Fig. 3: Anti-PD1-mIL12mut2 acquires enhanced bioactivity via αPD-1-mediated cis-binding. | Nature Communications

Fig. 3: Anti-PD1-mIL12mut2 acquires enhanced bioactivity via αPD-1-mediated cis-binding.

From: Anti-PD-1 cis-delivery of low-affinity IL-12 activates intratumoral CD8+T cells for systemic antitumor responses

Fig. 3

a HEK-mPD-1 cells were incubated with serially diluted hIgG1, αEGFR-mIL12mut2, αPD1-mIL12mut2, or anti-PD-1 antibody in vitro. Protein binding to HEK-mPD-1 cells was detected by flow cytometric analysis (n = 4 replicates). b The activity of αEGFR-mIL12mut2 or αPD1-mIL12mut2 was detected using HEK-mPD-1 cells. For PD-1 blockade, 20 μg/ml anti-PD-1 antibody was added into the incubation system of HEK-mPD-1 cells and αPD1-mIL12mut2 (n = 3 replicates). c Different concentration of anti-PD-1 antibody was added into the incubation system of HEK-mPD-1 cells and αPD1-IL12mut2. The activity of αPD1-mIL12mut2 was detected (n = 3 replicates). Cell culture medium without fusion protein was used to incubate HEK-mPD-1 cells as negative control. d Pre-activated WT or PD-1 KO CD8+T cells were incubated with αEGFR-mIL12mut2 or αPD1-mIL12mut2 for 0.5 h in vitro. The p-STAT4 in CD8+T cells was detected by flow cytometric analysis (n = 2). e Pre-activated WT CD8+T cells were labeled with CTV. An equal number of CTV-labeled WT CD8+T cells and non-labeled PD-1 KO CD8+T cells were mixed and then incubated with αEGFR-mIL12mut2 or αPD1-mIL12mut2 for 0.5 h in vitro. The p-STAT4 in CD8+T cells was detected by flow cytometric analysis (n = 4 mice/group). MFI, mean fluorescence intensity. f The activity of hIL12wt, αEGFR-hIL12mut2, or αPD1-hIL12mut2 was examined using the HEK or HEK-mPD-1 cells (n = 3 replicates). All data are shown as mean ± SD from two to three independent experiments. The P value was determined by two-way ANOVA (e).

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