Figure 1.

In vitro characterization of PD-L1 targeting BiTE. (A) Schematic representation of PD-L1 BiTEs and control BiTEs. VL and VH domains of single-chain variable fragment (scFv) or VH domain of nanobody (NB) targeting PD-L1 or irrelevant antigens were linked to VH and VL domains of anti-CD3ε scFv by flexible glycine-serine linkers. An immunoglobulin signal peptide (SP) and hexahistidine (His) affinity tag are added at N-terminal and C-terminal, respectively. PBMC-derived T cells were directed to kill DLD-1 carcinoma cells (5:1) using PD-L1 BiTEs at a dose of 40 nM. Cell cytotoxicity (B) was measured after 48 hours in the presence or absence of T cells. (C and D) BiTE-mediated induction of CD25 (C) and CD69 (D) cultured alone or in the presence of DLD-1 cells was measured by flow cytometry. (E and F) CD69 and CD25 were measured on CD4+ and CD8+ T cells by flow cytometry. (G) Percentage of interferon-gamma (IFN-γ) positive CD4+ and CD8+ T cells were measured after 6 hours in coculture with DLD-1 cells (5:1) and BiTE-containing supernatants. Degranulation of CD4+ and CD8+ T cells following addition of BiTE containing supernatants in coculture of DLD-1 and T cells was measured by CD107a externalization after 6 hours. Externalization was assessed by coculture with a CD107a-specific antibody followed by flow cytometry analysis (H). Secretion of granzyme B and perforin by BiTE-activated CD4+ and CD8+ T cells to mediate target cell killing by apoptosis was measured at 24 hours (I and J). (K) Cytokines released into supernatants were quantified by ELISA. Data show mean±SEM of biological triplicates. Statistical significance was assessed by two-way analysis of variance followed by Bonferroni post hoc analysis. Significance was assessed versus untreated cells within the relevant group (**p<0.01 and ***p<0.001). BiTE, bispecific T cell engager; IL, interleukin; PBMC, peripheral blood mononuclear cell; PD-L1, programmed death-ligand 1.