral neuropathies characterized by homogeneous immunoglobulins. , known as "paraproteins." They may or may not occur in association with hematologic malignancies. Abnormal clonal proliferation of B-lymphocytes or plasma cells that may result in excessive production of immunoglobulins Several disorders of the peripheral nervous system are caused by excessive production of abnormal immunoglobulins in the blood. PPN is closely related to the presence of antibodies in peripheral nerves, which interact with specific antigenic targets. Exemplary conditions that can be treated according to the present invention include: Major paraproteinaceous neuropathies include MMN, CIDP, and GBS.

[0041] "Multifocal motor neuropathy" - Multifocal motor neuropathy or MMN is a condition that affects It is a rare disorder with a prevalence of approximately 0.6 per 100,000 people, and men are affected more frequently than women (2. 7:1 ratio) (Harschnitz et al., J Clin Immunol 2014, 34:112-119). MMN is a type of It is a chronic immune-mediated neuropathy characterized by symmetric distal predominant muscle weakness. The hallmark of this disease is the presence of multifocal motor conduction block, and patients often IgM antibodies against glycosphingolipid GM1, abundantly expressed in the perinodal regions of peripheral nerves These autologous GM1 IgM antibodies have complement-activating properties and contribute to the severity of the disease. (Vlam et al., Neurol Neuroimmunol Neuroinflamm 2015, 25;2(4)) GM1 is abundantly expressed in peripheral motor neurons and is expressed in both the axon sheath and myelin of peripheral nerves. GM1 has several important functions necessary for maintaining action potential propagation and conduction velocity. Maximal GM1 expression is seen at the nodes of Ranvier and adjacent paranodes, where GM1 pins potassium channels and clusters sodium channels, providing a buffer for the paranodes. Furthermore, GM1 promotes neuritogenesis and apoptosis. It also acts as a receptor modulator of neurotrophic factors that control ptosis, and is involved in membrane signaling and They also function as part of multimolecular assemblies in lipid rafts for transport. Disruption of these functions The anti-GM1 IgM antibodies present in MMN patients , produced by activated B cells (plasma cells); however, the mechanism of this B cell activation is unclear. The system has not yet been established.

[0042] "Guillain-Barre Syndrome" - Guillain-Barre Syndrome or GBS is an infectious disease occurring at an incidence rate of 0.81 per 100,000 individuals. With an incidence rate of ~1.89 cases, men are affected more frequently than women (ratio 3:2) (Kieseier (Nature Reviews, 2018, 4:31). In 60-70% of cases, the first symptom of GBS is It is an acute infection that usually appears 1-3 weeks after an upper respiratory tract or gastrointestinal infection. The initial symptoms of GBS are It is usually a change in sensation or pain accompanied by muscle weakness that begins in the hands and feet. Autoantibodies against various gangliosides found in the axons are a common cause of GBS. They are used to aid in the diagnosis of certain subtypes. For example, anti-GM1 and anti-GD1a IgG antibodies , acute motor axonal neuropathy (AMAN) and acute motor and sensory axonal neuropathy (AMS) These antibodies bind to the nodes of Ranvier and are found in the serum of patients with AN. Thus, the antibody disrupts the fine structural sequences involved in sodium channel clustering, This results in slowing of axonal conduction and loss of function. Alternatively, the antibody binds to motor nerve terminals and inhibits signal transduction. It causes degeneration of prenatal nerve endings.

[0043] "Chronic inflammatory demyelinating polyneuropathy" - chronic inflammatory demyelinating polyneuropathy or CIDP is the most common immune-mediated neuropathy, with an incidence of 0.8-8.9 cases per 100,000 individuals. A range of incidence rates have been reported (Kieseier et al., Nature Reviews, 2018, 4:31). Men are affected more frequently than women (ratio 2:1). CIDP is closely related to GBS, It is considered the chronic counterpart of the acute disease. The most common symptoms of CIDP are pain in the legs, arms, and fingers. and weakness, numbness, and tingling in the hands. Other symptoms include fatigue, pain, and Some variants of CIDP involve the loop of Ranvier. These mutant forms show autoimmunity against paranodal proteins. Neurofascin-186, neurofascin-155, contactin-1, and caspr-1 This includes a subgroup of inflammatory neuropathies associated with IgG4 autoantibodies (Querol et al., Nat Rev Neu rol. 2017, 13(9):533-547). These proteins are expressed in the nodes of myelinated axons, the paranodes, and the nucleoids. Partitioning plays a vital role in the partitioning of the network into nodes, subnodes, and internodes. Voltage-gated sodium channels and voltage-gated potassium channels involved in the transmission of electric potentials These areas maintain separation and are therefore required for saltatory conduction. Destruction of these areas slows nerve conduction. or may result in blockage.

[0044] "Antagonists of the Complement System" - as used herein, or "complement antagonist" refers to an agent that inhibits the function of a complement factor or component of the complement cascade. Any agent capable of blocking or inhibiting complement activity and thereby inhibiting or reducing complement activity. Complement antagonists are the inhibitors of the classical complement pathway, the lectin complement pathway, and the alternative complement pathway. The complement pathway, or any combination thereof, may be blocked or inhibited. antagonists of complement factor 1 (CFR 1.1.2.2) target complement factors common to both of these pathways. Inhibits the classical and lectin complement pathways. Complement antagonists for this purpose inhibit the complement pathway upstream of complement factor C5. The inhibitor inhibits any component or factor of the complement pathway preceding C5 in the complement cascade. In other words, the compound acts as an antagonist, thereby inhibiting or reducing complement activity. This means that it does not directly inhibit C5 or inhibit any of the complement factors downstream of C5. For example, the antagonist may be a complement factor C1, C2, C3, or C4, or any of these. The function of the combination of complement factors can be inhibited. The factor inhibits the entire complement activation cascade even in the presence of its upstream activation signal. This means that the company is unable to fulfill its role in the

[0045] Antagonists for use in the present invention may take the form of any suitable agent. and capable of directly or indirectly blocking or inhibiting the function of a complement factor or component. Antagonists act by downregulating the expression of a target, for example by siRNA technology. In this regard, suitable antagonists include Examples of antagonists include inhibitory RNA species, such as siRNA or shRNA. By directly binding to a target, it can inhibit the function of that target; e.g., as an antagonist. The complement factor binds directly to its target, which then activates the next complement factor in the cascade. In a preferred embodiment, the antagonist is capable of inhibiting specific binding to its target. For example, a C2 antagonist may preferentially target the function of C2 compared to other molecular targets. Antagonists typically act by selectively binding to complement proteins, e.g. achieves the required level of specificity by interacting directly with its target Acting as an antagonist for use in the methods described herein. Suitable agents that may be obtained include: small molecule inhibitors; as well as inhibitory peptides, antibody mimetics, For example, affibodies, affilins, affitins, adnectins, atrimers, and evasins. , DARPins, Anticalins, Avimers, Finomers, Versabodies, and Duocalins. Examples of biological antagonists include, but are not limited to, those that In accordance with the present invention, the antagonist for use is an antibody or an antigen-binding fragment thereof.

[0046] "Antibody" or "Immunoglobulin" - As used herein, "immunoglobulin" means The term "antibody" refers to an antibody or antigen-binding domain that is a member of a human genome, regardless of whether it possesses any relevant specific immunoreactivity. The term "antibody" includes any polypeptide having a combination of two heavy chains and two light chains. The term "antibody" refers to such a population that has a significant, known specific immune reaction activity against a given antigen. 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" refers to five different types of immunoglobulins that can be biochemically distinguished. The class of antibodies includes IgG, which is an immunoglobulin that consists of two subunits with a molecular weight of about 23,000 daltons. It contains an identical light chain polypeptide and two identical heavy chains with molecular weights of 53,000 to 70,000. The chains are linked by disulfide bonds in a "Y" configuration. The light chains support the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. Light chains are classified as either kappa or lambda (κ, λ). Generally, the light and heavy chains are covalently linked to each other. The "tail" portions of the two heavy chains are what allow the immunoglobulin to be delivered to hybridomas, B cells, or When produced by either a genetically modified host cell, In the heavy chain, the amino acid sequence is The chains run from the N-terminus at the forked end of the Y to the C-terminus at the bottom of each chain. If the heavy chain is gamma, mu, alpha, delta, or epsilon (γ, μ, α , δ, ε), and there are several subclasses within them (e.g., γ1 to γ4). You may be aware that the "classes" of antibodies are IgG, IgM, IgA, IgD, or IgE. It is the nature of this chain that determines the immunoglobulin subclass (isotype). IgG1, IgG2, IgG3, IgG4, and IgA1 are well characterized and functionally specialized. Modified forms of each of these classes and isotypes are known to confer would be readily discernible to one of ordinary skill in the art in light of the present disclosure and therefore are within the scope of the present invention. .

[0048] As shown above, the variable region of an antibody allows the antibody to select an epitope on an antigen. In other words, the VL domain of the antibody can specifically recognize and bind to the target molecule. The VH and VH domains combine to form a variable region that defines a three dimensional antigen binding site. This four-chain antibody structure forms an antigen-binding site at the end of each arm of the Y. More specifically, the antigen-binding site is determined by three complementarity determining regions (CDRs) on each of the VH and VL chains. It is defined as follows.

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

[0050] "VHH antibody" - as used herein, referred to as "VHH antibody" or "heavy chain only antibody" The term is produced by species in the Camelidae family, which includes camels, llamas, and alpacas. Heavy-chain-only antibodies or VHH antibodies are composed of two heavy chains and no light chains. Each heavy chain has a variable domain at its N-terminus. These are classified as the variable domain of the heavy chain of the conventional heterotetrameric antibody, i.e., the VH domain. To distinguish them, they are called "VHH" domains.

[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 refers to the fact that certain portions of the variable domains, VH and VL, differ extensively in sequence among antibodies. It refers to the binding and specificity of each particular antibody to its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. This is the "hypervariable loop" in each of the VL and VH domains that form part of the antigen-binding site. The V domains are concentrated in three segments called "lambda light chain segments": the first, second, and third segments of the V light chain domains. and the third hypervariable loops are referred to herein as L1(λ), ​​L2(λ), and L3(λ), Residues 24 to 33 (L1(λ) consisting of 9, 10, or 11 amino acid residues) and 49 to 53 (3 10 residues) and 90-96 (5 residues) (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops are referred to herein as L1(κ), L2(κ), and L3(κ). κ), and L1 ( 6, 7, 8, 11, 12, or 13 residues in the VL domain. κ), 49–53 (L2(κ) consisting of three residues), and 90–97 (L3(κ) consisting of six residues). (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of this invention are referred to herein as H1, H2, and H3. The VH domain is divided into residues 25 to 33 (H1 consisting of 7, 8, or 9 residues), 52 to 56 (H2 consisting of 3 or 4 residues), and H2 (H1, H2O, H3O, H4O, H5O, H6O, H7O, H8O, H9O, H10O, H11O, H12O, H13O, H14O, H15O, H16O, H17O, H20O, H21O, H22O, H33O, H4O, H5O, H6O, H7O, H8O, H9O, H10O, (Morea et al., Methods 20:267-279 (2000)).

[0052] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first and second VL domains, respectively. Refers to the second and third hypervariable loops of both Vkappa and Vlamda isotypes The terms H1, H2, and H3 each refer to a hypervariable loop derived from a VH domain. The term refers to the first, second, and third hypervariable loops of a given amino acid sequence, including γ, ε, δ, α, or μ. The invention encompasses hypervariable loops from any of the known heavy chain isotypes.

[0053] The hypervariable loops L1, L2, L3, H1, H2, and H3 are each defined as a "complementarity determining region" as defined below. "Hypervariable Loops" and "Complementarity Determining Regions" The terms are not strictly synonymous, but they are used interchangeably because the hypervariable loops (HVs) are structurally defined. whereas complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., 2003). and Sequences of Proteins of Immunological Interest (Sequences of Proteins of Immunology). Cal Interest), 5th edition. Public Health Service, National Institutes of Health, Beth esda, MD., 1983), and because the boundaries of the HV and CDRs are different in some VH and VL domains. be.

[0054] The CDRs of the VL and VH domains typically consist of the following amino acids: residues 24 to 34 in the light chain variable domain (L CDR1), 50-56 (LCDR2), and 89-97 (LCDR3), as well as residues 31-35 or 32-36 in the heavy chain variable domain. is defined as including 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3). (Kabat et al., Sequences of Proteins of Immunological Interest, s of Immunological Interest), 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Therefore, HVs may be contained within the corresponding CDRs. and, unless otherwise indicated, the terms used herein for the "hypervariable loops" of the VH and VL domains. Any reference should be construed to encompass the corresponding CDRs, and vice versa.

[0055] The more highly conserved portions of the variable domains are in frame, as defined below. The variable domains of naturally occurring heavy and light chains each contain three hypervariable regions called work regions (FRs). The four FRs (FR1, FR2, FR3, FR4, FR5, FR6, FR7, FR8, FR9, FR11, FR12, FR13, FR14, FR15, FR16, FR17, FR18, FR19, FR111, FR192, FR193, FR194, FR195, FR196, FR197 The hypervariable loops of each chain are closely connected by FRs. These, together with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of the antibody. Structural analysis of the protein revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining region. (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); (J. Mol. Biol., 215:175-182 (1990)). Despite its high degree of sequence variability, Five of these six loops have small main-chain conformations called "canonical structures." These conformations are primarily determined by the length of the loop. and second, their packing, hydrogen bonding, or ability to adopt unusual main-chain conformations. The important residues at specific positions in the loops and framework regions that determine the three-dimensional structure are It is determined by the presence of groups.

