ABSTRACT
Targeted Radionuclide Therapies (TRT) involve the tailored combination of a therapeutic radionuclide and a targeting molecule, as for instance antibodies or fragments thereof. Despite their short shelf-life, these drug products must meet stringent regulatory standards before use. We introduce a novel, efficient method utilizing Bio-Layer Interferometry (BLI) for rapid identity testing of TRT drug products in less than five minutes. This approach not only reduces radioactive waste but also minimizes operator exposure to radiation. This label-free method has been successfully developed and validated for three different TRT products, ensuring compliance with Good Manufacturing Practices (GMP). Furthermore, we outline our strategic approach to the production and testing of custom biosensors for each product, firmly grounded in Quality-by-Design (QbD) principles.
Subject(s)
Interferometry , Interferometry/methods , Biosensing Techniques/methods , Radioisotopes/chemistry , Humans , Radiopharmaceuticals/chemistryABSTRACT
Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.
Subject(s)
Antibodies, Monoclonal/pharmacology , Hemophilia A/prevention & control , Immunoglobulin Fab Fragments/immunology , Protein C Inhibitor/pharmacology , Protein C/antagonists & inhibitors , Animals , Antibodies, Monoclonal/classification , Antibodies, Monoclonal/immunology , Bleeding Time , Cell Membrane Permeability/drug effects , Cells, Cultured , Crystallography, X-Ray , Hemophilia A/blood , Hemorrhage/prevention & control , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Immunoglobulin Fab Fragments/metabolism , Macaca fascicularis , Male , Protein C/chemistry , Protein C/immunology , Protein C/metabolism , Protein C Inhibitor/blood , Protein C Inhibitor/pharmacokineticsABSTRACT
Alzheimer's disease (AD) is a devastating disease affecting predominantly the aging population. One of the characteristic pathological hallmarks of AD are neuritic plaques, consisting of amyloid-ß peptide (Aß). While there has been some advancement in diagnostic classification of AD patients according to their clinical severity, no fully reliable method for pre-symptomatic diagnosis of AD is available. To enable such early diagnosis, which will allow the initiation of treatments early in the disease progress, neuroimaging tools are under development, making use of Aß-binding ligands that can visualize amyloid plaques in the living brain. Here we investigate the properties of a newly designed series of D-enantiomeric peptides which are derivatives of ACI-80, formerly called D1, which was developed to specifically bind aggregated Aß1-42. We describe ACI-80 derivatives with increased stability and Aß binding properties, which were characterized using surface plasmon resonance and enzyme-linked immunosorbent assays. The specific interactions of the lead compounds with amyloid plaques were validated by ex vivo immunochemistry in transgenic mouse models of AD. The novel compounds showed increased binding affinity and are promising candidates for further development into in vivo imaging compounds.
Subject(s)
Alzheimer Disease/diagnosis , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid/chemistry , Diagnostic Imaging/methods , Oligopeptides/chemistry , Peptide Fragments/chemistry , Amyloid/metabolism , Amyloid beta-Peptides/metabolism , Animals , Ligands , Mice , Neurites/metabolism , Neurites/pathology , Oligopeptides/metabolism , Peptide Fragments/metabolism , Protein BindingABSTRACT
Protein-ligand interactions characterise and govern the current state and fate of a living cell. The specificity of proteins is mainly determined by the relative affinities to each potential ligand. To investigate the consequences and potentials of ligands with increased specificity in comparison with ligands optimised solely for affinity, it was necessary to identify ligands that are optimised towards specificity instead of a barely optimised affinity to a given target. In the presented example, a modified phage display screening procedure yielded specific ligands for the LckSH3 domain. We found that increased specificity of one of the hereby obtained ligands for LckSH3 is achieved at the cost of a slightly reduced affinity to LckSH3 and a drastically reduced affinity to other SH3 domains. A surface plasmon resonance experiment simulating in vivo-like realistic competitive binding conditions exerted enhanced binding behaviour of the specific ligand under these binding conditions. The experimental data, together with a mathematical model describing the complex experimental situation, and theoretical considerations lead to the conclusion that increased specificity is achieved at the cost of reduced affinity, but after all, it pays if the ligand is applied under realistic, i.e. competitive, conditions.
