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Hanieh Khalili, Ashkan Khalili, Garima Sharma, Alastair Lockwood, Peng Khaw, Steve Brocchini; Bivalent anti-TNF-α Fab-PEG-Fab for ocular inflammation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6298.
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Infliximab (chimeric full IgG) and adalimumab (human full IgG) that bind to soluble TNF-α (sTNF-α) and transmembrane TNF-α (tmTNF-α) have been used off-label by ophthalmologists for the treatment of autoimmune inflammatory conditions such as uveitis via systemic administration. Our aim is to develop Fab-PEG-Fab (FpF) molecules from the Fabs derived from adalimumab and infliximab. FpFs would be expected to avoid any inflammation due to the antibody Fc to allow intravitreal injection. We have recently shown that anti-VEGF FpF molecules can act as mimetics of anti-VEGF IgG antibodies .
Adalimumab and infliximab, (5.0 mg/mL, 1.0 mL) were digested using immobilised papain (0.8 mL) and then purified using protein A to give the respective Fabs. The purified Fabs were treated with DTT (1.0 mg/mL) and the PEG reagent 1 was added to give the respective FpF molecules, which were purified by ion exchange chromategraphy (IEX). Affinity and binding analyses were performed using BIAcore.
Fab-infliximab and Fab-adalimumab were obtained after papain digestion of the respective parent IgG (Figure1, A, lane 1) and then treated with DTT (Figure 1, A, lane 2) followed by incubation with 1 eq PEG reagent 1 (10 kDa) (Figure 1, A, lane 3) to yield Fabinflix-PEG10-Fabinflix and Fabada-PEG10-Fabada respectively (Figure 1, A, lanes 9, 10). The band for each FpF appeared at about the 120 kDa molecular weight which is consistent with expectation due to the size of PEG when evaluated by SDS-PAGE. No formation of FpF was observed when a control reaction was conducted with Fab that had not been treated with DTT corroborating that conjugation occurred at the disulfide to link PEG in the same region as the natural hinge. Before conjugation of two Fabs to make FpF, binding of the Fabs derived from adalimumab (Humira) was studied in Biacore. NTA-chip was functionalized with NiCl2 followed by injection of the His-Tag TNF-α (Figure 1, B). Fab-ada bound to TNF- α with concentration dependent manner. The FpF-derived from adalimumab displayed binding to TNF-α
Anti-TNF-α FpF molecules were prepared from adalimumab and infliximab. Anti-TNF-α FpF adalimumab bound to TNF-α. Ongoing efforts are now focused to characterise these new FpFs and to evaluate their potential to inhibit ocular inflammation.
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