Targeting the NLRP3 inflammasome as a therapy for glaucoma

Abstract

Introduction:
Glaucoma is the largest cause of irreversible blindness worldwide and the focus of all glaucoma therapies is lowering intraocular pressure (IOP). Despite this, many patients still develop glaucoma and lose sight even when IOP is adequately controlled. Inflammation and in particular the NLRP3 inflammasome, has recently been proposed to play a role in the pathogenesis of the disease.
Aims:
The primary aims of this work were to investigate the role of theNLRP3 inflammasome in glaucoma and to test the efficacy of a newly developed and novel anti-NLRP3 antibody inhibitor.
Methods:
Three main studies were undertaken in this project; 1)Identification of NLRP3 inflammasome activation in glaucoma. Aqueous humour and plasma samples were collected from glaucoma (n=39) and nonglaucoma (cataract) (n=49) patients at Altnagelvin Area Hospital, Northern Ireland and the Mater Hospital, Dublin, Ireland. The glaucoma patients had primary open angle glaucoma (POAG) or pseudoexfoliation glaucoma(PXFG). Samples were tested using a multiplex proteomics method which measured 92 inflammatory related proteins using the proteomics extension assay technology (Olink). The machine learning algorithm, random forest was used to identify NLRP3 inflammasome related proteins and other biomarkers that could accurately distinguish between glaucoma and nonglaucoma patients. Immunohistochemistry was performed on glaucomatous human donor eyes (n=3) and control donor eyes (n=7) to measure and compare NLRP3 inflammasome expression. 2) Characterisation and humanisation of a novel antibody inhibitor of the NLRP3 inflammasome: InflamAb. 3) The therapeutic potential of InflamAb was assessed in in vitro models of inflammation, glaucoma relevant cell lines and an in vivo model of glaucoma.
Results:
Immunohistochemistry staining, and analysis of human glaucoma and non-glaucoma post-mortem eyes showed strong expression of NLRP3inflammasome proteins in human retinas, optic nerves and optic nerve heads. This expression also co-insides with an increased expression of microglial populations in these regions. Of the 92 proteins analysed in AH of POAG and PXFG patients, 53% were significantly increased in POAG and50% in PXFG. Panels of AH or plasma biomarkers related to the NLRP3Inflammasome pathway could accurately identify POAG patients(AUC=0.960, CI= 0.807, 0.988 and AUC=0.903, CI= 0.663, 0.972)respectively. A panel of biomarkers from the AH was also identified that could accurately distinguish between PXFG and non-glaucoma (AUC=0.944,CI= 0.769, 0.983). A humanised bispecific antibody, InflaMab was developed which targetsNLRP3 inhibiting inflammasome activation. InflamAb gains access into the cell via the IL-1R1 on the cell membrane to inhibit the NLRP3 target. A number of NLRP3 and IL-1R1 antibody variants were screened for therapeutic efficacy and the best chosen to be combined to make the final bispecific antibody. The NLRP3 inflammasome was activated in the lamina cribrosa cells of the ONH region and microglial cell lines and InflamAb inhibited NLRP3inflammasome activation in these cells and human macrophages (p<0.001).InflamAb also showed trends towards preserving RGC function and density in an in vivo model of glaucoma.
Conclusion:
Results support the validation of the role of the NLRP3inflammasome in glaucoma and support the targeting of this inflammasome as a potential therapy in glaucoma. A novel bispecific antibody, InflamAb, shows promising efficacy to inhibit the activation of the NLRP3inflammasome in glaucoma relevant cell lines and an in vivo model of glaucoma, however further preclinical work is required to fully validate its efficacy in glaucoma.

Thesis is embargoed until 31st March 2026

Date of Award	Mar 2024
Original language	English
Supervisor	Victoria McGilligan (Supervisor), Sarah Atkinson (Supervisor), Colin Willoughby (Supervisor) & Meredith Gregory-Ksander (Supervisor)