Maximilian Klement, Chengcheng Liu, Bernard Liat Wen Loo, Andre Boon-Hwa Choo, Dave Siak-Wei Ow, Dong-Yup Lee, Effect of linker flexibility and length on the functionality of a cytotoxic engineered antibody fragment, Journal of Biotechnology, Volume 199, 10 April 2015, Pages 90-97, ISSN 0168-1656, http://dx.doi.org/10.1016/j.jbiotec.2015.02.008.
Engineered antibody fragments often contain natural or synthetic linkers joining the antigen-binding domain and multimerization regions, and the roles of these linkers have largely been overlooked. To investigate linker effects on structural properties and functionality, six bivalent cytotoxic antibody fragments with of linkers of varying flexibility and length were constructed: (1) 10-AA mouse IgG3 upper hinge region, (2) 20-AA mouse IgG3 upper hinge region repeat, (3) 10-AA glycine and serine linker, (4) 20-AA glycine and serine linker repeat, (5) 21-AA artificial linker, and (6) no-linker control. Interestingly, a higher cytotoxicity was observed for fragments bearing the rigid short linkers compared to the flexible longer linkers. More importantly, amino acid composition related to the rigidity/flexibility was found to be of greater importance upon cytotoxicity than linker length alone. To further study the structure–function relationship, molecular modelling and dynamics simulation were exploited. Resultantly, the rigid mouse IgG3 upper hinge region was predicted to enhance structural stability of the protein during the equilibrium state, indicating the improved cytotoxicity over other combinations of fragments. This prediction was validated by measuring the thermal stability of the mouse IgG3 upper hinge as compared to the artificial linker, and shown to have a higher melting temperature which coincides with a higher structural stability. Our findings clearly suggest that appropriate linker design is required for enhancing the structural stability and functionality of engineered antibody fragments.
This research / project is supported by Bioprocessing Technology Institute, A*STAR, and by the Rural Development Administration (Republic of Korea), under its Next-Generation BioGreen21 Program (SSAC, No. PJ01109405)