MUC1 glycopeptide epitopes predicted by computational glycomics

Abstract

Bioinformatic tools and databases for glycobiology and glycomics research are playing increasingly important roles in functional studies. However, to verify hypotheses generated by computational glycomics with empirical functional assays is only an emerging field. In this study, we predicted glycan epitopes expressed by a cancer-derived mucin, MUC1, by computational glycomics. MUC1 is expressed by tumor cells with a deficiency in glycosylation. Although numerous diagnostic reagents and cancer vaccines have been designed based on abnormally glycosylated MUC1 sequences, the glycan and peptide sequences responsible for immune responses in vivo are poorly understood. The immunogenicity of synthetic MUC1 glycopeptides bearing Tn or sialyl-Tn antigens have been studied in mouse models, while authentic glyco-epitopes expressed by tumor cells remain unclear. To examine the immunogenicity of authentic cancer derived MUC1 glyco-epitopes, we expressed membrane bound forms of MUC1 tandem repeats in Jurkat, a mutant cancer cell line deficient of mucin-type core-1 β1–3 galactosyltransferase activity, and immunized mice with cancer cells expressing authentic MUC1 glyco-epitopes. Antibody responses to individual glyco-epitopes were determined by chemically synthesized candidate MUC1 glycopeptides predicted through computational glycomics. Monoclonal antibodies can be generated toward chemically synthesized glycopeptide sequences. With RPAPGS(Tn)TAPPAHG as an example, a monoclonal antibody 16A, showed 25-fold higher binding to glycosylated peptide (EC50=9.278±1.059 ng/ml) compared to its non-glycosylated form (EC50=247.3±16.29 ng/ml) as measured by ELISA experiments with plate-bound peptides. A library of monoclonal antibodies toward authentic MUC1 glycopeptide epitopes may be a valuable tool for studying glycan and peptide sequences in cancer, as well as reagents for diagnosis and therapy.

PSCA and MUC1 in non-small-cell lung cancer as targets of chimeric antigen receptor T cells

“…The clone 16A binds to glycopeptide RPAPGS(GalNAc)TAP-PAHG of MUC1 (MUC1-Tn) with high affinity. 52 The scFv of HMFG2 (used to engineer the CAR) can recognize a range of tumor-associated MUC1 glycoforms, such as Tn, STn, T and ST (binds to MUC1-Tn and MUC1-STn with higher affinity). HMFG2 has the broadest capacity for strong binding to tumorassociated MUC1 glycoforms.…”

Separation and detection of minimal length glycopeptide neoantigen epitopes centering the GSTA region of MUC1 by liquid chromatography/mass spectrometry

“…We chose to use two synthetic 13‐residue glycopeptides, RPAPGST (Tn)APPAHG and RPAPGS (Tn)TAPPAHG, as the starting materials for generating glycopeptides shorter than 10 amino acids. The chemical synthesis of glycopeptides was as described using fluorenylmethyloxycarbonyl (Fmoc)‐protected amino acids as the building blocks. The purity of the synthetic glycopeptides was examined by reversed‐phase HPLC and MS determination of molecular masses.…”

“…Identification of the glycosylation site on the above motifs is critical for dissecting the exact MUC1 antigenic epitopes for cancer diagnosis and therapy. For example, we have reported the expression of an abnormally glycosylated GSTA neoantigen motif in non‐small‐cell carcinoma cells . Biologically, the GSTA neoantigen motif is independent from the most widely studied PDTR motif within the tandem repeat sequence of MUC1.…”

“…For example, we have reported the expression of an abnormally glycosylated GSTA neoantigen motif in non-small-cell carcinoma cells. 3,4 Biologically, the GSTA neoantigen motif is independent from the most widely studied PDTR motif within the tandem repeat sequence of MUC1.…”

An antibody drug conjugate targeting a GSTA glycosite-signature epitope of mucin1 expressed by non-small cell lung cancer

“…Cell surface expression of MUC1 was assessed by flow cytometry staining. The cells were washed with 2% BSA in PBS and then incubated with the 16A 21…”