Complement system antagonists for use in the treatment of paraproteinemic neuropathy

Abstract

The present invention relates to a method for treating paraproteinemic neuropathies by administering antagonists of the complement system to an individual. These antagonists inhibit the upper complement system of complement factor C5. More specifically, the antagonists may be antibodies or antigen-binding fragments thereof that block or inhibit the complement system by inhibiting the C2b domain of complement factor C2. Paraproteinemic neuropathies that can be treated include, in particular, multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre's syndrome (GBS).

Description

Technology Field

[0001] The present invention relates to a method for treating paraproteinemic neuropathies using antagonists of the complement system. These antagonists block or inhibit the upper complement system of complement factor C5. Paraproteinemic neuropathies that can be treated include, in particular, multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre's syndrome (GBS). Background Technology

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

[0003] The response to infection must be rapid and comprehensive enough to prevent danger to the host, yet selective enough not to damage healthy cells. Complement achieves this delicate balance by employing a stepwise and closely regulated cascade system involving typically more than 30 soluble and surface-expressed proteins. Complement factors circulate in the blood as inactive precursor proteins. Activation of this system triggers an activation cascade in which one factor specifically proteolytically hydrolyzes a complement protein further down the cascade to activate the next factor. This cascade can ultimately result in: the production of anaphylatoxins that attract and activate macrophages and leukocytes; the formation of a lytic membrane attack complex (MAC); and opsonization for the phagocytosis and destruction of targets.

[0004] Activation of the complement system can occur through three pathways: the classical pathway; the lectin pathway; and the alternative pathway (see Fig. 1). Each pathway activates the central component, C3, which activates a common termination pathway leading to MAC formation (Muller-Eberhrd, Annu Rev Biochem1988, 57:321). The classical pathway is often referred to as antibody-dependent because it is strongly initiated by IgM or IgG clusters. This pathway is typically activated when hexamer C1q binds to the Fc site of an IgG or IgM molecule present in the antibody / antigen complex. After binding, C1s cause C4 to cleave to produce C4a and C4b. Subsequently, C2 causes surface-bound C4b (Mg 2+ It binds to (in the presence of) to form the C4bC2 complex, which is subsequently cleaved into two fragments by activated C1s: a small 30 kDa fragment, C2b, and a large 70 kDa fragment, C2a, which remains attached to C4b to form the classic C4bC2a pathway C3 convertase. This C3 convertase can cleave C3 into C3a (anaphylatoxin; enhances inflammation) and C3b, thereby initiating amplification and downstream effector functions.

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

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

[0007] These three pathways therefore converge at the central C3 converter. The C3 converter, C4bC2a or C3bBb, forms multimeric complexes with additional C3b molecules, producing the C5 converters, C4bC2aC3b and C3bBbC3b, respectively. These enzymes preferentially cleave complement factor C5 into C5a (anaphylatoxin; which enhances inflammation) and C5b fragments. C5b attracts and associates with C6 and C7; this complex inserts into the cell membrane and interacts with C8; this induces the binding of multiple units of the C9 molecule to form the C5b-9 membrane attack complex (MAC) or soluble terminal complement complex (TCC). MACs insert themselves into the cell membrane to form pores, resulting in the lysis of primarily non-nucleated cells (e.g., old red blood cells and certain Gram-negative bacteria). However, in nucleated cells, MAC formation is strictly regulated, and its lytic effects can be neutralized by ion pumps. Furthermore, sub-lytic levels of MAC can induce host cell damage or activation and can act as pro-inflammatory mediators. In addition to MAC-mediated cascade effects, anaphylatoxins, C3a and C5a, act as potent immunomodulators, enrolling immune cells to activation sites. Opsonins, C3b and C4b, can also bind to various complement receptors and can mediate the clearance of immune complexes, phagocytosis, or the stimulation of B-cell responses.

[0008] The complement system is further regulated by many complement regulatory proteins. These include C-1 inhibitors, which inhibit steps of the classical and lectin pathways; Factor H, which dissociates C3 convertase; Factor I, which degrades C4b and C3b; and plasma proteins vitronectin and clusterin, which inhibit MAC formation, as well as membrane proteins (Sahu et al., Immunol Res 1998, 17:109; Campbell et al., Annu Rev Immunol 1988, 6:161).

[0009] While complement forms an essential line of defense against pathogenic organisms, if not properly regulated, these defensive functions can shift toward host cells and induce or exacerbate immune, inflammatory, and degenerative conditions. When complement is overactivated, as seen in autoimmune diseases or in individuals with dysfunction of regulatory proteins, it induces severe inflammatory responses in numerous organs (Noris and Remuzzi, Semin Nephrol 2013, 33(6): 479-492). Given that the expression of complement proteins commonly occurs throughout the body, this complement system is considered to play a role in many diseases involving immune components, such as inflammatory diseases, degenerative diseases, cancer, and transplant rejection. This complement system is also becoming increasingly implicated in diseases of the central nervous system, such as Alzheimer's disease (Carpanini et al., Front Immunol 2019, 10:362).

[0010] The unique phase of complement as both an initial detector of external or compromised substances and a down-orchestrator of the immune response makes it an attractive therapeutic target. Indeed, clinical development programs for inhibitors targeting more than a dozen distinct complement targets are currently being reported.

[0011] Several soluble complement inhibitors have been produced. C1-INH (from various manufacturers) is currently approved for use in the treatment of hereditary angioedema and is being evaluated for other disorders such as sepsis and ischemia-reperfusion injury. However, C1-INH is a broad-spectrum serine protease inhibitor that blocks initiating proteases of both the classical and lectin pathways, as well as non-complement proteases of the coagulation and contact systems. Conversely, sutimlimab (also known as BIVV009, formerly TNT009) is a humanized, monoclonal antibody designed to selectively inhibit the classical complement pathway by targeting C1s, and shows promise in the treatment of hemolytic anemia and cold agglutinin disease. Other antibodies that inhibit key proteins of the cascade have also been developed. Annexon developed a monoclonal antibody (ANX005) that acts at the C1q level for neurodegenerative and autoimmune diseases, while Omeros developed a monoclonal antibody against MASP-2 (OMS721) as a clinical candidate for the treatment of atypical hemolytic uremic syndrome (aHUS).

[0012] Acting at the opposite end of the complement cascade is eculizumab (Soliris®), an anti-C5 antibody currently approved for use in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and anus. This antibody binds to a site on C5 that blocks activation by C5 converterase, thereby impairing the release of C5a and MAC formation. Many small molecule C5aRq antagonists (PMX53; PMX205; CCX186) showing potential as potential treatments for various disorders, as well as anti-C5a antibodies (IFX-1), have also been evaluated.

[0013] Antibodies targeting the C2 protein of the complement cascade have also been developed. International patent application number WO2014 / 189378 describes a binding molecule, for example, an antibody having properties specifically to inhibit C2 activity. Such a binding molecule is described as being useful for treating symptoms of various human diseases, such as inflammatory diseases or ischemia-reperfusion injury.

[0014] Despite meaningful efforts to develop therapeutic agents targeting the complement cascade, the clinical success of complement inhibitors remains limited. This is likely due to the complex nature of the complement cascade. To develop effective therapies based on complement inhibition, there remains a need to better understand the role of overactive complement activity in various disorders.

[0015] The inventors have discovered that targeting the complement cascade up to complement factor C5 would be an effective strategy for treating paraproteinemic neuropathies, particularly neuropathy such as multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre syndrome (GBS). Paraproteinemic neuropathies are peripheral neuropathy characterized by the presence of "paraproteins" in the serum. Paraproteins are specific types of monoclonal antibodies or immunoglobulins produced in relative excess by the abnormal clonal proliferation of B-lymphocytes or plasma cells. Unlike normal immunoglobulin antibodies, paraproteins are typically unable to fight infections.

[0016] As reported and exemplified here, the complement system plays a crucial role in the pathogenesis of paraproteinemic neuropathy. In particular, serum from patients with MMN, CIDP, and GBS has been found to activate complement through in vitro optionation of both Schwann cells and motor neurons. Importantly, the results shown here further demonstrate that Schwann cells and motor neurons express high levels of the complement regulatory protein CD59, a factor that prevents terminal polymerization of the membrane attack complex (MAC). These results also show that Schwann cells resist complement-mediated lysis. Taken together, this suggests that there is protection against complement-mediated lysis in paraproteinemic neuropathy, and that the observed pathology is likely MAC-independent, i.e., caused by complement proteins upstream of the MAC. Therefore, the present invention aims to treat paraproteinemic neuropathy by targeting the complement system upstream of complement factor C5.

[0017] As reported and exemplified here, the complement system plays a crucial role in the pathogenesis of paraproteinemic neuropathy. In particular, serum from patients with MMN, CIDP, and GBS has been found to activate the complement system through the in vitro optimization of both Schwann cells and motor neurons. Importantly, the results shown here further demonstrate that Schwann cells and motor neurons express high levels of CD59, a complement regulatory protein that prevents terminal polymerization of the membrane attack complex (MAC). These results also indicate that Schwann cells resist complement-mediated lysis. Taken together, this suggests that there is protection against complement-mediated lysis in paraproteinemic neuropathy, and that the observed pathology is likely MAC-independent, meaning it is caused by complement proteins upstream of the MAC. Therefore, the present invention aims to treat paraproteinemic neuropathy by targeting the complement system upstream of complement factor C5.

[0018] In a first aspect, the present invention provides a method for treating paraproteinemic neuropathy in an individual, the method comprising administering a complement system antagonist to the individual, wherein the antagonist inhibits the upper complement system of complement factor C5. The present invention also provides a complement system antagonist for use in treating paraproteinemic neuropathy in an individual, wherein the antagonist inhibits the upper complement system of complement factor C5.

[0019] In some embodiments, this antagonist inhibits the classical complement pathway and / or lectin pathway.

[0020] In some embodiments, paraproteinemic neuropathy is a demyelinating neuropathy.

[0021] In some embodiments, paraproteinemic neuropathy is characterized by the presence of IgM, IgA, or IgG immunoglobulin.

[0022] In some embodiments, paraproteinemic neuropathy is characterized by the presence of autoantibodies. The autoantibodies may be immunoglobulins of the IgM, IgA, or IgG class.

[0023] In some embodiments, paraproteinemic neuropathy is characterized by the presence of autoantibodies against a neural antigen. The neural antigen may be a ganglioside, or the neural antigen may be a myelin-associated glycoprotein (MAG). In embodiments where the neural antigen is a ganglioside, the ganglioside may be selected from GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GT3, and GQ1b. In some preferred embodiments, the ganglioside is GM1.

[0024] In some embodiments, paraproteinemic neuropathy is: multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre's syndrome (GBS), Miller Fisher syndrome, acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), chronic ataxic neuropathy-ophthalmoplegia-IgM paraprotein-cold agglutinins-disialosyl antibodies (CANOMAD) syndrome, distal acquired It is selected from distal acquired demyelinating symmetric (DADS) neuropathy, monoclonal gammopathy associated peripheral neuropathy, anti-MAG peripheral neuropathy, and POEMS syndrome. In some preferred embodiments, the paraproteinemic neuropathy is multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barré syndrome (GBS). The paraproteinemic neuropathy is preferably multifocal motor neuropathy (MMN).

[0025] In some embodiments, the antagonist inhibits the upper complement system of complement factor C3. In some embodiments, the antagonist inhibits C1, C1q, C1r, or C1s. In some embodiments, the antagonist inhibits complement factor C2, C2a, or C2b. In some embodiments, the antagonist inhibits complement factor C3, C3a, or C3b. In some embodiments, the antagonist inhibits complement factor C4, C4a, or C4b.

[0026] In some embodiments, the antagonist is selected from: an inhibitory RNA species, e.g., siRNA or shRNA; a small molecule inhibitor; a biological antagonist, e.g., an inhibitory peptide or an antibody mimic such as affibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, fynomer, versabody or duocalin; or an antibody or their antigen-binding sites.

[0027] In some embodiments, the antagonist is selected from: campstatin Cp40 (compstatin Cp40) (Amyndas); PEG-Cp40 (Amyndas); AMY-101 (Amyndas); AMY-201 (Amyndas); APL-1 and APL-2 (Apellis); Cinryze (Shire); CDX-1135 (Celldex); APT070 mirococept (APT070 Mirococept) (MRC); HC3-1496 (InCode); nafamostat (Torii Pharmaceutical), and vaccineinia virus complement control protein (VCP).

[0028] In some embodiments, the antagonist is an antibody or its antigen-binding fragment, preferably an IgG antibody or its antigen-binding fragment.

[0029] In some embodiments, the antigen-binding fragment is selected from: antibody light chain variable domain (VL), antibody heavy chain variable domain (VH), single chain antibody (scFv), F(ab')2 fragment, Fa fragment, Fd fragment, Fv fragment, one-armed (monovalent) antibody, diabodies, triabodidies, tetrabodies, unibodies, domain antibodies, and nanobodies.

[0030] In some embodiments, the antibody or its antigen-binding fragment binds to complement factor C1, C1q, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b.

[0031] In some embodiments, the antibody or its antigen-binding fragment is selected from: sutimlimab (Bioverativ); ANX005 (Annexon); mAb H17 (Elusys Therapeutics); and TNT003 (True North).

[0032] In some embodiments, an antibody or its antigen-binding fragment binds to complement factor C2. In some embodiments, an antibody or its antigen-binding fragment binds to the C2b domain of complement factor C2.

[0033] In some embodiments, the antibody or its antigen-binding fragment comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), said VH and VL domains comprising the following CDR sequences:

[0034] HCDR3 comprising or composed of sequence number 2 [EDDHDAFAY] ;

[0035] HCDR2 comprising or composed of sequence number 3 [DINPNYESTGYNQKFKG];

[0036] HCDR1 comprising or composed of sequence number 4 [DYNMD];

[0037] LCDR3 comprising or composed of sequence number 5 [QHSRELPYT];

[0038] LCDR2 comprising or composed of sequence number 6 [LASNLKS]; and

[0039] LCDR1 comprising or composed of sequence number 7 [RASKSVRTSGYNYMH].

[0040] In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 70% homology with SEQ ID NO. 8 or an amino acid sequence having at least 70% homology with it, or a VH domain composed thereof, and SEQ ID NO. 9 or an amino acid sequence having at least 70% homology with it, or a VL domain composed thereof. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence of SEQ ID NO. 8 or a VH domain composed thereof, and an amino acid sequence of SEQ ID NO. 9 or a VL domain composed thereof.

[0041] In some embodiments, the antibody or its antigen-binding fragment comprises a chain-fixing domain in human IgG.

[0042] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 16 and a light chain comprising the amino acid sequence of SEQ ID NO. 20.

[0043] In some embodiments, this method further includes administering IVIg to the individual.

