Figure S8 from Improving Intracellular Delivery of an Antibody–Drug Conjugate Targeting Carcinoembryonic Antigen Increases Efficacy at Clinically Relevant Doses In Vivo

Binding to CEACAM5 and CEACAM6. Binding of XAb and hMN14 were tested against CEACAM5 and CEACAM6. Mouse and human Fc binding beads (Bangs Laboratories) were incubated with XAb and hMN14, respectively. Then varying concentrations of biotinylated CEACAM5 or CEACAM6 were added, followed by streptavidin AF647 to quantify CEACAM binding (therefore, the affinity measured is monovalent). Fluorescence is normalized to adjust for different numbers of binding sites on mouse and human beads. The XAb binds both CEACAM5 and CEACAM6 (KD = 1.03 and 1.04 nM, respectively). hMN-14 only binds CEACAM5 (KD = 2.2 nM). However, XAb does not bind HT-1080 cells but binds HT-1080 cells transfected with CEACAM5 at a higher level than other CEACAM5 antibodies (Fig. S2, (1)). The results are consistent with XAb binding multiple epitopes on CEACAM5 to crosslink the receptor and induce rapid internalization. In addition, bivalent binding of two different antigens would not be anticipated to drive clustering-based internalization.