Rational design and synthesis of unnatural glycopeptides for detecting tumor-associated autoantibodies in pancreatic cancer

Cancer remains the second leading cause of death worldwide, with nearly 20 million newcases and 9.7 million cancer-related deaths reported in 2022 alone. By 2040, the annualincidence is projected to rise to 29.9 million. The ongoing need for innovative and effectivetreatment strategies is compounded by the critical importance of early detection, especiallyfor aggressive cancers such as pancreatic cancer, where survival rates are significantlyimproved through timely diagnosis.Mucin-1 (MUC1) is a heavily O-glycosylated glycoprotein expressed on the epithelial cellsurface. In healthy tissues, the peptide backbone is adorned with complex oligosaccharides,but in cancer cells, aberrant glycosylation results in truncated carbohydrates, exposing tumorassociated carbohydrate antigens (TACAs) like the Tn determinant (-O-GalNAc-Ser/Thr).1These antigens are immunogenic and recognized by anti-MUC1 antibodies, making thempromising biomarkers for early-stage cancer. However, current diagnostic methods often relyon natural antigens, which may lack the specificity and stability required for accuratedifferentiation.2 Furthermore, anti-MUC1 antibodies are found in both cancer patients andhealthy individuals, though at markedly lower levels in the latter, emphasizing the need forprecise synthetic antigen models that faithfully mimic tumor-associated epitopes of MUC1.In this work, we developed a diagnostic assay for pancreatic cancer that leverages tumorassociated autoantibodies against MUC1 using engineered glycopeptides displayed onnanoparticle probes. By employing a structure-guided approach, we designed non-naturalglycopeptides based on the MUC1 scaffold with enhanced binding affinity for specific antiMUC1 antibodies. These glycopeptides were tailored to recognize distinct tumor-associatedepitopes, as targeted by monoclonal antibodies SM3 and 5E5. The synthetic glycopeptideswere conjugated onto gold nanoparticles and integrated into a dot-blot assay to detectautoantibodies in serum samples from pancreatic cancer patients and healthy controls. Thisapproach highlights the potential of engineered peptide-based diagnostics in improving thespecificity and sensitivity of cancer detection.3