Endocytosis features of GD2-specific antibodies of different formats in tumor cells

Antibody-drug conjugates (ADCs) have emerged as one of the most promising classes of monoclonal antibody-based (mAb) targeted cancer therapies. To date, 15 drugs of this class have been approved for clinical use, while many more are undergoing more than 100 clinical trials. Similarly to unconjugated antibodies, ADCs target various tumor markers including carbohydrate antigens in glycosphingolipids. Among them, ganglioside GD2 is considered the most promising marker, and recent studies have shown significant potential for the use of anti-GD2 ADCs. Receptor internalization after antigen-antibody complex formation and its transport to cell lysosomes are important characteristics of the marker, on which the effectiveness of ADCs largely depends. The ability of GD2-specific antibodies to internalize and the mechanisms of endocytosis of their complexes with GD2 have been poorly studied. The study investigated the mechanisms of internalization of ganglioside GD2 complexes and the most relevant GD2-specific antibodies of various formats, including full-length antibodies, minibodies, and scFv fragments. It was demonstrated that all the antibody variants used are capable of internalizing into GD2-positive tumor cells and entering their lysosomal compartments. Full-length antibodies and minibodies demonstrate high efficiency of endocytosis in GD2-positive cells, which occurs through several pathways, primarily macropinocytosis and caveolae-mediated endocytosis. The obtained data may be of interest for development of more effective targeted drugs for the treatment of GD2-positive tumors.