Haley, Joanna Dawn; (2003) An investigation of the binding of human monoclonal antibodies to autoantigens by the modification and expression of cloned autoantibody cDNA. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Autoantibodies to a wide variety of antigens are associated with Systemic Lupus Erythematosus (SLE). High affinity IgG antibodies to double-stranded DNA (anti-dsDNA) are thought to be particularly closely related to tissue damage and disease activity in SLE. Sequence analysis of murine and human anti-DNA antibodies has shown that these binding properties are derived from the accumulation of replacement mutations and basic residues in the complementarity determining regions (CDRs) of such antibodies. This thesis describes the effects of particular sequence motifs, often sites of somatic mutation and/or containing arginine residues, on the binding to dsDNA and other autoantigens of a high affinity IgG anti-dsDNA mAb, B3. Using a transient expression system whole IgG containing B3 heavy chain (VH) paired with different light chain sequences (Vγ), all derived from human germline gene, 2a2 but with different patterns of somatic mutations, were produced. As predicted by computer-generated models, some mutations to VγCDR arginine residues enhanced binding to DNA whilst others blocked DNA binding. These CDR motifs also affect binding to histones and Sm antigen. It was concluded that it is not the presence of arginine residues in the CDRs but their precise position that is important in determining autoantigen-binding affinity. The VH also plays a role as pairing of the same 2a2-derived Vγ with the VH of a different anti-dsDNA antibody, 33.H11, gave reduced ability to bind DNA in comparison to B3VH. To study the pathogenic effects of these sequence motifs, a stable expression system was used to produce two cell lines, one producing wild type B3 and the other producing a B3 variant in which a VγCDR1 arginine critical to DNA binding had been reverted to a serine. The quantities of IgG produced were sufficient for such assays and also showed the same DNA binding affinities as the transient system IgG.

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