Fig. 6: Functional and structural characterization of the high-affinity cross-HLA-supertype antibody R302.

a SPR sensorgrams of the interaction between AETX-R302 and the indicated divarasib p*MHC antigens. Biotinylated divarasib p*MHCs were immobilized, and binding of soluble AETX-R302 was measured using single-cycle kinetics. Kinetic values of fitted data are shown in the table below. b Incucyte-based cytotoxicity analysis of A375^eGFP isogenic cell lines that were pulsed with diva-p₅, diva-p₇, p₅^WT, or p₇^WT peptides for 4 h prior to adding activated human T cells (10:1 E:T ratio) and AETX-R302 for 48 h. Plots show 3 technical replicates per condition; error bars indicate SD. The table shows EC₅₀ values for fitted curves (n.d. = not determined). c Cartoon representation of the structures of Fab R302 (only the variable chains are shown) in complex with diva-p₅/A*02 (left) and diva-p₇/A*03 (right). HLAs are depicted in purple (A*02) and white (A*03), β2m in gray, haptens in yellow, conjugated peptides in green (p₅) and dark blue (p₇), and variable chains of R023 in light blue (V_L) and blue (V_H). The same color scheme, reported in the center of the figure, applies to the structural representations of the following panels. d Surfaces of diva-p₅/A*02 (left) and diva-p₇/A*03 (right) buried upon the binding of Fab R302 are colored in light blue. The hapten is represented by sticks. The bottom of the panel shows the sequence alignment of HLA-A*02, HLA-A*03 and HLA-A*11 (residues 1 - 180), performed with BLASTp. The only non-conserved residue among the alleles located in R302 epitope (residue 62, on HLA-A*03 only) is indicated on the aligned sequences (black triangle) and on the structures. e Alignment of R302_diva-p₅/A*02 and R302_diva-p₇/A*03 structures. The right side shows a cartoon representation of the aligned HLAs (residues 1 – 180), the hapten-peptide conjugates (divarasib is represented as sticks), and the R302 variable chains. The left side shows the hapten-peptide conjugates only, represented as sticks. Anchor residues of the two peptides are indicated with a *. Notably, V8 and V9 in diva-p₅ and G10 and A11 in diva-p₇ are all included in the R302 binding interface. They all share similar chemical properties (small aliphatic sidechains) and the same conformation.