Pancreatic cancer is one of the most elusive tumors to both diagnose and treat, rendering it a disease with one of the highest mortality rates. Some of these difficulties are due to the lack of an early stage, detectable, and valid biomarker on the surface of pancreatic tumors. However, one cell-surface protein, Mucin 4 (MUC4) has been shown to be overexpressed in &gt; 80% of pancreatic tumors but absent in normal pancreatic tissue. MUC4 is a large glycoprotein containing a tandem repeat (TR) peptide sequence containing several serine and threonine residues that can be aberrantly glycosylated during tumorigenesis. These tumor-associated carbohydrate antigens (TACAs) are recognized as foreign to the immune system and have been used as immunogens in a variety of studies related to the development of tumor vaccines. One of these TACAs, the Thomsen-Friedenreich antigen (TF) is commonly found on tumor mucins and has been the basis for the design of anti-cancer immunotherapies. Our previous work had shown that nanoparticles coated with TF antigen-conjugated glycopeptides from the MUC4 TR sequence could generate an immune response in mice. One glycopeptide emerged as a lead for further analysis and this structure was used as an immunogen for antibody production. Immunization of rabbits and analysis of affinity-purified anti-MUC4 antibodies (cat#600-401-GY2, produced in collaboration with Rockland Immunochemicals, Inc.), showed preferential binding to the glycosylated MUC4 TR sequence over the non-glycosylated peptide. Flow cytometry experiments demonstrated specific recognition of MUC4+ cell lines over MUC4- cells. These results prompted us to investigate the specificity of binding using two microarray screening techniques: 1) A glycan microarray (GA) and, 2) a pancreatic tissue microarray (TMA). The GA revealed selective binding to only MUC4 constructs in an array composed of over 800 different glycans and glycopeptides. Staining of a TMA containing both normal tissue and pancreatic adenocarcinomas revealed specific binding to human pancreatic tumors. Additionally, there was evidence of intense staining in the surrounding intestines of what were deemed metastatic tumor emboli, indicating that the antisera may selectively stain more advanced stage tumors. Structural studies of the glycopeptide and antibody/glycopeptide complex are in progress to elucidate the features of the molecule that may be relevant to the development of a novel immunotherapy.

Developing immunotherapies for pancreatic cancers

256th National Meeting (2018)

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