Abstract 672: Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)
Authors: Smith Caroline Noel , Kihn Kyle , Williamson Zachary A. , Chow K. Martin , Hersh Louis B. , Korotkov Konstantin , Deredge Daniel , Blackburn Jessica S. 
Published: June 2022

Abstract
Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, and the only research antibody specific for PRL-3 can only recognize denatured protein. To address the lack of tools available to study PRL-3, we have developed alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3. Nanobodies have emerged as a valuable research tool and show promise as cancer therapeutics as they are ~15kD and lack light chains, allowing them to reach cavities within active sites that conventional antibodies cannot normally reach. Nanobodies also maintain high specificity and affinity for their antigens. We identified seven unique nanobodies that bind to PRL-3 with no activity towards PRL-1 and PRL-2, making our nanobodies one of the first tools to selectively target PRL-3 in its native state. We used biolayer interferometry and found the nanobody binding affinity for PRL-3 to be within a KD of 30 - 300 nM, similar to that of antibodies currently on the market. We identified PRL-3:nanobody interactions with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and showed binding outside the active site. These data were confirmed by analyzing the effects of nanobodies on PRL-3 phosphatase activity and substrate binding. Our anti-PRL-3 nanobodies specifically pulled down PRL-3 over PRL-1/-2 in immunoprecipitation experiments. Finally, we used these nanobodies to analyze PRL-3 localization in fixed immunofluorescence experiments in human cancer cells. We found that a C-terminal tag on PRL-3, such as FLAG or GFP, enhanced PRL-3 localization to the membrane, compared to untagged protein, which may have confounded previous PRL-3 functional studies. We are currently utilizing these nanobodies in two ways to understand PRL-3’s role in cancer. First, we will use the nanobody to stabilize PRL-3 for X-ray crystallography to develop higher resolution structures that could contribute to substrate identification and drug design. Secondly, we will examine PRL-3 function and trafficking during various cancer processes, such as proliferation, invasion, and stress, to determine how PRL-3 localization contributes to cancer progression.
Citation Format: Caroline Noel Smith, Kyle Kihn, Zachary A. Williamson, K. Martin Chow, Louis B. Hersh, Konstantin Korotkov, 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 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 672.