Fig. 2: Development and binding properties of the affinity-matured R114 antibody.
a Schematic representation of the structure guided affinity maturation of R023 to R114. The top panel shows a structural representation of the positions of R023 CDRs (shown as tubes) bound to soto-p7/A*11. The HLA moiety of the sotorasib p*MHC is shown in surface representation, while soto-p7 is represented as sticks (sotorasib) and tubes (p7). The side chains of R023 residues located at positions selected for the affinity maturation campaign are represented as spheres. The bottom panel shows CDR sequences of R023 and its descendant R114 clones, and a schematic representation of the affinity maturation process. R023 residues located at positions selected for the affinity maturation campaign are underlined, and mutations introduced in R114 are in bold. “Off-rate sorting” refers to Fluorescence-Activated Cell Sorting (FACS) of yeast libraries, performed upon staining aimed at the identification of binders with low dissociation from the target, as described below. b Binding of yeast-displayed scFv R023 and descendant matured clones to sotorasib p*MHC antigens in an “off-rate” setting. Target-bound yeast cells, after removal of unbound sotorasib p*MHCs, were incubated for 2 h with a 10-fold excess of a competitor molecule to prevent rebinding of dissociated target, and the signal from remaining bound molecules was measured. Different clones are indicated by colors (e.g., R023 is indicated by blue dots, and R114 is indicated by yellow dots). c Schematic representation of the TCE AETX-R114 in the (scFv)2-scFc format. d SPR sensorgrams of the interaction between AETX-TCEs and the indicated soto-p/MHC antigens. Biotinylated soto-p/MHCs were immobilized, and binding of soluble AETX-TCEs samples was measured using single-cycle kinetics. Kinetic values of fitted data are shown in the table below.
