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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., Competing Interests: B.J.D. and J.B.R. are shareholders of Integral Molecular. B.S., L.J.S., T.B., T.C., B.J.D., J.B.R., and R.C. have a patent related to the antibodies generated in this work, patent WO2020168059A1., (© 2022 The Author(s).)