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Abstract
Background Toripalimab is a PD-1 targeting humanized IgG4 monoclonal antibody (mAb) that is currently pending approval by US Food and Drug Administration (FDA) for first-line treatment of nasopharyngeal carcinoma (NPC) in combination with chemotherapy. While currently approved PD-1 mAbs have demonstrated significant clinical benefit particularly in patients with PD-L1 expressing tumors, in three on-going clinical trials, toripalimab in combination with chemotherapy has demonstrated good clinical efficacy irrespective of the PD-L1 status. Thus, we investigated the molecular and functional characteristics of toripalimab and compared it to pembrolizumab, a PD-1 mAb that is approved in the largest number of indications in the PD-1 mAb class.
Methods Post-hoc analysis was performed for three clinical trials namely JUPITER-02, JUPITER-06 and CHOICE-01, where patients were treated with toripalimab plus chemotherapy or placebo plus chemotherapy. Two PD-L1 scoring criteria: tumor proportion score (TPS) or combined positive score (CPS) were utilized. Binding affinity and kinetics for PD-1 mAbs toripalimab and pembrolizumab were determined by performing surface plasmon resonance (SPR) experiments. The ability of the PD-1 mAbs to activate T cells was characterized using T-cell based assays: 1) staphylococcal enterotoxin B (SEB) stimulated human peripheral blood mononuclear cells (PBMCs) and 2) anti-CD3 and anti-CD28 activated human CD8+ T cells. Efficacy was further tested in an ex-vivo system using dissociated tumor cells from treatment naïve non-small cell lung cancer (NSCLC) patients in the presence of anti-CD3/CD28 antibodies. Activation of PD-1 receptor by the PD-1 mAbs was determined using a Jurkat cell based SHP1/SHP2 recruitment assay.
Results Toripalimab plus chemotherapy improved overall survival (OS) irrespective of PD-L1 status in NPC (JUPITER-02), advanced NSCLC (CHOICE-01) and advanced esophageal squamous cell carcinoma (JUPITER-06) (table 1, figure 1). Toripalimab exhibited 12-fold higher binding affinity for PD-1 (figure 2) and significantly enhanced T cell activation in-vitro when compared to pembrolizumab (figure 3 and figure 4). Furthermore, toripalimab treatment showed an upregulated IFN-g gene signature in comparison to pembrolizumab in the ex-vivo system with NSCLC tumors (figure 5 and figure 6). Lastly, toripalimab demonstrated lower agonistic potential than pembrolizumab upon binding to PD-1, by recruiting lower levels of SHP-1 and SHP-2 in Jurkat-PD-1 cells (figure 7).
Conclusions Our study demonstrates that toripalimab is differentiated from pembrolizumab with stronger PD-1 binding, more potent in-vitro T cell activation and lower agonistic potential. These characteristics of toripalimab present it as a next generation PD-1 checkpoint inhibitor with potential for favorable clinical outcomes in treating cancer patients irrespective of their PD-L1 status.
Acknowledgements We thank the patients, their families and caregivers for participating in the toripalimab clinical studies; and all the investigators and their site personnel. We thank Marc Pondel, Rosh Dias, Nathalie Vandenkoornhuyse-Yanze and the Coherus Scientific Advisory Board members for critical review of the abstract.
Trial Registration NCT03581786, NCT03856411, NCT03829969
Ethics Approval The trial protocols were approved by an institutional review board or ethics committee at each participating site.
Post hoc analysis of the efficacy of treatment with toripalimab plus chemotherapy according to PD-L1 expressing subgroups in NPC, NSCLC and ESCC patients. Kaplan-Meier estimates of OS are shown to compare the toripalimab plus chemotherapy arm with the placebo plus chemotherapy arm in (A) NPC for PD-L1 TPS ≥ 1% and the PD-L1 TPS < 1% subgroups, (B) NSCLC for PD-L1 TPS ≥ 1% and the PD-L1 TPS < 1% subgroups, (C) ESCC for PD-L1 CPS ≥ 1 and the PD-L1 CPS < 1 subgroups. Censored patients are marked with ’I’ in the graph. Numbers of patients at risk at indicated time points are shown below the x axis. Number of events, median OS, hazard ratio for death and nominal p values are shown in the table below the Kaplan-Meier curves. CPS, combined positive score; ESCC, esophageal squamous cell carcinoma; HR, hazard ratio; NE, not estimated; NSCLC, non-small cell lung cancer: NPC, nasopharyngeal carcinoma; OS, overall survival; PD-L1, programmed cell death ligand 1; TPS, tumor proportion score.