[0056] "CDR" - As used herein, the term "CDR" or "complementarity determining region" refers to Discontinuous antigen binding within the variable regions of both the heavy and light chain polypeptides These specific regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6666. 16 (1977) and Kabat et al., Sequences of Proteins of Immunological Interest. in of Immunological Interest (1991), and Chothia et al., J. Mol. Biol. 196:90 1-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996). where these definitions, when compared with one another, have overlapping or non-overlapping amino acid residues. The above references include any of the above-cited references, including any subset thereof. The amino acid residues are shown for comparison. Preferably, the term "CDR" is used for sequence comparison. The CDRs are defined by Kabat based on the above. 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" - As used herein, a "framework region" or "FR region" refers to a The term "variable region" refers to a portion of a variable region (e.g., using the Kabat definition of a CDR), but not to a portion of a CDR. Therefore, the variable region framework contains about 100 to 120 amino acid residues. The length of the heavy chain variable domain is 10 amino acids, but includes only the amino acids outside the CDRs. For the example, and for the CDRs defined by Kabat et al., framework region 1 is framework region 1 corresponds to the domain of the variable region encompassing amino acids 1 to 30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 3 framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94; and framework region 4 corresponds to the variable region domain including amino acid 103 to The light chain framework region corresponds to the light chain variable region domain to the end of the region. The variable regions are similarly spaced apart by each of the CDRs. Using the definition for R, the framework region boundaries are defined by the ends of each CDR as above. In a preferred embodiment, the CDRs are as defined by Kabat. do.

[0058] In a natural antibody, the six CDRs present on each monomeric antibody are responsible for the way the antibody functions in an aqueous environment. specifically arranged to form an antigen-binding site when it assumes its three-dimensional shape; The remainder of the heavy and light variable domains are short, non-contiguous sequences of amino acids. They show less inter-molecular variability in sequence and are called framework regions. The cDNA is mainly in a β-sheet conformation, and the CDRs are connected to the β-sheet structure. , and in some cases, form loops that form part of it. The framework region orients the six CDRs by non-covalent interactions between the chains. The CDRs act to form a scaffold that allows antigen binding. The site defines a surface complementary to an epitope on the immunoreactive antigen. The location of the CDRs is within the skill of the art. can be more easily identified.

[0059] "Constant Region" - As used herein, the term "constant region" refers to a region that is a part of a variable domain. or the portion of an antibody outside the variable region. Immunoglobulin light chains are usually abbreviated as "CL" or "CL They have a single domain, the "constant region", called the VL domain. The immunoglobulin heavy chains are located at the C-terminus of the intermolecular chain. The heavy chains γ, α, and δ have different constant regions, which are composed of CH1 and CH2 domains. It consists of three immunoglobulin domains (CH1, CH2, and CH3) with a flexible hinge region separating the domains. The heavy chains μ and ε have a constant region consisting of four domains (CH1 to CH4). The heavy chain constant domain is located at the C-terminus of the VH domain.

[0060] The numbering of amino acids in heavy and light immunoglobulin chains begins with the N at the forked end of the Y. The constants of the heavy and light immunoglobulin chains run from the -terminus to the C-terminus at the bottom of each chain. Various numbering systems are used to define domains. According to the EU numbering system, IgG domains are The heavy chain constant domains of the polypeptide are defined as follows: CH1-amino acid residues 118 to 215; CH2-amino acid residues 118 to 215; CH3-amino acid residues 231-340; CH4-amino acid residues 341-446. According to the Kabat numbering system, the IgG molecule The heavy chain constant domains are defined as follows: CH1-amino acid residues 114 to 223; CH2-amino acid residues 114 to 223; CH3-amino acid residues 244 to 360; CH3-amino acid residues 361 to 477. An "Fc domain" or "Fc region" is typically The Fc region defines the portion of the constant region of the heavy chain that contains the CH2 and CH3 domains. The Fc region is made up of the hinge region and The "hinge region" may also include some residues from the CH1 domain and the CH2 domain. The hinge region comprises the portion of the heavy chain molecule that binds the ribosome. This hinge region contains approximately 25 residues and is flexible and therefore The hinge region is made up of three different The domains involved can be subdivided into upper, middle, and lower hinge domains (Roux KH (J. Immunol. 161:4083-90 1998). The body may 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 an intact or complete antibody. or a part or portion of an antibody or antibody chain that contains fewer amino acid residues than an antibody chain. The term "antigen-binding fragment" refers to a fragment that binds to an antigen or inhibits antigen binding. Immunoglobulins that compete with tactic antibodies (i.e., with the intact antibodies from which they were derived) As used herein, a "fragment" of an antibody molecule refers to a polypeptide fragment of an antibody or antibody. The term refers to an antigen-binding fragment of an antibody, such as an antibody light chain variable domain (VL), an antibody heavy chain variable Domain (VH), single chain antibody (scFv), F(ab')2 fragment, Fab fragment, Fd fragment, Fv fragment, one-arm ( monovalent) antibodies, diabodies, triabodies, tetrabodies, or antigen-binding fragments thereof Any antigen binding formed by the combination, assembly, or conjugation of As used herein, the term "antigen-binding fragment" includes unibody, domain, and It is further intended to encompass antibody fragments selected from the group consisting of: clonal antibodies, clonal antibodies, and nanobodies. Fragments can be, for example, chemical or non-chemical fragments of an intact or complete antibody or antibody chain. The polypeptide may be obtained by enzymatic treatment or by recombinant means.

[0062] "Specificity" and "Multispecificity" - for use in the methods described herein The antibodies bind to a target antigen in the complement system. The antibodies are said to "specifically bind" to that target antigen. ", where the term "specifically binds" refers to binding to a given target, e.g. This refers to the ability of any antibody to preferentially immunoreact with C1, C2, C3, or C4. Antibodies can be specific and contain one or more binding sites that specifically bind to a particular target. The antibodies can be incorporated into a "multispecific antibody" format, e.g., a bispecific antibody. In this case, a multispecific antibody binds to two or more target antigens. For this reason, a "multispecific antibody" typically comprises heavy and light chain polypeptides having different VH-VL pairs. Multispecific, and in particular bispecific, antibodies can be engineered to contain different combinations or pairings of the same. The subject may also use native antibodies, e.g., Fab antibodies of different specificities conjugated to the Fc region. The antibody can be engineered to adopt a Y-shaped overall conformation with a hamster sphingomyelin receptor. Alternatively, multispecific antibodies, e.g., bispecific antibodies, can be engineered to adopt non-native conformations. For example, variable domains with different specificities or The pair of variable domains are positioned at opposite ends of the Fc region.

[0063] "Modified Antibody" - As used herein, the term "modified antibody" refers to an antibody that is not naturally occurring. Synthetic forms of antibodies that have been modified to include, for example, at least two heavy chain portions, but not two an antibody that does not contain a complete heavy chain of the Multispecific forms engineered to bind to multiple antigens or to different epitopes on a single antigen antibodies of the same structure (e.g., bispecific, trispecific, etc.); 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 antibody" refers to an antibody in multivalent form (e.g., three or more copies of the same antibody). These include, but are not limited to, trivalent, tetravalent, etc. antibodies that bind to the same antigen. In an embodiment, the modified antibody of the invention comprises at least one heavy chain portion lacking a CH2 domain and and a polypeptide binding domain that contains the binding portion of one member of a receptor-ligand pair. It is a fusion protein.

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

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

[0066] "Germlined variant" - Also referred to as "germlined variant" or "germlined antibody." The term "humanizing substitution" refers to one or more amino acids present at a particular position in the VH or VL domain of an antibody. The amino acid residue and the equivalent position in a reference human VH or VL domain encoded by the human germline To specifically refer to "humanized variants" that result in substitutions of amino acid residues occurring at the As used herein, for any given "germlined variant," The amino acid residues to be substituted into the variants are those encoded by a single human germline. Humanized antibodies are generally derived exclusively or preferentially from the VH or VL domains. The terms "germline variant" and "germline variant" are often used interchangeably. One or more "humanizing substitutions" into a camelid-derived (e.g., llama-derived) VH or VL domain. The introduction of the VH or VL domains from camelids (llamas) also allows the production of "humanized variants" of the VH or VL domains from camelids (llamas). The amino acid residues to be substituted are preferentially or exclusively encoded by the human germline. When derived from a single VH or VL domain sequence derived from a camelid (llama), This could result in a "human germline variant" of the gene.

[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, wherein the affinity variant exhibits altered affinity for the target antigen compared to the reference antibody. For example, affinity variants may have altered affinity for a target when compared to a reference antibody. Preferably, the affinity variant exhibits a higher affinity for the target antigen when compared to the reference antibody. Affinity variants typically exhibit improved affinity for the CDRs when compared to a reference antibody. Such substitutions refer to one or more changes in the amino acid sequence at a given position in the CDR. The amino acid to be synthesized is a different amino acid, which may be a natural amino acid residue or a non-natural amino acid residue. The amino acid substitutions may be conservative or non-conservative. It may be objective.

[0068] "Subject" - As used herein, the term "subject" refers to a mammal, preferably a mammalian animal. The term refers to a human. The subject may be either male or female. The subject may be a person having a pathoproteinemia. In one embodiment, the subject may exhibit one or more symptoms consistent with neuropathy. wherein the patient is receiving medical treatment for the treatment of a paraproteinaceous neuropathy. Are receiving and / or actively seeking medical care for the treatment of paraproteinaceous neuropathy This is an individual that is in this state.

[0069] (B. Treatment method) The present invention provides a method for treating a paraproteinemic neuropathy. The method involves administering an antagonist of the complement system to the patient, wherein the antagonist inhibits complement factor C5. The present invention also provides a method for treating paraprotein neuropathy by inhibiting the upstream complement pathway. The present invention provides an antagonist of the complement system for use in a subject, the antagonist comprising It inhibits the complement pathway upstream of complement factor C5.

[0070] Paraproteinemic neuropathy is defined elsewhere herein as a homogeneous protein in serum. Peripheral neuropathies characterized by the presence of immunoglobulins or "paraproteins" Peripheral neuropathies involve motor, sensory, or vasomotor nerve fibers. Of particular interest are diseases or degenerative conditions of the peripheral nerves that are subject to immune-mediated Immune-mediated neuropathies represent a class of peripheral neuropathy caused by injury. In general, immune-mediated peripheral neuropathies are characterized by progressive muscle weakness. These disorders are often accompanied by sensory disturbances such as pain and numbness. It is triggered by autoreactive antibodies in serum that bind to components or proteins at the nodes of Ranvier. The common feature of these disorders is that the blood-nerve barrier (BNB) is compromised. This allows autoantibodies, complement components, and inflammatory cells access to the inner membrane of the nerve. This disruption of BNB induces the upregulation of circulating cytokines such as VEGF, TNFα, and IL1-β, as well as T cell proliferation and proliferation. It is thought that this is induced by metalloproteases secreted from the vesicles. and autoantibodies against myelin-associated glycoproteins present on myelin or at the junctions between axons, respectively. It can bind to neural antigens such as proteins (MAG) or gangliosides. As described elsewhere, the formation of antibody / antigen immune complexes is initiated by the recruitment of complement through C1q. The classical pathway of the system can be initiated.

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

[0072] As reported herein, patients with various paraproteinaceous neuropathies Immunoglobulins present in the serum of patients with PPN can activate complement, which This supports a role for complement-mediated tissue injury in the pathology seen in , eculizumab (Soliris™ - anti-C5 antibody) in the treatment of multifocal motor neuropathy (MMN) Previous clinical studies testing the drug, cerebrospinal fluid (CPE), have not shown efficacy (Fitzpatrick et al., J Perip her Nerv Syst, 2011, 16(2):84-91).

[0073] Importantly, we demonstrated that both Schwann cells and motor neurons undergo MAC-mediated lysis. showed that it upregulates the complement regulatory protein CD59, a protein that protects cells from This observation suggests that elements of the complement cascade upstream of C5 may play a more important role in PPN pathology. This fully overcomes the lack of efficacy previously seen with eculizumab. Without wishing to be bound by theory, it is possible that Complement activation in rheumatoid arthropathy has been shown to be expressed in Schwann cells and motor neurons. Induce the release of cytokines and / or chemokines through activation of the C3aR involved in the activation of the IL-1 receptor. Chemokines such as MCP-1 may attract inflammatory cells, thereby promoting neurological damage. May play a role in promoting scarring.

[0074] The present invention provides an improved treatment for PPN by targeting complement activity upstream of complement factor C5. This is what we are trying to achieve.

[0075] Neuropathies to be treated according to the methods described herein include those that are This includes all peripheral neuropathies classified as "proteinaceous neuropathies." This includes both acute and chronic disorders. Depending on whether the myelin and / or axons are damaged, the condition can be classified as demyelinating or axonal, or In one embodiment, the nifedipine that is to be treated can be classified into these combinations. The neuropathy is an axonal neuropathy. In one embodiment, the The neuropathy that results is a demyelinating neuropathy, e.g., chronic demyelinating neuropathy. be.

[0076] Paraproteinaceous neuropathies are often characterized by the presence of autoantibodies. Thus, in certain embodiments, those treated according to the methods described herein may The neuropathies that are the subject of this study are characterized by the presence of autoantibodies that recognize neural antigens. High titers of serum autoantibodies to peripheral sensory, motor, and sensorimotor neuropathies have been implicated. Antibodies occur in several forms. They are often derived from glycolipids, glycoproteins, and glycosaminoglycans. Reacts with glycosylated cell surface molecules, including aminoglycans, but not with intracellular proteins Antibodies have also been described. There are some correlations between antibody specificity and clinical symptoms, and neuropathies It has been suggested that the immune response may be caused by the antibody. Typically, the antibody is IgM or IgG. The autoantibodies of this type, as described elsewhere herein, are involved in the ancient regulation of the complement system in the peripheral nervous system. The access of autoantibodies and complement to peripheral nerves can lead to hemato-neuropathy. This is possible after the breakdown of the wall (BNB). In one embodiment, In one embodiment, the paraproteinase inhibitor is characterized by high titers of autoantibodies. The neuropathy is characterized by the presence of IgG, IgM, or IgA autoantibodies.