[0044] In some embodiments, this method further includes administering rituximab to the subject. Brief explanation of the drawing

[0045] Fig. 1 It is a schematic diagram showing the cell signaling cascades involved in the classical, lectin, and alternative pathways of the complement system. Figures 2a~2g This shows the expression profiles of various membrane proteins in living Schwann cells (sNF02.2). Using flow cytometry, Schwann cells were stained for different membrane proteins, particularly those associated with the complement cascade. The seven graphs show: (A) CD46, (B) CD55, (C) CD59, (D) CD64, (E) CD88, (F) GM1, and (G) MAG. Fig. 3 This shows the expression profiles of CRPs in sNF02.2 Schwann cells with or without treatment with phospholipase-C (PL-C). sNF02.2 Schwann cells were treated with different concentrations of PL-C (1 - 0.5 - 0.25 U / mL) and cultured at 37°C for 1 hour, and stained with antibodies to detect CD46 (A), CD55 (B), and CD59 (C), respectively. Fig. 4This demonstrates that Swan cells (sNF96.2) are resistant to complement-mediated lysis. Each graph in Figure 4 shows the left half of the graph as "not treated with PL-C" and the right half as "treated with PL-C." (A) PL-C treatment reduces the expression of both CD59 and CD55 through the cleavage of the GPI anchor. The expression of CD46, a transmembrane protein, was unaffected (right y-axis). PL-C also had no effect on the expression of GM1 (left y-axis, panel A). (B) Cells are essentially protected against C3 fixation. C3 fixation was observed in MMN patient serum without opsonization, and C3 fixation was slightly increased when opsonized in MMN patient serum. Cells treated with PL-C were more sensitive to C3 fixation, particularly after opsonization. Although only partial inhibition was observed after opsonization and PL-C treatment, ARGX-117 inhibited C3 fixation. This is likely due to the combined opsonization density of the anti-GM1 antibody and serum antibodies against other epitopes. (C) Similar results were observed when MAC fixation was quantified, as shown in Panel B. (D) Swan cells were protected from complement-mediated cell lysis, which is attributed to high levels of CD59 expression. Cells not treated with PL-C survived after incubation with opsonization and complement-activated serum. Cell lysis was observed after treatment with PL-C when Swan cells were opsonized with patient-derived anti-GM1 antibodies. This was inhibited to baseline levels by ARGX-117. Fig. 5This shows complement C3 fixation in sNF02.2 Swan cells induced by anti-HLA antibodies. Cells were opsonized with increasing concentrations of W6 / 32 (anti-HLA antibody) prior to the addition of HPS (human pooled serum) to activate the complement pathway. Both EDTA and TNT009 (480 μg / mL) were added to evaluate complement specificity. Fig. 6 (B) shows GM1 expression and IgM binding in Swan cells. sNF02.2 Swan cells were stained with CTb to detect GM1 expression using flow cytometry. (B) Detection of bound IgM in sNF02.2 Swan cells by flow cytometry after opsonization with MMN-patient serum (hatched bar) or after non-activation of complement (white bar). Fig. 7 Figure 1 shows the binding of IgM from MMN-patient serum to sNF02.2 Swan cells. Swan cells were opsonized with samples from other patients containing various GM1 titers and both showed IgM binding when cultured with sNF02.2 cells. (A) MF1 IgM staining (B) Percentage of IgM-positive Swan cells. Figures 8a–8cThis shows the optimization of C3 fixation on sNF02.2 Swan cells after opsonization with MMN-patient serum. 50,000 Swan cells were seeded into 96-well plates and opsonized with MMN-patient serum (1 hour, at room temperature). Each graph in Figures 8a–8c shows the left half of the graph as 'unopsonized' and the right half as 'opsonized'. (A) Complement was activated using different percentages of complement-activating serum: 10% (left graph), 5% (middle graph), or 2.5% (right graph). Complement-activating serum (black bar) versus cleared serum (= complement-activating serum pre-cultured with Swan cells) (gray bar) was investigated. (B) Summary of results comparing cleared serum (light gray bar) versus the serum (black bar) described in (A). (C) C3 fixation to Swan 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 10% (black bar) or 5% (gray bar) serum. The white bar represents the EDTA control, and all results were as expected. Fig. 9This demonstrates the C2 dependence of complement activation in Swan cells. Swan cells were seeded into 96-well plates and opsonized with MMN-patient serum (MMN-005) (1 hour at room temperature) and subsequently incubated with C2-depleted serum supplemented with increasing concentrations of rhC2, starting from 1.11 μg / mL to 30 μg / mL (physiological concentration). After 1 hour (37°C), the cells were stained with C3-BIO (LSBio, clone 6C9) and C3 fixation was measured using flow cytometry. Fig. 10 This demonstrates the dose-dependent inhibition of C3 fixation in sNF02.2 Swan cells opsonized with MMN-patient serum by ARGX-117. Swan cells were transferred to 96-well plates (50,000 cells / well), opsonized with MMN-patient serum (1 hour at room temperature), and subsequently cultured with 5% complement-activated serum pre-incubated with complement blocking antibodies or EDTA (20 minutes at room temperature). Detection of C3 fixation was performed by staining Swan cells with C3-BIO (LSBio, clone 6C9) and streptavidin-APC. (A) C3 fixation in Swan cells by the MFI value of APC. (B) Percent inhibition of C3 fixation on Swan cells was calculated from 0% inhibition to 5% serum set and 10 mM EDTA set to 100% inhibition. Figures 11a–11cFigure 2 shows cytokine secretion by sNF02.2 Swan cells following complement activation induced by MMN serum. Swan cells were seeded into 24-well plates, opsonized with MMN-patient serum (for 1 hour at room temperature), and complement-activated serum was added in the presence or without complement blocking antibodies. After 48 hours, the supernatant was collected, and cytokine secretion was measured using the Luminex platform. The three graphs show: (A) IL-6, (B) IL-8, and (C) MCP-1. Each graph in Fig. 11 shows bars in the following order (from left to right): no stimulation, IL-1b 10 ng / mL, IL-1b 5 ng / mL, IL-1b 2.5 ng / mL, TNF-α 50 ng / mL, TNF-α 25 ng / mL, TNF-α 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. Fig. 12 It shows the proposed mechanism for the complement event triggered by anti-GM1 autoantibodies present in MMN patients. Fig. 13shows the expression of membrane complement proteins, including complement regulatory proteins (CRPs), in fixed iPSC-MNs. Induced pluripotent stem cell-derived motor neurons (iPSC-MNs) were cultured and fixed on coverslips using 4% PFA prior to staining for expression markers. MGV = mean gray value. Fig. 14 This demonstrates the C2 dependence of C3 fixation. Complement activity was evaluated by measuring C3 fixation in iPSC-derived motor neurons opsonized with C2-depleted serum and reconstituted with increasing concentrations of purified human C2 (hC2). iPSC-MNs were cultured for 3 days prior to fixation and subsequently stained to detect GM1 expression and C3 deposition. Images were analyzed at 40x magnification, and the mean gray value (MGV) was calculated for GM1 only, C3 only, or the ratio between the two. Fig. 15 Figure 15 shows ARGX-117-blocked complement in other immune-mediated neuropathy. Serum from GBS or CIPD patients was used to opsonize motor neurons in the presence or without ARGX-117 (200 μg / mL). iPSC-derived motor neurons were cultured for 12–14 days prior to fixation and stained to detect GM1 expression and C3 deposition. Images were analyzed at 40x magnification, and the mean gray value (MGV) was calculated for GM1 only, C3 only, or the ratio between the two (C3 / GM1). Each graph in Figure 15 is a comparison of 'serum only' (left bar), serum + EDTA (center bar), and 'AR-117 200 μg / mL' (right bar). Fig. 16This shows the effect of IV Ig on C3 fixation using iPSC-MNs opsonized with MMN patient samples. iPSC-derived motor neurons were cultured for 12–14 days prior to fixation and subsequently stained for GM-1 expression and C3 deposition. Two different IVIg batches were examined: GammaQuin and Nanogam were both used at 50 mg / mL. Images were analyzed at 40x magnification, and mean contrast values ​​(MGV) were calculated for GM1 only, C3 only, or the ratio between the two (C3 / GM1). Each graph in Fig. 16 shows bars in the following order (from left to right): serum only, serum + EDTA, GammaQuin ops, GammaQuin ops + comp, GammaQuin comp, Nanogram ops, Nanogram ops + comp, Nanogram comp. Fig. 17 This demonstrates the anti-idiotypic effect of IVIg on GM1 binding by MMN patient serum. ELISA was performed with MMN patient serum, MMN005, and MMN073. GM1 was coated on 96-well plates and incubated with MMN patient serum with or without 50 μg / mL IVIg (2 batches: GammaQuin and Nanogam) using anti-human IgM antibodies before IgM was detected. Specific details for implementing the invention

[0046] A. Definition

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention applies. Without limitation, more explicit definitions of some terms used herein are provided below.

[0048] " paraproteinemia neuropathy ( Paraproteinemic neuropathy - As used herein, "paraproteinemic neuropathy" or "PPN" describes a set of peripheral neuropathy characterized by the presence of homogeneous immunoglobulins in the serum. Homogeneous immunoglobulins are known as "paraproteins." Abnormal clonal proliferation of B-lymphocytes or plasma cells, which may or may not be associated with blood cancer, produces an excess of immunoglobulins. Several disorders of the peripheral nervous system are closely associated with the presence of excess abnormal immunoglobulins in the blood. PPN can be caused by the interaction of antibodies with specific antigen targets on peripheral nerves or by the deposition of immunoglobulins. Examples of paraproteinemic neuropathy that can be treated according to the present invention include MMN, CIDP, and GBS.

[0049] Multifocal motor neuropathy (Multifocal motor neuropathy)" "Multifocal motor neuropathy," or MMN, is a rare disorder with a prevalence of approximately 0.6 per 100,000 people, and men are affected more frequently than women (ratio 2.7:1) (Harschnitz et al., J Clin Immunol2014, 34:112-119). MMN is a chronic, immune-mediated neuropathy characterized by asymmetric weakness, primarily in the distal extremities. A characteristic feature of this disease is the presence of multifocal blockade of motor conduction, and patients frequently exhibit high serum levels of IgM antibodies against glycosphingolipid GM1, which are abundantly expressed in the perinodal regions of peripheral nerves. These auto-GM1 IgM antibodies possess complement-activating properties and are a determinant of disease severity (Vlam et al., Neurol Neuroimmunol Neuroinflamm 2015, 25;2(4)). GM1 is abundantly expressed in peripheral motor neurons and is located in both the axolemma and myelin of peripheral neurons. GM1 possesses several important functions necessary for action potential propagation and the maintenance of propagation velocity. The highest GM1 expression is found around the nodes of Ranvier and paranodes, where potassium channels are anchored and sodium channels assemble to maintain tight junctions through paranode stabilization. Furthermore, GM1 is twofold: as a receptor regulator of neuroaffinity factors regulating neuritogenesis and apoptosis, and as part of a multi-molecule assembly in lipid rafts during membrane signaling and transport. Disruption of these functions results in the failure of conduction across the paranode. Anti-GM1 IgM antibodies present in MMN patients are produced by activated B cells (plasma cells); however, the mechanism of this B cell activation has not yet been established.

[0050] Guillain-Barre syndrome- Guillain-Barré syndrome, or GBS, has an incidence rate of 0.81–1.89 cases per 100,000 individuals, and men are affected slightly more frequently than women (a 3:2 ratio) (Kieseier et al., Nature Reviews ( , 2018, 4:31). In 60–70% of cases, the first symptoms of GBS appear between 1 and 3 weeks after an acute infection, usually an upper respiratory or gastrointestinal infection. Initial GBS symptoms begin in the feet and hands, accompanied by muscle weakness and typically changes in sensation and pain. This often spreads to the arms and upper body. Autoantibodies against various gangliosides found in the axons are used to aid in the diagnosis of specific subtypes of GBS. For example, anti-GM1 and anti-GD1a IgG antibodies are found in the serum of patients suffering from acute motor axonal neuropathy and acute motor and sensory axonal neuropathy (AMSAN). These antibodies bind to the nodes of Ranvier, where they disrupt the fine structural arrangements responsible for the sodium channel assembly, thereby slowing down axon conduction and causing loss of function. On the other hand, antibodies bind to motor nerve endings, causing degeneration of presynaptic nerve endings.

[0051] Chronic inflammatory demyelinating polyneuropathy Chronic inflammatory demyelinating polyneuropathy, or CIDP, is the most common immune-mediated neuropathy, with a reported prevalence ranging from 0.8 to 8.9 cases per 100,000 people (Kieseier et al., Nature Reviews ( , 2018, 4:31). Men are affected slightly more frequently than women (a ratio of 2:1). CIDP is closely associated with GBS and is considered the chronic counterpart of the acute disease. The most common symptoms of CIDP are weakness, numbness, and tingling in the legs, arms, fingers, and hands. Other symptoms include fatigue, pain, balance problems, and impaired ability to walk. Some variants of CIDP exhibit autoimmunity against proteins in Ranvier's nodules. These variants include a subgroup of inflammatory neuropathy with IgG4 autoantibodies against paranodal proteins neurofascin-186, neurofascin-155, contactin-1, and caspr-1. These proteins play a crucial role in the compartmentalization of myelinated axons into nodes, paranodes, and internodes. Compartmentalization is required for saltatory conduction because it maintains the separation of voltage-gated sodium and potassium channels involved in the transmission of action potentials. Disruption of this region can result in slowing or blocking nerve conduction.

[0052] " Composers Antagonist of the complement system- As used herein, "antagonist of the complement system" or "complement antagonist" refers to any agent capable of blocking or inhibiting the function of a complement factor or a component of the complement cascade, thereby inhibiting or reducing complement activity. An antagonist of the complement system may block or inhibit the classical complement pathway, the lectin complement pathway, the alternative complement pathway, or any combination thereof. Preferably, an antagonist of the complement system inhibits both pathways by targeting a complement factor common to both the classical complement pathway and the lectin complement pathway. A complement antagonist for use according to the method described herein inhibits the complement system upstream of complement factor C5. This means that the antagonist inhibits or reduces complement activity by inhibiting any component or factor of the complement pathway prior to C5 in the complement cascade; that is, the antagonist does not directly inhibit C5 or any complement downstream of C5 It means that the factor is not inhibited. For example, an antagonist can inhibit the function of C1, C2, C3, or C4, complement factors, or any combination thereof. Inhibition of complement factor function means that the complement factor is hindered from performing its role in the overall complement activation cascade, even in the presence of upstream activation signals.

[0053] The antagonists used in the present invention may take the form of any suitable formulation and may directly or indirectly block or inhibit the function of a complement factor or component. Antagonists may inhibit the function of a target by down-regulating the expression of the target, such as by siRNA technology. In this regard, suitable antagonists include repressive RNA species, e.g., siRNAs or shRNAs. Antagonists may inhibit the function of a target by directly binding to it; for example, an antagonist may directly bind to its target and interfere with the activation of the next complement factor in the cascade. In a preferred embodiment, the antagonist is specific to its target. For example, an antagonist for C2 will preferentially inhibit the function of C2 compared to other molecular targets. Antagonists will typically achieve the required level of specificity by interacting directly with their targets, for example, by selectively binding to complement proteins. Suitable formulations capable of serving as antagonists used in the methods described herein are, but are not limited to; small molecule inhibitors; and biological antagonists are included, comprising antibody mimetics such as inhibitory peptides, affibodies, affilins, afflictions, adnectins, atrimers, evasins, DARPins, anticalins, avimers, fynomers, versabodies, and duocalins. In a preferred embodiment, the antagonist used in the present invention is an antibody or an antibody-binding fragment thereof.

[0054] antibodies ( antibody or Immunoglobulin (Immunology)As used herein, the term “immunoglobulin (Immunology)” includes polypeptides having a combination of two heavy chains and two light chains, whether or not they possess any corresponding specific immunoreactivity. “Antibody” refers to such an aggregate having a specific immunoreactivity that is meaningfully known to any antigen of interest. Antibodies and immunoglobulins contain light chains and heavy chains, and may or may not have covalent bonds between the chains. The basic immunoglobulin structure in vertebrate systems is relatively well understood.

[0055] The general term “immunoglobulin” encompasses five distinct classes of antibodies that are biochemically distinguishable. As far as IgG is concerned, immunoglobulin comprises two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons, and two identical heavy chains with a molecular weight of 53,000–70,000. The four chains are connected by disulfide bonds to form a “Y” structure, where the light chains begin at the mouth of the “Y” and continue to the variable site, bracketing the heavy chains. The light chains of antibodies are classified as kappa or lambda (κ,λ). Each class of heavy chains can bind to either the kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bound to each other, and the “tail” portions of the two heavy chains are connected by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked ends of the Y structure to the C-terminus at the bottom of each chain. Experts in this field will know that heavy chains have some subclasses among them (e.g., g1–g4) and are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε). This is the property of these chains that determines the “class” of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well identified and are known to confer functional characteristics.Each modified form (version) of this class and isotype is easily distinguishable to an expert in consideration of immediate disclosure and, therefore, falls within the scope of an improvised invention.

[0056] As described above, the variable region of the antibody enables the antibody to selectively recognize and specifically bind to an antigen. That is, the antibody's VL domain and VH domain combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each Y arm. More specifically, the antigen-binding sites are defined by three complementary determining regions (CDRs) located in each VH and VL chain.

[0057] The term "antibody" used here is also intended to include "VHH antibodies" or "heavy-chain only antibodies."

[0058] " VHH Antibody - As used herein, "VHH antibodies" or "heavy-chain only antibodies" refer to antibodies against camels, llamas, and alpacas, including those of the Camelidae family ( Camelidae It refers to a type of antibody produced exclusively by species of the family. A heavy chain-only antibody, or VHH antibody, contains two heavy chains and has no light chain. Each heavy chain has a variable domain at the N-terminus, and this variable domain is referred to as the "VHH" domain to distinguish it from the heavy chain variable domain of traditional heterotetrameric antibodies, i.e., the VH domain described above.

[0059] variable part (Variable region) or "variable domain"The terms "variable region" and "variable domain" may be used interchangeably here and are considered to have the same meaning. The term "variable" implies that certain parts of the variable domains VH and VL differ significantly in sequence among antibodies, and that each specific antibody utilizes this for binding to its target antigen and specificity. However, this variability is not evenly distributed throughout the antibody's variable domain. Instead, it is concentrated in three parts, referred to as "hypervariable loops," within the VL and VH domains, respectively, which form part of the antigen-binding site. The first, second, and third highly variable loops of the V-lambda light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and can be defined as comprising residues 24-33 (composed of 9, 10, or 11 amino acid residues of L1(λ), ​​49-53 (composed of 3 residues of L2(λ), and 90-96 (composed of 5 residues of L3(λ)) in the VL domain (Morea et al., Methods 20:267-279 (2000). The first, second, and third highly variable loops of the V-kappa light chain domain are referred to herein as L1(κ), L2(κ), and L3(κ), and residues 25-33 (composed of 6, 7, 8, 11, 12, or 13 residues of L1(κ), in the VL domain), It can be defined as including 49-53 (L2(κ), consisting of 3 residues) and 90-97 (L3(κ), consisting of 6 residues) (Morea et al., Methods 20:267-279 (2000).The first, second, and third height-variable loops of the VH domain are referred to here as H1, H2, and H3 and can be defined as including residues 25–33 (composed of 7, 8, or 9 residues of H1), 52–56 (composed of 3 or 4 residues of H2), and 91–105 (H3, length highly variable) (Morea et al., Methods 20:267–279 (2000)).

