ABSTRACT
Monoacylglycerol lipase (MAGL) is one of the key enzymes in the endocannabinoid system. Inhibition of MAGL has been proposed as an attractive approach for the treatment of various diseases. In this study, we designed and successfully synthesized two series of piperazinyl pyrrolidin-2-one derivatives as novel reversible MAGL inhibitors. (R)-[18F]13 was identified through the preliminary evaluation of two carbon-11-labeled racemic structures [11C]11 and [11C]16. In dynamic positron-emission tomography (PET) scans, (R)-[18F]13 showed a heterogeneous distribution and matched the MAGL expression pattern in the mouse brain. High brain uptake and brain-to-blood ratio were achieved by (R)-[18F]13 in comparison with previously reported reversible MAGL PET radiotracers. Target occupancy studies with a therapeutic MAGL inhibitor revealed a dose-dependent reduction of (R)-[18F]13 accumulation in the mouse brain. These findings indicate that (R)-[18F]13 ([18F]YH149) is a highly promising PET probe for visualizing MAGL non-invasively in vivo and holds great potential to support drug development.
Subject(s)
Brain/diagnostic imaging , Enzyme Inhibitors/chemistry , Monoacylglycerol Lipases/metabolism , Neuroimaging/methods , Radiopharmaceuticals/chemistry , Animals , Brain/metabolism , Carbon Radioisotopes/chemistry , Crystallography, X-Ray , Drug Stability , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacokinetics , Mice , Molecular Conformation , Monoacylglycerol Lipases/chemistry , Positron-Emission Tomography , Radiopharmaceuticals/metabolism , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Wistar , Structure-Activity Relationship , Tissue DistributionABSTRACT
Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.
Subject(s)
Antibodies, Bispecific/biosynthesis , High-Throughput Screening Assays , Antibodies, Bispecific/isolation & purification , Automation , HEK293 Cells , Humans , Mutation/genetics , Protein MultimerizationABSTRACT
Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.
