IT delivery of anti-PD1 in nontumor-bearing mice
neurotoxicity
BRAIN
The safety of IT administration of anti-PD1 antibody was evaluated in nontumor-bearing immunocompetent mice. The dose of IT anti-PD1 used in the experiments was determined two ways. The first derivation was based on the use of a dose in mice that was equivalent by volume of CSF to 50 mg of IT anti-PD1 in humans, because 50 mg is a dose used with IT trastuzumab and IT rituximab, and in preliminary discussions was considered the maximum dose of IT nivolumab to be evaluated in the clinical trial. Based on differences in CSF volume of distribution, we determined that a dose of 6.5 μl (13 μg) of anti-PD1 would be equivalent to a dose of 50 mg of IT nivolumab in patients. The second approach was based on the differences in CSF volume of distribution between humans and mice (140 ml versus 36 µl), we determined that a dose of 13 µg of anti-PD1 in mice would be equivalent to a dose of 50 mg in patients. Murine IT dose based on this calculation was 6.5 μl of murine anti-PD1 concentration of 2,000 µl mlSixty-eight female only C57BL/6 mice were divided into three volume-matched (6.5 μl) treatment groups: (1) IT PBS control; (2) IT isotype control (13 µg; rat IgG2a; BioXCell, clone 2A3); and 3) IT mouse anti-PD1 antibody (13 μg; BioXCell, clone RMP1-14). Mice were killed at 48 h, 7 d and 14 d after IT injection. Brain and spinal cord tissue were collected from mice at all three time points for histological assessment for neurotoxicity. Brain regions and spinal cords were embedded in paraffin and sectioned at 5 µm and stained with H&E for histological observation. All IHC studies were performed on 5 µm sections. Samples were assessed for known inflammatory markers by IHC, including CD3 (SP7; Thermo Fisher, catalog no. RM-9107-S, dilution 1:100), CD8 (D4W2Z; Cell Signaling, catalog no. 98941, dilution 1:400), CD163 (polyclonal; ProteinTech, catalog no. 16646-1-A, dilution 1:200), CD56 (polyclonal; ProteinTech, catalog no. 14255-1-AP, dilution 1:3,000), CD15 (FUT4/1478R; Abcam, catalog no. ab218403, dilution 1:20), PD1 (D7D5W; Cell Signaling, catalog no. 84651, dilution 1:100) and PD-L1 (D5V3B; Cell Signaling, catalog no. 64988, dilution 1:100).