[0060] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third height-variable loops of the VL domain, respectively, and include height-variable loops obtained from both Vkappa and Vlambda isotypes. The terms H1, H2, and H3 refer to the first, second, and third height-variable loops of the VH domain, respectively, and include height-variable loops obtained from any known heavy chain isotype, including γ, ε, δ, α, or μ.

[0061] Hypervariable loops L1, L2, L3, H1, H2, and H3 may each contain a part of a “complementarity determining region” or “CDR” as defined below. “Hypervariable loops” and “complementarity determining region” are not strictly synonyms because hypervariable loops (HVs) are defined based on structure, whereas complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983) and the boundaries of HVs and CDRs may differ in some VH and VL domains.

[0062] The CDRs of the VL and VH domains can typically be defined as containing the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Therefore, HVs may be included within the corresponding CDRs, and a reference to the “highly variable loop” of the VH and VL domains here should be interpreted to include the corresponding CDRs, and vice versa, unless otherwise indicated.

[0063] The more highly conserved portion of the variable domain is called the framework region (FR) as defined below. Natural heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4, respectively) and are connected by three highly variable loops, adopting a predominantly beta-sheet (β-sheet) structure. In each chain, the highly variable loops are closely bound together by the FRs and, together with highly variable loops from other chains, contribute to the formation of the antibody's antigen-binding site. Structural analysis of antibodies has shown a correlation 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)); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high sequence variability, five of the six loops adopt only a small repertoire of main-chain conformations called "canonical structures." These conformations are determined first by the length of the loop and second by the presence of major residues at specific positions in the loop and framework regions, which determine the conformation through their packing, hydrogen bonding, or the ability to assume non-main-chain conformations.

[0064] " CDR"- As used herein, the term "CDRs" or "complementarity determining region" refers to non-continuous antigen-binding sites found within variable regions of both heavy and light chain polypeptides. These specific regions have been described by Kabat et al. (J. Biol. Chem. 252, 6609-6616 (1977)), Kabat et al. (Sequences of protein of immunological interest. (1991)), Chothia et al. (J. Mol. Biol. 196:901-917 (1987)), and MacCallum et al. (J. Mol. Biol. 262:732-745 (1996)), where the definitions include amino acid residues or sets of amino acid residues that overlap when compared to one another. Amino acid residues containing CDRs as defined by each of the cited references are presented for comparison. Preferably, the term "CDR" is as defined by Kabat based on sequence comparison.

[0065] CDR definition Kabat 1 Chothia 2 MacCallum 3 IN H CDR1 31-35 26-32 30-35 In H CDR2 50-65 53-55 47-58 In H CDR3 95-102 96-101 93-101 IN L CDR1 24-34 26-32 30-36 In L CDR2 50-56 50-52 46-55 In L CDR3 89-97 91-96 89-96

[0066] 1 Remaining numbering is Kabat, etc. (Kabat et al It follows the nomenclature of ., supra).

[0067] 2 Residual numbering is done by Chothia, etc. (Chothia et al It follows the nomenclature of ., supra).

[0068] 3 Remaining numbering is done by MacCallum et al. et al It follows the nomenclature of ., supra).

[0069] " framework Region (Framework region) - As used herein, the terms "framework region" or "FR region" include amino acid residues that are part of the variable region but are not part of the CDRs (e.g., using Kabat's definition of CDRs). Therefore, the variable region framework is approximately 100–120 amino acids long but contains only amino acids that are outside the CDRs. Kabat et al. et al In a specific example of a heavy chain variable domain and CDRs as defined by .), framework site 1 corresponds to the variable domain containing amino acids 1–30; framework site 2 corresponds to the variable domain containing amino acids 36–49; framework site 3 corresponds to the variable domain containing amino acids 66–94; and framework site 4 corresponds to the variable domain from amino acid 103 to the end of the variable domain. In the light chain, the framework sites are similarly separated by each light chain variable domain CDR. Similarly, Chothia et al. (Chothia et al .) or McCallum et al. (McCallum et al Using the definition of .) the framework region boundaries are separated by each CDR end as described above. In a preferred embodiment, the CDRs are as defined by Carvet.

[0070] In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short, discontinuous amino acid sequences specifically positioned to form antigen-binding sites, assuming the antibody assumes a three-dimensional form in a soluble environment. The remainder of the heavy and light chain variable domains exhibits less intermolecular variation in their amino acid sequences and are referred to as framework regions. Framework regions primarily adopt a beta-sheet form, and the CDRs form connecting loops, and in some cases, form parts of the beta-sheet structure. Therefore, these framework regions act to form a scaffold that provides the positions of the six CDRs in the correct orientation through chain-to-chain, non-covalent interactions. The antigen-binding sites formed by the positioned CDRs define surface complementarity to the epitopes on the immunoreactive antigen. The complementary surface facilitates the non-covalent binding of the antibody to the immunoreactive antigen epitopes. The positions of the CDRs can be easily identified by experts in the technical field.

[0071] Constant region -As used here, The "constant region" refers to the portion of the antibody located outside the variable domain or variable region. Immunoglobulin light chains have a single domain "constant region," typically referred to as the CL or CL1 domain. This domain is located at the C-terminus of the VL domain. Immunoglobulin heavy chains differ in their constant regions depending on the class of immunoglobulin (γ, μ, α, δ, ε). Heavy chains γ, α, and δ have a constant region consisting of three immunoglobulin domains (called CH1, CH2, and CH3), along with a flexible hinge region separating the CH1 and CH2 domains. Heavy chains μ and ε have a constant region consisting of four domains (CH1-CH4). The constant region of the heavy chain is located at the C-terminus of the VH domain.

[0072] The numbering of amino acids in heavy and light immunoglobulin chains flows from the N-terminus at the end of a Y-shaped fork to the C-terminus at the bottom of each chain. Different numbering schemes are used to define the anchoring domains of the immunoglobulin heavy and light chains. According to the EU numbering scheme, the heavy chain anchoring domains of an IgG molecule are identified as follows: CH1-amino acid residues 118–215; CH2-amino acid residues 231–340; CH3-amino acid residues 341–446. According to the Kabat numbering scheme, the heavy chain anchoring domains of an IgG molecule are identified as follows: CH1-amino acid residues 114–223; CH2-amino acid residues 244–360; CH3-amino acid residues 361–447. The "Fc domain" or "Fc region" is defined as a part of the anchorage site of a heavy chain, typically comprising CH2 and CH3 domains. The Fc region may also include some residues from the hinge region. The "hinge region" comprises a part of the heavy chain molecule that attaches the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thereby allowing the two N-terminal antigen-binding sites to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux KH et al . J. Immunol. 161:4083-90 1998). The antibody of the present invention containing a "fully human" hinge region may contain one of the hinge region sequences shown in Table 2 below.

[0073] Human hinge sequences IgG Upper hinge Middle hinge Lower hinge IgG1 EPKSCDKTHT (Sequence No.: 21) CPPCP (Sequence No.: 22) APELLGGP (Sequence No.: 23) IgG3 ELKTPLGDTTHT(Sequence No.: 24) CPRCP (EPKSCDTPPPCPRCP)3(Sequence No.: 25) APELLGGP (Sequence No.: 23) IgG4 ESKYGPP (Sequence No.: 26) CPSCP (Sequence No.: 27) APEFLGGP(Sequence No.: 28) IgG2 ERK (Sequence No.: 29) CCVECPPPCP(Sequence No.: 30) APPVAGP (Sequence No.: 31)

[0074] "fragmentThe terms "fragment" or "antigen-binding fragment" refer to a part or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. An "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes for antigen binding with an intact antibody (i.e., the intact antibody from which they originated). As used herein, the term "fragment" of an antibody molecule refers to an antigen-binding fragment of an antibody, e.g., antibody light chain variable domain (VL), antibody heavy chain variable domain (VH), single chain antibody (scFv), F(ab')2 fragment, Fab fragment, Fd fragment, Fv fragment, one-armed (monovalent) antibody, diabodies, triabodidies, It includes any antigen-binding molecule formed by tetrabodies or by a combination, assembly, or conjugation of such antigen-binding fragments. As used herein, the term “antigen-binding fragment” is intended to further include antibody fragments selected from the group consisting of unibodies, domain antibodies, and nanobodies. The fragments may be obtained, for example, through chemical or enzymatic treatment of intact or complete antibodies or antibody chains, or by recombinant methods.

[0075] Specificity ( Specificity) " and " Multispecific antibodies ( Multispecific antibodies)The antibodies for use in the methods described herein bind to target antigens within the complement system. It is desirable for the antibodies to "specifically bind" to their target antigens, where the term "specifically binds" means the ability of an antibody to respond preferentially to a given target, e.g., C1, C2, C3, or C4. The antibodies may be monospecific and contain one or more binding sites that specifically bind to a particular target. These antibodies may be combined in the form of "multispecific antibodies," e.g., bispecific antibodies, where the multispecific antibody binds to two or more target antigens. To achieve multispecificity, the "multispecific antibody" is typically engineered so that the heavy and light chain polypeptides contain different combinations or pairs of VH-VL pairs. Multispecific, notably bispecific antibodies are generally in the form of natural antibodies, e.g., Y-type antibodies in which antibodies with Fab arms of different specificities are conjugated to the Fc site. Antibodies can be engineered to adopt a non-natural form. On the other hand, multiple specificity antibodies, e.g., bispecificity antibodies, can be engineered to adopt a non-natural form, e.g., where a variable domain or a pair of variable domains with different specificities is located at opposite ends of the Fc region.

[0076] Modified antibody (Modified antibody)"-As used herein, “modified antibody” includes synthetic forms of antibodies modified to not naturally exist, for example, antibodies comprising at least two heavy chain portions but incomplete two heavy chains (such as domain-deleted antibodies or minibodies); multispecific forms of antibodies modified to bind to two or more different antigens or different epitopes of a single antigen (e.g., bispecific, trispecific, etc.); heavy chain molecules linked to scFv molecules and similars. scFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019. Additionally, the term “modified antibody” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, antibodies binding to three or more copies of the same antigen). In another embodiment, the modified antibody of the present invention is a fusion protein comprising a binding domain of a polypeptide comprising at least one heavy chain portion without a CH2 domain and a binding portion of one member of a receptor ligand pair.

[0077] " Humanization substitution ( Humanizing substitutions - As used here, " "Humanizing substitutions" refers to amino acid substitutions in which an amino acid residue located at a specific position in the antibody's VH or VL domain is replaced by an amino acid residue located at an equivalent position in the referenced human VH or VL. The referenced human VH or VL domain may be a VH or VL domain encoded by the human germline. Humanizing substitutions may be made in the framework region and / or the antibody's CDRs as defined herein.

[0078] humanized variant ( Humanized variants - As used herein, "humanized variant" or "humanized antibody" means a variant antibody containing one or more "humanizing substitutions" compared to a reference antibody, wherein a portion of the reference antibody (e.g., a portion thereof containing a VH domain and / or a VL domain or at least one CDR) has amino acids derived from a non-human species, and "humanizing substitutions" occur within an amino acid sequence derived from a non-human species.

[0079] Germ lineage variant ( Germlined variants)"-"The terms "germlined variants" or "germlined antibody" are used to specifically refer to "humanized variants" resulting from "humanizing substitutions," where one or more amino acid residues located at specific position(s) in the antibody's VH or VL domain are replaced with amino acids at equivalent positions in a reference human VH or VL domain encoded by the human germline. In any given "germlined variant," the substituted amino acid residues are typically derived entirely or predominantly from a single human germline-encoded VH or VL domain. "Humanized variant" and "germlined variants" are often used interchangeably. Camelid-derived (e.g., Introducing one or more "humanizing substitutions" into a llama-derived VH or VL domain results in the production of a "humanized variant" of a llama-derived VH or VL domain. If the substituted amino acid residues are entirely or predominantly derived from a single human germline-encoded VH or VL domain sequence, then the result may be a "human germline variant" of a llama-derived VH or VL domain.

[0080] "friendship variant ( Affinity variants"-As used herein, the term “affinity variants” refers to variant antibodies that show a change of one or more amino acid sequences compared to a reference antibody, wherein the affinity variant shows an altered affinity for a target antigen compared to the reference antibody. For example, the affinity variant will show an altered affinity for the target compared to the reference antibody. Preferably, the affinity variant, compared to the reference antibody, for the target antigen improved It will demonstrate affinity. Affinity variants typically show one or more changes in the amino acid sequences of CDRs compared to the reference antibody. Such substitutions may result in replacing the original amino acid present at a given position within the CDRs with another amino acid, which may be a naturally occurring or non-naturally occurring amino acid residue. Amino acid substitutions may be conservative or non-conservative.

[0081] Subject - As used herein, the term "Subject" means a mammal, preferably a human. The Subject may be male or female. The Subject may exhibit one or more symptoms consistent with paraproteinemic neuropathy. In some embodiments, the Subject may be a Patient, wherein the Patient is an individual who is under medical care and / or is actively seeking medical care for the treatment of paraproteinemic neuropathy.

[0082] B. Treatment Methods

[0083] The present invention provides a method for treating paraproteinemic neuropathy. The method involves administering a complement system antagonist to an individual, wherein the antagonist inhibits the upper complement system of complement factor C5. The present invention also provides a complement system antagonist for use in the treatment of paraproteinemic neuropathy, wherein the antagonist inhibits the upper complement system of complement factor C5.

[0084] Paraproteinemic neuropathy is defined elsewhere here and includes a class of peripheral neuropathy characterized by the presence of homogeneous immunoglobulin or "paraprotein" in the serum. Peripheral neuropathy is a disease or degenerative state of peripheral nerves in which motor, sensory, or vasomotor nerve fibers are affected. Of particular interest is immune-mediated neuropathy, which refers to a class of peripheral nerve disorders caused by immune-mediated damage to peripheral nerves. Generally, immune-mediated peripheral neuropathy is characterized by progressive muscle weakness and is often accompanied by sensory deficits such as pain and numbness. These disorders are suspected to be caused by autoreactive antibodies in the serum that bind to components of myelin or proteins located in the nodes of Ranvier. A common feature of these disorders is that they allow autoantibodies, complement components, and inflammatory cells to access the endoneurium of the nerve. This breakdown of the BNB is thought to be triggered by circulating cytokines such as VEGF, TNFα, and IL1-β, as well as metalloproteases secreted by T cells. Upon entering the nerve, autoantibodies can bind to neuroantigens, such as myelin-associated glycoproteins (MAGs) or gangliosides, which are present in the myelin sheath or at the junctions between axons, respectively. As described elsewhere, the formation of antibody / antigen immune complexes can initiate the classical pathway of the complement system through the induction of C1q.

[0085] Paraproteinemic neuropathy is a group of immune-mediated neuropathy characterized by an excess of immunoglobulin in the serum. PPNs are frequently associated with the presence of autoantibodies. Existing treatment strategies for this class of neuropathy currently include intravenous immune globulin (IVIg), plasmapheresis, corticosteroids, azathioprine, rituximab, chlorambucil, fludarabine, melphalan, and others (Rison and Beydoun. BMC Neurology. (See 2016) 16:13)).

[0086] As reported here, immunoglobulins present in the serum of patients with other paraproteinemic neuropathy can activate complement, and this supports the role of complement-mediated tissue damage in the pathology seen in PPN patients. However, eculizumab (Soliris TM Previous clinical studies testing anti-C5 antibodies for the treatment of multifocal motor neuropathy (MMN) have not demonstrated efficacy (Fitzpatrick et al., J Peripher Nerv Syst , 2011, 16(2):84-91).

[0087] Importantly, the inventors have shown that both Schwann cells and motor neurons upregulate CD59, a complement regulatory protein that protects cells from MAC-mediated lysis. This observation suggests that elements of the complement cascade up to C5 play a more significant role in PPN pathology. This may well explain the previously observed lack of efficacy with eculizumab. Without being bound by theory, it is thought that complement activation in paraproteinemic neuropathy induces the release of cytokines and / or chemokines through the activation of C3aR, which has been found to be expressed in Schwann cells and motor neurons. Chemokines such as MCP-1 may play a role in attracting inflammatory cells, thereby promoting neurological damage.

[0088] The present invention seeks to improve PPN treatment by targeting the upstream complement activity of complement factor C5.

[0089] Neuropathies treated according to the method described herein include all peripheral neuropathy classified as "paraproteinemic neuropathies." This includes both acute and chronic disorders. Paraproteinemic neuropathies may be classified as demyelinating, axonal, or a combination thereof, depending on whether myelin and / or axons are damaged. In some embodiments, the neuropathy to be treated is an axonal neuropathy. In some embodiments, the neuropathy to be treated is a demyelinating neuropathy, e.g., chronic demyelinating neuropathy.

[0090] Paraproteinemic neuropathy is frequently characterized by the presence of autoantibodies. Therefore, in some embodiments, the neuropathy to be treated according to the method described herein is characterized by the presence of autoantibodies. High titers of serum autoantibodies recognizing neurogenic antigens occur in several forms of peripheral sensory, motor, and sensorimotor neuropathy. Antibodies frequently react with glycosylated cell surface molecules, including glycolipids, glycoproteins, and glycosaminoglycans; however, antibodies against intracellular proteins have also been described. There are some correlations between antibody specificity and clinical symptoms, implying that neuropathy may be caused by antibodies. Autoantibodies, typically IgM or IgG isotypes, have the ability to activate the classical pathways of the complement system in peripheral nerves, as described elsewhere here. Access to peripheral nerves by autoantibodies and the complement system is possible after the destruction of the blood-nerve-barrier (BNB). In some embodiments, paraproteinemic neuropathy is characterized by high-titre autoantibodies. In some embodiments, paraproteinemic neuropathy is characterized by the presence of IgG, IgM, or IgA autoantibodies.

