Combinatory enhancement of innate and adaptive immune responses is a promising strategy in immunotherapeutic drug development. Bifunctional macromolecules that simultaneously target two mechanisms may provide additional advantages over the combination of targeting two single pathways. Interferon alpha (IFNα) has been used clinically against viral infection such as the chronic infection of hepatitis B virus (CHB) as well as some types of cancers. OX40 is a costimulatory immune checkpoint molecule involved in the activation of T lymphocytes. To test whether simultaneously activating IFNα and OX40 signaling pathway could produce a synergistic therapeutic effect on CHB and tumors, we designed a bifunctional fusion protein composed of a mouse OX40 agonistic monoclonal antibody (OX86) and a mouse IFNα4, joined by a flexible (GGGGS)₃ linker. This fusion protein, termed OX86-IFN, can activate both IFNα and OX40. We demonstrated that OX86-IFN could effectively activate T lymphocytes in the peripheral blood of mice. Furthermore, we showed that OX86-IFN had superior therapeutic effect to monotherapies in HBV hydrodynamic transfection and syngeneic tumor models. Collectively, our data suggests that simultaneously targeting interferon and OX40 signaling pathways by bifunctional molecule OX86-IFN elicits potent antiviral and antitumor activities, which could provide a new strategy in developing therapeutic agents against viral infection and tumors.

先天性和适应性免疫应答的组合增强是免疫治疗药物开发中的一种有前途的策略。与靶向两个单一途径的组合相比，同时靶向两个机制的双功能大分子可以提供额外的优势。干扰素α（IFNα）已在临床上用于抵抗病毒感染，例如乙型肝炎病毒（CHB）的慢性感染以及某些类型的癌症。OX40是参与T淋巴细胞活化的共刺激免疫检查点分子。为了测试同时激活IFNα和OX40信号通路是否可以对CHB和肿瘤产生协同治疗作用，我们设计了一种双功能融合蛋白，该蛋白由小鼠OX40激动性单克隆抗体（OX86）和小鼠IFNα4组成，并通过柔性（GGGGS）连接_3个链接器。这种融合蛋白称为OX86-IFN，可以激活IFNα和OX40。我们证明了OX86-IFN可以有效激活小鼠外周血中的T淋巴细胞。此外，我们发现OX86-IFN在HBV流体动力学转染和同基因肿瘤模型中具有优于单一疗法的治疗效果。总体而言，我们的数据表明，双功能分子OX86-IFN同时靶向干扰素和OX40信号通路会引起有效的抗病毒和抗肿瘤活性，这可能为开发抗病毒感染和肿瘤的治疗剂提供新策略。