Abstract 2305: Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)
Authors: Caroline N. Smith, K. Martin Chow, Louis B. Hersh, Daniel Deredge, Jessica S. Blackburn
Journal: Cancer Research (2021)

Abstract Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and has been linked to poor patient prognosis in a variety of tumor types. The mechanisms by which PRL-3 promotes tumor progression are not well understood, which is in part due to lack of tools to study this protein. There is an intense need for research-grade antibodies in the PRL field. However, the development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is both shallow and hydrophobic. The most specific research antibody against PRL-3 only interacts with denatured protein, which limits its use experimentally. There is currently a humanized monoclonal antibody, PRL-3-zumab, that has good specificity for PRL-3 in cell culture and in vivo, and is in Phase 2 clinical trial for treating gastric and hepatocellular carcinomas; unfortunately, PRL-3-zumab is not currently available for the research community to use. To address this, we have designed, purified, and tested alpaca-derived PRL-3 single domain antibodies, or nanobodies. Nanobodies have emerged as a useful research tool and show promise as a cancer therapeutic. Nanobodies are only ~15kD, and lack light chains which allows them to fit into cavities in target proteins that conventional antibodies cannot normally reach. Other advantages of nanobodies include their stability under stringent conditions, lack of immunogenicity, and a high specificity and affinity for their antigens. We have identified 7 unique nanobodies that bind to PRL-3 in ELISA with no activity towards PRL-1 and PRL-2. Nanobodies were found to have variable binding affinity for PRL-3 using biolayer interferometry. Anti-PRL-3 nanobodies immunoprecipitated PRL-3 from HEK293T cell lysates both when overexpressed and using endogenous protein levels, with most nanobodies showing no cross-reactivity with PRL-1 and PRL-2. Each nanobody was also utilized to define PRL-3 localization in human colon cancer cells in immunofluorescence cell staining, which showed PRL-3 localized to the plasma membrane and the nucleus. Following validation of nanobody specificity for PRL-3, we began determining the binding site for each nanobody to PRL-3 as well as determining how nuclear PRL-3 contributes to cancer progression. These anti-PRL-3 nanobodies are the first tool that allow for the study of PRL-3 in multiple cell-based assays without the issue of cross-reactivity with other PRLs, a tool that has yet to exist in the PRL field. They can now be utilized to study the structure, function, and localization of PRL-3 in both normal and cancer cells to determine and define the oncogenic role that PRL-3 plays in multiple types of cancer. Finally, nanobodies against PRL-3 will also serve as a useful first step in the development of biologics to target PRL-3 in clinic. Citation Format: Caroline N. Smith, K. Martin Chow, Louis B. Hersh, Daniel Deredge, Jessica S. Blackburn. Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2305.