[0077] In one embodiment, the paraproteinemic neuropathy is caused by autoantibodies to neural antigens. In one embodiment, the neural antigen is located at the node of Ranvier. In another embodiment, the neural antigen is a protein in 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. Antibodies are usually associated with the presence of IgM monoclonal proteins. 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 neural antigen is a ganglioside. Neuropathy-associated autoantibody The gangliosides most commonly recognized by are GM1, GD1a, GD1b, and GQ1b. In some embodiments, the gangliosides are GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3 In a preferred embodiment, the ganglioside is selected from the group consisting of GT1a, GT1b, GT3, and GQ1b. The ganglioside is GM1. Individual patients may have genotypes directed against a single ganglioside or against multiple gangliosides. Thus, in one embodiment, the patient may have antibodies against the paraproteinase Neuropathies are characterized by the presence of autoantibodies directed against one or more neural antigens. In certain embodiments, the peripheral neuropathy is characterized by the presence of autoantibodies against one or more gangliosides. In one embodiment, the neural antigen is a paranodal protein. In some embodiments, the neural antigen is selected from contactin 1, NF155, NF186, and NF140. The paranodal protein may be selected from the group consisting of those that have been associated with various peripheral neuropathies. Autoantibodies known to be associated with IL-1, IL-2, and IL-1, are listed in Table 3 below. For example, IgG4 autoantibodies against G, usually of the IgM isotype, have been identified in patients with CIDP. Detection of M1 antibodies is associated with multifocal motor neuropathy and GM1 IgM is a monoclonal IgM paraprotein that is associated with inflammatory bowel disease and lower motor neuropathy. It can exist as monoclonal or polyclonal IgM. Table 3. Autoantibodies in peripheral neuropathies [Table 3]

[0079] The specific paraprotein-related neuropathies to be treated according to the methods described herein The neuropathies are classified into multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIMD), and DP), Guillain-Barré syndrome (GBS), Miller Fisher syndrome, acute motor axonal neuropathy Acute motor and sensory axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), chronic ataxic neuropathy Qi-ophthalmoplegia-IgM paraprotein-cold agglutinin-disialosyl antibody (CANOMAD) syndrome, distal Acquired demyelinating symmetric (DADS) neuropathy, monoclonal gammopathy-associated peripheral neuropathy In a preferred embodiment, the peripheral neuropathy may be selected from the group consisting of peripheral neuropathy, anti-MAG peripheral neuropathy, and POEMS syndrome. Paraprotein neuropathy is classified into multifocal motor neuropathy (MMN), chronic inflammatory neuropathy (CIAN), and The present invention is selected from the group consisting of chronic intracranial neuropathy (CIDP), Guillain-Barre syndrome (GBS), and idiopathic pulmonary fibrosis (IPF). The methods described herein are particularly preferred for the treatment of multifocal motor neuropathy (MMN).

[0080] The methods described herein include administering to a subject in need thereof a therapeutically effective amount of a deficiency in amyloidosis. The subject is preferably a human subject. wherein the patient is receiving medical treatment for the treatment of a paraproteinaceous neuropathy. Are receiving and / or actively seeking medical care for the treatment of paraproteinaceous neuropathy The subject to be treated is typically, but not limited to, an individual suffering from numbness. gradual onset of pain; tingling or tingling in the feet or hands that may radiate up the legs and arms a tingling sensation; a sharp, stabbing, throbbing, freezing pain, or Burning pain; extreme sensitivity to touch; lack of coordination and falling; twitching and muscle Paraprotein neuropathy, including: spasms; muscle thinning (atrophy); muscle weakness or paralysis The subjects to be treated exhibit one or more symptoms consistent with any standard evaluation criteria. Thus, they may have been previously diagnosed with a paraprotein neuropathy. Subjects may have been identified or previously identified based on excess immunoglobulins in serum. Alternatively or in addition, the subject may have been diagnosed with one or more neurological disorders. The patient may have autoantibodies against the antigen.

[0081] The subject to be treated must already be undergoing treatment for a proteinogenic neuropathy. Patients may have had previous treatment for a paraproteinaceous neuropathy. In some embodiments, the subject has previously received IVIg or is receiving IVIg. In other embodiments, the subject has previously received rituximab. In some embodiments, the patient has or is receiving rituximab. The subject has undergone or is undergoing plasma exchange. The elephant may have not responded to previous treatment or its condition may have worsened. Patients may have developed resistance to previous treatments that led to the disease.

[0082] The methods of treatment described herein include complete or partial relief of one or more symptoms associated with PPN. Improvement in performance can be measured by any suitable standard for evaluating PPN treatment. It can be measured using objective criteria.

[0083] The methods of the invention include administering to the patient one or more additional therapeutic agents for the treatment of a paraproteinemic neuropathy. The one or more additional therapeutic agents may include, as a combination therapy, a complement antagonist. Alternatively, the one or more additional agents may be administered simultaneously with a complement antagonist. The drug may be administered before or after administration of the agent, i.e., the drugs may be administered sequentially. Additional therapeutic agents that may be administered according to the methods of the present invention include IVIg, rituximab, colchicine, and cholangiocarcinoma. Thiosteroids, azathioprine, chlorambucil, fludarabine, melphalan, cyclosporine Rofosfamide / prednisone, melphalan, gabapentin, pregabalin, valproate ate, dextromethorphan, tramadol, duloxetine, amitriptyline, and venlafaxine.

[0084] C. Complement Antagonists The results presented herein demonstrate that the final step of the complement cascade, in particular MAC-mediated lysis, is a critical step in the pathogenesis of PPN. This indicates that Schwann Cells and motor neurons express high levels of the complement regulatory protein CD59, which prevents MAC-mediated lysis. Therefore, it is believed that the protein-mediated neuropathies described herein are related to The complement-associated pathology associated with C5 may be mediated by factors upstream of C5, e.g., C3aR. Therefore, the present invention aims to prevent abnormal complement activation by inhibiting the complement pathway upstream of C5. A method for treating proteinaceous neuropathy is provided.

[0085] The method includes administering to the subject an antagonist of the complement system, wherein the antagonist Inhibits the complement pathway upstream of complement factor C5. The antagonist is complement factor C5, either by itself or in combination with the membrane attack complex. It does not directly inhibit any of the downstream factors (C6, C7, C8, C9). In fact, it inhibits the upstream complement factor C5. Antagonists that inhibit the complement system in the liver are known to inhibit factors in the complement cascade prior to complement factor C5 or Complement factors upstream of C5 include C1, C2, C4, and C3. Thus, in certain embodiments, the antagonist is any of C1, C2, C4, and C3. In one embodiment, the complement system is inhibited by inhibiting one or more of the functions of the complement system. The agonist is selected from C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, and C4b. Inhibiting any one of these functions inhibits the complement system.

[0086] In one embodiment, the antagonist inhibits the complement pathway upstream of complement factor C3. Complement factors upstream of include C1, C4, and C2. Thus, in one embodiment, The antagonist inhibits the function of any one of C1, C2, and C4. In one embodiment, the antagonist inhibits C1, C1q, C1r, C1s, C 2. Complement by inhibiting the function of any one of C2a, C2b, C4, C4a, or C4b Inhibits the system.

[0087] The initiator of the classical complement pathway is complement factor C1. Thus, in some embodiments, Thus, the antagonist inhibits complement factor C1. In other embodiments, the antagonist Inhibiting the complement cascade with complement factor C1 inhibits complement factors C1q, C1r, or C1s. and thereby preventing or reducing cleavage of C1-specific targets C4 and C2, and complement This makes it possible to specifically inhibit the activation of complement via the classical pathway of the complement system. Reduces deposition of the opsonin C4b, thereby targeting cells for phagocytosis and destruction Furthermore, inhibition of C1 prevents the formation of the C3 convertase C4bC2a and C4 and C2 are cleaved by MASP enzymes via the lectin pathway. However, the total level of C3 convertase is not known to be related to the complement factor C1. This can also result in significant inhibition.

[0088] In certain embodiments, the antagonist inhibits complement factor C4. In this case, the antagonist inhibits complement factors C4a or C4b. This prevents the formation of the C3 convertase C4bC2a and inhibits the classical and lectin pathways of the complement system. This makes it possible to inhibit complement activation via both pathways.

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

[0090] In certain embodiments, the antagonist inhibits complement factor C3. In this case, the antagonist inhibits complement factors C3a or C3b. Inhibition of C3 inhibits the C5 convertase Prevents or reduces the formation of C3bBbC3b, thereby inhibiting anaphylatoxin C5a and MAC production Deposition of factor C5b can be reduced.

[0091] Depending on the point in the complement cascade at which the antagonist inhibits the complement system, inhibits the classical complement pathway, the lectin complement pathway, the alternative complement pathway, or a combination of these pathways In certain embodiments, the antagonist inhibits the classical and lectin complement pathways. It inhibits the complement pathway but does not affect the alternative complement pathway. It targets C2 and C4. The antagonists inhibit the classical and lectin complement pathways while leaving the alternative pathway intact. In some cases, this important weapon of the innate immune system is not fully functional. It may be beneficial to keep one of the complement pathways intact to prevent Alternatively or additionally, the antagonist may inhibit complement to achieve a therapeutic effect. It may only be necessary to produce a partial inhibition of activity.

[0092] Antagonists for use in the methods of treatment described herein are those that inhibit complement activity. Inhibiting or reducing factors or components of the complement system upstream of complement factor C5 As reported herein, it inhibits the activity of complement factors that precede the formation of the MAC complex. To achieve this, it is beneficial to target the complement cascade upstream of C5. Antagonists for use in accordance with the disclosed methods include those that are biologically active complement-derived peptides. by inhibiting the production of peptides such as C4a, C4b, C3a, C3b, and C5a, thereby inhibiting complement activity. Antagonists can reduce or inhibit, at least in part, cell and It can prevent the damaging effects of complement-derived peptides on tissues. The drug reduces the deposition of opsonins by reducing the deposition of anaphylatoxins. By increasing the concentration of IL-1 in the blood, the production or secretion of cytokines and / or chemokines is reduced. by reducing phagocytosis, by reducing immune cell recruitment, and Any combination of these can inhibit or reduce complement activity. In embodiments, antagonists of the complement system inhibit the deposition of anaphylatoxins. In certain embodiments, the antagonist inhibits deposition of C3a or C5a. In certain embodiments, the antagonist inhibits deposition of opsonins. The agonist inhibits deposition of C3b or C4b. In one embodiment, the antagonist Inhibits the production and / or secretion of inflammatory cytokines and / or chemokines. In an embodiment, the antagonist inhibits the production and / or secretion of MCP-1.

[0093] Antagonists for use in the present invention inhibit the function of a complement factor, thereby inhibiting the function of a complement factor. The agent may be any agent capable of inhibiting or reducing the activity of the As described above, an antagonist for use in the present method can have a specific binding domain for its target. This specificity is usually achieved by directly binding to the target and by detecting the function of the target. This is achieved by using antagonists that block the activity of the receptor.

[0094] In some embodiments, the antagonist is an inhibitory RNA species, e.g., siRNA or sh RNA; small molecule inhibitors; inhibitory peptides; antibody mimetics, e.g., affibodies, affinocytes, and the like. Phosphatase, Affitin, Adnectin, Atrimer, Evasin, DARPin, Anticalin, Avi biological antagonists, including Mar, Finomer, Versabody, and Duocalin; Antibodies and antigen-binding fragments thereof.

[0095] In a preferred embodiment, the antagonist is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof binds to complement factors C1, C1q, C1s, C1r, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27 In one embodiment, the antibody or The antigen-binding fragment binds to complement factors C1, C1q, C1s, C1r, C2, C2a, C2b, C4, C4a, or C4b. In a preferred embodiment, the antibody or antigen-binding fragment binds to C2, preferably C2b. Combine.

[0096] The antibodies and antigen-binding fragments for use in the methods described herein can be used for therapeutic administration to humans. They are intended for use in immunosuppressants and therefore usually include IgA, IgD, IgE, IgG, and IgM types, many of which are is of the IgG type, which in this case is divided into four subclasses IgG1, IgG2a and b, IgG3 In a preferred embodiment, the antibody can belong to either IgG The antibody, optionally an IgG1 antibody, may be monoclonal, polyclonal, or multispecific. The antibodies can be antibodies (e.g., bispecific antibodies) that are specific for their target. Monoclonal antibodies are preferred because they exhibit appropriate immunological specificity. This is because it is specific and directed against a single antigenic site.

[0097] An antigen-binding fragment as described herein typically comprises a portion of a full-length antibody, usually a fragment that binds to its antigen. 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 antibody fragments Examples of suitable antibodies include multispecific antibodies formed using the methods described in Hollige, which is incorporated herein by reference. (See R. and Hudson (2005) Nature Biotechnol. 23:1126-36).

[0098] The antibodies or antigen-binding fragments for use in accordance with the methods described herein are highly human Such antibody molecules having high human homology can be VH and VL of native non-human antibodies that show sufficiently high % sequence identity to the germline sequences In some embodiments, the antibody molecule comprises a non-human Humanized or germline variants of the antibody.

[0099] In non-limiting embodiments, the antibody comprises a CH1 domain and / or a CL domain (respectively, The amino acid sequences may be fully or substantially human. For antibody molecules intended for therapeutic use in humans, the entire antibody constant region or Typically, at least a portion of the amino acid sequence is completely or substantially human. Therefore, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, and the CL domain are One or more of the CH4 domains (and CH5 domain, if present) or any combination thereof It may be completely or substantially human in terms of amino acid sequence. CH1 domain, hinge region, CH2 domain, CH3 domain, and / or CL domain (and / or CH4 domain, if present) The main antibody is a human antibody, preferably a human IgG antibody, more preferably an IgG1, IgG2, or Ig The antibody may be derived from a human IgG1 antibody, G3, or IgG4.

[0100] Advantageously, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain and the CL domain (and and, if present, the CH4 domain) all have completely 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 a nucleic acid sequence that is at least 90%, or at least 92%, human. or at least 95%, or at least 97%, or at least 99% amino acid sequence identity In this context, the term "human amino acid sequence" refers to a rearranged and somatic cell proliferation The antigens encoded by human immunoglobulin genes, including naturally mutated germline genes, It refers to the amino acid sequence.

[0101] In one embodiment, the antibody or antibody fragment is a polypeptide that increases the serum half-life of the antibody or antibody molecule. Such optimization includes a human Fc domain that contains one or more mutations designed to enhance the Efforts are aimed at improving antibody circulation in vivo. Examples of mutations in the human IgG Fc domain include His433Lys+Asn434Phe(NHance); Arg435Hi s; Asn434Ala; Met252Tyr+Ser254Thr+Thr256Glu(YTE); Met428Leu +Asn434Ser(LS); Th r252Leu+Thr253Ser+Thr254Phe(LSF); Glu294delta+Thr307Pro+Asn434Tyr(C6A-66); T hr256Asn+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 ranging from one day to one month between doses. .

[0102] In another embodiment, the Fc domain is an Fc domain that is capable of inhibiting an effector function of the Fc domain. Such mutations are well known to those of skill in the art. In the case of human IgG1, the antibody has been modified to disable or impair its effector functions. Such Fc domain mutations typically include a human Fc domain containing a 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 2 at least one amino acid from the heavy chain constant region of 35, 236, 237, 297, 318, 320, or 322 thereby altering effector function while retaining antigen binding. Examples of mutations in the human IgG Fc domain that impair effectors include The following are the amino acid sequences: Leu234Ala+Leu235Ala (designated LALA); Leu234Ala+Leu235Ala+Pro329Gly (designated LALA) -PG); Ser228Pro+Leu235Glu; Pro331Ser+Leu234Glu+Leu235Phe in IgG4; and and Pro331Ser+Leu234Ala+Leu235Ala.

[0103] Examples of antagonists suitable for use in the present invention that inhibit complement factor C1 include Cinr yze (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 Pustatin Cp40 (Amyndas), PEG-Cp40 (Amyndas), AMY-101 (Amyndas), AMY-201 (Amyndas), APL-1 and APL-2 (Apellis), CDX-1135 (Celldex), APT070 Mirococept (MRC), HC3-1496 (In Code), humanized monoclonal antibody H17 (Elusys Therapeutics), or vaccinia virus Complement control proteins (VCPs) are included.

[0105] D. Anti-C2 Antibodies and Antigen-Binding Fragments Particularly preferred complement antagonists for use in the therapeutic methods described herein are: In a preferred embodiment, for use in therapy, A complement antagonist for this purpose is an antibody or antigen-binding fragment thereof that binds to C2. Suitable anti-C2 antibodies and antigen-binding fragments for use in the described methods include those described herein. The antibodies and antibodies identified in International Patent Application WO2014 / 189378, which is incorporated herein in its entirety, The original binding fragment is included.

[0106] C2 is a 90-100 kDa glycoprotein involved in the classical and lectin pathways of complement activation. As mentioned above, C2 is activated either by C1s in the classical pathway or by activated MASP2 in the lectin pathway. C2 binds to surface-bound C4b (Mg 2+ (in the presence of The 4bC2 complex is then cleaved by activated C1s or MASP2 to form two Fragment: A larger 70 kDa fragment, C2a, which remains bound to C4b and forms the C3-convertase, C4bC2a and a smaller 30 kDa N-terminal fragment, C2b, which is released into the fluid phase. Upon binding to C4b, C2a binds to the C3 and C5 converters, which can cleave C3 and C5, respectively. It constitutes the catalytic subunit of tase.

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

[0108] In one embodiment, the antibody or antigen-binding fragment binds C2b and has a variable heavy (VH) domain. and a variable light (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] LCDR1 comprising or consisting of Includes.

[0109] In certain embodiments, the antibody or antigen-binding fragment binds to C2b and has at least one sequence similar to SEQ ID NO:8. contains sequences that are at least 70%, at least 80%, at least 90%, or at least 95% identical to A variable heavy chain (VH) domain consisting of or at least 70% identical to SEQ ID NO: 9, or a sequence that is 80%, at least 90%, or at least 95% identical to the sequence In one embodiment, the antibody or antigen that binds to C2b comprises a variable light chain (VL) domain comprising The binding fragment comprises a variable heavy domain (VH domain) comprising or consisting of SEQ ID NO:8 and and a variable light domain (VL domain) comprising or consisting of SEQ ID NO:9.

[0110] In one embodiment, the antibody or antigen-binding fragment binds C2b and has a variable heavy (VH) domain. and a variable light (VL) domain, wherein the VH domain has at least the sequence of SEQ ID NO: 8. contain sequences that are 70%, at least 80%, at least 90%, or at least 95% identical to each other or consisting of said sequence, wherein the VL domain comprises the CDR sequence: [ka] LCDR3 comprising or consisting of: [ka] LCDR2 comprising or consisting of: [ka] LCDR1 comprising or consisting of Includes.

[0111] In one embodiment, the antibody or antigen-binding fragment binds C2b and has a variable heavy (VH) domain. and a variable light (VL) domain, wherein the VH domain has the amino acid sequence of SEQ ID NO:8. and the VL domain comprises or consists of the CDR sequence: [ka] LCDR3 comprising or consisting of: [ka] LCDR2 comprising or consisting of: [ka] LCDR1 comprising or consisting of Includes.

[0112] In one embodiment, the antibody or antigen-binding fragment binds C2b and has a variable heavy (VH) domain. and a variable light (VL) domain, wherein the VH domain has the CDR sequences: [ka] HCDR3 comprising or consisting of: [ka] HCDR2 comprising or consisting of: [ka] HCDR1 comprising or consisting of and the VL domain has at least 70%, at least 80%, at least 9 0%, or at least 95% identical to the sequence.

[0113] In one embodiment, the antibody or antigen-binding fragment binds C2b and has a variable heavy (VH) domain. and a variable light (VL) domain, wherein the VH domain has the CDR sequences: [ka] HCDR3 comprising or consisting of: [ka] HCDR2 comprising or consisting of: [ka] HCDR1 comprising or consisting of and the VL domain comprises or consists of the amino acid sequence of SEQ ID NO:9. become.

[0114] A domain of an antibody or antigen-binding fragment has a particular percentage sequence identity to a reference sequence. For the embodiment defined by, the VH and / or VL domain is present in the reference sequence. Alternatively, the mutation may retain the CDR sequences identical to those of the corresponding CDRs in the framework region, such that the mutation is It exists only within the region. Table 4. VH and VL domain sequences [Table 4]

[0115] In certain embodiments, the anti-C2b antibody is a human antibody, in particular a human IgG1, IgG2, IgG3, or IgA antibody. The CH1 domain, hinge domain, CH2 domain, and / or CH3 domain of gG4. In one embodiment, the anti-C2b antibody comprises a C H1 domain, a hinge domain, a C H2 domain, and a C H3 domain of human IgG1. and a CH3 domain and containing substitutions L234A and L235A in the CH2 domain, The positions are defined according to the EU numbering. Alternatively or additionally, an anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG1; and the substitutions H433K and N434F in the CH3 domain, where said positions are defined according to EU numbering. The EU numbering is based on Edelman, GM et al., Proc. Natl. Acad. Sci. USA, 63: 7 8-85 (1969); and Kabat et al., "Sequences of Proteins of Immunological Interest," "Proteins of Immunological Interest," US Dept. Health and Human Services, Refers to the convention for the Fc region described in the 5th edition, 1991.

[0116] In one embodiment, the anti-C2b antibody comprises a C H1 domain, hinge domain, C H2 domain, and C H3 domain of human IgG4. In one embodiment, the antibody comprises a CH1 domain of human IgG4, and a CH3 domain. The amino acid sequence includes a main domain, a hinge domain, a CH2 domain, and a CH3 domain, and in the hinge domain Contains the substitution S228P.

[0117] In one embodiment, the anti-C2b antibody comprises a C H1 domain, hinge domain, C H2 domain, and C H3 domain of human IgG4. domain, and a CH3 domain, and containing the substitution L445P in the CH3 domain.

[0118] In one embodiment, the anti-C2b antibody comprises a C H1 domain, hinge domain, C H2 domain, and C H3 domain of human IgG4. domain, and the CH3 domain, and has the substitutions S228P in the hinge domain and S228P in the CH3 domain. and substitution L445P.

[0119] In one embodiment, the anti-C2b antibody comprises a C H1 domain, hinge domain, C H2 domain, and C H3 domain of human IgG4. domain, and a CH3 domain, and containing substitutions H433K and N434F in the CH3 domain.

[0120] In one embodiment, the anti-C2b antibody comprises a C H1 domain, hinge domain, C H2 domain, and C H3 domain of human IgG4. domain, and the CH3 domain, and has the substitution S228P in the hinge domain and the substitution S228P in the CH3 domain. The amino acid sequence includes the substitutions H433K and N434F.

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

[0122] In some embodiments, the heavy chain constant domain comprises a human IgG4 heavy chain constant domain. In one embodiment, the heavy chain constant domain consists of a human IgG4 heavy chain constant domain. In embodiments, the heavy chain constant domain is set forth in any one of SEQ ID NOs: 12, 13, or 14. The present invention relates to a method for producing a human IgG4 heavy chain constant domain comprising the amino acid sequence of .

[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 at least the amino acid sequence set forth in SEQ ID NO:20. at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% A light chain having sequence identity to the following: (i) a sequence identical to the amino acid sequence set forth in SEQ ID NO: 15, which is at least 90%, at least 95%, or at least 9 a heavy chain having 7%, at least 98%, or at least 99% sequence identity; (ii) a sequence identical to the amino acid sequence set forth in SEQ ID NO: 16, which is at least 90%, at least 95%, or at least a heavy chain having 97%, at least 98%, or at least 99% sequence identity; (iii) a sequence identical to the amino acid sequence set forth in SEQ ID NO: 17, which is at least 90%, at least 95%, or at least a heavy chain having at least 97%, at least 98%, or at least 99% sequence identity; (iv) an amino acid sequence that is at least 90%, at least 95%, or at least a heavy chain having 97%, at least 98%, or at least 99% sequence identity; and (v) a sequence identical to the amino acid sequence set forth in SEQ ID NO: 19, which is at least 90%, at least 95%, or at least 9 heavy chains having 7%, at least 98%, or at least 99% sequence identity or consisting of said light chain and said heavy chain.

[0125] In one embodiment, the anti-C2b antibody has a light chain having the amino acid sequence of SEQ ID NO:20 and a sequence It includes a heavy chain having an amino acid sequence selected from sequence numbers 15 to 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 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] TIFF2025041724000033.tif168170

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

[0129] The heavy and / or light chains of the antibody are characterized by a certain percentage sequence identity to a reference sequence. For embodiments defined in the above, the heavy and / or light chains may be expressed as those present in the reference sequence. The CDR sequences may be identical, so that mutations are only present outside the CDR regions. .

[0130] Unless otherwise specified in this application, the percent sequence identity between two amino acid sequences is the optimal can be determined by comparing these two sequences aligned in a suitable manner, The amino acid sequences to be compared are aligned in the order of optimal alignment between these two sequences. The percentage of identity may include additions or deletions with respect to the reference sequence. determining the number of identical positions where the amino acid residues are identical between the two sequences; Divide by the total number of positions in the comparison window to get the percentage of identity between these two sequences. For example, the BLAST program The BLAST 2 sequence program can be used (see Tatusova et al., "Blast 2 Sequences"). A new tool for comparing protein and nucleotide sequences (Blast 2 sequences - a "new tool for comparing protein and nucleotide sequences)", FEMS Microbiol Lett . 174:247-250), and the parameters used are those given by default (in particular, For the parameters, "Open Gap Penalty": 5 and "Extended Gap Penalty": 2. The matrix to be selected is, for example, the matrix proposed by this program. The percentage of identity between the two sequences being compared. is calculated directly by this program.

[0131] The anti-C2 antibody is modified in the Fc region to enhance binding to the neonatal receptor FcRn, preferably human FcRn. The increased binding affinity can be achieved at acidic pH (e.g., at about pH 7.0 or below). 5.5 to about pH 6.0). The increase in binding affinity may be measurable at neutral pH (e.g., It may be possible to measure at pH 6.9 to approximately pH 7.4. This refers to an increase in the binding affinity for FcRn compared to the binding affinity of the intact Fc region. Typically, The unmodified Fc region has the wild-type amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. In such an embodiment, the binding of an antibody molecule having a modified Fc region to FcRn. The increased affinity is achieved by increasing the affinity of wild-type IgG1, IgG2, IgG3, or IgG4 to FcRn, preferably human FcRn. It is measured relative to the binding affinity of G4.