[0091] In some embodiments, paraproteinemic neuropathy is characterized by the presence of autoantibodies against neural antigens. In some embodiments, the neural antibody is a protein located in the nodes of Ranvier. In other embodiments, the neural antibody is a protein located in the myelin sheath. Myelin-associated glycoproteins (MAGs) are components of peripheral and central nervous myelin. High-titer IgM antibodies against MAGs are associated with sensorimotor demyelinated peripheral neuropathy. MAG antibodies are usually associated with the presence of IgM monoclonal proteins. In some embodiments, the neural antigen is myelin-associated glycoprotein (MAG).

[0092] Gangliosides are a group of glycosphingolipids widely distributed in the membrane components of the nervous system. In some embodiments, the neuroantigen is a ganglioside. The gangliosides most commonly recognized by neuropathy-associated autoantibodies are GM1, GD1a, GD1b, and GQ1b. In some embodiments, the ganglioside is selected from GM1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GT3, and GQ1b. In a preferred embodiment, the ganglioside is GM1. Individual patients may possess antibodies against a single ganglioside or multiple gangliosides. Thus, in some embodiments, paraproteinemic neuropathy is characterized by the presence of autoantibodies against one or more neuroantigens. In some embodiments, peripheral neuropathy is characterized by the presence of autoantibodies against one or more gangliosides. In some embodiments, the neuroantigen is a paranodal protein. In some embodiments, the neuroantigen may be a paranodal protein selected from contactin 1, NF155, NF186, and NF140. Autoantibodies known to be associated with various peripheral neuropathy are described in Table 3 below. For example, IgG4 autoantibodies against paranodal proteins have been identified in patients with CIDP. Detection of GM1 antibodies, usually IgM isotypes, is associated with multifocal motor neuropathy and lower extremity motor neuropathy, characterized by muscle weakness and atrophy. GM1 IgM may exist as monoclonal IgM paraproteins or as polyclonal IgM.

[0093] Autoantibodies in peripheral neuropathy disease Antigen Antibody isotype GBS subtype AMAN GM1, GM1b, GD1a IgG AMSAN GM1, GD1a IgG Miller-Fisher syndrome GQ1b, GT1a IgG CIDP (and subtypes) MAG, Contactin 1, NF155, NF186, NF140 IgG4 CANOMAD GD3, GD1b, GT1b, GQ1b IgM DADS MAG IgM MMN GM1 IgM Anti-MAG neuropathy MAG IgM POEMS Unknown IgG / IgA

[0094] The specific paraproteinemic neuropathy to be treated according to the methods described herein is multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP) and Guillain-Barre syndrome (GBS), Miller Fisher syndrome, acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), chronic ataxic neuropathy-ophthalmoplegia-IgM paraprotein-cold agglutinins-disialosyl antibodies (CANOMAD) syndrome, and distal acquired It may be selected from distal acquired demyelinating symmetric (DADS) neuropathy, monoclonal gammopathy-associated peripheral neuropathy, anti-MAG peripheral neuropathy, and POEMS syndrome. In some preferred embodiments, paraproteinemic neuropathy is selected from multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barré syndrome (GBS). The method described herein is particularly desirable for the treatment of multifocal motor neuropathy (MMN).

[0095] The method described herein is for the treatment of paraproteinemic neuropathy in individuals requiring it. The individual is preferably a human individual. The individual may be a patient, wherein the patient is a patient who is under medical care and / or actively seeking medical care for the treatment of paraproteinemic neuropathy. The individual to be treated will typically, but not exclusively, exhibit one or more symptoms consistent with paraproteinemic neuropathy, including: onset of progressive numbness; prickling or tingling of the feet or hands that may spread to the legs or arms; sharp, jabbing, throbbing, freezing, or burning pain; extreme sensitivity to touch; lack of coordination and falls; tremors and muscle spasms; muscle wasting; muscle weakness and paralysis. The individual to be treated may have previously been diagnosed with paraproteinemic neuropathy by any standard evaluation criteria. The individual may have been identified or previously diagnosed based on an excess of immunoglobulin in the serum. On the other hand, or additionally, the individual may possess autoantibodies against one or more neuroantigens.

[0096] The subject to be treated may already be receiving treatment for paraproteinemic neuropathy or may have previously received treatment for paraproteinemic neuropathy. In some embodiments, the subject has previously received or is receiving IVIg. In other embodiments, the subject has previously received or is receiving rituximab. In some embodiments, the subject has received or is receiving plasma exchange. The subject may have been unresponsive to previous treatments or may have developed resistance to previous treatments, resulting in worsening symptoms.

[0097] The treatment methods described herein may completely or partially resolve one or more symptoms associated with PPN. Behavioral improvement can be measured using any standard evaluation criterion for assessing PPN treatment.

[0098] The methods of the present invention may include the administration of one or more additional therapeutic agents for the treatment of paraproteinemic neuropathy. One or more additional therapeutic agents may be administered simultaneously with a complement antagonist as a combination therapy. Alternatively, one or more additional agents may be administered before or after the administration of the complement antagonist, that is, the agents are administered sequentially. Additional therapeutic agents that may be administered according to the present invention include, but are not limited to, IVIg, rituximab, corticosteroids, azathioprine, chlorambucil, fludarabine, melphalan, cyclophosphamide / prednisone, melphalan, gabapentin, pregabalin, valproate, dextromethorphan, tramadol, duloxetine, amitriptyline, and venlafaxine.

[0099] C. Composers Complement antagonists

[0100] The results presented herein suggest that the end stage of the complement cascade, particularly MAC-mediated lysis, may not play a significant role in the pathology of PPN. This is because Swan cells and motor neurons express high levels of the complement regulatory protein CD59, which protects against MAC-mediated lysis. This, therefore, follows the fact that the complement-related pathology associated with paraproteinemic neuropathy described herein can be mediated by upstream factors of C5, for example, through C3aR. Therefore, the present invention provides a method for treating paraproteinemic neuropathy by inhibiting the C5 upstream complement pathway.

[0101] This method involves administering an antagonist to the complement system to an individual, wherein the antagonist inhibits the complement system upstream of complement factor C5. An antagonist that inhibits the complement system upstream of complement factor C5 does not directly inhibit complement factor C5 itself or any of the downstream factors (C6, C7, C8, C9) that merge to form the membrane attack complex. Instead, an antagonist that inhibits the complement system upstream of complement factor C5 targets a factor or a component of the complement cascade prior to complement factor C5. The complement factors upstream of C5 include C1, C2, C4, and C3. Therefore, in some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C2, C4, and C3. In some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b.

[0102] This method involves administering an antagonist to the complement system to an individual, wherein the antagonist inhibits the complement system upstream of complement factor C5. An antagonist that inhibits the complement system upstream of complement factor C5 does not directly inhibit complement factor C5 itself or any of the downstream factors (C6, C7, C8, C9) that merge to form the membrane attack complex. Instead, an antagonist that inhibits the complement system upstream of complement factor C5 targets a factor or a component of the complement cascade prior to complement factor C5. The complement factors upstream of C5 include C1, C2, C4, and C3. Therefore, in some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C2, C4, and C3. In some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b.

[0103] In some embodiments, the antagonist inhibits the upper complement system of complement factor C3. The upper complement factors of C3 include C1, C4, and C2. Therefore, in some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C2, and C4. In some embodiments, the antagonist inhibits the complement system by inhibiting the function of any one of C1, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, or C4b.

[0104] The initiator of the classical complement pathway is complement factor C1. Therefore, in some embodiments, the antagonist inhibits complement factor C1. In other embodiments, the antagonist inhibits complement factors C1q, C1r, or C1s. By inhibiting the complement cascade at complement factor C1, it is possible to prevent or reduce the cleavage of C4 and C2, which are C1-specific targets, and to specifically inhibit complement activation through the classical pathway of the complement system. Inhibition of C1 can reduce the deposition of opsonin C4b, thereby reducing targeting of cells for phagocytosis and destruction. Additionally, inhibition of C1 can prevent or reduce the formation of C3 convertase, C4b, and C2a. C4 and C2 can also be cleaved by MASP enzymes via the lectin pathway; However, the total level of C3 converterase can still be significantly inhibited by the targeting of complement factor C1.

[0105] In some embodiments, the antagonist inhibits complement factor C4. In other embodiments, the antagonist inhibits complement factor C4a or C4b. By targeting complement factor C4, it is possible to prevent the formation of C3 convertase, C4b, and C2a, and to inhibit complement activation through both the classical and lectin pathways of the complement system.

[0106] In some embodiments, the antagonist inhibits complement factor C2. In other embodiments, the antagonist inhibits complement factor C2a or C2b. Inhibition of C2 will also prevent or reduce the formation of C3 convertase, and thus reduce the deposition of anaphylatoxin C3a and opsonin C3b, and other complement-activating products downstream of C2. An antagonist of C2 can prevent the formation of C3 convertase by directly inhibiting C2a. On the other hand, the antagonist can prevent C2 from initially binding to surface-bound C4b by inhibiting C2b. An antagonist of C2b may leave the binding of C2a to C4b intact. Nevertheless, C2 activity may be significantly inhibited by an antagonist of C2b.

[0107] In some embodiments, the antagonist inhibits complement factor C3. In other embodiments, the antagonist inhibits complement factor C3a or C3b. Inhibition of C3 can prevent or reduce the formation of C5 converterase, C3bBbC3b, and thus reduce the deposition of aniphylatoxin C5a and MAC generator, C5b.

[0108] Depending on the point in the complement cascade where the antagonist inhibits the complement system, the antagonist may inhibit the classical complement pathway, the lectin complement pathway, the alternative complement pathway, or a combination of these pathways. In some embodiments, the antagonist inhibits the classical complement pathway and the lectin complement pathway but does not affect the alternative complement pathway. Antagonists targeting C2 and C4 may inhibit the classical complement pathway and the lectin complement pathway, while leaving the alternative pathway intact. In some cases, it may be advantageous to leave one of the complement pathways intact so that a critical point of the innate immune system is not entirely impaired. On the other hand, or additionally, to achieve a therapeutic effect, it may be necessary to bring about only partial inhibition of complement activity.

[0109] The antagonist for use in the therapeutic method described herein inhibits factors or components of the upstream complement system of complement factor C5 to inhibit or reduce complement activity. As reported herein, it is advantageous to target the complement cascade upstream of C5 to inhibit the activity of complement factors prior to MAC complex formation. The antagonist for use according to the method described herein can reduce or inhibit complement activity by inhibiting the production of biologically active complement-derived peptides such as C4a, C4b, C3a, C3b, and C5a. The antagonist can, at least partially, prevent the damaging effects of complement-derived peptides on cells and tissues. Antagonists of the complement system may inhibit or reduce complement activity by reducing anaphylatoxin deposition, by reducing opsonin deposition, by reducing the production or secretion of cytokines and / or chemokines, by reducing phagocytosis, by reducing the induction of immune cells, and any combination thereof. In some embodiments, the antagonist of the complement system inhibits anaphylatoxin deposition. In some embodiments, the antagonist inhibits the deposition of C3a or C5a. In some embodiments, the antagonist inhibits the deposition of opsonins. In some embodiments, the antagonist inhibits the deposition of C3b or C4b. In some embodiments, the antagonist inhibits the production and / or secretion of inflammatory cytokines and / or chemokines. In one embodiment, the antagonist inhibits the production and / or secretion of MCP-1.

[0110] The antagonist for use in the present invention may be any agent capable of inhibiting the function of a complement factor and thereby inhibiting or reducing complement activity. As described elsewhere herein, it is desirable for the antagonist for use in this method to exhibit specificity toward its target. This specificity is typically achieved by an antagonist that directly binds to its target and inhibits the function of the target.

[0111] In a preferred embodiment, the antagonist is an antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment binds to complement factors C1, C1q, C1s, C1r, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, or C4b. In some embodiments, the antibody or its 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 its antigen-binding fragment binds to C2, preferably C2b.

[0112] The antibody and its antigen-binding fragment for use in the method described herein are intended for therapeutic use in humans and are therefore typically of the IgA, IgD, IgE, IgG, or IgM type, often of the IgG type, in which case it may belong to any one of the four subclasses IgG1, IgG2a and b, IgG3, or IgG4. In a preferred embodiment, the antibody is an IgG antibody, optionally an IgG1 antibody. The antibody may be a monoclonal, polyclonal, or multispecific (e.g., a bivalent specific antibody) antibody, provided that they exhibit appropriate immunological specificity for the target. Monoclonal antibodies are preferred because they are highly specific and target a single antigen site.

[0113] The antigen-binding fragments described herein will typically comprise a portion of a full-length antibody, generally its antigen-binding or variable domain. Examples of antibody fragments include Fab, Fab', F(ab')2, divalent-specific Fab's (bi-specific Fab's), and Fv fragments, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments (Holliger and Hudson (2005), incorporated herein by reference). Nature Biotechnol . 23:1126-36, see reference)

[0114] The antibody or antigen-binding fragment for use according to the method described herein may exhibit high human homology. Such antibody molecules having high human homology may include antibodies comprising the VH and VL domains of natural non-human antibodies that exhibit sufficiently high % sequence homology with human germline sequences. In some embodiments, the antibody molecule is a humanized or germline-series variant of a non-human antibody.

[0115] In non-limiting embodiments, the antibody may comprise an amino acid sequence that is entirely or substantially human, comprising a CH1 domain and / or a CL domain (from the heavy chain and light chain, respectively). In antibody molecules intended for use in human therapeutic purposes, it is typical for the entire anchorage site of the antibody, or at least a portion thereof, to have an entirely or substantially human amino acid sequence. Therefore, one or more of the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and, if present, a CH4 domain), or any combination thereof, may be entirely or substantially human with respect to amino acid sequence. The CH1 domain, hinge region, CH2 domain, CH3 domain, and / or CL domain (and / or, if present, a CH4 domain) may be derived from a human antibody, preferably a human IgG antibody, more preferably a human IgG1 antibody of subtype IgG1, IgG2, IgG3, or IgG4.

[0116] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and, if present, the CH4 domain) may all have entirely or substantially human amino acid sequences. In the context of a humanized or chimeric antibody, or an antibody fragment's anchorage site, the term "substantially human" means that the amino acid sequence homology with the human anchorage site is at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99%. In this context, "human amino acid sequence" means the amino acids encoded by human immunoglobulin genes, including germline, rearranged, and somatically mutated genes.

[0117] In one embodiment, the antibody or antibody fragment comprises a human Fc domain, which contains one or more mutations designed to increase the serum half-life of the antibody or antibody molecule. Such optimization efforts are intended to improve the circulation of the antibody in vivo. Examples of mutations affecting the half-life in the human IgG Fc domain include His433Lys + Asn434Phe (NHance); Arg435His; Asn434Ala; Met252Tyr + Ser254Thr + Thr256Glu (YTE); Met428Leu + Asn434Ser (LS); Thr252Leu + Thr253Ser + Thr254Phe (LSF); Glu294delta + Thr307Pro + Asn434Tyr (C6A-66); Thr256Asn + Ala378Val + Ser383Asn + Asn434Tyr (C6A-78); Glu294delta (del) is included. Depending on the serum half-life of the complement inhibitor, it may be administered as a single dose or as multiple doses at intervals of 1 day to 1 month between subsequent doses.

[0118] In other embodiments, the Fc domain may contain one or more mutations designed to impair the effector function of the Fc domain. Such mutations are well known to those skilled in the art. In the case of human IgG1, the antibody may contain a human Fc domain modified to eliminate or impair its effector function. Such Fc domain mutations generally involve at least one amino acid change at the heavy chain anchorage sites 234, 235, 236, 237, 297, 318, 320, or 322, thereby causing an alteration in effector function while maintaining its antigen binding. Examples of effector-impairing mutations in the human IgG Fc domain include Leu234Ala + Leu235Ala (referred to as LALA); Leu234Ala + Leu235Ala + Pro329Gly (referred to as LALA-PG); IgG4 includes Ser228Pro + Leu235Glu; Pro331Ser + Leu234Glu + Leu235Phe; and Pro331Ser + Leu234Ala + Leu235Ala.

[0119] Examples of antagonists suitable for use in the present invention for inhibiting complement factor C1 include Cinryze (Shire), sutimlimab—also known as TNT009 and BIV009 (Bioverativ), TNT003 (True North), ANX005 (Annexon), and nafamostat (Torii Pharmaceutical).

[0120] Examples of antagonists suitable for use in the present invention for inhibiting complement factor C3 include compstatin Cp40 (Amyndas), PEG-Cp40 (Amyndas), AMY-101 (Amyndas), AMY-201 (Amyndas), APL-1 and APL-2 (Apellis), CDX-1135 (Celldex), APT070 Mirococept (MRC), HC3-1496 (InCode), humanized monoclonal antibody H17 (Elusys Therapeutics), or vaccineinia virus complement control protein (VCP).

[0121] D. Anti-C2 antibodies and antigen-binding fragments

[0122] Particularly preferred complement antagonists for use in the therapeutic method described herein are those that inhibit complement factor C2. In a preferred embodiment, the complement antagonist for use in the treatment is an antibody that binds to C2 or an antigen-binding fragment thereof. Suitable anti-C2 antibodies and antigen-binding fragments for use in the therapeutic method described herein include the antibodies and antigen-binding fragments identified in International Patent Application No. WO2014 / 189378, the full text of which is incorporated herein.

[0123] C2 is a 90–100 kDa glycoprotein participating in the classical and lectin pathways of complement activation. As described above, C2 can be activated by C1s of the classical pathway or by activated MASP2 of the lectin pathway. C2 is surface-bound C4b (Mg 2 +It binds to (in the presence of) to form C4bC2, which is then cleaved into two fragments by activated C1s or MASP2: the larger 70 kDa fragment C2a, which remains attached to C4b to form C3-convertase C4bC2a, and the smaller 30 kDa N-terminal fragment C2b, which is released into the liquid phase. Once activated and bound to C4b, C2a constitutes the catalytic subunits of C3 and C5 convertases capable of cleaving C3 and C5, respectively.

[0124] The amino acid sequence of human C2 is known (GenBank Accession No. NM_000063) and is shown below as Sequence No. 1.

[0125] Human C2 amino acid sequence (Sequence No. 1):

[0126] MGPLMVLFCLLFLYPGLADSAPSCPQNVNISGGTFTLSHGWAPGSLLTYSCPQGLYPSPASRLCKSSGQWQTPGATRSLSKAVCKPVRCPAPVSFENGIYTPRLGSYPVGGNVSFECEDGFILRGSPVRQCRPNGMWDGETAVCDNGAGHCPNPGISLGAVRTGFRFGHGDKVRYRCSSNLVLTGSSE RECQGNGVWSGTEPICRQPYSYDFPEDVAPALGTSFSHMLGATNPTQKTKESLGRKIQIQRSGHLNLYLLLDCSQSVSENDFLIFKESASLMVDRIFSFEINVSVAIITFASEPKVLMSVLNDNSRDMTEVISSLENANYKDHENGTGTNTYAALNSVYLMMNNQMRLLGMETMAWQEIRHAIILLTD GKSNMGGSPKTAVDHIREILNINQKRNDYLDIYAIGVGKLDVDWRELNELGSKKDGERHAFILQDTKALHQVFEHMLDVSKLTDTICGVGNMSANASDQERTPWHVTIKPKSQETCRGALISDQWVLTAAHCFRDGNDHSLWRVNVGDPKSQWGKEFLIEKAVISPGFDVFAKKNQGILEFYGDDIAL LKLAQKVKMSTHARPICLPCTMEANLALRRPQGSTCRDHENELLNKQSVPAHFVALNGSKLNINLKMGVEWTSCAEVVSQEKTMFPNLTDVREVVTDQFLCSGTQEDESPCKGESGGAVFLERRFRFFQVGLVSWGLYNPCLGSADKNSRKRAPRSKVPPPPRDFHINLFRMQPWLRQHLGDVLNFLPLPL

[0127] In some embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH and VL domains comprise a CDR sequence:

[0128] - HCDR3 containing or composed of sequence number 2 [EDDHDAFAY];

[0129] - HCDR2 containing or composed of sequence number 3 [DINPNYESTGYNQKFKG];

[0130] - HCDR1 containing or composed of sequence number 4 [DYNMD];

[0131] - LCDR3 comprising or composed of sequence number 5 [QHSRELPYT];

[0132] - LCDR2 comprising or composed of sequence number 6 [LASNLKS]; and

[0133] - LCDR1 containing or composed of Sequence No. 7 [RASKSVRTSGYNYMH].

[0134] In some embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain comprising or composed of a sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology with SEQ ID NO. 8 and a variable light chain (VL) domain comprising or composed of a sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology with SEQ ID NO. 9. In some embodiments, the antibody or antigen-binding fragment binding to C2b comprises a variable heavy chain domain (VH domain) comprising or composed of SEQ ID NO. 8 and a variable light chain domain (VL domain) comprising or composed of SEQ ID NO. 9.

[0135] In some embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises or is composed of a sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology with SEQ ID NO. 8 and the VL domain comprises a CDR sequence:

[0136] LCDR3 comprising or composed of sequence number 5 [QHSRELPYT];

[0137] LCDR2 comprising or composed of SEQ ID NO. 6 [LASNLKS]; and

[0138] LCDR1 containing or composed of sequence number 7 [RASKSVRTSGYNYMH].

[0139] In some embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises a CDR sequence:

[0140] HCDR3 comprising or composed of sequence number 2 [EDDHDAFAY];

[0141] HCDR2 containing or composed of sequence number 3 [DINPNYESTGYNQKFKG];

[0142] HCDR1 containing or composed of sequence number 4 [DYNMD], and the VL domain contains or is composed of a sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology with sequence number 9.

[0143] In some embodiments, the antibody or antigen-binding fragment binds to C2b and comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises a CDR sequence:

[0144] HCDR3 comprising or composed of sequence number 2 [EDDHDAFAY];

[0145] HCDR2 containing or composed of sequence number 3 [DINPNYESTGYNQKFKG];

[0146] HCDR1 containing or composed of sequence number 4 [DYNMD], and the VL domain contains or is composed of the amino acid sequence of sequence number 9.

[0147] In an embodiment where the domain of the antibody or antigen-binding fragment is defined by a specific percentage of sequence homology with the reference sequence, the VH and / or VL domains may retain a CDR sequence identical to the sequence present in the reference sequence so that the variation exists only within the framework region.

[0148] Variable heavy chain (VH) and variable light chain (VL) domain sequences Variable domain (Variable domain) order (Sequence) Sequence number (SEQ ID NO:) Variable heavy chain (VH) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSS 8 Variable light chain (VL) DNVLTQSPDSLAVSLGERATISCRASKSVRTSGYNYMHWYQQKPGQPPKLLIYLASNLKSGVPDRFSGSGSGTDFTLTISSLQAEDAATYYCQHSRELPYTFGQGTKLEIK 9

[0149] In some embodiments, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and / or CH3 domain of a human antibody, particularly human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG1 and comprises substitutions of L234A and L235A in the CH2 domain, wherein these positions are defined according to EU numbering. On the other hand or additionally, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG1 and comprises H433K and N434F substitutions in the CH3 domain, wherein these positions are defined according to EU numbering. EU numbering is Edelman et al. (Edelman, GM et al., Proc . Natl . Acad . Sci . USA, 63: 78-85 (1969)); and refers to precedents for the Fc region described in Kabat et al. (in "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, 5th edition, 1991).

[0150] In some embodiments, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG4. In some embodiments, the antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG4 and comprises an S228P substitution in the hinge domain.

[0151] In some embodiments, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG4 and includes an L445P substitution in the CH3 domain.

[0152] In some embodiments, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG4 and both S228P substitution in the hinge domain and L445P substitution in the CH3 domain.

[0153] In some embodiments, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG4 and includes H433K and N434F substitutions in the CH3 domain.

[0154] In some embodiments, the anti-C2b antibody comprises the CH1 domain, hinge domain, CH2 domain, and CH3 domain of human IgG4, and S228P substitution in the hinge domain, and H433K and N434F substitutions in the CH3 domain.

[0155] In some embodiments, the anti-C2b antibody comprises a human IgG heavy chain fixation domain. In some embodiments, the heavy chain fixation domain comprises a human IgG1 heavy chain fixation domain. In some embodiments, the heavy chain fixation domain consists of a human IgG1 heavy chain fixation domain. In some embodiments, the heavy chain fixation domain comprises a human IgG1 heavy chain fixation domain comprising or composed of the amino acid sequence presented as SEQ ID NO. 10 or 11.

[0156] In some embodiments, the heavy chain fixation domain comprises a human IgG4 heavy chain fixation domain. In some embodiments, the heavy chain fixation domain comprises a human IgG4 heavy chain fixation domain comprising or composed of the amino acid sequence presented in any one of SEQ Nos. 12, 13, or 14.

[0157] The heavy chain fixed domains are shown in Table 5 below.

[0158] 중 쇄 고정 도메인 (Heavy Chain Constant Domains) ID 서열 서열 번호 인간 IgG1 (UniProt) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10 인간IgG1 LALA NHance (ARGX-117) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPG 11 인간IgG4 (UniProt) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 12 인간IgG4 S228P L445P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK 13 인간IgG4 S228P NHance L445P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALKFHYTQKSLSLSPGK 14

[0159] In some embodiments, the anti-C2b antibody comprises or consists of a light chain having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence homology with the amino acid sequence shown in SEQ No. 20, and a heavy chain selected from the following:

[0160] (i) a heavy chain having at least 90%, at least 95%, at least 97%, or at least 98%, or at least 99% sequence homology with the amino acid sequence shown as sequence number 15;

[0161] (ii) a heavy chain having at least 90%, at least 95%, at least 97%, or at least 98%, or at least 99% sequence homology with the amino acid sequence shown as SEQ ID NO. 16;

[0162] (iii) a heavy chain having at least 90%, at least 95%, at least 97%, or at least 98%, or at least 99% sequence homology with the amino acid sequence shown as SEQ ID NO. 17;

[0163] (iv) a heavy chain having at least 90%, at least 95%, at least 97%, or at least 98%, or at least 99% sequence homology with the amino acid sequence shown as SEQ ID NO. 18 and

[0164] (v) A heavy chain having at least 90%, at least 95%, at least 97%, or at least 98%, or at least 99% sequence homology with the amino acid sequence shown as SEQ ID NO. 19.

[0165] In some embodiments, the anti-C2b antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 20 and a heavy chain having an amino acid sequence selected from SEQ ID NOs 15-19.

[0166] In some embodiments, the anti-C2b antibody comprises a light chain having the amino acid sequence of SEQ ID NO. 20 and a heavy chain having the amino acid sequence of SEQ ID NO. 16.

[0167] The heavy chain and light chain sequences are shown in Table 6 below.

[0168] 중 쇄 및 경 쇄 (Heavy Chains and Light Chains) ID 서열 서열 번호 인간IgG1 (UniProt) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 인간IgG1 LALA NHance (ARGX-117) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPG 16 인간IgG4 (UniProt) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 17 인간IgG4 S228P L445P EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK 18 인간IgG4 S228P NHance L445P EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQATGQGLEWIGDINPNYESTGYNQKFKGRATMTVDKSISTAYMELSSLRSEDTAVYYCAREDDHDAFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALKFHYTQKSLSLSPGK 19 경 쇄 (ARGX-117) DNVLTQSPDSLAVSLGERATISCRASKSVRTSGYNYMHWYQQKPGQPPKLLIYLASNLKSGVPDRFSGSGSGTDFTLTISSLQAEDAATYYCQHSRELPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20

[0169] In a preferred embodiment, the anti-C2b antibody is a monoclonal IgG antibody.

[0170] In an embodiment where the heavy chain and / or light chain of the antibody is defined by a specific percentage of sequence homology with the reference sequence, the heavy chain and / or light chain may retain a CDR sequence identical to the CDR sequence present in the reference sequence, so that the variation exists only outside the CDR site.

[0171] Unless otherwise stated in this application, % sequence homology between two amino acid sequences may be determined by comparing the two most optimally aligned sequences, wherein the amino acid sequences to be compared may include insertions or deletions relative to a reference sequence for optimal alignment between the two sequences. The percentage of homology is calculated by determining the number of identical positions where amino acid residues are identical between the two sequences, dividing this number of identical positions by the total number of positions within the comparison window, and multiplying the result obtained by 100 to obtain the percentage of homology between the two sequences. For example, BLAST programs, "BLAST 2 sequences" (Tatusova et al, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett It is possible to use . 174:247-250), and the coefficients used are those given by default (in particular, the coefficients are "open gap penalty": 5, and "extension gap penalty": 2; the selected matrix is, for example, the matrix "BOSUN 6" suggested by the program), and the percentage of homology between the two sequences being compared is calculated directly by the program.

[0172] Anti-C2 antibodies may be modified within the Fc site to increase binding affinity for the neonatal receptor FcRn, preferably human FcRn. Increased binding affinity can be measured at acidic pH (e.g., from approximately pH 5.5 to approximately pH 6.0). Increased binding affinity can also be measured at neutral pH (e.g., from approximately pH 6.9 to approximately pH 7.4). "Increased binding affinity" refers to increased binding affinity for FcRn compared to the binding affinity of the unmodified Fc site. Typically, the unmodified Fc site will possess the wild-type amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. In such an embodiment, the increased binding affinity of the antibody molecule having the modified Fc site for FcRn will be measured against the binding affinity of wild-type IgG1, IgG2, IgG3, or IgG4 for FcRn, preferably human FcRn.

[0173] E. Pharmaceutical compositions

[0174] Antagonists for use in the therapeutic methods described herein, in particular antibodies and their antigen-binding fragments, can be prepared as pharmaceutical compositions for administration to individuals.

[0175] Pharmaceutical compositions may be prepared with any known adventit or excipient according to such traditional techniques as disclosed in Remington (Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995), as well as with pharmaceutically acceptable carriers or diluents. The term “pharmaceutically acceptable carrier” relates to a carrier or excipient that is non-toxic in nature. Examples of such excipients include, but are not limited to, physiological saline, Ringer’s solution, dextrose solution, and Hanks’ solution. Non-aqueous excipients such as fixed oils and ethyl oleate are also used.

[0176] The pharmaceutical composition must typically be sterile and stable under manufacturing and storage conditions. The composition may be formulated as a solution, a micro-emulsion, a liposome, or other ordered structure suitable for high drug concentrations. Examples of suitable water-soluble and non-water-soluble carriers applicable to the pharmaceutical composition include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and similar ones), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity may be maintained, for example, by using a coating material such as lecithin, by maintaining the required particle size in the case of a dispersant, and by using a surfactant.

[0177] The pharmaceutical composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Preventing the presence of microorganisms can be ensured by both the sterilization process and the inclusion of various antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, sorbic acid, and similar substances. Additionally, it is desirable to include polyalcohols such as sugar, mannitol, sorbitol, and glycerol, or isotonic agents such as sodium chloride in the composition.Pharmaceutically acceptable antioxidants also include, for example, (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and similar ones; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and similar ones; (3) Metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and similar substances may be included.

[0178] The pharmaceutical composition may be administered through any suitable mode of administration. For example, administration may be parenteral, preferably intravenous (IV) or subcutaneous (sc) injection or infusion. The phrases “parenteral administration” and “administered parenterally” as used herein mean a mode of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intraperitoneal, subcutaneous, intramuscular, nitrobacterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0179] Merging into references

[0180] Various papers have been cited in the preceding description and the examples below, the full text of each of which is included here in the bibliography.

[0181] Example of implementation

[0182] The present invention will be further understood by referring to the following non-limiting embodiments.

[0183] line Example 1. Complement inhibition in an in vitro model of multifocal motor neuropathy using living Schwann cells

[0184] Swan cells are myelin-secreting glial cells that spiral around the axons of the peripheral nervous system to form a myelin sheath. These cells are attached to the axons by the protein GM1. Their most important function is the myelination of axons to increase the saltatory conduction of neurons, and they also aid in neuronal survival and signal transmission. Dysfunction of these cells causes demyelination, which reduces signal transmission. Consequently, Swan cells are associated with several demyelinating disorders, such as MMN. A human Swan cell line—sNF02.2—exists and originated from a lung metastasis in a patient diagnosed with a malignant peripheral nerve sheath tumor. These cells were established from primary tumor material through numerous passages until a homogeneous Schwann cell-like population formed in culture, which exhibits clonal morphology positive for Schwann cell markers S100 and p75. This cell line, purchased from ATCC®, was used in the experiments described herein.

[0185] A. Method

[0186] 1.1 Protocol for culturing Schwann cells

[0187] sNF02.2 (ATCC® CRL-2885 TM) or sNF96.2 (ATCCⓒ CRL-2884) Swan cells were treated with 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured in DMEM medium supplemented with 10% FCS at 37°C under 5% CO2. Twice a week, when cell confluency was >80%, cells were subcultured or used for experiments. The medium was discarded, and cells were washed with 10 mL of PBS. To detach the cells, 3 mL (T75) or 5 mL (T175) of Accutase cell detachment solution (eBioscience TM Thermo Fischer Scientific (Cat. N° 00-455-56) was added, and the cells were incubated at 37°C for 5 minutes or until the cells were completely detached. Afterwards, culture medium (7 mL for T75 and 10 mL for T175) was added, and the cells were transferred to 15 mL tubes and centrifuged (125x g (10 min). The pellet was resuspended in 5 mL of culture medium and counted using trypan blue to distinguish living cells from dead cells. Then, the cells were adjusted to the desired concentration and inoculated into culture flasks (10 mL in T75, 20 mL in T175) or used for FACS experiments.

