Anticardiolipin (aCL) autoantibodies are associated with thrombosis, recurrent fetal loss, and thrombocytopenia. Only aCL found in autoimmune disease require the participation of the phospholipid binding plasma protein ␤2 glycoprotein I (␤2GPI) for antibody binding and now are called anti-␤2GPI. The antigenic specificity of aCL affinity purified from 11 patients with high titers was evaluated in an effort to better understand the pathophysiology associated with aCL. Seven different recombinant domain-deleted mutants of human ␤2GPI, and full length human ␤2GPI (wildtype), were used in competition assays to inhibit the autoantibodies from binding to immobilized wild-type ␤2GPI. Only those domain-deleted mutants that contained domain 1 inhibited the binding to immobilized wild-type ␤2GPI from all of the patients. The domain-deleted mutants that contained domain 1 inhibited all aCL in a similar but not identical pattern, suggesting that these aCL recognize a similar, but distinguishable, epitope(s) present on domain 1.

Autoantibodies against β2-glycoprotein I (β2GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in β2GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole β2GPI containing single point mutations in the first domain. The binding to β2GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40–43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-β2GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-β2GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40–43 contributes to a dominant target epitope.

Growing evidence indicates that the affinity of monoclonal antibodies (mAbs) for CD16 (Fc␥RIII) plays a central role in the ability of the mAb to mediate antitumor activity. We evaluated how CD16 polymorphisms, and mAb with modified affinity for target antigen and CD16, affect natural killer (NK) cell phenotype when CD20 ؉ malignant B cells were also present. The mAb consisted of rituximab (R), anti-CD20 with enhanced affinity for CD20 (AME-B), and anti-CD20 with enhanced affinity for both CD20 and CD16 (AME-D).Higher concentrations of mAb were needed to induce CD16 modulation, CD54 up-regulation, and antibody-dependent cellular cytotoxicity (ADCC) on NK cells from subjects with the lower affinity CD16 polymorphism. The dose of mAb needed to induce NK activation was lower with AME-D irrespective of CD16 polymorphism. At saturating mAb concentrations, peak NK activation was greater for AME-D. Similar results were found with measurement of CD16 modulation, CD54 up-regulation, and ADCC. These data demonstrate that cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism, and they provide further evidence for the clinical development of such mAbs with the goal of improving clinical response to mAb.

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