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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.

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4(+) T cell responses to protection against influenza infection.

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.

Influenza virus remains a significant health concern, with current circulating strains that affect millions each year plus the threat of newly emerging strains, such as swine-origin H1N1 and avian H5N1. Our hypothesis is that influenza-derived HLA-class I-restricted epitopes can be identified for use as a reagent to monitor and quantitate human CD8(+) T-cell responses and for vaccine development to induce protective cellular immunity. Protein sequences from influenza A virus strains currently in circulation, agents of past pandemics and zoonotic infections of man were evaluated for sequences predicted to bind to alleles representative of the most frequent HLA-A and -B (class I) types worldwide. Peptides that bound several different HLA molecules and were conserved among diverse influenza subtypes were tested for their capacity to recall influenza-specific immune responses using human donor PBMC. Accordingly, 28 different epitopes antigenic for human donor PBMC were identified and 25 were 100% conserved in the newly emerged swine-origin H1N1 strain. The epitope set defined herein should provide a reagent applicable to quantitate CD8(+) T cell human responses irrespective of influenza subtype and HLA composition of the responding population. In addition, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.