[0188] 1.2 Swan Cell Staining Protocol

[0189] Cells were cultured as described above, and after counting, 50,000 cells were transferred to V-bottom plates. Cells were washed once with FACS buffer (PBS 1% BSA 0.01% sodium azide) and stained with the corresponding antibody diluted in FACS buffer for 45 minutes in the dark on ice (see Table 7 for FACS staining antibodies). If necessary, cells were washed once with the addition of 100 μL FACS buffer and then stained 125x for 5 minutes. g Centrifuged at [a certain level]. Then, the cells were incubated with the secondary antibody on ice in the dark for 45 minutes. After incubation, 100 μL of FACS buffer was added and centrifuged (125x [amount]). g (5 min). Finally, the cell pellet was re-suspended in 100 μL of FACS buffer and analyzed using FACS Canto II and accompanying software.

[0190] FACS staining antibodies antibodies (Antibody) Label / Fluorochrome (Label / Fluorochrome) company (Company) Catalog Number (Cat. N°) clone (Clone) CD46 FITC BD 555949 E4.3 Mouse IgG2a FITC BioLegend 400207 MOPC-173 CD55 FITC BioLegend 311306 JS11 Mouse IgG1 FITC BD 556028 CD59 APC eBioscience 17-0596 OV9A2 Mouse IgG1 APC Biolegend 400119 MOPC-21 CD64 AF647 Sony 2125060 10.1 Mouse IgG1 AF647 Biolegend 400155 MOPC-21 CD88 (C3aR) PE Biolegend 345804 hC3aRZ8 Mouse IgG2b PE BD 555058 37-35 Mouse IgG2b PE BD 559529 MCP-11 CholeraToxinB subunit AF488 Thermofisher C34775 Rabbit anti-MAG Thermofisher PA5-49646 Goat anti-rabbit AF488 Life technologies A11070 Rabbit anti-RBC LSBio LS-C347937 C3 Biotin LSBio LS-C62849 6C9 C3 FITC LSBio LS-C62855 6C9 Anti-human IgM F(ab)'2 FITC Southern Biotech 2022-02 CD46 APC Thermofisher A15711 MEM-258 CD55 APC Biolegend 311312 JS11 CD35 Alexa Fluor® 647 BD 565329 E11 CD11b APC Invitrogen 17-0118-42 ICRF44 CD11c APC BD 333144 C3aR APC Biolegend 345805 hC3aRZ8 C5aR FITC GeneTex GTX75734 P12 / 1 Streptavidin APC eBioscience 17-4317-82

[0191] 1.3 Complement activation assay on living Schwann cells

[0192] When complement activation was evaluated, 50,000 cells were transferred to a 96-well V-bottom plate and opsonized with 20 μL of each opsonizing agent diluted in VB++ (at room temperature for 1 hour). Then, 100 μL of VB++ was added and centrifuged (125x g (at 5 minutes). Then, the supernatant was discarded, and the cells were incubated for 1 hour at 37°C with 100 μL of complement-activated serum (pre-incubated with EDTA, MgEDTA, or Abs for 15 minutes at room temperature). Then, the cells were 125x g It was centrifuged for 5 minutes, and the supernatant was discarded. Subsequently, staining for complement activation was performed according to the staining protocol described above.

[0193] 1.4 Cytokine secretion assay

[0194] sNF02.2 cells were cultured as described above. After accutase treatment and cell count, cells were transferred to 1 ml of sNF02.2 culture medium in 24-L plates at a density of 10,000 cells / well. After 2 days, the culture medium was discarded, and the cells were opsonized with 100 μL of heat-inactivated MMN patient serum diluted 1:50 in VB++ for 1 hour at room temperature. Then, 100 μL of 15% complement-activated serum (with MgEDTA or antibody, pre-incubated at room temperature for 15 minutes) was added to the opsonized cells, followed by incubation at 37°C for 1 hour. Subsequently, 300 μL of culture medium was added to the cells, resulting in a final volume of 500 μL / well. After 24 and 48 hours, 200 μL of supernatant was collected for self-analysis at the multiplex core facility of the University Medical Centre in Utrecht (MC Utrecht).

[0195] B. Results

[0196] 1.5 Expression of complement receptors by living Schwann cells

[0197] To investigate MMN biology using Swan cells, the expression of complement regulatory factors was evaluated. Therefore, sNF02.2 Swan cells were cultured and FACS staining for expression markers was performed. The results are shown in Figures 2a–2g and summarized in Table 8 below.

[0198] Expression of complement regulatory proteins on sNF02.2 Schwann cells name Marker (Marker) Swan cell (Schwann cells) MCP CD46 ++ DAF CD55 ++ Protectin ( Protecting ) CD59 +++ CR1 CD35 - C5aR CD88 - C3aR + GM1 ++ MAG + FcγRI CD64 ++

[0199] These results indicate that complement regulatory proteins (CD46, CD55, and CD59) are expressed in Swan cells, along with high expression of CD59, a major regulator of the terminal pathway. High CD59 expression in Swan cells suggests that complement-mediated lysis is localized to these cells because MAC formation is inhibited. Instead, inflammation is induced in neural compartments via cellular signaling. Additionally, Swan cells show expression of Fcγ Receptor 1 (CD64), which is involved in phagocytotic clearance. CR1 expression in Swan cells occurs at the onset of myelination, and since these cultured sNF02.2 Swan cells lack myelin, the detection of CR1 (CD35) was not observed. (Terenghi F et al., Neurology , 2004, 62: 666-668). Only C3aR showed low expression, and C5aR was absent. In conclusion, Swan cells highly express CD59, suggesting that the sub-lytic formation of MAC triggers inflammation within neurons.

[0200] To investigate the sensitivity of complement regulatory proteins (CRP) in Swan cells to complement-mediated lysis, sNF02.2 cells were treated with different concentrations of phospholipase-C (PL-C), which cleaves glycosylphosphatidylinositol-anchored proteins including CD59 (Fitzpatrick A, Mann C et al., J Peripher Nerv Syst , 2011, 16(2): 84-91). The results are shown in Figure 3 and demonstrate that both CD55 and CD59 are sensitive to complement-mediated lysis, as the expression levels of both CRPs decrease upon PL-C treatment.

[0201] 1.6 Resistance of Schwann cells to complement-mediated cell lysis

[0202] Previous experiments have shown high expression of CD59 and CD55 in Swan cells (sNF92.2 and sNF02.2). To investigate the functional significance of this expression, anti-GM1 mediated C3 and MAC fixation in sNF96.2 Swan cells was evaluated with or without CD59 and CD55. Additionally, the effect of complement activation on cell survival was investigated. For this purpose, Swan cells were cultured and transferred to V-bottom plates for further experimental analysis. Treatment with phospholipase C (PL-C) is commonly used to remove GPI-linked proteins, such as CD59 and CD55, from the cell surface. Indeed, PL-C treatment resulted in the removal of CD59 and CD55, while the surface expression of CD46 and GM1 remained unaffected. (Fig. 4A). PL-C treatment To evaluate the effect on the sensitivity of C3 and MAC fixation, Swan cells were opsonized with MMN-patient serum containing an anti-GM1 antibody or veronal buffer (VB) as a control (1 hour, room temperature). Subsequently, cells were incubated with 5% complement-activated serum or 5% complement-activated serum pre-incubated with 480 μg / ml ARGX-117 at 37°C for 1 hour. Finally, C3 and MAC fixation were detected using biotin-labeled antibodies against C3 and MAC, respectively, and stained with APC-conjugated streptavidin. The results are shown in Figure 4A, where it was found that C3 fixation was slightly increased after opsonization with MMN-05 serum compared to opsonization with VB alone in non-PL-C treated Swan cells. However, ARGX-117 completely inhibited C3 fixation in both conditions. In the VB control group, classical pathway-mediated complement activation was likely due to antibodies (e.g., anti-HLA antibodies) present in complement-activating serum. Upon PL-C treatment, increased C3 fixation was observed, and ARGX-117 inhibited, though not completely, C3 fixation induced by MMN-005 serum in PL-C-treated cells. (Fig. 4B) A high opsonization density is expected under these conditions and was achieved by IgM / IgG antibodies against non-GM1 targets regulated by strong complement activation, as there are no IgM antibodies against GM1 or mCPRs. A similar observation was found in MAX fixation, where untreated cells showed low levels of MAC fixation, which also increased slightly after opsonization with MMN_005 serum. PL-C treatment caused an increase in MAC fixation after opsonization with MMN-005 serum, which was inhibited by ARGX-117. (Fig. 4C).Finally, to evaluate the induction of complement-mediated cytotoxicity (CDC), viability was measured by staining with 7AAD combined with Annexin-V, and double-positive cells were considered late apoptotic / dead. When using cells expressing normal levels of complement-activating proteins, CDC was not detected even after opsonization with MMN-005 serum. However, PL-C treatment increased apoptosis in MMN-005 opsonized cells, and the addition of ARGX-117 resulted in complete protection against complement-mediated cell lysis. (Fig. 4 D). In conclusion, Swan cells are internally protected against complement-mediated lysis due to high levels of CD59 and CD55 expression. In the absence of mCRPs, CDC was detected, which can be (partially) prevented by ARGX-117.

[0203] 1.7 Detection of complement activation using Schwann cells

[0204] To investigate complement activation in living Swan cells, cells were transferred to 96-well V-bottom plates (50,000 cells / well) and opsonized with an anti-HLA antibody (W6 / 32 - BioLegend; Cat N° 311402) for 30 minutes at room temperature. Subsequently, complement-activating serum (pre-incubated with EDTA, MgEGTA, or T NT009 for 15 minutes at room temperature) was added to the cells to a final concentration of 5% serum. After 1 hour of incubation (37°C), cells were transferred to 96-well V-bottom plates, and C3 fixation was measured after staining (45 minutes on ice). The results are shown in Figure 5.

[0205] These results demonstrate C3 fixation after complement activation with 5% HPS (human pooled serum). Furthermore, since C3 fixation is reduced to baseline levels upon the addition of EDTA 1 (0 mM) or TNT009 (480 mg / mL), C3 fixation is complement-specific. Moreover, since C3 is also blocked in the presence of MgEDTA, we rule out the possibility that it occurred primarily through the alternative complement pathway. Notably, C3 fixation was independent of opsonization with W6 / 32.

[0206] 1.8 Binding of MMN-patient derived autoantibodies to cultured Schwann cells

[0207] Before determining the pathogenicity of IgM anti-GM1 antibodies, the binding of these antibodies to human Swan cells was investigated in vitro. For this purpose, sNF02.2 Swan cells were cultured and transferred to 96-well round-bottom plates (50,000 cells / well) containing 50 μL of sNF02.2 culture medium. Then, the cells were stained with cholera toxin B-AF488 to detect GM1 expression. Fig. 6A The results shown demonstrate that human sNF0.2.2 Swan cells cultured in vitro express GM1. Furthermore, culture with MMN-patient serum resulted in higher anti-GM1 expression (containing autoantibodies against GM1), and more importantly, binding of IgM to Swan cells was detected after opsonization with MMN-patient serum. (Fig. 6B). Different serums from MMN patients were tested with a wide range of IgM anti-GM1 antibody titers, and all showed that IgM binds to Swan cells. Furthermore, a titration effect of IgM binding to Swan cells was detected in the serums of all investigated patients. (Fig. 7).

[0208] Next, the complement activation potential of the patient's IgM anti-GM1 antibody on Swan cells was evaluated. For this purpose, human sNF02.2 Swan cells were transferred to 96-well V-bottom plates (50,000 cells / well) and opsonized with MMN-patient serum (for 1 hour at room temperature). Then, complement-activated serum was added (for 1 hour at 37°C), and C3 was detected using a FITC-conjugated antibody. Initially, only limited C3 fixation was observed due to insufficient C3 detection with the antibody used. Therefore, a different approach was employed to increase C3 detection and reduce background complement activity. First, different percentages (10%, 5%, and 2.5%) of complement-activated serum were tested and pre-incubated with 200 μg / mL anti-C5 mAb (1h, 37°C) to inhibit terminal complement pathway activation and thereby prevent lysis of sNF02.2 Swan cells. To lower the complement background, conditions with clear serum (complement-activated serum incubated four times at 4°C for 10 minutes to remove antibodies in the serum) were also tested.

[0209] FIGS. 8A and FIGS. 8B The results shown in the figure indicate no difference between complement-activated serum (black bar) and cleared serum (gray bar), suggesting that antibodies still present in the serum do not contribute to complement activation and C3 fixation. EDT (white bar) was used as a control. However, since the window between the non-opsonized and opsonized samples is the largest, the use of 5% complement-activated serum appears to be optimal. Secondly, anti-C3 antibodies were tested to increase the detection window. The results Fig. 8CIt is shown that using biotinylated anti-C3 (LSBio, clone 6C9) to detect C3 fixation in Swan cells improves the detection range from 10% to 25%.

[0210] Therefore, optimal detection of C3 fixation in Swan cells was observed when complement-activated serum was used at a final concentration of 5% and biotinylated anti-C3 antibodies from LSBio (clone 6C9) were used. In conclusion, C3 fixation was detected in Swan cells after opsonization with MMN patient serum. This suggests that IgM anti-GM1 antibodies present in the serum of MMN patients can activate the classical complement pathway.

[0211] 1.9 C2-dependency

[0212] C2 dependence was evaluated to demonstrate the importance of complement factor C2 in the pathogenesis of MMN. sNF02.2 Swan cells were opsonized with MMN patient serum and C3 fixation was evaluated by converting them into C2-depleted serum (Complement Technology; Cat N° A312) reconstituted with recombinant human C2, rhC2 (U-protein express; Cat N°: C001, 1987). The results were Fig. 9 It was observed that C2-depleted serum alone showed relatively high mean fluorescence intensity (MFI) values—the MFI signal was higher compared to the EDTA control, resulting in a small experimental window; regardless of this, serum reconstituted with 30 μg / mL rhC2 restored C3 fixation to normal levels in Swan cells. Furthermore, C3 fixation was recovered at approximately 5–10 μg / mL rhC2, corresponding to 20–40% of physiological C2 levels. These results suggest that low levels of C2 do not result in C3 fixation or complement activation.

[0213] Various inhibitory monoclonal antibodies against complement factors have been generated, and some of them have been approved for clinical application. For example, TNT009 (BIVV009) is an anti-C1 humanized antibody that specifically blocks the classical complement pathway ((Jager U, D'Sa et al., Blood , 2019, 133(9): 893-901), on the other hand, OMS646 targets MASP-2 and thereby blocks the lectin pathway. Eculizumab inhibits C5 and therefore blocks MAC deposition induced by all three pathways (Brodsky R, Young N et al., Blood , 2018, 111: 1840-1847).

[0214] ARGX-117 is a monoclonal antibody targeting C2, and it is unique in that it inhibits the classical and lectin pathways while leaving the alternative pathway intact. The above results demonstrate that Ig anti-GM1 autoantibodies in the serum of MMN patients can specifically bind to sNF02.2 Swan cells. Furthermore, autoantibodies can activate the complement cascade, resulting in C3 fixation in Swan cells. To evaluate the therapeutic potential of this antibody, the effect of ARGX-117 on C3 fixation mediated by MMN serum was investigated. For this purpose, Swan cells were cultured and transferred to V-bottom plates (50,000 cells / well), opsonized with MMN-patient serum (1 hour, room temperature), and then cultured with 5% complement-activated serum pre-incubated with a complement blocking antibody or EDTA (20 minutes, room temperature). Detection of C3 fixation was performed by staining Swan cells with C3-BIO (LSBio, clone 6C9) and streptavidin-APC. The results Fig. 10 It shows in.

[0215] These results demonstrate that C3 fixation is dose-dependently inhibited by both ARGX-117 and TNT009. However, the inhibition of both antibodies did not reach that of the EDTA control. TNT009 can block C3 fixation up to 53 μg / mL, whereas a higher concentration of ARGX-117 (160 μg / mL) is required to block it to the same extent. Importantly, anti-C5 was not added to the cells, which had previously been performed to block the terminal complement pathway in order to enable the detection of C3 fixation. Surprisingly, the cells were not lysed, which implies that they are internally protected against complement-mediated lysis, likely due to the high expression of CD59 in Swan cells. In conclusion, ARGX-117 can dose-dependently block C3 fixation in Swan cells, and these cells are internally protected against complement-mediated lysis.

[0216] 1.11 Cytokine Secretion Essay ( Cytokine secretion assay)

[0217] To elucidate the pathophysiology regarding the origin of neurological damage in MMN patients, complement-mediated cytokine production was measured. Swan cells were seeded into 24-well plates (10,000 cells / well), opsonized with MMN-patient serum (1 hour at room temperature), and complement-activated serum was added either in the presence of complement blocking antibodies or without them. After 48 hours, the supernatant was collected, and cytokine secretion was measured using the Luminex platform. The results Fig. 11 It is shown that stimulating Swan cells with IL1-β or TNF-α results in increased secretion of IL-6, IL-8, and MCP-1. No increase in IL-6 or IL-8 was observed in MMN-patient serum. Nevertheless, the level of MCP-1 in cells cultured with MMN-patient serum was doubled compared to the control group ( Fig. 11C This increase could be blocked with the addition of ARGX-117 or T NT009, whereas eculizumab could only partially block MCP-1 secretion.

[0218] MCP-1 plays a role in recruiting inflammatory immune cells, monocytes, and macrophages to the site of infection and is also involved in the pathogenesis of several diseases, including neuro-inflammatory processes characterized by neurodegeneration. For example, levels of circulating MCP-1 increase during the progression of GBS (Orlikowski et al, J of neuroimmunol, 2003, 134 (118-27))). In conclusion, MCP-1 is produced by sNF02.2 Swan cells following opsonization into MMN patient serum and subsequent complement activation. ARGX-117 was able to block MCP-1 production.

