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. 2021 Oct 28;9(11):e003036. doi: 10.1136/jitc-2021-003036

Figure 1.

Figure 1

Production of Fc-deficient anti-PD-L1- F(ab′)2–IR700 and evaluation in vivo, and evaluation of in vitro NIR-PIT with anti-PD-L1- F(ab′)2–IR700 on various murine tumor cells. (A) Intravenously injected anti-PD-L1-IgG (100 μg) or anti-PD-L1- F(ab’)2 (100 μg) did not alter the percentage of antigen-presenting cells (APCs) in the spleen CD45-positive cells 1 day after administration. Data are the mean±SEM (n=5; p≥0.05, Student’s t-test). (B) (Left) EGFR expression was evaluated on high EGFR-expressed A431-luc-GFP cells with pan-IR700 by flow cytometry. (Right) In vitro EGFR-targeted NIR-PIT with pan-IR700 was measured by luciferase activity, which decreased in a NIR light dose-dependent manner. Data are the mean±SEM (n=4, ****p<0.0001, Student’s t-test). With a light dose of 4 (J/cm2), in vitro EGFR-targeted NIR-PIT destroyed all A431-luc-GFP cells. (C) (Left) PD-L1-expression was evaluated on MC38-luc with PD-L1-F(ab’)2-IR700 by flow cytometry. (Right) In vitro PD-L1-targeted NIR-PIT with PD-L1-F(ab’)2-IR700 on MC-38-luc was measured by luciferase activity, which decreased in a NIR light dose-dependent manner. Data are the mean±SEM (n=4, **p<0.01, Student’s t-test). Even with a light dose of 128 (J/cm2), in vitro PD-L1-targeted NIR-PIT destroyed only 40%–50% of MC38-luc cells. In vitro PD-L1-targeted NIR-PIT with PD-L1-F(ab’)2-IR700 on other murine tumor cells (LL/2-luc, Tramp-C2-luc, B16F0) was also evaluated (online supplemental figure 2B). (D) Microscopic observations before and after PD-L1-targeted NIR-PIT. MC38-luc cells were incubated with anti-PD-L1-F(ab’)2-IR700 for 6 hours and observed with a fluorescence microscope before and after NIR light irradiation (20 J/cm2). Photo-induced necrotic cell death was observed after exposure to NIR light at 20 min after NIR-PIT (scale bar, 10 µm). Other murine tumor cells (LL/2-luc, Tramp-C2-luc) was also observed as the same way as MC38-luc (online supplemental figure 2C). EGFR, epidermal growth factor receptor; NIR-PIT, near-infrared photoimmunotherapy; PD-L1, programmed death-ligand 1.