Abstract 6350: Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy
Authors: Bover Laura C. , Muhsin Ahmed , Vien Long T. , Manzanares Felipe Amaya , Wu Zhuang , Johnson Janis D. , Reyes Diego Castro , Moreno-Smith Myrthala , Labanca Estefania , Sorin Irene , Saucedo Silvia , Gazzaniga Silvina , Bravo Alicia I. 
Published: June 2023

Abstract
BST2, a raft-associated type II transmembrane protein, functions as a restriction factor and inhibits HIV release by cross-linking virions onto infected cell surfaces. It also engages ILT7, a specific inhibitory receptor on plasmacytoid dendritic cells, which are associated with poor prognosis when infiltrate patients’ breast tumors. Two groups have identified BST2 isoforms of different lengths, generated by post-transcriptional modification. Based on these findings and our work on protein interaction of ILT7-BST2, we hypothesized that the expression of BST2 isoforms differed in normal and cancer tissues. To demonstrate that, we generated and characterized anti-long BST2 isoform monoclonal antibodies (mAbs). Results were compared with our previously developed mAb anti-BST2 full length molecule, 26F8, that recognizes a common epitope to all BST2 isoforms. By comparing the amino acid sequences of different isoforms of BST2, we identified a region of 24 residues on the extracellular domain of BST2, restricted to its long isoform. We then generated mAbs by hybridoma technology, immunizing BALB/c mice with the 24 mers peptide-KLH conjugated. Screening and positive selection of several clones were performed following our lab SOP. One of the mAbs, LA5, was selected based on positive ELISA against the immunogen and FACS staining of recombinant HEK293 cells expressing full-length BST2 protein. Pharmacokinetic determined using OCTET platform, revealed a KD of 4.61+/- 0.074 × 10−9 M for LA5. Epitope mapping of LA5 on BST2 overlapping peptides, confirmed specific recognition of full-length BST2 protein and the peptide used as immunogen. LA5 heavy and light chains were sequenced and analysis was performed by IgBlast and IMGTV. Binding of LA5 and 26F8 to the myeloma cell lines MM1 (B-lymphoblast) and U266 (B-lymphocyte) by flow cytometry, indicated 97-100% positive cells with both mAbs, with higher MFI values observed for clone 26F8: 5.8 and 1.8-fold-increase in MM1 and U266 cells respectively. IHC on formalin-fixed paraffin embedded tissues of human ductal breast carcinoma, revealed that LA5 staining was able to identify infiltrating carcinoma cells but not the normal surrounding acinar cells. In contrast, 26F8 staining did not discriminate between BST2+ malignant and surrounding normal acinar cells. Currently, we are analyzing a panel of &gt;105 breast cancer tissue samples of different molecular classifications. Initial data revealed a high-level of expression of BST2 (by 26F8) on the epithelial area of the tumors, lower in the TME (tumor microenvironment-infiltrating immune cells) and almost negative expression in the surrounding fibroblasts rich stroma. Evaluation of IHC staining by LA5 is being processed and yet to be established. These IHC findings indicate a potential diagnostic value of LA5 to distinct between tumor and normal cells in patients’ specimens. Treatment prospects may arise during the process.
Citation Format: Laura C. Bover, Ahmed Muhsin, Long T. Vien, Felipe Amaya Manzanares, Zhuang Wu, Janis D. Johnson, Diego Castro Reyes, Myrthala Moreno-Smith, Estefania Labanca, Irene Sorin, Silvia Saucedo, Silvina Gazzaniga, Alicia I. Bravo. Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6350.