Effect of monovalency on anti-contactin-1 IgG4

Abstract

Introduction: Autoimmune nodopathies (AN) have been diagnosed in a subset of patients fulfilling criteria for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who display no or poor response to intravenous immunoglobulins. Biomarkers of AN are autoantibodies, mainly IgG4, directed against the ternary paranodal complex composed by neurofascin-155, contactin-1 (CNTN1), and Contactin-associated-protein-1 (CASPR1) or against the nodal isoforms of neurofascin. IgG4 can undergo a Fab-arm exchange (FAE) which results in functionally monovalent antibody. This phenomenon differentially affects the pathogenicity of IgG4 depending on the target of autoantibodies. Here, we have evaluated this issue by examining the impact of valency on anti-CNTN1 IgG4 which induces paranodal destruction through a function blocking activity.

Methods: Sera were obtained from 20 patients with AN associated with anti-CNTN1 antibodies. The proportion of monospecific/bispecific anti-CNTN1 antibodies was estimated in each patient by ELISA by examining the ability of serum antibodies to cross-link untagged CNTN1 with biotinylated CNTN1. To determine the impact of monovalency, anti-CNTN1 IgG4 were enzymatically digested into monovalent Fab and tested in vitro on cell aggregation assay. Also, intraneural injections were performed to determine whether monovalent Fab and native IgG4 may penetrate paranode, and antibody infiltration was monitored 1- and 3-days post injection.

Results and discussion: We found that the percentage of monospecific antibodies were lower than 5% in 14 out of 20 patients (70%), suggesting that IgG4 have undergone extensive FAE in situ. The levels of monospecific antibodies correlated with the titers of anti-CNTN1 antibodies. However, no correlation was found with clinical severity, and patients with low or high percentage of monospecific antibodies similarly showed a severe phenotype. Native anti-CNTN1 IgG4 were shown to inhibit the interaction between cells expressing CNTN1/CASPR1 and cells expressing neurofascin-155 using an in vitro aggregation assay. Similarly, monovalent Fab significantly inhibited the interaction between CNTN1/CASPR1 and neurofascin-155. Intraneural injections of Fab and native anti-CNTN1 IgG4 indicated that both mono- and bivalent anti-CNTN1 IgG4 potently penetrated the paranodal regions and completely invaded this region by day 3. Altogether, these data indicate anti-CNTN1 IgG4 are mostly bispecific in patients, and that functionally monovalent anti-CNTN1 antibodies have the pathogenic potency to alter paranode.

Keywords: GBS; Schwann; auto-immune; axon; demyelination; immunoglobulin; myelin.

Figure 1

Fab-arm exchange occurs in patient with AN. (A) The capacity of serum antibodies to cross-link untagged CNTN1 with biotinylated CNTN1 was measured by sandwich ELISA in healthy controls (HC; n = 23), seronegative CIDP patients (CIDP -; n = 43), Nfasc155+ autoimmune nodopathy (AN Nfasc155 +; n = 21) and CNTN1+ autoimmune nodopathy (AN CNTN1+; n = 20). Antibodies from HC, seronegative patients or Nfasc155+ did not cross-link CNTN1. By contrast, CNTN1+ AN patients significantly cross-linked CNTN1 (*** P<0.001 by one-way ANOVA followed by Bonferroni’s post-hoc tests). (B) The percentage of monospecific antibodies was calculated by interpolating the data from a calibration curve obtained with anti-CNTN1 IgG3. The percentage of monospecific anti-CNTN1 antibodies in CNTN1+ AN (n = 20) was significantly smaller compared to the percentage of monospecific anti-Nfasc155 antibodies in Nfasc155+ AN (n = 21) (** P<0.005 by unpaired two-tailed Student’s t tests). (C) The titers of anti-CNTN1 IgG4 correlated with the percentage of monospecific CNTN1 antibodies calculated in each patient. (D) The clinical severity (ONLS) is plotted against the percentage of monospecific antibodies in each patient (n = 16). No significant correlation was found. P value, Spearman’s correlation coefficient (r), R square (R²) and 95% confidence band (dotted lines) are indicated on the graph. n.s.: non-significant, ONLS: Overall Neuropathy Limitation Score.

Figure 2

Monovalent Fab are sufficient to inhibit the interaction between CNTN1/CASPR1 and Nfasc155. (A-D) These are representative images of cell aggregation assays. HEK293T cells were transfected with CASPR1-GFP/CNTN1 (green) and were incubated with cells expressing mcherry tagged Nfasc155 (red) in presence of control IgG4 from a healthy donor (CTL; B), native CNTN1-reactive IgG4 from patient AN1 (C) or monovalent Fab-reactive to CNTN1 (D). As negative controls, cells expressing GFP (green) were incubated with cells expressing mcherry tagged Nfasc155 (A). The right panels represent the distribution of the percentage of green cells per aggregate in each aggregate (N = 4 distinct experiments for each condition). (E) The native IgG4 and monovalent Fab were tested from two AN patients reactive against CNTN1 (AN1 and AN2). The number of cell aggregates per visualization field was counted in ten images and averaged (N = 4 distinct experiments per condition). Both native IgG4 and monovalent Fab reactive against CNTN1 significantly inhibited the interaction between CASPR1/CNTN1 and Nfasc155 (**** P<0.0001 by one-way ANOVA followed by Bonferroni’s post-hoc tests). No significant differences were observed between the effect of monovalent or native IgG4 for both patients. Native anti-CNTN1 IgG4 do not promote CNTN1/CASPR1 antigen clustering (C). Bars represent mean +/- S.E.M. Scale bar: 10 µm.

Figure 3

Mono- and bivalent IgG4 infiltrate paranodal regions. (A) These are teased sciatic nerve fibers from adult Lewis rats which have received a single intraneural injection of native CNTN1 reactive IgG4 (top panels; n = 4 at each time point) or monovalent Fab (lower panels; n = 4 at each time point). Nerves have been collected 1- or 3-days post-injection (dpi) and have been stained for CNTN1 (red) and IgG (green). (B) The percentage of IgG infiltration was measured in each animal at 1 and 3 dpi, and the mean percentage of infiltration was calculated (n = 4 for each condition). The paranodal infiltration of native IgG4 reactive to CNTN1 was significantly higher at 3 dpi compared to 1 dpi (**** P<0.0001, by one-way ANOVA followed by Bonferroni’s post-hoc tests). The percentage of infiltration of monovalent Fab was also significantly higher at 3 dpi compared to 1 dpi (** P<0.01 by one-way ANOVA followed by Bonferroni’s post-hoc tests). No significant difference was seen between monovalent Fab and native IgG4 infiltration at both times. Bars represent mean and S.D. Scale bar: 5 µm.