7.

NanoBondy covalent conjugation at the cell surface. (A) Western blotting of NanoBondy reaction at the cell surface. Anti-CD45 NanoBondy at 5 μM was incubated with YTS cells or Expi293F cells for 1 h at 37 °C ± calcium. Covalent conjugation was evaluated by Western blot using an anti-VHH polyclonal antibody to detect the NanoBondy. Anti-IgG NanoBondy or hydroxylamine to react with the anhydride provided negative controls. Western blot to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the loading control. The complete GAPDH blot is presented in Figure S6A. The experiment was conducted once. (B) YTS cells were stained as in (A), except with 25 μM anti-CD45 NanoBondy, and analyzed by Western blot using an anti-CD45 antibody. The CD45 band demonstrates an upward shift upon covalent conjugation with the a-CD45 NanoBondy. The full-length GAPDH blot is presented in Figure S6B. The experiment was conducted once. (C) Western blotting of the DuoBondy reaction on CD8⁺ T cells. DuoBondy (WT) or DuoBondy (DA) at 1 μM was incubated with CD8⁺ T cells for 40 min at 37 °C ± calcium. Covalent conjugation was evaluated by Western blot using an anti-VHH polyclonal antibody to detect the DuoBondy. Representative blot from two independent experiments. (D) DuoBondy consists of a nanobody binder (Nb102c3) to PD-1 (dark blue) fused N-terminally to the established covalently reacting anti-CD45 NanoBondy (purple) with SPM in orange.