Figure 1.

Engineered mouse anti-PD-1 mAbs retain equivalent in vitro binding properties and function. (A) Mouse PD-1-transfected HEK293F cells were incubated with the indicated anti-PD-1 mAb isotypes at a range of concentrations prior to staining with a PE- or APC-labeled secondary antibodies. Data show mean fluorescence intensity (MFI) as a percentage of maximum. (B) Cells were incubated with anti-PD-1 mAb as in (A) in the presence of 1 µg/mL AF488-conjugated rat anti-PD-1 mAb. Data are presented as MFI of the rat anti-PD-1 relative to the concentration of competitive mouse mAb. Data in (A) and (B) show one representative experiment of two. Bars represent mean±SEM of triplicates. (C) Surface plasmon resonance analysis illustrating anti-PD-1 mAb binding to mouse PD-1 and blockade of PD-L1/2. Mouse anti-PD-1 mAbs (100 µg/mL) were passed over His-tagged PD-1 captured with an anti-His mAb. Recombinant PD-L1 or PD-L2-Fc were passed over (25 µg/mL) to demonstrate PD-1 binding or blockade. (D) Purified CD8 T cells from C57BL/6 mice were incubated with 1 µg/mL plate-bound anti-CD3, 5 µg/mL plate-bound PD-L1-Fc or irrelevant controls, and 5 µg/mL soluble mouse anti-PD-1 mAbs or irrelevant isotypes. Proliferation was assessed by [3H]-thymidine incorporation. Data show combined means from two independent experiments. Bars represent mean±SD. (E) Activated CFSE-labeled murine splenic T cells were opsonized with anti-PD-1 isotypes (filled bars) or irrelevant controls (open bars) prior to coculture with BMDMs. Experiment performed twice in triplicates. Bars show mean±SD. Student T-test, **p<0.01. BMDM, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; mAb, monoclonal antibodies; PD-1, programmed cell-death.