Abstract 5457: Dual-antigen bridging ELISAs to validate bispecific molecules with CD3-directed T cell engager function

Bispecific antibodies (bsAbs), designed to recognize two distinct antigens or epitopes, represent a promising class of next-generation biological therapeutics. These macromolecules, capable of engaging multiple targets, have gained significant interest over the past decade for their potential to harness the immune system against specific targets. With additive or synergistic mechanisms of action, bsAbs and other engineered dual-targeting molecules often exhibit greater potency than monoclonal antibodies (mAbs) or combination therapies. By simultaneously bridging two targets, bsAbs provide additional clinical benefits, such as enhanced target cell killing. Therefore, assessing their ability to bind both targets concurrently is essential for advancing their therapeutic potential. CD3-bispecific antibodies (CD3-bsAbs) have emerged as a promising approach in cancer immunotherapy, particularly for hematological cancers. These bsAbs activate T cells by binding both a tumor-associated antigen (TAA) on tumor cells and CD3 on T cells, leading to tumor cell destruction. Recent advancements in bsAb biology and technologies have enabled the development of diverse CD3-bsAb formats, ranging from small fragments with dual variable domains to larger structures incorporating additional domains linked to conventional immunoglobulins. Here, we present dual-target bridging ELISAs for bispecific molecules designed to engage T cells via CD3 binding. These assays are simple, cost-effective, and require no elaborate equipment, making them accessible to most laboratories. By leveraging the bridging capability of bispecific molecules, the method uses an immobilized capture recombinant antigen 1 and a CD3-containing detection reagent (antigen 2) to assess both binding events simultaneously, regardless of molecule format, isotype, or species of origin. Our assays feature low background, high signal-to-noise ratios, and compatibility with purified proteins, human serum, or cell culture supernatants, making them ideal for evaluating bsAb bridging abilities in drug discovery and high-throughput screenings (HTS). Additionally, the all-in-one HRP-conjugated CD3 complex simplifies the protocol by reducing steps what makes these assays less time consuming and more cost effective when compared to surface plasmon resonance (SPR) or cell-based bioassays. Citation Format: Connie Zhang, Katarzyna Zientara-Rytter, Moji Samoudi, Sean Green, Kimberly Skuster, Charlene Chang, Mohammad Awad, Veronique Baron, Michelle Kinbara, Jonathan Mikolosko, Pavel Shashkin. Dual-antigen bridging ELISAs to validate bispecific molecules with CD3-directed T cell engager function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5457.