An investigation of the importance of somatic mutations and basic residues in the binding of antiphospholipid antibodies to cardiolipin
Sequence analysis of human antiphospholipid antibodies (aPL) has shown that their binding properties are derived from the accumulation of replacement mutations and basic residues in their complementarity determining regions (CDRs). This thesis describes the effects of particular CDR motifs, often sites of somatic mutation and/or containing arginine residues, on the binding to cardiolipin (CL) of two human IgG monoclonal aPL, IS4 and CL24. Following sequence analysis of IS4 and CL24 a transient expression system was used to express whole IgG containing variants of IS4 and CL24 produced by chain shuffling experiments with three other monoclonal antibodies, UK4, B3 and 33H11. The light chain sequences (VL) of four of these five mAbs were encoded by the same germline VL gene and hence differed only in their pattern of somatic mutations. The heavy chain (Vh) of IS4 was dominant in conferring the ability to bind CL in a direct ELISA, whilst the identity of the paired Vl was important in determining the strength of CL binding. Computer-generated models revealed surface exposed arginine residues in the CDRs of IS4Vh and those VL sequences that were particularly favourable for binding to CL. Seven new Vl sequences and six new Vh sequences were produced by using CDR exchange and site-directed mutagenesis to alter the patterns of somatic mutation and arginine residues in the wild-type VL and Vh sequences of these antibodies. Alteration of specific arginine residues in B3VL CDR1 and IS4VH CDR3 led to a significant reduction in CL binding. It was concluded that it is not just the presence but precise locations of specific arginine residues in the CDRs of pathogenic aPL, which are important in determining binding affinity for CL. In order to produce larger quantities of these expressed Vh/Vl combinations, a stable expression system was developed. Three stable cell lines were produced, expressing IS4VH with three different Vl sequences. The IgG produced showed the same CL binding characteristics as those produced in the transient system.