Selective in vivo removal of pathogenic anti-MAG autoantibodies, an antigen-specific treatment option for anti-MAG neuropathy

Significance

Anti-MAG (myelin-associated glycoprotein) neuropathy is a rare but disabling autoimmune disorder affecting the peripheral nervous system. The pathogenicity of anti-MAG IgM autoantibodies that target the HNK-1 glycoepitope is well established. Current therapies are mostly immunosuppressive but so far are neither approved nor sufficiently effective. Therefore we designed a glycopolymer that acts as an autoantibody scavenger by mimicking the natural HNK-1 glycoepitope and demonstrated that the glycopolymer neutralizes disease-causing antibodies in patient sera. Moreover, pathogenic antibodies were removed efficiently in an immunological mouse model of anti-MAG neuropathy. Because clinical improvement of patients’ neuropathic symptoms correlates with reduced serum levels of anti-MAG antibodies, the glycopolymer represents a promising antigen-specific therapeutic option for the treatment of this neuropathy.

Abstract

Anti-MAG (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal IgM autoantibodies that recognize the carbohydrate epitope HNK-1 (human natural killer-1). This glycoepitope is highly expressed on adhesion molecules, such as MAG, present in myelinated nerve fibers. Because the pathogenicity and demyelinating properties of anti-MAG autoantibodies are well established, current treatments are aimed at reducing autoantibody levels. However, current therapies are primarily immunosuppressive and lack selectivity and efficacy. We therefore hypothesized that a significant improvement in the disease condition could be achieved by selectively neutralizing the pathogenic anti-MAG antibodies with carbohydrate-based ligands mimicking the natural HNK-1 glycoepitope 1. In an inhibition assay, a mimetic (2, mimHNK-1) of the natural HNK-1 epitope blocked the interaction of MAG with pathogenic IgM antibodies from patient sera but with only micromolar affinity. Therefore, considering the multivalent nature of the MAG–IgM interaction, polylysine polymers of different sizes were substituted with mimetic 2. With the most promising polylysine glycopolymer PL₈₄(mimHNK-1)₄₅ the inhibitory effect on patient sera could be improved by a factor of up to 230,000 per epitope, consequently leading to a low-nanomolar inhibitory potency. Because clinical studies indicate a correlation between the reduction of anti-MAG IgM levels and clinical improvement, an immunological surrogate mouse model for anti-MAG neuropathy producing high levels of anti-MAG IgM was developed. The observed efficient removal of these antibodies with the glycopolymer PL₈₄(mimHNK-1)₄₅ represents an important step toward an antigen-specific therapy for anti-MAG neuropathy.

Anti-MAG (myelin-associated glycoprotein) neuropathy is a disabling demyelinating peripheral neuropathy with an autoimmune etiology and a prevalence of about 1 in 100,000 (1). It is slowly progressive, affecting sensory and motor nerves (2–4). Cardinal clinical symptoms are sensory ataxia with impaired gait, paresthesias, distal muscle weakness, and tremor (5). Monoclonal IgM autoantibodies recognize the HNK-1 (human natural killer-1) trisaccharide epitope 1 that is present on MAG as well as on other glycoconjugates of the peripheral nervous system (PNS) (6). There is strong evidence that these IgM antibodies have a pathogenic role in the development of demyelination and neuropathy (4, 5). Histopathological studies of nerve biopsies from patients showed demyelinating lesions and widening of myelin lamellae as well as deposits of anti-MAG IgM on myelin sheaths (3, 7). Moreover, localization of anti-MAG IgM antibodies to areas of widened myelin lamellae indicates their role in myelin disintegration (8). In addition to IgM deposits, some studies report the presence of the complement factor C3d on myelin, suggesting an inflammatory element in demyelination (7, 9, 10). Strong evidence for the pathogenic role of anti-MAG antibodies is provided by the damage of peripheral nerve myelin observed in experimental animals after the passive transfer of patients’ anti-MAG antibodies (11, 12). Additionally, active immunization of cats with the HNK-1–containing glycolipid sulfoglucuronyl paragloboside (SGPG) induced autoantibodies against the HNK-1 epitope and caused an ataxic sensory neuropathy resembling the human disease (13).

The term “HNK-1 epitope” denotes the sulfated trisaccharide SO₃-3-GlcA(β1–3)Gal(β1–4)GlcNAc(1) (14), present in the PNS on the glycoprotein MAG, protein zero (P0), peripheral myelin protein 22 (PMP22), and on the glycolipids SGPG and SGLPG (sulfoglucuronyl-lactosaminyl-paragloboside) (Fig. 1A) (6). MAG belongs to the family of sialic acid-binding Ig-like lectins (siglecs) (15, 16) and is located mainly in periaxonal membranes of oligodendroglial cells in the CNS (17). In Schwann cells of the PNS, MAG is localized mainly in paranodal loops and Schmidt–Lantermann incisures, where the anti-MAG autoantibodies of patients could be detected (7, 8, 18, 19). MAG is involved in adhesion and signaling processes at the axon–glia interface and exhibits a regulatory effect on axonal caliber (6). In addition to complement activation, anti-MAG antibodies are thought to interfere directly with MAG’s adhesion and signaling function (4). Several observations suggest that MAG is the main target for the IgM antibodies: (i) deposits of patients’ antibodies to PNS sites are colocalized with MAG (19); (ii) MAG is selectively lost from PNS myelin in patients with high antibody titers (20); (iii) the pathology of MAG-knockout mice (21, 22) shows similarities to the human nerve pathology (23, 24); and (iv) human MAG has a higher HNK-1 epitope density (25) than any other myelin glycoconjugate, leading to strong IgM antibody binding (26). Based on in vitro studies, it was postulated that patients’ IgM autoantibodies increase the permeability of the microvascular endothelial structure, presumably through the binding of endothelial sulfoglucuronyl glycosphingolipids, and thereby gain access to the PNS parenchyma via a leaky blood–nerve barrier (27).

Fig. 1.

