Abstract
Purpose :
Inflammation is a key process in the progression of retinal diseases. However, it is inadequately targeted by current standard-of-care agents. RG6179 is a recombinant monoclonal antibody that potently inhibits all forms of IL-6 signaling. This abstract reports preclinical data examining the mechanistic action of RG6179.
Methods :
To characterize the inhibitory effects of RG6179 on cis-IL-6 signaling, it was tested on HEK-Blue IL-6 reporter cells (Invivogen #hkb-hil6). Cells were incubated in ±RG6179 in an 8-point dilution series for 1 hour at 37 ○C prior to the addition of recombinant human IL-6 (R&D Systems #206-IL/CF) at 3 ng/ml (n=4). To characterize the effects of RG6179 on trans-IL-6 signaling, RG6179 was tested in HUVEC cells stimulated with a combination of human IL-6 plus recombinant human IL-6R (R&D Systems #3227-SR-025). IL-6 and IL-6R were incubated together for 1 hour prior to addition to allow for IL-6/IL-6R complex formation. RG6179 was added to cell media in an 8-point dilution series for 1 hour prior to addition of the IL-6/IL-6R complex. HUVECs were incubated in at 37 ○C for 72 hours and ICAM1 cell surface expression was measured via flow cytometry (n=4). To examine the effect of RG6179 on trans-IL-6-mediated barrier breakdown in HUVECs, a transendothelial electrical resistance (TEER) assay was used.
Results :
To measure the effect of RG6179 on cis-IL-6 signaling, HEK-Blue IL-6 reporter cells were simulated with human IL-6 and subsequent SEAP production was measured. RG6179 potently inhibited cis-IL-6 mediated SEAP production in a concentration dependent manner (IC50 = 40.8 ± 1.8 pM). The effect of RG6179 on trans-IL-6 signaling was assessed by induced ICAM1 cell surface expression in HUVEC cells following stimulation with IL-6/IL-6R complex. Stimulation of HUVEC with IL-6/IL-6 receptor led to trans-IL-6 induced expression of ICAM1 on the cell surface, an effect that was inhibited in a concentration-dependent manner by RG6179 (IC50 = 1.0 ± 0.1 nM). In a TEER assay, the IL-6/IL-6R led to a significantly reduced barrier capacity after 12 hours, which could be rescued in a concentration dependent manner following addition of RG6179.
Conclusions :
This data supports the effectiveness of RG6179 at inhibiting both the cis-IL-6 and trans-IL-6 signaling pathways in vitro. Furthermore, it demonstrates the ability of RG6179 to restore trans-IL-6 mediated barrier breakdown in vitro.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.