Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
J Immunother Cancer ; 10(9)2022 09.
Article in English | MEDLINE | ID: mdl-36100308

ABSTRACT

BACKGROUND: Immune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI therapy. CD27 is a member of the tumor necrosis factor receptor superfamily that plays a critical role in promoting responses of T cells, B cells and NK cells. METHODS: Anti-CD27 antibodies were generated and selected for agonist activity using NF-кB luciferase reporter assays. Antibodies were humanized and characterized for agonism using in vitro T-cell proliferation assays. The epitope recognized on CD27 by MK-5890 was established by X-ray crystallography. Anti-tumor activity was evaluated in a human CD27 knock-in mouse. Preclinical safety was tested in rhesus monkeys. Pharmacodynamic properties were examined in mouse, rhesus monkeys and a phase 1 dose escalation clinical study in patients with cancer. RESULTS: Humanized anti-CD27 antibody MK-5890 (hIgG1) was shown to bind human CD27 on the cell surface with sub-nanomolar potency and to partially block binding to its ligand, CD70. Crystallization studies revealed that MK-5890 binds to a unique epitope in the cysteine-rich domain 1 (CRD1). MK-5890 activated CD27 expressed on 293T NF-κB luciferase reporter cells and, conditional on CD3 stimulation, in purified CD8+ T cells without the requirement of crosslinking. Functional Fc-receptor interaction was required to activate CD8+ T cells in an ex vivo tumor explant system and to induce antitumor efficacy in syngeneic murine subcutaneous tumor models. MK-5890 had monotherapy efficacy in these models and enhanced efficacy of PD-1 blockade. MK-5890 reduced in an isotype-dependent and dose-dependent manner circulating, but not tumor-infiltrating T-cell numbers in these mouse models. In rhesus monkey and human patients, reduction in circulating T cells was transient and less pronounced than in mouse. MK-5890 induced transient elevation of chemokines MCP-1, MIP-1α, and MIP-1ß in the serum of mice, rhesus monkeys and patients with cancer. MK-5890 was well tolerated in rhesus monkeys and systemic exposure to MK-5890 was associated with CD27 occupancy at all doses. CONCLUSIONS: MK-5890 is a novel CD27 agonistic antibody with the potential to complement the activity of PD-1 checkpoint inhibition in cancer immunotherapy and is currently undergoing clinical evaluation.


Subject(s)
Neoplasms , Tumor Necrosis Factor Receptor Superfamily, Member 7 , Animals , Antibodies, Monoclonal/therapeutic use , Cell Count , Epitopes , Humans , Immunotherapy , Macaca mulatta , Mice , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor
2.
Mol Cancer Ther ; 19(6): 1298-1307, 2020 06.
Article in English | MEDLINE | ID: mdl-32229606

ABSTRACT

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/pharmacokinetics , Leukocytes, Mononuclear/drug effects , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , T-Lymphocytes/drug effects , Animals , Antineoplastic Agents, Immunological/pharmacokinetics , Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , Female , Humans , Immune Checkpoint Inhibitors/pharmacokinetics , Immune Checkpoint Inhibitors/pharmacology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Macaca fascicularis , Mice , Mice, Inbred BALB C , Neoplasms/immunology , Neoplasms/pathology , Programmed Cell Death 1 Ligand 2 Protein/antagonists & inhibitors , Programmed Cell Death 1 Ligand 2 Protein/immunology , Programmed Cell Death 1 Ligand 2 Protein/metabolism , Programmed Cell Death 1 Receptor/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Tissue Distribution , Toxicity Tests
3.
Anal Biochem ; 386(2): 172-80, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19111520

ABSTRACT

We demonstrate the use of label-free real-time optical biosensors in competitive binding assays by epitope binning a panel of antibodies. We describe three assay orientations that we term in tandem, premix, and classical sandwich blocking, and we perform each of them on three platforms: ForteBio's Octet QK, Bio-Rad's ProteOn XPR36, and GE Healthcare's Biacore 3000. By testing whether antibodies block one another's binding to their antigen in a pairwise fashion, we establish a blocking profile for each antibody relative to the others in the panel. The blocking information is then used to create "bins" of antibodies with similar epitopes. The advantages and disadvantages of each biosensor, factors to consider when deciding on the most appropriate blocking assay orientation for a particular interaction system, and tips for dealing with ambiguous data are discussed. The data from our different assay orientations and biosensors agree very well, establishing these machines as valuable tools for characterizing antibody epitopes and multiprotein complexes of biological significance.


Subject(s)
Antibodies/chemistry , Biosensing Techniques/methods , Biological Assay , Biosensing Techniques/instrumentation , Epitopes/chemistry , Kinetics , Models, Biological , Sensitivity and Specificity
4.
Anal Biochem ; 377(2): 209-17, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18405656

ABSTRACT

ForteBio's Octet optical biosensor harnesses biolayer interferometry to detect and quantify molecular interactions using disposable fiber-optic biosensors that address samples from an open shaking microplate without any microfluidics. We recruited a monoclonal antibody against a panel of peptides to compare the Octet directly with Biacore's well-established 3000 platform and Bio-Rad's recently launched ProteOn XPR36 array system, which use surface plasmon resonance (SPR) to detect the binding of one analyte over four surfaces and six analytes over six surfaces, respectively. A sink method was used to prevent analyte from rebinding the ligand-coated Octet tips and enabled us to extract accurate kinetic rate constants, as judged by their close agreement with those determined by SPR. Although the Octet is not sensitive enough to detect the binding of small molecules directly, it can access their affinities indirectly via solution competition experiments. We conducted similar experiments on the SPR instruments to validate these measurements. The Octet is emerging as a versatile complement to other more sophisticated biosensors, and the ProteOn provides high-quality data near the sensitivity of Biacore but in a more multiplexed format. Our results provide a benchmark for assessing the performance of the above-mentioned sensors.


Subject(s)
Biosensing Techniques/methods , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Benchmarking , Biosensing Techniques/instrumentation , Calcitonin Gene-Related Peptide/immunology , Calcitonin Gene-Related Peptide/metabolism , Humans , Kinetics , Mice , Protein Binding , Rats , Sensitivity and Specificity , Staining and Labeling , Surface Plasmon Resonance , Time Factors
5.
Protein Sci ; 17(8): 1326-35, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18505735

ABSTRACT

We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.


Subject(s)
Antibodies, Monoclonal/metabolism , Biosensing Techniques/methods , Nerve Growth Factor/metabolism , Binding, Competitive , Humans , Kinetics , Models, Biological , Nerve Growth Factor/immunology , Nerve Tissue Proteins/metabolism , Protein Binding , Receptor, trkA/metabolism , Receptors, Nerve Growth Factor/metabolism
6.
J Am Chem Soc ; 126(36): 11156-7, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15355089

ABSTRACT

The discovery, from nature, of a diverse set of microbial epoxide hydrolases is reported. The utility of a library of epoxide hydrolases in the synthesis of chiral 1,2-diols via desymmetrization of a wide range of meso-epoxides, including cyclic as well as acyclic alkyl- and aryl-substituted substrates, is demonstrated. The chiral (R,R)-diols were furnished with high ee's and yields. The discovery of the first microbial epoxide hydrolases providing access to complementary (S,S)-diols is also described.


Subject(s)
Alcohols/chemistry , Epoxide Hydrolases/chemistry , Epoxy Compounds/chemistry , Catalysis , Epoxide Hydrolases/metabolism , Stereoisomerism
SELECTION OF CITATIONS
SEARCH DETAIL