Jeffrey C. Gildersleeve

Carbohydrate array analysis of anti-Tn antibodies and lectins reveals unexpected specificities : Implications for diagnostic and vaccine development

The Tn antigen is a carbohydrate antigen expressed in most carcinomas, during embryogenesis, on pathogenic parasites, and on HIV. It has been evaluated extensively as a potential diagnostic marker and several Tn‐based vaccines are in clinical trials. Based on discrepancies in the literature regarding Tn expression, we began to question whether antibodies and lectins used routinely to detect the Tn antigen were providing accurate information. To investigate this possibility, a carbohydrate microarray and a highly sensitive assay were developed and three frequently used Tn receptors (HBTn1, Bric111, and VVL‐B4) were evaluated. Carbohydrate‐array analysis revealed unexpected cross‐reactivity with other human carbohydrate epitopes. VVL‐B4 bound the Tn antigen, GalNAcα1‐6Gal, and GalNAcα1‐3Gal. Bric111 bound the Tn antigen, blood group A, GalNAcα1‐6Gal, and GalNAcα1‐3Gal. HBTn1 showed the best selectivity, but still displayed moderate binding to blood group A. Implications for the development of Tn‐based diagnostics and vaccines are discussed.

Resolving conflicting data on expression of the Tn antigen and implications for clinical trials with cancer vaccines

The tumor-associated Tn antigen has been investigated extensively as a biomarker and therapeutic target. Cancer vaccines containing the Tn antigen as a single tumor antigen or as a component of a polyvalent vaccine have progressed into phase I and II clinical trials. One major focus of Tn-based vaccines is the treatment of prostate cancer patients. Although expression of the antigen on prostate tumors is a critical prerequisite, previous reports investigating Tn expression in prostate tumors have produced conflicting results. Using a combination of immunohistochemistry and carbohydrate microarray profiling, we show that only 4% to 26% of prostate tumors express the Tn antigen. Based on our results, the majority of prostate cancer patients do not express the appropriate antigen. Therefore, efforts to preselect the subset of prostate cancer patients with Tn-positive tumors or apply Tn vaccines to other cancers with higher rates of antigen expression could significantly improve clinical response rates. Because conflicting information on carbohydrate expression is a general problem for the field, the approach described in this article of analyzing antigen expression with multiple antibodies and using carbohydrate microarray profiles to interpret the results will be useful for the development of other carbohydrate-based cancer vaccines and diagnostics.[Mol Cancer Ther 2009;8(4):971–9)

GalNAcα1-3Gal, a new prognostic marker for cervical cancer

Cancer cells undergo significant changes in carbohydrate expression, and these alterations can be useful as biomarkers and therapeutic targets. In this study, we investigated the expression of carbohydrate antigens containing a terminal GalNAcα1‐3Gal or GalNAcα1‐6Gal on human cervix and cervical carcinoma. Monoclonal antibodies to each of these carbohydrates were generated by immunizing rabbits with the corresponding antigen conjugated to KLH followed by hybridoma production. Antibodies were screened and evaluated using a combination of carbohydrate microarray profiling, ELISA, dot blot and immunohistochemical staining to verify specificity. Antibody 132‐3 was found to selectively recognize GalNAcα1‐3Gal with little cross‐reactivity to other structurally similar antigens such as GalNAcα1‐6Gal, blood group A, Forssman antigen and the Tn antigen on both solution assays and human tissue. Although GalNAcα1‐6Gal expression was not detected, GalNAcα1‐3Gal expression was found on 55% of squamous cell carcinomas. Expression in normal tissue was observed but was restricted to the suprabasal epithelial layer. Importantly, we found expression of the antigen on cervical cancer had a statistically significant correlation with the 5‐year survival rate of the patients (48 vs. 85% for antigen negative vs. positive, p = 0.017). Expression of GalNAcα1‐3Gal did not correlate with other clinical factors including tumor stage, size and lymph node metastasis, indicating the antigen is a new, independent biomarker for the prognosis of cervical cancer. Published 2009 UICC.