2021, Vol. 48
2. 复旦大学附属中山医院肝癌研究所 上海 200032
2. Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China
程序性细胞死亡分子1（programmed cell death-1，PD-1）/程序性细胞死亡分子配体1（programmed cell death ligand-1，PD-L1）信号通路所介导的肿瘤免疫逃逸是肿瘤发生和发展的重要机制之一。在肿瘤微环境中，肿瘤细胞可表达PD-L1，通过与PD-1相互作用进而影响效应T细胞的抗肿瘤活性。抑制或阻断PD-1/PD-L1通路可显著增强T细胞介导的免疫杀伤活性，进而提高抗肿瘤效应。目前包括Nivolumab、Pembrolizumab、Atezolizumab和Avelumab在内的针对PD-1/PD-L1的抑制性抗体已展现出显著的临床效益[1-2]，但总体缓解率仍然较低[3-4]。PD-1/PD-L1介导的免疫抑制与其本身蛋白质表达水平及蛋白质翻译后修饰密切相关。许多重要的细胞内和细胞外因子，如干扰素γ（intrferon γ，IFN-γ）、转化生长因子β（tranforming growth factor β，TGF-β）、miR-33a和miR-200，可通过转录和转录后调控影响PD-1/PD-L1的表达[5-7]。此外，PD-1/PD-L1存在多个N-糖基化位点，其糖基化修饰显著影响免疫治疗的疗效[8-9]。
糖基化修饰是一种常见的蛋白质翻译后修饰，通过糖基转移酶的作用将糖类转移到蛋白质，并和蛋白质上的氨基酸残基形成糖苷键的过程。糖基化修饰的主要类型包括N-糖基化和O-糖基化。N-糖基化是指聚糖与蛋白质肽链中天冬酰胺的自由氨基连接，从而将糖链组装在蛋白质上的过程。O-糖基化则是指聚糖与蛋白肽链的丝氨酸或苏氨酸进行连接来修饰蛋白质。蛋白质糖基化修饰可帮助免疫细胞进行正确的定位与迁移[10]。异常的糖基化修饰与肿瘤的发生、增殖、侵袭、转移和免疫逃逸等密切相关[11-12]。因此，阐明糖基化修饰对PD-1/PD-L1分子表达及功能的影响，可以为提高肿瘤免疫治疗的疗效提供新策略。目前国内针对PD-1/PD-L1糖基化修饰的相关综述有限，本文将在此基础上对PD-1/PD-L1糖基化修饰在肿瘤免疫治疗中的作用进行综述。
PD-1糖基化修饰 PD-1为免疫球蛋白超家族中的一员，其主要表达在活化的T细胞和B细胞表面，是一个具有268个氨基酸残基的Ⅰ型跨膜蛋白。PD-1与其配体PD-L1结合，通过去磷酸化影响下游信号通路上的关键分子，从而起到负性免疫应答调节的作用[13]。在正常生理状态下，PD-1信号的激活可抑制过激炎症反应，预防自身免疫性疾病的发生[14]。在肿瘤中，PD-1信号通路的激活可抑制T细胞免疫应答，从而导致肿瘤细胞免疫逃逸[15]。目前为止，PD-1报道共有4个N-糖基化位点，分别为N49、N58、N74和N116[16]（图 1）。阻断任意一个糖基化位点，均会导致PD-1稳定性下降以及表达水平的下调，提示PD-1糖基化对维持其稳定性以及表达水平密切相关[9]。此外，PD-1糖基化可以增强PD-1/PD-L1的结合能力，阻断上述任一位点糖基化均会导致PD-1/PD-L1结合能力下降[9]。Wang等[17]利用重组人源PD-1免疫小鼠后，通过抗体人源化获得了针对PD-1的特异性单克隆抗体MW11-h317。该单克隆抗体对PD-1表现出高度亲和力，并能够有效阻断PD-1与PD-L1/L2的相互作用，诱导T细胞介导的免疫应答，抑制肿瘤生长。对MW11-h317与PD-1的结合位点进行分析后发现，其主要针对PD-1的N58糖基化位点。mAb059c是一种针对PD-1的单克隆抗体，Liu等[18]发现其重链互补决定区（complementarity-determining region，CDR）1/2区域主要识别PD-1的N58糖基化位点，突变N58糖基化位点可以明显降低mAb059c和PD-1的结合能力。此外，Sun等[9]构建了针对PD-1的N58糖基化位点的特异性单克隆抗体STM418，该抗体相比于Nivolumab和Pembrolizumab具有更高的亲和力，同时能够显著抑制PD-1/PD-L1结合，促进T细胞激活，提升抗肿瘤效应。
