There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. It was shown that incorporation of pH-dependent antigen binding into therapeutic antibodies enhanced their efficacy that was driven by rapid dissociation of the antigen in the acidified endosome (pH 6.0) and the recycling of free antibody [1-2]. Previous studies emphasized the synergistic effects of multiple histidine groups for sharp pH-sensitive binding profiles [1-2, 4]. Here, we describe a novel strategy for incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both high affinity binding at pH 7.4 and pH-sensitivity and excludes conventional negative selection steps. As prove of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary determining regions of TNFalpha-specific adalimumab.
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