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David A Copland, Hanieh Khalili, Richard W J Lee, Peng Tee Khaw, Steve Brocchini, Andrew D Dick; Novel anti-TNF-α antibody mimetic to treat ocular inflammation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3210. doi: https://doi.org/.
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Systemic administration of Infliximab, a chimeric mAb that neutralizes TNF-α, is used off-label for the treatment of autoimmune ocular inflammatory diseases such as uveitis. Our aim is to develop a Fab-PEG-Fab (FpF) molecule using Fab fragments derived from Infliximab, with the expectation that FpFs will subvert Fc-mediated inflammation and permit intravitreal administration.
To test the in vivo efficacy of Infliximab, EAU was induced in B10.RIII mice and disease severity determined by clinical assessment and flow cytometric analysis of retinal infiltrate. Groups of mice were treated following onset of clinical disease with a single intravitreal dose (2.5μl, 15μg/eye) of Infliximab or control vehicle alone. Retinal infiltrate was examined and enumerated by flow cytometry, 4 days following treatment. The FpF-infliximab was prepared using the Fab from the proteolytic digestion of infliximab and a PEG di(bis-sulfone) reagent which site specifically underwent conjugation with the accessible disulphide on two Fabs to yield the FpF which was then purified by ion exchange chromatography (IEX). Affinity and binding analyses were performed using BIAcore NTA chip.
The assessment of retinal infiltrate in mice receiving a single intravitreal administration of Infliximab reveals a significant reduction in CD45+ cells with reduced CD11b+, Ly6G+, CD4+ & CD8+ populations. Clinical assessment demonstrates that Infliximab suppresses disease progression, with some eyes maintaining only the initial signs of retinal inflammation. Using the PEG di(bis-sulfone) which has the bis-alkylating moiety on each terminus to link two Fab-Infliximab, the FpF molecule was successfully prepared and purity evaluated by SDS-PAGE analysis. Purified FpF-Infliximab maintains binding affinity for TNF-α, and kinetic studies demonstrate a lower dissociation rate constant compared to whole Infliximab, suggesting a tighter TNF-αinteraction with FpF-Infliximab.
The chimeric Infliximab mAb successfully suppresses ocular infiltrate with reduced clinical disease severity in EAU. FpF-Infliximab molecules can be synthesized, and importantly possess a similar binding affinity and lower dissociation rate constant as the parent IgG. Together the data supports ongoing experiments to evaluate FpF-infliximab’s potential as a novel therapeutic for treatment of ocular inflammation.
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