Electrophoretically Snagging Viral Genomes in Wormlike Micelle Networks Using Peptide Nucleic Acid Amphiphiles and dsDNA Oligomers.

We demonstrate that the attachment of 30-170 bp dsDNA oligomers to ssDNA viral genomes gives a significant additional mobility shift in micelle-tagging electrophoresis (MTE). In MTE, a modified peptide nucleic acid amphiphile is attached to the viral genome to bind drag-inducing micelles present in capillary electrophoresis running buffers. Further attachment of 30-170 bp dsDNA oligomers drastically shifts the mobility of the 5.1 kB ssDNA genome of mouse minute virus (MMV), providing a new mechanism to improve resolution in CE-based analysis of kilobase nucleic acids. A model based on biased-reptation electrophoresis, end-labeled free-solution electrophoresis, and Ferguson gel-filtration theory is presented to describe the observed mobility shifts.

Design and Synthesis of Cathepsin-K-Activated Osteoadsorptive Fluorogenic Sentinel (OFS) Probes for Detecting Early Osteoclastic Bone Resorption in a Multiple Myeloma Mouse Model.

We describe the design and synthesis of OFS-1, an Osteoadsorptive Fluorogenic Sentinel imaging probe that is adsorbed by hydroxyapatite (HAp) and bone mineral surfaces, where it generates an external fluorescent signal in response to osteoclast-secreted cathepsin K (Ctsk). The probe consists of a bone-anchoring bisphosphonate moiety connected to a Förster resonance energy transfer (FRET) internally quenched fluorescent (IQF) dye pair, linked by a Ctsk peptide substrate, GHPGGPQG. Key structural features contributing to the effectiveness of OFS-1 were defined by structure-activity relationship (SAR) and modeling studies comparing OFS-1 with two cognates, OFS-2 and OFS-3. In solution or when preadsorbed on HAp, OFS-1 exhibited strong fluorescence when exposed to Ctsk (2.5-20 nM). Time-lapse photomicrographs obtained after seeding human osteoclasts onto HAp-coated well plates containing preadsorbed OFS-1 revealed bright fluorescence at the periphery of resorbing cells. OFS-1 administered systemically detected early osteolysis colocalized with orthotopic engraftment of RPMI-8226-Luc human multiple myeloma cells at a metastatic skeletal site in a humanized mouse model. OFS-1 is thus a promising new imaging tool for detecting abnormal bone resorption.

Kinetic models towards an enhanced understanding of diverse ADC conjugation reactions.

The conjugation reaction is the central step in the manufacturing process of antibody-drug conjugates (ADCs). This reaction generates a heterogeneous and complex mixture of differently conjugated sub-species depending on the chosen conjugation chemistry. The parametrization of the conjugation reaction through mechanistic kinetic models offers a chance to enhance valuable reaction knowledge and ensure process robustness. This study introduces a versatile modeling framework for the conjugation reaction of cysteine-conjugated ADC modalities-site-specific and interchain disulfide conjugation. Various conjugation kinetics involving different maleimide-functionalized payloads were performed, while controlled gradual payload feeding was employed to decelerate the conjugation, facilitating a more detailed investigation of the reaction mechanism. The kinetic data were analyzed with a reducing reversed phase (RP) chromatography method, that can readily be implemented for the accurate characterization of ADCs with diverse drug-to-antibody ratios, providing the conjugation trajectories of the single chains of the monoclonal antibody (mAb). Possible kinetic models for the conjugation mechanism were then developed and selected based on multiple criteria. When calibrating the established model to kinetics involving different payloads, conjugation rates were determined to be payload-specific. Further conclusions regarding the kinetic comparability across the two modalities could also be derived. One calibrated model was used for an exemplary in silico screening of the initial concentrations offering valuable insights for profound understanding of the conjugation process in ADC development.