Abstract 3107: SMASH: Single molecule antibody screening with high-throughput imaging system
Authors: Jo Jihye , Chun Changju , Sohn Byeong-Kwon , Yu Booyoung , Lee Jiyu 
Published: March 2024

Abstract
Larger, more diverse antibody libraries are being screened in recent years. Despite enabling the high-throughput screening of extensive libraries, the process for identification and characterization of antibodies remains laborious and time-intensive. Here we report a platform that not only compiles hit finding and validation processes, but also provides high-resolution binding affinity as well as rank order among the library being screened. We streamlined the library construction and affinity screening via microscale transient expression in HEK293T cells with designed antibody sequence and direct quantification of antibody-antigen binding affinity which reduced the timeline from multiple weeks to a single week. We constructed the single-chain fragment antigen binding (scFab) libraries by replacing the duplex DNA fragment in CDR of interest while preserving the human framework of Fab. We have demonstrated that the SPID technique allows to detect antibody-antigen interactions from just dozens of pg of scFabs in total. This allowed us to bypass the conventional DNA cloning or even PCR amplification to achieve the minimum amount of DNA plasmids for expressing scFabs to be analyzed such as BLI and SPR. Therefore, we assembled the DNA plasmids through in vitro ligation with high efficiency and directly introduced it to HEK293T in 96-well microplate with microscale (200 ul) for transient transfection, which only takes three days for library generation. Next, we utilized SPID to quantify the number of single molecules between free and antigen-bound fractions of scFab, resulting in occupancy at the antibody-antigen binding equilibrium. This technique facilitated the determination of binding affinity (KD) for 200 antibody-antigen pairs within a 6-hour TAT encompassing both measurement and analysis. In total, we utilized 4,000 times less antibody for characterization while achieving enhanced sensitivity compared to both BLI and SPR methods. As a proof-of-concept, we generated a library consisting of 720 variants of Trastuzumab in scFab format to probe the landscape of CDRs affecting affinities against HER2 showing nM to low uM dynamic range of KD. Out of 720 analyzed variants, 5% were deleterious, 38% provided substantial changes, 40% showed little/no change, 17% enhanced affinity to HER2. To validate the robustness of the platform, we extensively expanded the library screening, assessing over 2,000 antibody variants for their affinity against more than three different therapeutic targets. To conclude, we demonstrated an efficient approach for monitoring changes in affinity resulting from single mutations in scFab. This rapid screening of site-saturated libraries would lead to a combinational variant library that our platform can screen. Ultimately, our platform aims to identify potential candidates for therapeutic antibody development throughput the phases of antibody discovery, maturation, and engineering.
Citation Format: Jihye Jo, Changju Chun, Byeong-Kwon Sohn, Booyoung Yu, Jiyu Lee. SMASH: Single molecule antibody screening with high-throughput imaging system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3107.