Abstract 6006: The discovery and development of a CD3 agonistic single domain antibody to accelerate the development of CD3 T cell engager multi-specific antibodies

Abstract CD3 T cell engager (TCE) bispecific antibodies (BsAbs) is a clinically well validated and dominant therapeutic modality of approved bispecific antibodies, contributing to 10 out of 14 marketed BsAb drugs. To develop a TCE BsAb with desired potency and safety profile, a CD3 agonist Ab with appropriate potency and ideal developability is critical. Currently, all approved TCE BsAbs are based on a CD3 agonist Ab in the format of conventional monoclonal antibodies containing both light chain and heavy chain, which may limit the flexibility in multi-specific antibody construction and development. In contrast, considering these limitations, single domain antibodies (sdAb, or VHH) are highly favored in the construction and development of bi- or multi-specific antibodies, mainly due to their smaller size, good stability and developability. Therefore, to ease the development of TCE multi-specific antibodies, we set forth to discover an agonist sdAb of CD3. We first identified a parental CD3 sdAb from an immunized camel phage display library. Through successful humanization and affinity maturation, we obtained a series of variants with a spectrum of binding affinity to CD3 and potency in activating T cells. These CD3 sdAb variants were then used to construct a series of bi- or multi-specific antibodies with ease and flexibility, no matter the TAA (tumor associated antigen) arm is a conventional monoclonal antibody (CD20, HER2, etc.), or sdAb (GPRC5D, etc.). The optimization process focused on fine-tuning the affinity, valency, and geometry of both the CD3 arm and TAA arm. This resulted in multi-specific antibodies with enhanced yield and developability, potent tumor cell killing capabilities, and with reduced cytokine release in vitro comparing to clinical benchmark antibodies. In preclinical in vivo studies, these TCE multi-specific antibodies showed favorable pharmacokinetics (PK) and safety profiles along with robust tumor growth inhibition. In summary, we have successfully discovered and developed a series of CD3 agonist sdAbs, showing a spectrum of affinity to CD3 and potency in T cell activation. These CD3 sdAbs have demonstrated a great potential in supporting and accelerating the development of diverse TCE multi-specific antibodies with enhanced ease and flexibility. Citation Format: Yu Liang, Yingyu Li, Yi Zhou, Cheng Luo, Xin Wang, Yang Xin, Shishuai Shao, Tingting Yang, Mingzhu Shao, Jin Chen, Ziyu Chen, Sujuan Hong, Hai Huang, Cheng Chen, Hui Wang, Miaomiao Song, Yuanxin Zhang, Shuai Yang, Xiaojie Tu, Sujuan Wang, Li Chen. The discovery and development of a CD3 agonistic single domain antibody to accelerate the development of CD3 T cell engager multi-specific antibodies [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 6006.