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Abstract：
Immune checkpoint inhibitors (ICIs) therapy targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) shows promising clinical benefits. However, the relatively low response rate highlights the need to develop an alternative strategy to target PD-1/PD-L1 immune checkpoint. Our study focuses on the role and mechanism of annexin A1 (ANXA1)-derived peptide A11 degrading PD-L1 and the effect of A11 on tumor immune evasion in multiple cancers.
Binding of A11 to PD-L1 was identified by biotin pull-down coupled with mass spectrometry analysis. USP7 as PD-L1\'s deubiquitinase was found by screening a human deubiquitinase cDNA library. The role and mechanism of A11 competing with USP7 to degrade PD-L1 were analyzed. The capability to enhance the T cell-mediated tumor cell killing activity and antitumor effect of A11 via suppressing tumor immune evasion were investigated. The synergistic antitumor effect of A11 and PD-L1 mAb (monoclonal antibody) via suppressing tumor immune evasion were also studied in mice. The expression and clinical significance of USP7 and PD-L1 in cancer tissues were evaluated by immunohistochemistry.
A11 decreases PD-L1 protein stability and levels by ubiquitin proteasome pathway in breast cancer, lung cancer and melanoma cells. Mechanistically, A11 competes with PD-L1\'s deubiquitinase USP7 for binding PD-L1, and then degrades PD-L1 by inhibiting USP7-mediated PD-L1 deubiquitination. Functionally, A11 promotes T cell ability of killing cancer cells in vitro, inhibits tumor immune evasion in mice via increasing the population and activation of CD8+ T cells in tumor microenvironment, and A11 and PD-1 mAb possess synergistic antitumor effect in mice. Moreover, expression levels of both USP7 and PD-L1 are significantly higher in breast cancer, non-small cell lung cancer and skin melanoma tissues than those in their corresponding normal tissues and are positively correlated in cancer tissues, and both proteins for predicting efficacy of PD-1 mAb immunotherapy and patient prognosis are superior to individual protein.
Our results reveal that A11 competes with USP7 to bind and degrade PD-L1 in cancer cells, A11 exhibits obvious antitumor effects and synergistic antitumor activity with PD-1 mAb via inhibiting tumor immune evasion and A11 can serve as an alternative strategy for ICIs therapy in multiple cancers.
Binding of A11 to PD-L1 was identified by biotin pull-down coupled with mass spectrometry analysis. USP7 as PD-L1\'s deubiquitinase was found by screening a human deubiquitinase cDNA library. The role and mechanism of A11 competing with USP7 to degrade PD-L1 were analyzed. The capability to enhance the T cell-mediated tumor cell killing activity and antitumor effect of A11 via suppressing tumor immune evasion were investigated. The synergistic antitumor effect of A11 and PD-L1 mAb (monoclonal antibody) via suppressing tumor immune evasion were also studied in mice. The expression and clinical significance of USP7 and PD-L1 in cancer tissues were evaluated by immunohistochemistry.
A11 decreases PD-L1 protein stability and levels by ubiquitin proteasome pathway in breast cancer, lung cancer and melanoma cells. Mechanistically, A11 competes with PD-L1\'s deubiquitinase USP7 for binding PD-L1, and then degrades PD-L1 by inhibiting USP7-mediated PD-L1 deubiquitination. Functionally, A11 promotes T cell ability of killing cancer cells in vitro, inhibits tumor immune evasion in mice via increasing the population and activation of CD8+ T cells in tumor microenvironment, and A11 and PD-1 mAb possess synergistic antitumor effect in mice. Moreover, expression levels of both USP7 and PD-L1 are significantly higher in breast cancer, non-small cell lung cancer and skin melanoma tissues than those in their corresponding normal tissues and are positively correlated in cancer tissues, and both proteins for predicting efficacy of PD-1 mAb immunotherapy and patient prognosis are superior to individual protein.
Our results reveal that A11 competes with USP7 to bind and degrade PD-L1 in cancer cells, A11 exhibits obvious antitumor effects and synergistic antitumor activity with PD-1 mAb via inhibiting tumor immune evasion and A11 can serve as an alternative strategy for ICIs therapy in multiple cancers.
摘要：
背景：针对程序性细胞死亡1（PD-1）/程序性细胞死亡配体1（PD-L1）的免疫检查点抑制剂（ICIs）疗法显示出有希望的临床益处。然而,相对较低的应答率凸显了开发针对PD-1/PD-L1免疫检查点的替代策略的必要性.我们的研究重点是膜联蛋白A1（ANXA1）衍生肽A11降解PD-L1的作用和机制，以及A11对多种癌症中肿瘤免疫逃避的影响。
方法:通过结合质谱分析的生物素下拉法鉴定A11与PD-L1的结合。通过筛选人去泛素酶cDNA文库，发现USP7作为PD-L1的去泛素酶。分析了A11与USP7竞争降解PD-L1的作用和机制。研究了A11通过抑制肿瘤免疫逃避增强T细胞介导的肿瘤细胞杀伤活性和抗肿瘤作用的能力。还在小鼠中研究了A11和PD-L1mAb（单克隆抗体）通过抑制肿瘤免疫逃避的协同抗肿瘤作用。采用免疫组织化学方法评价USP7和PD-L1在癌组织中的表达及临床意义。
结果：A11通过泛素蛋白酶体途径降低乳腺癌中PD-L1蛋白的稳定性和水平，肺癌和黑色素瘤细胞。机械上,A11与PD-L1去泛素酶USP7竞争结合PD-L1，然后通过抑制USP7介导的PD-L1去泛素化降解PD-L1。功能上,A11促进T细胞体外杀伤癌细胞的能力，通过增加肿瘤微环境中CD8+T细胞的数量和激活来抑制小鼠的肿瘤免疫逃避，A11和PD-1mAb在小鼠体内具有协同抗肿瘤作用。此外，USP7和PD-L1的表达水平在乳腺癌中明显更高，非小细胞肺癌组织和皮肤黑色素瘤组织比相应的正常组织和癌组织呈正相关,预测PD-1mAb免疫治疗疗效和患者预后的两种蛋白均优于单个蛋白。
结论：我们的结果表明，A11与USP7竞争结合并降解癌细胞中的PD-L1，A11具有明显的抗肿瘤作用，并通过抑制肿瘤免疫逃避与PD-1mAb具有协同抗肿瘤活性，A11可作为多种癌症ICIs治疗的替代策略。
方法:通过结合质谱分析的生物素下拉法鉴定A11与PD-L1的结合。通过筛选人去泛素酶cDNA文库，发现USP7作为PD-L1的去泛素酶。分析了A11与USP7竞争降解PD-L1的作用和机制。研究了A11通过抑制肿瘤免疫逃避增强T细胞介导的肿瘤细胞杀伤活性和抗肿瘤作用的能力。还在小鼠中研究了A11和PD-L1mAb（单克隆抗体）通过抑制肿瘤免疫逃避的协同抗肿瘤作用。采用免疫组织化学方法评价USP7和PD-L1在癌组织中的表达及临床意义。
结果：A11通过泛素蛋白酶体途径降低乳腺癌中PD-L1蛋白的稳定性和水平，肺癌和黑色素瘤细胞。机械上,A11与PD-L1去泛素酶USP7竞争结合PD-L1，然后通过抑制USP7介导的PD-L1去泛素化降解PD-L1。功能上,A11促进T细胞体外杀伤癌细胞的能力，通过增加肿瘤微环境中CD8+T细胞的数量和激活来抑制小鼠的肿瘤免疫逃避，A11和PD-1mAb在小鼠体内具有协同抗肿瘤作用。此外，USP7和PD-L1的表达水平在乳腺癌中明显更高，非小细胞肺癌组织和皮肤黑色素瘤组织比相应的正常组织和癌组织呈正相关,预测PD-1mAb免疫治疗疗效和患者预后的两种蛋白均优于单个蛋白。
结论：我们的结果表明，A11与USP7竞争结合并降解癌细胞中的PD-L1，A11具有明显的抗肿瘤作用，并通过抑制肿瘤免疫逃避与PD-1mAb具有协同抗肿瘤活性，A11可作为多种癌症ICIs治疗的替代策略。