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| Both PD-1 and PD-L1 are glycoproteins.There are four N-glycosylated sites of PD-1, including N49, N58, N74 and N116. PD-L1 also has four N-glycosylation sites, N35, N192, N200 and N219. FUT8 can catalyze the core fucosylation of PD-1. Monoclonal antibodies targeting N58 glycosylation site of PD-1 can effectively block the PD-1/PD-L1 interaction.In addition, blocking N74 glycosylation site of PD-1 of CAR-T cells can enhance its killing effect. On the other hand, inhibition or promotion of the glycosylation of PD-L1 can affect PD-1/PD-L1 interaction and result in certain biological effects. 图 1 PD-1/PD-L1糖基化修饰调控关系模式图 Fig 1 Regulation of the glycosylation of PD-1/PD-L1 in cancer |
Okada等[16]利用规律成簇间隔短回文重复结构（clustered regularly interspaced short palindromic repeats，CRISPR）/CRISPR相关蛋白9（CRISPR-associated protein 9，Cas9）全基因组敲除技术筛选出一系列同PD-1表达密切相关的分子，发现其中核心岩藻糖基转移酶8（fucosyltransferase 8，FUT8）能够催化PD-1岩藻糖基化并调节PD-1在T细胞上的表达。基因敲除或抑制FUT8表达均可明显降低PD-1糖基化修饰水平，同时下调PD-1表达，从而增强T细胞介导的抗肿瘤免疫效应。Agrawal等[19]同时发现在黑色素瘤中，FUT8可以介导肿瘤转移，抑制FUT8可以显著降低黑色素瘤细胞的侵袭和转移能力。此外，在经过PD-1抑制剂Pembrolizumab治疗的转移性黑色素瘤的患者中，高表达FUT8的患者预后相对较差[9]。以上研究表明，FUT8不仅对PD-1的糖基化修饰和表达起着至关重要的作用，同时也影响肿瘤转移和抗PD-1免疫治疗的作用，具有重要的研究前景。
嵌合抗原受体T细胞（chimeric antigen receptor T-cell，CAR-T）免疫疗法是近年来一种新兴的治疗肿瘤的精准靶向疗法，但在实体肿瘤中，CAR-T细胞免疫疗法疗效却不尽人意[20]。一项针对CAR-T细胞PD-1糖基化修饰的研究表明，抑制CAR-T细胞PD-1的N74位点的糖基化修饰可以显著提升CAR-T细胞的杀伤效应，从而提升其在实体肿瘤中的抗癌作用[21]。综上，靶向PD-1糖基化修饰在肿瘤治疗中具有巨大的潜力。
PD-L1糖基化修饰 PD-L1可在肿瘤细胞表面表达，其能与活化T细胞表面PD-1结合，从而抑制T细胞的增殖、活化、迁移和细胞毒性作用。经糖基化修饰的PD-L1相对分子质量为45 000~55 000，而未糖基化修的PD-L1相对分子质量为33 000。抑制PD-L1的N-糖基化修饰，可以明显改变PD-L1的分子量大小[22]。而抑制O-糖基化修饰则并不出现上述改变，提示PD-L1同PD-1一样，主要表现为高度N-糖基化修饰[23]。通过质谱法鉴定，PD-L1共有4个N-糖基化位点，包括N35、N192、N200和N219。其中N192、N200和N219的糖基化修饰对于维持PD-L1蛋白稳定性至关重要（图 1）[22]。非糖基化修饰的PD-L1较不稳定，其可与糖原合成酶激酶3β（glycogen synthase kinase 3β，GSK3β）结合并被磷酸化后通过泛素/蛋白酶体系统降解，而N192、N200和N219位点的的糖基化修饰则可产生空间阻遏效应，阻碍GSK3β和PD-L1的相互作用，进而起到稳定PD-L1的作用[22]。异常的PD-L1糖基化水平同样会导致PD-L1表达水平的改变。二甲双胍可诱导AMP激活蛋白激酶（5’ AMP-activated protein，AMPK）同PD-L1结合并使其磷酸化修饰，导致PD-L1的异常糖基化，进而导致其降解[24]。