Abstract LB-002: A fully human antibody binds Tn and sTn carbohydrate antigens specifically on serine residues, without need for polypeptide interaction
Authors: Rainey G Jonah , Sawada Ritsuko , Ostrowski Jacek , Kahn Ayesha , Marinkovic-Petrovic Aleksandra , Mudd Kevin , Scholz Wolfgang , Maffuid Paul W. 
Published: August 2018

Abstract
Background: Thompsen-nouvelle (Tn) antigen is an O-linked glycan that is associated with a broad array of tumors. Tn is a single alpha-linked GalNAc added to Ser or Thr as the first step of a major O-linked glycosylation pathway. In cancer, the glycosylation machinery can generate mucins with glycans aberrantly truncated at the initial GalNAc (Tn), or singly modified by addition of a sialic acid (sTn) on variable number 20 amino acid tandem repeat (VNTR) sequences. A mounting body of evidence supports that the modified residue (Ser or Thr) has a major impact on attached Tn structure. Accordingly, reports have described monoclonal antibodies and lectins that differentiate among contexts. Here we describe a fully human antibody that interacts preferentially with Ser-Tn/sTn, and shows broad tumor reactivity.
Methods: 2F3 anti-Tn/sTn was generated from a patient who received a vaccine that incorporated a MUC1 VNTR with Tn on both Ser and Thr. B cells were isolated, hybridomas generated, and supernatants screened by ELISA for reactivity with polymeric Tn on a synthetic backbone. Hits were screened by ELISA for reactivity to related glycans and binding to Tn+ cells was confirmed by FACS. A lead antibody, 2F3, was selected and used to determine target prevalence on tumor microarrays. 2F3 was affinity matured by error prone PCR. Hits were screened for specificity by glycan array and a panel of Tn glycopeptides through collaboration with the Consortium for Functional Glycomics to determine the preferred Tn context recognized by 2F3.
Results: Five hybridomas secreted antibodies that bound Tn/sTn by ELISA. 2F3 bound Tn+ cells by FACS. 2F3 demonstrated strongest binding to multimeric Tn/sTn, and weaker binding to monomeric Tn. Tumor microarray studies demonstrated binding to 47% of ovarian and 75% of breast (including 89% of triple negative) cancer tissues. While MUC1 VNTR peptides with no Tn or with Tn on two different Thr residues showed no binding by ELISA, strong binding was observed to a MUC1 VNTR peptide modified on all 5 Ser/Thr. In order to determine whether spacing, number of Tn modifications, specific residues modified, or sequence context contributed to the specificity of binding by 2F3, we examined a panel of ~50 Tn glycopeptides. We observed binding only to peptides that contained Ser-Tn, and only those that were at least 5 amino acids from the attachment point to the solid matrix. Ser-Tn near free termini and multiple Ser-Tn in a single peptide increased binding, in this preliminary study. Finally, glycan array analysis demonstrated high specificity for Tn/sTn with no significant binding observed to almost 600 glycan structures found in mammals.
Conclusions: 2F3 anti-Tn/sTn antibody recognizes Tn preferentially on Ser residues that are in accessible contexts. 2F3 exhibits a pharmaceutically useful pattern of binding because high percentages of primary ovarian and breast cancer samples are recognized. We are now in preclinical exploration of the optimal MoA to exploit the specificity and selectivity of this candidate drug.
Citation Format: G Jonah Rainey, Ritsuko Sawada, Jacek Ostrowski, Ayesha Kahn, Aleksandra Marinkovic-Petrovic, Kevin Mudd, Wolfgang Scholz, Paul W. Maffuid. A fully human antibody binds Tn and sTn carbohydrate antigens specifically on serine residues, without need for polypeptide interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-002.