Recombinant human hexamer-dominant IgM monoclonal antibody to Ganglioside GM3 for treatment of melanoma

2406 Ganglioside GM3 is the most common ganglioside found on human melanoma cells. It is expressed at much higher densities and frequencies on melanoma cells than on normal cells. A human B-lymphoblastoid cell line that was established by immortalization of human B-cells by Epstein-Barr virus produces a human IgM antibody to GM3 (L612). L612 binds to GM3 and kills GM3-positive human melanoma cells in the presence of complement. L612 has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (~20%), pentameric IgM (~74%), and other minor IgM molecules. Because hexameric IgMs have been shown to activate complement more effectively than pentameric IgMs, we tested whether hexameric L612 plays a dominant role in the antitumor activity. Chinese hamster ovary (CHO) cells were transfected with L612 heavy (H)- and light (L)-chain genes, with or without the joining (J)-chain gene, that resulted in generation of pentamer dominant- and hexmaner dominant-IgM, respectively. Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was ~80% hexameric, whereas recombinant IgM secreted from CHO cells transfected with joining-chain genes (designated CJ45) was ~90% pentameric. CA19 and CJ45 were compared for their anti-melanoma activities by in vitro and in vivo experiments. Both CA19 and CJ45 IgMs caused complement-dependent cytotoxicity (CDC) against GM3 rich M-1 human and B16F10 mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller as compared to that of CJ45. Neither L612 nor its two recombinant forms (CA19 and CJ45) induced CDC against a GM3-negative A549 lung cancer cells, showing an antigen-specific binding and killing by these antibodies.
 The in vivo antitumor effects of the recombinant IgMs were evaluated using B16F10 cell line in a nude rat xenograft model. The nude rat’s complement had been shown to be as effective as human complement with respect to induction of CDC by these human IgM antibodies against the mouse melanoma. Intravenous injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing B16F10 xenograft. Compared to vehicle, CA19, CJ45, and L612 prolonged survival by 238%, 162%, and 155%, respectively. These results suggest that a hexamer-dominant human IgM against GM3 may provide a more potent treatment option for GM3-positive melanoma.