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 0.93–59.5 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 0.63–20.3 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 dissociation and kinetic rate constants (ka, kd, and KD) from three replicate experiments for PD-1 binding to toripalimab and pembrolizumab.
Differences in levels of cytokines released by SEB activated human peripheral blood mononuclear cells in the presence of anti-PD-1 antibodies. PBMCs from nine healthy donors were cultured with 100 ng/mL SEB in the presence of 10, 3.3 or 1.1 μg/mL anti-PD-1 antibody (Ab): pembrolizumab (pembro) or toripalimab (tori) or isotype Ab control (Ctrl) in triplicate. After 3 days, cell supernatants were collected to examine IFN-y (A) and IL-2 (B) levels by ELISA. Graphs indicate relative fold change (mean ± SEM, n = 9) in cytokine secretion in the presence of pembro or tori relative to Ctrl. Supernatants from PBMCs cultured with 100 ng/mL SEB in the presence of 3.3 μg/mL Ab: pembro or tori or Ctrl in triplicate were tested by performing Luminex assays to examine Th1 cytokines (C), Th2 cytokines (D), Th17 cytokines (E), myeloid derived cytokines (F), IL-9, IL-10 (G) and ratio of IFN-/IL-10 and TNF-/1L-10 (H). Each point in the graphs represents an individual donor, Mean + SEM, n = 9. Statistical analysis was performed using One-way ANOVA with Tukey’s multiple comparisons tests for A-G and using paired t test for H. P < 0.05 is considered significant.
Toripalimab is more potent than pembrolizumab in enhancing IFN-y secretion in CD3/CD28 activated naïve human CD8+ T cells. Naïve CD8+ T cells from seven healthy donors were activated with human anti-CD3 (0.5 μg/mL) and human anti-CD28 (0.5 μg/mL) immobilized on the plate surface. 10 μg/mL of isotype control Ab (Ctrl), pembrolizumab (pembro) or toripalimab (tori) in duplicate wells. IFN-y levels in cell culture supernatant was quantified on day 3 of activation using ELISA. (A) IFN-y levels from seven donors. (B) Fold change in concentration of IFN-y relative to the Ctrl. p values in A were calculated using one-way ANOVA followed by Tukey’s multiple comparison test and in B were calculated via paired t test. P < 0.05 is considered significant.
Toripalimab more positively modulates genes associated with IFNG production than pembrolizumab in dissociated NSCLC tumor cells isolated from treatment naïve patients at the 24-hour time point. (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 genesets. The normalized expression scores (NES) from pathways for each PD-1 Ab (tori or pembro) treatment compared to control at each timepoint 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 hr timepoint for ’Positive regulation of IFNG production’ with the PD-1 Abs. (D) A Venn diagram comparing the core-enrichment genes from the 24 hr timepoint 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). 7 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).
Toripalimab induces an elevated IFN-y gene signature in dissociated NSCLC tumor cells isolated from treatment naïve patients at the 24-hour time point. (A) Custom immune-cell and interferon response pathway analysis. All NES scores were filtered by an exploratory p.adjust < 0.15. (B) Leading edge plots of ’Hallmark_interferon_response’ following toripalimab (tori, top) or pembrolizumab (pembro) treatment at 24 hr. (C) Heatmap of 94 core-enrichment genes from the interferon response pathway after 6 and 24 hr treatments with pembro and tori. NES, normalized expression score; p.adj, p-adjusted, (Bonferonni-Hochberg); log2FC, log2-fold-change; iso6, isotype at 6 hr, iso24, isotype at 24 hr; Core.enrichment, core-enrichment genes from the leading edge’s subset of genes from the geneset are those genes that drive the enrichment score in the GSEA analysis. T06, 6 hr timepoint; T024, 24 hr timepoint.
Toripalimab recruits lower levels of SHP-1 and SHP-2 In PathHunter Jurkat PD-1 cells. (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.
Patient demographics and trial registration numbers for the clinical trials in figure 1
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