Specific immunotherapy of experimental myasthenia gravis by a novel mechanism

Abstract

Objective: Myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG), are antibody (Ab)-mediated autoimmune diseases, in which autoantibodies bind to and cause loss of muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. To develop a specific immunotherapy of MG, we treated rats with ongoing EAMG by intraperitoneal injection of bacterially-expressed human muscle AChR constructs.

Methods: Rats with ongoing EAMG received intraperitoneal treatment with the constructs weekly for 5 weeks beginning after the acute phase. Autoantibody concentration, subclassification, and specificity were analyzed to address the underlying therapeutic mechanism.

Results: EAMG was specifically suppressed by diverting autoantibody production away from pathologically relevant specificities directed at epitopes on the extracellular surface of muscle AChRs toward pathologically irrelevant epitopes on the cytoplasmic domain. A mixture of subunit cytoplasmic domains was more effective than a mixture containing both extracellular and cytoplasmic domains or than only the extracellular domain of alpha1 subunits.

Interpretation: Therapy using only cytoplasmic domains, which lack pathologically relevant epitopes, avoids the potential liability of boosting the pathological response. Use of a mixture of bacterially-expressed human muscle AChR cytoplasmic domains for antigen-specific immunosuppression of myasthenia gravis has the potential to be specific, robust, and safe.

Figure 1. EAMG was substantially suppressed in rats treated i.p. with 5 mg doses of a mixture of human α1, β1, γ, δ, and ε subunits at weekly intervals starting on day 14 following disease induction

All rats, except adjuvant control (closed circles) which received only adjuvant, were immunized with 35 μg of Torpedo californica AChR in TiterMax adjuvant at day 0. Treatments (i.p., 5 mg/dose) were initiated after the acute phase, 14 days after EAMG induction, and thereafter once a week for 5 weeks until the end of the experiments. The EAMG control rats received no treatment (closed squares). Data represent the mean ± SE of two independent experiments (n = 12 for each point, from 6 rat groups in each of two experiments). (A) The mean clinical scores of the rats treated with the subunit mixture (closed triangles) were significantly lower when compared to those of the EAMG control rats (closed squares) at all time points after day 30 (p < 0.01). (B) The effect of the treatment on clinical symptoms was corroborated by changes in the rats’ body weight. Relative weight loss was calculated as follows: % relative weight loss = 100 − (body weight on day X/average body weight of adjuvant control on day X) × 100. The insert indicates the statistical significance of the difference between groups on day 49 by the t-test.

Figure 2. EAMG was not suppressed in rats treated i.p. with the same 5 mg doses of OVA, α4 AChR subunits, or α1-ECD on the same weekly dosage schedule starting on day 14

All rats were immunized as described in Fig 1. Adjuvant control rats (open squares) received only adjuvant. Each group consisted of six rats. The EAMG control rats (closed squares) were the weakest. Control therapies of EAMG, also at 5 mg/dose i.p., including treatments with OVA (closed circle), human AChR α4 subunits (closed triangles), and α1-ECD (closed diamonds), were not statistically different from the EAMG control in clinical score (A) or weight loss (B). All groups showed similar acute phases with a peak around day 10.

Figure 3. Treatment with the subunit mixture provided a significant dose-dependent therapeutic benefit with the 5 mg/dose being the most beneficial

All rats were immunized as described in Fig 1. Adjuvant control rats (closed circles) received only adjuvant. Intraperitoneal treatments of EAMG consisted of 5 doses of 1 mg (closed reverse triangles), 2 mg (closed diamonds) or 5 mg subunit mixture (closed triangles) at weekly intervals starting on day 14. The insert indicates the statistical significance of the differences between the EAMG control group (closed squares) and groups treated with increasing doses of the subunit mixture on day 48 by the t-test.

Figure 4. Treatments greatly increased titers of antibodies to the constructs used for therapy but reduced or had little effect on titer to rat muscle AChRs

These are the rats used in Fig 2 and 3. Sera of individual rats from 6 rat groups were collected 7 weeks after the induction of EAMG. (A) Antibody titer to rat muscle AChR was evaluated by immunoprecipitation of AChRs labeled with 4 nM ¹²⁵I-αBgt. Treatment with 5 mg doses of the subunit mixture reduced autoantibody titer by half, while treating with 1 or 2 mg doses had no significant effect on the titers. Control therapy with 5 mg doses of OVA, which resulted in no clinical benefit, also decreased antibody titer to rat muscle AChR by half. The other control therapy, with 5 mg doses of <1-ECD, had no effect on autoantibody titer. Thus, antibody titer to rat muscle AChR, including antibodies to extracellular and cytoplasmic domains, was not correlated with the clinical state of the rats. Rats immunized with OVA, but not Torpedo AChR, then treated i.p. with 5 mg doses of the AChR subunit mixture, developed very low titers to rat muscle AChRs (less than 10% of that of the EAMG control rats). (B) Antibody titers to the subunit mixture (open bar) and to the <1-ECD (closed bar) were evaluated by ELISA assays. Untreated EAMG, as expected, resulted in some titer to both antigens. Treatment of EAMG with i.p. OVA had little effect on these titers. Therapies with either the subunit mixture or the <1-ECD greatly increased antibody responses to the constructs used for therapy. Rats immunized with ovalbumin, but not Torpedo AChR, then treated i.p. with subunits, developed low titers to the subunit mix, but no detectable response to <1 extracellular domain. This indicates that those antibodies from rats treated with the subunit mixture were primarily directed against cytoplasmic domains.

