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 H 3-23 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, ␣-actinin, cardiolipin, ovalbumin, ␤ 2 -glycoprotein I (␤ 2 GPI), and the N-terminal domain of ␤ 2 GPI (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 ␣-actinin, ovalbumin, ␤ 2 GPI, and domain I of ␤ 2 GPI. The combination B3 (R53S) V H /B3 V L bound human, but not bovine, ␤ 2 GPI.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.

Pathogenic Autoantibodies in Systemic Lupus Erythematosus Are Derived from Both Self-Reactive and Non-Self-Reactive B Cells

“…Only four human IgG anti-dsDNA antibodies from patients with SLE have been analyzed previously and all of these also fail to bind DNA in the germline configuration (7)(8)(9). Since the number of sequenced dsDNA-binding antibodies derived from lupus patients is still very limited compared with those isolated from different autoimmune mouse models, it remains a question whether conclusions drawn from murine models can be applied fully to the human dsDNAantibody repertoire.…”

“…Whether nucleosomes, DNA, or phospholipid antigens released by apoptotic cells, or alternatively, foreign antigens trigger the response is unclear. Back mutation of four human IgG anti-DNA antibodies derived from lupus patients demonstrates that in their germline configuration these antibodies may fail to bind DNA (7)(8)(9). This observation would suggest that polyclonal activation is not the mechanism for the generation of pathogenic autoantibodies and that self-DNA is a critical eliciting antigen.…”

Thrombin Binding Predicts the Effects of Sequence Changes in a Human Monoclonal Antiphospholipid Antibody on Its In Vivo Biologic Actions

“…On the day of the experiment, wells were treated for 4 h at 37°C with 500 g/ml test monoclonal IgG or a control monoclonal IgG (a recombinant mAb with no binding to a number of Ags, previously described in Ref. 31), which lacked aPL reactivity (0 GPLU), or treated with 3 g/ml LPS (Sigma-Aldrich). Cells were then harvested with trypsin-EDTA and centrifuged at 100 ϫ g for 5 min, then incubated for 30 min at 4°C with mouse anti-human mAbs against E-selectin, VCAM-1, ICAM-1, and tissue factor (BD Biosciences).…”

Examining the non-linear relationship between monoclonal antiphospholipid antibody sequence, structure and function

“…74 These studies are relevant to the work described above with IS4, because B3 has cross-reactivity with CL and β 2 GPI. We found that a single change (germline reversion) from Arg to Ser in B3VHCDR2 caused a dramatic Table 1 Overall binding characteristics of the 14 heavy/light chain combinations…”