在前列腺癌和三阴性乳腺癌细胞中，Maher等[25]发现Sigma1分子可以通过促进PD-L1的糖基化而上调PD-L1的表达水平。抑制Sigma1可抑制IFN-γ介导的PD-L1的表达。在胶质瘤中，PD-L1共同分子伴侣FKBP51s同样可以通过促进PD-L1的糖基化而上调PD-L1的表达水平。抑制FKBP51s可以显著降低PD-L1的表达[26]。而促进PD-L1的糖基化水平，可以明显提高其表达水平，提示PD-L1糖基化同时对其表达起正性调控作用[27]。研究还发现，上皮间质转化（epithelial-mesenchymal transition，EMT）可通过β-catenin信号轴诱导肿瘤干细胞高表达N-糖基转移酶STT3，进而增加PD-L1糖基化，导致肿瘤干细胞PD-L1上调和肿瘤免疫逃逸[28]。KYA1797K抑制剂则可以抑制β-catenin/STT3信号轴而影响PD-L1糖基化，从而进一步抑制免疫逃逸并诱导肿瘤干细胞凋亡[29]。在此基础上，Chan等[30]发现IL-6可以诱导JAK1磷酸化PD-L1的Y112位点，而该位点的磷酸化有助于STT3A催化PD-L1糖基化，而阻断IL-6/JAK1信号轴可以抑制STT3A同PD-L1结合，导致PD-L1降解。
PD-L1的糖基化修饰和其与PD-1的结合以及免疫抑制密切相关，阻断PD-L1糖基化可以显著提升抗肿瘤效应。利用N-糖苷酶（PNGase F）水解PD-L1的糖链后，PD-1/PD-L1结合能力丧失[23]。将T细胞同表达糖基化和表达非糖基化PD-L1的乳腺癌细胞进行共培养后，发现表达非糖基化PD-L1的乳腺癌细胞凋亡更为显著。进一步研究发现在BALB/c小鼠体内，野生型PD-L1细胞相比于PD-L1糖基化位点突变细胞生长更为迅速，而将上述细胞接种到免疫缺陷小鼠后生长速度相似，表明糖基化对PD-L1的免疫抑制功能十分重要[23]。阻断PD-L1的糖基化同时还可以影响PD-L1正确转运至细胞膜上。Verdura等[31]发现白藜芦醇可以靶向破坏PD-L1糖链并影响其二聚化，从而导致其无法被正确转移至细胞膜上。在三阴乳腺癌中，糖基转移酶B3GNT3表达显著升高且与预后密切相关。B3GNT3受到上游EGF/EGFR信号的激活而促进PD-L1的N192和N200位点糖基化，从而确保PD-1/PD-L1结合[32]。通过构建单克隆抗体药物偶联复合物STM108-ADC针对PD-L1的N192和N200糖基化位点，可以显著抑制肿瘤活性[32]。此外，Liu等[33]发现在B细胞淋巴瘤中，糖基转移酶GLT1D1同样可以促进PD-L1的N-糖基化水平，并损害细胞毒性T细胞对肿瘤细胞的杀伤功能。Shao等[34]发现2-脱氧葡萄糖（2-deoxyglucose）可以作为葡萄糖类似物降低PD-L1的糖基化水平，从而拮抗PARP抑制剂诱导的糖化PD-L1表达。在此基础上，Kim等[35]发现2-脱氧右旋葡萄糖（2-deoxy-D-glucose）可以同样可诱导PD-L1去糖基化，并增强4‐1BB介导的抗肿瘤效应。
常规情况下，PD-L1表达小于1%的患者一般不推荐使用PD-L1治疗[36]。由于PD-L1糖基化的存在，通常会造成临床检查假阴性出现。移除PD-L1糖基化修饰可以提高检测率，更好地指导临床用药，同时在一定程度上对患者的疗效进行预测[37-38]。以上研究表明，针对PD-L1糖基化修饰的抗肿瘤治疗具有一定的临床应用前景。
结语 近年来，基于阻断PD-1/PD-L1的免疫治疗手段飞速发展，已经逐步在临床推广和应用，但其治疗缓解率仍然相对较低。随着针对PD-1/PD-L1糖基化修饰研究的不断深入，人们发现糖基化修饰对PD-1/PD-L1的稳定性和相互作用起着至关重要的作用，同时也是肿瘤细胞免疫逃逸的重要机制之一。不仅如此，一些能够催化PD-1/PD-L1糖基化修饰的糖基转移酶也被发现参与肿瘤侵袭和转移，并且与肿瘤患者的预后密切相关。因此，开发针对PD-1/PD-L1糖基化为靶点的治疗手段具有明显优势，有望成为肿瘤治疗的新途径和新方法。
作者贡献声明 李胤 论文构思、撰写和修订，文献回顾，图表绘制。陈一苇，陈芳华 文献回顾，论文撰写和修订。张舒 论文构思和修订。卢春来 论文构思、撰写和修订。
利益冲突声明 所有作者均声明不存在利益冲突。
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