Repertoire cloning of a human IgG inhibitor of αIIBβ3 function. The OG idiotype
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文摘
A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion following the production of IgG antibodies (Ab1) specific for the integrin subunit β3. We generated recombinant VH and VL cDNA libraries using IgG-specific mRNA isolated from OG peripheral blood B-lymphocytes that had been selected for binding to antigen (αIIbβ3 adsorbed to plastic dishes). These antigen-specific B-lymphocytes contain rearranged VH DNA segments that belong exclusively to the VH4 gene family. Recombinant Fab were expressed on the surface of filamentous phage coinfected with VH and VL segments cloned into the phagemid pHEN1 or the phage fd-tet-DOG1. To facilitate selection of the desired recombinant Ab1 Fab, we developed a rabbit polyclonal antibody specific for affinity-purified OG anti-β3 Fab (Ab2). Ab2 reacts specifically with Ab1, and this interaction is inhibited by purified αIIbβ3. Following three rounds of phage selection on Ab2 adsorbed to plastic dishes and random reassociation of heavy and light chains, we isolated Ab1 Fab and tested their binding to αIIbβ3. Five Id-positive Fab were selected for further characterization. These Fab use one of two VH genes (H21 or H23) complexed with one of three Vλ genes. Subsequent sequence data demonstrated that all three lambda genes are the same clone L22 which uses a germline Vλ gene segment. Fab using H23 bind to αIIbβ3, while those using H21 do not. Based on sequence homology, both H21 and H23 use VH gene segments belonging to the VH4 gene family. Thus, the idiotype OG is restricted to the VH4 gene family and is the first sequenced prototype of human antibodies that bind close to or at a functional epitope(s) of αIIbβ3.