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Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：OX40已被广泛研究为具有激动剂抗体的免疫疗法的靶标，这些抗体已进入癌症的临床试验，但尚未显示出实质性功效。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，目的是开发用于癌症合理联合治疗的改进抗体。
方法：在许多体内模型中评估了抗mOX40和抗hOX40mAb，包括在hOX40敲入（KI）小鼠和同基因肿瘤模型中的OT-I过继转移免疫模型。在缺乏Fcγ受体(FcγR)的hOX40KI小鼠中评估FcγR接合的影响。此外,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)检查可能的抗hOX40mAb作用机制的体外实验。
结果：临床相关mAbGSK3174998的同种型变体显示出不同机制的免疫调节作用；mIgG1介导直接T细胞激动，而mIgG2a间接起作用，可能通过激活FcγRs消耗调节性T细胞（Tregs）。在OT-I和EG.7-OVA模型中，hIgG1是最有效的人类同种型，能够直接和通过Treg消耗作用。抗hOX40hIgG1与抗mPD-1协同以改善EG.7-OVA模型中的治疗结果。最后,人外周血单核细胞（hPBMC）的体外测定，抗hOX40hIgG1也显示了T细胞刺激和Treg消耗的潜力。
结论：这些发现强调了理解同种型在治疗性单克隆抗体作用机制中的作用的重要性。作为hIgG1，抗hOX40mAb可以引发多种作用机制，可以帮助或阻碍治疗结果，依赖于微环境。在设计潜在的组合伙伴及其FcγR要求时，应考虑到这一点，以实现最大的益处并改善患者的预后。
方法：在许多体内模型中评估了抗mOX40和抗hOX40mAb，包括在hOX40敲入（KI）小鼠和同基因肿瘤模型中的OT-I过继转移免疫模型。在缺乏Fcγ受体(FcγR)的hOX40KI小鼠中评估FcγR接合的影响。此外,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)检查可能的抗hOX40mAb作用机制的体外实验。
结果：临床相关mAbGSK3174998的同种型变体显示出不同机制的免疫调节作用；mIgG1介导直接T细胞激动，而mIgG2a间接起作用，可能通过激活FcγRs消耗调节性T细胞（Tregs）。在OT-I和EG.7-OVA模型中，hIgG1是最有效的人类同种型，能够直接和通过Treg消耗作用。抗hOX40hIgG1与抗mPD-1协同以改善EG.7-OVA模型中的治疗结果。最后,人外周血单核细胞（hPBMC）的体外测定，抗hOX40hIgG1也显示了T细胞刺激和Treg消耗的潜力。
结论：这些发现强调了理解同种型在治疗性单克隆抗体作用机制中的作用的重要性。作为hIgG1，抗hOX40mAb可以引发多种作用机制，可以帮助或阻碍治疗结果，依赖于微环境。在设计潜在的组合伙伴及其FcγR要求时，应考虑到这一点，以实现最大的益处并改善患者的预后。
