In vitro glycoengineering of IgG1 and its effect on Fc receptor binding and ADCC activity.

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity

Summary Table—Impact of increasing galactose / sialic acid levels of IgG1 on FcγRI, IIa, and IIIa binding and ADCC activity.

<p>Summary Table—Impact of increasing galactose / sialic acid levels of IgG1 on FcγRI, IIa, and IIIa binding and ADCC activity.</p

Effect of Fc glycoengineering on ADCC activity.

<p>The ADCC activities are quantified relative to a reference material set to 100% by full curve parallel line analysis. For each sample, the box plot represents 5 independent measures of duplicates. The dashed lines indicate the 95% confidence interval (CI) of the bulk material.</p

FcγRIIIa column assay analysis.

<p>Normalized UV chromatograms of the hypo- and hyper-galactosylated as well as the mono-sialylated samples are exemplarily shown (A). UV absorbance was measured at 280 nm. Retention times for all batches are compared for the fucosylated (early eluting) peak (B) and the partly/non-fucosylated (late eluting) peak (C).</p

Production workflow for the different glycan variants of IgG1.

<p>Numbers in parenthesis represent the days needed for sample preparation. Starting material denoted as “bulk” is material obtained from the production process after regular fermentation and purification steps.</p

SPR analysis on FcγRI (A), FcγRIIa (B), and FcγRIIIa binding (C) in ascending intensity.

<p>Bulk material is used as reference and set to 100%. All samples are measured in triplicates.</p

Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans

ABSTRACTThere are few treatments that slow neurodegeneration in Alzheimer’s disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood–brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aβ) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aβ40 and Aβ plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4–18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial