Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

Abstract

Inhibitors of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have dramatically changed the treatment landscape for patients with cancer. Clinical activity of anti-PD-(L)1 antibodies has resulted in increased median overall survival and durable responses in patients across selected tumor types. To date, 6 PD-1 and PD-L1, here collectively referred to as PD-(L)1, pathway inhibitors are approved by the US Food and Drug Administration for clinical use. The availability of multiple anti-PD-(L)1 antibodies provides treatment and dosing regimen choice for patients with cancer. Here, we describe the nonclinical characterization of dostarlimab (TSR-042), a humanized anti-PD-1 antibody, which binds with high affinity to human PD-1 and effectively inhibits its interaction with its ligands, PD-L1 and PD-L2. Dostarlimab enhanced effector T-cell functions, including cytokine production, in vitro. Since dostarlimab does not bind mouse PD-1, its single-agent antitumor activity was evaluated using humanized mouse models. In this model system, dostarlimab demonstrated antitumor activity as assessed by tumor growth inhibition, which was associated with increased infiltration of immune cells. Single-dose and 4-week repeat-dose toxicology studies in cynomolgus monkeys indicated that dostarlimab was well tolerated. In a clinical setting, based on data from the GARNET trial, dostarlimab (Jemperli) was approved for the treatment of adult patients with mismatch repair-deficient recurrent or advanced endometrial cancer that had progressed on or following prior treatment with a platinum-containing regimen. Taken together, these data demonstrate that dostarlimab is a potent anti-PD-1 receptor antagonist, with properties that support its continued clinical investigation in patients with cancer.

Keywords: Anti-PD-1 antibody; TSR-042; cancer; characterization; dostarlimab; immune checkpoint; solid tumors.

Conflict of interest statement

All authors are present or former employees of GlaxoSmithKline or AnaptysBio, Inc.

Figures

Figure 1.

Dostarlimab binding to native PD-1 receptor expressed on human and cynomolgus monkey CD3+ T cells. Dostarlimab binds with high affinity to native human and cynomolgus monkey PD-1 expressed on the surface of CD3+ T cells. Occupancy of dostarlimab on cynomolgus monkey and human CD3+ T cells was assessed by preincubating PBMCs from either (a) cynomolgus monkey or (b) human donors with dostarlimab at varying concentrations and then incubating with either a saturating concentration (20 μg/mL) of dostarlimab or IgG4 isotype control before detection with either FITC-anti-CD3 or PE-anti-IgG4 antibodies and analysis by flow cytometry. The number of CD3+/IgG4+ cells was quantified, and percentage receptor occupancy was calculated as described in Materials and Methods. FITC: fluorescein isothiocyanate; IgG: immunoglobulin G; PBMC: peripheral blood mononuclear cells; PD-1: programmed cell death protein 1; PE: phycoerythrin

Figure 1.

Dostarlimab binding to native PD-1 receptor expressed on human and cynomolgus monkey CD3+ T cells. Dostarlimab binds with high affinity to native human and cynomolgus monkey PD-1 expressed on the surface of CD3+ T cells. Occupancy of dostarlimab on cynomolgus monkey and human CD3+ T cells was assessed by preincubating PBMCs from either (a) cynomolgus monkey or (b) human donors with dostarlimab at varying concentrations and then incubating with either a saturating concentration (20 μg/mL) of dostarlimab or IgG4 isotype control before detection with either FITC-anti-CD3 or PE-anti-IgG4 antibodies and analysis by flow cytometry. The number of CD3+/IgG4+ cells was quantified, and percentage receptor occupancy was calculated as described in Materials and Methods. FITC: fluorescein isothiocyanate; IgG: immunoglobulin G; PBMC: peripheral blood mononuclear cells; PD-1: programmed cell death protein 1; PE: phycoerythrin

Figure 2.

Dostarlimab disrupts binding of PD-1 to its ligands. Dostarlimab potently blocks the interaction of PD-1 with its ligands PD-L1 and PD-L2 and inhibits PD-1/PD-L1-driven signaling. (a) Dostarlimab inhibits the binding of labeled PD-L1-mFc-DyL650 and PD-L2-mFc-DyL650 to CHO-K1 cells stably expressing recombinant human PD-1. (b) Dostarlimab relieves PD-1/PD-L1-mediated repression of an NFAT-luciferase reporter downstream of TCR signaling. For a detailed description of the assay, see Materials and Methods. CHO: Chinese hamster ovary; EC50: half-maximal effective concentration; IC50: half-maximal inhibitory concentration; NFAT: nuclear factor of activated T cells response element; PD-1: programmed cell death protein 1; PD-L1(2): programmed cell death ligand 1(2); RLU: relative light units; TCR: T-cell receptor

Figure 2.

Dostarlimab disrupts binding of PD-1 to its ligands. Dostarlimab potently blocks the interaction of PD-1 with its ligands PD-L1 and PD-L2 and inhibits PD-1/PD-L1-driven signaling. (a) Dostarlimab inhibits the binding of labeled PD-L1-mFc-DyL650 and PD-L2-mFc-DyL650 to CHO-K1 cells stably expressing recombinant human PD-1. (b) Dostarlimab relieves PD-1/PD-L1-mediated repression of an NFAT-luciferase reporter downstream of TCR signaling. For a detailed description of the assay, see Materials and Methods. CHO: Chinese hamster ovary; EC50: half-maximal effective concentration; IC50: half-maximal inhibitory concentration; NFAT: nuclear factor of activated T cells response element; PD-1: programmed cell death protein 1; PD-L1(2): programmed cell death ligand 1(2); RLU: relative light units; TCR: T-cell receptor

Figure 3.

Functional activity of dostarlimab in primary T-cell–based assays. The functional activity of dostarlimab was evaluated in several in vitro assays, including (a) allogeneic MLR, (b) SEB activation, and (c) flu/PPD/TT activation assays. In the MLR assay, dendritic cells and allogeneic CD4+ T cells were incubated in the presence of dostarlimab or isotype control at the indicated concentrations for 48 hours, and activation of T cells was determined by quantifying the level of IL-2 secretion. Representative data from 4 donor combinations is shown in (a). PBMCs from 5 and 3 donors, respectively, were incubated as described in Materials and Methods with either SEB or flu/PPD/TT in the presence of dostarlimab or isotype control at the indicated concentrations for 72 hours and 5 days, respectively. Activation of T cells was determined by measuring the level of IL-2 or IFN-γ secretion. Results shown are the normalized mean ± SEM values across all donors. IFN: interferon; IL: interleukin; MLR: mixed lymphocyte reaction; PBMC: peripheral blood mononuclear cell; PPD: purified protein derivative from Mycobacterium tuberculosis tuberculin; SEB: staphylococcal enterotoxin B; SEM: standard error of the mean; TT: tetanus toxoid antigen

Figure 3.

Functional activity of dostarlimab in primary T-cell–based assays. The functional activity of dostarlimab was evaluated in several in vitro assays, including (a) allogeneic MLR, (b) SEB activation, and (c) flu/PPD/TT activation assays. In the MLR assay, dendritic cells and allogeneic CD4+ T cells were incubated in the presence of dostarlimab or isotype control at the indicated concentrations for 48 hours, and activation of T cells was determined by quantifying the level of IL-2 secretion. Representative data from 4 donor combinations is shown in (a). PBMCs from 5 and 3 donors, respectively, were incubated as described in Materials and Methods with either SEB or flu/PPD/TT in the presence of dostarlimab or isotype control at the indicated concentrations for 72 hours and 5 days, respectively. Activation of T cells was determined by measuring the level of IL-2 or IFN-γ secretion. Results shown are the normalized mean ± SEM values across all donors. IFN: interferon; IL: interleukin; MLR: mixed lymphocyte reaction; PBMC: peripheral blood mononuclear cell; PPD: purified protein derivative from Mycobacterium tuberculosis tuberculin; SEB: staphylococcal enterotoxin B; SEM: standard error of the mean; TT: tetanus toxoid antigen

Figure 4.

Antitumor and pharmacodynamic activity of dostarlimab monotherapy in a humanized mouse model of non–small cell lung cancer. (a) Study schema: dostarlimab or IgG4 isotype control (200 µg/mouse, twice weekly) was dosed to established A549 tumors (80–120 mm3) grown as xenografts in humanized NOG-EXL mice (Taconic), and (b) antitumor activity and (c) pharmacodynamic effects in the tumor. Human CD4+, CD8+, NK/NKT, and activated CD8+ (GZMB, CD8+) cells in the tumors were quantified by flow cytometry as percentage of human CD45+ cells. *p < .05 (unpaired t test). PD-1: programmed cell death protein 1; CD-8: cluster of differentiation-8; GZMB: Granzyme B; IgG: immunoglobulin G; NK: natural killer; Treg: regulatory T cells

Figure 4.

Antitumor and pharmacodynamic activity of dostarlimab monotherapy in a humanized mouse model of non–small cell lung cancer. (a) Study schema: dostarlimab or IgG4 isotype control (200 µg/mouse, twice weekly) was dosed to established A549 tumors (80–120 mm3) grown as xenografts in humanized NOG-EXL mice (Taconic), and (b) antitumor activity and (c) pharmacodynamic effects in the tumor. Human CD4+, CD8+, NK/NKT, and activated CD8+ (GZMB, CD8+) cells in the tumors were quantified by flow cytometry as percentage of human CD45+ cells. *p < .05 (unpaired t test). PD-1: programmed cell death protein 1; CD-8: cluster of differentiation-8; GZMB: Granzyme B; IgG: immunoglobulin G; NK: natural killer; Treg: regulatory T cells

Figure 5.

Antitumor activity of dostarlimab monotherapy in a humanized mouse model of breast cancer. (a) Study schema; dostarlimab or IgG4 isotype control (200 μg/mouse, twice weekly) was dosed to already established MDA-MB-436 tumors (80–120 mm3) grown as xenografts in humanized NOG-EXL mice (Taconic), and (b) antitumor activity. PD-1: programmed cell death protein 1; IgG: immunoglobulin G

Figure 5.

Antitumor activity of dostarlimab monotherapy in a humanized mouse model of breast cancer. (a) Study schema; dostarlimab or IgG4 isotype control (200 μg/mouse, twice weekly) was dosed to already established MDA-MB-436 tumors (80–120 mm3) grown as xenografts in humanized NOG-EXL mice (Taconic), and (b) antitumor activity. PD-1: programmed cell death protein 1; IgG: immunoglobulin G

Figure 6.

Concentration–time profiles of single-dose dostarlimab. Mean serum concentration–time profiles of dostarlimab after single intravenous infusion of dostarlimab to male and female cynomolgus monkeys. Concentration–time profiles were plotted through the first post-dose time point that was below the limit of quantification, if applicable. The lower limit of quantification was 31.0 ng/mL