[0132] E. Pharmaceutical Compositions Antagonists, particularly antibodies and The antibodies and antigen-binding fragments can be formulated as pharmaceutical compositions for administration to a subject.

[0133] Pharmaceutical compositions may be formulated according to conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy. eScience and Practice of Pharmacy, 19th ed., edited by Gennaro, Mack Publishing Co., Eas In accordance with the techniques disclosed in the above, a pharma- ceutically acceptable carrier or diluent may be used. and any other known adjuvants and excipients. The term "pharmaceutically acceptable carrier" refers to a carrier or excipient that is essentially non-toxic. Examples of such excipients include saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous vehicles such as, but not limited to, fixed oils and ethyl oleate. It can also be used.

[0134] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be in the form of a solution, microemulsion, liposome, or other suitable formulation for high drug concentration. Suitable pharmaceutical compositions may be formulated as any other suitable ordered structure. Examples of suitable aqueous and non-aqueous carriers include water, ethanol, polyols (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. Suitable fluidity can be achieved, for example, by the use of coating materials such as lecithin. In the case of dispersions, by maintaining the required particle size and by the use of surfactants. This can be maintained.

[0135] Pharmaceutical compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be achieved by sterilization procedures and the use of various antibacterial and antifungal agents, e.g. Guaranteed by both the inclusion of raben, chlorobutanol, phenol, sorbic acid, etc. In addition, sugars, polyhydric alcohols, for example, mannitol, sorbitol, glycol It may also be desirable to include an isotonicity agent, such as ceramide or sodium chloride, in the composition. Pharmaceutically acceptable antioxidants include, for example, (1) water-soluble antioxidants, such as ascorbyl phosphate, Bisulfite, Sodium Metabisulfite, Sodium Sulfite (2) Oil-soluble antioxidants, e.g., ascorbyl palmitate, butylated hydroxypropyl Cyanisol (BHA), Butylated Hydroxytoluene (BHT), Lecithin, Propyl Gallate, α-tocopherol, etc.; and (3) metal chelating agents, e.g., citric acid, ethylenediamine. Tetraethylorthoacetate (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like may also be included.

[0136] The pharmaceutical composition can be administered by any suitable mode of administration. For example, administration can be by or parenterally, preferably by intravenous (iv) or subcutaneous (sc) injection or infusion. As used herein, the terms "parenteral administration" and "parenterally administered" refer to The phrase refers 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, pneumatic This includes intrathecal, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. nothing.

[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] (Example) The invention will be further understood with reference to the following non-limiting examples.

[0139] Example 1 In vitro model of multifocal motor neuropathy (MMN) using live Schwann cells Complement Inhibition in Schwann cells are myelin cells that wrap around axons in the peripheral nervous system to form the myelin sheath. These cells are erythroblasts, secreting erythroblasts (ER) and secrete erythroblasts (Gln) that secrete erythroblasts. These cells are attached to axons by the protein GM1. Its most important function is to provide myelin sheaths for axons to increase saltatory conduction of neurons. They also aid in the survival and signaling of neurons. Insufficiency leads to demyelination, which results in reduced signaling. Thus, Schwann cells It has been associated with several demyelinating disorders such as MMN. present, derived from a pulmonary metastasis of a patient diagnosed with malignant peripheral nerve sheath tumor. The cells were homogenous, exhibiting a clonal morphology positive for Schwann cell markers S100 and p75. Primary tumor material was passaged in culture for many times until a Schwann cell-like population was formed. This cell line, purchased from ATCC®, was used in the experiments described herein. Used in.

[0140] (A. Method) (1.1 Protocol for culturing Schwann cells) sNF02.2 (ATCC® CRL-2885™) or sNF96.2 (ATCC® CRL-2884) Schwabing The cells were cultured in 10% FCS buffer containing 100 U / mL penicillin, 100 μg / mL streptomycin, and The cells were cultured at 37°C and 5% CO2 in DMEM medium supplemented with 100% ethanol. The cells were cultured twice a week until the confluency reached >80. When the cell culture medium reached 10%, the cells were either passaged or used in an experiment. Wash with PBS. To dissociate the cells, add 3 mL (T75) or 5 mL (T175) of Accutase cell detachment solution. Add the solution (eBioscience™, Thermo Fischer Scientific; Catalog No. 00-455-56) and The cells were incubated at 37°C for 5 min or until the cells were completely detached. Culture medium (7 mL for T75 and 10 mL for T175) was added, the cells were transferred to a 15 mL tube, and then centrifuged (1 The pellet was resuspended in 5 ml of culture medium and incubated for 1 h at 25 × g for 10 min to distinguish between live and dead cells. The cells were then counted using trypan blue. The cells were then adjusted to the desired concentration and plated in culture flasks. (10 mL in a T75, 20 mL in a T175) or used in FACS experiments.

[0141] (1.2 Protocol for staining Schwann cells) Cells were cultured as above and, after counting, 50,000 cells were transferred to v-bottom plates. Wash once in FACS buffer (PBS 1% BSA 0.01% sodium azide) and then place in FACS buffer. Staining was performed with each diluted antibody for 45 minutes on ice in the dark (see Table 7 for FACs staining antibodies). If necessary, wash the cells once by adding 100 μL of FACS buffer. The cells were then incubated with the secondary antibody for 4 h on ice in the dark. After incubation for 5 minutes, 100 μL of FACS buffer was added. The cells were centrifuged (125 × g for 5 min). Finally, the cell pellet was transferred to 100 μL of FACS buffer. Cells were resuspended 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 incubated with VB+ + 20 μL of each opsonization agent diluted in 1:1000 (1 h, RT). Then, 100 μL of VB++ was added and the sample was centrifuged (125×g for 5 min). The supernatant was then discarded and the cells were The cells were preincubated with 100 μL of complement-activating serum (EDTA, MgEDTA, or Ab) for 15 min at RT. The cells were then incubated with 100 mM NaCl (prepared with 10 mM NaCl) at 37°C for 1 h. The cells were then centrifuged at 125 × g for 5 min. The cells were then separated and the supernatant was discarded. Staining for complement activation was then performed according to the staining protocol described above. Therefore, it was carried out.

[0143] 1.4 Cytokine Secretion Assay sNF02.2 cells were cultured as described above. After Accutase treatment and counting, the cells were resuspended in 1 mL of sNF0 2.2 The cells were transferred to a 24-well plate at a density of 10,000 cells / well in culture medium. After 2 days, the cells were cultured in culture medium. Discard the medium and incubate the cells in 100 µL of heat-inactivated MMN patient serum at a dilution of 1:50 in VB++ for 1 h at RT. Then, 100 μL of 15% complement-activated serum (MgEGTA or antibody) was added at RT for 15 min. (preincubated for 1 h) was added to the opsonized cells, and then incubated at 37°C for 1 h. Afterwards, 300 μL of culture medium was added to the cells, resulting in a final volume of 500 μL. After 24 and 48 h, 200 μL of the supernatant was transferred to the University of Utrecht Medical Center. by several core facilities of the University Medical Centre in Utrecht (MC Utrecht) Collected for in-house analysis.

[0144] (B.Result) 1.5 Expression of complement receptors by live Schwann cells To investigate MMN biology using Schwann cells, we assessed the expression of complement regulatory factors. Therefore, we cultured sNF02.2 Schwann cells and performed FACS staining of the expression markers. The results are shown in Figure 2. and summarized in Table 8 below. Table 8: Expression of complement regulatory proteins on sNF02.2 Schwann cells. [Table 8]

[0145] These results suggest that complement regulatory proteins (CD46, CD55, and CD59) are important regulators of the terminal pathway. They found that it is expressed in Schwann cells, with high expression of the nodal factor CD59. High CD59 expression on Wan cells inhibits complement-mediated lysis by inhibiting the formation of MAC. These results suggest that inflammation is restricted by these cells. Schwann cells also express Fcγ receptor 1 ( The expression of CR1 in Schwann cells occurs with the onset of myelination, and these Because cultured sNF02.2 Schwann cells lack myelin, detection of CR1 (CD35) was not observed. (Terenghi F et al., Neurology, 2004, 62: 666-668). Only C3aR showed low expression, whereas C5aR In conclusion, Schwann cells highly express CD59 and are involved in the differentiation of the neural compartment. This suggests sublytic MAC formation that induces inflammation.

[0146] To examine the susceptibility of complement regulatory proteins (CRP) on Schwann cells to complement-mediated lysis To investigate this, sNF02.2 cells were cultured with glycosylphosphatidylinositol anchor proteins containing CD59. The cells were treated with various concentrations of phospholipase C (PL-C), which cleaves the protein (Fitzpatrick A, Ma (Nnn C et al., J Peripher Nerv Syst, 2011, 16(2): 84-91). The results are shown in Figure 3. Both CD55 and CD59 are complement-dependent, since the expression levels of both CRPs were decreased by PL-C treatment. This indicates susceptibility to mediated lysis.

[0147] 1.6 Resistance of Schwann cells to complement-mediated lysis Previous experiments showed high expression of CD59 and CD55 in Schwann cells (sNF92.2 and sNF02.2). To investigate the functional significance of this expression, we cultured 100% IL-16 T cells with or without CD59 and CD55. Anti-GM1-mediated C3 and MAC fixation onto sNF96.2 Schwann cells was evaluated. To investigate the effect of complement activation on the expression of Schwann cells, we cultured Schwann cells and performed further experimental analyses. The cells were transferred to a v-bottom plate for analysis. GPI-linked proteins such as CD59 and CD55 were isolated from the cell surface. Treatment with phospholipase C (PL-C) is commonly used to remove phospholipase C from the nucleosomes. The treatment resulted in the depletion of CD59 and CD55, while leaving surface expression of CD46 and GM1 unaffected. (Figure 4A). Effect of PL-C treatment on susceptibility to C3 and MAC fixation To investigate this, Schwann cells were cultured in serum from MMN patients containing anti-GM1 antibodies or in the presence of Veronar as a control. The cells were then opsonized with 5% complement-activated serum or 480 μg The mice were incubated with 5% complement-activated serum preincubated with 1 µg / ml of ARGX-117 at 37°C for 1 h. Finally, biotin-labeled antibodies against C3 and MAC were used to detect C3 and MAC, respectively. 3 and MAC fixation were detected and stained with APC-conjugated streptavidin. , as shown in Figure 4A , which indicates that C3 fixation is conserved only in the VB in non-PL-C-treated Schwann cells. was slightly increased after opsonization with MMN-05 serum compared with opsonization with However, ARGX-117 was not able to complete C3 fixation in both conditions. The classical pathway-mediated complement activation in the VB control was probably due to the complement-activating serum This is caused by antibodies (e.g., anti-HLA antibodies) present in the C3 Increased fixation was observed, and ARGX-117 enhanced the C-cell fixation induced by MMN-005 serum on PL-C-treated cells. 3 fixation was inhibited, although not completely, by the antibody (Figure 4B). The density is predicted and achieved by IgM antibodies against GM1 and IgM / IgG antibodies against non-GM1 targets. The absence of mCRP mediates strong complement activation. Similar observations have been made regarding MAC fixation. In this case, untreated cells showed low levels of MAC fixation, again in contrast to MMN-005 serum. The PL-C treatment increased the expression of IgG1-related IgG1-related IgG1-related IgG1-related IgG2 ... This resulted in increased MAC fixation after activation, which was inhibited by ARGX-117 (Figure 4C). To assess the induction of complement-mediated cytotoxicity (CDC), viability was subsequently assessed using annexin-V and Double positive cells were classified as late apoptosis / death as measured by combined 7AAD staining. Using cells expressing normal levels of complement regulatory proteins, MMN-005 serum No CDC was detected even after opsonization with . However, PL-C treatment did not result in CDC in M Addition of ARGX-117 increased apoptosis in MN-05 opsonized cells and inhibited complement-mediated In conclusion, Schwann cells express CD59 and CD Due to the high expression levels of 55, the cells are essentially protected from complement-mediated lysis. detected CDC 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 cultured in 96-well v-bottom plates (50 The cells were then transferred to a well (1,000 cells / well) and incubated with anti-HLA antibody (W6 / 32 - BioLegend; Cat. No. 311402) for 3 h at RT. The cells were then opsonized for 10 min with either complement-activating serum (EDTA, MgEGTA, or TNT009). Both were preincubated at RT for 15 min and added to the cells at a final concentration of 5% serum. After incubation for 2 h (37°C), cells were transferred to 96-well V-bottom plates and stained (45 min). After 1 min (0.5 min, 0.5 min, 1 ...

[0149] These results demonstrate that C3 fixation occurs after complement activation with 5% HPS (pooled human serum). Furthermore, after addition of EDTA (10 mM) or TNT009 (480 mg / mL), C3 fixation was reduced to basal levels. Therefore, C3 fixation was complement specific. Furthermore, C3 was blocked in the presence of MgEDTA, They ruled out that this occurs primarily through the alternative complement pathway. The determination was independent of opsonization with W6 / 32.

[0150] 1.8 Binding of autoantibodies from MMN patients to cultured Schwann cells Before determining the pathogenicity of IgM anti-GM1 antibodies, we investigated the effect of these antibodies on human Schwann cell proliferation in vitro. To this end, sNF02.2 Schwann cells were cultured in 96-well round-bottom plates. The cells were then transferred to 50 μL of sNF02.2 culture medium in a 100-well dish (50,000 cells / well). The cells were then incubated with cholera toxin B. GM1 expression was detected by staining with -AF488. The results shown in Figure 6A were consistent with those of in vitro culture. We have shown that human sNF0.2.2 Schwann cells express GM1. Incubation with 100 μg / ml of 100% GM1-specific autoantibodies resulted in higher anti-GM1 expression. More importantly, IgM binding to Schwann cells was associated with opsonization by sera from MMN patients. We investigated various sera from MMN patients with a wide range of IgM anti-GM1 antibody titers. All showed IgM binding to Schwann cells. Furthermore, the titer of IgM binding to Schwann cells was The results were detected in all patient sera examined (Figure 7).

