Development of anti-drug antibodies following intravitreal administration of anti-C3b Fabs in cynomolgus monkeys and rabbits

Abstract

Purpose : Antibodies were designed to target neo-epitopes on C3b, in order to inhibit the C3/C5 convertases of the complement pathway. In vivo effects of the antibodies were characterized in cynomolgus (cyno) monkeys and rabbits after intravitreal (IVT) administration.

Methods : Human Fab antibodies (NVS1 and NVS2), which are specific for C3b, were generated using phage-display technology. The selection criteria was based on assessment of potency in complement functional assays, affinity, and selectivity for C3b. Anti-C3b antibodies (50μL/eye) were intravitreally injected into cyno monkeys (n=4; 1 and 5 mg/eye) and rabbits (n=12; 0.5 and 5 mg/eye). Eyes were analyzed by OCT, cytospin, histology, and measured for complement activation and cytokine production.

Results : Anti-C3b antibodies bound neo-epitopes found on C3b/iC3b/C3c with high affinity (human: 4-20pM, cyno: 45-125pM), did not cross-react with C3 or C5, and were potent inhibitors of the alternative complement pathway by blocking binding to Factor B. A single IVT administration of anti-C3b Fabs at 5 mg/eye produced severe inflammation in cyno monkeys and rabbits. Anti-drug antibodies (ADA) were detected in the serum of all dosed animals 14 days after injection. Animals dosed at 5 mg/eye also exhibited hyper-reflective features in the vitreous, retinal degeneration, inflammatory cell infiltrates, cytokine production, and intraocular complement activation. Severity of the ocular toxicity correlated with high titers of ADA in the vitreous. Notably, none of the eye pathologies, including ocular ADA, were observed in the untreated contralateral eye. In vitro, serum from monkeys with high ADA titers was able to initiate classical pathway activation in response to drug.

Conclusions : Antibodies selective for C3b were potent in vitro inhibitors of the alternative pathway of complement. However, in vivo administration of Fabs resulted in severe intraocular complement activation. The binding of the C3b:Fab complex to antigen presenting cells may have resulted in the generation of an immune response against the Fabs. Binding of anti-drug antibodies to C3b:Fab complexes resulted in activation of complement through the classical pathway, and contributed to the retinal inflammation observed in vivo.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.