Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.     

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.     

Design, synthesis, and conformational study of biologically active photolabeled analogues of the main immunogenic region of the acetylcholine receptor

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding to acceptor molecules, which provides important insights for mapping the bimolecular interfaces. The autoimmune disease myasthenia gravis is caused by autoantibodies against the acetylcholine receptor (AChR). The majority of the anti-AChR antibodies bind to the "main immunogenic region" (MIR) of the AChR. To identify the contact points between the complementarity determining regions of the anti-MIR antibodies that recognize the MIR contact sites of the AChR, we present here three photoreactive dodecapeptide MIR analogues containing the photolabel p-benzoyl-L-phenylalanine (Bpa) moiety, either in position 1 or 11. The structure of the produced 12-mers was analyzed using two-dimensional (1)H-NMR spectroscopy, whereas their binding to anti-MIR monoclonal antibodies (mAbs) was determined by immunochemical assays. In all cases the modifications resulted in conservation of the beta-turn conformation of the N-terminus, which has been proved essential for antibody recognition and increased anti-MIR binding relative to the MIR decapeptide.     

Antigenic modulation of human myotube acetylcholine receptor by myasthenic sera. Serum titer determines receptor internalization rate

Antibodies to the acetylcholine receptor (AChR) added to AChR-bearing muscle cells cross-link the receptors, thus increasing their internalization and degradation rate (antigenic modulation). This mechanism contributes to AChR loss in myasthenia gravis. Until recently, antigenic modulation has been studied in animal tissues, where only a small fraction of human anti-AChR antibodies bind. In the present study, we examined the antigenic modulation of AChR by using patients' sera and cultures of human muscle cells. We aimed to see whether antigenic modulation correlates better with disease severity or with antibody titer. Antibody-containing sera from 29 myasthenic patients in various states of the disease and with different antibody titers against AChR were tested. Control sera from six healthy individuals were also tested. Our experiments showed that all myasthenic sera affected the overall AChR content on the human myotube surface, causing a 49 to 82% loss, whereas control sera had no effect. Although at fixed serum volumes there was some correlation between disease severity and AChR loss, this effect was clearly due to differences in antibody titers. In fact, the antigenic modulation depended mainly on the final concentration of the antibody present. Thus, intrinsic factors other than antibodies to AChR may determine or influence the patients' susceptibility to the disease.     

Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198

Myasthenia gravis is a neuromuscular disorder caused by an antibody-mediated autoimmune response to the muscle-type nicotinic acetylcholine receptor (AChR). The majority of monoclonal antibodies (mAbs) produced in rats immunized with intact AChR compete with each other for binding to an area of the alpha-subunit called the main immunogenic region (MIR). The availability of a complex between the AChR and Fab198 (Fab fragment of the anti-MIR mAb198) would help understand how the antigen and antibody interact and in designing improved antibody fragments that protect against the destructive activity of myasthenic antibodies. In the present study, we modeled the Torpedo AChR/Fab198 complex, based primarily on the recent 4A resolution structure of the Torpedo AChR. In order to computationally dock the two structures, we used the ZDOCK software. The total accessible surface area change of the complex compared to those of experimentally determined antigen-antibody complexes indicates an intermediate size contact surface. CDRs H3 and L3 seem to contribute most to the binding, while L2 seems to contribute least. These data suggest mutagenesis experiments aimed at validating the model and improving the binding affinity of Fab198 for the AChR.     

Structural analysis of immunotherapeutic peptides for autoimmune Myasthenia gravis

Myasthenia gravis (MG) and its animal model, experimental MG (EAMG), are autoimmune disorders in which major pathogenic antibodies are directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (nAChR). In an earlier attempt to develop peptide mimotopes capable of preventing the anti-MIR-mediated pathogenicity, the peptide Pep.1 was initially identified from phage display, and subsequently, Cyclic extended Pep.1 (Cyc.ext.Pep.1), which incorporates eight additional residues into the Pep.1 sequence and has an affinity for the anti-MIR antibody mAb198 3 orders of magnitude greater than that of Pep.1, was developed. In an animal model, Pep.1 shows no ability to inhibit mAb198-induced EAMG, whereas Cyc.ext.Pep.1 successfully blocks anti-MIR antibody 198 (mAb198)-induced EAMG. Our aim in this study was to identify the structural characteristics related to the different affinities for mAb198 of Pep.1 and Cyc.ext.Pep.1 using NMR spectroscopy and alanine scanning analysis. The NMR structural analysis revealed that Pep.1 is very flexible in solution, whereas Cyc.ext.Pep.1 is highly rigid within a region containing several turn structures. Interestingly, TRNOE experiments revealed that mAb198-bound Pep.1, particularly in the region between Asn7 and Glu11, shows significant structural similarity to the region between Asn10 and Glu14 of Cyc.ext.Pep.1, which is critical for interaction with mAb198. We therefore conclude the higher affinity of Cyc.ext.Pep.1 for mAb198 reflects the fact that incorporation of additional residues producing a single disulfide bond endows Pep.1 with a conformational rigidity that mimics the structure of mAb198-bound Pep.1. Furthermore, our results suggest that cyclic extended peptides could be utilized generally as useful tools to optimize the affinity of phage library-derived peptide antigens.     

