Domain binding and isotype dictate the activity of anti-human OX40 antibodies

Abstract

BackgroundPrevious data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit.MethodsThis study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb.ResultsBinding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype—with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope—with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms.ConclusionThese findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering

“…Our observations regarding the influence of mAb isotype on agonism are concordant with those reported for other TNFRSF members (CD40, 4-1BB and OX40) 17 , 18 , 27 , 29 . For CD40, the m1 format, which preferentially engages FcγRIIb is highly agonistic, whereas m2a is inactive 41 .…”

“…Although it is clear that mAb isotype plays a central role in providing agonism, it is also evident that epitope location matters. Murine OX40 mAb binding to membrane-proximal epitopes mediate superior agonism over those binding more distally 28 , with similar findings for human OX40 (hOX40) mAb 29 . In contrast, for hCD40 mAb, strong agonism is observed with mAb binding to the most distal epitopes 18 .…”

“…For CD40, the m1 format, which preferentially engages FcγRIIb is highly agonistic, whereas m2a is inactive 41 . Whilst a similar observation was made for 4-1BB and OX40, the m2a format was not inactive but was able to induce antigen-specific CD8 + T-cell expansion as a consequence of Treg depletion as opposed to direct agonism in order to deliver tumour control 17 , 29 . Cumulatively, all of these findings including those made by Wasiuk et al 32 enable us to draw the following conclusions: The therapeutic efficacy of an agonistic immunostimulatory mAb and its mechanism of action is influenced by its isotype and in turn by (1) the abundance of FcγRIIb in the tumour microenvironment, (2) the relative expression of the mAb target on individual cell types, and (3) the reliance of the tumour model on Treg cells, when they express the relevant TNFRSF.…”

TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity

“…However, a wealth of information has been generated on the functional requirements of agonistic mAb. For example, we previously demonstrated that the complex interplay between epitope, isotype and FcγR availability determines the agonistic activity of anti-CD40 mAbs, and similar principles are being applied to other TNFRs [7][8][9]47 . Fluorescence microscopy has shown that TNF ligands and agonistic mAbs induced TNFR clustering on the cell surface; however, the requirement for secondary crosslinking remains unresolved 46,[48][49][50] .…”

“…ostimulatory tumour necrosis factor receptors (TNFRs) play important roles in immune activation and represent promising targets for next-generation cancer immunotherapeutics [1][2][3] . Selective TNFR activation has been shown to augment antitumor immunity and confer robust therapeutic benefits in animal models 1 with TNFR-targeting mAbs and recombinant ligands showing promising antitumor activities in early phase trials 1,4,5 , although they remain behind checkpointblocking reagents in their development, likely due to a poorer understanding of how the activity is achieved and their multiple potential mechanisms of action 6,7 . These clinical candidates display a range of agonistic activities resulting from differential TNFR activation, with their level of agonism correlated positively with their pharmacodynamic properties in clinical trials [8][9][10][11][12][13] .…”