Assessing the Fc-mediated effector functions on the basis of anti-TNFR2 antibody isotypes

Abstract

The advent of monoclonal antibodies (mAbs) has revolutionised the field of immunotherapies and immunodiagnostics. Advancements in antibody engineering have allowed the researchers to fine-tune the antibodies to achieve favourable effects and efficacy, for instance half-life of the antibody and the Fc-mediated effector functions can be modified by altering the Fc region of the antibody. The Fc tail of the antibody by interacting with the Fc-receptors (FcRs) dictates the effector functions which mainly include antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP) and cytokine secretion. These Fc-mediated effector functions critically contribute to the efficacy of the treatment especially where the antibody-mediated cell death is beneficial, for example, in cancers for depleting the tumour cells. However, it may be advantageous to have a silent Fc domain to prevent unwanted side effects from Fc-mediated effector functions in therapeutic approaches where the mechanism of action is solely dependent on the Fab arms. To study the physiological relevance of these different antibody isotypes (with active and silent Fc domains), Tumour necrosis factor receptor-2 (TNFR2) was selected as a target because of its crucial role in autoimmune diseases and cancers. Regulatory T cells (Tregs) that are known for their suppressive functions on the immune system, highly express TNFR2, particularly within the tumour microenvironment. Stimulation of TNFR2 on Tregs leads to activation and expansion of strongly suppressive Tregs which are beneficial in autoimmune diseases to keep unwarranted immune response under control. On the contrary, expansion of Tregs is undesirable in cancers as the tumour cells can easily evade the immune system. Thus, targeting TNFR2 with different antibody isotypes can modulate Treg activity and be instrumental in autoimmune diseases and cancers.

Three mouse anti-TNFR2 antibodies (candidate 15, 25 and 30) with active and silent Fc isotypes were produced. All the antibody candidates with active isotypes were capable of depleting the target cells owing to their Fc-mediated effector functions, whereas the silent isotypes did not deplete the target cells as they had no Fc-mediated effector functions. Moreover, antibody candidate 25 demonstrated TNFα blocking activity. Thus, the active isotype of antibody candidate 25 was able to block the binding of TNFα to TNFR2 as well as deplete the target cells. Furthermore, all the antibody candidates demonstrated antagonistic activity and were able to prolong the survival of mice in the tumour mice models.