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

Abstract

In the autoimmune disease myasthenia gravis (MG), antibodies against the muscle nicotinic acetylcholine receptor (AChR) cause loss of functional AChR in the neuromuscular junction. To isolate AChR‐specific human antibody fragments (Fab), a phage‐display library was constructed from an MG patient's thymic B lymphocytes. The first Fab isolated had a low affinity for human AChR, but two sequential antibody chain shufflings using the MG donor heavy and light chain gene repertoires resulted in isolating two new Fab with an approximately 30‐fold higher binding ability. The selected Fab contained extensively mutated heavy and light chains and probably represent intraclonal variants of a common progenitor having diverged in vivo by somatic hypermutation. Interestingly, the isolated Fab bound to an extracellular highly immunogenic region located either on an α‐subunit site affected by the γ/ϵ‐subunits or on the interface between α‐ and γ/ϵ‐subunits. This region is not the previously described "main immunogenic region" (MIR), although it seems to be close to it, as one improved Fab and an anti‐MIR mAb competed for AChR binding with distinctly different subpopulations of MG sera. Furthermore, this Fab protected surface AChR in cell cultures against MG autoantibody‐induced antigenic modulation, suggesting a potential therapeutic use in MG, especially in combination with a human anti‐MIR Fab.

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

“…Several anti-MIR human Fab fragments were isolated by phage display antibody libraries constructed from thymic B lymphocytes of MG patients [10,11] and more recently, human Fab were isolated against an extracellular highly immunogenic epitope located close to the MIR [12]. It would be interesting to investigate the precise location of these two immunogenic epitopes with regard to the MIR region as well as the abundance of MG autoantibodies against these epitopes.…”

“…Despite the different techniques for human mAb production that have evolved during the last years, only a very limited number of reports concern high-affinity AChR-protective human antibodies, and most are directed against the extracellular domain of the asubunit [10][11][12]. All these antibodies have been isolated from phage display human antibody libraries.…”

“…However, the immunogenicity of rodent antibodies in humans prevents their application in clinical procedures. To overcome this problem, a humanized scFv antibody fragment was constructed [9], and several human Fab, mainly against the AChR asubunit, were isolated from MG thymic lymphocytes by phage display libraries [10][11][12]. Transgenic mice expressing human Ig genes provide an alternative method for isolating human mAb of potential therapeutic value [13][14][15][16].…”

The Use of Phage Display in Neurobiology

“…This work has been most extensively carried out with thyroid disease (Portolano et al, 1993(Portolano et al, , 1995McIntosh et al, 1996McIntosh et al, , 1997aMcIntosh and Weetman, 1997;Kakinuma et al, 1997), with the antibodies selected from such libraries having similar specificities to those found in patients' serum and with the high affinities characteristic of immune libraries. Similar experiments have been carried out with systemic lupus erythematosis (Barbas et al, 1995;Marchbank and Deutscher, 1995;Roben et al, 1996), celiac disease (Sblattero et al, , 2002(Sblattero et al, , 2004Marzari et al, 2001), Sjogren's syndrome (Suzuki et al, 1997;Maruyama et al, 2004), paraneoplastic encephalomyelitis (Graus et al, 1998a), diabetes , and myasthenia gravis (MG) (Farrar et al, 1997;Graus et al, 1997Graus et al, , 1998bRey et al, 2000;Fostieri et al, 2005). The antibodies isolated from patients with MG recognized the AChR in or close to the major immunogenic region, a finding confirmed by the fact that two of the selected antibodies were able to inhibit donor serum anti-AchR antibodies as well as those in unrelated MG patients.…”

Guidelines for pre-clinical assessment of the acetylcholine receptor-specific passive transfer myasthenia gravis model—Recommendations for methods and experimental designs

“…The sequences of a few AChR specific antibodies have been published (Graus et al, 1995; Farrar et al, 1997; Graus et al, 1997; Matthews et al, 2002; Fostieri et al, 2005; Vrolix et al, 2014) and these can be used to construct corresponding antibody expression vectors for heterologous production.…”