Fig. 1: ProCTLA-4 antibody shows improved toxicity.

a Schematic structure of ProCTLA4 antibody, which was created with BioRender (https://BioRender.com/90qugj2). b Human CTLA-4-overexpressing MC38 cells (MC38-hCTLA-4) were incubated with serial dilutions of Ipilimumab (Ipi), ProCTLA-4 antibody and MMP-digested ProCTLA-4 antibody (ProCTLA-4(MMP)). Protein binding to MC38-hCTLA-4 was detected by flow cytometry. c–e A549 tumor-bearing male NSS mice were transferred 5 million human peripheral blood mononuclear cells (PBMC) via intravenous injection (i.v.) on day 8 after tumor inoculation and were intraperitoneally (i.p.) treated with PBS or 200 μg Ipi or equimolar ProCTLA4 (n = 5/group) on days 9, 12 and 15 after tumor inoculation. The percentage of body weight change (c), the percentage of survival curve (d), and the level of IFN-γ in serum (e) were measured. f–i 10-day-old female and male hCTLA-4 KI mice were treated i.p. with anti-PD-1, anti-PD-1 plus Ipi, or anti-PD-1 plus ProCTLA-4 (n = 10/group), respectively, at a dose of 100 μg on days 10, 13, 16, and 19 after birth. Mice were euthanized on day 21 after the first treatment for analysis. The body weight change after treatment was measured (f). TNF (g) in serum was analyzed by CBA. Composite histopathology scores of the multi-tissues (h) are shown. Representative images of H&E-stained paraffin sections from the pancreas, colon and lung (i) are shown (anti-PD-1 (n = 10), anti-PD-1 plus Ipi (n = 7), or anti-PD-1 plus ProCTLA-4 (n = 10)). Scale bar, 50 μm. All data are representative of two or three independent experiments. Statistical analysis was performed using non-linear best fits (b), two-way ANOVA with Tukey’s multiple comparisons test (c, f), log-rank (Mantel-Cox) test (d) or ordinary one-way ANOVA with multiple comparisons test (e, g, h).