The HNK-1 carbohydrate epitope and its synthetic mimetics. (A) The HNK-1 glycoepitope 1 in the PNS is highly expressed by myelin glycoproteins, such as MAG (with up to eight HNK-1 epitopes), and glycolipids, such as SGPG and SGLPG. This trisaccharide epitope with its characteristic sulfated glucuronic acid at the nonreducing end is subject to an autoimmune attack in anti-MAG neuropathy and binds to monoclonal anti-MAG IgM autoantibodies. (B) Four HNK-1–related disaccharides were prepared. GlcAβ1–3Gal, sulfated in the 3′ position and equipped with an aromatic aglycone, represents a mimetic of the natural HNK-1 trisaccharide epitope (2, mimHNK-1). Compound 3 represents the desulfated version thereof, whereas compound 4 represents the minimal epitope recognized by IgM autoantibodies. For the multivalent presentation of the HNK-1 epitope mimetic 2, derivative 5 modified with an amino linker for polymer coupling was prepared. (C, a) TMSOTf, 4 Å MS, DCM, 0 °C → rt, 86%. (b) LiOH, THF/H₂O, 89%. (c, 1) Ac₂O, 80 °C. (2) DMAP, Pyr. (d) NaOAc, MeOH, 73% over two steps. (e) SO₃·Pyr, DMF, 91%. (f) LiOH, THF/H₂O. (g) H₂, Pd(OH)₂/C, MeOH/H₂O, 78% over two steps. (h) γ-Thiobutyrolactone, DTT, Et₃N, DMF, 85 °C, 59%. (i) (ClCH₂CO)₂O, 2,6-lutidine/DMF, 96%; (j, 1) DBU, DMF/H₂O. (2) Thioglycerol, Et₃N, 70%.

The goals of current therapies are to reduce pathogenic autoantibodies, decrease expanded autoantibody-producing B-cell clones, and/or interfere with antibody-effector mechanisms (4). Corticosteroids (e.g., prednisone), cytostatics (e.g., cyclophosphamide), IFN alpha-2a, plasma exchange, i.v. Ig, and the CD20⁺ B-cell–depleting agent rituximab have been used for the therapy of anti-MAG neuropathy (4, 5, 28). However, these approaches lack efficiency and selectivity, and thus so far no satisfactory treatment is available (28). Indeed, with the most promising agent, rituximab (4, 5), acute exacerbations of symptoms were observed in some patients (29, 30). Because clinical improvement of patients’ neuropathic symptoms correlates with reduced serum levels of anti-MAG antibodies (4, 31–34), and disease worsening is associated with increasing anti-MAG levels during treatment follow-up (5, 35), a more efficient and safer therapy might be achieved with antigen-specific agents that selectively target anti-MAG IgM antibodies or antibody-producing B cells.

In this study, biodegradable poly-l-lysine–based glycopolymers containing a disaccharide glycomimetic of the natural HNK-1 glycoepitope were synthesized. It could be shown that these glycopolymers prevent binding of anti-MAG IgM antibodies to MAG at low-nanomolar epitope concentrations in vitro. More importantly, in vivo data clearly demonstrated the efficient removal of pathogenic anti-MAG antibodies in an immunological mouse model for anti-MAG neuropathy. In summary, multivalent HNK-1 glycomimetics exhibit a considerable therapeutic potential for an antigen-specific treatment of anti-MAG neuropathy.

Results

Mimetics of the HNK-1 Glycoepitope 1 Block Anti-MAG Autoantibodies in Patients’ Sera.

Tokuda et al. (36) reported binding of IgM antibodies from patients with anti-MAG neuropathy and also mouse monoclonal HNK-1 antibody (20) to derivatives of the glycolipid SGPG containing only the terminal disaccharide SO₃-3-GlcA(β1–3)Gal. Therefore, we synthesized three different inhibitors, the disaccharides 2, 3, and 4 (Fig. 1B), and tested their potential to inhibit the binding of patients’ anti-MAG antibodies to MAG in an ELISA. In disaccharide 2, the GlcNAc moiety at the reducing end of the natural HNK-1 trisaccharide 1 is replaced by a para-methoxyphenyl aglycone, and compound 3 represents the unsulfated version thereof. Finally, the sulfated disaccharide 4 with a methyl aglycone represents the minimal epitope recognized by patients’ anti-MAG IgM as well as by the mouse monoclonal HNK-1 antibody (36). With disaccharide 2, inhibition of autoantibody binding to MAG was achieved at micromolar concentrations, whereas the derivatives 3 (missing the 3′ sulfate) and 4 (with a methyl instead of a para-methoxyphenyl aglycone) inhibited binding only at high-micromolar to millimolar concentrations (Table 1). For a further characterization of the disaccharides 2–4, the binding to serum samples with confirmed high anti-MAG IgM antibody titers from four patients (MK, DP, KH, and SJ) diagnosed with an anti-MAG neuropathy was studied. Under standard assay conditions, the sera were diluted to achieve OD₄₅₀ values close to 1. Although serum from patient MK exhibited a much higher binding to the sulfated ligand 2 than to the unsulfated disaccharide 3 (∼230-fold), serum from patient SJ showed only a 12.6-fold stronger binding to the sulfated ligand 2 (Table 1). Moreover, the para-methoxyphenyl aglycone in disaccharide 2 improves binding to pathogenic IgM autoantibodies. Its replacement by a methyl aglycone (→ disaccharide 4) led to an affinity drop for sera from patients MK and SJ by a factor of 6.9 and 1.6, respectively (Table 1). In summary, disaccharide 2 turned out to be the most potent autoantibody ligand and therefore the most suitable mimetic of the natural HNK-1 glycoepitope. Consequently, in further studies, it was used as the mimetic of the HNK-1 epitope (mimHNK-1).

Table 1.

IC₅₀ values of compounds 2, 3, and 4 and the polymer PL_40–60(mimHNK-1)₄₅ for MAG binding by the sera of four patients

Patient serum	Compound 2, IC50, µM	Compound 3, IC50, µM	Compound 4, IC50, µM	PL40–60kDa (mimHNK-1)45, IC50, µM
MK	124.0 ± 9.5	28,967.3 ± 533.0	860.0 ± 58.2	0.0046 ± 0.0001
DP	536.1 ± 23.5	N.d.	N.d.	N.d.
KH	614.2 ± 20.1	N.d.	N.d.	0.0245 ± 0.0025
SJ	793.1 ± 24.0	9,981.1 ± 1002.2	1,237.0 ± 56.0	0.0316 ± 0.0067

N.d., not determined.

Synthesis of Glycopolymers Comprising the MimHNK-1 Epitope 5.

Given the multivalent nature of the IgM–MAG interaction (26), we hypothesized that a multivalent presentation of the mimHNK-1 epitope 2 might increase antibody binding affinity substantially. To enable coupling to the polymer, the mimHNK-1 epitope 2 was equipped with a tyramine-based thiol-linker (→ 5) (Fig. 1C). For its synthesis acceptor 7 was glycosylated with the glucuronic acid derivative 6 to yield disaccharide 8. To obtain the 3′-unprotected alcohol 9, a three-step procedure according to Kornilov et al. (37) was applied. The glucuronic acid-[3,6]-lactone was formed by saponification followed by reflux in acetic anhydride. After acetylation of the remaining hydroxyls followed by methanolysis of the lactone, the desired alcohol 9 was obtained. Finally, sulfation of the 3′-hydroxyl group (→ 10), saponification, hydrogenolysis, and azide reduction yielded the unprotected disaccharide 11. Nucleophilic opening of γ-thiobutyrolactone with amine 11 yielded thiol 5, ready for coupling in substoichiometric amount to the chloroacetylated poly-l-lysine 13. To improve the water solubility of the glycopolymer, the remaining chloroacetyl groups were capped with thioglycerol. In the final glycopolymer PL_y(mimHNK-1)_x, y defines the degree of polymerization of the backbone in kilodaltons, and x stands for the percentage of epitope loading, as determined by ¹H NMR spectroscopy.

To evaluate two important polymer parameters, namely epitope loading and degree of polymerization, two series of glycopolymers were prepared. The inhibitory activities of these polymers were determined in a MAG-binding inhibition assay with the mouse monoclonal anti–HNK-1 IgM antibody (20) as competitor (Fig. 2 A and B and Table 2). Inhibitory activity increased gradually when 10–45% of the lysine side chains of a 40- to 60-kDa poly-l-lysine polymer (PL_40–60) were loaded with epitope 5. However, when the loading was increased further to 50 and 75%, a drop of inhibitory activity was observed. With the most potent polymer (45% loading) an IC₅₀ of 53.7 ± 10.8 nM was obtained. Consequently, a loading of 45% was used for the evaluation of the degree of polymerization. Poly-l-lysines of different size, i.e., molecular masses ranging from 4–15 kDa (average of 45 lysines) up to 150–300 kDa (average of 1,075 lysines) (Fig. 2B and Table 2) were used. The two most potent polymers had a molecular mass of 75–150 kDa (Sigma Aldrich) and 84 kDa (Alamanda Polymers). These glycopolymers, PL_75–150(mimHNK-1)₄₅ and PL₈₄(mimHNK-1)₄₅, exhibited IC₅₀ values of 5.9 ± 5.1 nM and 5.4 ± 1.2 nM, respectively. Because PL₈₄(mimHNK-1)₄₅ presents a narrower molecular mass distribution (45% epitope loading, 55% thioglycerol capping leading to a calculated molecular mass of 217 kDa), it was chosen as the lead candidate for further studies.

Fig. 2.

In vitro inhibitory activity of mimHNK-1 glycopolymers on MAG binding of anti-MAG IgM. (A) Ten to seventy-five percent of the side chains of a poly-l-lysine (hydrobromide salt) polymer with a molecular mass of 40–60 kDa were loaded with the mimHNK-1 epitope 5. A MAG-binding inhibition ELISA was used to determine the inhibitory capacity of the different glycopolymers. With a loading of 45% the strongest inhibition of MAG-binding by a mouse monoclonal anti–HNK-1 IgM diluted 1:1,000 was observed [IC₅₀ for PL_40–60(mimHNK-1)₄₅ = 53.7 ± 10.8 nM]. (B) Inhibitory activity of a polymer series with an epitope loading of 45% and different backbone sizes was analyzed by the MAG-binding inhibition ELISA. A polymer with a poly-l-lysine backbone with a molecular mass of 70–150 kDa (Sigma Aldrich), equivalent to 360–720 l-lysines, and a polymer with a backbone with a molecular mass of 84 kDa (Alamanda Polymers), equivalent to 400 l-lysines, showed comparable affinities: IC₅₀ for PL_75–150(mimHNK-1)₄₅ = 5.9 ± 5.1 nM; IC₅₀ for PL₈₄(mimHNK-1)₄₅ = 5.4 ± 1.2 nM. (C) Serum samples (1:1,000) from anti-MAG neuropathy patients KH, SJ, HF, MK, and DP showed high MAG binding as determined by ELISA, whereas control serum samples (1:1,000) from five patients with other neurological disorders showed no MAG-binding. (D) The binding of serum anti-MAG IgM from patients KH, SJ, HF, MK, and DP (1:7,500–1:45,000) to MAG was inhibited by PL₈₄(mimHNK-1)₄₅ with an average IC₅₀ of 3.6 ± 0.4 nM. (E) Anti-MAG IgM antibodies in patients’ sera (n = 15; 1:1,000) were inhibited from binding to MAG by PL₈₄(mimHNK-1)₄₅ with an epitope concentration of 1 µM. (F) A control polymer PL_40–60(mimHNK-1)₀ (100% thioglycerol-capped control polymer) showed no inhibition of MAG binding up to a thioglycerol-lysine concentration of 10 mM per unit. Results in A, B, and D are shown as mean ± SD; results in C are shown as single values with the mean; values in E are shown as median + 95% CI; and values in F are shown as mean + SD.

Table 2.

IC₅₀ values of PL_y(mimHNK-1)_x polymers with different degrees of polymerization (y, kDa) and epitope loadings (x, % of epitope loading) for MAG binding by mouse monoclonal anti–HNK-1 IgM antibody

PLy(mimHNK-1)x	IC50, nM
	10	25	31	45 ± 1	50	75
4–15				29,406.2 ± 11,149.8*
15–30				1,404.0 ± 577.1
30–70				345.7 ± 53.5
40–60	3,892.0 ± 644.2	324.7 ± 41.0	124.8 ± 24.1	53.7 ± 10.8	561.9 ± 59.6	935.5 ± 53.9
70–150				5.9 ± 5.1
150–300				14.1 ± 5.2
84				5.4 ± 1.2

^*In the polymer series with different backbone sizes epitope loading was 45% for all polymers except for the smallest PL_4–15(mimHNK-1)_x polymer, where epitope loading was only 38%.

In Vitro Validation of PL₈₄(mimHNK-1)₄₅ with Patients’ Sera.

To determine the inhibitory activity of the lead glycopolymer PL₈₄(mimHNK-1)₄₅ by ELISA, serum samples from five patients (MK, DP, KH, SJ, and HF) with a confirmed clinical diagnosis of anti-MAG neuropathy and high anti-MAG antibody titers were used. These serum samples showed strong MAG binding at a 1:1,000 dilution, whereas sera from patients with other neurological diseases (n = 5), two of them with a confirmed monoclonal gammopathy without anti-MAG antibodies, showed no binding to MAG at the same dilution (Fig. 2C). For inhibition assays, the patient serum samples were diluted to yield OD₄₅₀ values close to 1. With the sera of five anti-MAG neuropathy patients, the glycopolymer PL₈₄(mimHNK-1)₄₅ exhibited a mean IC₅₀ value of 3.6 ± 0.4 nM, comparable to the value (IC₅₀ = 5.4 ± 1.2 nM) we obtained with the mouse monoclonal anti–HNK-1 antibody (Fig. 2D). The 100% thioglycerol-capped, epitope-free control polymer PL_40–60(mimHNK-1)₀ did not inhibit binding of anti-MAG antibodies of sera from patients KH, MK, and SJ to MAG (Fig. 2F). To evaluate the therapeutic applicability of PL₈₄(mimHNK-1)₄₅, i.e., whether the response is broad or restricted to sera from specific patients, MAG-binding was determined by ELISA with an additional set of 10 serum samples from anti-MAG patients at a uniform dilution of 1:1,000 and a glycopolymer epitope concentration of 1 µM. Compared with PBS-treated controls, PL₈₄(mimHNK-1)₄₅ significantly reduced MAG binding in all samples (Fig. 2E).

