OX40 作为免疫疗法的靶点已被广泛研究，激动剂抗体已进入癌症临床试验，但尚未显示出实质性疗效。在这里，我们研究了抗小鼠 (m) OX40 和抗人 (h) OX40 抗体的潜在作用机制，包括临床相关的单克隆抗体 (mAb) (GSK3174998)，并评估了同种型如何改变这些机制，旨在开发改进的抗体用于癌症的合理联合治疗。抗 mOX40 和抗 hOX40 mAb 在多种体内模型中进行了评估，包括 hOX40 敲入 (KI) 小鼠和同基因小鼠的 OT-I 过继转移免疫模型肿瘤模型。在缺乏 Fc γ 受体 (FcγR) 的 hOX40 KI 小鼠中评估了 FcγR 参与的影响。此外，还评估了使用抗小鼠程序性细胞死亡蛋白 1 (mPD-1) 的联合研究。还进行了使用外周血单核细胞 (PBMC) 的体外实验，检查可能的抗 hOX40 mAb 作用机制。临床相关 mAb GSK3174998 的同种型变体显示出机制不同的免疫调节作用； mIgG1 介导直接 T 细胞激动，而 mIgG2a 则间接发挥作用，可能是通过激活 FcγR 消耗调节性 T 细胞 (Treg)。在 OT-I 和 EG.7-OVA 模型中，hIgG1 是最有效的人类同种型，能够直接发挥作用并通过 Treg 耗竭发挥作用。抗 hOX40 hIgG1 与抗 mPD-1 协同作用，可改善 EG.7-OVA 模型的治疗结果。最后，用人外周血单核细胞 (hPBMC)、抗 hOX40 hIgG1 进行的体外测定也显示了 T 细胞刺激和 Treg 耗竭的潜力。这些发现强调了了解同种型在治疗药物作用机制中的作用的重要性单克隆抗体。作为 hIgG1，抗 hOX40 mAb 可以引发多种作用机制，有助于或阻碍治疗结果，具体取决于微环境。在设计潜在的组合伙伴及其 FcγR 要求时应考虑这一点，以实现最大利益和改善患者治疗结果。© 作者（或其雇主）2024。CC BY-NC 允许重复使用。不得商业再利用。请参阅权利和权限。英国医学杂志出版。

OX40 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.Anti-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.Isotype 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.These 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.© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.