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

Keywords: HNK-1 glycoepitope; IgM autoantibodies; demyelinating peripheral neuropathy; glycosylated polylysine; myelin-associated glycoprotein.

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%.

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.

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.

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.