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Reversible antibodies for real-time monitoring or proteins

Congratulations to Christian Fercher and our collaborators Martina Jones and Steve Mahler for completing what was a real tour-de-force project. Christian designed an in situ mutagenesis approach coupled with a phage library screen to select anti-EGFR scFv mutants which demonstrated significantly faster off-rates in comparison to the wildtype (panitumumab) VH/VL sequences. The difficulty was in finding an approach to rapidly screen the kinetics of several hundred clones, narrowing these down for Biacore analysis, and eventually selecting a clone with 30-fold faster off-rate. Christian then used this mutant to monitor protein concentration in a flowing stream, while varying the level up and down over 12 hours or so. Very cool!

Call me simple - but the most surprising thing to me was that while increasing the speed of the off-rate effectively "matches" the on-rate, I expected this to reduce the overall affinity and increase non-specific binding. However, while the affinity dropped somewhat, we did not detect non-specific binding based on using plasma in the solutions, or using related growth factors as targets. There are lots more biosensors to build now that we have this method down pat!

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