Abstract
Purpose :
Recent preclinical data and post hoc analysis of clinical data suggest the importance of new approaches that inhibit the generation of pathological levels of VEGF while maintaining physiological levels of VEGF in ocular neovascular disease (NV). Here, we investigated the novel function of a fully human monoclonal IgG anti-c-kit antibody (NN2101) in inhibiting hypoxia and pathological angiogenesis while maintaining trophic factor homeostasis.
Methods :
HRMEC, RPE, MIO-M1, and Y79 cells were used in vitro to demonstrate the inhibitory effect of NN2101. Proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins in hypoxia-conditioned media. Also, db/db mice were treated every 2 weeks with NN2101 via intravitreal injection for 16 weeks.
Results :
Hypoxia-induced expression of c-kit, HIF, and VEGF in Muller glial, photoreceptor, and RPE cells was reduced by NN2101. Interestingly, NN2101 reduced HIF-1α protein stability via the ubiquitin-proteasome pathway. In addition, NN2101 decreased tube formation, migration, and proliferation of hypoxic endothelial cells. Consistently, in vivo Matrigel plug assay showed NN2101 inhibits neovascularization. HRMEC/RPE co-culture exhibited that HIF-1α inhibition by NN2101 effectively blocks VEGF secretion from RPE, similar to Eylea. Also, secretome analysis under hypoxia showed proangiogenic factors, including HIF target genes related to AMD, RVO, and DR were up-regulated, an effect significantly reduced by NN2101 vs Eylea. In muller glial and photoreceptor cells, NN2101 treatment maintained VEGF at basal levels even under the hypoxic conditions, whereas Eylea reduced VEGF to 10% of basal levels. In db/db mice, NN2101 rescued apoptosis in the ganglion cell layer, whereas Eylea-treated mice showed increased apoptotic cell death compared to vehicle control. Analysis of ERG waveforms and immunofluorescence staining demonstrated that the function of photoreceptors, amacrine, and Muller glial cells were significantly improved by NN2101 in the db/db mouse. Also, the number of degenerative capillaries was significantly decreased by NN2101 treatment compared to control.
Conclusions :
We demonstrated that c-kit inhibition with NN2101 efficiently suppresses pathological vessels by reducing expression of multiple angiogenic cytokines via destabilization of HIF in the retina.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.