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

IntroductionWe have recently developed a novel T cell engager concept by utilizing gamma 9 delta 2TCR 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 gamma delta TCR to an anti-CD3 single chain variable fragment (scFv) (gamma delta ECTO-alpha CD3), here we explore alternative designs with the aim to enhance GAB effectivity. MethodsThe first alternative design was made by linking the variable domains of the gamma and delta chain to an anti-CD3 scFv (gamma delta VAR-alpha CD3). The second alternative design was multimerizing gamma delta VAR-alpha CD3 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 gamma delta ECTO-alpha CD3, in T cell activation and cytotoxicity assays. Results and discussionThe gamma delta VAR-alpha CD3 proteins were poorly expressed, and while the addition of stabilizing mutations based on finding for alpha beta single chain formats increased expression, generation of meaningful amounts of gamma delta VAR-alpha CD3 protein was not possible. As an alternative strategy, we explored the natural properties of the original GAB design (gamma delta ECTO-alpha CD3), and observed the spontaneous formation of gamma delta ECTO-alpha CD3-monomers and -dimers during expression. We successfully enhanced the fraction of gamma delta ECTO-alpha CD3-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 gamma delta ECTO-alpha CD3-dimers and -monomers, that gamma delta ECTO-alpha CD3-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 gamma delta ECTO-alpha CD3-dimer based GAB design has great potential, though protein production needs to be further optimized before preclinical and clinical testing.