Y.-J. Schneider

Metabolic design of macroscopic bioreaction models: application to Chinese hamster ovary cells

The aim of this paper is to present a systematic methodology to design macroscopic bioreaction models for cell cultures based upon metabolic networks. The cell culture is seen as a succession of phases. During each phase, a metabolic network represents the set of reactions occurring in the cell. Then, through the use of the elementary flux modes, these metabolic networks are used to derive macroscopic bioreactions linking the extracellular substrates and products. On this basis, as many separate models are obtained as there are phases. Then, a complete model is obtained by smoothly switching from model to model. This is illustrated with batch cultures of Chinese hamster ovary cells.

In vitro identification of targeting-ligands of human M cells by Phage Display.

To improve transport of vaccine-loaded nanoparticles, the phage display technology was used to identify novel lead peptides targeting human M cells. Using an in vitro model of the human follicle-associated epithelium (FAE) which contains both Caco-2 and M cells, a T7 phage display library was screened for its ability either to bind the apical cell surface of or to undergo transcytosis across Caco-2 cells or FAE. The selection for transcytosis across both enterocytes and FAE identified three different peptide sequences (CTGKSC, PAVLG and LRVG) with high frequency. CTGKSC and LRVG sequences enhanced phage transport across M-like cells. When polymeric nanoparticles were grafted with the sequences CTGKSC and LRVG, their transport by FAE was significantly enhanced. These peptides could therefore be used to enhance the transport of vaccine-loaded nanoparticles across the intestinal mucosal barrier.

Drug Targeting in Human Cancer Chemotherapy

Drug targeting aims to restrict the access of pharmacological agents to selected cells. Theoretically, such a method should on one hand, decrease unsuitable side effects resulting from an interaction of the drugs with non target cells and, on the other hand, enhance the pharmacological activity by increasing the proportion of the administered drug found within the target cells. Our conceptual approach to this problem consists in linking drugs through a covalent bond to macromolecular carriers which are recognized by receptors or antigens present at the cell surface of the target cells and thereafter endocytosed to allow the release of the drug after hydrolysis of the covalent linkage by lysosomal enzymes.

Immunohistochemical Reaction of Three Monoclonal Antibodies to Human Milk Fat Globule Membrane with Human Normal and Tumoral Tissues

Abstract The reactivity of three monoclonal antibodies to human milk fat globule membrane with human normal and tumoral tissues has been studied by indirect immunohistochemical methods. Results show that one of them reacts with 19 out of 19 ductal breast carcinoma as well as with lymph node metastasis but also with some normal epithelial cells. The two others react respectively with 12 and 13 out of 19 breast tumors examined. However some weak reaction with normal tissues has been observed as well. These results suggest that such antibodies could be used as diagnostic tool but also to restrict partially the access of attached drug to breast tissue, decreasing therefore toxicity of the drug.

Covalent Linkage of Anthracyclines to Macromolecular Carriers

Abstract Daunorubicin (DNR) and doxorubicin (DOX) have been covalently linked to protein carriers such as human serum albumin (SA), galactosylated serum albumin (gal SA) and antibodies. The drug is transformed into a succinylated tetrapeptide derivative and is linked to the amino group of the protein via an activated ester derivative. Molar drug to protein ratios of 4 to 12 have been reached in the drug albumin conjugates whereas in the drug antibodies conjugates its value never raised above 3. Monomeric conjugates were obtained in good yields with over 99.7% of the drug covalently attached to the carrier.

Binding, Uptake and Subcellular Distribution of Two Monoclonal Antibodies to Human Milk Fat Globule Membrane Studied on Human Mcf 7 Breast Carcinoma Cells

Abstract Binding, uptake and subcellular distribution of two monoclonal antibodies 7F11C7 (IgG 2b) and 9H4 (IgG 3) raised against Human Milk Fat Globule Membrane (HMFGM) have been studied on cultured MCF-7 cells, a human breast carcinoma line. Eventhough the kinetics of interaction is different for the two 3H-labelled antibodies, both are interiorized by endocytosis, gain access to lysosomes and their resulted degradation products are released from the cells. For 9H4, recycling of the antigenic sites after antibody unloading inside lysosomes is assumed. After 24 hours of incubation, cell fractionation experiments localize the 7F11C7 antibody in lysosomes whereas 9H4 is found associated with both plasma membranes and lysosomes.

Drug Targeting with Monoclonal Antibody for Human Hepatomas

Abstract Binding, uptake and subcellular distribution of monoclonal antibody to surface antigen of hepatitis B virus (HbsAg) or to alphafoetoprotein (AFP) have been studied in cultured human hepatoma cells. 3H labelled anti-HbsAg IgG is taken up selectively by PLC/PRF/5 cells. In constrast anti-AFP IgG is not selectively taken up by three cell lines. Cell fractionation studies indicate that these antibodies, as control IgG, gain access to lysosomes wherein they are digested.

Evidence for Endocytosis of Iron Loaded Transferrin by Cultured Rat Embryo Fibroblasts

The interaction of 59Fe loaded 3H labelled transferrin with cultured rat fibroblasts has been studied. At 4°C, 3H transferrin binds to a specific receptor but also to low affinity sites. At 37°C, cells take up 59Fe from 3H transferrin in a process continuous with time and nearly saturable with transferrin concentration; iron is incorporated for a large extent into ferritin. 3H label is accumulated by the cells, mainly in lysosomes; degradation products are released from the cells into the culture medium. The uptake of 3H transferrin proceeds continuously with time, is not saturable with transferrin concentration and is much lower than that of 59Fe. Short term experiments demonstrate that there is a mechanism whereby after iron release, the iron depleted protein is returned intact to the culture medium. Methylamine and chloroquine considerably reduce the uptake of iron by fibroblasts. These agents inhibit the delivery of transferrin to lysosomes and the rate at which iron depleted transferrin is released from the cells. We propose that transferrin is taken up by receptor-mediated endocytosis, that iron is detached inside the lysosomes as a consequence of the low pH, and that iron depleted protein is recycled back to the cell surface and released into the extracellular medium. The effect of chloroquine could be attributed to an increase of the lysosomal pH and that of methylamine to the inhibition of membrane recycling.

Binding, Uptake and Processing of Polymeric IgA by Cultured Rat Hepatocytes

The binding, uptake and processing of polymeric IgA (pIgA) by cultured rat hepatocytes have been studied in conditions where the cells reassociate into hepatic-like trabeculae and reform bile caniculi.

Daunorubicin Conjugated to Galactosylated Neoglycoprotein as Drug Carrier Conjugate for Human Hepatomas

Abstract Human serumalbumin to which galactose residues have been linked is selectively taken up via receptor mediated endocytosis by hepatocytes and hepatoma cells. Daunorubicin has been linked to this neoglycoprotein via a succinylated tetrapeptide arm. This conjugate is selectively taken up by cultured rat hepatocytes and human hepatoma Hep G2 cells in a time and concentration dependent process. Our results strongly suggest that the conjugate is endocytosed and that within the lysosomes, the drug is liberated intact whereas the protein is digested. These results indicate that daunorubicin-galactosylated serumalbumin could be used for the treatment of human hepatoma.