A new anti-human Fc method to capture and analyze ADCs for characterization of drug distribution and the drug-to-antibody ratio in serum from pre-clinical species.

Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. They combine monoclonal antibody specificity for over-expressed tumor antigens and the high cytoxicity of small molecular drugs (SMDs) and can therefore selectively kill tumor cells while minimizing toxicity to normal cells. Nevertheless, the premature deconjugation of ADCs in the circulation may trigger off target toxicity in patients. The released free drug level must be low in circulation for an extended period of time as well as the de-conjugation rate to ensure an acceptable therapeutic window. As a result, the assessment of the stability of the linker between payload and mAb in the systemic circulation is of paramount importance before entering in clinical trial. Here we report a new universal method to immunocapture and analyze by LC-MS the stability and distribution of ADCs in sera from relevant preclinical species (mouse, rat and cynomolgus monkey). Furthermore we demonstrated that this workflow can be applied to both ADCs with cleavable and non cleavable linkers. Last but not least, the results obtained in cynomolgus serum using immunoprecipitation and LC-MS analysis were cross validated using an ELISA orthogonal method. As the ligand used for immunoprecipitation is targeting the Fc part of mAb (CaptureSelect™ Human IgG-Fc PK Biotin), this protocol can be applied to analyze the stability of virtually all ADCs in sera for preclinical studies without the need to prepare specific molecular tools.

Absolute and multiplex quantification of antibodies in serum using PSAQ™ standards and LC-MS/MS.

BACKGROUND: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies. MATERIALS & METHODS: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared. RESULTS: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.

Glomerular proliferation during early stages of diabetic nephropathy is associated with local increase of sphingosine-1-phosphate levels.

In this study, the effects of short-term diabetes (4 days) on rat renal glomerular cells proliferation and the potential involvement of sphingolipids in this process were investigated. Immunohistochemical analysis showed that streptozotocin (STZ)-induced diabetes promoted increased intra-glomerular hyperplasia, particularly marked for mesangial cells. This was associated with a concomitant increase in neutral ceramidase and sphingosine-kinase activities and the accumulation of the pro-proliferative sphingolipid sphingosine-1-phosphate, in glomeruli isolated from kidney cortex of STZ-treated rats. These results suggest a possible involvement of sphingolipid metabolites in the glomerular proliferative response during the early stages of diabetic nephropathy.

a-Series gangliosides mediate the effects of advanced glycation end products on pericyte and mesangial cell proliferation: a common mediator for retinal and renal microangiopathy?

Advanced glycation end products (AGEs) are involved in the development of microvascular complications, including alterations of retinal pericyte and renal mesangial cell growth occurring during diabetic retinopathy and diabetic nephropathy, respectively. Because gangliosides are implicated in the regulation of cell proliferation, we hypothesized that AGEs could exert cellular effects in part by modulating ganglioside levels. Results of the present study indicate that AGEs caused an inhibition of both bovine retinal pericyte (BRP) and rat renal mesangial cell (RMC) proliferation, associated with an increase of a-series gangliosides consecutive to GM3 synthase activity increase and GD3 synthase activity inhibition. Similar modifications were also found in the renal cortex of diabetic db/db mice compared with controls. Treatment of BRP and RMC with exogenous a-series gangliosides decreased proliferation and blockade of a-series gangliosides with specific antibodies partially protecting the two cell types from the AGE-induced proliferation decrease. Further, inhibition of GM3 synthase using specific SiRNA partially reversed the AGE effects on mesangial cell proliferation. These results suggest that a-series gangliosides are mediators of the adverse AGE effects on BRP and RMC proliferation. They also raise the hypothesis of common mechanisms involved in the development of diabetic retinopathy and diabetic nephropathy.