SBIR-STTR Award

Glaucoma is one of the leading causes of blindness in the world. This chronic and progressive optic neuropathy is characterized by loss of axons of the retinal ganglion cells (RGC) that constitute the optic nerve. Reduction of elevated intraocular pressure, the only modifiable causative factor, slow the onset and progression of the disease, yet no treatment is available to restore optic nerve damage (8, 9). Nerve growth factor (NGF) is an endogenous neurotrophin that exerts trophic and differentiating activity on neurons of the central and peripheral nervous systems with protective and/or regenerative effects observed in degenerative diseases or following injury. NGF applied topically to the eye has been shown to significantly prevent RGC degeneration in experimental rat models of glaucoma. In 3 patients with advanced glaucoma, treatment with topical NGF (eye drop) improved visual acuity, contrast sensitivity, and electrophysiological functions without undesired side effects. NGF binds to both tyrosine kinase receptor TrkA and receptor P75NTR (TNF receptor superfamily). Importantly, the binding of NGF to TrkA alone promotes RGC?s survival and proliferation. In contrast, binding of NGF to p75NTR leads to apoptosis. rNGF (recombinant NGF) is currently produced in non- human cell systems. Due to the importance of pro-sequence for efficient folding or refolding, the in vitro (trypsin) or in vivo (furin) post-proteolytic modifications of pro-NGF, and the requirement of forming disulfide bonded monomer and non-covalent homodimer, expression yields from current manufacturing process are low and the NGF protein was in low quality. We have developed a cost-effective and scalable expression system to produce therapeutic human proteins from a proprietary HEK293 cell line. In the preliminary studies, we have established a cell pool of NGF which exhibited >10-fold higher yield than current expression systems with the ex vivo activity comparable to that of the murine wildtype NGF (wtNGF). In this study, we will first optimize the NGF to selectively activate TrkA receptor without compromising the expression yield or protein stability. We will then select top stable HEK293 cell clones for TrkA selective NGF mutant, which will be suitable for future large-scale cGMP manufacturing. Since there are substantial similarities between the rodent and human eyes, we will validate the efficacy and safety in an established rat model of glaucoma. Specific aims include: 1: To optimize rNGF as a TrkA-specific agonist and select top stable HEK293 cell clones suitable for future large-scale cGMP manufacturing. 2: To determine whether topical treatment with TrkA selective NGF mutant more effectively preserves retinal integrity and function compared to the wtNGF in a rat glaucoma model of episcleral vein by hypertonic saline injections.

Glaucoma is a leading cause of irreversible vision loss, which is characterized by progressive degenerationof retinal ganglion cells (RGC) and their optic nerve axons. While age is a key risk factor, elevated intraocularpressure (IOP) is the only modifiable risk factor, with topical IOP-lowering drugs as the first line treatment.However, RGC degeneration and vision loss continues in half the patients taking these medications. By the timewhen characteristic visual field defects are detected, 30-50% of the RGCs have already been lost. Thus, thereis an urgent need to develop novel therapies, independent of IOP reduction, which protect RGCs fromdegeneration and boost the function of RGCs challenged in the disease. The binding of nerve growth factor (NGF) to TrkA attenuates reactive astrocytosis and promotes RGCsurvival and proliferation. In contrast, the binding to p75NTR leads to astrocytosis and RGC apoptosis. Serumlevels of NGF are reduced in early and moderate glaucoma patients compared to healthy controls. Wildtypehuman NGF (OxervateTM, Cenegermim, Dompe) was approved by FDA in 2018 as eye drops to treatneurotrophic keratitis. In a recent Phase 1b clinical trial, it has been shown that topical use of Cenegermim wassafe and tolerable in open-angle glaucoma patients. However, wildtype NGF treatment failed to provideneuroprotection in preclinical models of established glaucoma, antagonized by up-regulated p75NTR activity.Moreover, inefficient manufacturing of Cenegermim using an E coli expression system compromises proteinstability and quality at a prohibitive cost for chronic treatment. In the Phase 1 study, we successfully reached all the milestones by designing and validating a human NGFmutein, HC201. The mutein preserves protein stability and expression with enhanced TrkA receptor activitiesbut abrogated p75NTR binding and signaling. In rat models of glaucoma induced by episcleral vein cauterization,topical treatment with HC201 robustly protected RGCs. In contrast, wildtype NGF was not effective. HC201efficacy was also observed in diabetic corneal ulcer and dry eye disease models. Meanwhile, we have developeda highly cost-effective and scalable process to produce HC201 in mammalian cells. In the Phase II SBIRapplication, we will continue to validate the efficacy and elucidate mechanism of action in the inducible microbeadocclusion model of glaucoma in rats and squirrel monkeys. The use of different approaches to elevate IOP intwo species will significantly improve the success for translation to human clinical trials. With a highly experienceddrug development team, we also will initiate critical activities necessary to enable IND filing. Specific Aims include: 1) Determine whether topical treatment with HC201 preserves retinal integrity andfunction compared to the placebo control using the microbead occlusion model of glaucoma in rats and squirrelmonkeys; 2) Manufacture cGMP grade HC201 from 100L engineering run; 3) Evaluate nonclinical safety ofrepeat dose HC201. The long-term goal is to develop HC201, alone or in combination with current hypotensive medication, topreserve and even restore vision in glaucoma patients.

Public Health Relevance Statement:
NARRATIVE The efficacy and safety of an optimized human nerve growth factor will be evaluated in clinically relevant animal models of glaucoma.

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