Abstract 974: Fully customizable multiplex immunofluorescent (mIF) assay as proof-of-concept lab-developed test (LDT) for cancer immunotherapy: Using direct conjugation of antibodies with commercially-available dyes
Authors: Lim Jeffrey Chun Tatt , Yusoff Nur Zahirah M , Neo Zhen Wei , Meng Jia , Lim Chwee Ming , Tan Yee Joo , Yeong Joe Poh Sheng 
Published: April 2023

Abstract
We presented a fully customizable multiplex immunofluorescent (mIF) assay as proof-of-concept lab-developed test (LDT) for cancer immunotherapy using direct conjugation of antibodies with commercially-available dyes. The benefits of cancer immunotherapy are currently limited to about 20% of all cancer patients due to the lack of patient suitability test for precision medicine. Its existing gold standard test, immunohistochemistry (IHC) may insufficiently stratify patients, whereas adoption of Tyramide Signal Amplification-based multispectral mIF (ms-mIF) into clinical practice has been slow. We have established ms-mIF using clinical autostainers in the Anatomical Pathology Lab of Singapore General Hospital for almost a decade. A ms-mIF-based LDT assay was employed to simultaneously detect and quantify PD-L1 labelling with multiple antibody clones, for accurate assessment of tumor and immune cells. The test has since been embraced by private diagnostic companies and hospitals in the region. While ms-mIF can be a better patient screening method, its high requisite expertise, requirement of proprietary reagents from multiple suppliers and expensive specialized equipment have made its adoption challenging. Taking advantage of the repertoire of optimized IHC antibodies in the clinical lab, we conjugated these commercial primary antibodies using commercial fluorescent labelling kits to develop a proof-of-concept one-stop mIF solution to simultaneously detect multiple biomarkers. Owing to the common practice of LDT in Southeast Asia, we also generated in-house monoclonal antibodies, such as PD-L1 and CD39 to formulate customizable mIF assay. The staining was visualized using Zeiss Axioscan 7, validated using IHC and assessed in a cohort of immunotherapy-treated samples. Direct staining in mIF enables simultaneous detection of multiple biomarkers on a single tissue section in one labelling step and faster turnover time. It also provides flexibility in the choice of antibody host species and dye usable, making mIF a fully customizable assay. This one-stop assay could potentially be packaged into a ready-to-use kit to include antibodies, reagents and protocols easily implementable in clinical labs. It overcomes technical barriers of ms-mIF as it requires less prerequisite expertise and increases accessibility of clinical labs to the assay for precision medicine. In summary, to our knowledge this is the first report on direct conjugation for multiplex biomarker detection for clinical application. It provides insight and vision for clinical labs to implement mIF as LDT and to develop their own LDT assay. Our future work includes development of 2-step mIF using fluorescent-conjugated secondary antibodies to improve detection signal. [J.L. and N.Y. contributed equally to this work.]
Citation Format: Jeffrey Chun Tatt Lim, Nur Zahirah M Yusoff, Zhen Wei Neo, Jia Meng, Chwee Ming Lim, Yee Joo Tan, Joe Poh Sheng Yeong. Fully customizable multiplex immunofluorescent (mIF) assay as proof-of-concept lab-developed test (LDT) for cancer immunotherapy: Using direct conjugation of antibodies with commercially-available dyes [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 974.