Myasthenia Gravis: Effect on Antibody Binding of Conservative Substitutions of Amino Acid Residues Forming the Main Immunogenic Region of the Nicotinic Acetylcholine Receptor

Abstract

In Myasthenia Gravis most anti-acetylcholine receptor (AChR) antibodies are against a highly conserved area of the AChR α-subunit called the Main Immunogenic Region (MIR). Amino acid residues critical for MIR formation have been located within the sequence α67–76. In the present study, binding of anti-AChR monoclonal antibodies (mAbs) to synthetic peptide analogues of the sequence α67–76 of human and Torpedo AChRs containing conservative single-residue substitutions identified the amino acid residues most important to the antigenicity of the MIR sequence, and offered clues to its tridimensional structure.

Conservative substitutions of residues Asn₆₈ and Asp₇₁ greatly diminished mAb binding, identifying them as critical contact residues for anti-MIR mAbs. Substitutions at Asp₇₀ and Tyr₇₂ moderately affected binding. Cross-reactive mAbs originally raised against Electrophorus AChR bound single residue-substituted synthetic peptides in a manner consistent with the possibility that Electrophorus AChR may have a glutamic acid residue at position α70 or α71. Substitutions at residues Asp/Ala₇₀ and Val/Ile₇₀ between human and Torpedo α-subunits may be size-compensating, suggesting these amino acids in the native AChR may be in closer proximity than proposed in previous models of the MIR.     

Prevention of passively transferred experimental autoimmune myasthenia gravis by an in vitro selected RNA aptamer

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are mainly caused by autoantibodies directed against acetylcholine receptors (AChR) located in the postsynaptic muscle membrane. Previously, we isolated an RNA aptamer with 2'-fluoropyrimidines using in vitro selection techniques that acted as an effective decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and a significant fraction of patient autoantibodies with MG. To investigate the therapeutic potential of the RNA, we tested the ability of the RNA aptamer to protect the receptors in vivo from mAb198. Clinical symptoms of EAMG in rats engendered by passive transfer of mAb198 were efficiently inhibited by a truncated RNA aptamer that was modified with polyethylene glycol, but not by control scrambled RNA. Moreover, the loss of AChR in the animals induced by the antibody was also significantly blocked with the modified RNA aptamer. These results suggested that RNA aptamers could be applied for antigen-specific treatment for autoimmune diseases including MG.     

The third-dimensional structure of the complex between an Fv antibody fragment and an analogue of the main immunogenic region of the acetylcholine receptor: a combined two-dimensional NMR, homology, and molecular modeling approach

Binding of autoantibodies to the acetylcholine receptor (AChR) plays a major role in the autoimmune disease Myasthenia gravis (MG). In this paper, we propose a structure model of a putative immunocomplex that gives rise to the reduction of functional AChR molecules during the course of MG. The model complex consists of the [G(70), Nle(76)] decapeptide analogue of the main immunogenic region (MIR), representing the major antigenic epitope of AChR, and the single chain Fv fragment of monoclonal antibody 198, a potent MG autoantibody. The structure of the complexed decapeptide antigen [G(70), Nle(76)]MIR was determined using two-dimensional nmr, whereas the antibody structure was derived by means of homology modeling. The final complex was constructed using calculational docking and molecular dynamics. We termed this approach "directed modeling," since the known peptide structure directs the prestructured antibody binding site to its final conformation. The independently derived structures of the peptide antigen and antibody binding site already showed a high degree of surface complementarity after the initial docking calculation, during which the peptide was conformationally restrained. The docking routine was a soft algorithm, applying a combination of Monte Carlo simulation and energy minimization. The observed shape complementarity in the docking process suggested that the structure assessments already led to anti-idiotypic conformations of peptide antigen and antibody fragment. Refinement of the complex by dynamic simulation yielded improved surface adaptation by small rearrangements within antibody and antigen. The complex presented herein was analyzed in terms of antibody-antigen interactions, properties of contacting surfaces, and segmental mobility. The structural requirements for AChR complexation by autoantibodies were explored and compared with experimental data from alanine scans of the MIR peptides. The analysis revealed that the N-terminal loop of the peptide structure, which is indispensable for antibody recognition, aligns three hydrophobic groups in a favorable arrangement leading to the burial of 40% of the peptide surface in the binding cleft upon complexation. These data should be valuable in the rational design of an Fv mutant with much improved affinity for the MIR and AChR to be used in therapeutic approaches in MG.     

