Single amino acid substitutions in V(H) CDR2 are sufficient to generate or enhance the specificity of two forms of an anti-arsonate antibody variable region for DNA.
We previously showed that a variety of amino acid substitutions at positions 58 and 59 in the V(H) CDR2 of an anti-arsonate (Ars) antibody Fab simultaneously resulted in increased or unaltered affinity for Ars and substantially enhanced affinity for DNA. To test the generality of these observations, we generated and characterized several antibody phage display libraries of this Fab containing random amino acid substitutions at V(H) CDR2 position 55. Position 55 was randomized in two contexts; in the unmutated V region, and in a previously isolated V(H) CDR2 position 58 and 59 mutant that displayed binding to both Ars and ssDNA. In the unmutated V region context, mutants that displayed strong binding to both Ars and DNA nearly exclusively contained Arginine residues at position 55. In the context of the 58 and 59 mutations, a variety of amino acid residues were observed at position 55 among mutants that bound strongly to both Ars and DNA, including Arginine, Lysine and Serine. None of these position 55 mutations measurable altered affinity for Ars. These data substantiate the view that "dual reactive" antibodies--specific for both a foreign and an autoantigen--are frequently generated in vivo via hypermutation during immune responses driven by the foreign antigen.
Department of Microbiology and Immunology and the Kimmel Cancer Institute, Thomas Jefferson Medical College, Philadelphia, PA 19107, USA.
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D019943:Amino Acid Substitution / Q000276:immunology*; D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics*; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000276:immunology; D004247:DNA / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*
D000974:Antibodies, Antinuclear; D001323:Autoantibodies; D001324:Autoantigens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D004247:DNA