Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to antiDNA antibodies by alanine scanning

Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to antiDNA antibodies by alanine scanning. affinity was determined by surface plasmon resonance. Enzymelinked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or ALW) which binds to all PL911 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL911 antiDNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to antiDNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal antiDNA antibodies to autoantigensin vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block antiDNA antibody binding to renal tissue. Keywords:antiDNA antibodies, lupus nephritis, phage display library, SLE == Introduction == Among the pathogenic autoantibodies found commonly in the sera of patients with systemic lupus erythematosus (SLE), immunoglobulin (Ig)G antiDNA antibodies attract particular attention for their key role in the pathogenesis of lupus nephritis1. AntiDNA antibodies contribute to renal injury by indirect binding to the glomerular basement membrane mediated by DNA/nucleosomes or by direct binding to glomerular antigens, the latter of which is mediated by crossreactivity2. The crossreaction between antiDNA IgGs and particular selfantigens may explain the specificity of tissue damage in lupus nephritis. Besides complement activation, binding of antiDNA antibodies to live cells can modulate gene expression and cell metabolism, enhance cellular proliferation and induce phenotypical changes which are found in glomerular resident cells in lupus kidneys3,4,5,6,7. Therefore, blocking the crossreaction of antiDNA IgGs with glomerular antigens may be a valuable approach to the management of lupus nephritis initiated and/or perpetuated by circulating autoantibodies. Previously, using classswitchingin vitro, we generated IgG1, IgG2a and IgG2b versions of the murine monoclonal HDAC5 PL911 IgG3 antiDNA antibody8, which all share an identical light chain and heavy chain Hydroflumethiazide variable region, but differ from each other in the heavy chain constant region (i.e. isotype)9. Surprisingly, we identified significant differences in both antigenic specificity and renal pathogenicity between these PL911 isotypes, due to the effects of the different constant regions on antigenantibody interactions10. These findings may also explain why certain subclasses of lupus antiDNA antibodies are associated more closely with pathogenic potential11,12. Thus, the blocking of multiple antiDNA antibody subsets, especially highaffinity or more pathogenic isotypes, may be a worthy therapeutic goal. Screening phage display libraries with antibodies is a useful Hydroflumethiazide method for selecting peptides that mimic the antigenic partners with which these antibodies interact. Using pathogenic antiDNA antibodies as the bait and isolating peptide Hydroflumethiazide mimics by a phage display approach has been suggested as a promising approach for identifying novel therapies that can protect target organs from antibodymediated injury in SLE13,14,15,16,17,18,19,20,21. Here, we screened a phage peptide display library with each of the four isotypes of the pathogenic PL911 antiDNA antibody. Our goals were to discover Hydroflumethiazide both common and isotype specific dsDNA mimics, and identify peptides that would inhibit the crossreaction of lupus autoantibodies with DNA and glomerular antigens. == Materials and methods == == Scanning of a phage display library == Monoclonal antibodies derived from the PL911 parent clone, having differential affinity to dsDNA (IgG3 > IgG2a > IgG1 > IgG2b), were purified from culture supernatants, as described9. The Ph.D.12phage display library (New England Biolabs, Ipswich, MA, USA) was used for peptide selection22. Bound phages selected by the members of the PL911 antibody panel were isolated (11 distinct phages by IgG1, 19 phages by IgG2a, 18 phages by IgG2b and 12 phages by IgG3; Supporting information, Figure S1). From these phage clones, a peptide with the sequence of ALWPPNLHAWVP (abbreviated as ALW) was chosen for further study as it was the sequence isolated most frequently by all four isotypes. Biotinlabelled or unlabelled ALW peptides and two sizeidentical peptide controls (P1 peptide, SPNQHTPPWMLK; PLP peptide, PLPHNPWVLAAW, scrambled randomly from ALW) were synthesized at the Proteomics Resource Center of Rockefeller University (NY, USA). PLP was used as the primary control peptide, as this peptide is identical in amino acid composition (but not in sequence) and length to ALW. P1 is the same length as ALW, and also binds to an irrelevant IgG3 monoclonal antibody (clone 3E5).