TNFR family targeting agonistic antibodies
Recently, the Fc tail of agonistic mAbs that target specific members of the Tumour Necrosis Factor Receptor (TNFR) family has been shown to play a critical role in their therapeutic efficacy. This class of mAbs is designed to either activate death receptors such as DR4, DR5 and FAS on cancer cells in order to induce cell death, or to activate co-stimulatory receptors such as CD40, 41BB, OX40, GITR and CD27 on immune cells in order to improve anti-tumour immune responses.
TNFRs require trimerisation in order to initiate their associated signalling cascade77. Therefore, bivalent engagement of these receptors with Fab arms is usually not sufficient for their activation and additional cross-linking is required. For these antibodies, the interaction with FcγRs functions as an effective scaffold for clustering. Specifically, it has been shown that FcγRIIb represents a dominant scaffold for antibody mediated TNFR crosslinking and activation of downstream signalling because of its relatively high expression78,79. Consequently, in vivoagonistic antibody activity was found to be highly dependent on successful FcγRIIb engagement80,81 and Fc-engineered antibodies with improved FcγRIIb binding showed stronger anti-tumour activity82,83. However, the expression of FcgRIIb is dynamic and can be downregulated by particular cytokines84, leaving the success of FcγRIIb-mediated cross-linking for receptor clustering unpredictable. In addition, effective FcR-engagement by agonistic antibodies was found to be associated with serious hepatotoxicity85–87, which could potentially be explained by the high expression of FcγRIIb on certain subsets of liver cells88. Therefore, new strategies have been explored to improve the agonistic activity of these mAbs independent of FcγR engagement. One of these strategies is the use of hIgG2(B), whose unique disulphide bonds rearrangement in the hinge region89 provides it with a compact and highly agonistic conformation90. In line with this finding, the agonistic effect of anti-CD40 hIgG2 antibodies was demonstrated to be FcγR-independent both in vitro and in vivo , confirming that the use of hIgG2(B) is a viable strategy for improving the agonistic activity of mAbs targeting TNFR family members91. Furthermore, isotype switching from hIgG1 to hIgG2 was sufficient to convert an immunosuppressive anti-CD40 antagonistic antibody into a potent agonist with anti-tumour activity92. These findings constitute one of the most striking examples of how the choice of the isotype can completely change the activity of a mAb.
Another approach to improve the agonistic activity of TNFR family targeting mAbs, independent of FcγR engagement, is the recently developed HERA platform. HERA is an artificial chimeric molecule which, instead of Fab-arms, has two trimeric TNFR binding domains, fused to an IgG1 Fc backbone with abrogated FcγR binding. The resulting hexavalent molecule is capable of exerting its agonistic activity without FcγR-mediated crosslinking. So far, two HERA molecules targeting CD27 and CD40 have shown promising anti-tumour activity, without significant toxicological signs in pre-clinical mouse models93,94. These findings suggest that agonistic HERA molecules may offer improved safety combined with unaltered efficacy and thus an advantageous clinical profile.
The strategies described to improve agonistic activity in a FcγR-independent manner could have an additional advantage as they prevent unwanted depletion of immune cells expressing the target molecule. However, experiments in mice suggest that the therapeutic effect of some TNFR family targeting agonistic antibodies (such as anti-GITR95, anti-OX4096 or anti-4-1BB97) also involved Treg depletion, suggesting that, analogous to anti-CTLA4, a functional Fc tail might be advantageous. Similarly, some Fc-mediated downstream effector functions may be useful for agonistic mAbs targeting death receptors on cancer cells, as Fc-mediated cytotoxicity and ADCP would act as an additional tumour cell depleting mechanism and might facilitate cross-presentation inducing an adaptive anti-tumour response.
A few solutions have been proposed to combine the divergent properties, as mentioned above, in a single Ig molecule. For instance, a pentameric IgM antibody with high complement activation capacity has been used to successfully induce DR5 clustering via multivalent interaction, inducing tumour regression in preclinical models98. An alternative approach which takes advantage of Ig multimerisation, but avoids IgM manufacturing issues, is the so called HexaBody technology. It is based on a single point mutation (E430G) in the Fc domain of IgG1 that enhances Fc-Fc interactions upon binding to membrane-bound targets99. Consequently, these antibodies have a strong tendency to form hexamers on the target cell, ultimately leading to both high agonistic activity and improved CDC100. A combination of different HexaBodies targeting two different epitopes on DR5 is currently in an early clinical testing (NCT03576131). Given this enhanced complement activation of HexaBodies, this antibody form could furthermore be attractive whenever tumour cell lysis is intended, such as for classical tumour antigen-targeting antibodies, such as anti-CD20; suggesting for the design of an entirely novel type of tumour antigen-targeting antibodies.
In addition to HexaBodies, a highly agonistic anti-4-1BB recombinant Ig with potent Fc-effector function was achieved by combining human IgG2 CH1 and hinge locked in B conformation, with murine IgG2a CH2 and CH3 (the IgG subclass with the highest A/I ratio in the mouse)97. In mice, tumour treatment with this chimeric construct induced both Teff stimulation in lymph nodes (strong 4-1BB agonism) and Fc-mediated Treg depletion within tumours, leading to increased intra-tumoural Teff/Treg ratio and enhanced survival compared to a wild-type mIgG2a construct97. By analogy with the mouse example, a chimera of hIgG2(B) and hIgG1 might be applicable in humans.
In conclusion, important breakthroughs have been made in the design of TNFR agonistic antibodies by making their activity FcγR independent. It is precisely the FcγR independency that may overcome initial problems seen in the clinic such as severe toxicity and modest efficacy. However, the contribution of Fc-mediated cell-depletion to the therapeutic efficacy represents an important consideration for the optimal design of a specific agonistic antibody (fig.4c ).