厦门大学 生命科学学院 公共卫生学院 国家传染病诊断试剂与疫苗工程技术研究中心 分子疫苗学和分子诊断学国家重点实验室，福建 厦门 361102
收稿日期：2018-10-25；接收日期：2018-12-28
基金项目：国家自然科学基金(No. 31870925)，传染病重大专项(No. 2017ZX10202203-001)资助
摘要：Claudins蛋白家族是组成紧密连接(Tight junctions，TJs)必不可少的骨架蛋白，在维持上皮和内皮细胞中的细胞极性、细胞间的粘附固定、细胞旁路的离子运输等发挥重要作用。近年来大量的研究结果证明，claudins在许多人类恶性肿瘤中异常表达。因此，claudins也被作为癌症治疗的潜在靶标。文中就claudin蛋白家族在肿瘤中的表达情况及其相关药物的研究进展进行阐述。
关键词：claudin肿瘤梭菌肠毒素抗体靶向治疗
Advances in the application of claudins to tumor therapy
Siyuan Chen, Xue Liu, Wenxin Luo
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Sciences, Xiamen University, Xiamen 361102, Fujian, China
Received: October 25, 2018; Accepted: December 28, 2018
Supported by: National Natural Science Foundation of China (No. 31870925), Major Projects of Infectious Diseases (No. 2017ZX10202203-001)
Corresponding author: Wenxin Luo. Tel: +86-592-2188657; Fax: +86-592-2181258; E-mail: wxluo@xmu.edu.cn.
Abstract: Claudin proteins are the most crucial components of tight junctions, and play an essential role in maintaining cell polarity, regulating cell permeability and the intercellular ion. In recent years, many studies have shown that abnormality of claudins expression is implicated in the tumor progression. The expression correlates with tumor prognosis and can serve as a biomarker of prognosis and potential therapeutic targets. This review summarizes the current knowledge regarding claudin dysregulation in cancer and highlights the progress in claudin-based treatments.
Keywords: claudintumorClostridium perfringens enterotoxinantibodytargeted therapy
紧密连接(Tight junctions，TJs)又叫occluding junctions或zonulae occludentes，是细胞黏附结构的重要连接形式之一。紧密连接包括跨膜蛋白occludins和claudins (CLDNs)以及膜外周蛋白如ZO-1[1-3]，其缝合了相邻极化的上皮细胞间或内皮细胞间的缝隙，使之具有屏障功能，同时调控着离子、水、大分子甚至癌细胞通过旁细胞途径的转运[4]。
Claudins的异常表达使得TJs功能受损，屏障功能降低，从而导致组织通透性提高，最终导致包括遗传性、过敏性疾病，各个系统感染性疾病乃至肿瘤等多种疾病的发生[5]。Claudins表达水平的上调或下调对肿瘤的发生发展具有促进或抑制作用，在肿瘤的增殖、侵袭、迁移与转移过程中扮演重要角色，其在肿瘤诊断和治疗中具有潜在价值，可作为诊断标志，又可作为免疫治疗的靶点[6]。本文主要就claudin蛋白家族的结构功能、在恶性肿瘤中的异常表达情况及其作为靶标的肿瘤抗体的研究进展进行了综述，为研究者进一步研究claudins提供参考。
1 Claudin蛋白家族及其结构与功能Claudin蛋白家族最早在1998年由Furuse Mikio从鸡肝中克隆得到并为之命名[7]。从线虫到人类，claudins的结构十分相似，高度保守。迄今为止，claudins已经发现有27个成员的蛋白家族，其中有24种在哺乳动物中表达，分子量从20–35 kDa不等，此外，人类和黑猩猩缺少CLDN13[4]。根据不同claudins的序列同源性的高低可以将其分为两组，分别是经典型claudins和非经典型claudins (图 1)[8]。
