Esther Aboud-Pirak

Cytotoxic activity of daunorubicin or vindesin conjugated to a monoclonal antibody on cultured MCF-7 breast carcinoma cells.

Conjugates were constructed between daunorubicin or vindesin and a monoclonal antibody to human milk fat globule membrane associated antigen. This antibody recognizes a high molecular weight glycoprotein present at the cell surface of human normal and tumour epithelial cells; after specific binding to plasma membrane of cultured MCF-7 human breast carcinoma cells, it is endocytosed and gains access to lysosomes, wherein it is broken down (Aboud-Pirak et al., Cancer Res 48: 3188-3196, 1988). Covalent linkage of daunorubicin (through a succinylated tetrapeptide arm) or of vindesin (through a hemisuccinate arm) yields conjugates with maximal molar ratios (drug molecule/specific IgG under monomeric form, i.e. unaggregated) or 2.0 and 4.5 respectively. The conjugate with daunorubicin inhibits the binding of the 3H labelled antibody to MCF-7 cells as efficiently as the native unconjugated antibody, whereas the conjugate with vindesin inhibits it only by 56%. Both conjugates are entirely stable in plasma and serum; after 24 hr incubation at pH 4.8 in the presence of rat liver lysosomal enzymes, 60 and 33% of daunorubicin and vindesin respectively are released from the conjugates. Adherent non-confluent cultures of cells recognized (MCF-7) or not (Hep-G2, human hepatocarcinoma cells) by the antibody were incubated from 1 hr to 6 days with different concentrations of daunorubicin or vindesin, free or conjugated to the specific or to a control monoclonal antibody. LD50, defined as the drug concentration required to reach 50% of the amount of cell associated protein obtained in the absence of drug were determined at the end of 6 days continuous incubation or after shorter incubation followed by reincubation in drug free medium up to 6 days. Both cell lines are almost equally susceptible to the free drugs. The conjugate between daunorubicin and the antibody appears inactive, even at saturating concentrations of antibody. This could result from the extrusion out of the cells of daunorubicin molecules released from the conjugate, impairing the drug to reach the intracellular concentration required for cytotoxicity. In contrast, conjugation of vindesin to the specific but not to a control antibody restricts the activity of the drug to cells selectively recognized by the specific antibody. However, even after corrections for the loss of immunoreactivity and for the incomplete release of vindesin from the conjugate, cytotoxicity is achieved at higher concentrations or requires longer exposure to the conjugated than to the free drug.(ABSTRACT TRUNCATED AT 400 WORDS)

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.