Antibody specificity against highly conserved membrane protein Claudin 6 driven by single atomic contact point

The tight junction protein claudin 6 (CLDN6) is differentially expressed on cancer cells with almost no expression in healthy tissue. However, achieving therapeutic MAb specificity for this 4 transmembrane protein is challenging because it is nearly identical to the widely expressed CLDN9, with only 3 extracellular amino acids different. Most other CLDN6 MAbs, including those in clinical development are cross-reactive with CLDN9, and several trials have now been stopped. Here we isolated rare MAbs that bind CLDN6 with up to picomolar affinity and display minimal cross-reactivity with CLDN9, 22 other CLDN family members, or across the human membrane proteome. Amino acid-level epitope mapping distinguished the binding sites of our MAbs from existing clinical-stage MAbs. Atomic-level epitope mapping identified the structural mechanism by which our MAbs differentiate CLDN6 and CLDN9 through steric hindrance at a single molecular contact point, the γ carbon on CLDN6 residue Q156.