Figure 5. Sera from successfully treated rats were much less effective at passive transfer of EAMG

Three rats per group were injected i.p. with pooled serum containing 70 pmol of autoantibodies to rat muscle AChR at time 0, and then were examined every 12–24 h for weight loss, muscular weakness and fatigability. This amounted to 0.79 ml of serum from rats treated with 5 mg doses of the subunit mixture (closed triangles) and 0.53 ml from untreated rats with EAMG (closed squares). 0.91 ml of serum from adjuvant control group was used as negative control (closed circles). Serum from rats treated with the subunit mixture had little ability to passively transfer EAMG when compared to that of untreated rats with EAMG (p < 0.04). The effect of injection of serum on clinical symptoms (A) was corroborated by changes in body weight of the rats (B).

Figure 6. Treatment with only cytoplasmic domains was even more effective in suppressing ongoing EAMG than using both extracellular and cytoplasmic domains

All rats, except adjuvant control (closed circles), were immunized with 70 μg of Torpedo AChR in TiterMax adjuvant at day 0. This is twice the dose used in Fig 1–5. Treatments of EAMG consisted of 5 i.p. doses of 5 mg of the subunit mixture (closed triangles) or a mixture of the cytoplasmic domain constructs in the weight ratio 1:1 (closed diamonds) at weekly intervals starting on day 14. Data represent the mean ± SE of two independent experiments (n = 12 for each point, from 6 rat groups in each of two experiments). (A) The mean clinical scores of both the rats treated with the subunit mixture (closed triangles) and those treated with the cytoplasmic domain mixture were significantly lower when compared to those of the untreated rats (closed squares) at all time points after day 29 (p < 0.01 and p< 0.005, respectively). (B) The effect of the treatment on clinical symptoms was corroborated by changes in body weight. The rats treated with the cytoplasmic domain mixture had less weakness and weight loss than those treated with the subunit mixture. The insert indicates the statistical significance of the differences between untreated EAMG group (closed square) and groups treated with either the subunit mixture or the cytoplasmic domain mixture on day 49 by the t-test. Detailed features of the rats in these experiments are presented in Table 2.

Figure 7. Successful suppression of EAMG greatly increased antibody titers to the constructs used for therapy, and increased the fraction of autoantibodies to rat muscle AChR cytoplasmic domains, while modestly reducing titer to rat muscle AChR, and slightly changing autoantibody isotypes

These are rats used in Fig 6. Sera of individual rats from 6 rat groups were collected 7 weeks after the induction of EAMG. (A) These sera were assayed for their immunoprecipitation titer to ¹²⁵I-αBgt labeled rat muscle AChRs. Treatment with the subunit mixture decreased the titer to rat muscle AChR slightly (18%). Treatment with the cytoplasmic domain mixture substantially reduced the response (56%). (B) Anti-AChR IgG isotypes were determined by ELISA assays. These data represent the proportion of each IgG isotype in rat serum. Both therapies increased the proportion of IgG1 and decreased IgG2b. (C) These sera were also assayed by ELISA for reaction with subunit constructs containing both extracellular and cytoplasmic sequences, containing only the α1 extracellular domain, or only cytoplasmic domain sequences. Treatments with either the subunit mixture or the cytoplasmic domain mixture provoked large amounts of antibodies to the constructs used for therapy. Antibodies from the treated rats were primarily directed against the cytoplasmic domains. In particular, unlike treating with the subunit mixture, treatment with the cytoplasmic domain mixture not only avoided provoking antibody response to the α1 extracellular domain, but actually reduced antibodies to the α1 extracellular domains as compared with both the untreated EAMG rats and, especially, rats treated with the subunit mixture. (D) The fraction of AChR-specific autoantibodies which were directed against cytoplasmic domains was also assayed by inhibition of rat serum binding to ¹²⁵I αBgt labeled rat muscle AChR by 50 nM of the cytoplasmic domain constructs. The proportion of autoantibodies to the cytoplasmic domain is calculated as follows: % Abs to cytoplasmic domain = (1 - titer in the presence of the cytoplasmic constructs/titer in the presence of OVA) × 100. Both treatments significantly increased the fraction of autoantibodies specific for cytoplasmic domains.

Figure 8. Sera from successfully treated rats had reduced ability to cause antigenic modulation of AChR in TE671 cells

IgG antibodies were purified by Protein G Sepharose affinity chromatography from pooled serum from rats used in Fig 6. (A) Serially diluted IgG antibodies purified from the untreated EAMG rat serum pool were incubated for 4 hours with TE671 cells in 12-well tissue culture plates (Corning, Lowell, MA) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Then antigenic modulation, measured as loss of αBgt binding sites, was determined after 3 hours incubation with 4 nM ¹²⁵I-<Bgt. Background radioactivity was determined using a 100-fold excess of unlabeled <Bgt. IgG purified from normal rat serum was used as negative control to determine the total amount of surface AChRs. All measurements were in triplicate. The percent loss of surface AChR by antigenic modulation was calculated as follows: % loss of surface AChR = (1-Δcpm in the presence of antibodies/Δcpm in the presence of normal IgGs) ×100. Antigenic modulation was dose-dependent within tested antibody concentration range. (B) Based on the dose-dependence curve, the relative pathogenicity of the autoantibodies from the treated groups was calculated by the equation: % relative pathogenicity of the autoantibodies = (Anti-AChR_EAMG/Anti-AChR_treated) × 100. Anti-AChR_treated corresponds to the autoantibody titer of IgG from the treated groups used in antigenic modulation. Anti-AChR_EAMG corresponds to the autoantibody titer of IgG from the EAMG control rat serum, which cause an equivalent extent of antigenic modulation in TE671 cells. Treatment with 5 mg doses of the subunit mixture caused a 54% loss of pathologically significant autoantibodies, while treating with the same doses of the cytoplasmic domain mixture caused a 71% loss of pathologically significant autoantibodies.