Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

Abstract

Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed “inhibitors”. A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2–5 µg/ml (∼1–33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG4 antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG1, IgG4, IgG1 & IgG4, and IgG1, IgG2 & IgG4. An IgG1-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects’ immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40–80% of FVIII-specific antibodies in most samples were directed against this domain.

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

“…69 Because they measure both inhibitory and non-inhibitory antibodies, they are not equivalent to and cannot be substituted for functional inhibitor assays. They may be used to screen specimens for those requiring inhibitor assays 65 or to confirm the presence of specific antibodies.…”

Role of B Cells in Breaking and Maintaining Tolerance to Clotting Factor VIII in Congenital and Acquired Hemophilia A

“…The majority of patients with severe inhibitors develop a polyclonal response consisting of primarily IgG1 and IgG4 subclasses of antibody that inactivate FVIII via a type I pattern with linear, second order kinetics [10]. A recent study using sensitive surface plasmon resonance (SPR) to investigate subclass specific binding to FVIII demonstrated that early anti-FVIII immune response in AH patients is characterized by primarily IgG1 while the IgG4 subtype is produced later, suggesting the gradual development of a polyclonal response caused by repeated doses of antigen over time [13]. Interestingly, epitope specificity does not appear to correlate with specific isotypes, rather, it has been suggested that IgG class switching occurs after epitope specificity is determined [13,14].…”

“…Despite the difference in pathological outcome, anti-FVIII auto-antibodies isolated from patients with acquired hemophilia appear to be remarkably similar to allo-antibodies directed against FVIII. The polyclonal response is also characterized by IgG1 and IgG4 subclasses, and these share the same epitopes as anti FVIII allo-antibodies [13,16,17]. It has been suggested that the differences in FVIII binding kinetics and the manifestation of bleeding might be related to a preponderance of antibody specific for one FVIII epitope, rather than the mix of many epitopes observed in patients with congenital hemophilia that develop inhibitors [13,16].…”

“…The polyclonal response is also characterized by IgG1 and IgG4 subclasses, and these share the same epitopes as anti FVIII allo-antibodies [13,16,17]. It has been suggested that the differences in FVIII binding kinetics and the manifestation of bleeding might be related to a preponderance of antibody specific for one FVIII epitope, rather than the mix of many epitopes observed in patients with congenital hemophilia that develop inhibitors [13,16]. It is also possible that a much larger plasma cell population exists in patients with acquired hemophilia.…”

“…Experimental evidence strongly suggests this repopulation must occur concurrent with physiologically relevant levels of FVIII [68]. In patients whose inhibitor returned following rituximab treatment, the newly synthesized antibodies displayed changes in subclass distribution and epitope specificity compared to pre-rituximab antibodies, suggesting that they were derived from B cells that repopulated the periphery postrituximab treatment, and not generated from an existing pool of long-lived plasma cells that escaped deletion [13,14]. Hence, lack of a long-term response to rituximab might represent a failure to re-establish peripheral tolerance to FVIII, rather than incomplete depletion of specific memory B cells.…”