We have synthesized various formulations that have potential for active specific immunotherapy (ASI) of human cancers. Sialyl-Tn (STn) is a potentially important target structure for ASI because its expression on mucins is a strong, independent predictor of poor prognosis, suggesting that it may have functional significance in the metastatic process. In this first pilot study of synthetic sialyl-Tn hapten conjugated to keyhole limpet hemocyanin (STn-KLH), with Detox adjuvant, toxicity and humoral immunogenicity were assessed in 12 patients with metastatic breast cancer. Toxicity was minimal, restricted to local cutaneous reactions (apart from transient nausea and vomiting following single low-dose cyclophosphamide treatment). Using STn-conjugated human serum albumin in a solid-phase enzyme-linked immunosorbent assay, it was shown that all patients developed IgM and IgG specific for the synthetic STn hapten. Following immunization, most patients were shown to develop increased titres of complement-mediated cytotoxic antibodies, partially inhibited by synthetic STn hapten, but not by the related TF hapten. We also detected IgM and IgG antibodies reactive with natural STn determinants expressed on ovine submaxillary mucin, the STn specificity of this reactivity being confirmed by hapten inhibition. Evaluation of clinical efficacy in a small pilot study is difficult. Five patients are alive 12 or more months after entry, and another 4 patients are alive 6 or more months after entry into the study. All 3 patients with known widespread bulky disease progressed despite ASI, 2 having died from widespread cancer. Two patients had partial responses, each lasting 6 months. While several patients had disease stability for 3-10 months, 1 patient with pulmonary metastases remains stable 15 months after entry into the program.

MUC1 mucin is a large transmembrane glycoprotein, of which the extracellular domain is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is underglycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above) as well as the normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc), and TF (Gal beta1-3 GalNAc) carbohydrates. In the present study, NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRP core epitope region to the recognition and binding of a monoclonal antibody, Mab B27.29, raised against the intact tumor-associated MUC1 mucin. Four peptides were studied: a MUC1 16mer peptide of the sequence Gly1-Val2-Thr3-Ser4-Ala5-Pro6-Asp7-Thr8-Arg9-Pro10-Ala11-Pro12-Gly13-Ser14-Thr15-Ala16, two singly Tn-glycosylated versions of this peptide at either Thr3 or Ser4, and a doubly Tn-glycosylated version at both Thr3 and Ser4. The results of these studies showed that the B27.29 MUC1 B-cell epitope maps to two separate parts of the glycopeptide, the core peptide epitope spanning the PDTRP sequence and a second (carbohydrate) epitope comprised of the Tn moieties attached at Thr3 and Ser4. The implications of these results are discussed within the framework of developing a glycosylated second-generation MUC1 glycopeptide vaccine.

Synthetic human MUC1 peptides are important candidates for therapeutic cancer vaccines. To explore whether a human MUC1 peptide BP25 (STAPPAHGVTSAPDTRPAPGSTAPP) can be rendered immunogenic by incorporation in liposomes, the effects of physical association of the peptide with liposomes on immune responses were investigated. Lipid conjugated and nonconjugated MUC1 peptides were incorporated in liposomes with a composition of distearoylphosphatidylcholine/cholesterol/dimyristoylphosphatidylglyc erol (3:1:0.25, molar ratio) containing monophosphoryl lipid A (1% w/w of the total lipids). Liposomes were characterized for peptide retention by HPLC and for surface peptide display of MUC1 epitopes by flow cytometry. C57BL/6 mice were immunized with lipopeptide alone, peptide mixed with peptide-free liposomes, and peptide associated with liposomes in entrapped or surface-exposed forms. T cell proliferative responses, cytokine patterns, and antibody isotypes were studied. Results showed that immune responses were profoundly influenced by the liposome formulations. Physically associated, either encapsulated or surface-exposed, peptide liposomes elicited strong antigen-specific T cell responses, but not lipopeptide alone or peptide mixed with peptide-free liposomes. Analysis of the cytokines secreted by the proliferating T cells showed a high level of IFN-gamma and undetectable levels of IL-4, indicating a T helper type 1 response. Thus, physical association of the peptide with liposomes was required for T cell proliferative responses, but the mode of association was not critical. On the other hand, the nature of the association significantly affected humoral immune responses. Only the surface-exposed peptide liposomes induced MUC1-specific antibodies. A domination of anti-MUC1 IgG2b over IgG1 (94 versus 6%) was observed. Our results support the hypothesis that different immune pathways are stimulated by different liposome formulations. This study demonstrated that a liposome delivery system could be tailored to induce either a preferential cellular or humoral immune response.

Purpose: To determine the clinical toxicities and antibody response against sTn and tumor cells expressing sTn following immunization of high-risk breast cancer patients with clustered sTn-KLH

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