Generation of an Immunological Mouse Model for Anti-MAG Neuropathy.

An extract enriched with the two PNS glycolipids, SGPG and its higher homolog SGLPG, was isolated from bovine cauda equina (38). Both glycolipids share the HNK-1 glycoepitope 1 (Fig. 1A). The purity of the extracts was analyzed by TLC and staining of the separated glycolipids. Furthermore, the recognition of the HNK-1 glycoepitope 1 (Fig. 1A), present in the two enriched glycolipids, was confirmed by TLC immunostaining with the mouse anti–HNK-1 IgM antibody (Fig. 3A). For the immunization of 6- to 8-wk-old BALB/c wild-type mice the SGPG/SGLPG extract together with adjuvants was used. In the immunized animals, anti-MAG IgM antibody levels reached a plateau 50–70 d postimmunization (Fig. 3B). In the control group immunized with adjuvants in the absence of SGPG/SGLPG extract (vehicle), no MAG antibody reactivity could be detected. The isotype of the anti-MAG antibodies and their glycoepitope specificity were analyzed in plasma samples of four mice. Both IgG and IgM antibodies were detected, but the latter isotype was predominant (Fig. 3C). In addition, mouse plasma reacted specifically with the HNK-1 glycoepitope 1 present on MAG and SGPG but not with other neuronal/myelin glycoepitopes (i.e., GM1, GM2, GD1a, GD1b, and GQ1b) (Fig. 3C). A comparison of the mouse plasma samples with serum samples from patients with anti-MAG neuropathy (n = 4) showed a similar epitope reactivity pattern (Fig. 3D). As expected, no IgG antibody reactivity could be determined in patient serum samples. Moreover, Western blot analysis of antibody reactivity against MAG in a human CNS myelin extract showed comparable reactivity in patient serum (n = 2) and mouse plasma (n = 2) with an upper band at 100 kDa corresponding to MAG and a lower band at 90 kDa corresponding to dMAG, a protease breakdown product consisting of only the soluble extracellular domain of MAG (Fig. 3E) (3, 6). In a control experiment with rabbit anti–l-MAG antibody recognizing the C-terminal intracellular part of l-MAG (39), the extracellular dMAG could not be detected.

Fig. 3.

Generation and analysis of an immunological mouse model for anti-MAG neuropathy. (A) A mixture of the glycolipids SGPG/SGLPG was isolated from bovine cauda equina. The analysis of the extracts E1 and E2, by both TLC mostain staining (Left) and TLC immunostaining with 1:400 diluted mouse HNK-1 IgM antibody (Right) is shown. The SGPG/SGLPG ratio varied in different extracts, and some contamination was still observed after purification. E1 was subjected to an additional purification step with silica column chromatography; E2 was not. (B) Five BALB/c wild-type mice (6–8 wk old) were injected s.c. with E1 in PBS together with the immunogenic KLH and TiterMax Gold as adjuvant at days 0, 14, and 28. A control group (n = 5) was treated without E1 at the same time points. Anti-MAG IgM levels were followed up over time by ELISA and increased until a plateau was reached after about day 70. (C) Sera (diluted 1:100) of four immunized BALB/c mice showed both anti-MAG and anti-SGPG IgM and IgG antibodies binding specifically to the HNK-1 glycoepitope on MAG and SGPG but not to five gangliosides that are relevant myelin/nerve glycoepitopes. (D) Serum samples (1:1,000) from four patients with anti-MAG neuropathy showed a high specificity for the HNK-1 glycoepitope on MAG and SGPG. In contrast to the immunized BALB/c mice, which developed both anti-MAG IgM and IgG, patients exhibit only anti-MAG IgM antibodies. (E, Left) Western blot analysis of MAG reactivity in serum and plasma samples was performed using a human CNS myelin extract. Serum samples (1:800) from two patients (MK and DP) and plasma samples (1:400) from two mice at day 70 after immunization tested positive for anti-MAG IgM, whereas plasma samples (1:400) from the same two mice did not show any MAG reactivity before immunization. (Right) A rabbit anti-human l-MAG antibody was used in a control experiment (1:1,000). Results in B are shown as mean ± SD, and results in C and D are shown as mean + SD.

Evaluation of PL₈₄(mimHNK-1)₄₅ in a Surrogate Anti-MAG Neuropathy Mouse Model.

Subsequently, the generated mouse model was used to explore the therapeutic potential of PL₈₄(mimHNK-1)₄₅. Initially, two important pharmacokinetic parameters were of interest. The first was the thermodynamic solubility of PL₈₄(mimHNK-1)₄₅ in PBS, which was determined to be at least 150 mg/mL, confirming a favorable high water solubility. The second was the plasma half-life after a single i.v. dose of 10 mg/kg (n = 5), which turned out to be in the range of 17 min (t_1/2, 16.9 ± 5.5 min) (Fig. 4A). Thereafter, the therapeutic potential of the glycopolymer PL₈₄(mimHNK-1)₄₅ was explored by administering a dose of 10 mg/kg (in PBS) into the tail vein of immunized mice (n = 5); the control groups were treated with PBS or with only control polymer PL_40–60(mimHNK-1)₀ (n = 5 in both experiments) (Fig. 4 B and C). Three hours before and after administration of the glycopolymer, blood samples were taken and analyzed, showing a significant decrease of anti-MAG IgM levels upon treatment (n = 5). Moreover, with one administration of 10 mg/kg PL₈₄(mimHNK-1)₄₅, significantly decreased levels were observed for up to 5 d, and at day 7 the decrease was still present, although nonsignificant (Fig. 4D). Importantly, both anti-MAG IgM and anti-MAG IgG levels were significantly reduced 3 h after treatment with 10 mg/kg of PL₈₄(mimHNK-1)₄₅ (n = 4) (Fig. 4E). With a dose of 1 mg/kg of the glycopolymer (Fig. 4F), anti-MAG IgM antibody levels were significantly reduced 3 and 24 h after treatment but returned to pretreatment levels after 3 d (Fig. 4F). Weekly treatment with 10 mg/kg PL₈₄(mimHNK-1)₄₅ for 5 wk progressively reduced the rebound of anti-MAG IgM antibody levels at the end of each treatment week (n = 6). Anti-MAG IgM levels were significantly reduced compared with pretreatment only for 3 d after the first drug administration, but after the fifth administration antibody levels remained significantly lowered for 14 additional days (Fig. 4G). With Western blot analysis of mouse plasma samples taken 3 h after treatment with 1 mg/kg of glycopolymer, antibody reactivity against MAG using a human CNS myelin extract could no longer be detected (Fig. 4H).

