Bacterial expression of a single‐chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies

Abstract

Monoclonal antibodies (mAb) against the main immunogenic region (MIR) of the acetylcholine receptor (AChR) are very potent in inducing antigenic modulation of the AChR in animals and in muscle cell cultures. A recombinant antibody fragment of the rat anti-MIR mAb198 was cloned by polymerase chain reaction and expressed as soluble single-chain Fv fragment (scFv198) in E. coli and affinity purified. DNA sequencing was used to define the VH (IB) and VL (K2) chain gene usage. scFv198 was found immunologically and biologically active. Its binding affinity for the Torpedo AChR (KD = 2 +/- 0.6 nM) was very similar with that of the intact mAb198 (KD = 1.8 +/- 0.6 nM) while for the human AChR (KD = 80.7 +/- 16.6 nM) it was about four times lower than that of the intact mAb198 (KD = 21.6 +/- 6.6 nM). This fragment was capable of efficiently protecting the AChR in human cell cultures, against antigenic modulation caused by the intact mAb198 or by the antibodies from a myasthenic patient. The produced scFv198 fragment is, therefore, potentially useful in therapeutic applications for myasthenia gravis after appropriate genetic manipulations.

Crystallization and preliminary crystallographic study of an Fab fragment of a pathogenic rat monoclonal antibody against the nicotinic acetylcholine receptor

“…functional single chain Fv fragment has been constructed and expressed in Escherichia coli (Mamalaki et al, 1993).…”

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

“…The prominent pathogenicity of anti-MIR mAb has been extensively documented by in vitro and in vivo studies (reviewed in [6]). It has been shown that univalent anti-MIR antigenbinding fragments (Fab) or scFv antibody fragments can efficiently inhibit a large portion of autoantibodies from binding to the AChR [7,8]. Furthermore, Fab and scFv fragments can efficiently protect the receptor against antigenic modulation induced by anti-MIR mAb or MG sera [7,8] and thus are good candidates for a therapeutic approach.…”

“…It has been shown that univalent anti-MIR antigenbinding fragments (Fab) or scFv antibody fragments can efficiently inhibit a large portion of autoantibodies from binding to the AChR [7,8]. Furthermore, Fab and scFv fragments can efficiently protect the receptor against antigenic modulation induced by anti-MIR mAb or MG sera [7,8] and thus are good candidates for a therapeutic approach. However, the immunogenicity of rodent antibodies in humans prevents their application in clinical procedures.…”

Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies

“…hAChR antigenic modulation and protection on CN21 cells hAChR antigenic modulation and protection were performed as described [8]. CN21 cells grown to confluency in 48-well tissue culture plates were incubated, in the presence of 40 lg/ ml cycloheximide, first with 10 lg/ml of anti-hAChR Fab, or the negative control Fab TT [26], or culture medium, and then with either 10 lg/ml of goat anti-human F(ab') 2 antibody or an MG serum dilution resulting in 90% of maximal antigenic modulation in the absence of competitor.…”

“…Analysis of the isolated antibody clones would also extend existing information on the human anti-AChR autoreactive repertoire. Anti-MIR antibody fragments have been previously used to protect AChR from pathogenic autoantibody action [7][8][9]. However, the isolation of a panel of AChR "protectors" directed against the most dominant immunogenic AChR epitopes is probably required for the development of an efficient therapeutic pool.…”