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| 图 1 Claudin蛋白家族成员 Fig. 1 Claudin protein family members. The image is made by using Neighbor-joining method in MEGA 7.0.14 software, and the claudins' sequences come from GenBank. |
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Claudins成员之间具有相似的结构(图 2)，包括胞内一个短的N端(Amino-terminal)，四次穿膜形成一大一小的两个胞外loop区，以及一个胞内的C端(Carboxy-terminal)[8]。在较大的胞外loop区上含有带电的氨基酸，可以调节细胞旁路对阴阳离子的选择透过性。在这个loop上，claudins有两个高度保守的半胱氨酸残基(Cysteine)。较小的loop则和其他的细胞膜上的claudins通过芳香族残基的疏水作用形成二聚体。除此之外，CLDN3、4、6、9的小loop是梭菌肠毒素Clostridium perfringens enterotoxin (CPE)的受体。C端是claudins中差异最大的部分。除了CLDN12之外，其他的claudins成员在C端都有PDZ结构域(Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1) and Zonula occludens-1 protein (ZO-1))，这一区域使得claudins可以和包括TJs相关的ZO family的适配体在内的细胞质支架蛋白直接互相作用[8]。这一特点使其在细胞信号转导中发挥重要作用，同时也使claudins能与肌动蛋白间接作用，对TJs间的稳定性和选择透过性起重要作用[9]。Claudins在TJs中的正确定位需要PDZ结合元件的上游的C端序列。这一区域含有的氨基酸残基有利于翻译后的各种修饰(图 2)，影响claudins的定位和功能[3, 10-14]。
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| 图 2 Claudins蛋白结构及关键区段 Fig. 2 Structure and key segments of claudins. |
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2 Claudins在肿瘤中的表达及可能的机理Claudins的异常表达可导致上皮细胞、内皮细胞的结构破坏及功能受损，其在多种上皮来源的肿瘤中异常表达，表明claudins可能在肿瘤的侵袭和转移中发挥着重要的作用。细胞间的吸附消失被认为和肿瘤的侵略性有关，因此有假说认为，在肿瘤代谢中，claudins表达水平的下调加强了肿瘤的转移和入侵。但另一方面，也有研究发现有些claudins的过量表达可能导致在某些肿瘤中蛋白的定位和功能异常，从而促进了肿瘤侵袭和转移能力。因此，在上皮/内皮相关肿瘤发生过程中，很可能看到claudins的异常表达[4]。
如表 1所示，一些claudins在癌症中表达下调，这被认为是由于这些claudins在TJs形成和细胞黏附的屏障作用，从而抑制了肿瘤发生和发展。例如，在胸腺癌中，CLDN7在原位和浸润性导管癌的表达降低，并且与肿瘤的组织化程度呈负相关，在高度病变肿瘤中CLDN7的表达明显降低。在siRNA介导敲除CLDN7的TE食管鳞状细胞系中，细胞的生长和转移增加，伴随钙粘蛋白(E-cadherin)的表达降低。而CLDN7的高表达逆转了这些表型，使得细胞间黏附更强而入侵能力更弱，同时钙粘蛋白表达增加[45]。类似的现象也有在CLDN1有关的一些癌症中发现，在人胃癌细胞中敲除CLDN1促进了肿瘤的发生[46]，而在肺癌中CLDN1的过表达抑制肿瘤的分裂以及肿瘤细胞的迁移、入侵和代谢[15]。相关研究认为，一种细胞膜蛋白激酶RON可能在胸腺癌中CLDN1的下调中有重要作用[47]。肿瘤发生过程中上/内皮细胞向间充质细胞转化(Epithelial/endothelial to mesenchymal transition，EMT)途径的激活被提出用于解释这些现象。原发性肿瘤细胞的相关的claudins表达受到抑制从而降低，细胞极性被破坏同时细胞黏附性降低，肿瘤侵入性增强，透过基底膜入侵进入血流。接着这些循环的肿瘤细胞退出血液进行间充质细胞向上/内皮细胞转化(Mesenchymal to epithelial/endothelial transition，MET)形成微转移[48] (图 3)。
表 1 部分claudins在一些癌症组织中的表达变化Table 1 Changes in expression of several claudins in some cancer tissues
