(1) Vaccination with GM2/BCG induced immunoglobulin M (IgM) antibodies in most patients. (2) GM2 antibody production was associated with a prolonged disease-free interval and survival. (3) Comparison of the two arms of this trial as randomized fails to show a statistically significant improvement in disease-free interval or survival for patients treated with GM2/BCG vaccines.

GM2, GD2, and GD3 gangliosides are expressed on the surface of human melanoma cells and represent potential targets for immunological control of melanoma growth by monoclonal antibodies and active immunization. The immunogenicity of GM2 was investigated by analyzing the humoral immune response of melanoma patients to vaccination with cell lines selected for high GM2 expression and with vaccines containing purified GM2. The whole-cell vaccine and vaccines containing purified GM2 and bacillus CalmetteGuerin (BCG) elicited GM2 antibody in a high proportion of patients, particularly in GM2/BCG-vaccinated patients pretreated with cyclophosphamide and given a GM2/BCG booster immunization. Vaccines containing purified GM2 and Salmonella minnesota R595 as the adjuvant were also effective, but only in patients pretreated with cyclophosphamide. GM2 antibodies in vaccinated patients were of the IgM class and were cytotoxic for GM2-positive targets in the presence of human complement.Gangliosides are prominent cell-surface constituents of melanoma and other tumors of neuroectodermal origin. Three gangliosides, the monosialoganglioside GM2 and the disialogangliosides GD2 and GD3, are of particular interest to tumor immunologists because of their potential as targets for passive immunization with monoclonal antibodies (mAbs) and for active immunization with cancer vaccines. Despite the presence of GM2, GD2, and GD3 in normal brain and other tissues (1), these gangliosides are immunogenic in mice and humans; mouse mAbs have been generated against GM2 (2), GD2 (3, 4), and GD3 (5-8), and human sera and human mAbs with reactivity for GM2 (9-12), GD2 (11,13,14), and GD3 (15) have been identified.Over the past decade we have immunized sequential groups of melanoma patients with a variety of melanoma cell vaccines (16)(17)(18)(19)(20). These vaccine trials were based on our seriological analysis of the humoral immune response of melanoma patients to cell-surface antigens of autologous and allogeneic melanomas (21), and each vaccine was constructed to contain melanoma surface antigens known to be immunogenic in humans. Although vaccinated patients readily produced antibody to HLA-related alloantigens and heterologous serum components in the vaccine, only rarely was antibody elicited to more restricted melanoma antigens, such as class 1 (unique), or GD2 or other class 2 (shared) melanoma antigens. In parallel vaccine studies in the mouse, we have identified immunizing procedures that facilitate the serological response to tumor antigens (22-24). In the case of GM2, immunization with GM2-expressing tumor cells or purified GM2 only infrequently induced GM2 antibody in mice, whereas vaccines containing GM2 with adjuvants such as bacillus Calmette-Gudrin (BCG) or Salmonella minnesota R595 were far more effective (24).In the present study, we have examined the immunogenicity of GM2-containing vaccines in stage III melanoma patients. Two types of vaccines were used: a whole-cell vaccine containing high levels of GM2 and vaccines con...

GD3 is the ganglioside most abundantly expressed on the cell surface of human melanoma, and treatment with a murine MAb recognizing GD3 has induced major responses in a small proportion of patients with melanoma. We have therefore attempted to induce production of GD3 antibodies in melanoma patients by active immunization. We found, however, that vaccination with GD3-expressing melanoma cells or purified GD3 does not result in antibody production. We describe here attempts to overcome the poor immunogenicity of GD3 in patients with melanoma by chemical modification. GD3 lactones, GD3 amide and GD3 gangliosidol were synthesized, and the humoral immune response to these derivatives was analyzed. Immunization of melanoma patients with these GD3 derivatives resulted in production of IgM antibodies and, in the case of GD3 amide, also of IgG antibodies. The antibodies to the GD3 derivatives did not cross-react with GD3. This is in contrast to observations in the mouse, where GD3 lactone I induced antibodies that showed cross-reactivity with GD3. Thus, the human immune response was specifically directed toward the modified epitope, rather than to the native structure.

Gangliosides expressed in malignant melanoma are potential targets for immunotherapy. Immunization of melanoma patients with vaccines containing purified GM2 ganglioside has resulted in induction of GM2 antibodies, and high titers of GM2 antibodies have correlated with increased survival. Melanoma ganglioside 9-O-acetyl GD3 is another candidate for ganglioside vaccine construction because of its limited expression in normal human tissues. As purification of 9-O-acetyl GD3 from human melanoma (9-O-acetylated on the terminal sialic acid) is not practical for broad application, we investigated the antibody response of melanoma patients to O-acetyl GD3 from several additional sources: hamster melanoma (7-O-acetyl GD3), bovine buttermilk (mixture of 7-O-acetyl GD3, 9-O-acetyl GD3 and 7,9-di-O-acetyl GD3) and chemically modified GD3 from bovine brain (9-O-acetylated on the subterminal sialic acid). Only immunization with the buttermilk-derived O-acetyl GD3 preparation resulted in consistent production of IgM antibodies. However, the induced antibodies reacted with the immunogen and with 7-O-acetyl GD3 derived from hamster melanoma but not with 9-O-acetyl GD3 or human melanoma cells expressing 9-O-acetyl GD3 on their cell surface. In contrast, all O-acetyl GD3 derivatives used for immunization were recognized by murine MAbs that reacted with 9-O-acetyl GD3, and immunization of mice with buttermilk-derived O-acetyl GD3 resulted in the production of antibodies that reacted with human melanoma cells expressing 9-O-acetyl GD3. Apparently, the human and murine immune systems preferentially recognize different epitopes on these molecules.

The gangliosides GM2, GD2 and GD3 are differentiation antigens expressed on the cell surface of human melanomas and other cancers of neuroectodermal origin. We have compared the antibody response after vaccination with gangliosides GM1, GM2, GM3, GD2 and GD3 in the mouse. Purified gangliosides were injected subcutaneously alone or attached to Salmonella minnesota mutant R595 after pretreatment of the mice with low-dose cyclophosphamide. Spontaneous GM1 antibodies, but not antibodies against the other gangliosides, were detected in many mice, the incidence increasing with age. Purified gangliosides injected alone (in saline) induced no antibody response. R595/GM1 and R595/GD3 vaccination induced consistent high-titer antibody responses. R595/GM2 and R595/GD2 induced occasional antibody responses, and R595/GM3 induced no antibody response. Comparison of the antibody responses induced against these gangliosides in the mouse with those in man reveals that GM1, GM3 and GD2 have a similar immunogenicity in both species while the relative immunogenicity of GM2 and GD3 is reversed. To understand better the basis for these differences, the antibody responses against the five gangliosides in man and the mouse were compared with their known expression in normal tissues. No correlation was detected between ganglioside expression in normal brain and immunogenicity, consistent with this being a cloistered site. The antibody responses did correlate inversely with expression in normal non-brain human and murine tissues. Variations between species of ganglioside immunogenicity may reflect variations in ganglioside expression in normal tissues.