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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Abstract

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
Volume13
DOIs
Publication statusPublished - 5 Jan 2023

Keywords

  • 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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