Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome

Abstract

Objective Previous studies have shown the importance of somatic mutations and arginine residues in the complementarity‐determining regions (CDRs) of pathogenic anti–double‐stranded DNA (anti‐dsDNA) antibodies in human and murine lupus, and in studies of murine antibodies, a role of mutations at position 53 in V_H CDR2 has been demonstrated. We previously demonstrated in vitro expression and mutagenesis of the human IgG1 monoclonal antibody B3. The present study was undertaken to investigate, using this expression system, the importance of the arginine residue at position 53 (R53) in B3 V_H. Methods R53 was altered, by site‐directed mutagenesis, to serine, asparagine, or lysine, to create 3 expressed variants of V_H. In addition, the germline sequence of the V_H3–23 gene (from which B3 V_H is derived) was expressed either with or without arginine at position 53. These 5 new heavy chains, as well as wild‐type B3 V_H, were expressed with 4 different light chains, and the resulting antibodies were assessed for their ability to bind to nucleosomes, α‐actinin, cardiolipin, ovalbumin, β₂‐glycoprotein I (β₂GPI), and the N‐terminal domain of β₂GPI (domain I), using direct binding assays. Results The presence of R53 was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of R53 reduced binding to α‐actinin, ovalbumin, β₂GPI, and domain I of β₂GPI. The combination B3 (R53S) V_H/B3 V_L bound human, but not bovine, β₂GPI. Conclusion The fact that the R53S substitution significantly alters binding of B3 to different clinically relevant antigens, but that the alteration is in opposite directions depending on the antigen, implies that this arginine residue plays a critical role in the affinity maturation of antibody B3.