Improvement of RNA aptamer activity against myasthenic autoantibodies by extended sequence selection.

Myasthenia gravis (MG) is mainly engendered by autoantibodies directed against acetylcholine receptors (AChRs) located in the postsynaptic muscle cell membrane. Previously, we isolated an RNA aptamer with 2'-amino pyrimidines using in vitro selection techniques that acted as a decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and patient autoantibodies with MG (1). However, low affinity of this RNA to mAb198 relative to that of AChR might limit potential of the RNA as an inhibitor of the autoantibodies. To improve decoy activity of the RNA aptamer against autoantibodies, here we employed in vitro selection methods with RNA libraries containing extra random nucleotides extended to the 3' end of previously selected RNA sequences. RNAs isolated in this study showed significant increases in the binding affinities to mAb198 as well as bioactivities protecting AChRs on human cells from both mAb198 and patient autoantibodies, compared with the previous RNA aptamers. These results have important implications for the development of antigen-specific modulation of autoimmune diseases including MG.     

Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponin-permeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltage-dependent sodium channel alpha-subunit. This may represent a general pattern for multisubunit membrane proteins during development.     

Direct Proof of the In Vivo Pathogenic Role of the AChR Autoantibodies from Myasthenia Gravis Patients

Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG.     

Decrease in acetylcholine-receptor content of human myotube cultures mediated by monoclonal antibodies to alpha, beta and gamma subunits

One of the two main causes of acetylcholine-receptor loss in myasthenia gravis is antigenic modulation, i.e. accelerated internalization and degradation rate by antibody-crosslinking. This phenomenon has been studied only in animal tissues. Therefore, we tested antigenic modulation of the acetylcholine receptor on human embryonic myotubes in cultures. Several monoclonal antibodies to the alpha, beta and gamma subunits of the receptor reduced its concentration, in some cases down to one-third of the control. Some of these antibodies only form complexes of one antibody with two receptor molecules; consequently such small complexes are sufficient to accelerate internalization of the human acetylcholine receptor. This technique might be proved valuable for clinical screening of sera from myasthenic patients.     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies

In myasthenia gravis a highly conserved area of the nicotinic receptor (AcChR) dominates the autoantibody response (main immunogenic region, MIR), and it is formed by residues within the sequence segment 67-76 of the AcChR alpha-subunit. We have studied the binding of eight anti-MIR mAb to synthetic peptides containing the sequence segment 67-76 of the human alpha-subunit, and peptide analogues containing single residue substitutions of this sequence. We used also a peptide where both Asp70 and Asp71 were substituted by glycine residues. The binding of six anti-MIR mAb was strongly influenced by several substitutions. All these mAb required residues Asn68, and Pro69 for binding. Five of them required also Asp71 and Tyr72. Substitution of Asp70, which is an Ala residue in Torpedo AcChR, was irrelevant for the binding of an anti-Torpedo and an anti-Electrophorus mAb, and moderately reduced the binding of an anti-human mAb (no. 203). Substitution of Trp67 moderately reduced the binding of some of these mAbs. A mAb of this group (the antihuman mAb no. 198) bound in a manner only slightly influenced by ionic strength, whereas the binding of the other five mAb of this group was very sensitive to the ionic strength. Two anti-Electrophorus MIR mAb bound similarly to all peptide analogues in low ionic strength. At high ionic strength only the peptide analogue where Asp 70 was changed to a Gly residue bound significantly. This may indicate that the Electrophorus MIR has an uncharged residue at this position, as does Torpedo AcChR. Residues at position 73, 74, 75, and 76 were of little or no importance for the binding of all anti-MIR mAb. A free amino terminus was essential for the binding of most mAb. The results of competition experiments between different peptides and native AcChR for mAb binding were consistent with those obtained in direct binding experiments.     

