Characterization of the Anti–PD-1 Antibody REGN2810 and Its Antitumor Activity in HumanPD-1Knock-In Mice

Abstract

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4 (S228P) high-affinity anti-PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cellbased assays, REGN2810 reverses PD-1-dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knockin mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy.

Preclinical Development of the Anti-LAG-3 Antibody REGN3767: Characterization and Activity in Combination with the Anti-PD-1 Antibody Cemiplimab in Human PD-1xLAG-3–Knockin Mice

“…S1. The hIgG4 (S228P) anti-human PD-1 antibody cemiplimab (REGN2810), that binds to both human and monkey PD-1 proteins and blocks PD-1 interactions with PD-L1 and PD-L2, was described previously (35).…”

“…Briefly, a Lag3-targeting vector was engineered that replaced 1750 bp of the extracellular portion of the mouse Lag3 gene (containing exons 2-4) with the corresponding 1741 bp region of the human gene (exons 2-4). Dual humanized mouse embryonic stem cells (ESC) were created by electroporation of the Lag3-targeting vector into C57BL/6N mouse ESCs that contained a humanized Pdcd1 gene encoding the extracellular portion of human PD-1 and the transmembrane and intracellular portion of mouse Pdcd1 (35). Correct gene targeting in ESC clones was identified by a loss of allele assay (41).…”

“…MC38.Ova cells were described previously (35) and authenticated by short tandem repeat profiling in 2016 (IDEXX BioResearch). MC38.Ova cells (5 Â 10 5 ) were injected subcutaneously in the hind flank of 8-10 weeks old female human PD-1xLAG-3 knockin mice.…”

“…We have previously generated cemiplimab, a human anti-PD-1 antibody blocking PD-1/PD-L1-mediated T-cell inhibition (35). Here we describe the generation of REGN3767, a fully human high-affinity anti-LAG-3 antibody, using VelocImmune mice containing human immunoglobulin gene segments (36,37).…”

Harnessing the immune system via FcγR function in immune therapy: a pathway to next‐gen mAbs

“…Indeed, checkpoint inhibitors, such as mAbs targeting CTLA‐4 or the PD‐L1/PD‐1 interaction for the suppression of inhibitory signals that contribute to immune tolerance in the tumor microenvironment, are formatted on an IgG4 backbone. Pembrolizumab, nivolumab and cemiplimab are all anti‐PD‐1 antibodies currently used for cancer therapy and have been formatted on an IgG4 backbone95‐97 with a stabilized core hinge (S 228 P) to prevent half‐IgG4 exchange. Similarly, the checkpoint inhibitor tremelimumab is an anti‐CTLA‐4 antibody formatted on an IgG2 backbone to avoid potential ADCC killing of target cells 98…”