A root cause analysis to identify the mechanistic drivers of immunogenicity against the anti-VEGF biotherapeutic brolucizumab.

Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.

An Afucosylated Anti-CD32b Monoclonal Antibody Induced Platelet-Mediated Adverse Events in a Human Fcγ Receptor Transgenic Mouse Model and Its Potential Human Translatability.

To assess the safety and tolerability of NVS32b, a monoclonal, afucosylated, anti-CD32b (FCGR2B) antibody, we used a humanized transgenic (Tg) mouse model that expresses all human Fc gamma receptors (FCGRs) while lacking all mouse FCGRs. Prior to its use, we extensively characterized the model. We found expression of all human FCGRs in a pattern similar to humans with some exceptions, such as low CD32 expression on T cells (detected with the pan CD32 antibody but more notably with the CD32b-specific antibody), variation in the transgene copy number, integration of additional human genes, and overall higher expression of all FCGRs on myeloid cells compared to human. Unexpectedly, NVS32b induced severe acute generalized thrombosis in huFCGR mice upon iv dosing. Mechanistic evaluation on huFCGR and human platelets revealed distinct binding, activation, and aggregation driven by NVS32b in both species. In huFCGR mice, the anti-CD32b antibody NVS32b binds platelet CD32a via both Fc and/or complementarity determining region (CDR) causing their activation while in human, NVS32b binding requires platelet preactivation and interaction of platelet CD32a via the Fc portion and an unknown platelet epitope via the CDR portion of NVS32b. We deemed the huFCGR mice to be overpredictive of the NVS32b-associated human thrombotic risk.