Cancer Biomarkers Defined by Autoantibody Signatures to Aberrant O-Glycopeptide Epitopes

Abstract

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.

Microvesicle Cargo of Tumor-Associated MUC1 to Dendritic Cells Allows Cross-presentation and Specific Carbohydrate Processing

“…In carcinomas that express MUC1, the tumor-associated glycoforms of MUC1 have been shown to be immune targets in vivo (27). In patients with cancer, CD8 þ T cells directed against MUC1 peptide epitopes were detected before and after vaccination (28)(29)(30), and the IgG auto-antibodies against specific tumor-associated glycoforms were found in patients with cancer with early-stage disease (5,31). Early work showed that MUC1 following internalization is blocked in the endolysosomal compartment of dendritic cells (9), suggesting that only a tolerizing CD4 þ T-cell response could be generated against this glycoantigen.…”

Engineering Mammalian Mucin-type O-Glycosylation in Plants

“…Our interest in engineering O-glycosylation from scratch stems from our previous identification of immunodominant aberrant O-glycopeptide epitopes in the cancer-associated MUC1 mucin, which are not covered by immunological tolerance (41,61,62). Vaccination with MUC1 glycopeptides with the truncated GalNAc-Ser/Thr O-glycoform produced by GalNAc-T2 and -T4 in combination produce IgG antibodies with cancer-specific reactivity (61), and spontaneous IgG antibodies to the same epitope are found in many cancer patients at time of diagnosis (3,63). Recombinant production of such vaccines will require a host cell that produces similar truncated O-glycans, and glycoengineered plants as reported here may provide such a system.…”

“…with respect to design of vaccines aimed at eliciting immunity to specific glycoforms found on virus particles and virus-infected cells or on cancer cells (2). We have previously shown that the cancer-associated mucin MUC1 contains immunodominant aberrant O-glycopeptide epitopes to which cancer-specific IgG antibodies can be elicited in man (3), and we have developed a chemoenzymatic strategy for synthesis of such vaccine glycopeptides (3). However, sustainable recombinant expression systems for production of vaccines targeting cancer cells with immature O-glycosylation or envelope virus glycoproteins are still in demand.…”

“…O-Glycosylation is controlled by a family of up to 20 UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) 3 ; the repertoire of GalNAc-Ts present in a given cell defines the pattern and density with which proteins are decorated with O-glycans. Normal mammalian cells typically produce elongated and/or branched O-glycans capped with sialic acids or different blood group-related structures.…”

Autoanticorps et diagnostic précoce des cancers

“…Les autoanticorps comme source de biomarqueurs Bien que peu de données soient disponibles concernant l'origine de la production d'autoanticorps en réponse à la présence tumorale, il est maintenant bien établi que les patients atteints de cancers produisent des autoanticorps contre des protéines mutées [4], mal repliées [5], surexprimées [6], dégradées de manière aberrante [7], glycosylées de manière aberrante [8,9], ou exprimées de manière ectopique [10]. Ces réponses spontanées sont fréquemment détectées chez les patients, de 5 à 30 % des patients pour un seul autoantigène.…”