The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library

Abstract

Molecular cloning techniques and V gene phage display have revolutionised the production of human monoclonal antibodies. Antibodies of a defined specificity can be obtained by selecting phage display libraries on antigen in a process known as panning. We have applied these techniques to the isolation of three HLA-A2-specific single chain variable domain fragments (scFv) from a patient alloimmunised by blood transfusion. Analysis of specificity with cells of HLA genotyped donors revealed the following: i) in addition to the major reactivity with HLA-A2, cross-reactivity with the HLA-A28 epitope; and ii) inhibition of scFv binding to the antigen by the patients' antibodies. The heavy chain variable genes of all three were derived from the germline gene Cos-3, carry the hallmarks of somatic hypermutation, and are most likely derived from clonally related B cells. The light chain variable domains were encoded by DPK1 and DPK8 from the VkappaI family. These data show that phage display can be used to clone HLA-specific alloantibodies that recognise the native antigen from alloimmunised patients.