ABSTRACT
In the tumor microenvironment, multiple inhibitory checkpoint receptors can suppress T-cell function, thereby enabling tumor immune evasion. Blockade of one of these checkpoint receptors, PD-1, with therapeutic antibodies has produced positive clinical responses in various cancers; however, the efficacy of this approach can be further improved. Simultaneously targeting multiple inhibitory checkpoint receptors has emerged as a promising therapeutic strategy. Here, we report the development and characterization of REGN3767, a fully human IgG4 antibody targeting LAG-3, another inhibitory receptor on T cells. REGN3767 binds human and monkey LAG-3 with high affinity and specificity and blocks the interaction of LAG-3 with its ligand, MHC class II. In an engineered T-cell/antigen-presenting cell bioassay, REGN3767 alone, or in combination with cemiplimab (REGN2810, human anti-PD-1 antibody), blocked inhibitory signaling to T cells mediated by hLAG-3/MHCII in the presence of PD-1/PD-L1. To test the in vivo activity of REGN3767 alone or in combination with cemiplimab, we generated human PD-1xLAG-3 knockin mice, in which the extracellular domains of mouse Pdcd1 and Lag3 were replaced with their human counterparts. In these humanized mice, treatment with cemiplimab and REGN3767 showed increased efficacy in a mouse tumor model and enhanced the secretion of proinflammatory cytokines by tumor-specific T cells. The favorable pharmacokinetics and toxicology of REGN3767 in nonhuman primates, together with enhancement of antitumor efficacy of anti-PD-1 antibody in preclinical tumor models, support its clinical development.
Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antigens, CD/chemistry , Antigens, CD/genetics , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/genetics , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Antigens, CD/metabolism , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Female , Gene Knock-In Techniques , Haplorhini , Histocompatibility Antigens Class II/metabolism , Humans , Mice , Neoplasms/genetics , Protein Binding/drug effects , Signal Transduction/drug effects , Tumor Microenvironment/drug effects , Xenograft Model Antitumor Assays , Lymphocyte Activation Gene 3 ProteinABSTRACT
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 cell-based 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, knock-in 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. Mol Cancer Ther; 16(5); 861-70. ©2017 AACR.
Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Cell Proliferation/drug effects , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/genetics , Animals , Antibodies, Monoclonal, Humanized/immunology , Cell Line, Tumor , Gene Knock-In Techniques , Humans , Immunotherapy , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Kinesin spindle protein (KSP) is a mitotic kinesin that is expressed only in proliferating cells and plays a key role in spindle pole separation, formation of a bipolar mitotic spindle, as well as centrosome separation and maturation. Inhibition of KSP has the potential to provide anti-tumor activity while avoiding peripheral neuropathy associated with some microtubule-targeted drugs. Based on MK-0731 and related heterocyclic compounds targeting the KSP monastrol binding site, structurally constrained spiro-cyclic KSP inhibitors were designed. In particular, rapid evaluation and optimization of the novel spiro 1,3,4-thiadiazolines resulted in a series of potent KSP inhibitors demonstrating mechanism based activities in cells, including induction of the mitotic marker phospho-histone H3 and induction of monaster spindle formation. Further optimization of the pharmacokinetic (PK) properties afforded MK-8267 as a potent, orally bioavailable and brain penetrant KSP inhibitor which showed anti-tumor activity in preclinical xenograft models.