Auto-antibodies to cancer antigens hold great promise as sensitive amplified biomarkers for early detection of cancer. Most high through-put strategies to discover such auto-antibodies largely fail to allow identification of antibodies specific for cancer-associated posttranslational modified variants of normal proteins. We hypothesized that aberrant processed proteins are likely auto-antibody targets. MUC1 is over-expressed and aberrantly glycosylated in many cancers and we sought to evaluate the potential of natural cancer-induced auto-antibodies to such aberrant O-glycoforms of MUC1 as sensitive diagnostic biomarkers of disease. We first demonstrated, using an antibody-based glycoprofiling ELISA, that circulating mucins in cancer patients exclude truncated aberrant cancer-associated glycoforms. We then developed an O-glycopeptide microarray and used this to demonstrate detection of IgG antibodies to MUC1 aberrant O-glycopeptide epitopes in patients vaccinated with 25Tn-MUC1-106-mer conjugated to KLH. Finally, screening of sera from breast, ovarian and prostate cancer patients led to identification of three distinct aberrant MUC1 O-glycopeptide epitopes that are targeted by cancer-associated IgG auto-antibodies. The results suggest that auto-antibodies to aberrant O-glycopeptide epitopes may represent a fruitful and previously unaddressed source of sensitive biomarkers for early detection of cancer. The methods developed for chemoenzymatic synthesis of O-glycopeptides in combination with microarrays allow for broader data-mining of the entire cancer O-glycopeptidome.

One of the most widely used and potent immunological adjuvants is a mixture of soluble triterpene glycosides purified from the soap bark tree (Quillaja saponaria). Despite challenges in production, quality control, stability and toxicity, the QS-21 fraction from this extract has exhibited exceptional adjuvant properties for a range of antigens. It possesses an ability to augment clinically significant antibody and T-cell responses to vaccine antigens against a variety of infectious diseases, degenerative disorders and cancers. The recent synthesis of active molecules of QS-21 has provided a robust method to produce this leading vaccine adjuvant in high purity as well as to produce novel synthetic QS-21 congeners designed to induce increased immune responsiveness and decreased toxicity.

CD1d-restricted T cells have been impli-

BackgroundEvidence indicates that Bax functions as a “lipidic” pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.Methodology/Principal FindingsMCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1–2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight “pore-forming” oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.Conclusions/SignificanceOur recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

Several novel, fully synthetic, carbohydrate-based antitumor vaccines have been assembled. Each construct consists of multiple cancer-related antigens displayed on a single polypeptide backbone. Recent advances in synthetic methodology have allowed for the incorporation of a complex oligosaccharide terminating in a sialic acid residue (i.e., GM2) as one of the carbohydrate antigens. Details of the vaccine synthesis as well as the results of preliminary immunological investigations are described herein.

SummaryCarbohydrates have established themselves as the most clinically relevant antigens of those tested and subsequently developed for vaccines against infectious diseases. However, in cancer patients, many of the defined carbohydrate antigens are really altered 'self' antigens and for unclear reasons, the body does not react to them immunologically. Although these self antigens have been found to be potentially suitable targets for immune recognition and killing, the development of vaccines for cancer treatment is actually more challenging compared with those for infectious diseases mainly because of the difficulty associated with breaking the body's immunological tolerance to the antigen. These antigens lack the inherent immunogenicity associated with bacterial antigens and, therefore, methods to enhance immunological recognition and induction of immunity in vivo are under investigation. These include defining the appropriate tumour-associated antigen, successfully synthesizing the antigen to mimic the original molecule, inducing an immune response, and subsequently enhancing the immunological reactivity so that all components can work together. This has been successfully accomplished with several glycolipid and glycoprotein antigens using carriers such as keyhole limpet haemocyanin (KLH) together with a saponin adjuvant, QS-21. Not only can high titre IgM and IgG antibodies be induced, which are specific for the antigen used for immunization, but the antibodies can mediate complement lysis. The approaches for synthesis, conjugation, clinical administration and immunological potential are discussed.

We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.

Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability, and an enigmatic molecular mechanism of action. Herein, we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained at the injection site and nearest draining lymph nodes preferentially compared to attenuated variants. Overall, these studies have yielded critical insights into saponin structure–function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.