Exploiting CD38-mediated endocytosis for immunoliposome internalization

Abstract

CD38 appears to be a promising candidate in antibody therapy; it is upregulated on cell surfaces in many lymphoid tumors and undergoes rapid internalization after interaction with antibodies. The receptor-mediated endocytosis allows conjugating toxins/drugs that promote suicide only of the malignant cells. Here, we describe the preparation of CD38-immunoliposomes and test their functionality by incubating them with CD38+/- cells. Liposomes were prepared by extrusion of a lipid mixture containing a biotinylated polyethylene glycol-phospholipid and loaded with 5(6)-carboxyfluorescein. The anti-CD38 antibody (IB4) was biotinylated and then linked to streptavidin molecules; streptavidin acts like a bridge between the antibody and the biotinylated lipid of the liposomes. CD38+/- cells were incubated either with liposomes or immunoliposomes and analyzed by fluorescence microscopy and cytofluorimetry. The results indicated a specific mechanism of internalization, owing to CD38-mediated endocytosis, where CD38+ cells incubated with immunoliposomes scored top fluorescence levels. This coupling strategy, based on the use of a streptavidin bridge to prepare immunoliposomes, does not interfere with the cellular functionality and its broad potential use represents a great advantage. Here IB4, a murine monoclonal anti-CD38 antibody, was used to simplify the experiments, but the coupling procedure may be suitable also with human antibodies, against CD38 or other human markers.