Fig. 4.

Therapy studies with F8-4-1BBL in format 5. (A) Preventive therapy in WEHI-164 fibrosarcoma-bearing mice was started on day 5 when the tumors reached a volume of 40 mm³. The tumor sizes are shown as mean + SD (n = 5). The body weight data are represented as mean body weight change ± SD for each group. (B) Therapy in WEHI-164 fibrosarcoma-bearing mice was started on day 7 when the tumor volume was >80 mm³. The tumor sizes are shown as mean + SD (n = 5). The statistical results of regular two-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism 8.4.1 on day 13 are shown. ns, not significant; *P = 0.0427. The body weight data are represented as mean body weight change ± SD for each group. (C) In CT26-colon carcinoma-bearing mice, therapy was started on day 7 when the tumor volume exceeded 80 mm³. The tumor sizes are shown as mean + SD (n = 5). The result of a regular two-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism 8.4.1 revealed that as of day 13, the tumor size was significantly smaller in the mice treated with PD-1 blockade compared with the mice treated with saline (***P = 0.0004). The body weight data are represented as mean body weight change ± SD for each group. (D) In MC-38 colon carcinoma-bearing mice, therapy was started on day 7 when the tumor volume exceeded 75 mm³. The tumor sizes are shown as mean + SD (n = 5 or 6). The result of a regular two-way ANOVA followed by a Tukey’s multiple comparison test using GraphPad Prism 8.4.1 is shown for day 12 (***P < 0.001). The body weight data are represented as mean body weight change ± SD for each group. (E) GL-261 was implanted orthotopically in C57BL/6 mice. Subsequently, mice were treated i.v. with saline, F8-4-1BBL, αPD-1, or the combination starting on day 5. Tumor size was assessed at day 12 after tumor implantation. (Left) MRI with tumors outlined in red. (Right) Quantification of tumor perimeters (n = 5). The survival data are presented as Kaplan–Meier plots (n = 5). Results from a Gehan–Breslow–Wilcoxon test reveal significant (P = 0.0036) differences between the survival curves of the different treatment groups. The body weight data are represented as mean body weight change ± SD for each group. Black arrows denote injections of the single agents, and gray arrows denote injection of F8-4-1BBL in the combination treatment. CR, complete response.