PD-1 and CTLA-4 antibodies offer great hope for cancer immunotherapy. However, many patients are incapable of responding to PD-1 and CTLA-4 blockade and show low response rates due to insufficient immune activation. The combination of checkpoint blockers has been proposed to increase the response rates. Besides, antibody drugs have disadvantages such as inclined to cause immune-related adverse events and infiltration problems. In this study, we developed a cyclic peptide C25 by using Ph.D.-C7C phage display technology targeting LAG-3. As a result, C25 showed a relative high affinity with human LAG-3 protein and could effectively interfere the binding between LAG-3 and HLA-DR (MHC-II). Additionally, C25 could significantly stimulate CD8⁺ T cell activation in human PBMCs. The results also demonstrated that C25 could inhibit tumor growth of CT26, B16 and B16-OVA bearing mice, and the infiltration of CD8⁺ T cells was significantly increased while FOXP3⁺ Tregs significantly decreased in the tumor site. Furthermore, the secretion of IFN-γ by CD8⁺ T cells in spleen, draining lymph nodes and especially in the tumors was promoted. Simultaneously, we exploited T cells depletion models to study the anti-tumor mechanisms for C25 peptide, and the results combined with MTT assay confirmed that C25 exerted anti-tumor effects via CD8⁺ T cells but not direct killing. In conclusion, cyclic peptide C25 provides a rationale for targeting the immune checkpoint, by blockade of LAG-3/HLA-DR interaction in order to enhance anti-tumor immunity, and C25 may provide an alternative for cancer immunotherapy besides antibody drugs.

PD-1和CTLA-4抗体为癌症免疫治疗带来了巨大希望。但是，由于免疫激活不足，许多患者无法对PD-1和CTLA-4阻滞作出反应，并且反应率较低。已提出将检查点阻止程序组合使用以提高响应率。此外，抗体药物具有诸如引起免疫相关不良事件和渗透问题的缺点。在这项研究中，我们通过使用针对LAG-3的Ph.D.-C7C噬菌体展示技术开发了环肽C25。结果，C25显示出与人LAG-3蛋白的相对高亲和力，并且可以有效地干扰LAG-3与HLA-DR（MHC-II）之间的结合。此外，C25可以显着刺激CD8 ⁺人PBMC中的T细胞活化。结果还表明，C25可以抑制携带CT26，B16和B16-OVA的小鼠的肿瘤生长，并且在肿瘤部位CD8 ⁺ T细胞的浸润明显增加，而FOXP3 ⁺ Tregs则明显减少。此外，促进了CD8 ⁺ T细胞在脾脏，引流淋巴结中，特别是在肿瘤中分泌IFN- γ。同时，我们利用T细胞耗竭模型研究了C25肽的抗肿瘤机制，结果与MTT分析相结合，证实C25通过CD8 ⁺发挥抗肿瘤作用。T细胞但不能直接杀死。总之，环肽C25通过阻断LAG-3 / HLA-DR相互作用以增强抗肿瘤免疫性，为靶向免疫检查点提供了依据，并且C25除抗体药物外还可以为癌症免疫治疗提供替代方案。