Targeting the 4-1BB costimulatory molecule through single chain antibodies promotes the human T-cell response

Adoptive T-cell therapy (ACT) using autologous tumor-reactive T lymphocytes has considerable potential for cancer immunotherapy. In ACT, T cells are isolated from cancer patients and then stimulated and expanded in vitro by cytokines and costimulatory molecules. 4-1BB is an important costimulatory protein belonging to the TNF receptor superfamily. It is involved in T-cell survival, proliferation and activation. Agonistic anti-4-1BB monoclonal antibodies have been introduced as appropriate tools for ACT. Here, various single-chain fragment variable (scFv) antibodies were used to activate T cells isolated from peripheral blood via immune magnetic isolation. The T cells were stimulated with IL-2 and anti-CD-3 mAb and then treated with agonistic anti-4-1BB scFvs. The results showed the remarkable effects of anti-41BB scFvs on the functional properties of T cells, including their activation, proliferation and cytokine production. The flow cytometry analysis revealed a considerable increase in the expression of the T-cell activation marker CD69. Moreover, T-cell proliferation was evidenced in treated cells by CFSE labeling compared to the control groups. Anti-4-1BB scFvs significantly increased IFN-γ and IL-2 mRNA and protein expression in T cells, but exhibited no stimulatory effect on IL-4 expression. These findings show that anti-4-1BB scFvs could evoke a Type I immune response. Our results demonstrate that targeting the 4-1BB molecule using agonistic scFvs could be an effective strategy for T-cell stimulation as part of an ACT approach to cancer treatment.