| Claudin | Cancer site | Expression change | Reference |
| CLDN1 | Lung | Down | [15] |
| Colorectal | Down | [16] | |
| Prostate | Down | [17-18] | |
| Breast | Down | [19] | |
| CLDN2 | Prostate | Down | [17] |
| CLDN3 | Ovarian | Up | [20-22] |
| Prostate | Up | [17-18] | |
| Breast | Up | [23] | |
| Pancreas | Up | [24] | |
| CLDN4 | Ovarian | Up | [20-22] |
| Prostate | Up | [17-18] | |
| Breast | Up | [25-26] | |
| Pancreas | Up | [27] | |
| CLDN5 | Lung | Up | [28] |
| pancreas | Up | [24] | |
| Prostate | Down | [17] | |
| Cervical | Down | [29] | |
| CLDN6 | Ovarian | Up | [30] |
| Lung | Down | [31] | |
| CLDN7 | Ovarian | Up | [20] |
| Lung | Down | [32] | |
| Breast | Down | [33] | |
| Prostate | Down | [18] | |
| CLDN8 | Cervical | Up | [29] |
| Biliary tract | Down | [34] | |
| Colorectal | Down | [35] | |
| Prostate | Down | [17] | |
| CLDN9 | Cervical | Down | [29] |
| CLDN10 | Hepatocellular | Up | [36] |
| Biliary tract | Down | [34] | |
| CLDN11 | Breast | Down | [37] |
| CLDN12 | Melanoma | Up | [38] |
| Colorectal | Up | [35] | |
| CLDN15 | Mesothelioma | Up | [39] |
| CLDN16 | Breast | Down | [40] |
| CLDN18 | Gastric | Up | [41] |
| Pancreas | Up | [42] | |
| CLDN20 | Breast | Up | [43] |
| CLDN23 | Colorectal | Down | [44] |
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| 图 3 Claudins有关的肿瘤细胞转移 Fig. 3 Tumor cell migration associated with claudins. |
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另一方面，也可以在人卵巢癌表面上皮细胞中过量表达CLDN3与CLDN4，使其在转移、入侵和生存能力上都有提升[49]。CLDN4的过表达促进肿瘤细胞的入侵能力，这在肠道癌细胞系Caco-2中也有被发现，而这被认为可能与基质金属蛋白酶(Matrix metalloproteinase，MMP)中的MMP-2和MMP-9的激活有关[50]，这二者有能力降解基底膜的三重螺旋的Ⅳ和Ⅴ型胶原蛋白，从而导致恶性肿瘤的侵袭和转移[51]。在人肾上皮细胞293T细胞上的一些实验表明包括CLDN1、3、4、5在内的许多种claudins可以介导pro-MMP2的激活，从而促进肿瘤的侵袭和转移。同时另有研究表明胃癌中CLDN4在MMP-9的表达中或许十分重要，并且可能决定了弥漫性胃癌中肠型的发展[52-53]。在肝癌细胞中，CLDN10的表达促进了细胞的存活、运动和入侵，同时MMP-2表达上调[54]。除此之外，也有研究发现，肝转移中的CLDN2表达水平升高可以促进肿瘤细胞/细胞外基质粘附或促进肿瘤细胞与驻留肝细胞之间的相互作用，从而增强乳腺癌细胞的存活[55]。
3 以claudins为肿瘤治疗靶点的相关研究在许多癌症中claudins的异常表达和其在质膜上的定位，使他们成为癌症治疗的潜在靶标候选。其中，CLDN3、4、6、18.2在许多癌症中表达量都有显著增高，是肿瘤靶向治疗的热门靶标(表 2)。人们越来越关注在癌症恶化过程中针对claudins制定策略，有很多种针对肿瘤细胞表达claudins的策略值得期待，包括：1) CPE结合介导肿瘤细胞溶解；2) C-CPE偶联药物递送细胞毒性药物或小分子抑制剂，或作为示踪试剂；3)以claudins为靶点的抗体或抗体偶联药物(Antibody-drug conjugates，ADC)作为递送细胞毒性药物或小分子抑制剂的载体，或连接荧光基团作为示踪试剂；4)以claudins为靶点的嵌合抗原受体T细胞免疫疗法(Chimeric antigen receptor T-cell immunotherapy，CAR-T) (图 4)。
