Preclinical evaluation of the efficacy, pharmacokinetics and immunogenicity of JS-001, a programmed cell death protein-1 (PD-1) monoclonal antibody

Abstract

JS-001 is the first monoclonal antibody (mAb) against programmed cell death protein-1 (PD-1) approved by the China Food and Drug Administration (CFDA) into the clinical trails. To date, however, no pre-clinical pharmacological and pharmacokinetic (PK) data are available. In this study, we investigated the efficacy of JS-001 and conducted a preclinical PK study, including the monitoring of anti-drug antibodies (ADAs). We found that JS-001 specifically bound to PD-1 antigen with an EC₅₀ of 21 nmol/L, and competently blocked the binding of PD-1 antigen to PD-L1 and PD-L2 with IC₅₀ of 3.0 and 3.1 nmol/L, respectively. Furthermore, JS-001 displayed distinct species cross-reactivity: it could bind to the PD-1 antigen on the peripheral blood mononuclear cells (PBMCs) of humans and cynomolgus monkeys, but not to those of mice and woodchucks; the K_d values for the interaction between JS-001 and PD-1 antigens on CD8⁺ T cells of human and cynomolgus monkey were 2.1 nmol/L and 1.2 nmol/L, respectively. In vitro, treatment with JS-001 (0.01-10 μg/mL) dose-dependently stimulated human T cell proliferation, as well as IFN-γ and TNF-α secretion. In HBsAg-vaccinated cynomolgus monkeys, the expression of PD-1⁺/CD4⁺ and PD-1⁺/CD8⁺ was significantly elevated, intramuscular injection of JS-001 (1 and 10 mg/kg) resulted in dramatic decreases in PD-1⁺/CD4⁺ and PD-1⁺/CD8⁺ expression in a dose-dependent manner, which was supported by PD-1 receptor occupancy (RO) results. In the PK study, 18 cynomolgus monkeys treated with single, ascending doses of 1, 10, and 75 mg/kg, and another 6 cynomolgus monkeys received 10 mg/kg successive administration. The plasma clearance of JS-001 followed a linear PK profile with single administration in the 1 and 10 mg/kg groups and a non-linear PK profile in the 75 mg/kg group. In the successive 10 mg/kg administration group, no drug accumulation was observed. But the AUC from the last exposure was lower than that of the first administration, which was probably due to the production of ADAs, as demonstrated in immunogenicity study. These non-clinical data are encouraging and provide a basis for the efficacy and safety of JS-001 in clinical trials.

Figure 1

In vitro activity of JS-001. (A) In vitroFCA experiments demonstrate that JS-001 specifically binds to the PD-1 antigen and (B) efficiently blocks the PD-1/PD-L1 and PD-1/PD-L2 interactions. Apparent affinity was determined using median fluorescence intensity (MFI). (C) The T cell proliferation response was performed using flow cytometry to determine the CFSE intensity. *P<0.05, **P<0.01 vs hIgG4. ^#P<0.05, ^##P<0.01 vs Nivolumab. (D) IFN-γ and (E) TNF-α levels were determined using ELISA. Nivolumab, positive control; hIgG4, negative control. *P<0.05, **P<0.01 vs hIgG4. ^#P<0.05, ^##P<0.01 vs Nivolumab. Data are shown as the mean±SD from 3 independently analyzed experiments.

Figure 2

Species cross-reactivity of JS-001 and its affinity to PD-1 molecules in humans and cynomolgus monkeys. (A) An ELISA assay was used to determine the species cross-reactivity of JS-001 with antigens derived from different species. FCA analysis confirmed the affinity of JS-001 and PD-1 molecules to human and cynomolgus monkey PBMCs. (B) JS-001 binds to the PD-1 molecules on human PBMCs and (C) cynomolgus monkey PBMCs (CD4⁺ and CD8⁺ T cells). Data are given as the mean±SD from 3 independently analyzed experiments.

Figure 3

JS-001 decreased the expression of PD-1⁺/CD4⁺ and PD-1⁺/CD8⁺T cells in an HBsAg-immunized primate model. HBsAg vaccination was performed at d 0 and the HBsAb and PD-1 expression levels were evaluated. At d 14 post-HBsAg vaccination, the PD-1 expressions on CD4⁺ (A) and CD8⁺ (B) cells were observed in the control and HBsAg immunization groups using FCA. (C) HBsAb determination at d 14 post-HBsAg vaccination. *P<0.05, **P<0.01 vs C (n=3 for control and n=9 for HBsAg immunization, 3 per HBsAg dose group). Data are shown as the mean±SD from 3 independently analyzed monkeys. At d 28, the HBsAg and PD-1 antibodies were simultaneously administered for another 28 d of observation. The nine monkeys immunized with the HBsAg vaccine were randomly divided into three groups, H, HP1, and HP10, and the indexes for the preparation of H, HP1, and HP10 group were then tested. H: HBsAg vaccination only; HP1: HBsAg vaccination+JS-001 (1 mg/kg); HP10: HBsAg vaccination+JS-001 (10 mg/kg). PBMCs were harvested at d 3, 14, 21 and 28 post-HBsAg vaccination and PD-1 antibody blockade. (D) PD-1-expressing CD4⁺ and (E) PD-1-expressing CD8⁺ cell in the four groups were determined by flow cytometry. Data are shown as the mean±SD from 3 independently analyzed monkeys. *P<0.05, **P<0.01 vs H. ^#P<0.05, ^##P<0.01 vs HP1.

Figure 4

PD-1 receptor occupancy and PK profiles. (A) PD-1 RO experiment was performed by FCA; a detailed description is available in Materials and Methods. Long-term PD-1 occupancy analysis in the four groups. *P<0.05, **P<0.01 vs H. ^#P<0.05 vs HP1. Data are shown as the mean±SD from 3 independently analyzed monkeys. (B) Drug concentration time curves of cynomolgus macaques after a single vd administration of JS-001 at low, mid, and high dosages (mean±SD, n=6). (C) Drug concentration-time curves of cynomolgus macaques after successive vd administrations of 10 mg/kg JS-001 (mean±SD, n=6).