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

Abstract

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.

Incidence and Risk factors of Intraocular Inflammation after Brolucizumab Treatment in Japan: A Multicenter AMD Study

“…Some of these factors include stability issues with brolucizumab, immune responses to the resulting substance, preexisting antidrug antibody responses, and immune complexes (drug/antidrug antibody) that may be associated with endothelial activation and/or induce cytokine release and platelet aggregation. 28 The incidence of IOI is presumably higher in women because of the sex difference in inflammatory stress mechanisms and in the functioning and response of the immune system. 29 This leads to variations in how males and females react to infections and vaccinations.…”

Reducing Immunogenicity by Design: Approaches to Minimize Immunogenicity of Monoclonal Antibodies

“…For example, the immunogenicity of brolucizumab, an antibody used for the treatment of neovascular age-related macular degeneration, resulted in ADAs that were associated with the development of retinal vasculitis/retinal vascular occlusion (RV/RO) in some patients. Formation of immune complexes with brolucizumab is a proposed mechanism for RV/RO induced in some patients through cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release [ 15 ].…”

“…Although epitope mapping to determine ADA binding is challenging due to the polyclonal nature of ADAs, knowledge of potential B cell epitopes can be used to reduce the immunogenicity of future protein therapeutics. For example, a short amino acid sequence within brolucizumab similar to that of a bacterial protein antigen was identified through a peptide-scanning approach [ 15 ]. This suggests that the ADA response toward brolucizumab was due to preexisting antibodies against this bacterial epitope.…”

“…This suggests that the ADA response toward brolucizumab was due to preexisting antibodies against this bacterial epitope. This result emphasized the need for considering sequence similarity to bacterial proteins in the design of protein therapeutics [ 15 , 121 ]. Additionally, it demonstrated the importance of epitope mapping for a better understanding of ADA targets and for better design of mAbs for therapy.…”

Layer-by-Layer Microneedle-Mediated rhEGF Transdermal Delivery for Enhanced Wound Epidermal Regeneration and Angiogenesis

“…Moreover, after treatment with this LBL MN-rhEGF patch, the wound bed contracted in good condition and the intact epithelium layer formed. In addition, CD31, known as platelet endothelial cell adhesion molecule-1 (PECAM-1), 47 is a biomarker of vascular endothelial differentiation and becomes employed to indicate the existence of endothelial tissue and evaluate angiogenesis. 48 The immunohistochemical results of CD31 (Figure 6b) showed that treatment methods of all three groups induced the formation of blood microvessels from the wound tissue of the rats back at different degrees.…”