Chronic experimental autoimmune myasthenia gravis induced by monoclonal antibody to acetylcholine receptor: biochemical and electrophysiologic criteria

To determine whether the chronic presence of antibody to acetylcholine receptor (AChR) can account for the neuromuscular abnormalities in myasthenia gravis (MG), rats injected repeatedly with monoclonal antibody (mAb) to AChR were compared with those injected with control mAb. In a previous report, those receiving anti-AChR mAb, studied ultrastructurally, had grossly simplified endplates when compared with normal controls. In this report, animals injected once or chronically for 9 to 12 wk had reduced content of muscle AChR. The chronically injected animals also had diminished miniature endplate potential amplitudes, but to a lesser extent than the reduction in AChR content. These studies establish the pathogenetic role of antibody to AChR in the induction of the ultrastructural, biochemical, and electrophysiologic hallmarks of MG.     

Amino Acid Residues Within the Sequence Region α55–74 of Torpedo Nicotinic Acetylcholine Receptor Interacting with Antibodies to the Main Immunogenic Region and with Snake α-Neurotoxins

Abstract

The sequence region 55–74 of the α-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment—residues α67–76—forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence α55–74 of Torpedo AChR binds α-bungarotoxin (αBTX), suggesting that amino acid residues within this sequence region may contribute to formation of an αBTX binding site.

Using single-residue substituted synthetic analogues of the sequence α55–74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with α-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg₅₅, Arg₅₇, Trp₆₀, Arg₆₄, Leu₆₅, Arg₆₆, Trp₆₇, or Asn₆₈ strongly inhibited α-toxin binding, whereas substitutions of Ile₆₁, Val₆₃, Pro₆₉, Ala₇₀, Asp₇₁, or Tyr₇₂ had marginal effects. Substitutions within the region α68–72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp₆₀ substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp₆₂ slightly but consistently affected binding of mAbs 6 and 22.     

Specificity of monoclonal antibodies against human thyroglobulin; comparison with autoimmune antibodies.

Ten monoclonal antibodies (mAb) directed against human thyroglobulin (hTgb) were produced, purified and characterized. The mAb avidity for hTgb ranged from 10(-10) to 10(-6) M. The species specificity of the mAb was as follows: eight mAb reacted with monkey Tgb, three with dog Tgb and one with pig Tgb; none with bovine and ovine Tgb. The binding of mAb to hTgb was not significantly inhibited in the presence of Tgb carbohydrate moieties, tyrosine, iodotyrosines and iodothyronines. The topology of the antigenic determinants recognized by the 10 mAb on hTgb was explored by inhibition of Tgb binding of radiolabeled mAb by the other antibodies. Six distinct clusters of reactivity were described. Localization of the antigenic determinants recognized by mAb on hTgb was attempted using tryptic fragments of hTgb to inhibit the binding of mAb to hTgb. The inhibitory effect of hydrolysis products was different for each mAb but exhibited partial analogies between mAb of the same cluster of reactivity. Anti-hTgb autoimmune antibodies (aAb) purified from sera of Graves patients cross-reacted essentially with mAb of one out of the six clusters. These results demonstrate that the large number of antigenic determinants presented by the hTgb are not disseminated on the molecule but are clustered in antigenic regions. Furthermore, from the six antigenic regions evidenced in this paper, only one is involved in autoimmune antibody production in Grave's disease.     

Paratope- and framework-related cross-reactive idiotopes on anti-acetylcholine receptor antibodies

Cross-reactive idiotopes are a possible target for therapeutical interventions in autoimmune diseases. To investigate their role in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) we analyzed the Id of rat anti-AChR mAb 6, 35, 61, 65 and a control myeloma protein IR27. Anti-Id 6, 35, 61, 65 bound in a direct binding assay with various affinity to all rat anti-AChR mAb that were tested. Anti-Id IR27 recognized none of the anti-AChR mAb. The specificity of these crossreactions was confirmed by inhibition studies with anti-AChR mAb and two control rat myeloma proteins (IR27 and IR241). In addition, the Id expression on mAb D6, a mouse anti-human AChR mAb was recognized by anti-Id 6, 35, and 65. Anti-Id, except anti-Id IR27, bound to affinity purified IgG from the sera of rats with EAMG, but not to preimmune Lewis IgG. These results suggest extensive sharing of idiotopes among anti-AChR mAb, which are also present in EAMG serum. Anti-AChR mAb against the main immunogenic region (6, 35, 65) from different rat strains, shared at least one paratope-related cross-reactive idiotopes. In the view of the fact that anti-main immunogenic region antibodies might form a predominant fraction of the polyclonal response against AChR, it is conceivable that an anti-Id recognizing these antibodies could have therapeutical applications as for example a selective immune absorbent or in immunotoxin therapy.