表 2 Claudins相关临床治疗试验进展Table 2 Clinical trial progress related with claudins
| Clinical trials. gov identifier: | Phase | Recruitment status | Conditions | Intervention/ treatment | Start/Completion date |
| NCT01630083 | 2 | Active, not recruiting | CLDN18.2-positive adenocarcinomas of the stomach, the esophagus or the gastroesophageal junction. | Zolbetuximab (IMAB362), epirubicin, oxaliplatin, capecitabine | 2012.06–2018.12 |
| NCT01671774 | 1 | Completed | Advanced adenocarcinoma of the stomach, the lower esophagus or the gastro- esophageal junction (CLDN18.2) | IMAB362, zoledronic acid, interleukin-2 | 2012.08–2014.10 |
| NCT02054351 | 1 | Completed | Ovarian Cancer (CLDN6) | IMAB027 | 2013.12–2017.03 |
| NCT03159819 | – | Recruiting | Advanced gastric adenocarcinoma and pancreatic adenocarcinoma (CLDN18.2) | CAR-CLDN18 T cells | 2017.04–2019.12 |
| NCT03504397 | 3 | Recruiting | Claudin (CLDN) 18.2 positive, HER2- negative, locally advanced unresectable or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma | Zolbetuximab (IMAB362), placebo, oxaliplatin, leucovorin, fluorouracil | 2018.06–2021.02 |
| NCT03505320 | 2 | Recruiting | Claudin (CLDN) 18.2-Positive, metastatic or locally advanced unresectable gastric or gastroesophageal Junction (GEJ) adenocarcinoma | Zolbetuximab (IMAB362), oxaliplatin, leucovorin, fluorouracil | 2018.06–2020.04 |
| NCT03653507 | 3 | Not yet recruiting | Claudin (CLDN) 18.2-Positive, HER2-negative, locally advanced unresectable or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma (GLOW) | Zolbetuximab (IMAB362), oxaliplatin, capecitabine | 2018.10–2021.08 |
| All the experimental information above was obtained from the ClinicalTrials.gov website and updated until October 10, 2018. | |||||
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| 图 4 靶向claudins的治疗策略 Fig. 4 Treatment strategies targeting claudins. |
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3.1 以claudins为肿瘤治疗靶点的CPE相关研究CPE是一个35 kDa的蛋白，其C端在与受体claudins第二个loop结合的过程中诱导穿孔的形成，可导致细胞膜的通透和上皮细胞的溶解，继而引导caspase-3途径的细胞凋亡或细胞胀裂[56]。CPE蛋白可以被分为两部分，N端为细胞毒性域，和寡聚化及穿孔形成有关，C端为受体结合域，即C-CPE[56-57] (图 5)。CPE特异地与游离的CLDN结合而极少与整合入TJs的claudins结合。许多研究发现，CLDN3、4、6、7、9是具有高亲和力CPE/C-CPE受体，CLDN1、2、8、19是低亲和力CPE/C-CPE受体[58-66]。
