Determination of Anti-Factor VIIII Antibody Titers, Isotypes and Epitopes by SPR Analysis of Human Plasma

Abstract 202

Development of neutralizing anti-factor VIII (FVIII) allo-antibodies (“inhibitors”) occurs in a significant proportion of congenital Hemophilia A (HA) patients receiving exogenous FVIII, and autoimmune responses to FVIII can also occur. Accurate early detection of these immune responses, including low-titer or non-neutralizing antibodies, could have prognostic value and thus assist clinicians in evaluating therapeutic options. Rapid, sensitive, high-information-content surface plasmon resonance (SPR) assays have been developed to quantify the titer, IgG subtype distribution and domain/epitope specificity of anti-FVIII antibodies by direct analysis of pre-treated plasma samples. Samples were injected directly onto the biosensor of a Biacore T-100 instrument, on which recombinant FVIII was captured via a covalently immobilized anti-FVIII monoclonal antibody (mAb), and the increase in resonance units (RU) was converted to antibody titers. The lower limit of detection was 100ng/mL (∼1nM ∼0.01 Bethesda units/mL), allowing characterization of anti-FVIII antibodies that could not be detected by the Bethesda assay. Subsequent serial injections of isotype-specific anti-human mAbs yielded quantitative measurements of the fractions of anti-FVIII antibodies with specific subtypes. Plasma samples from 200 HA subjects age 2 and above (with and without a measurable inhibitor) as well as samples from 3 autoimmune adults were analyzed. Distinct IgG subtype distributions were observed, with most inhibitor subjects exhibiting mixed IgG₁ + IgG₄, or IgG₁ + IgG₂ + IgG₄ responses. Serial samples from 1 subject following initial FVIII infusions showed transient class switching with the appearance of anti-FVIII-IgG₃.

Epitope mapping was next carried out via competition studies utilizing (a) a panel of 11 monoclonal antibodies (mAbs) whose epitopes on the FVIII-C2 domain have been mapped at high resolution and (b) recombinant FVIII-C2 proteins. The mAb-competition assays allowed the (FVIII-C2) epitope specificity of anti-FVIII antibodies in plasma to be mapped at a similar resolution, identifying specific clusters of surface-exposed FVIII residues that comprise or overlap immunodominant epitope(s) recognized by human antibodies. Complementary solution-phase competition SPR assays utilized wild type FVIII-C2 protein and selected proteins from a panel of 60 FVIII-C2 point mutants (muteins) that were identified during epitope mapping of the mAbs by SPR. This second approach alleviated several technical issues associated with the earlier blocking studies that utilized mAbs to compete the polyclonal plasma IgG (e.g. dissociation of the blocking mAb and steric effects) and provided reproducible quantitative results. Because saturating concentrations of FVIII-C2 proteins were used, changes in the number of RUs measured before and after addition of WT-FVIII-C2 indicated the fraction of polyclonal anti-FVIII antibodies that recognized this domain. Notably, the IgG subtype distribution of all FVIII-C2 specific antibodies reflected the total IgG subtype distribution, indicating that affinity maturation of B-cell epitopes preceded antibody class switching. Interestingly, saturating amounts of either FVIII-C2 or the high-affinity anti-FVIII-C2 mAb BO2C11 completely competed binding of antibodies from 1 subject (with HA genotype A2201P) to FVIII captured on the biosensor surface, indicating that his antibody response was to a single B-cell epitope or else to closely overlapping epitopes. However, although rare subjects had a completely FVIII-C2 specific response or no binding to FVIII-C2, between 30–50% of the anti-FVIII antibodies in the vast majority of inhibitor-positive samples targeted the FVIII-C2 domain. This was true for inhibitor subjects with severe HA due to the intron 22 inversion, for subjects with other HA genotypes resulting in severe HA and for the 3 autoimmune subjects.

Results of these competition SPR assays are directly translatable to our laboratory's efforts to design FVIII muteins that retain adequate pro-coagulant activity but can avoid neutralization by anti-FVIII antibodies in inhibitor patients' blood. FVIII muteins having amino acid substitutions that eliminate immunodominant B-cell epitopes are expected to have potential for development as less immunogenic/antigenic bypass therapies for patients who develop a high-titer inhibitor.