Recombinant Anti-Human EGFR VHH Single Domain Antibody (EgA1)

Figure 1 Functional characterization of purified EgA1 ATTACK.

Biolayer interferometry (BLI)-derived sensorgrams (in black) for the interaction between immobilized EGFR-Fc and EgA1-based antibodies.

Harwood, S. L., Alvarez-Cienfuegos, A., Nuñez-Prado, N., Compte, M., Hernández-Pérez, S., Merino, N., ... & Mikkelsen, K. (2018). ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy. Oncoimmunology, 7(1), e1377874.

Figure 2 Functional characterization of purified EgA1 ATTACK.

The binding to EGFR on the cell surface of HeLa cells by cetuximab- and EgA1-based antibodies at 0.1, 0.32, 1, and 10 nM, measured by FACS and normalized to the binding at 10 nM.

Harwood, S. L., Alvarez-Cienfuegos, A., Nuñez-Prado, N., Compte, M., Hernández-Pérez, S., Merino, N., ... & Mikkelsen, K. (2018). ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy. Oncoimmunology, 7(1), e1377874.

Figure 3 Functional characterization of purified EgA1 ATTACK.

The binding to CD3 on the cell surface of Jurkat cells by OKT3- and EgA1-based antibodies at 0.1, 1, 3.2, and 10 nM, measured by FACS and normalized to the binding at 10 nM.

Harwood, S. L., Alvarez-Cienfuegos, A., Nuñez-Prado, N., Compte, M., Hernández-Pérez, S., Merino, N., ... & Mikkelsen, K. (2018). ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy. Oncoimmunology, 7(1), e1377874.

Figure 4 EGa1-L cause a massive sequestration of cell surface EGFR.

Immediately after a 4 h incubation, cells were collected and stained for cell surface EGFR.

Oliveira, S., Schiffelers, R. M., van der Veeken, J., van der Meel, R., Vongpromek, R., en Henegouwen, P. M. V. B., ... & Roovers, R. C. (2010). Downregulation of EGFR by a novel multivalent sdAb-liposome platform. Journal of Controlled Release, 145(2), 165-175.

Figure 5 EGa1-L induce downregulation of EGFR. 14C cells were incubated for 4 h at 37 °C with liposomes containing two different ratios of sdAb to TL (0.4 and 0.8 nmol/ μmol TL) at 0.5 mM TL concentration.

Ratio's of the intensity of EGFR bands normalized to β-actin bands (non-treated set to 100%), from three independent experiments, are shown for lysates prepared after 2 days of chase following the 4 h pulse incubation.

Oliveira, S., Schiffelers, R. M., van der Veeken, J., van der Meel, R., Vongpromek, R., en Henegouwen, P. M. V. B., ... & Roovers, R. C. (2010). Downregulation of EGFR by a novel multivalent sdAb-liposome platform. Journal of Controlled Release, 145(2), 165-175.