Pharmacokinetic and toxicity study

One female and one male cynomolgus monkeys were recruited to conduct the single-dose pharmacokinetic study and toxicity in NHPs. The monkeys received a dosage of 3 mg/kg 2MW4691 through a 0.5-h intravenous infusion. For the pharmacokinetics study, serums were collected immediately after infusion, and at 2, 8, 24, 48, 72, 96, 120, 168, 336, and 504-h post-administration. For the toxicity study, blood samples were collected before treatment and at 24, 72, and 168 h after transfusion. PBMCs were isolated from peripheral blood and then stained with specific markers of CD4 T cells, CD8 T cells, and Tregs for population analysis by FACS. Besides, the routine clinical observation, ethology, weight, and food consumption were monitored regularly.

The concentration of the target antibody in serum was measured using two different ELISA methods. Method A involved coating an ELISA plate with anti-human Fab (Sigma-Aldrich, USA) to capture serially diluted positive serum. This was then detected using HRP-labeled goat anti-human Fc (Jackson ImmunoResearch, USA). Method B comprised coating ELISA plate with anti-human Fab, adding serial dilutions of positive serum to the plate, adding biotin-labeled hCTLA-4 (ECD, his tag), detecting the reaction with streptavidin HRP (Thermo Fisher, USA). Concurrently, a standard binding response-concentration curve was established to calculate the serum antibody concentration.