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BackgroundOX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we investigated potential mechanisms of action of anti-mouse (m) OX40 and anti-human (h) OX40 antibodies, including a clinically relevant monoclonal antibody (mAb) (GSK3174998) and evaluated how isotype can alter those mechanisms with the aim to develop improved antibodies for use in rational combination treatments for cancer.MethodsAnti-mOX40 and anti-hOX40 mAbs were evaluated in a number of in vivo models, including an OT-I adoptive transfer immunization model in hOX40 knock-in (KI) mice and syngeneic tumor models. The impact of FcγR engagement was evaluated in hOX40 KI mice deficient for Fc gamma receptors (FcγR). Additionally, combination studies using anti-mouse programmed cell death protein-1 (mPD-1) were assessed. In vitro experiments using peripheral blood mononuclear cells (PBMCs) examining possible anti-hOX40 mAb mechanisms of action were also performed.ResultsIsotype variants of the clinically relevant mAb GSK3174998 showed immunomodulatory effects that differed in mechanism; mIgG1 mediated direct T-cell agonism while mIgG2a acted indirectly, likely through depletion of regulatory T cells (Tregs) via activating FcγRs. In both the OT-I and EG.7-OVA models, hIgG1 was the most effective human isotype, capable of acting both directly and through Treg depletion. The anti-hOX40 hIgG1 synergized with anti-mPD-1 to improve therapeutic outcomes in the EG.7-OVA model. Finally, in vitro assays with human peripheral blood mononuclear cells (hPBMCs), anti-hOX40 hIgG1 also showed the potential for T-cell stimulation and Treg depletion.ConclusionsThese findings underline the importance of understanding the role of isotype in the mechanism of action of therapeutic mAbs. As an hIgG1, the anti-hOX40 mAb can elicit multiple mechanisms of action that could aid or hinder therapeutic outcomes, dependent on the microenvironment. This should be considered when designing potential combinatorial partners and their FcγR requirements to achieve maximal benefit and improvement of patient outcomes.

BackgroundIn recent years, a regulatory network involving nectin/nectin-like immune receptors has emerged as a potential point of manipulation for cancer immunotherapy. Central to this axis, CD226 (DNAM-1) is a T and NK cell co-stimulatory receptor that competes for ligand (CD155 and CD112) binding with multiple inhibitory receptors (TIGIT, CD96, and PVRIG [CD112R]). Despite a large body of literature for TIGIT, detailed cellular characterization of the entire axis is still lacking. Therefore, we used mass cytometry (CyTOF) to systematically evaluate expression of the CD226 axis in tumors from a range of indications.MethodsTo thoroughly characterize the CD226 axis in the tumor microenvironment, we immunophenotyped approximately 100 tumor samples derived from a variety of cancer types using a bespoke 46-parameter CyTOF panel. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. Using a suite of high-dimensional analytical tools, including FlowSOM, UMAP, and tSNE, we revealed distinct expression profiles for each receptor; a finding that was previously obscured due to a lack of sufficient resolution.ResultsWe observed a notable divergence in expression profiles between the CD226 axis members across tumor indications. For example, TIGIT expression was found to be highest on activated CD4+ regulatory T (Treg) cells, where its expression correlated strongly with ICOS, FoxP3, CD25, and CCR8. By contrast, CD96 and PVRIG exhibited broad expression across intratumoral T and NK cell populations. Other receptors (e.g., CD226) demonstrated variegated expression profiles across T and NK cell subsets. Finally, despite relatively consistent expression profiles of certain CD226 axis (i.e., TIGIT on Treg cells) across tumors, we also found several cell subsets/clusters unique to specific indications.ConclusionsUsing high-parameter CyTOF analysis, we were able to thoroughly characterize the CD226 axis (CD226, TIGIT, CD96, PVRIG) and related immune receptors across a range of tumor indications. These analyses revealed divergent expression profiles for each CD226 axis member, suggesting distinct/contextual biological role(s) for each receptor. However, future studies will need to dissect the importance of the distinct cellular representation for each CD226 axis member.Ethics ApprovalAll samples were purchased from Discovery Life Sciences (DLS). DLS represents and warrants that it has ownership of all Products available for sale and has properly obtained, where required under HHS/OHRP 45 CFR 46.102 (d) (f), IRB approval (or appropriate research approval for institutions outside the U.S.) for study protocols and informed consent documents for all human subject derived biological materials.