Antitumor effects of a novel monoclonal antibody with high binding affinity to ganglioside GD3

Abstract

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface human tumors of neuroectodermal origin, has been studied as a target molecule for passive immunotherapy. We established ten kinds of anti-GD3 monoclonal antibodies (mAb) of the mouse IgG3 subclass by immunization with purified GD3 and melanoma cells. One of the established mAb, KM641, showed major reactivity with GD3 and minor reactivity with GQ1b out of 11 common gangliosides in an enzymelinked immunosorbent assay. Immunostaining of gangliosides, separated on thin-layer chromatography plates, using KM641 revealed that most of the melanoma cell lines contained immunoreactive GD3 and GD3-lactone at a high level, but only the adrenal gland and the urinary bladder out of 21 human normal tissues had immunoreactive GD3. In immunofluorescence, KM641 bound to a variety of living tumor cell lines especially melanoma cells, including some cell lines to which another anti-GD3 mAb R24, established previously, failed to bind. High-affinity binding of KM641 to a tumor cell line was quantified by Scatchard analysis (K _d = 1.9×10⁻⁸ M). KM641 exerted tumor-killing activity in the presence of effector cells or complement against melanoma cells expressing GD3 at a high level. Not only natural killer cells but also polymorphonuclear cells were effective as the effector cells in antibody-dependent cellular cytotoxicity. Intravenous injection of KM641 markedly suppressed the tumor growth of a slightly positive cell line, C24.22 (7.2×10⁵ binding sites/cell), as well as a very GD3-positive cell line, G361 (1.9×10⁷ binding sites/cell), inoculated intradermally in nude mice. KM641, characterized by a high binding affinity for GD3, has the potential to be a useful agent for passive immunotherapy of human cancer.