OBJECTIVES The addition of RANKL/RANK blockade to immune checkpoint inhibitors (ICIs) such as anti-PD-1/PD-L1 and anti-CTLA4 antibodies is associated with increased anti-tumor immunity in mice. Recent retrospective clinical studies in patients with advanced melanoma and lung cancer suggest the addition of anti-RANKL antibody to ICI increases the overall response rate relative to ICI treatment alone. Based on this rationale, we developed a novel bispecific antibody (BsAb) co-targeting RANKL and PD-1. METHODS We characterized target binding and functional activity of the anti-RANKL/PD-1 BsAb in cell-based assays. Anti-tumor activity was confirmed in experimental lung metastasis models and in mice with established subcutaneously transplanted tumors. RESULTS The anti-RANKL/PD-1 BsAb retained binding to both RANKL and PD-1 and blocked the interaction with respective counter-structures RANK and PD-L1. The inhibitory effect of anti-RANKL/PD-1 BsAb was confirmed by demonstrating a complete block of RANKL-dependent osteoclast formation. Monotherapy activity of anti-RANKL/PD-1 BsAb was observed in anti-PD-1 resistant tumors and, when combined with anti-CTLA-4 mAb, increased anti-tumor responses. An equivalent or superior anti-tumor response was observed with the anti-RANKL/PD-1 BsAb compared with the combination of parental anti-RANKL plus anti-PD-1 antibodies depending upon the tumor model. DISCUSSION Mechanistically, the anti-tumor activity of anti-RANKL/PD-1 BsAb required CD8+T cells, host PD-1 and IFNγ. Targeting RANKL and PD-1 simultaneously within the tumor microenvironment (TME) improved anti-tumor efficacy compared with combination of two separate mAbs. CONCLUSION In summary, the bispecific anti-RANKL/PD-1 antibody demonstrates potent tumor growth inhibition in settings of ICI resistance and represents a novel modality for clinical development in advanced cancer.

中文翻译：

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双特异性抗体对 RANKL 和 PD-1 的双重靶向提高了抗肿瘤免疫力。

目的 将 RANKL/RANK 阻断添加到免疫检查点抑制剂 (ICI)，如抗 PD-1/PD-L1 和抗 CTLA4 抗体与小鼠抗肿瘤免疫增加有关。最近对晚期黑色素瘤和肺癌患者的回顾性临床研究表明，相对于单独的 ICI 治疗，在 ICI 中添加抗 RANKL 抗体可提高总体反应率。基于这个原理，我们开发了一种新的双特异性抗体 (BsAb)，共同靶向 RANKL 和 PD-1。方法 我们在基于细胞的测定中表征了抗 RANKL/PD-1 BsAb 的靶标结合和功能活性。在实验性肺转移模型和已建立皮下移植肿瘤的小鼠中证实了抗肿瘤活性。结果 抗 RANKL/PD-1 BsAb 保留了与 RANKL 和 PD-1 的结合，并阻断了与各自的反结构 RANK 和 PD-L1 的相互作用。抗 RANKL/PD-1 BsAb 的抑制作用通过完全阻断 RANKL 依赖性破骨细胞形成得到证实。在抗 PD-1 耐药肿瘤中观察到抗 RANKL/PD-1 BsAb 的单药治疗活性，并且当与抗 CTLA-4 mAb 联合使用时，抗肿瘤反应增加。根据肿瘤模型，与亲本抗 RANKL 加抗 PD-1 抗体的组合相比，使用抗 RANKL/PD-1 BsAb 观察到等效或更好的抗肿瘤反应。讨论 从机制上讲，抗 RANKL/PD-1 BsAb 的抗肿瘤活性需要 CD8+T 细胞、宿主 PD-1 和 IFNγ。与两种单独的 mAb 的组合相比，在肿瘤微环境 (TME) 中同时靶向 RANKL 和 PD-1 提高了抗肿瘤功效。结论 总之，双特异性抗 RANKL/PD-1 抗体在 ICI 耐药的情况下表现出有效的肿瘤生长抑制作用，代表了晚期癌症临床开发的一种新模式。