title: Effect of linker flexibility and length on the functionality of a cytotoxic engineered antibody fragment.
abstract: 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.
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore; Department of Chemical and Biomolecular Engineering, Synthetic Biology Research Consortium, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Klement
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
authors.forename: Chengcheng
authors.initials: C
authors.lastname: Liu
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
authors.forename: Bernard Liat Wen
authors.initials: BL
authors.lastname: Loo
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
authors.forename: Andre Boon-Hwa
authors.initials: AB
authors.lastname: Choo
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore. Electronic address: dave_ow@bti.a-star.edu.sg.
authors.forename: Dave Siak-Wei
authors.initials: DS
authors.lastname: Ow
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore; Department of Chemical and Biomolecular Engineering, Synthetic Biology Research Consortium, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore. Electronic address: cheld@nus.edu.sg.
authors.forename: Dong-Yup
authors.initials: DY
authors.lastname: Lee
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins
keywords: Engineered antibody fragments; Molecular dynamics simulation; Peptide linkers; Recombinant fusion proteins; Synthetic protein engineering