[0151] Next, we evaluated the complement activation ability of the patient's IgM anti-GM1 antibodies in Schwann cells. Human sNF02.2 Schwann cells were transferred to 96-well v-bottom plates (50,000 cells / well) and cultured in vitro with 100% MMN patients. The cells were opsonized with serum (1 h, RT), followed by addition of complement-activating serum (1 h, 37°C). C3 was detected using a FITC-conjugated antibody.

[0152] Initially, only limited C3 fixation was observed due to poor detection of C3 by the antibodies used. Therefore, to enhance C3 detection and reduce background complement activation, To determine the complement activity of sera, we used a variety of approaches. Tested (10%, 5%, and 2.5%) and pre-incubated with 200 μg / mL anti-C5 mAb (1 h, 37° C.). by incubating the sNF02.2 Schwann cells with α-glucosamine to prevent terminal complement pathway activation, thereby preventing lysis of sNF02.2 Schwann cells. To reduce complement background, serum stripping (= on Schwann cells at 4°C) The serum was incubated for 4 x 10 minutes at 4 °C to remove the antibodies in the serum. was also tested.

[0153] The results shown in Figures 8A and 8B show that the complement activation (black bars) and depletion (gray bars) serum This indicates that there is no difference in the number of antibodies present in serum, and that the antibodies still present in serum are responsible for complement activation, Therefore, it was suggested that EDTA did not contribute to C3 fixation. EDTA (white bar) was used as a control. However, the window between non-opsonization and opsonization was the largest. Therefore, the use of 5% complement-activated serum seemed to be optimal. Next, various anti-C3 antibodies were detected. An experiment was conducted to increase the window. The results are shown in Figure 8C. Biotinylated anti-C3 (LSBio, clone 6C9) was used to detect C3 fixation in Ewan cells. A 25% window improvement was demonstrated.

[0154] Therefore, optimal detection of C3 fixation on Schwann cells was achieved by adding complement-activating serum at a final concentration of 5%. and when used with a biotinylated anti-C3 detection antibody (clone 6C9) from LSBio. In conclusion, C3 fixation was detected in Schwann cells after opsonization with serum from MMN patients. This results in the activation of the classical complement pathway by IgM anti-GM1 antibodies present in the serum of MMN patients. It has been shown that this is possible.

[0155] (1.9 C2 dependency) To confirm the importance of complement factor C2 in the pathogenesis of MMN, we assessed C2 dependency. 2. Schwann cells were opsonized with serum from MMN patients and treated with increasing concentrations of recombinant human C2, rhC Complement 2 (U-protein express; Catalog number: C001, 1987) was used to reconstitute C2-depleted serum. The results are shown in Figure 9. This resulted in relatively high mean fluorescence intensity (MFI) values ​​only in C2-depleted serum - MFI signal It was found that the EDTA control had a higher solubility and a smaller experimental window. Nevertheless, serum reconstituted with 30 μg / mL rhC2 did not significantly inhibit C3 fixation in Schwann cells. Furthermore, C3 fixation restored physiological C2 levels by 20–40%. These results suggest that low levels of C2 can also restore C3 fixation. It has also been shown not to result in 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 produced, some of which Several have been approved for clinical use. For example, TNT009 (BIVV009) specifically inhibits the classical complement pathway. It is a humanized anti-C1s antibody that selectively blocks C1s (Jager U, D'Sa et al., Blood, 2019, 133(9): 89 3-901), whereas OMS646 targets MASP-2, thereby blocking the lectin pathway. Clizumab 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 inhibits both the classical and lectin pathways. These results are unique because they inhibit the inflammatory pathway while leaving the alternative pathway intact. IgM anti-GM1 autoantibodies in human serum can specifically bind to sNF02.2 Schwann cells Furthermore, this autoantibody activates the complement cascade and inhibits Schwann cells. To evaluate the therapeutic potential of this antibody, we investigated the effect of IgG on MMN serum. We investigated the effect of ARGX-117 on C3 fixation mediated by Schwann cells. The cells were cultured, transferred to v-bottom plates (50,000 cells / well), and opsonized with MMN patient serum (1 h). RT) and then preincubated with complement blocking antibodies or EDTA (20 min, RT)5 Schwann cells were incubated with 100% complement activity serum from C3-BIO (LSBio, clone 6C 9) and detection of C3 fixation was performed by staining with streptavidin-APC. , as shown in FIG.

[0158] These results show that C3 fixation is inhibited dose-dependently by both ARGX-117 and TNT009. However, the inhibition by both antibodies did not reach that of the EDTA control. T009 was able to block C3 fixation up to 53 μg / mL, but it was more effective at blocking the same extent. A high concentration of ARGX-117 was required (160 μg / mL). Importantly, no anti-C5 was added to the cells. , which was previously reported to block the terminal complement pathway so that C3 fixation could be detected. Surprisingly, the cells were not lysed and the cells were, presumably, Schwann cells. This means that due to their high expression of CD59, they are essentially protected from complement-mediated lysis. In addition, ARGX-117 was able to block C3 fixation in Schwann cells in a dose-dependent manner, and these 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 patients with MMN, we investigated complement-mediated cytokines. Tokine production was measured. Schwann cells were seeded in 24-well plates (10,000 cells / well). (1 h, RT), and complement-activated serum in the presence of complement-blocking antibodies. After 48 hours, the supernatants were harvested and analyzed using the Luminex platform. Cytokine secretion was measured. The results are shown in FIG. 11. It has been shown that stimulation of Dewan cells results in increased secretion of IL-6, IL-8, and MCP-1. Neither elevated levels of IL-6 nor IL-8 were observed in the serum of patients with MN. MCP-1 levels in cells incubated with N patient serum were 2-fold higher than in controls. This increase could be blocked by the addition of ARGX-117 or TNT009. However, 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 sites of infection. Several diseases involving neuroinflammatory processes play a key role and are characterized by neurodegeneration. For example, circulating levels of MCP-1 increase during GBS progression (Orlikowski et al., 2003). J of neuroimmunol, 2003, 134(118-27)). In conclusion, MCP-1 is expressed in the blood of MMN patients. Produced by sNF02.2 Schwann cells after opsonization with serum and subsequent complement activation. ARGX-117 was able to block MCP-1 production.

[0161] (C. Conclusion) The regulation of the complement system was evaluated using sNF02.2 Schwann cells. It has been shown that these cells express high levels of the complement regulatory proteins CD46, CD55, and CD59. Furthermore, we demonstrated that IgM anti-GM1 antibodies from MMN patients activate the classical complement pathway and C3 It was shown that fixation was dependent on the presence of C2 in the MMN model systems examined. , efficiently blocked complement activation in sNF02.2 cells sensitized with anti-GM1 antibodies.

[0162] These results suggest that complement events induced by anti-GM1 autoantibodies contribute to the pathology in MMN patients. This suggests a novel mechanism by which IgM may contribute to the Anti-GM1 autoantibodies activate the classical complement pathway, but due to high expression of CD59 on Schwann cells, Therefore, direct complement-dependent cytotoxicity (CDC) is not possible in this It is an unlikely mechanism that contributes to neuronal damage under these conditions. C complex formation triggers cellular activation that can induce the production and secretion of inflammatory mediators. In this study, we demonstrated that sNF02.2 cells stimulated the complement-dependent uptake of the chemokine MCP-1. Eculizumab, an anti-C5 antibody, inhibited MMN production in one of the two MMN sera tested. The sNF02.2 cells secreted MCP-1, but the sNF02.2 cells secreted MCP-1 in a sublytic manner. These results suggest that this is induced by a mechanism upstream of sexual MAC formation.

[0163] Complement activation by anti-GM1 antibodies induces the release of cytokines and / or chemokines via C3aR These are likely to induce , which was found to be expressed in these cells. Chemokines, such as MCP-1, play a role in attracting inflammatory cells and mediating further neuronal functions. Here, complement activation by anti-GM1 antibodies in Schwann cells can result in dysfunction and damage. leads to MCP-1 secretion, which is inhibited by ARGX-117, a possible explanation for the action of this antibody. In conclusion, the pathology of neuronal destruction and demyelination in MMN patients is Physiology appears to occur upstream of MAC formation, which may lead to a disruption of the complement cascade upstream of the MAC. ARGX-117 is an attractive therapeutic target for treating this disease. These target proteins and therefore represent examples of suitable molecules for treating this indication.

[0164] Example 2 In vitro diagnosis of multifocal motor neuropathy (MMN) using fixed Schwann cells Complement inhibition in a mouse model (A. Method) 2.1 Protocol for culturing and fixing Schwann cells on glass coverslips Schwann cells were cultured with 100 U / mL penicillin, 100 μg / mL streptomycin, and The cells were cultured in DMEM medium supplemented with 10% FCS at 37°C and 5% CO2. When the cell viability reached >80%, the cells were passaged or used in experiments. The medium was discarded and the cells were cultured for 10 To dissociate the cells, add 3 mL (T75) or 5 mL (T175) of Accutase Cell Dissociator. Release solution was added and cells were incubated at 37°C for 5 min or until 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. The pellet was resuspended in 5 ml of culture medium and the viable and dead cells were separated. Trypan blue was used to count the cells to identify the cells. The cells were then adjusted to the desired concentration. and seeded into culture flasks (10 mL in T75, 20 mL in T175) or placed in 24-well plates. The cells were seeded onto a glass cover slip.

[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. The cells were incubated with 4% P Fix with FA (10 min at 4 °C), wash once with 500 μL of PBS, and place the coverslips in a 24-well plate. To minimize non-specific staining, cells were washed with 100 μL of NH4Cl. Quench for 5 min at RT, wash once with 100 μL PBS, then rinse with 100 μL PBS + 2% BSA After washing with 100 μL of PBS, the cells were diluted 1:5 in PBS + 2% BSA and blocked for 2 h at RT. The plates were then incubated face down with 0.0 diluted heat-inactivated MMN patient serum for 60 minutes. Wash the cells once (500 μL of PBS + 2% BSA) and resuspend in 15% complement-active serum (pre-treated with complement-blocking antibodies). Incubate face down at RT for 30 min with or without incubation, then wash once. The cells were then washed (100 μL of PBS + 2% BSA). The cells were then incubated with 100 μL of primary antibody (diluted in PBS + 2% BSA). After staining and incubation face down (1 h, RT, dark), in 100 μL of PBS + 2% BSA The cells were then washed with 100 μL of streptavidin-APC (diluted 1:100 in PBS + 2% BSA). Incubate in the dark at RT with the plate facing down for 1 h, then wash once with 100 µL of PBS. The coverslips were dried on tissue and then washed once with 100 μL of MilliQ water. Pipette 7 μL of ProLong™ Diamond Antifade Mountant containing the API onto the coverslip. The lenses were then fixed with nail polish after drying overnight at 4°C. Cells were analyzed using a Z1 microscope with the following settings: 40x magnification, 25% LED, Alexa Fluor ( trademark) 400ms for the 488 channel, 100ms for the APC channel, and 100ms for the DAPI channel. The antibodies used for staining are shown in Table 9 below. Table 9 Staining antibodies [Table 9] (1) Reacts with both human C3a and C3b (2) Anti-C5b-9 clone aE11 binds to a neoepitope exposed on C9 when taken up into TCC. It is the opposite.

[0166] (B.Result) Experiments performed with fixed Schwann cells were compared with those performed with live Schwann cells. Results very similar to those reported in Example 1 above were observed.

[0167] (2.3 Expression of Complement Receptors) sNF02.2 Schwann cells were cultured and fixed on glass coverslips, and then the expression markers were analyzed. The results are shown in Table 10 below, which shows that all complement regulatory proteins It has been shown that CD59 is expressed in Schwann cells. A key regulator of the terminal pathway, protecting motor neurons from MAC-mediated lysis CD46, CD55, and C3aR showed moderate expression in Schwann cells, and On the other hand, C5aR was highly expressed in the cell bodies of Schwann cells. CD35, CD11b, and CD11c were all , was not present in Schwann cells. Table 10. Expression of complement regulatory proteins in fixed Schwann cells [Table 10]

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

[0169] sNF02.2 Schwann cells were cultured on glass coverslips in 1 mL of sNF02.2 culture medium (50,000 mL) for 3 days. 0 cells / coverslip) and then fixed with 4% PFA. Coverslips were then washed in PBS. The cells were then quenched with NH4Cl4 (5 min, RT) and then blocked with PBS-2% BSA for 2 h. To detect 1 expression, staining with cholera toxin B-Alexa488 was performed. Not only cultured human sNF0.2.2 Schwann cells but also mouse neuroblastoma-derived N2a cells expressed GM1. It was shown that this could be achieved.

[0170] Incubation with MMN patient sera produced prominent anti-GM1 staining that colocalized with IgM staining. We examined various sera from MMN patients with a wide range of IgM anti-GM1 antibody titers and found that all were Schwarzenegger-related. Furthermore, excess soluble unlabeled cholera toxin inhibited IgM binding to MMN serum-derived IgM. The binding of IgM antibodies to human Schwann cells was GM-1 specific, since it blocked the binding of GM-1 antibodies. Preincubation with 100 μg / mL of unlabeled cholera toxin inhibited the binding of cholera toxin to GM1 cancer. Anti-GM1 antibody binding to Schwann cells is suppressed by competition between anti-GM1 antibody and gliosides. This effectively prevented the merger.

