Stabilization of antibody VH-domains by proteolytic selection

Single variable domains of antibodies represent the smallest antigen binding fragments but are less stable than when associated with their cognate variable domains. Here we have attempted to improve the thermodynamic stability of a model heavy chain variable domain (VH) by “proteolytic selection” a method whereby the protease-resistance of the displayed protein is coupled to the infectivity of a filamentous bacteriophage. The gene encoding the heavy chain variable domain was taken from the anti-lysozyme antibody HyHEL-10, mutated at random by error-prone PCR, and displayed on filamentous bacteriophage by fusion between the domains of the phage p3 protein. As the entire p3 protein is required for phage infectivity, treatment of the phage library with trypsin at an elevated temperature (which leads to cleavage of p3 fusions with unfolded variable domains) selects for infectious phages bearing the more stable variable domains. After several rounds of selection, a mutant (S65G/T70S/D99N) was obtained with improved stability (Tm=58.5 °C and ΔG25 °C=6.3 kcal/mol compared to 51.6 °C and 4.2 kcal/mol for the parent domain). These mutations are conservative and the mutant domain retains the ability to pair with its cognate light chain variable domain in an Fv fragment and to mediate binding to lysozyme. Our results show that the thermodynamic stability of antibody single domains can be improved by “proteolytic selection” and this may represent a step towards making useful antibody single domains for biotechnological application.