Evaluating Antibody Pharmacokinetics as Prerequisite for Determining True Efficacy as Shown by Dual Targeting of PD-1 and CD96.

One important prerequisite for developing a therapeutic monoclonal antibody is to evaluate its in vivo efficacy. We tested the therapeutic potential of an anti-CD96 antibody alone or in combination with an anti-PD-1 antibody in a mouse colon cancer model. Early anti-PD-1 treatment significantly decreased tumor growth and the combination with anti-CD96 further increased the therapeutic benefit, while anti-CD96 treatment alone had no effect. In late therapeutic settings, the treatment combination resulted in enhanced CD8+ T cell infiltration of tumors and an increased CD8/Treg ratio. Measured anti-PD-1 concentrations were as expected in animals treated with anti-PD-1 alone, but lower at later time points in animals receiving combination treatment. Moreover, anti-CD96 concentrations dropped dramatically after 10 days and were undetectable thereafter in most animals due to the occurrence of anti-drug antibodies that were increasing antibody clearance. Comparison of the anti-PD-1 concentrations with tumor growth showed that higher antibody concentrations in plasma correlated with better therapeutic efficacy. The therapeutic effect of anti-CD96 treatment could not be evaluated, because plasma concentrations were too low. Our findings strongly support the notion of measuring both plasma concentration and anti-drug antibody formation throughout in vivo studies, in order to interpret pharmacodynamic data correctly.

Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle.

Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aß antibody, which uses a monovalent binding mode to the TfR, increases ß-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.

The SpoMBe pathway drives membrane bending necessary for cytokinesis and spore formation in yeast meiosis.

Precise control over organelle shapes is essential for cellular organization and morphogenesis. During yeast meiosis, prospore membranes (PSMs) constitute bell-shaped organelles that enwrap the postmeiotic nuclei leading to the cellularization of the mother cell's cytoplasm and to spore formation. Here, we analysed how the PSMs acquire their curved bell-shaped structure. We discovered that two antagonizing forces ensure PSM shaping and proper closure during cytokinesis. The Ssp1p-containing coat at the leading edge of the PSM generates a pushing force, which is counteracted by a novel pathway, the spore membrane-bending pathway (SpoMBe). Using genetics, we found that Sma2p and Spo1p, a phospholipase, as well as several GPI-anchored proteins belong to the SpoMBe pathway. They exert a force all along the membrane, responsible for membrane bending during PSM biogenesis and for PSM closure during cytokinesis. We showed that the SpoMBe pathway involves asymmetric distribution of Sma2p and does not involve a GPI-protein-containing matrix. Rather, repulsive forces generated by asymmetrically distributed and dynamically moving GPI-proteins are suggested as the membrane-bending principle.

Cytokinesis in yeast meiosis depends on the regulated removal of Ssp1p from the prospore membrane.

Intracellular budding is a developmentally regulated type of cell division common to many fungi and protists. In Saccaromyces cerevisiae, intracellular budding requires the de novo assembly of membranes, the prospore membranes (PSMs) and occurs during spore formation in meiosis. Ssp1p is a sporulation-specific protein that has previously been shown to localize to secretory vesicles and to recruit the leading edge protein coat (LEP coat) proteins to the opening of the PSM. Here, we show that Ssp1p is a multidomain protein with distinct domains important for PI(4,5)P(2) binding, binding to secretory vesicles and inhibition of vesicle fusion, interaction with LEP coat components and that it is subject to sumoylation and degradation. We found non-essential roles for Ssp1p on the level of vesicle transport and an essential function of Ssp1p to regulate the opening of the PSM. Together, our results indicate that Ssp1p has a domain architecture that resembles to some extent the septin class of proteins, and that the regulated removal of Ssp1p from the PSM is the major step underlying cytokinesis in yeast sporulation.

Identification of neurotoxic chemicals in cell cultures.

In order to identify the neurotoxic potential of drugs or chemicals in vitro, a combination of different in vitro cell culture tests is required. Depending on the type and use of a given test compound, a sequential exposure of freshly isolated and cultured hepatocytes and chicken brain cells is suitable. In order to find out more about the stability of liver-derived metabolites, co-cultures are appropriate. In order to determine how metabolites enter the brain, a combination of an in vitro system which mimics the blood-brain barrier and choroid plexus is proposed.

Differential requirement for phospholipase D/Spo14 and its novel interactor Sma1 for regulation of exocytotic vesicle fusion in yeast meiosis.

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.

[Development of hepatocyte cultures in toxicity testing]

The liver plays a key role in drug and xenobiotic metabolism. The probability of detecting the toxicity of unknown chemicals in vitro is therefore highest in liver cell cultures. In culture however, enzymes involved in xenobiotic metabolism are preferentially degraded within one to two days. In order to improve this situation, investigations were focused on the maintenance of a tissue-like oxygen tension and the maintenance of xenobiotic metabolism by means of heterotypic cell cultures. In conventional culture dishes, as a function of cell density, the oxygen diffusion is delayed and depleted. Using teflon membrane culture dishes, a stable, incubator-controlled, tissue like oxygen tension of 4% and 13% O2 respectively, was achieved. In co-cultures of hepatocytes, the auxiliary cells selected from livers of 10 day old rats maintained the liver cell-specific cytochrome P-450 dependent aldrin epoxidase up to one week, to 40% of the original value. The analysis of cellular DNA and protein content in hepatocytes by flow cytometry revealed specific ploidy shifts inducible by low oxygen tension, by fetal calf serum, by phenobarbital and dimethylsulfoxide. Chemically induced alterations in ploidy might be an indicator to detect compounds which interfere with growth and differentiation of hepatocytes in culture.