The objective of this study is to produce and characterize human monoclonal anti-ganglioside antibodies for the treatment of human cancer. Our laboratory has produced and now houses three stable human B-lymphoblastoid cell lines that secrete IgM monoclonal antibodies to three of the four ganglioside antigens commonly expressed by melanoma, GD2, GM2, and GM3, respectively. We have demonstrated the anti-tumor effect of these human monoclonal antibodies (HuMAbs) through in vitro assays, an in vivo nude mouse model, and in human melanoma via intralesional injection. Complete regression has been achieved in several patients without any noticeable side effects. In this renewal proposal we will develop HuMAbs to the fourth ganglioside, GD3, a major ganglioside in melanoma. 9-0-acetylated GD3 which is a GD3 containing sialic acid with an o-acetyl ester on carbon 9 and more immunogenic than GD3 in man, will be used as an antigen source during HuMAb development. Human antibody to 9-0-AcGD3 has been shown to cross react with GD3. The conventional EBV-transformation technique as well as the repertoire cloning method will be employed in the generation of HuMAbs. Development of HuMAbs to various ganglioside molecules is necessary since the ganglioside profile differs significantly from tumor to tumor and antigen-negative tumors do not respond to HuMAb therapy. HuMAbs developed against ganglioside antigens are primarily of the IgM class. In an effort to generate IgG isotype HuMAbs, we have been experimenting with recombinant DNA technology using the variable region of IgM HuMAbs and IgG human constant domains. Polymerase chain reaction (PCR) is used to amplify DNA encoding V regions of the parental HuMAbs. It is hoped that production of IgG by myeloma cells transfected with these genes is greater than IgM production by EBV-transformed human B-- lymphoblasts. In addition, methods to purify IgG on a large scale are well established while IgM purification is still very difficult and costly. We are hopeful that these recombinant IgG chimeric antibodies will exhibit the ability to lyse tumor cells not only via the complement dependent route but also through cellular cytotoxicity. Different subclasses of IgG antibodies to GD2, GM3, GM2, and 9-0-AcGD3 will be investigated. Finally, we will evaluate the therapeutic usefulness of these chimeric antibodies and compare it to that of native IgM antibodies, and select one or more subclasses most suited for the treatment of human melanoma. In the future studies will extend to the application of these HuMAbs to other histological types of cancer which express ganglioside antigens.
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