Kd ≠ IC50

A mistake I've seen more than once: Kd ≠ IC50

A well-known concept to most, but one that gets confused in practice — often leading to the wrong interpretation of an in vitro assay result.

The story: in an SPR assay, molecule A was 3x more potent than molecule B. The engineering team decided to affinity-optimize molecule B for use as the clinical candidate. But in a concentration-based binding assay (ELISA), molecules A and B looked equivalent. The team was confused — why keep optimizing B if they're the same? It turned out the target concentration in the assay was so high that affinity no longer mattered — both molecules simply hit IC50 at half the target concentration. After adjusting the assay's target concentration for the next run, the team moved forward with further affinity maturation on molecule B.

Binding assays need to reflect the affinity of the molecule — which means the target concentration needs to be in range of the KD. If target concentration >> KD, you fall into a concentration-dependent regime that no longer reflects true affinity.

Of course, there are plenty of caveats: mono- vs. multivalent binding, whether the recombinant SPR reagent reflects the natural target (which can vary by company and batch), avidity effects, and more. But whenever I have both SPR data and data from another assay (often on-cell binding), I do at least a back-of-envelope check to see if the results align — and whether we're in a concentration-dependent regime for the binding assay.

This matters in the clinic as well — the inherent target concentration and how it relates to the therapeutic's affinity can both determine the "danger" of TMDD as well as whether affinity maturation is necessary.

What am I missing? Do you have a preferred binding assay for in vivo translation?

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#PKPD #affinitymaturation #KD #IC50 #DrugDiscovery #DrugDevelopment #TMDD