Figure 4. GFRA1 is a quickly internalized target suited to antibody-drug conjugate targeting.

(A) An internalization assay was performed following conjugation of 10H9 or IgG1 control with a pH-sensitive GFP dye and incubation with Ad293-GFRA1 and parental cells. Cells were imaged over a 5-hour time course to reveal the fast kinetics of 10H9 internalization and trafficking to lysosomes in target-positive cells. (B) In vitro efficacy of SG3249-conjugated 10H9 (GFRA1-PBD) was evaluated in multiple GFRA1-positive cell-line models to demonstrate target-mediated cell-killing. Representative experiments are shown and the values indicate mean + SEM. (C) Bystander activity of the GFRA1-PBD conjugate was assessed by FACS following 3 days of target-positive (FITC positive) and negative (PE positive) cell co-culture and ADC treatment. FACS plots for FITC and PE expression and total cell number at harvest are displayed for multiple titration points. ADC, antibody-drug conjugate; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; PBD; pyrrolobenzediazepine dimer; PE, proline-glutamate.