T cell engagers (TCEs) are an increasingly validated class of multispecific antibodies. By pairing one arm targeting CD3 with another arm targeting a tumor-associated antigen, TCEs force a T cell and tumor cell into close proximity, triggering T cell activation and tumor cell killing. There are currently eight FDA-approved TCEs, with six of these having been approved since 2022. Adimab's existing anti-CD3 IgG/Fab/scFv panel spans a wide affinity range with human–cynomolgus cross-reactivity, and bispecific CD3 × HER2 TCEs generated with this panel have demonstrated cytotoxicity in cell-based assays. To further expand this CD3 binder offering and reduce the manufacturing complexity of TCEs, Adimab scientists developed a panel of anti-CD3 heavy chain-only antibodies (HCAbs), sometimes called VHHs or nanobodies, discovered from llama immune libraries using our yeast-based platform. 

Approach and outcomes 

• VHH genes from llamas immunized with CD3 were used to construct yeast immune libraries. Iterative rounds of FACS-based selection against human CD3, an off-target antigen, and cynomolgus CD3 enabled the isolation of anti-CD3 heavy-chain-only antibodies with desirable properties. 

• Approximately 10% of discovered HCAbs stimulated T cells, as measured by IL-2 secretion and CD69 upregulation in Jurkat T cells. Those stimulating HCAbs also displayed favorable developability profiles comparable to or better than benchmark binders UCHT1 and Ablynx 60E11. 

• Six VHH leads were chosen for humanization. These spanned a wide range of human T cell binding and monovalent affinities, with KD values of 4–122 nM against human CD3εδ-Fc. Crystal structures obtained for two leads demonstrate binding to CD3δε with an overlapping binding patch with UCHT1 but with a different angle of approach, and specificity for human CD3ε. 

• In functional assays, the VHH panel demonstrated activity in both IgG-based bispecific and TCR fusion formats. CD3 VHH × CD20 bispecific T cell engagers induced cytotoxicity in Raji lymphoma cells, and CD3 VHH–TCR fusion T cell engagers induced cytotoxicity in MeWo melanoma cells. Multiple leads performed comparably to the clinically validated benchmark UCHT1 scFv (tebentafusp). 

• Adimab's conventional IgG/FAb/scFv anti-CD3 panel spans a wide affinity range with human–cynomolgus cross-reactivity, and bispecific CD3 × HER2 TCEs generated with this panel facilitated cytotoxicity in a breast cancer cell line (SKOV3). 

Why it matters 

Adimab's anti-CD3 VHHs offer a simplified manufacturing path for TCE development while delivering functional activity comparable to clinically validated benchmarks across multiple bispecific formats. CD3 VHHs are accessible as part of Adimab's non-exclusive TCE offering.

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