Bacterial expression of a single-chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies.
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.
Mamalaki
A
A
Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
Trakas
N
N
Tzartos
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D009157:Myasthenia Gravis / Q000276:immunology*; D051381:Rats; D011950:Receptors, Cholinergic / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D004247:DNA