[0171] We evaluated the complement activation ability of patient IgM anti-GM1 antibodies against Schwann cells. Toshwan cells were cultured in heat-inactivated serum from MMN patients (containing autoreactive IgM anti-GM1 antibodies) and in vitro. The cells were incubated with HPS (pooled human serum) which served as a complement source from the Using specific antibodies, the deposition of complement factors, e.g., C4 and C3 formation, was determined. The results showed that C4 fixation was detected in 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 the coverslips with various anti-C3 antibodies. Schwann cells opsonized with human serum showed C3 fixation on these cells. However, high anti-IgM GM1 titers did not necessarily correlate with high C3 deposition. were all 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 used to detect the effects of opsonization with serum from MMN patients on fixed sera. This was detected in Ewan cells, suggesting that IgM anti-GM1 antibodies from MMN patients activate the classical complement pathway. This shows that it is possible.

[0175] (2.5 C2 dependency) To confirm the importance of complement factor C2 in the pathogenesis of MMN, we assessed C2 dependency. Therefore, Schwann cells were opsonized with C2-depleted serum and then exposed to increasing concentrations of purified human C2 (hC2 ) to assess C3 formation. The results revealed that C3 fixation was absent in C2-depleted serum. However, the addition of C2 restored C3 fixation in a concentration-dependent manner. Interestingly, complete blockade of C2 was not necessary to block C3 fixation; (approximately 20%) of target levels, demonstrating little C3 formation.

[0176] In summary, C3 fixation was dependent on the presence of C2. Thus, complete inhibition of C2 was not required to prevent C3 formation in Schwann cells.

[0177] 2.6 Efficacy of ARGX-117 on fixed Schwann cells Schwann cells were cultured on cover slips for 3 days and then fixed. The cells were then washed and The cells were then quenched, blocked, and opsonized with patient serum. The cells were incubated for 20 min at RT with complement-activating serum in the presence or absence of various complement-blocking antibodies or IVIg. Finally, the 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. It was done.

[0179] Whereas C3 inhibition was observed with ARGX-117 and TNT009, both used at 200 μg / mL, As expected, no effect was observed with either eculizumab or OMS646 at a concentration of 12.5 mg / mL. Only IVIg at a high concentration (200 μg / mL) showed partial inhibition of C3 fixation in Schwann cells. g / mL) of complement inhibitory antibodies block C4 and C3 fixation in this in vitro disease model of MMN. I was able to do it.

[0180] To determine the differential effect of anti-complement antibodies, titrations were performed and downstream complement events were analyzed. Fixed Schwann cells were opsonized with MMN patient serum, starting at 15 μg / mL and then Increasing concentrations of complement blocking antibodies (TNT009, eculizumab, and ARGX-117) up to 480 μg / mL ) were used to activate complement. Because eculizumab inhibits downstream of C3, the results were consistent with those of eculizumab. ARGX-117 did not show any inhibitory effect on C3 fixation by crizumab. Up to 100% of the subjects showed complete inhibition of C3 fixation.

[0181] (C. Conclusion) The complement inhibitory effect of ARGX-117, which targets human C2b, was evaluated using fixed Schwann cells. This provides an in vitro model that mimics the pathophysiology of MMN. showed that IL-1 cells expressed high levels of the complement regulatory proteins CD46, CD55, and CD59. Furthermore, IgM anti-GM1 antibodies from MMN patients activate the classical complement pathway and C3 fixation. In the MMN model, however, it was shown that ARGX-117 efficiently inhibited complement activation by The study showed that IL-16 effectively blocked IL-16 receptor agonist (ALA)-dependent agonist (EGA)-dependent agonist (TNT009) and was superior to both eculizumab and TNT009.

[0182] Example 3 In Vitro Diagnosis of Multifocal Motor Neuropathy (MMN) Using Induced Pluripotent Stem Cells (iPSCs) Complement inhibition in a mouse model (A. Method) 3.1 Protocol for differentiation of spinal motor neurons from iPSCs Induced pluripotent stem cell (iPSC)-derived motor neuron-like cells (MNs) were generated as described in the literature (Harschnitz O et al. As described in the literature, J Clin Immunol. 2014, Jul;34 Suppl 1:S112-9) and Dr. L van der Pol from the Department of Neurology, UMCU, The Netherlands, and co-researchers Briefly, human fibroblasts were cultured under conditions approved by the Institutional Review Board. These cells were obtained from skin biopsies from healthy individuals under a approved protocol. DMEM GlutaMAX supplemented with 1% fetal bovine serum and 1% penicillin / streptomycin The cells were cultured at 37°C and 5% CO2 in mouse embryonic fibroblast (MEF) medium containing Following the protocol, human fibroblasts were reprogrammed within the first five passages. Cells were plated in 6-well dishes at a density of 10,000 cells per well and incubated for 24 h in MEF medium. After that, MEF medium, 4 mg / mL hexadimethrine bromide, Oct4, Klf4, and S In a mixture containing lentiviral vectors expressing ox2 and c-Myc, After 24 h of incubation, cells were washed three times with PBS, pH 7.4. The cells were then incubated with trypsin-EDTA for 5 days. The cells were pre-coated with 0.1% gelatin and confluent with irradiated MEFs. The culture medium was DMEM-F12, Knockout Serum replacement, 1% penicillin / streptomycin, L-glutamine, non-essential amino acids, β- Contains mercaptoethanol and 20ng / mL recombinant human fibroblast growth factor-basic After 3–6 weeks, the cells were cultured in human embryonic stem cell (huES) medium for further expansion and characterization. To obtain the iPSCs, colonies were manually picked. The iPSCs were maintained in huES medium and after 4–6 passages, iPSCs were cultured on irradiated MEFs in huES medium and stored in liquid nitrogen. iPSCs were passaged manually. Feeder cultures of iPSCs were grown 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 and 5% CO2 for 12-14 days in the MN orientation. Fix the iPSC-MNs with 50% PFA (10 min at 4 °C), wash three times with 500 µL of PBS, and place the coverslips on 2 Removed from 4-well plates. To minimize non-specific staining, and fixative. To neutralize, cells were quenched with 100 µL of 50 mM NH4Cl for 5 min at RT and diluted with 1% PBS. Wash twice (all washes were by immersion of the coverslip) and then add 100 μL of PB After washing with PBS, the cells were diluted 1:1 in PBS+2%BSA for 2 h at RT. The plates were incubated face down for 60 minutes with 1:50 dilution of heat-inactivated MMN patient serum. Then, cells were washed once in PBS and incubated with 15% complement-active serum (preincubated with complement-blocking antibodies). Incubate with 100 µL of PBS (with or without PBS) face down for 30 min at RT and wash once in PBS. The cells were then stained with 100 μL of primary antibody (diluted in PBS + 2% BSA) and placed face down on the in- After incubation (1 h, RT, in the dark), the cells were washed in PBS. Then, the cells were transferred to 100 μL of streptomycin. Implant the cells face down with putavidin-APC (diluted 1:100 in PBS + 2% BSA) for 1 h at RT in the dark. After incubation, the plates were washed once with PBS and once with MilliQ water (similarly, the coverslips were washed once with PBS and once with MilliQ water). After drying the coverslip on the tissue, 7 μL of ProLong 100 μL containing DAPI was added. Pipette Diamond Antifade Mountant onto the objective glass and then place the cover glass. It 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 LEDs at the following settings: (unless otherwise indicated) 40x or 20x magnification (depending on experiment), 25% LED, Alexa Fluor™ For 488 channels 400 ms for the APC channel, 100 ms for the DAPI channel, and 50 ms for the DAPI channel. Four photographs were taken for each condition across the body. All photographs were taken for the positive and negative controls. The images were normalized and recorded in uncompressed 8-bit TFTs in single and composite channels using ZEN 2012 software. The images were exported to IFF format. The mean gray value of each single channel was calculated using ImageJ (Fiji). Ratios were calculated using Microsoft Excel 2010 and visualized using GraphPad Prism 7. The antibodies used for the colors are shown in Table 12 below. Table 12 Staining antibodies [Table 12] (1) Reacts with both human C3a and C3b (2) Anti-C5b-9 clone aE11 binds to a neoepitope exposed on C9 when taken up into TCC. It is the opposite.

[0184] 3.3 Protocol for determining GM1 using ELISA NUNC maxisorp plates were coated with GM1 (5 μg / mL) in methanol. The solution was allowed to evaporate for + / - 2.5 hours in a laminar flow cabinet. The wells were then filled with 200 μL of 1% BSA-P The cells were blocked with 1% BSA-PBS for 2 hours at RT. MMN patient serum was added to 1% BSA-PBS (both were plated with IVIg). The plate was diluted with 100 μL of 100 mM NaCl and incubated at 4°C for 24 hours. The plate was then washed six times with PBS and then incubated for 1 h. The sections were incubated with the primary antibody in 1% BSA-PBS (1 h at RT). Six washes with PBS were then performed. After a washing step, the wells were incubated with 100 μL of HRP-conjugated secondary antibody for 1 h at RT. After six washing steps with PBS, TMB was added and the reaction was stopped with hydrochloric acid. Plates were analyzed at 415 nm using a BioRad ELISA reader.

[0185] (B.Result) Currently, no animal models of MMN are available, so we investigated the pathogenicity of anti-GM1 IgM antibodies in MMN patients. To investigate this, we developed an in vitro model of MMN. These were then generated from fibroblasts and differentiated into motor neurons as described above. Motor neurons were fixed on coverslips using paraformaldehyde (PFA) and NH4Cl The cells were then quenched with MMN patient serum containing anti-GM1 IgM autoantibodies. The cells were opsonized for 1 h to allow the binding of these autoantibodies to GM1. After washing, the cells were subjected to complement blockade. Complement was activated for 30 min using human pooled serum (HPS) in the presence or absence of blocking antibodies. Finally, cells were stained with antibodies against complement factors to assess complement activity. The results are set out 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 receptors and IL-1, IL-2, and IL-1 expression in iPSC-MNs was evaluated. Therefore, iPSC-MNs were cultured and cultured in 4% PFA. After fixation on cover slips, cells were stained for expression markers. The results are shown in Table 13 and Figure 13. As shown. Table 13. Expression of complement regulatory proteins on iPSC-MNs [Table 13]

[0187] These results suggest that complement regulatory proteins (CD46, CD55, and CD59) are all expressed in immobilized motility CD59 is highly expressed in motor neurons. These results suggest that CD46 and CD55 are localized to the cell body and protect against MAC-mediated lysis. Both C3R and C5R were expressed in motor neurons, and C5aR was expressed in cells. CD11b and CD11c were both absent. and CD35 expression was low.

[0188] 3.5 Binding of MMN patient-derived autoantibodies to iPSC-MNs The binding of IgM anti-GM1 antibodies to iPSC-MNs was examined in vitro. For this purpose, iPSC-MNs were cultured at 1200 μl for 1 h. Culture the cells on glass coverslips in L. hMN medium for 12–14 days (80,000–150,000 cells / 13 mm cover). The coverslips were then washed in PBS and fixed with NH4Cl4. The cells were quenched (5 min, RT) and then blocked with PBS-2% BSA for 2 h. To identify the cellular components of the iPSC-derived motility cells, staining with cholera toxin B-Alexa 488 was performed. Motor neurons express GM1, and incubation with MMN patient serum colocalizes with IgM staining. We showed that the antibody produced significant anti-GM1 staining in the presence of IgG.

[0189] We also assessed the complement activation ability of patient IgM anti-GM1 antibodies in iPSC-derived motor neurons. Human iPSC-derived motor neurons were then incubated with heat-inactivated serum from an MMN patient (autoreactive IgM anti-GM1 antibody). The cells were incubated with HPS, which served as an exogenous complement source. The deposition of complement factors such as C4 and C3 was determined using specific antibodies. The results showed that C4 and C3 fixation was associated with iP However, high anti-IgM GM1 titers were not associated with high C3 deposition. In conclusion, C4 and C3 fixation were not necessarily correlated with serum from MMN patients. After opsonization, it was detected in fixed iPSC-MNs, which were identified using IgM anti-GM1 antibodies derived from MMN patients. This shows that the body can activate the classical complement pathway.

[0190] (3.6 C2 dependency) To confirm the importance of complement factor C2 in the pathogenesis of MMN, we assessed C2 dependency. The derived motor neurons were opsonized with C2-depleted serum and then treated with increasing concentrations of purified human C2 (hC2 ) and complement activation was assessed by measuring C3 fixation. The results are shown in Figure 14. It was revealed that C3 fixation was not observed in C2-depleted serum. Furthermore, ARGX-117 restored C3 fixation in a concentration-dependent manner. Constituted C2-depleted serum completely inhibited C3 fixation.

[0191] In summary, C3 fixation is dependent on the presence of C2. This is because the presence of hC2 inhibits C3 fixation. This is demonstrated by the ability of ARGX-117 to block the classical complement pathway and prevent C3 formation. , complete inhibition of C2 does not appear to be necessary.

[0192] 3.7 Efficacy of ARGX-117 in preventing complement fixation in fixed iPSC-MNs As described above in Section 1.9, various inhibitory monoclonal antibodies against complement factors are available. Antibodies have been produced, some of which have been approved for clinical use. The effect of anti-inflammatory antibodies was examined on fixed iPSC-MNs. For this purpose, iPSC-MNs were grown on coverslips. After 12-14 days of culture, the cells were fixed. Then, the cells were washed, quenched, blocked, and incubated for 12-14 days. The cells were then incubated in the presence of various complement blocking antibodies or with opsonization with patient serum. The cells were then incubated for 20 min at RT in the absence or presence of complement-activating serum. , and imaging was performed using 40x or 20x magnification.

