Analysis of autoimmune bone marrow by antibody-phage display - Somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes

Objective. To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibod ies from the bone marrow of individuals with systemic lupus erythematosus ( SLE). Methods. A library of single-chain variable fragments (scFv) was constructe d from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotyp e by cloning into the pHENIX phagemid vector. The Library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, an d their heavy (H)- and light (L)-chain recombinants, were prepared, purifie d, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. Results. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respective ly) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of repl acement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both sin gle-stranded DNA and dsDNA. These 2 Ig subunits contained third complementa rity-determining region arginines and had acquired the majority of replacem ent mutations. Conclusion. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patie nts exploit diverse V genes and cationic V-D-J and V-J junctions for DNA bi nding, and accumulate replacement mutations that enhance binding.