[0219] C. Conclusion

[0220] The regulation of the complement system was evaluated using sNF02.2 Swan cells. The data show that Swan cells express high levels of complement regulatory proteins CD46, CD55, and CD59. Furthermore, IgM anti-GM1 antibodies from MMN patients activate the classical complement pathway, and C3 fixation was shown to be dependent on the presence of C2 in the studied MMN model system. ARGX-117 effectively blocked complement activity in sNF02.2 cells sensitized with anti-GM1 antibodies.

[0221] These results suggest a novel mechanism in which complement events are triggered by anti-GM1 autoantibodies and contribute to the pathology of M MN patients. This is schematically do 12This is described in [the text]. IgM anti-GM1 autoantibodies activate the classical complement pathway; however, due to the high expression of CD59 in Swan cells, soluble MACs are not formed. Direct complement-dependent cytotoxicity (CDC) therefore does not appear to be a mechanism contributing to neurological damage in this condition. However, sub-lytic MAC complex formation, which causes cellular activation capable of inducing the production and secretion of inflammatory mediators, has been described. In this study, the production of the complement-dependent chemokine MCP-1 by sNF02.2 cells was observed. Eculizumab, anti-C5, was unable to inhibit MCP-1 secretion from one of the two MMN antibodies tested, suggesting that the secretion of MCP-1 by sNF02.2 cells is triggered by a upstream mechanism of sub-lytic MAC formation.

[0222] Complement activation by anti-GM1 antibodies appears to induce the release of cytokines and / or chemokines via C3aR, which has been found to be expressed in these cells. Chemokines, such as MCP-1, may play a role in attracting inflammatory cells and further leading to neurological dysfunction and damage. Here, complement activation by anti-GM1 antibodies in Swan cells results in MCP-1 secretion that is inhibited by ARGX-117, suggesting a possible therapeutic mechanism for this antibody. In conclusion, the pathophysiology of neuronal destruction and demyelination in MMN patients appears to occur upstream of MAC formation, making the components of the complement cascade upstream of MAC more interesting therapeutic targets for the treatment of this disease. ARGX-117 targets C2 complement proteins and is therefore an example of a molecule suitable for treating this condition.

[0223] Example 2. Complement inhibition in an in vitro model of multifocal motor neuropathy (MMN) using fixed Swan cells in vitro model of Multifocal Motor Neuropathy (MMN) using fixed Schwann cells)

[0224] A. Method

[0225] 2.1 Protocol for culturing and fixing Schwann cells on cover slips

[0226] Swan cells were treated with 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured in DMEM medium supplemented with 10% FCS at 37°C under 5% CO2. Twice a week, when cell confluency was >80%, cells were subcultured or used for experiments. The medium was discarded, and the cells were washed with 10 mL of PBS. To detach the cells, 3 mL (T75) or 5 mL (T175) of Accutase cell detachment solution was added, and the cells were incubated at 37°C for 5 minutes, or until the cells were completely detached. Subsequently, culture medium (7 mL for T75 and 10 mL for T175) was added, and the cells were transferred to a 15 mL tube and centrifuged (125x). g , 10 min). The pellet was resuspended in 5 mL of culture medium and counted using trypan blue to distinguish living cells from dead cells. Then, the cells were adjusted to the desired concentration and inoculated into culture flasks (10 mL for T75, 20 mL for T175) or onto coverslips placed in 24-well plates.

[0227] 2.2 In Vitro ARGX-117 Evaluation Protocol (Protocol for in vitro evaluation of ARGX-117)

[0228] sNF02.2 Swan cells were placed on a coverslip at 37°C, 5% CO2 Cells were cultured for 3 days. Cells were fixed using 4% PFA (at 4°C for 10 minutes), washed once with 500 μL PBS, and coverslips were removed from the 24-well plate. To minimize non-specific staining, cells were cooled with 100 μL NH4Cl at room temperature for 5 minutes, washed once with 100 μL PBS, and then blocked with 100 μL PBS + 2% BSA at room temperature for 2 hours. After washing with 100 μL PBS, cells were incubated top-down with heat-inactivated MMN patient serum diluted 1:50 in PBS + 2% BSA for 60 minutes. Next, the cells were washed once (500 μL PBS + 2% BSA), incubated top-up in 15% complement-activated serum (pre-incubated with - / + complement blocking antibodies) at room temperature for 30 minutes, and washed once (100 μL PBS + 2% BSA). Subsequently, the cells were diluted with 100 μL primary antibody (diluted in PBS + 2% BSA), incubated top-down (for 1 hour at room temperature, in the dark), and washed with 100 μL PBS + 2% BSA. Then, the cells were incubated top-down in 100 μL Streptavidin-APC (diluted 1:100 in PBS + 2% BSA) at room temperature in the dark for 1 hour, followed by washing once with 100 μL PBS and 100 μL MilliQ water. After drying the coverslip on the tissue, 7 μL of ProLong Diamond Antifade Mountant (ProLong TMNext, the cells were washed once with Diamond Antifade (500 μL PBS + 2% BSA), pipetted with 15% complement active ingredient Mountant (DAPI) onto a coverslip, dried overnight at 4°C, and fixed with nail polish. The cells were analyzed using a Zeiss Z1 microscope equipped with Colibri LEDs with the following settings: 40x magnification, 25% LED, Alexa Fluor 488. TM 488) 400 ms in the APC channel, 100 ms in the DAPI channel, and 50 ms in the DAPI channel. The antibodies used for staining are shown in Table 9 below.

[0229] Staining antibodies antibodies (Antibody) Label / Fluorochrome (Label / Fluorochrome) company (Company) Catalog number (Cat. N°) expansion factor (Dilation Factor) Polyclonal chlorine anti-human C4 (Polyclonal goat anti-human C4) Biotin MyBioSource MBS560216 1:100 Polyclonal anti-human C3 (Polyclonal sheep anti-human C3 (1) ) Biotin MyBioSource MBS560642 1:100 Anti-C5b-9 (Anti-C5b-9 (2) ) Biotin Novus Bio NBP2-23494 1:500 Streptavidin (Streptavidin) APC ThermoFishereBioscience TM 17-4317-82 1:100 Cholera Toxin Western Unit B (Cholera Toxin Subunit B) Alexa Fluor TM 488 ThermoFisher C34775 1:500 Hing-Human C3 Clone 9 (Anti-human C3 clone 9) Biotin Sanquin MW1830 1:100 Hing-Human C3 Clone 28 (Anti-human C3 clone 28) Biotin Sanquin MW1860 1:100 Chlorine anti-human IgM μ-chain (Goat anti-human IgM μ-chain) Biotin Sigma Aldrich B1265 1:300 CD46 APC ThermoFisher A157711 1:50 CD55 APC BioLegend 311312 1:25 CD59 APC Life technologies 17-0596-42 1:50 Goat anti-mouse IgG (Goat anti-mouse IgG) AF647 Invitrogen A21235 1:400 GM1 AF488 Invitrogen C34775 1:500

[0230] (1) Reacts with both human C3a and C3b

[0231] (2) Anti-C5b-9 clone aE11 opposes the new epitope exposed to C9 when merged into TCC.

[0232] B. Results

[0233] In experiments performed using fixed Swan cells, results very similar to those reported in Example 1 above using live Swan cells were observed.

[0234] 2.3 Composers Expression of complement receptors

[0235] sNF02.2 Swan cells were cultured and fixed to coverslips prior to staining for marker expression. The results are shown in Table 10 below, indicating that all complement regulatory proteins are expressed in Swan cells. CD59 is highly expressed, suggesting that motor neurons are protected against MAC-mediated lysis, as CD59 is a major regulator of the terminal pathway. CD46, CD55, and C3aR showed moderate expression in Swan cells, whereas C5aR was highly expressed in the cell bodies of Swan cells. CD35, CD11b, and CD11c were all absent in Swan cells.

[0236] Expression of complement regulatory proteins in fixed Swan cells (Expression of complement regulatory proteins on fixed Schwann cells) Marker (Marker) name (Name) Swan cell (fixed) Schwann cells (fixed) CD46 MCP ++ CD55 DAF ++ CD59 Protecting +++ CD35 CR1 - CD88 C5aR ++ C3aR + GM1 ++ CD11b CR3 - CD11c CR4 -

[0237] 2.4 Fixed Swan to the cell MMN Binding of patient-derived autoantibodies MMN patient-derived autoantibodies to fixed Schwann cells)

[0238] The binding of MMN patient-derived autoantibodies to fixed Swan cells was studied and results similar to those reported in Example 1.8 above were observed.

[0239] sNF02.2 Swan cells were cultured on coverslips (50,000 cells / coverslip) in 1 mL of sNF02.2 culture medium for 3 days and then fixed with 4% PFA. Subsequently, the coverslips were washed in PBS and NH4Cl4 was used before blocking with PBS-2% BSA for 2 hours. The reaction was stopped at room temperature for 5 minutes. Cholera toxin B - Alexa488 staining was performed to detect G1 expression. The results show that human sNF0.2.2 Swan cells cultured in vitro express GM1, as well as neuroblastoma-derived mouse N2a cells.

[0240] Incubation with MMN patient serum resulted in anti-GM1 staining at the same location as IgM staining. Other serums from MMN patients were investigated with a wide range of IgM anti-GM1 antibody titers and all showed IgM binding to Swan cells. Furthermore, incubation with an excess of soluble unlabeled cholera toxin interfered with the binding of IgM antibodies from MMN serum, so the binding of IgM antibodies to human Swan cells was GM-1 specific. Since the binding of cholera toxin and anti-GM1 antibodies to GM1 gangliosides is competitive, pre-incubation with 100 μg / mL of unlabeled cholera toxin effectively interferes with the binding of anti-GM1 antibodies to Swan cells.

[0241] Human Swan cells were cultured with heat-inactivated serum from MMN patients (containing autoantibodies IgM and anti-GM1 antibodies) and HPS (human pooled serum) functioning as an exogenous complement source. The deposition of complement factors, such as C4 and C3 formation, was measured using specific antibodies. The results showed that C4 fixation could be detected in Swan cells and was associated with the anti-GM1 titer. However, high IgM anti-GM1 titers were not always associated with high C4 deposition.

[0242] C3 detection was evaluated, and coverslips were stained with different anti-C3 antibodies. Subsequently, Swan cells opsonized with serum from other MMN patients showed C3 fixation in these cells. However, high IgM anti-GM1 titers were not always associated with high C3 deposition. All EDTA controls were negative.

[0243] Table 11 below is a summary of different complement factor expression levels in different MMN patient samples.

[0244] C3 and C4 fixation of other MMN patient samples (C3 and C4 fixation of different MMN-patient samples) patient Anti-IgM GM1 titre* C3 fixation C4 fixation MMN-005 1:800 height height MMN-014 1:0 lowness lowness MMN-017 1:800 lowness lowness MMN-024 1:6400 Medium / High height MMN-035 1:100 Low / Increased lowness MMN-042 1:1600 middle height MMN-052 1:25600 Low Low / medium MMN-073 1:25600 Medium / High height

[0245] * Measured based on GM1 ELISA

[0246] In conclusion, C4 and C3 fixation were detected in Swan cells fixed after opsonization with MMN patient serum. This suggests that IgM anti-GM1 antibodies from MMN patients can activate the classical complement pathway.

[0247] 2.5 C2 dependency

[0248] C2 dependence was evaluated to demonstrate the importance of complement C2 in the pathogenesis of MMN. Therefore, Swan cells were opsonized with C2-depleted serum and reconstituted with increasing concentrations of purified human C2 (hC2) to evaluate C3 formation. The results showed that there was no C3 fixation with C2-depleted serum. However, the addition of C2 restored C3 fixation in a concentration-dependent manner. Interestingly, total blockade of C2 was not necessary to block C3 fixation; 4.58 μg / mL hC2 (approximately 20% of physiological levels) showed almost no C3 fixation.

[0249] In summary, C3 fixation depended on the presence of C2. Complete inhibition of C2 was not required to block the classical complement pathway and thereby inhibit C3 formation in Swan cells.

[0250] 2.6 Fixed Swan In cells ARGX -117 Efficacy of ARGX -117 on fixed Schwann cells)

[0251] Swan cells were cultured on coverslips for 3 days prior to fixation. Subsequently, the cells were washed, the reaction was stopped and blocked, and then opsonized with patient serum. Afterward, the cells were incubated at room temperature for 20 minutes with complement-activated serum, with or without other complement-blocking antibodies or IVIg. Finally, the cells were stained and imaged using 40x magnification.

[0252] C4 fixation to Swan cells was inhibited by TNT009 (200 μg / mL) as well as by 12.5 mg / mL IVIg treatment.

[0253] C3 inhibition was observed with both ARGX-117 and T NT009 at 200 μg / mL, whereas it was not observed with eculizumab or OMS646 as expected. IVIg showed partial inhibition of C3 fixation in Swan cells only at 12.5 mg / mL. Therefore, in this in vitro disease model system for MMN, high concentrations (200 μg / mL) of complement-inhibitory antibodies were able to block C4 and C3 fixation.

[0254] To measure the other effects of anti-complement antibodies, titrations were performed and downstream complement events were analyzed. Fixed ring cells were opsonized with MMN-patient serum, and complement was activated in the presence of increasing concentrations of complement blocking antibodies (TNT009, eculizumab, and ARGX-117), starting from 15 μg / mL and up to 480 μg / mL. The results showed that eculizumab inhibited sub-C3, indicating no inhibitory effect on C3 fixation. ARGX-117 showed complete inhibition of C3 fixation up to a concentration of approximately 30 μg / mL.

[0255] C. Conclusion

[0256] The complement inhibitory effect of ARGX-117, targeting human C2b, was evaluated in an in vitro model mimicking the pathophysiology of MMN using immobilized Swan cells. Data showed that Swan cells express high levels of complement regulatory proteins, CD46, CD55, and CD59. Furthermore, IgM anti-GM1 antibodies from MMN patients activated the classical complement pathway, and C3 immobilization was shown to depend on the presence of C2 in the MMN model system. ARGX-117 effectively blocked complement activation and outperformed both eculizumab and TNT009.

[0257] Example 3. Induced pluripotent stem cells ( iPSCs In a test tube using ) Multifocal work out neurosis model Composers Complementation inhibition in an in vitro model of Multifocal Motor Neuropathy ( MMN ) using induced pluripotent stem cells ( iPSCs )

[0258] A. Method

[0259] 3.1 iPSCs from Protocol for differentiation of spinal motor neurons iPSCs )

[0260] Motor neuron-like cells (MNs) from induced pluripotent stem cells (iPSCs) have been described in the literature (Harschnitz O et al. J Clin ImmunolIt was prepared as described in . 2014, Jul;34 Suppl 1:S112-9) and in collaboration with Dr. L van der Pol and his coworkers, the Department of Neurology, UMCU, The Netherland. Briefly, human fibroblasts were obtained from skin biopsy tissues from healthy individuals under protocols approved by the Institutional Review Board. These cells were cultured at 37°C in 5% CO2 in mouse embryonic fibroblast (MEF) medium containing DMEM GlutaMAX supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were reprogrammed within the first 5 passages according to the protocol. Human fibroblasts were plated in 6-well dishes at a density of 10,000 cells per well and cultured in MEF medium for 24 hours. Subsequently, viral transduction was performed using a mixture containing MEF medium, 4 mg / mL hexadimethrine bromide, a lentiviral vector expressing Oct4, Klf4, and Sox2, and c-Myc. After 24 hours of culture, the cells were washed 3 times with PBS, pH 7.4, and cultured in MEF medium for an additional 5 days. Afterward, the cells were cultured with trypsin-EDTA and transferred to 10 cm dishes containing a confluent layer of MEFs pre-coated with 0.1% gelatin and irradiated in MEF medium.The culture medium was replaced with human embryonic stem cell (huES) medium containing DMEM-F12, knockout serum replacement, 1% penicillin / streptomycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, and 20 ng / mL recombinant human fibroblast growth factor-basic. Colonies of iPSCs were hand-picked after 3 to 6 weeks for further confirmation and characterization. The iPSCs were maintained in huES medium, cryopreserved after 4 to 6 passages, and stored in liquid nitrogen. The iPSCs were cultured in irradiated MEFs in huES medium and hand-passed. Feeder-free culture of iPSCs was performed on Geltrex and maintained in mTeSR1 medium. Feeder-free cultured iPSCs were enzymatically subcultured using accutase.

