Effect of linker flexibility and length on the functionality of a cytotoxic engineered antibody fragment.
J Biotechnol
; 199: 90-7, 2015 Apr 10.
Article
de En
| MEDLINE
| ID: mdl-25697559
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.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Protéines de fusion recombinantes
/
Fragments d'immunoglobuline
/
Ingénierie des protéines
/
Anticorps
Type d'étude:
Prognostic_studies
Limites:
Animals
Langue:
En
Journal:
J Biotechnol
Sujet du journal:
BIOTECNOLOGIA
Année:
2015
Type de document:
Article