[0193] As expected, C4 fixation in motor neurons was inhibited by TNT009 (200 μg / mL) and was not affected by other m It was not inhibited by Ab.

[0194] Whereas C3 inhibition was observed with ARGX-117 and TNT009, both used at 200 μg / mL, Eculizumab, OMS646, and rituximab used at the same concentrations were ineffective. Therefore, ARGX-117, which blocks C2, at a high concentration (200 μg / mL) was able to inhibit this in vitro disease of MMN. We were able to block downstream C3 fixation in a disease model system.

[0195] To determine the differential effect of anti-complement antibodies, titrations were performed and downstream complement events were analyzed. Fixed iPSC-derived motor neurons were opsonized with MMN patient serum. Starting at 3 μg / mL, Then, increasing concentrations of complement blocking antibodies (TNT009, OMS646, and ARGX) were administered up to 200 μg / mL. OMS646 was preincubated with lectin-117 and then added to iPSC-MNs. The results showed no inhibitory effect on C3 fixation by OMS646. 17 showed complete inhibition of C3 fixation up to a concentration of approximately 12 μg / mL. The same results were obtained with TNT009. In this case, C3 deposition was also blocked up to 12 μg / mL. Thus, ARGX-117 and TNT009 are effective in preventing MM Equivalent fixation of C3 fixation in iPSC-derived motor neurons opsonized with N patient serum It worked well.

[0196] 3.8 Complement inhibition by ARGX-117 in other immune-mediated neuropathies Autoimmune peripheral neuropathies are characterized by motor and / or sensory symptoms of disease severity. MMN represents a group of rare, disabling, clinically heterogeneous disorders characterized by These are autoimmune reactivity mediated by specific IgM antibodies targeting GM1. Direct evidence. Chronic inflammatory demyelinating polyneuropathy (CIDMP) as the most common disorder. IDP) and other immune-mediated nephropathy, including Guillain-Barré syndrome (GBS) as the most acute disorder. ARGX-117 has been identified as a treatment for other immune-mediated neuropathies. To evaluate the efficacy, the in vitro model described above was used. Instead of opsonizing iPSC-derived motor neurons with serum, we used IgG1-specific ... The serum from both patients was used. The results are shown in Figure 15. The autoantibodies present activate complement, measured as C3 fixation on motor neurons. This was shown to be blocked by the addition of 200 μg / mL ARGX-117. Thus, in addition to MMN, ARGX-117 may also be useful in disease models of other immune-mediated neuropathies. Complement was blocked with .

[0197] 3.9 Effect of IVIg on complement inhibition using the in vitro MMN assay IVIg is a biologic agent that is often used to treat a variety of autoimmune diseases. In HIV-1 infection, IVIg is the FDA-approved first-line treatment. Several mechanisms are involved in the pathogenesis of HIV-1 infection. IVIg, which includes direct neutralization of immune immunoglobulins, FcR blockade, and modulation of several immune cells IgG present in IVIg binds to C3, thereby inhibiting C3 IVIg also acts to inhibit complement activation by removing it from serum and blocking the terminal complement pathway (T Erenghi 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 inhibits the anti-GM1-GM1 complex. (Bhatheja K, Field, J. Int JB iOchem Cell Biol. 2006;38(12):1995). The standard treatment for MMN is IVIg; however, These treatments are not effective when administered over a long period of time. The complement inhibitory effect of IVIg was investigated using an in vitro MMN model using neuron. To investigate this, iPSC-derived motor neurons were cultured on glass coverslips, fixed, and inoculated with serum from MMN patients. After psoriasis, complement-activating serum was added to activate the complement cascade. g (GammaQuin and Nanogam) and various approaches to IVIg treatment were also evaluated. First, 50 mg / mL of IVIg was added in the opsonization step, in which IVIg inhibited the motility of motor neurons. These results suggest that the IL-16 receptor agonist, ... and IL-16 receptor agonist could not block C3 fixation by opsonization and complement activation, respectively. Addition of IVIg during the first 24 h of C3 fixation or only during complement activation was performed. GammaQuin showed the strongest effect on complement inhibition compared to Nanogam (Figure 16 (see ).

[0198] To evaluate the anti-idiotypic effect of IVIg, we used serum from MMN patients to investigate the competition against GM1. An ELISA was performed. The results are shown in Figure 17, which shows the effect of GM1 binding in this assay. In summary, IVIg treatment inhibits complement activation. They found that IVIg blocks C3 fixation when added at approximately 10 min after injection, confirming that IVIg removes C3 from serum. However, IVIg had no effect on idiotypic antibodies.

[0199] (C. Conclusion) Using iPSC-derived motor neurons, thereby mimicking the possible pathophysiology of MMN The complement inhibitory effect of ARGX-117, which targets human C2b, was evaluated in an in vitro model. The data show that motor neurons in this line express complement regulatory proteins and that expression of CD59 is Furthermore, we showed that IgM anti-GM1 antibodies from MMN patients inhibited the classical complement pathway. ARGX-117 was shown to activate MM cells and that C3 deposition was dependent on the presence of C2. Complement activation in N patients as well as in CIDP and GBS patient samples was efficient Furthermore, IVIg only blocked complement at the complement activation step, not the anti-idiotype. There was no effect on type antibodies. The present application provides the following aspects of the invention. (Aspect 1) A method of treating a paraproteinemic neuropathy in a subject, comprising administering to the subject an inhibitor of the complement system. The method includes administering an antagonist to a complement factor C5 upstream of the complement factor C5. The method of inhibiting the system. (Aspect 2) 2. The method of claim 1, wherein the antagonist inhibits the classical complement pathway and / or the lectin complement pathway. How to. (Aspect 3) The method according to claim 1 or 2, wherein the paraproteinaceous neuropathy is a demyelinating neuropathy. Method of posting. (Aspect 4) The paraproteinaceous neuropathy is characterized by the presence of IgM, IgA, or IgG immunoglobulins. The method according to any one of embodiments 1 to 3, characterized by: (Aspect 5) 2. The method of claim 1, wherein the paraproteinaceous neuropathy is characterized by the presence of autoantibodies. The method according to any one of claims 1 to 4. (Aspect 6) The paraprotein neuropathy is characterized by the presence of autoantibodies against neural antigens. The method according to any one of embodiments 1 to 5, wherein (Aspect 7) According to embodiment 6, the neuroantigen is a ganglioside or a myelin-associated glycoprotein (MAG). How to. (Aspect 8) The gangliosides are GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, GT1b, G The method of embodiment 7, wherein the antibody is selected from the group consisting of T3, T4, and GQ1b. (Aspect 9) The method of embodiment 8, wherein the ganglioside is GM1. (Aspect 10) The paraprotein neuropathy is: multifocal motor neuropathy (MMN), chronic inflammatory demyelination Myeloproliferative Polyneuropathy (CIDP), Guillain-Barre Syndrome (GBS), Miller Fisher Syndrome group, acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMS AN), chronic ataxic neuropathy-ophthalmoplegia-IgM paraprotein-cold agglutinin-disialosyl Antibody (CANOMAD) syndrome, distal acquired demyelinating symmetric (DADS) neuropathy, monoclonal a condition selected from amphoteric pulmonary fibrosis, anti-MAG peripheral neuropathy, and POEMS syndrome; The method according to any one of claims 1 to 9. (Aspect 11) 11. The method of embodiment 10, wherein said paraproteinaceous neuropathy is MMN, CIDP, or GBS. . (Aspect 12) The method of embodiment 10, wherein said paraproteinaceous neuropathy is MMN. (Aspect 13) 13. Any one of aspects 1 to 12, wherein the antagonist inhibits the complement pathway upstream of complement factor C3. The method described in section . (Aspect 14) Any one of aspects 1 to 13, wherein the antagonist inhibits C1, C1q, C1r, or C1s. The method described. (Aspect 15) Any of aspects 1 to 13, wherein the antagonist inhibits complement factors C2, C2a, or C2b. The method described in claim 1. (Aspect 16) Any of aspects 1 to 12, wherein the antagonist inhibits complement factors C3, C3a, or C3b. The method described in claim 1. (Aspect 17) Any of aspects 1 to 13, wherein the antagonist inhibits complement factors C4, C4a, or C4b. The method described in claim 1. (Aspect 18) The antagonist may be: an inhibitory RNA species, e.g., siRNA or shRNA; a small molecule inhibitor; biological antagonists, e.g., inhibitory peptides or antibody mimetics, e.g., Fibodies, affilins, affinins, adnectins, atrimers, evasins, DARPins, Anticalins, Avimers, Finomers, Versabodies, or Duocalins; or antibodies or an antigen-binding fragment thereof. (Aspect 19) The antagonist is: compstatin Cp40 (Amyndas); PEG-Cp40 (Amyndas); AMY-101 (Amyndas) myndas); AMY-201(Amyndas); APL-1 and APL-2(Apellis); Cinryze(Shire); CDX-1135(Cel ldex); APT070Milococept (MRC); HC3-1496 (InCode); Nafamostat (Torii Pharmace and vaccinia virus complement control protein (VCP). The method according to any one of the preceding claims. (Aspect 20) 18. The method according to any one of claims 1 to 17, wherein the antagonist is an antibody or an antigen-binding fragment thereof. Method of posting. (Aspect 21) 21. The method of embodiment 20, wherein the antibody is an IgG antibody. (Aspect 22) The antigen-binding fragment may comprise: an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody variable domain (VL), scFv, F(ab')2 fragment, Fab fragment, Fd fragment, Fv fragment, one-arm (monovalent) antibody, diabody Select from di-, triabodies, tetrabodies, unibodies, domain antibodies, and nanobodies. 21. The method of embodiment 20, wherein (Aspect 23) The antibody or antigen-binding fragment thereof binds to complement factors C1, C1q, C1s, C2, C2a, C2b, C3, C3a, C 23. The method of any one of embodiments 20 to 22, wherein the antibody binds to C3b, C4, C4a, or C4b. (Aspect 24) The antibody or antigen-binding fragment thereof is: stimulimab BIV009 / TNT009 (Bioverativ); ANX005 (A nnexon; mAb H17 (Elusys Therapeutics); and TNT003 (True North). 23. The method described in claim 23. (Aspect 25) 23. The method according to any one of aspects 20 to 22, wherein the antibody or antigen-binding fragment binds to complement factor C2. method. (Aspect 26) 26. The method of embodiment 25, wherein the antibody or antigen-binding fragment binds to the C2b domain of complement factor C2. . (Aspect 27) 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: - an 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 27. The method of embodiment 26, comprising: (Aspect 28) the antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain; wherein the VH domain comprises or consists of the amino acid sequence of SEQ ID NO:8. and wherein the VL domain has the CDR sequence: - 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 28. The method of embodiment 27, comprising: (Aspect 29) The antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 8 or a sequence identical to at least 70 amino acids therein. % identity to the VH domain or consisting of the amino acid sequence. 9 or an amino acid sequence having at least 70% identity thereto. 29. The method of embodiment 27 or embodiment 28, comprising a VL domain comprising or consisting of said amino acid sequence. (Aspect 30) The antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:8 or and a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO:9. 30. The method of embodiment 29, comprising an L domain. (Aspect 31) Any of aspects 27 to 30, wherein the antibody or antigen-binding fragment comprises a human IgG heavy chain constant domain. The method described in claim 1. (Aspect 32) The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. The method of any one of embodiments 25 to 27, comprising a light chain comprising a sequence of 20 amino acids. (Aspect 33) The method of any one of embodiments 1 to 32, further comprising administering IVIg to said subject. (Aspect 34) 34. The method of any one of claims 1 to 33, further comprising administering rituximab to the subject. How to. (Aspect 35) Complement system antagonists for use in treating paraproteinemic neuropathy in a subject - Patents.com an antagonist of the complement pathway, the antagonist inhibiting the complement pathway upstream of complement factor C5; Antagonist of.

Claims

1. 1. A pharmaceutical composition comprising an anti-C2 antibody or antigen-binding fragment comprising a variable heavy (VH) domain and a variable light (VL) domain for use in treating a paraproteinemic neuropathy in a subject, comprising: The VH domain comprises the CDR sequences: - an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 2; - an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and - HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4 and The VL domain comprises the CDR sequences: - an LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:5; - an LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO:6; and LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7 Including, The pharmaceutical composition, wherein the antibody or antigen-binding fragment binds to the C2b domain of C2, and the paraproteinaceous neuropathy is chronic inflammatory demyelinating polyneuropathy (CIDP) or Guillain-Barre 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.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antigen-binding fragment is selected from a single chain antibody (scFv), a F(ab')2 fragment, and a Fab fragment.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the VH domain comprises or consists of an amino acid sequence which is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO:

8.

11. The pharmaceutical composition of claim 10, wherein the VH domain comprises or consists of the amino acid sequence of SEQ ID NO:

8.

12. The pharmaceutical composition according to any one of claims 1 to 9, wherein the VL domain comprises or consists of an amino acid sequence which is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO:

9.

13. The pharmaceutical composition of claim 12, wherein the VL domain comprises or consists of the amino acid sequence of SEQ ID NO:

9.

14. 10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the VH domain comprises or consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 8, and the VL domain comprises or consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

9.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment comprises a human IgG heavy chain constant domain.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 16, and a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:

20.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the treatment further comprises administering IVIg to the subject.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the treatment further comprises administering rituximab to the subject.

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