InflaMab: A novel NLRP3 Inflammasome inhibitor tested in a mouse model of glaucoma

Background: Recent evidence shows that activation of the NLRP3 inflammasome plays an essential role in glaucoma and we previously reported that NLRP3 knockout mice were protected from axon degeneration and death of RGCs in a microbead-induced mouse model of glaucoma. These data support pharmacologic inhibition of NLRP3 as a potential neuroprotective therapy in glaucoma and several small molecule inhibitors of NLRP3 have been developed. However, these types of inhibitors often lack specificity and their short half-lives make them a poor candidate for local intravitreal administration, the ideal route of administration for glaucoma. The aim of this study is to characterize the anti-inflammatory effects of a novel biologic that targets the NLRP3 inflammasome and determine whether local administration of this biologic can provide sustained neuroprotection in an inducible mouse model of glaucoma. Methods: InflaMab is a bi-specific antibody that was developed to gain access to the intracellular compartment and target NLRP3. In vitro studies were performed using the THP1 macrophage cell line and murine bone marrow-derived macrophages (BMDMs) to assess the anti-inflammatory potential of InflaMab. IL-1β secretion was measured using ELISA and Caspase-1 activation was assessed by staining cells with a non-cytotoxic Fluorescent Labeled Inhibitor of Caspase-1 (FLICA). ELISA was also used to measure TNFα in order to demonstrate specificity of InflaMab. In vivo, the neuroprotective effect of InflaMab was assessed in a microbead-induced mouse model of glaucoma. WT C57BL/6J mice received one intravitreal injection of InflaMab (250 ng/ml) on Day 0 (just prior to injection of microbeads). IOP was monitored by rebound tonometry and at Day 28 post microbead injection pattern electroretinogram (pERG) was performed to assess RGC function. Following euthanasia the retina was processed for RGC counts. Results: In vitro, InflaMab significantly inhibited IL-1β secretion in THP1 cells and BMDMs (p<0.05) and had no inhibitory effect on TNFα production, demonstrating specificity. Confocal microscopy revealed a significant reduction of caspase-1 activation in THP1 cells treated with InflaMab as opposed to non-treated cells. In vivo, a single intravitreal injection of InflaMab provided significant neuroprotection in terms pERG amplitude and RGC densities when compared to untreated controls. Conclusions: These preliminary data demonstrate that (i) InflaMab inhibits inflammasome activation and (ii) a single intravitreal administration of InflaMab provides sustained neuroprotection in an inducible mouse model of glaucoma.