Developing Methotrexate-Induced Immune Tolerance in a Rabbit Model of Persistent Retinal Vascular Leak

Abstract

Purpose : Preclinical testing of biologics is complicated by immunogenicity, which differs from immunogenicity in humans. It is necessary to mitigate immunogenic responses in order to effectively test the pharmacodynamics and pharmacokinetics of novel biologics, especially for repeat administration. The persistent retinal vascular leak (PRVL) rabbit model is a valuable tool for testing intravitreal (IVT) therapies; however, rabbits frequently mount a strong anti-drug antibody (ADA) response. We developed an antigen-independent immune tolerance protocol to 1) reduce ocular inflammation, and 2) reduce systemic production of ADAs while maintaining the model’s vascular phenotype.

Methods : Dl-α-aminoadipic acid was administered IVT in rabbit eyes to induce the PRVL model (Rodrigues et al., 2018, IOVS). Daily administration of methotrexate (MTX; 30mg/kg s.c.; N=10) or PBS (N=10) was initiated 3 days prior to IVT challenge with a biologic and continued for 21 days. Ocular examinations, fundus angiography, and blood collections were conducted from weeks 2 through 12 to evaluate the effect of MTX on systemic ADA levels and incidence of ocular inflammation. Eyes were enucleated and fixed for histopathological assessment.

Results : The MTX dosing regimen was well tolerated. Incidence of moderate-severe ocular inflammation was eliminated in MTX-treated rabbits, which coincided with significantly reduced systemic ADA titer levels compared to PBS-treated controls. Histologic evaluation determined that 4/10 eyes of PBS-treated rabbits showed substantial lymphoplasmacytic infiltrate following IVT administration of the biologic while eyes of MTX-treated rabbits did not.

Conclusions : The reduction of systemic ADAs in the MTX group demonstrates that induction of immune tolerance in the PRVL rabbit model is feasible. A coinciding decrease in incidence of gross ocular inflammation was observed in vivo, and histologic results confirmed that MTX treatment eliminated infiltrate signatures typically associated with activation of adaptive immunity. This immune tolerance protocol can be used in future studies to reduce ocular inflammation, minimize study attrition, and increase confidence in drug exposure levels.

This is a 2021 ARVO Annual Meeting abstract.