Background: Thomsen-nouvelle (Tn) and its sialylated form (sTn) are carbohydrate antigens reported to be present on a broad array of tumor types, including breast, lung, and ovarian, but are not found on the cell surface of normal tissues. Tn is a single sugar residue that is added to Ser or Thr residues during the first step of O-glycosylation. In normal cells, subsequent saccharides are added to make a complex mature glycan. In many cancers, however, O-glycoproteins with the immature precursor (Tn) find their way to the cell surface where they can be exploited as tumor markers. Methods: Discovery: Fully human anti-Tn monoclonal antibodies were isolated from B cell hybridomas derived from patient samples post-vaccination. The human vaccine was directed against three glycan targets, including Tn. Target validation: Tumor microarrays were used to determine the frequency of candidate antibody reactivity with cancers. Lead optimization: The original lead, an IgM, was converted to IgG1 and affinity matured. Higher-affinity variants were generated and tested for target selectivity, tumor binding, ADCC, and internalization by FACS and ELISA-based methods. Results: Several clones were identified that recognized synthetic antigen related to the vaccine. One lead, mAb2F3, also bound to Tn-positive cells and was selected for further evaluation. EC50 values for binding of mAb2F3 are 7 nM (cells) and 9 nM (synthetic antigen). mAb2F3 binds to Tn displayed on diverse synthetic scaffolds, as well as a short peptide derived from MUC-1, a heavily O-glycosylated protein associated with carbohydrate tumor antigens. mAb2F3 also binds STn, albeit to a lower level. mAb2F3 did not show binding to TF, a closely related core glycan. mAb2F3 was used to stain microarrays of tissue cores form a panel of primary human tumors. 75% of breast (including >80% of triple negative), 47% of ovarian, and 30% of colon tumor samples stained positive with mAb2F3. Affinity variants were obtained using a combined method of error-prone PCR and parsimonious mutagenesis. Mutants were screened by ELISA against a panel of Tn-containing antigens and confirmed by FACS. Mutants were identified with improved affinity resulting in >25-fold improvement in ELISA binding and 10-fold increase in cell binding. Original and matured antibodies were clustered by antigen and bound to CD16a as a measure of their ability to drive ADCC. Higher-affinity variants were able to more potently drive CD16a association by ELISA. Finally, in order to address the suitability of mAb2F3 for use as a targeting agent for a toxin or radioisotope therapeutic adjunct, internalization into T47-D breast cancer cells was measured. 20% of surface-bound antibody was internalized within an hour, with >50% internalization by 3 hours at 37 degrees. Conclusions: We have identified a fully human antibody that potently binds and recognizes Tn in multiple contexts, suggesting that Tn is the entire epitope, and adjacent protein carrier interaction is not required. mAb2F3 does not bind TF, a closely related glycan, suggesting that it is highly specific for Tn and STn. Affinity maturation improved binding activity to synthetic antigens and cells by >10-fold. Finally, mAb2F3 internalizes into cells, making it an attractive targeting agent for clinical use in difficult-to-treat cancers such as triple-negative breast and ovarian.

Citation Format: G Jonah Rainey, Ritsuko Sawada, Toni Jun, Aleksandra Marinkovic-Petrovic, Ayesha Kahn, Wolfgang Scholz, Paul W. Maffuid. A therapeutic antibody candidate against the broadly expressed Tn and STn carbohydrate cancer antigens [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B104.