Fig. 1

Tumor control by Bispecific AcTaferon shows ample potential and is completely safe. A Design and layout of the Bispecific AcTaferon (AFN) construct. Targeting domains are N-terminally connected to the mutated cytokine. A HIS-tag was added for easy purification. B Schematic representation of the experimental setup. Mice were s.c. inoculated with 6 × 10⁵ B16 tumor cells. When palpable tumor was detected (day 7 after tumor inoculation) mice were treated 10 times with a perilesional administration with PBS (grey), 30 μg of the Bisp-AFN Clec9A-PDL1-AFN (green) or 30 μg of the Clec9A-PDL1-IFN wild type (red) according to the indicated arrows above the timeline. C, D Figures show tumor growth (C) as well as time to reach a B16 tumor volume of 150 mm³ (D). One representative experiment out of three is shown (6/group/experiment). E–F Weight change (E) and body temperature (F) are depicted at day 14 after B16 tumor inoculation. Values are relative to day 7, representing the start of the treatment schedule. The graphs show individual values ± SEM of 3 independent experiments (6/group/experiment). G One day after the last treatment in the B16 model, blood was collected, and hematological analysis (Hemavet) was performed. Leukocyte analysis is shown as a summary of individual values ± SEM of three independent experiments (6/group/experiment). H Schematic representation of the experimental layout using a Humanized Immune System (HIS) mouse model. Mice were s.c. inoculated with RL tumor cells. When palpable tumor was detected mice were treated with a perilesional administration with PBS (grey) or 30 μg of the fully humanised Bisp-AFN Clec9A-PDL1-AFN (green) according to the indicated arrows above the timeline. Intraperitoneal administration of Flt3L is indicated by the blue arrows underneath the timeline. I-J Figures show tumor growth (I) as well as time to reach an RL tumor volume of 150 mm³ (J) in an HIS setting. K The graph shows RL tumor growth in absence of a human immune system (Non-HIS NSG mice). One representative experiment out of two is shown (5–6/group/experiment). Tumor growth (C) was analyzed using Two-way ANOVA with Tukey’s multiple comparisons test. For the HIS/non-HIS model (I, K), tumor growth was analyzed at day 24 using unpaired two-tailed student t-test. Black lines underneath the X-axis depict the treatment time. Time to reach a specific tumor size (D, J) is represented in a Kaplan Meier plot compared by log-rank (Mantel-Cox) test. Bar plots (E, F, G) were analyzed using One-way ANOVA Kruskal–Wallis test with Dunn’s multiple comparisons test. * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001