Development of HC-HA/PTX3 as a Biologic for Proliferative Vitreoretinopathy (PVR) Treatment

Abstract

Purpose : PVR is characterized by membranes developed on the retinal surface after retinal detachments, mainly due to proliferation and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE). Despite surgical intervention, the visual outcome remains poor. We would like to develop HC-HA/PTX3, a unique matrix purified from human amniotic membrane with anti-inflammatory and anti-scarring activities, as a biologic for PVR.

Methods : HC-HA/PTX3 was manufactured from human amniotic membrane by 2 – 4 runs of CsCl gradient ultracentrifugation in the presence of 4 M Guanidine HCl. The identity and purity were verified by HA and protein quantitation, Western blot, agarose gel electrophoresis, and the potency was verified with M2 macrophage polarization assay using the cloned monocyte precursor (expression of monocyte markers: CCR2, CD62L, CX3CR1, Ly6C) from RAW264.7 cells. The cell viability was determined by WST-1 assay, and the EMT assay was performed by pSmad2/3 immunostaining and fibronectin ELISA using ARPE-19 cells with TGF-β stimulation.

Results : We have purified and qualified HC-HA/PTX3 under GMP for IND-enabling pre-clinical studies and used it as the Reference Material for the potency assay. We have validated that purified and lyophilized HC-HA/PTX3 was stable after storage at -20°C for at least two years. Multiple clones of CCR2^l/CX3CR1^h/Ly6C1⁺ RAW264.7 cells were obtained and exhibited differentiation into M1 macrophage with IFN-γ/LPS stimulation suitable for the M2 polarization assay. We prepared a two-tiered bank (Master Cell and Working Cell) from the selected clone and validated that these cells (up to passage 4) were stable (no significant changes in viability and differentiation capacity) in liquid nitrogen for at least four years. In vitro, HC-HA/PTX3 (up to 200 µg/ml) was not toxic to human RPE cells and down-regulated nuclear translocation of SMAD2/3 and fibronectin production. In vivo, intravitreal injection of HC-HA/PTX3 (up to 150 µg/per eye) was safe to rabbit eyes as judged by histopathology and ERG and intravitreal injection of HC-HA/PTX3 (37.5 - 150 µg per eye) reduced RPE-induced PVR in rabbits.

Conclusions : HC-HA/PTX3 manufactured under GMP is safe and effective in both in vitro testing and pre-clinical studies. Hence, these findings will allow us to further develop it as a novel biologic for managing PVR through human trials.

This is a 2020 ARVO Annual Meeting abstract.