GD3 Vaccines for Melanoma: Superior Immunogenicity of Keyhole Limpet Hemocyanin Conjugate Vaccines I

Cell surface gangliosides show altered patterns of expression as a consequence of malignant transformation and have therefore been of interest as potential targets for immunotherapy, including vaccine construction. One obstacle has been that some of the gangliosides that are overexpressed in human cancers are poorly immunogenic in humans. A case in point is 13o3, a prominent ganglioside of human malignant melanoma. Using an approach that has been effective in the construction of bacterial carbohydrate vaccines, we have succeeded in increasing the immunogenicity of GD3 in the mouse by conjugating the ganglioside with immunogenic carHers. Several conjugation methods were used. The optimal procedure involved ozone cleavage of the double bond of GD3 in the ceramide backbone, introducing an aldehyde group, and coupling to aminolysyl groups of proteins by reductive amination. Conjugates were constructed with a synthetic multiple antigenic peptide expressing repeats of a malarial T-cell epitope, outer membrane proteins of Neisseria meningitidis, cationized bovine serum albumin, keyhole limpet hemocyanin, and polylysine. Mice immunized with these conjugates showed a stronger antibody response to GD3 than mice immunized with unconjugated GD3. The strongest response was observed in mice immunized with the keyhole limpet hemocyanin conjugate of the Gm aldehyde derivative and the adjuvant QS-21. These mice showed not only a long-lasting high-titer IgM response but also a consistent high-titer IgG response (predominantly IgG1), indicating recrnitment of T-cell help, although the titers of IgM and IgG antibodies following booster immunizations were not as high as they are in the response to classical T-cell-dependent antigens. This method is applicable to other gangliosides, and it may be useful in the construction of immunogenic ganglioside vaccines for the immunotherapy of human cancers expressing gangliosides on their cell surface. I N T R O D U C ~ O N Gangliosides are glycolipid constituents of the cell membrane. The term was coined in 1942 to refer to lipids of the central nervous system that contained sialic acid, to signify their prime location in ganglion cells and their glycosidic nature (1). Their lipophilic component, the ceramide (an amide-linked long-chain sphingoid base and a fatty acid), is thought to be embedded in the outer membrane of the cell membrane lipid bilayer. The carbohydrate portion of the molecule is oriented toward the outside of the cell. Malignant transformation appears to activate enzymes involved in ganglioside glycosylation, resulting in altered patterns of ganglioside expression in tumors such as astrocytoma, neuroblastoma, and malignant melanoma (2). In normal melanocytes, for example, the predominant ganglioside is GM3 .3 Other gangliosides including GD3, GM2, GDla, and GT1 b constitute less than 10% of the total (3). In malignant melanoma, increased Received 7/30/93; accepted 11/1/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA33049 and CA 08478, USPHS Grant NS-11853-19, and the Perkin Foundation. 2 To whom requests for reprints should be addressed, at Immunology Program, Memorial Sloan-Kettering Cancer Center, 425 East 68th Street, Room 820 K, New York, NY 10021. 3 The designations GM3, GM2, GM1, GD3, GDla and Golb are used in accordance with the abreviated ganglioside nomenclature proposed by Svennerholm (40). expression of GD3, GD2, and GM2 has been observed (4, 5), and these gangliosides have therefore been considered potential targets for immunotherapy. One approach to ganglioside-targeted immunotherapy has been the use of mAbs. 4 Treatment of patients with melanoma or neuroblastoma with mAb recognizing GD3, GD2, or GM2 has resulted in tumor regression in some cases (6-9). The other approach has been to immunize patients with ganglioside vaccines in attempts to induce production of ganglioside antibodies by the patients themselves. These attempts have been successful so far only with GM2 vaccines. Patients with American Joint Committee on Cancer Stage III malignant melanoma, after complete resection of all tumor, have been shown to produce anti-GM2 antibodies in response to vaccination with GM2 and Bacillus Calmette-Gu~rin (after pretreatment with low-dose cyclophosphamide to reduce suppressor activity), and the disease-free interval and overall survival were longer in patients producing GM2 antibodies (10). GD3 and Go2, on the other hand, were found to be only rarely immunogenic when administered in the same way to patients with melanoma (11). Even with the GM2 vaccines, the antibody response showed the characteristics of a T-cell-independent response, that is to say, IgM production of short duration, rare conversion to IgG production, and lack of a booster effect (12, 13). Similar difficulties have been encountered in the development of effective vaccines against bacterial carbohydrate antigens. One approach that has been successful in overcoming these problems is conjugation of the antigen with immunogenic protein carriers. For example, a conjugate vaccine that links the Haemophilus influenzae type b capsular polysaccharide to the outer-membrane protein complex of Neisseria meningitidis serogroup B was recently shown to induce the production of antibodies and a high rate of protection against invasive disease caused by Haemophilus influenzae type b in infants (14), and similar results were reported for a conjugate vaccine using a nontoxic mutant diphtheria toxin as carrier (15). We have explored this approach in attempts to increase the immunogenicity of melanoma gangliosides. We report here the effects of conjugating GD3 with several protein carriers on its immunogenicity in the mouse. M A T E R I A L S A N D M E T H O D S Gangliosides. GM3, GM2 and GDlb, extracted from bovine brain, were provided by.Fidia Research Laboratory (Abano Terme, Italy). GD2 was made from Grab by enzymatic cleavage with /3-galactosidase from bovine testes (16). GD3 (reel) was isolated from human melanoma tissue (17), GD3 (bbm) and GT3 were isolated from bovine buttermilk (18), and disialyllactose (GD3 oligosaccharide) was isolated from bovine colostrum as previously described (19). Reagents. HPTLC silica gel plates were obtained from E. Merck (Darmstadt, Germany); 4-chloro-l-naphthol, p-nitrophenyl phosphate disodium, and sodium cyanoborohydride were from Sigma Chemical Co. (St. Louis, MO); 4 The abbreviations used are: mAb, monoclonal antibody; MAP, multiple antigenic peptide; OMP, outer membrane protein; cBSA, cationized bovine serum albumin; ITLC, immune thin-layer chromatography; HPTLC, high-performance thin-layer chromatography; ELISA, enzyme-linked immunosorbent assays; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; bbm, bovine buttermilk.