[0261] 3.2 ARGX Protocol for In Vitro Evaluation of -117 in vitro evaluation of ARGX -117)

[0262] iPSCs were cultured on coverslips toward MNs at 37°C, 5% CO2, for 12–14 days. Subsequently, iPSC-MNs were fixed using 4% PFA (at 4°C for 10 minutes), washed three times with 500 μL PBS, and the coverslips were removed from the 24-well plate. To minimize non-specific staining and neutralize the fixative, the cells were stopped at room temperature for 5 minutes using 100 μL 50 mM NH4Cl, washed once with PBS (during all washing steps involving coverlip immersion), and then blocked at room temperature for 2 hours with 100 μL PBS + 2% BSA. After washing with PBS, the cells were incubated top-down for 60 minutes with heat-inactivated MMN-patient serum diluted 1:50 in PBS + 2% BSA. Next, the cells were washed once with PBS, incubated top-up in 15% complement-activated serum (pre-cultured with - / + complement blocking antibodies) for 30 minutes at room temperature, and washed once with PBS. Subsequently, the cells were diluted with 100 μL of primary antibody (diluted in PBS + 2% BSA), incubated top-down (for 1 hour at room temperature in the dark), and washed with PBS. Then, the cells were incubated top-down in 100 μL of Streptavidin-APC (diluted 1:100 in PBS + 2% BSA) for 1 hour at room temperature in the dark, followed by washing once with PBS and MilliQ water (the coverslip was also immersed). After drying the coverslip over the tissue, 7 μL of ProLong Diamond Antifade Mountant (ProLong TMDiamond Antifade Mountant (DAPI) was pipetteed into the object glass and covered with a top-down coverslip. The mixture was dried overnight at room temperature and secured with nail polish. Cells were analyzed using a Zeiss Z1 microscope equipped with Colibri LEDs under the following settings: 40x or 20x magnification (depending on the experiment), 25% LED, and Alexa Fluor 488. TM 488) 400 ms for the channel, 100 ms for the APC channel, and 50 ms for the DAPI channel (unless otherwise indicated). Four images were taken per condition over the entire coverslip. All images were normalized against positive and negative controls and exported in non-compressed 8-bit TIFF format in single and combined channels using ZEN 2012 software. The mean gray value for each single channel was calculated in ImageJ (Fiji). Ratios were calculated in Microsoft Excel 2010 and visualized using GraphPad Prism 7. The antibodies used for staining are shown in Table 12 below.

[0263] staining antibodies antibodies (Antibody) Label / Fluorochrome company (Company) Catalog number (Cat. N°) Dilation Factor Polyclonal goat anti-human C4 Biotin MyBioSource MBS560216 1:100 Polyclonal sheep anti-human C3 (1) ) Biotin MyBioSource MBS560642 1:100 Anti-C59-9 (Anti-C5b-9 (2) ) Biotin Novus Bio NBP2-23494 1:500 Streptavidin APC ThermoFishereBioscience TM 17-4317-82 1:100 Cholera Toxin Subunit B Alexa Fluor TM 488 ThermoFisher C34775 1:500 Anti-human C3 clone 9 Biotin Sanquin MW1830 1:100 Anti-human C3 clone 28 Biotin Sanquin MW1860 1:100 Goat anti-human IgM μ Chain Biotin Sigma Aldrich B1265 1:300 CD46 APC ThermoFisher A15711 1:50 CD55 APC BioLegend 311312 1:50 CD59 APC Life technologies 17-0596-42 1:50 Goat anti-mouse IgG AF647 Invitrogen A21235 1:400 GM1 AF488 Invitrogen C34775 1:500 CD35 APC BD 565329 1:50 CD11b APC Invitrogen 17-0118-42 1:50 CD11c APC BD 333144 1:50 C3aR APC Biolegend 345805 1:50 C5aR FITC GeneTex GTX75734 1:50 Polyclonal rabbit anti-human IgM Dako A0425 1:1000 Poly Swine anti-Rabbit Ig HRP Dako P0217 1:10000

[0264] (1) Reacts with both human C3a and C3b.

[0265] (2) Anti-C5b-9 clone aE11 is opposed to the new epitope exposed to C9 when merged into TCC.

[0266] 3.3 Protocol to determine GM1 using ELISA

[0267] NUNC maxisorp plates were coated with GM1 (5 μg / mL) in methanol. The methanol was evaporated in a laminar flow cabinet for + / - 2.5 hours. Subsequently, the wells were blocked with 200 μL of 1% BSA-PBS at room temperature for 2 hours. MMN patient serum was diluted in 1% BSA-PBS (or pre-incubated with IV Ig) and 100 μL was added to each well, followed by overnight incubation at 4°C. Next, the plates were washed 6 times with PBS and then incubated in 1% BSA-PBS with a primary antibody detecting human IgM (for 1 hour at room temperature). After washing 6 times with PBS, the wells were incubated with 100 μL of HRP-conjugated secondary antibody for 1 hour at room temperature. After washing 6 times with PBS, TMB was added, and the reaction was stopped using hydrochloric acid. The plates were analyzed at 415 nm using a BioRad ELISA reader.

[0268] B result

[0269] To investigate the pathogenesis of anti-GM1 IgM antibodies in MMN patients, an in vitro model of MMN was developed as there are currently no animal models of MMN. Briefly, human induced pluripotent stem cells were generated from fibroblasts and differentiated into motor neurons as described above. These motor neurons were then immobilized on coverslips with paraformaldehyde (PFA) and stopped with NH4Cl. Subsequently, the cells were opsonized for 1 hour using MMN patient serum containing anti-GM1 IgM autoantibodies to allow these autoantibodies to bind to GM1. After washing, complement was activated for 30 minutes using human pooled serum (HPS) in the presence or without complement blocking antibodies. Finally, the cells were stained with antibodies against complement factors to evaluate complement activation. The results of this study are described below.

[0270] 3.4 iPSC Derived motor nerve cells Composers Expression of complement receptors iPSC derived motor neurons)

[0271] To better understand the role of complement in the pathophysiology of MMNs, the expression of complement regulatory proteins and complement receptors was evaluated in iPSC-MNs. Therefore, iPSC-MMS were cultured and fixed to coverslips using 4% PFA prior to staining the expression markers. The results of this study graph 13 and Fig. 13 It shows in.

[0272] Expression of complement regulatory proteins in iPSC-MNs (Expression of complement regulatory protein on iPSC-MNs) Marker (Marker) Name iPSC-derived motor neurons (iPSC-derived motor neurons) CD46 MCP ++ CD55 DAF ++ CD59 Protecting +++ CD35 CR1 + CD88 C5aR ++ C3aR ++ GM1 +++ CD11b - CD11c -

[0273] These results show that all complement regulatory proteins (CD46, CD55, and CD59) are expressed in stationary motor neurons. CD59 is highly expressed, suggesting that motor neurons are protected against MAC-mediated lysis. CD46 and CD55 were located in the cell body and showed moderate expression. Both C3R and C5R are expressed in motor neurons, while C5aR is expressed only in the cell body and not in the neurites. Both CD11b and CD11c were absent, while CD35 expression was very low.

[0274] The binding of IgM anti-GM1 antibodies to iPSC-MNs was investigated in vitro. For this purpose, iPSC-MNs were cultured on coverslips (80,000–150,000 cells / 13 mm coverslip) in 1200 μL of hMM medium for 12–14 days and subsequently fixed with 4% PFA. The coverslips were then washed in PBS, and the reaction was stopped with NH4Cl4 (5 min at room temperature) before blocking with PBS-2% BSA for 2 hours. To detect GM1 expression, staining with Cholera toxin B-Alexa 488 was performed. The results showed that motor neurons derived from iPSCs expressed GM1, and culture with MMN patient serum resulted in significant anti-GM1 staining co-located with IgM staining.

[0275] The potential of patient IgM anti-GM1 antibodies to complement-activate iPSC-derived motor neurons was evaluated. For this purpose, human iPSC-derived motor neurons were cultured with heat-inactivated serum from MMN patients (containing autoactivated IgM anti-GM1 antibodies) and HPS, which function 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 could be detected in iPSC-MNs. However, high anti-IgM GM1 titers were not always correlated with high C3 deposition. In conclusion, C4 and C3 fixation were detected in iPSC-MNs fixed after opsonization with MMN patient serum. This suggests that IgM anti-GM1 antibodies from MMN patients can activate the classical complement pathway.

[0276] C2 dependence was evaluated to demonstrate the importance of complement C2 in MMN pathogenesis. iPSC-derived motor neurons were opsonized with C2-depleted serum and reconstituted with increasing concentrations of purified human C2 (hC2) to assess complement activity by measuring C3 fixation. The results Fig. 14 It was shown that there was no C3 fixation with C2-depleted serum. However, the addition of C2 restored C3 fixation in a concentration-dependent manner. Furthermore, ARGX-117 completely blocked C3 fixation in C2-depleted serum reconstituted with physiological amounts of hC2.

[0277] As described in Section 1.9 above, various inhibitory monoclonal antibodies against complement factors have been generated, and some of them have been approved for clinical use. The effects of these inhibitory antibodies were studied in fixed iPSC-MNs. For this purpose, iPSC-MNs were cultured on coverslips for 12–14 days prior to fixation. Subsequently, the cells were washed, inhibited, and blocked, and then opsonized with patient serum. The cells were then incubated with complement-activated serum at room temperature for 20 minutes, either in the presence of or without other complement-blocking antibodies. Finally, the cells were stained and imaged using 40x or 20x magnification.

[0278] C4 fixation to motor neurons was inhibited by TNT009 (200 μg / mL) and, as expected, was not inhibited by other mAbs.

[0279] C3 inhibition was observed in both ARGX-117 and T NT009 at 200 μg / mL, whereas eculizumab, OMS646, and rituximab, used at the same concentration as expected, showed no effect. Therefore, high concentrations (200 μg / mL) of ARGX-117, which blocks C2, were able to block C3 sub-in vitro in this MMN disease model system.

[0280] To determine the differential effects of anti-complement antibodies, titrations were performed and sub-complement events were analyzed. Immobilized iPSC-derived motor neurons were opsonized with MMN-patient serum. Before addition to iPSC-MNs, the complement-activating serum was pre-incubated with complement-blocking antibodies (TNT009, OMS646, and ARGX-117) at increasing concentrations, starting from 3 μg / mL to 200 μg / mL. The results showed that OMS646 had no inhibitory effect on C3 fixation because it inhibits the lectin pathway. ARGX-117 was shown to completely inhibit C3 fixation up to a concentration of approximately 12 μg / mL. The same results were obtained with TNT009, where C3 deposition was also blocked up to 12 μg / mL. Therefore, ARGX-117 and TNT009 performed equally well in inhibiting C3 fixation in motor neuron-derived iPSCs opsonized with MMN-patient serum.

[0281] 3.8 In other immune-mediated neuropathy ARGX by -117 Composers Complement inhibition by ARGX-117 in other immune-mediated neuropathies

[0282] Autoimmune peripheral neuropathy represents a clinically heterogeneous group of rare and disorderable diseases characterized by disease severity in terms of motor and / or sensory symptoms. In MMN, there is direct evidence of autoimmune reactivity mediated by specific IgM antibodies targeting GM1. Other immune-mediated neuropathy has been identified. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is included as the most common, and Guillain-Barre's Syndrome (GBS) is included as the most acute disorder. To evaluate the therapeutic effects of ARGX-117 on other immune-mediated neuropathy, the in vitro models described above were used. However, instead of opsonizing iPSC-derived motor neurons with MMN patient serum, serum from GBS and CIDP patients was used, respectively. The results Fig. 15 The autoantibodies present in the serum of both patients and were shown to be able to activate complement when measured as C3 fixation on motor neurons, and this was blocked by the addition of 200 μg / mL ARGX-117. Therefore, in addition to MNN, ARGX-117 also blocked complement in other immune-mediated neuropathy disease models.

[0283] 3.9 In vitro MMN Using an essay Composers Regarding inhibition IVIg's Effect of IVIg on complement inhibition using the in vitro MMN assay)

[0284] IVIg is a biological agent frequently used to treat various autoimmune diseases. In MMN, IVIg is an FDA-approved first-line treatment. Several mechanisms are involved in the immunomodulation of IVIg, including the direct neutralization of pathogenic immunoglobulins, FcR blockade, and the modulation of several immune cells. IVIg also acts as an IgG to inhibit complement activation; it can bind to C3, thereby depleting C3 from the serum and inhibiting the terminal complement pathway (Terenghi F et al., Neurology , 2004, 62: 666-668; Fitzpatrick A, Mann C et al., J Peripher Nerv Syst , 2011, 16(2): 84-91). In MMN, IVIg was shown to reduce the pathological deposition of the anti-GM1-GM1 complex (Bhatheja K, Field J. Int J Biochem Cell Biol. 2006;38(12):1995). The standard treatment for MMN is IVIg; however, this treatment is not effective when administered over a long period. Therefore, the complement inhibitory effect of IVIg was investigated using an in vitro MMN model and iPSC-derived motor neurons. For this purpose, iPSC-derived motor neurons were cultured in coverslips, fixed, and opsonized with MMN-patient serum before the addition of complement-activating serum to activate the complement cascade. Two brands of IVIg (GammaQuin and Nanogam) were tested, and different approaches to IVIg treatment were also evaluated. First, 50 mg / mL IVIg was added during the opsonization phase, where IVIg was unable to block C3 fixation in motor neurons. Second, IVIg administration was performed during opsonization and complement activation, or only during complement activation, respectively. Both resulted in reduced C3 fixation, and GammaQuin showed the strongest effect on complement inhibition compared to Nanogam. (Fig. 16 reference).

[0285] To evaluate the anti-idiotypic effect of IVIg, a competitive ELISA against GM1 was performed using MMN patient serum. Fig. 17 The results shown in this essay demonstrated that there was no competitive effect of IVIg on GM1 binding. In conclusion, IVIg treatment blocked C3 fixation when added during the complement activation phase, confirming that IVIg depletes C3 from the serum. However, IVIg had no effect on idiotypic antibodies.

[0286] C. Conclusion

[0287] The complement inhibitory effect of ARGX-117, targeting human C2b, was evaluated in an in vitro model using iPSC-derived motor neurons, thereby mimicking the possible pathophysiology of MMN. The data show that in this system, motor neurons express complement regulatory proteins with the highest expression of CD59. Furthermore, IgM anti-GM1 antibodies from MMN patients activated the classical complement pathway, and C3 deposition was shown to be dependent on the presence of C2. ARGX-117 not only effectively blocked complement activation in MMN patients but also blocked complement activation in CIDP and GBS patient samples. Additionally, IVIg blocked complement only during the complement activation phase, and anti-ideotype antibodies had no effect.

Claims

Claim 1 A pharmaceutical composition for treating paraproteinemic neuropathies in an individual, wherein the pharmaceutical composition comprises an anti-C2 antibody or an antigen-binding fragment thereof comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises or consists of the amino acid sequence of SEQ ID NO. 8, and the VL domain comprises the following CDR sequences: - LCDR3 consisting of the amino acid sequence of SEQ ID NO. 5; - LCDR2 consisting of the amino acid sequence of SEQ ID NO. 6; and - LCDR1 consisting of the amino acid sequence of SEQ ID NO. 7, wherein the antibody or the antigen-binding fragment thereof binds to the C2b domain of C2. Claim 2 A pharmaceutical composition according to claim 1, wherein the anti-C2 antibody or its antigen-binding fragment inhibits the classical complement pathway or the lectin complement pathway. Claim 3 A pharmaceutical composition according to claim 1, wherein the paraproteinemia neuropathy is a demyelinating neuropathy. Claim 4 A pharmaceutical composition according to claim 1, wherein the paraproteinemic neuropathy is characterized by the presence of IgM, IgA, or IgG immunoglobulins. Claim 5 A pharmaceutical composition according to claim 1, wherein the paraproteinemia neuropathy is characterized by the presence of autoantibodies. Claim 6 A pharmaceutical composition according to claim 1, wherein the paraproteinemic neuropathy is characterized by the presence of autoantibodies against a neural antigen. Claim 7 A pharmaceutical composition according to claim 6, wherein the neural antigen is a ganglioside or a myelin-associated glycoprotein (MAG). Claim 8 A pharmaceutical composition according to claim 7, wherein the ganglioside is selected from the group consisting of M1, GM1b, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GT3, and GQ1b. Claim 9 A pharmaceutical composition according to claim 8, wherein the ganglioside is GM1. Claim 10 In claim 1, the paraproteinemic neuropathy is multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), Miller Fisher syndrome, acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), chronic ataxic neuropathy-ophthalmoplegia-IgM paraprotein-cold agglutinins-disialosyl antibodies (CANOMAD) syndrome, distal acquired A pharmaceutical composition selected from distal acquired demyelinating symmetric (DADS) neuropathy, monoclonal gammopathy associated peripheral neuropathy, anti-MAG peripheral neuropathy, or POEMS syndrome. Claim 11 A pharmaceutical composition according to claim 10, wherein the paraproteinemia neuropathy is MMN, CIDP, or GBS. Claim 12 A pharmaceutical composition according to claim 10, wherein the paraproteinemia neuropathy is MMN. Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 A pharmaceutical composition according to claim 1, wherein the antibody or its antigen-binding fragment comprises an amino acid sequence of SEQ ID NO. 8 or a VH domain formed therefrom and an amino acid sequence of SEQ ID NO. 9 or a VL domain formed therefrom. Claim 31 A pharmaceutical composition according to claim 1, wherein the antibody or its antigen-binding fragment comprises a human IgG heavy chain fixation domain. Claim 32 A pharmaceutical composition according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO. 16 and a light chain having the amino acid sequence of SEQ ID NO.

20. Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 A pharmaceutical composition according to claim 1, wherein the anti-C2 antibody or its antigen-binding fragment inhibits the classical complement pathway and the lectin complement pathway.

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