The making of multivalent gamma delta TCR anti-CD3 bispecific T cell engagers
van Diest, Eline; Nicolasen, Mara J T; Kramer, Lovro; Zheng, Jiali; Hernández-López, Patricia; Beringer, Dennis X; Kuball, Jürgen
(2023) Frontiers in Immunology, volume 13
(Article)
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
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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.
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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, Immunology and Allergy, Immunology, Journal Article
ISSN: 1664-3224
Publisher: Frontiers Media S. A.
Note: Funding Information: Funding for this study was provided by KWF grant numbers 6790, 11393, 12586, 13043, 13493 to JK and 11979 to DB and JK. Acknowledgments Publisher Copyright: Copyright © 2023 van Diest, Nicolasen, Kramer, Zheng, Hernández-López, Beringer and Kuball.
(Peer reviewed)