title: Constructing an adenocarcinoma vaccine: immunization of mice with synthetic KH-1 nonasaccharide stimulates anti-KH-1 and anti-Le(y) antibodies.
abstract: There is mounting evidence to suggest that immunization-based strategies can be used to mobilize the human immune system against specific carbohydrate antigens displayed on the surface of cancer cells. Following isolation and identification, such antigens can be administered as conjugate vaccines. The tumor-associated carbohydrate antigen KH-1 is 1 such antigen and may serve as a potential target for immunization against adenocarcinoma. However, a serious impediment to the application of a vaccine-based approach involving this antigen is that its availability from natural sources is severely limited. In order to overcome this limitation, we have developed an efficient total synthesis of this complex glycolipid. We have extended our synthesis to reach a structurally related analog in which the ceramide portion of KH-1 is replaced with an allyl substituent. These synthetic advances have led to the preparation of 2 potential vaccine constructs, each based on the conjugation of the KH-1 nonasaccharide and the carrier protein keyhole limpet hemocyanin (KLH). In 1 construct (KH-1-Et-KLH), the nonasaccharide is conjugated to KLH via a simple ethyl linkage, while in the other (KH-1-MMCCH-KLH), conjugation is mediated by a 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) cross-linker. We report here the immunological properties of these 2 constructs. Mice were immunized with either of the 2 KH-1-KLH vaccine candidates or the KH-1 ceramide, along with the immunological adjuvant QS-21. Immunization with the ceramide served as a negative control and, as expected, failed to stimulate the production of antibodies against the KH-1 glycolipid. The construct in which the KH-1 nonasaccharide is linked to KLH via a simple alkyl chain stimulated significant quantities of IgM antibodies, whereas the construct linked to KLH by MMCCH induced high titers of both IgM and IgG antibodies. Inhibition data demonstrated that antibodies generated in response to immunization with the KH-1-KLH constructs recognize not only the KH-1 antigen but also the Lewis(y) (Le(y)) antigen, which, from a structural perspective, is similar to the 4 residues located at the non-reducing end of the KH-1 nonasaccharide. Thus, the KH-1-KLH constructs elicit an immune response that successfully targets 2 adenocarcinoma markers. As assessed by FACS analysis, the antibodies raised were strongly reactive with the KH-1/Le(y) positive cell line MCF-7 but not with KH-1 and Le(y) negative melanoma cell lines. Based on the results of our study, a KH-1-KLH plus QS-21 vaccine is being prepared for clinical evaluation.
authors.affiliation: Laboratory of Tumor Vaccinology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. ragupatg@mskcc.org
authors.forename: Govindaswami
authors.initials: G
authors.lastname: Ragupathi
authors.forename: Prashant P
authors.initials: PP
authors.lastname: Deshpande
authors.forename: Don M
authors.initials: DM
authors.lastname: Coltart
authors.forename: Hyunjin M
authors.initials: HM
authors.lastname: Kim
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Williams
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Danishefsky
authors.forename: Philip O
authors.initials: PO
authors.lastname: Livingston
mesh_terms: D000230:Adenocarcinoma / Q000276:immunology* / Q000517:prevention & control; D000276:Adjuvants, Immunologic; D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000276:immunology*; D019496:Cancer Vaccines / Q000138:chemical synthesis* / Q000276:immunology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002518:Ceramides / Q000737:chemistry / Q000276:immunology; D003432:Cross-Linking Reagents; D005260:Female; D005434:Flow Cytometry; D006433:Hemocyanins / Q000276:immunology; D007114:Immunization; D007074:Immunoglobulin G / Q000096:biosynthesis; D007075:Immunoglobulin M / Q000096:biosynthesis; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000276:immunology; D012503:Saponins / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D015295:Antigens, Tumor-Associated, Carbohydrate; D019496:Cancer Vaccines; D002518:Ceramides; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen; D009844:Oligosaccharides; D012503:Saponins; C105737:antigen KH-1; C078785:saponin QA-21V1; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin