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Background T cell-engagers (TCEs) are a class of multispecific antibodies that have shown great promise as immunotherapeutic molecules to treat cancers. TCEs can redirect T cells to recognize and kill cancer cells through co-engagement of a tumor associated antigen and a T cell antigen, often CD3. Despite their clinical efficacy, the molecular complexity of current TCEs poses challenges for drug manufacturability, developability, and achieving desirable PK/PD properties. Currently, all approved TCE multispecifics rely on anti-CD3 binders derived from conventional antibodies, containing both a heavy and light chain.Heavy chain-only antibodies (HCAbs) featuring a unique single-domain (VHH) binding moiety are attractive alternatives to traditional IgGs in immunotherapeutic molecules. The absence of a light chain in HCAbs notably simplifies multispecific development. To our knowledge, no anti-CD3 HCAbs with properties comparable to best-in-class clinically approved IgGs have been reported.Methods Camelids naturally produce two classes of antibodies: conventional (HC+LC) and HCAbs (HC-only). 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. Leads were subsequently humanized and optimized to exhibit desirable drug developability and potency profiles. The functional potency of these lead molecules was assessed and compared to clinically validated TCEs in two bispecific formats: 1+1 anti-CD3 X anti-CD20 (targeting Raji cells) and as a VHH-TCR fusion using a TCR specific for the gp100 peptide HLA complex (targeting SK-MEL-5 and MeWo cancer cell lines).Results Llama immunization, yeast-based HCAb discovery, and HCAb humanization resulted in a panel of six functional lead anti-CD3 TCEs. These humanized lead molecules predominantly competed with the UCHT1-binding epitope; recognizing the CD3ε subunit of the human T cell receptor (TCR)/CD3 complex with weak to strong affinities and robust drug developability profiles. As TCE bispecific molecules, the panel displayed a promising functional potency range; with two molecules displaying comparable in vitro tumor cell killing efficacy to clinically validated molecules such as tebentafusp.Conclusions In this work, we showcase the discovery and engineering of novel anti-CD3 HCAbs, which demonstrate T cell cytotoxicity comparable to clinically validated TCEs when paired with IgG or TCR modalities. This introduces a flexible new tool for advancing the TCE class of biologics.