T cell engagers (TCEs) are gaining clinical traction rapidly, but their success hinges on a CD3-binding arm that is potent, manufacturable, and compatible with complex multi-specific architectures. Heavy-chain only antibodies (HCAbs) lack the light chain, reducing assembly complexity while preserving function. In this poster, Adimab scientists established a humanized panel of anti-CD3 HCAbs with tunable activity, mapped epitopes, and strong developability to streamline next-generation TCE design.

Key findings and highlights

• Hundreds of llama-derived anti-CD3 HCAbs were discovered via immunization and yeast immune library selections, from which six functional lineages were selected for humanization.
• Humanized progeny retained T cell stimulation within ~10% of parental activity in CD69/NFAT reporter assays.
• Epitope analysis showed most functional HCAbs compete with the UCHT1 epitope while none bind the CD3 N-terminal peptide epitope; therefore, activity is restricted to human T cells.
• When reformatted, anti-CD3 × CD20 bispecifics produced robust redirected T-cell cytotoxicity (RTCC) against CD20⁺ Raji cells, and VHH–T cell receptor (VHH–TCR) fusions mediated killing of SK-MEL-5 melanoma cells; potency tracked with monovalent CD3 affinities spanning ~0.18–100 nM.
• The developability assessment of the anti-CD3 HCAb panel showed low polyspecificity scores and short HIC retention times.

Our study addresses a central bottleneck in TCE programs—the need for compact, developable, and potency-tunable CD3 binders—by delivering a humanized HCAb panel with defined epitope bins and validated function across bispecific and VHH–TCR formats. The panel is available through Adimab’s non-exclusive partnering model to accelerate modular multi-specific design.

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