The making of multivalent gamma delta TCR anti-CD3 bispecific T cell engagers

Eline van Diest, Mara J T Nicolasen, Lovro Kramer, Jiali Zheng, Patricia Hernández-López, Dennis X Beringer, Jürgen Kuball

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INTRODUCTION: We have recently developed a novel T cell engager concept by utilizing γ9δ2TCR as tumor targeting domain, named gamma delta TCR anti-CD3 bispecific molecule (GAB), targeting the phosphoantigen-dependent orchestration of BTN2A1 and BTN3A1 at the surface of cancer cells. GABs are made by the fusion of the ectodomains of a γδTCR to an anti-CD3 single chain variable fragment (scFv) (γδECTO-αCD3), here we explore alternative designs with the aim to enhance GAB effectivity.

METHODS: The first alternative design was made by linking the variable domains of the γ and δ chain to an anti-CD3 scFv (γδVAR-αCD3). The second alternative design was multimerizing γδVAR-αCD3 proteins to increase the tumor binding valency. Both designs were expressed and purified and the potency to target tumor cells by T cells of the alternative designs was compared to γδECTO-αCD3, in T cell activation and cytotoxicity assays.

RESULTS AND DISCUSSION: The γδVAR-αCD3 proteins were poorly expressed, and while the addition of stabilizing mutations based on finding for αβ single chain formats increased expression, generation of meaningful amounts of γδVAR-αCD3 protein was not possible. As an alternative strategy, we explored the natural properties of the original GAB design (γδECTO-αCD3), and observed the spontaneous formation of γδECTO-αCD3-monomers and -dimers during expression. We successfully enhanced the fraction of γδECTO-αCD3-dimers by shortening the linker length between the heavy and light chain in the anti-CD3 scFv, though this also decreased protein yield by 50%. Finally, we formally demonstrated with purified γδECTO-αCD3-dimers and -monomers, that γδECTO-αCD3-dimers are superior in function when compared to similar concentrations of monomers, and do not induce T cell activation without simultaneous tumor engagement. In conclusion, a γδECTO-αCD3-dimer based GAB design has great potential, though protein production needs to be further optimized before preclinical and clinical testing.

Original languageEnglish
Article number1052090
JournalFrontiers in Immunology
Publication statusPublished - 5 Jan 2023


  • Antigens, CD
  • Butyrophilins
  • CD3 Complex/metabolism
  • Humans
  • Lymphocyte Activation
  • Neoplasms/drug therapy
  • Receptor-CD3 Complex, Antigen, T-Cell
  • Single-Chain Antibodies/genetics
  • gamma delta TCR
  • tumor immunology
  • Gamma Delta T cells
  • protein engineering
  • bispecific T cell engager


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