Fig 1: Toripalimab positively modulates genes associated with IFN-γ production in dissociated NSCLC tumor cells isolated from treatment naïve patients. A Schematic diagram of expression profiling of dissociated tumor cells treated with toripalimab (tori), pembrolizumab (pembro) or isotype control antibody. B GSEA analysis of Gene Ontology Biological Process gene sets. The normalized expression scores (NES) from pathways for each PD-1 Ab (tori or pembro) treatment compared to control at each time point are shown. Pathways were filtered with p.adjust < 0.05 followed by combinatorial or unique filtering by the various treatment comparisons. C A Venn diagram comparing the core enrichment genes from the 24 h time point for “Positive regulation of IFNG production” with the PD-1 Abs. D A Venn diagram comparing the core enrichment genes from the 24 h time point for “Leukocyte differentiation.” A table of these core enrichment genes is included in the Supplement Fig. S4. E Leading edge plots comparing “Positive regulation of IFNG production” by tori (top) and pembro (bottom). F Heatmap of the core enrichment genes of the “Positive regulation of IFNG production” pathway after tori and pembro treatment. Sixteen of the 41 combined core enrichment genes are common to both PD-1 Ab treatments (cyan). Seven of the core enrichment genes are unique to pembro treatment (light blue) while 18 of the core enrichment genes are unique to tori treatment (tan)
Fig 2: Representative SPR analysis of human PD-1 binding to PD-1 antibodies. A Biacore sensorgrams of PD-1 binding to covalently immobilized toripalimab. PD-1 was injected in triplicate for 3 min in a range from 0.93 to 59.5 nM with dissociation followed for 5 min. B PD-1 at the highest concentration of 59.5 nM was injected in triplicate for 3 min with dissociation followed for 90 min. C Sensorgrams of PD-1 binding to covalently immobilized pembrolizumab. PD-1 was injected in triplicate for 1 min in a range from 0.63 to 20.3 nM with dissociation followed for 3 min. The equilibrium dissociation constants and kinetic rate constants for each interaction are noted in the panels. All sensorgrams were globally fit (red lines) to a 1:1 interaction model including a term for mass transport. D Average kinetic rate and equilibrium dissociation constants (ka, kd and KD) from three replicate experiments for PD-1 binding to toripalimab and pembrolizumab
Fig 3: Toripalimab recruits lower levels of SHP1 or SHP2 than pembrolizumab in PathHunter® Jurkat PD-1 cell lines. A Schematic representation of the experimental system. PathHunter® Jurkat PD-1 cell lines expressing the SHP1 or SHP2 signaling assay system were cocultured with U2OS cells opsonized with increasing doses of isotype Ab (Ctrl), pembrolizumab (pembro) or toripalimab (tori) (dose range 0.01–10 µg/mL) in triplicate. Chemiluminescence signal detected as relative luminescent units (RLU) indicates SHP1 or SHP2 recruitment to PD-1. EA Enzyme acceptor, ED enzyme donor. B Representative dose–response curve for SHP1 and SHP2 recruitment in the Jurkat PD-1 SHP1 and SHP2 signaling cell lines; C Graphical representation of the EC50 and EC90 values calculated from dose–response curves from 5 independent experiments. Data are shown as mean ± SEM, p < 0.05 considered significant
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