Fig. 4.

Treatment of SGPG-immunized mice with the PL₈₄(mimHNK-1)₄₅ glycopolymer. (A) PL₈₄(mimHNK-1)₄₅ was determined in plasma samples of five mice by ELISA after a single i.v. bolus injection of 10 mg/kg and revealed a drug half-life of ∼17 min (t_1/2, 16.9 ± 5.5 min). (B) An i.v. bolus injection of 10 mg/kg PL₈₄(mimHNK-1)₄₅ resulted in a significant (93%) decrease of anti-MAG IgM antibody levels 3 h after administration of PL₈₄(mimHNK-1)₄₅ compared with pretreatment levels (n = 5; **P ≤ 0.01). (C) Treatment of immunized mice (n = 5) with PBS and treatment with the control polymer PL_40–60(mimHNK-1)₀ (n = 5) did not result in any changes in anti-MAG antibody levels. (D) A dose of 10 mg/kg PL₈₄(mimHNK-1)₄₅ led to a sustained decrease in anti-MAG antibodies, which was significant up to 5 d after injection (n = 5; ***P ≤ 0.001). (E) An i.v. bolus injection (10 mg/kg) of PL₈₄(mimHNK-1)₄₅ significantly depleted anti-MAG IgG in the mice (n = 4) 3 h posttreatment (***P ≤ 0.001). (F) A lower dose of PL₈₄(mimHNK-1)₄₅ (1 mg/kg) also depleted anti-MAG IgM antibodies (n = 5; ***P ≤ 0.001), which recovered 72 h after injection. (G) Weekly treatment with 10 mg/kg PL₈₄(mimHNK-1)₄₅ for 5 wk resulted in a decreasing anti-MAG IgM antibody rebound toward the end of each week. Compared with pretreatment levels, anti-MAG IgM levels remained significantly lowered for up to 14 d after the fifth administration on day 28 (n = 6; *P ≤ 0.05). (H) Western blot analysis of MAG reactivity in a human CNS myelin extract with plasma samples (1:400) taken from mice (n = 2) before immunization, on day 82 after immunization (postimmunization), and 3 h after treatment with 1 mg/kg PL₈₄(mimHNK-1)₄₅ on day 82. Plasma of treated mice showed no MAG reactivity 3 h after treatment compared with plasma before treatment (postimmunization). Sera (1:800) from two patients with anti-MAG neuropathy (MK and DP) and a rabbit anti-human l-MAG antibody (1:1,000) were used in control experiments. (I) Nonimmunized mice (n = 4) underwent daily i.v. treatment with 10 mg/kg PL₈₄(mimHNK-1)₄₅ for 10 consecutive days (drug treated). ADAs were detected in the treated group. In the ADA-positive control group (immunized ctrl.), which was immunized s.c. with PL₈₄(mimHNK-1)₄₅ together with the immunogenic KLH and TiterMax Gold (n = 4), the IgG ADA response was more pronounced than the IgM ADA response. Except for A and I, in which results are shown as mean ± SD, results are shown as median + 95% CI.

When wild-type BALB/c mice were treated daily with 10 mg/kg PL₈₄(mimHNK-1)₄₅ for 10 consecutive days, plasma samples did not show any formation of antidrug antibodies (ADAs) of the IgG or IgM isotype for a follow-up period of 50 d. An ADA-positive control group was obtained when wild-type BALB/c mice were immunized s.c. with PL₈₄(mimHNK-1)₄₅, immunogenic Keyhole limpet hemocyanin (KLH), and TiterMax Gold. Interestingly, immunization induced a much stronger response to IgG ADA than to IgM ADA (Fig. 4I). These IgG ADA did not show reactivity with the backbone of the glycopolymer or with MAG but reacted only with PL₈₄(mimHNK-1)₄₅.

Discussion

The pathogenesis of the anti-MAG neuropathy as an antibody-mediated autoimmune disease is widely accepted, and therapies are aiming at a reduction of levels of autoantibodies and/or autoantibody-producing B-cell clones through immunomodulation and immunosuppression (4, 5). Nonetheless, there is a strong need for personalized, new disease-modifying agents devoid of nonspecific immunosuppression. To develop an antibody-specific treatment, Page et al. raised an anti-idiotypic antibody against the variable regions of a monoclonal IgM antibody derived from a patient with anti-MAG neuropathy (40). It successfully inhibited the binding of the individual patient’s IgM antibodies to MAG in vitro but turned out to be ineffective with four other patients’ IgM antibodies, indicating idiotypic heterogeneity among patients’ autoantibodies. Despite this microheterogeneity, antibodies from different patients bind to the HNK-1 epitope, suggesting that all anti-MAG antibodies can be targeted by mimetics of their shared glycoepitope (41). The monoclonal anti-MAG IgMs of neuropathy patients bind specifically to the HNK-1 carbohydrate epitope present on MAG and on the PNS-specific glycolipids SGPG and SGLPG (5). The minimal carbohydrate epitope recognized by the autoantibodies is the SO₃-3-GlcA(β1–3)Gal disaccharide (36, 37). After MAG deglycosylation, the patients’ autoantibodies lose their MAG reactivity (42). Saturation transfer difference (STD) NMR experiments revealed that the terminal trisaccharide SO₃-3-GlcA(β1–3)Gal(β1–4)GlcNAc, i.e., the HNK-1 epitope, of the glycolipid SGPG interacts with the mouse monoclonal anti–HNK-1 IgM and that the most significant contribution is made by the terminal disaccharide SO₃-3-GlcA(β1–3)Gal (43).

In this study, we synthesized a glycomimetic of the natural HNK-1 trisaccharide epitope. Our data suggest that the aromatic aglycone of 2 (mimHNK-1) mimics the “hydrophobic face” (44) of the reducing end GlcNAc moiety and that the sulfate group in the 3′ position of the GlcA moiety is crucial for antibody binding (Table 1).

Ogino et al. (26) have shown that decavalent IgM antibodies can establish multivalent interactions with MAG, which presents up to eight HNK-1 epitopes on its extracellular domain (45, 46). Thus, to improve the low affinity of the sulfated disaccharide 2, a multivalent presentation on a poly-l-lysine backbone was explored (45). Poly-l-lysine is biodegradable and therefore is suitable for therapeutic applications (47). A careful optimization of the degree of polymerization and epitope density yielded the tailor-made glycopolymer PL₈₄(mimHNK-1)₄₅, exhibiting maximal inhibitory activity. MAG binding by anti-MAG IgM antibodies was inhibited by this glycopolymer in all tested patient sera. Compared with the monomer 2, the inhibition of MAG binding was improved by a factor of 38,000–230,000, strongly supporting the multivalent nature of the antigen–antibody interaction.

Next, the potential of PL₈₄(mimHNK-1)₄₅ to deplete anti-MAG IgM was studied in a mouse model for anti-MAG antibodies. Several animal models for anti-MAG neuropathy have been reported and are based on passive transfer of patients’ IgM antibodies into healthy experimental animals, including cats (11, 48) and chickens (12), and, more recently, on active immunization of cats with SGPG leading to a sensory ataxic neuropathy (13). Although these animal models partly mimic characteristics of the human myelin and nerve pathology of anti-MAG neuropathy, rodents do not display a clear neuropathic phenotype after immunization with SGPG (49, 50). This lack of neurological symptoms is a consequence of the following findings: (i) during early neurogenesis, HNK-1 expression in neural crest cells is very low in rats and is absent in mice (51); (ii) MAG in rodents lacks the HNK-1 epitope (27, 42); and (iii) SGPG/SGLPG expression is about one order of magnitude lower in the peripheral nerves of rats and mice than in higher mammals, such as cats and dogs (52). To obtain a surrogate immunological model for anti-MAG neuropathy, BALB/c mice, frequently used for immunological studies, were selected for active immunization. Because the clinical improvement in patients correlates with the reduction of their anti-MAG IgM antibody levels (4, 31–33), this mouse model is a suitable surrogate model for studying the depletion of anti-MAG IgM antibodies by therapeutic agents. Importantly, the mouse IgM antibodies are comparable with their human counterparts, because they recognized both human MAG and SGPG, although reactivity toward other nerve and myelin glycoepitopes is absent (Fig. 3 C–E).

The i.v. administration of 1 and 10 mg/kg of PL₈₄(mimHNK-1)₄₅ to anti-MAG IgM–positive mice effectively reduced antibody levels in a dose-dependent manner. In addition to anti–HNK-1 IgM, anti–HNK-1 IgG also could be removed efficiently. Upon repetitive weekly administration of 10 mg/kg of the glycopolymer, the decreasing rebound of anti-MAG antibody levels suggests the existence of a potential immunomodulatory treatment effect. Furthermore, the relatively short plasma half-life of PL₈₄(mimHNK-1)₄₅ of ∼17 min after i.v. injection in mice implies a total elimination within 2 h. Thus, the reduced level of anti-MAG antibodies for up to 2 wk after treatment (Fig. 4G) results from the removal rather than the neutralization of anti-MAG antibodies (53, 54). Furthermore, the short half-life is comparable to that of a previously described glycopolymer (47, 54) and might be crucial for low immunogenicity. Indeed, daily treatment of nonimmunized mice with PL₈₄(mimHNK-1)₄₅ did not trigger the formation of IgM and IgG ADAs (Fig. 4I), suggesting low immunogenicity of PL₈₄(mimHNK-1)₄₅. In addition, no signs of intolerance were observed upon repetitive administration, a result that is in accordance with a polymer developed by Duthaler et al. (47), which efficiently eliminated both anti-αGal IgM and IgG in nonhuman primates and showed neither immunogenicity nor toxicity. Regarding off-target effects, the glycopolymer may interact with HNK-1–binding molecules, such as the proinflammatory cytokines IL-6 (55) and HMGB1, the extracellular matrix components laminin-1 and -2, and lecticans (56). However, PL₈₄(mimHNK-1)₄₅ is not expected to interact with the HNK-1 receptors in the nervous system, where HNK-1 is mainly expressed; because of the large size and high charge of the glycopolymer, it is not anticipated to penetrate the blood–nerve or blood–brain barrier.

The concept of anti-glycan antibody removal has been explored previously both in vivo, with anti-idiotypic antibodies (40, 57), peptide glycomimetics (58), monomeric glycans (59), and glycopolymers (47, 54, 59), and ex vivo using immunoaffinity columns (59, 60). Glycopolymers have been used thus far only for in vivo removal of natural anti-glycan antibodies, e.g., polymers of αGal or ABH blood group glycoantigens (47, 54, 59). Here we describe the selective in vivo removal of an anti-glycan autoantibody using a carbohydrate-based therapeutic.

The PL₈₄(mimHNK-1)₄₅ glycopolymer potentially enables an antigen-specific therapy for anti-MAG neuropathy and therefore is of considerable clinical interest. Furthermore, glycopolymers might be generally effective as therapeutic agents for the depletion of pathogenic anti-carbohydrate autoantibodies in other antibody-mediated diseases such as multifocal motor neuropathy or Guillain–Barré syndrome.

Materials and Methods

Statistical Analysis.

Unless otherwise stated, results are given as mean ± SD or median + 95% CI of three independent experiments. Comparisons between two conditions were performed using either Student’s t test or one-way ANOVA with Dunnett’s multiple comparison posttest with a 0.05 confidence level accepted for statistical significance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Synthesis of the Glycomimetics and Glycopolymers.

See SI Appendix for the preparation and analysis of compounds 2–5 and the glycopolymers.

Patient and Control Serum Samples.

Sera from patients with positive anti-MAG IgM titers and control sera from patients with neurological disorders other than anti-MAG neuropathy (no anti-MAG IgM reactivity) were obtained from the University Hospital of Basel. Patients MK, DP, KH, SJ, and HF had a clinical diagnosis of a monoclonal IgM gammopathy and anti-MAG neuropathy with high antibody titers (≥70,000 Bühlmann titer units) as measured by anti-MAG ELISA (Bühlmann Laboratories AG). The additional 10 patient sera used in our study also tested positive by anti-MAG ELISA for high levels of anti-MAG IgM antibodies, which are highly indicative of anti-MAG neuropathy. Control sera tested negative for anti-MAG IgM antibodies. Two of the five control sera originated from patients with a monoclonal IgM gammopathy without anti-MAG reactivity. The use of patient sera was approved by the Ethics Committee of Northwestern and Central Switzerland (EKNZ). Informed consent was obtained from all nonanonymized participants.

ELISA.

The potential of HNK-1 mimetics (compound 2–4 and polyvalent derivatives of 5) to inhibit binding of anti-MAG IgM (mouse or human) to immobilized MAG was determined in an anti-MAG ELISA. For that purpose, 96-well plates coated with human MAG (EK-MAG; Bühlmann Laboratories AG) were used. The assay was performed according to the manufacturer’s protocol. Briefly, test compound, sera from patients or mouse monoclonal anti-HNK-1 IgM antibody (20), and incubation buffer (provided with the kit) were added to a final volume of 50 µL per well. The assay was run in triplicate. In case of MAG-binding assays, antibodies (in the absence of test compounds) were incubated in the same volume. If not indicated otherwise, human serum and mouse plasma samples (single or duplicate) were diluted 1:1,000 and 1:100, respectively. For detection of human anti-MAG IgM or IgG, anti-human IgM or IgG secondary antibodies conjugated to HRP were used. The mouse HNK-1 IgM antibody (20) and anti-MAG IgM from plasma of immunized mice were detected with goat anti-mouse IgM HRP conjugate (A8786; Sigma Aldrich) diluted 1:10,000. Anti-MAG IgG in mouse plasma was detected with goat anti-mouse IgG HRP conjugate (A4416; Sigma Aldrich) diluted 1:10,000. The OD of the colorimetric signal was measured at 450 nm on a microplate reader (Spectramax 190; Molecular Devices). The IC₅₀ values of the tested compounds were calculated using Prism 5.0 software (GraphPad Software, Inc.). IC₅₀ values of polymers were based not on the average molecular mass (MW) of the polymers but on the equivalent weight per glycoepitope. The equivalent weight was calculated by the formula [(MW_{glycoepitope-lysine-unit})⋅x + (MW_{thioglycerol-lysine-unit})⋅(1 − x)]/x. It is independent of the degree of polymerization (n) but is dependent on the fraction x of glycosylated lysine units (as measured by ¹H NMR). This calculation allowed a direct comparison between the inhibitory activity of the mimHNK-1 epitope monomer and the same molecule as part of a polymer. For the 100% thioglycerol-capped control polymer, only the MW_{thioglycerol-lysine-unit} was used for concentration calculation. For the evaluation of glycoepitope specificity of mouse and patient antibodies, both an anti-SGPG ELISA and GanglioCombi MAG ELISA (EK-GCM; Bühlmann Laboratories AG) were performed according to the manufacturer’s instructions. In our assay, 50 µL of patient sera at 1:1,000 (patients MK, SJ, SP, and KH) and mouse plasma at 1:100 dilution (plasma samples from three mice at day 43 and one mouse at day 62 after first immunization) were incubated.

Purification of SGPG/SGLPG from Bovine Cauda Equina.

An extract of glycolipids enriched with SGPG and SGLPG was isolated from bovine cauda equina similar to the protocol of Burger et al. (38). In contrast to Burger’s protocol, ion exchange chromatography of the isolated glycolipid pellet was performed using a DEAE-Sephadex A-25 column (6 g, 3 × 6 cm) that was run with 75 mL of each of the following eluents: 0.05 M NaOAc, 0.1 M NaOAc, 0.2 M NaOAc, and 0.5 M NaOAc in MeOH (flow rate 1 mL/min). SGPG and SGLPG were recovered in the 0.5 M NaOAc fraction, which was evaporated to dryness. The residue was suspended in 10 mL of water (pH 2.5) and desalted on a RP-18 silica column using a CombiFlash companion (Teledyne Isco) (10 mL per fraction, flow rate 8 mL/min). After removal of NaOAc with 80 mL of water (pH 2.5), the glycolipids were eluted with 120 mL of MeOH and were recovered in fractions containing the two glycolipids as analyzed by TLC. MeOH was evaporated, and the extract was lyophilized. To remove impurities, an additional chromatography on silica was performed. A CH₂Cl₂/MeOH gradient (30 min 15–45% MeOH; 10 min 45% MeOH, 10 min 100% MeOH) was used (10 mL per fraction, 8 mL/min) and fractions with purified SGPG/SGLPG were collected and analyzed by TLC. Again, the solvent was evaporated, and the extract was lyophilized.

TLC and TLC Immunostaining.

TLC was performed on Silica gel 60 F₂₅₄-coated glass plates (Merck) for 25–30 min using chloroform/methanol/0.2% KCl (50:40:10) as the eluent. Glycolipids were visualized by charring at 156 °C with mostain (0.02 M solution of ammonium cerium sulfate dihydrate and ammonium molybdate tetrahydrate in aqueous 10% H₂SO₄). TLC immunostaining was performed as described by Ilyas et al. (50). The plate was blocked in 0.1% poly(isobutyl methacrylate) in hexane for 60 s, then was blocked for 30 min in blocking buffer (1% BSA, 0.1% Tween 20 in PBS), and finally was incubated for 2 h with 1:400 diluted HNK-1 antibody (20) in blocking buffer. After two washing steps with PBS, the plate was incubated for 1 h with 1:4,000 diluted anti-mouse IgM alkaline phosphatase (AP) (µ-chain) (A3437; Sigma Aldrich) conjugate in blocking buffer. The plate was washed twice with PBS and incubated with 3 mL of 1:200 diluted nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) in development buffer [0.1 M Tris base (pH 8.8), 0.1 M NaCl, 5 mM MgCl]. The color reaction was stopped after 20 min, and the plates were visualized with a Gel Doc XR+ Reader (Bio-Rad Laboratories).

Animals.

Immunization was performed based on the protocol of Ilyas et al. (13). Groups of four to six male BALB/c wild-type mice at the age of 6–8 wk were injected s.c. at multiple sites on the lower back with a total of 100 µg of purified SGPG/SGLPG (in PBS) mixed with KLH (1.4 mg/mL final concentration) and emulsified with an equal volume of TiterMax Gold. Two booster injections were performed after 2 and 4 wk with 20 µg of purified SGPG/SGLPG mixed with KLH and TiterMax Gold. Control mice were injected with the same mixture but without SGPG/SGLPG. The same immunization protocol was applied for the induction of ADAs (immunogenicity assay), but mice were immunized with PL₈₄(mimHNK-1)₄₅, together with KLH and TiterMax Gold. Blood samples were taken by puncture of the tail vein, were transferred to tubes containing 1 µL of 0.5 M EDTA, and were centrifuged for 15 min at 1,800 × g. The supernatant (plasma) was transferred to new tubes and stored at −55 °C. The glycopolymer (or control polymer), dissolved in PBS, was administered by i.v. injection of the tail vein. Animal experiments were conducted in accordance with Permit no. 2778 of the Animal Research Authorities of the Canton Basel-Stadt, Switzerland.

In Vivo Pharmacokinetics.

