Abstract 318: Atomic-level specificity of Claudin 6 monoclonal antibodies isolated for treating solid tumors

The tight junction protein Claudin 6 (CLDN6) is differentially expressed on cancer cells with almost no expression in healthy tissue, making it a valuable therapeutic target for many solid tumor cancers. Despite their potential as cancer therapeutics, very few CLDN6 monoclonal antibodies (MAbs) are in development, because MAbs with high affinity and specificity for CLDN6 are difficult to isolate. CLDN6 is structurally complex, with 4 transmembrane domains and 95% extracellular sequence conservation between human and mouse. Achieving MAb specificity for CLDN6 is especially challenging because its extracellular region strongly resembles those of 23 other human CLDN family members. In particular, the widely expressed CLDN9 differs from CLDN6 by only 3 extracellular residues. Using MAb discovery strategies specifically tailored to complex membrane proteins, including the use of virus-like particles (Lipoparticles), divergent species (chicken) immunization, and optimized phage display panning, we isolated 6 rare MAbs that recognize the native structure of CLDN6 with as low as picomolar affinity. The MAbs were screened against a Membrane Proteome Array containing ~6,000 membrane proteins and demonstrated specificity for CLDN6 with minimal cross-reactivity for CLDN9 or other CLDN family members. Epitope mapping using Shotgun Mutagenesis alanine scanning across the 220 residue CLDN6 sequence distinguished the binding sites of the MAbs from clinical-stage benchmarks. Atomic-level epitope mapping using comprehensive site-specific mutagenesis identified the γ carbon on CLDN6 residue Q156 as the critical structural mechanism enabling these MAbs to differentiate between CLDN6 and CLDN9 with high specificity. The CLDN6 MAbs identified here can be used to study CLDN6-positive cancers, including ovarian, endometrial, lung, and testicular cancer, and have the potential to be developed into highly selective therapeutics. Characterization of highly specific CLDN6 MAbs isolated for treatment of solid tumors MAb IM301 IM302 Benchmark (IMAB027, Astellas) VH CDR3 length (Kabat) 18 18 8 CLDN protein binding: Biosensor KD ± error, nM CLDN6 (target) 0.6 ± 0.03 < 0.001 0.5 ± 0.01 CLDN9 No binding No binding 3.6 ± .09 CLDN3 No binding No binding No binding CLDN4 No binding 146 ± 20 153 ± 6 Mouse CLDN6 binding Yes Yes Yes Cyno CLDN6 binding Yes Yes Yes Conformational epitope Yes Yes Yes Epitope topology Yes Yes Yes Critical CLDN6 epitope residues E48, E154, R158 E154, R158 F35, G37, S