Fig. 2
Generation of Nb-TriTE and Nb-BiTE. Schematic drawing of a Nb-BiTE molecule (A), a Nb-TriTE molecule (D) and the protein model for them was proposed by using the SWISS-MODEL website tool. (www.ExPASy.org/resources/swiss-model). The Nb-BiTE molecule consists of a FAP Nb that targets CAFs and a CD3ε Nb that engages T cells. The Nb-TriTE molecule was based on the Nb-BiTE molecule with the addition of a PD-1 Nb that blocks the PD-1/PD-L1 axis. B SDS‒PAGE analysis of purified Nb-BiTE protein was visualized by Coomassie blue staining. Lanes: M- molecular weight marker, 1- Nb-BiTE protein. C Western blot identification of purified Nb-BiTE protein was probed with anti-His-tag antibody, M- molecular weight marker; 1- Nb-BiTE protein. E SDS‒PAGE analysis of purified Nb-TriTE protein was visualized by Coomassie blue staining. Lanes: M- molecular weight marker, 1- Nb-TriTE protein. F Western blot identification of purified Nb-TriTE protein was probed with anti-His-tag antibody, M- molecular weight marker; 1-Nb-TriTE protein. G Binding analysis of Nb-BiTE to FAP-expressing HepG2-FAP cells and CD3-expressing human T cells at a concentration of 2 μg/mL measured by flow cytometry. H Binding analysis of Nb-TriTE to FAP-expressing HepG2-FAP cells, PD-1-expressing 293T-PD-1 cells and CD3-expressing human T cells at a concentration of 2 μg/mL measured by flow cytometry. Blocking assay using recombinant target antigens that substantially interfere with binding ability, representative histograms for each group. I The binding affinity of the Nb-TriTE was determined by SPR analysis. J Nb-TriTE simultaneous binding ability to two target antigens (hCD3ε/hFAP, hPD-1/hFAP and hCD3ε/hPD-1) was assessed by a two-step sandwich ELISA assay. K The effect of Nb-TriTE on blocking PD-1/PD-L1 interaction was assessed by ELISA. All data represent the mean ± standard deviation from 3 independent experiments