A single dose (10 mg/kg, in PBS) of PL₈₄(mimHNK-1)₄₅ was administered via the tail vein in control-immunized BALB/c mice administered KLH and TiterMax Gold without SGPG/SGLPG antigen. Blood samples were taken by tail vein puncture at multiple time points after injection (i.e., 5, 15, 30 min, 1, 2, 4, 8, 12, and 24 h) and were transferred into tubes containing 1 µL of 0.5 M EDTA. The plasma was isolated by centrifugation at 1,800 × g for 15 min and was stored at −55 °C. To detect the glycopolymer by sandwich ELISA, we used two recombinant monoclonal antibody (Fab) fragments targeting the fully thioglycerol capped poly-l-lysine backbone [PL_40–60(mimHNK-1)₀], i.e., a capture Fab fragment (AbD27381; Bio-Rad Laboratories) and a detection Fab fragment (AbD27389; Bio-Rad Laboratories). MaxiSorp plates (442404; Thermo Fisher,) were coated overnight at 4 °C with 50 µL (5 ng/µL) of the capture Fab fragment in PBS. Plates were blocked for 2 h at room temperature with 5% BSA in PBS containing 0.1% Tween20 (PBST). Plasma samples were diluted in PBST to obtain a signal in the linear range (1:5–1:16,000) and were incubated at room temperature for 2 h (50-μL working volume). PL₈₄(mimHNK-1)₄₅ in PBST (0.001–8 ng/µL) was used as a standard. The detection Fab fragment, which was conjugated to HRP with the LYNX rapid HRP antibody conjugation kit (LNK006P; Bio-Rad Laboratories), was diluted in 0.5% BSA/PBST at a concentration of 2 ng/μL and incubated at room temperature for 1 h (50-μL working volume). After each step, the plate was washed five times with PBST (250 µL). Finally, 50 µL of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (34028;Thermo Fisher Scientific) was added, and after 5 min incubation the color reaction was stopped with 1 M H₂SO₄. OD₄₅₀ was measured on a microplate reader (SpectraMax 190; Molecular Devices). Data analysis was performed with GraphPad Prism 5.0 software (GraphPad Software, Inc.) and PK Solver (61). The plasma half-life was calculated with PK Solver using the one-compartment (i.v. bolus) model.

Immunogenicity Assay (ADA Detection ELISA).

Nonimmunized mice underwent daily treatment with 10 mg/kg of PL₈₄(mimHNK-1)₄₅ (i.v.) for 10 consecutive days. Blood samples were taken weekly via the tail vein starting 24 h after the last injection, and plasma was prepared as described earlier for further analysis. As a positive control, blood samples were taken from mice immunized with PL₈₄(mimHNK-1)₄₅ and adjuvants, according to the immunization protocol described above. For the detection of ADAs, MaxiSorp plates (442404; Thermo Fisher) were coated overnight at 4 °C with 50 µL (0.1 µg/mL) of PL₈₄(mimHNK-1)₄₅ in PBS. They were washed with 0.1% PBST and blocked with 5% BSA in 0.1% PBST for 2 h at room temperature. Plasma samples were diluted 1:100 in 2.5% BSA/PBST and were incubated (50 µL) on the plate for 2 h at room temperature immediately after blocking (without washing). Next, ADA detection was done by a 2-h incubation at room temperature with a goat anti-mouse IgM HRP conjugate (A8786l; Sigma Aldrich) or with a goat anti-mouse IgG HRP conjugate (A4416; Sigma Aldrich) diluted 1:10,000 in 1% BSA/PBST. Finally, plates were washed and incubated with 50 µL of TMB substrate for 30 min, after which the color reaction was stopped with 1 M of H₂SO₄. OD was measured at 450 nm with a microplate reader, and data analysis was performed with Prism software.

Myelin Preparation.

Freshly frozen human postmortem corpus callosum (white matter) was homogenized with a 12-mm Polytron (2 × 10 s) in ice-cold 0.25 M sucrose, 10 mM Hepes (pH 7.4), 2 mM EGTA with a protease inhibitor mixture (1 µg/mL aprotinin, 2 µg/mL leupeptin, 1 µg/mL pepstatin, 100 µg/mL PMSF). The suspension was brought up to an end concentration of 1.4 M sucrose, 10 mM Hepes (pH 7.4), and 2 mM EGTA. A four-step discontinuous gradient was set up in a 14 × 95 mm Polyallomer Beckmann tube with 1.1 mL of 2 M sucrose, 10 mM Hepes (pH 7.4), 2 mM EGTA solution at the bottom of the tube; then with 8.5 mL of 1.4 M sucrose, 10 mM Hepes (pH 7.4), and 2 mM EGTA brain suspension layered carefully above; then 2.2 mL 0.85 M sucrose, 10 mM Hepes (pH 7.4), and 2 mM EGTA; and finally 0.75 mL of 0.25 M sucrose, 10 mM Hepes (pH 7.4), and 2 mM EGTA on the top. After centrifugation for 20 h at 25,000 rpm and 4 °C (Beckmann Coulter L-70K free-swing rotor ultra-centrifuge), myelin membranes were enriched in the 0.25 M sucrose fraction, and plasma membranes were located at the interface to the 0.85-M sucrose fraction. Myelin membranes were collected and homogenized in 15 volumes of cold 10 mM Hepes (pH 7.4), 2 mM EGTA and were centrifuged (25,000 rpm for 2 h at 4 °C). The supernatant was discarded, and the pellet was resuspended in sterile water and stored at −80 °C.

SDS/PAGE and Western Blotting.

The protein concentration of the human CNS myelin extract was determined with a bicinchoninic acid assay (Sigma Aldrich). Samples (15 µg) were separated by Tris/glycine SDS/PAGE on 8% gels and were analyzed by Western blotting using Protran BA85 nitrocellulose membranes (GE Healthcare). Membranes were blocked for 1.5 h in blocking buffer [3% BSA, 20 mM Tris base (pH 7.4), 0.1 M NaCl, 0.05% sodium azide]. The same buffer was used to dilute patient serum samples, mouse plasma samples, and the control antibody. Patient sera (patients MK and DP) were diluted to 1:800, and plasma from immunized mice was diluted to 1:400. A rabbit anti-human l-MAG antibody (1:1,000) was used as control (39). The membrane was incubated with the antibody overnight at 4 °C. The secondary antibodies, the AP-conjugated anti-human IgM (1:20,000; A3437; Sigma Aldrich), goat anti-mouse IgM (1:20,000; A9688; Sigma Aldrich), and goat anti-rabbit IgG (1:40,000; A3687; Sigma Aldrich), were incubated with the membranes for 1 h at room temperature. Membranes were developed for 15–30 min in NBT/BCIP diluted 1:200 in development buffer. The membranes were imaged with a Gel Doc XR+ reader (Bio-Rad Laboratories).