Fig 2: CLDN6 MAbs are cell membrane localizedHEK-293F cells were transfected with CLDN6, stained with the MAbs shown, as well as with DAPI nuclear stain, and visualized by immunofluorescence microscopy. Scale bar corresponds to 50 µm. IMAB027, IMAB206, and AE49-11 served as benchmark MAbs.

Fig 3: Epitope mapping at single amino acid resolution(A) Critical residues for MAb binding were identified by shotgun mutagenesis alanine scanning across CLDN6. MAb binding was assessed by flow cytometry of each MAb against each of 219 individual mutations expressed in HEK-293T cells. A residue was considered critical if it resulted in <35% of binding signal relative to wild-type CLDN6, with control MAbs binding to the same mutation at wild-type levels. A commercially available MAb served as positive control (+CTRL). IMAB027, IMAb206, and AE49-11 served as benchmarks. Error bars depict half the range (highest minus lowest binding value) of at least two measurements.(B) Epitope residues are shown on a CLDN6 model based on the CLDN9 crystal structure (PDB# 6OV2, extracellular domain shown).

Fig 4: Isolation of highly specific CLDN6 MAbs(A) CLDN6 is highly expressed in cancerous tissues and absent from healthy tissues. The Gene Expression Profiling Interactive Analysis (GEPIA) database17 was queried for CLDN6 RNAseq expression data in cancerous and healthy tissue samples. Each datapoint represents one patient. Expression was measured in number of sequenced fragments per kilobase of transcript per million mapped reads (FPKM).(B) Human protein sequences were retrieved from UniProt and aligned using ClustalOmega.18 Simple Phylogeny was used to generate a tree based on the alignment, which was then displayed using iTol.19(C) Isolated scFvs were tested for CLDN6 target specificity by flow cytometry. Individual clones displaying more than a 5:1 signal-to-background (S:B) ratio for CLDN6 are shown in red.(D) Forty clones were screened in IgG format for CLDN6:CLDN9 selective binding by flow cytometry. Red bars show the ratio of CLDN6:CLDN9 binding signal for each individual antibody (left Y axis). Gray bars show S:B for CLDN6 binding of each individual antibody (right Y axis).

Fig 5: Membrane proteome-wide testing shows specificity of CLDN6 MAbs(A) MAbs were screened for specificity using Integral Molecular’s Membrane Proteome Array, consisting of ~6,000 human membrane proteins in their native state in unfixed cells. Antibody binding was detected by flow cytometry, and hits were defined as a binding signal more than 3 standard deviations higher than background and validated in follow-up assays. Unlabeled black dots represent hits that were below the defined threshold or that did not validate on retesting.(B) MAb binding to ABCC3 is intracellular. IM301, IM302, and a control antibody (anti-P2X3) were tested for binding to ABCC3 endogenously expressed in A549 cells using 8 serial 3-fold antibody dilutions starting at 30 µg/mL (red). To detect intracellular binding, cells were permeabilized before testing for reactivity. Binding was measured using flow cytometry with a fluorescently labeled secondary antibody diluted 1:500.