Why might higher affinity reduce performance in a cell-based assay?

Why

Affinity maturation may reduce dissociation rate but also alter paratope chemistry, polyreactivity, aggregation or target residence time. In solid tissues, very tight binding near vessels can sometimes limit deeper distribution, while some receptor functions require internalization or reversible engagement. In a competitive functional assay, an antibody could bind tightly yet block the wrong epitope or stabilize an unhelpful conformation.

Test affinity and kinetic parameters separately from cellular occupancy, internalization, pathway effect and viability across representative expression levels. For example, an optimized binder with a slower off-rate might look excellent on immobilized recombinant antigen but drive little biological response if it does not block the functional receptor interface. The proper endpoint depends on therapeutic mechanism, dose and tissue context.