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Background CD3-directed T cell engagers (TCE) have become a popular therapeutic modality with ten approvals in oncology and recent interest in extending their use into autoimmune indications. To develop an optimal TCE bispecific antibody (bsAb), a CD3 agonist antibody with appropriate potency to minimize cytokine release while maintaining target cytotoxicity is critical. Cyno cross reactivity is also of great benefit, particularly in autoimmune indications, as one can be better informed of the TCE’s safety profile through preclinical monkey studies. Moreover, it is important that the CD3 binding arm has ideal developability and flexibility for bsAb engineering. Currently, all approved TCE bsAbs are based on a conventional CD3 antibody containing both heavy and light chains, which limits its flexibility in bsAb engineering. In contrast, VHH antibodies are highly favored in bsAb engineering due to their single domain nature, good stability, and conformational flexibility; therefore, to simplify TCE development, we aim to develop a cyno cross-reactive, CD3 VHH agonist antibody.Methods A parental CD3 VHH antibody from an immunized camel phage display library was identified. Subsequent humanization and affinity maturation resulted in a set of VHHs with varying CD3 binding affinities and T cell activation potencies. These CD3 VHH variants were then used to construct a series of TCEs with ease and flexibility irrespective of the format of the tumor-targeting arm (i.e., Fab or VHH). The optimization process focused on fine-tuning the affinity, valency, and geometry of both the CD3 and tumor-targeting arms.Results Over 40 geometric TCE configurations were engineered and tested using our CD3 VHHs. Optimized configurations against hematological (CD20, GPRC5D, BCMA, etc.) and solid tumor (HER2, ROR1, etc.) targets consistently showed equal or greater in vitro and/or in vivo function related to T cell activation, target cytotoxicity, and antitumor activity compared to clinical benchmarks. Our TCEs also demonstrated reduced CRS/ICANS-associated cytokine (IL2, TNFα, IFNγ, etc.) release in vitro compared to the benchmarks and no apparent in vivo toxicities, suggesting a favorable safety profile.Conclusion We have successfully developed a series of cyno cross-reactive, CD3 agonist VHHs displaying a range of CD3 affinity and T cell activation potency. TCEs incorporating these CD3 VHHs and various tumor-targeting arms demonstrate ease and efficiency in engineering multiple geometric configurations for subsequent functional assessment. Compared to clinical benchmarks, our TCEs demonstrated less cytokine release with comparable or greater cytotoxicity in vitro and in vivo, thereby validating these CD3 VHHs as an ideal platform for TCE engineering.