Figure 2 |. Selection against carbonic anhydrase IX, hit validation and conversion of ligands to CAR-T cell activators.

a, NF-DEL high-throughput DNA sequencing results represented as three-dimensional fingerprints before and after selections against CAIX. The x and y axes represent the two building blocks while the color heat map and z-axis correspond to the DNA sequence counts. Selections were performed in triplicate (see Supplementary Figure 21). In order to show reproducibility of selection results, two CAIX prints are shown in this Figure. Cut-off values equal to 20 (for CAIX selection #1) and 15 (for CAIX selection #2) were applied to the fingerprints. No cut-off was applied for the naïve library fingerprint. The most enriched combination is indicated with an arrow (average EF=23±5 × 10²). For the calculation of enrichment factors (EF) see Supplementary Tables 2,3 and Equation 1 (S29). b, off-DNA affinity measurements of selected compounds by FP and small-molecule ELISA, see also Supplementary Table 6. Error bars indicate standard deviation of three measurements. c, FACS with 8 and 10 on SK-RC-52 cells (renal cell carcinoma, in blue), on HEK293T (human embryonic kidney cells, control, in green), on SK-RC-52 cells alone (renal cell carcinoma, control, in red). d, Schematic representation of the immunological synapse of a fluorescein-specific universal CAR-T cell with a CAIX-presenting SK-RC-52 tumor cell, established by cross-linking the cells with the heterobifunctional crosslinker (blue). Killing was monitored after 24 h. % SK-RC-52 lysis is given as a function of ligand 8 (dark blue) and 10 (light blue) concentration. % SK-RC-52 lysis is also given for controls I-IV and the functional combination V(anti CAIX CAR-T). Error bars indicate standard deviation of three measurements e, Chemical structures of compounds 7, 8, 9, 10, 11, 12 and of tripeptide linker (LH, LFluo). f, Evaluation of the tumor-targeting performance of compound 8 after i.v. administration for human SK-RC-52 xenografted in mice, using ex vivo immunofluorescence detection of the fluorescein moiety. Images were taken 60 min after intravenous injection of 65 μg of compound 8 (green = ligand, blue = DAPI staining. Scale bar = 100 μm).