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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.

CD134 (OX40) is a member of the tumour necrosis factor receptor superfamily (TNFRSF). It acts as a costimulatory receptor on T cells, but its role on NK cells is poorly understood. CD137, another TNFRSF member has been shown to enhance the anti-tumour activity of NK cells in various malignancies. Here, we examine the expression and function of CD134 on human and mouse NK cells in B-cell lymphoma. CD134 was transiently upregulated upon activation of NK cells in both species. In contrast to CD137, induction of CD134 on human NK cells was dependent on close proximity to, or cell-to-cell contact with, monocytes or T cells. Stimulation with an agonistic anti-CD134 mAb but not CD134 ligand, increased IFNγ production and cytotoxicity of human NK cells, but this was dependent on simultaneous antibody:Fcγ receptor binding. In complementary murine studies, intravenous inoculation with BCL 1 lymphoma into immunocompetent syngeneic mice resulted in transient upregulation of CD134 on NK cells. Combination treatment with anti-CD20 and anti-CD134 mAb produced a synergistic effect with durable remissions. This therapeutic benefit was abrogated by NK cell depletion and in Fcγ chain −/− mice. Hence, anti-CD134 agonists may enhance NK-mediated anti-tumour activity in an Fcγ receptor dependent fashion.

It has been well established that for an effective and controlled immune response multiple inputs are integrated. For example, the interaction of the T cell receptor (TCR) with an MHC-peptide complex alone is insufficient to cause complete T cell activation and concomitant interaction of co-stimulatory T cell receptors with their ligands is required. OX40 (CD134) and its ligand, OX40L (CD252), are members of the tumour necrosis factor receptor/tumour necrosis factor (TNFR/TNF) superfamily. Investigation into targeting such receptors with monoclonal antibodies (mAb) for use in cancer immunotherapy is ongoing, and in recent years OX40 has been shown to be a promising therapeutic target. This thesis explores the development, characterisation and therapeutic potential of a panel of novel antihOX40 mAb in a unique hOX40 knock-in (KI) mouse model. The Antibody and Vaccine Group at the University of Southampton developed a number of anti-hOX40 mAb within the Southampton Antibody Programme (SAP) using hybridoma technology. These mAb were characterised in terms of binding domain and affinity using techniques such as flow cytometry and surface plasmon resonance (SPR) It was found that the panel of mAb all possessed a high affinity for the hOX40 receptor and binding spanned over all four extracellular domains (ECD). Their activity was then determined in vitro using proliferation assays and in vivo using a novel hOX40 KI mouse expressing human ECD and mouse intracellular domains. The immunostimulatory potential of these reagents was first assessed in an CD8+ OT-I transfer model. Subsequently, the therapeutic efficacy of these mAb were assessed using a number of mouse tumour models. Collectively the data highlighted both the importance of mAb isotype but also specific domain binding in relation to the type and strength of the anti-hOX40 mAb effector function; with mAb binding to the membrane proximal region delivering stronger agonism (T cell expansion) as mIgG1 mAb and stronger regulatory T cell (Treg) depletion as mIgG2a. Intriguingly, mAb of both isotypes were seen to eradicate established tumours, albeit through different mechanisms. Together the work in this thesis demonstrates the desired characteristics, functional effects and mechanisms of action of anti-hOX40 mAb and provides encouragement for their translation towards the clinic as potential cancer therapeutics.

Previous data suggests that anti-OX40 mAb can elicit anti-tumour effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. The contributions of these different effector mechanisms has not previously been systematically evaluated, particularly for mAb directed to human OX40. Therefore, we generated a novel human OX40 knock-in (KI) mouse to evaluate a panel of anti-hOX40 mAb and show 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 tumour models by engaging these different mechanisms. These 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 preferable according to the composition of different tumours. Competing Interest Statement MSC is a retained consultant for BioInvent International and has performed educational and advisory roles for Baxalta and Boehringer Ingleheim. He has received research funding from Roche, Gilead, Bioinvent International and GSK. BF, IT, LM and MS are employees of Bioinvent International.