Fig 2: CD40 binding and activation retain the same after removing the Fc region of MAB273. n = 3, mean ± SEM. A Cartoon shows the process of generating Fab and F(ab’)2 fragments of MAB273. B ELISA results show the CD40 binding capacity of complete MAB273, its Fab or F(ab’)2 fragments, the readouts of OD values are displayed by using anti-Fab/F(ab’)2 (left) or anti-Fc (right) secondary antibodies, respectively. C Surface expression levels of CD40 on human PBMCs were evaluated by flow cytometry after exposure with complete MAB273, its F(ab’)2 or Fab fragments. The concentrations used were converted to nM for equal comparison. D–E. Human PBMCs were stimulated with complete MAB273, F(ab’)2, Fab or TLR7/8L (positive control). Costimulatory markers CD80, CD70 and LN homing marker CCR7 on B cells (D) and MDCs (E) were evaluated by flow cytometry. F Human PBMCs were stimulated with complete MAB273, F(ab’)2, Fab or CpG (positive control) for 5 days. B cell proliferation is displayed as the number of CellTrace Violet negative B cells per 106 B cells. Representative flow cytometry plots are shown

Fig 3: MAB273 shows potent CD40 binding and activation capacities in rhesus macaque PBMCs in vitro. Rhesus PBMCs were stimulated with MAB273, CP-870,893, isotype control antibody, the F(ab’)2 fragment of MAB273 (similar molar concentrations to complete MAB273) or TLR7/8L (positive control). A–D Surface expression levels of CD40 and costimulatory marker CD80 on B cells (A) and MDCs (C) were evaluated by flow cytometry. n = 3, mean ± SEM. Representative flow cytometry histograms of CD80 on B cells (B) and MDCs (D) are shown. E Rhesus PBMCs were stimulated with MAB273, CP-870,893, isotype control antibody, the F(ab’)2 fragment of MAB273 (similar molar concentrations to complete MAB273) or CpG (positive control) for 5 days. B cell proliferation is displayed as the number of CellTrace Violet negative B cells per 106 B cells. Representative flow cytometry plots are shown. n = 3 + 3, mean ± SEM. F Levels of cytokines (IL-12 p40, IL-6, TNF and IFN-γ) were measured by ELISA, supernatants used were taken from (A) and (C). n = 3, mean ± SEM

Fig 4: In vivo biodistribution of MAB273. A Rhesus macaques (n = 3) were administered 0.1 mg/kg s.c. of Alexa Fluor 680-MAB273 in the skin above the left quad and 0.9% saline solution s.c. in the skin above the right quad. The cartoon shows the sites of immunization and sampling performed after 24 h (n = 1) or 48 h (n = 2). B Histograms show Alexa Fluor 680-MAB273 signal on different cell populations in different tissues of one representative animal. Control = peripheral blood B cells from the same animal before immunization with labeled MAB273. C MAB273 + CD45 + cells normalized by counting beads at site of injection or draining LNs. n = 3, mean ± SEM. Compiled graphs show one animal that was sampled at 24 h (open circle), and two animals that were sampled at 48 h (closed circle). D Pie charts show proportion of different CD45 + immune cells targeted with MAB273 at the injection site, the primary and secondary draining LNs. The values are the average of three animals. E–F Expression of CD40 at site of injection and draining LNs (E) as well as PBMCs (F). Compiled data were evaluated by flow cytometry. Geometric mean fluorescence intensity (MFI) is shown. n = 3, mean ± SEM

Fig 5: In vivo innate immune activity in rhesus macaques. A Outline for toxicity and safety study in rhesus macaques administered 1 mg/kg i.v., 0.1 mg/kg i.v., or 0.1 mg/kg s.c. (n = 2 per group). B Levels of MAB273 in plasma over time (left), AUC (area under curve, right) is calculated after normalizing (left) to linear axes. n = 2 per group. C–E Surface expression levels of CD40 (C), costimulatory marker CD80 and LN homing marker CCR7 on B cells (D) and MDCs (E) were evaluated by flow cytometry over time. n = 2 per group. F Systemic levels of pro-inflammatory cytokines (IL-12 p40, IL-6, IFN-γ) in plasma over time. n = 2 per group