Design and Synthesis of Monobody Variants with Low Immunogenicity

Abstract

Mirror-image proteins (d-proteins) are promising scaffolds for drug discovery because of their high proteolytic stability and low immunogenic properties. Facile and reproducible processes for the preparation of functional d-proteins are required for their application in therapeutic biologics. In this study, we designed and synthesized a novel monobody variant with two cysteine substitutions that facilitate the synthetic process via sequential native chemical ligations and improve protein stability by disulfide bond formation. The synthetic anti-GFP monobody in this model study exhibited good binding affinity to the target enhanced green fluorescent protein. In vivo administration of the synthetic anti-GFP monobody (l-monobody) to mice induced antidrug antibody (ADA) production, whereas no ADA production was observed following immunization with the mirror-image anti-GFP monobody (d-monobody). These results suggest that the synthetic d-monobody is a non-antibody protein scaffold with low immunogenic properties.

High-affinity mirror-image monobody targeting MCP-1 generated via TRAP display and chemical protein synthesis

“…On the other hand, the plasma samples from 9D-immunized mice did not show any ELISA signals even at day 35. These results are totally consistent with the previous study showing less immunogenicity of D-monobody than that of L-monobody 58 . Given that antibody production begins with degradation of protein antigens into peptides in antigen-presenting cells, the low immunogenicity of mirror-image protein could be attributed to the high proteolytic stability.…”

“…Therefore, we assume that the monobody established in this study could automatically fold into the proper conformation. CD spectrometry of these synthetic monobodies dissolved in PBS buffer without a folding procedure indicated the existence of monobody-like β-sheet structure as observed in the previous study 58 (Figure 4B). Importantly, 9D and 9L showed mutually inverted spectra, suggesting that 9D possesses the mirror-image conformation of 9L.…”

“…Recently, we developed a chemically synthesized an anti-GFP monobody variant containing two Cys substitutions, which simplify the synthesis of the monobody from three peptide segments and eliminate the need for a desulfurization step after peptide assembly 58 . Importantly, this synthetic L-configured monobody variant maintained its affinity for the target protein, GFP.…”

“…Next, the immunogenicity of 9D was assessed in comparison with 9L according to the previously reported procedure 58 . Intraperitoneal immunization of BALB/c mice was performed with administration of 9D or 9L emulsified in Freund's complete adjuvant at day 0, and incomplete adjuvant at days 14 and 28.…”

Engineering a Low-Immunogenic Mirror-Image VHH against Vascular Endothelial Growth Factor

“…However, even approved drugs, such as a fully human antibody “adalimumab” and the humanized VHH “ozoralizumab”, are associated with some risk of ADA production. , Remarkably, ADA was not detected in any of the mice treated with d -VHH­[B1] in this study. Thus, similar to the encouraging results obtained with d -protein scaffolds, ,,,, the use of d -VHHs may represent a compelling approach for mitigating the adverse immunogenic events associated with conventional antibody therapy.…”