Abstract 1552: Novel anti-CD3 heavy chain-only antibodies for use in T-cell engaging therapeutics

Abstract T-cell engagers (TCEs) display increasing potential as an immunotherapeutic treatment for both hematologic and solid cancers. These multi-specific antibody-based therapeutics consist of a CD3-targeting arm and one or more tumor associated antigen (TAA) targeting arms. The multimeric nature of these therapeutics can present antibody engineering and manufacturing challenges. Developing an anti-CD3 heavy chain-only antibody (HCAb) reduces the design complexity of the final TCE therapeutic. Functional anti-CD3 HCAbs were discovered by combining llama immunization and a novel yeast-based HCAb presentation and discovery technology. Following several rounds of yeast-based HCAb selection, T-cell stimulating anti-CD3 HCAbs were identified that predominantly competed with UCHT1. Stimulating CD3-specific HCAbs were reformatted into CD3 X CD20 bispecifics, which displayed potent T-cell activation and Raji cell killing in a redirected T-cell cytotoxicity (RTCC) assay. This panel was further validated as a bispecific VHH-TCR fusion using a TCR specific for the gp100 peptide HLA complex; donor T-cell activation and target killing was observed against both SK-MEL-5 and MeWo cancer cell lines. The panel was subsequently humanized while maintaining functional activity and favorable drug developability profiles. These humanized anti-CD3 HCAbs are promising candidates for replacing conventional anti-CD3 IgG Fab arms in next-generation multispecific TCE antibodies. Citation Format: Paul Khalifé, Hannah C. Watkins, Elizabeth A. McGurk, Elizabeth R. Parker, Cameron M. Henkel, Todd M. Boland, Hailey M. Heston, Whitney E. Renaud, Christin R. Strong, Jingfu Zhou, Carissa Metzger, Michael E. Brown, Jessica P. Dawson, Andrew D. Avery, C Garrett Rappazzo, Beth H. Sharkey, Morgan B. Morrill, Cory L. Ahonen, Michael B. Battles, Noel T. Pauli. Novel anti-CD3 heavy chain-only antibodies for use in T-cell engaging therapeutics [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 1552.