Resolution of disulfide heterogeneity in Nogo receptor 1 fusion proteins by molecular engineering

NgR1 (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgR1 have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgR1 are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgR1(310) Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgR1 truncation site or the spacing between the NgR1 and Fc domains, or replacing cysteines within the NgR1 or IgG hinge regions. One variant, which incorporates replacements of Cys(266) and Cys(309) with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.