Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit

Abstract

Blockade of immune checkpoint pathways by programmed cell death protein 1 (PD-1) antibodies has demonstrated broad clinical efficacy against a variety of malignancies. Sintilimab, a highly selective, fully human monoclonal antibody (mAb), blocks the interaction of PD-1 and its ligands and has demonstrated clinical benefit in various clinical studies. Here, we evaluated the affinity of sintilimab to human PD-1 by surface plasmon resonance and mesoscale discovery and evaluated PD-1 receptor occupancy and anti-tumor efficacy of sintilimab in a humanized NOD/Shi-scid-IL2rgamma (null) (NOG) mouse model. We also assessed the receptor occupancy and immunogenicity of sintilimab from clinical studies in humans (9 patients with advanced solid tumor and 381 patients from 4 clinical studies, respectively). Sintilimab bound to human PD-1 with greater affinity than nivolumab (Opdivo®, MDX-1106) and pembrolizumab (Keytruda®, MK-3475). The high affinity of sintilimab is explained by its distinct structural binding mode to PD-1. The pharmacokinetic behavior of sintilimab did not show any significant differences compared to the other two anti-PD-1 mAbs. In the humanized NOG mouse model, sintilimab showed superior PD-1 occupancy on circulating T cells and a stronger anti-tumor effect against NCI-H292 tumors. The strong anti-tumor response correlated with increased interferon-γ-secreting, tumor-specific CD8+ T cells, but not with CD4+ Tregs in tumor tissue. Pharmacodynamics testing indicated a sustained mean occupancy of ≥95% of PD-1 molecules on circulating T cells in patients following sintilimab infusion, regardless of infusion dose. Sintilimab infusion was associated with 0.52% (2/381 patients) of anti-drug antibodies and 0.26% (1/381 patients) neutralizing antibodies. These data validate sintilimab as a novel, safe, and efficacious anti-PD-1 mAb for cancer immunotherapy.

Keywords: PD-1; antibody; cancer immunotherapy; immunogenicity; receptor occupancy; sintilimab.

Figures

Figure 1.

Higher binding affinity of sintilimab to human PD-1 compared with MDX-1106 and MK-3475. (a) Binding affinity and kinetics of anti-PD-1 mAbs to human PD-1 determined by surface plasma resonance. Sintilimab, MDX-1106 and MK-3475 were performed with human PD-1 in multi-cycle kinetics and raw data were fitted using 1:1 binding model. (b) Meso Scale Discovery analyses of the binding between human PD-1 and anti-PD-1 mAbs. (c) Individual residue of sintilimab forming the hydrophobic core was mutated to alanine. SPR analysis reveals residue mutations impair sintilimab binding to the hPD-1 protein.

Figure 2.

Sintilimab showed in vitro and in vivo higher levels of PD-1 receptor occupancy. Human PBMC were stimulated to express PD-1 before incubation with sintilimab, MDX-1106 or MK-3475. Flow cytometry results showing proportions of CD3+ T cells that bind with different anti-PD-1 mAbs (a) and the mean fluorescence intensity of PD-1 (b). Data are expressed as the means ± SE of three independent experiments. (c) The effects of anti-PD-1 mAbs on mixed lymphocyte reaction (MLR) response. CD4+ T cells isolated from human PBMC were co-cultured with mature monocyte-derived dendritic cells at a ratio of 10:1 in the presence of different concentrations of anti-PD-1 mAbs. Twelve hours later, unbound mAbs was removed. Cells were co-cultured for 4 more days and the concentration of IL-2 in cultural supernatant was detected by Cisbio kit. In NOG mice reconstituted with human immune cells, PD-1 receptor occupancy on circulating CD3+ T cells 24 h (d) and 72 h (e) after anti-PD-1 mAbs intraperitoneal injection at doses of 1, 3 and 10 mg/kg (n ≥ 3 mice/group). (f) Mean (± SE) serum concentration-time profiles following a single IV administration of 10 mg/kg sintilimab, MDX-1106 or MK-3475 to hPD-1 knock-in mice (n = 3 animals per group).

Figure 3.

Better tumor control and T cell response of sintilimab treatment in NCI-H292 tumor mouse model reconstituted with human immune cells. Six days after human PBMC intravenous implantation, NCI-H292 tumor cells were implanted subcutaneously into the right flanks of NOG mice. (a) Tumor growth of NCI-H292 tumor-bearing mice treated with human IgG (10 mg/kg), sintilimab (0.1, 1 and 10 mg/kg), MDX-1106 (10 mg/kg) or MK-3475 (10 mg/kg). (b) Tumor weights at the end of the study. Three weeks after NCH-H292 cells implantation, tumors were collected and total counts of CD3+ T cells per gram tumor (c), CD8+ T cells per gram tumor (d), CD8/Treg ratios (e), IFN-γ producing tumor-specific CD8+ cells per gram tumor (f) were quantified (n ≥ 5 mice/group).

Figure 4.

PD-1 receptor occupancy on circulating CD3+ T cells in patients with advanced solid tumors following infusion with sintilimab. Sintilimab was administered at doses of 1, 3 or 10 mg/kg on study day 0. Blood was collected on day 0, 1, 7, 13 and 27.