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| 图 5 CPE的结构功能区域和细胞裂解作用(改自Hashimoto等[56]) Fig. 5 Structural functional areas and cytolysis effects of CPE (adapted from Hashimoto et al[56]). |
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研究显示，CLDN3和CLDN4在几个卵巢癌病例中的表达量是正常上皮细胞的83–109倍[66]。此外，DNA微阵列分析显示，CLDN3和CLDN4是卵巢癌中表达差异最大的5个基因中的2个[67]。CLDN6和CLDN7在卵巢癌中过表达[39, 68]，意味着CPE敏感的几个claudins在卵巢癌中的表达往往是增多的[69]。此外，有研究报道化疗耐药或复发性卵巢癌的CLDN3和CLDN4的表达水平高于化疗敏感性卵巢癌[69-70]，这为CPE用于卵巢癌的靶向治疗提供了希望。
有****尝试直接使用CPE治疗肿瘤。腹腔注射CPE的抗化疗原发性卵巢癌细胞的小鼠移植模型肿瘤生长得到抑制，且呈剂量依赖，并且并未引发明显的副作用[69, 71]。在胰腺癌细胞Panc-1移植的小鼠体内注射CPE则完全抑制肿瘤生长，导致肿瘤生长明显受抑和坏死，同时未造成任何不良影响[27]。
由于CLDN3/4在肺、胃等正常组织中也有表达，因此也有研究者为了减少CPE潜在的毒副风险，同时也为了使其具有更小的抗原特性，尝试使用C-CPE作为抗肿瘤药物穿透的增强剂并应用于癌症诊断。反复腹腔注射紫杉醇联合C-CPE，在植入皮下肿瘤的小鼠产生了显著的协同抗肿瘤作用，同时发现C-CPE诱导了卵巢癌细胞的形态学改变[72]。对腹腔移植了腹腔转移卵巢癌细胞OSPC-ARK-1的小鼠静脉注射C-CPE联合荧光染料，能够检测到以往视觉观察难以发现的小至1 mm2区域的肿瘤组织[73]。用111共轭谷胱甘肽S-转移酶和C-CPE结合的断层显像研究显示，CLDN4阳性的乳腺癌细胞MDA-MB-468移植小鼠的肿瘤组织中，放射性示踪物的累积与CLDN4阴性移植小鼠对比明显增加[74]。C-CPE联合荧光素在CLDN4阳性的胰腺癌细胞Capan-1小鼠中的累积与CLDN4阴性移植小鼠相比更高[75]。
3.2 以claudins为肿瘤治疗靶点的抗体及CAR-T研究目前已有许多以claudins为抗原筛选得到的抗体，抗原包含了claudins的短肽、细胞、DNA或是病毒颗粒类似物。另一种靶向肿瘤claudins的治疗策略是特异性抗体的应用，CLDN3、4、6、18.2是该研究中的热门靶标，有许多研究团队已经筛选了特异性针对肿瘤细胞高表达的claudins胞外区的抗体。
如前文所述，CLDN3和CLDN4在许多卵巢癌病例中的表达量异常增高，因此靶向CLDN3/4抑或二者的抗体有作为治疗手段的潜力，一些抗体也被证明在临床前动物模型上有抗肿瘤作用，更是有抗体已经进入了临床阶段(表 2)。筛选得到能够识别并结合CLDN4胞外第2个loop的鼠抗KM3900并人源化后得到的抗体KM3934，在体外实验中表现出抗体依赖性的细胞毒性(Antibody dependent cytotoxicity，ADCC)和补充依赖性细胞毒性(Complement dependent cytotoxicity，CDC)，并在移植人卵巢癌细胞系MCAS或人胰腺癌细胞系CFPAC-1的免疫缺陷小鼠体内能够显著抑制肿瘤的生长[76]。CLDN6在一定比例的晚期卵巢癌中高水平表达，在正常成人组织中没有发现。IMAB027是一株特异性结合CLDN6的单克隆抗体。临床前实验证明这种抗体有抑制肿瘤生长和杀死癌细胞的ADCC和CDC效应，目前正在进行复发性晚期卵巢癌患者的临床Ⅰ期试验(NCT02054351)。早期的数据表明，IMAB027具有良好的耐受性[77]。CLDN18.2参与肿瘤的发展和进展，暴露在细胞外的loop可用于单克隆抗体结合。这些生物学特性表明它是靶向治疗的理想分子，是目前claudins相关肿瘤临床治疗研究中最热门的靶标。除了胃内层的正常组织中，CLDN18.2在70%–90%的胃、胰、胆管癌中高表达。IMAB362就是一株抗CLDN18.2的单克隆抗体[78]，能够诱导ADCC和CDC效应，及介导肿瘤被破坏。来自二期临床研究的发现表明，单纯化疗相比，将实验性抗体IMAB362 (Ganymed药物)添加到标准化疗中，可以提高以前未经治疗的胃癌患者的整体生存3–5个月，而在CLDN18.2表达超过70%的肿瘤上，甚至能由9个月提高至16.7个月[79]。
如表 2所示，目前也有针对CLDN18.2的CAR-T细胞疗法的研究在一期临床招募阶段。CAR-T细胞设计的基本原理涉及结合抗原结合和T细胞激活功能的重组受体，从人身上取出T细胞，通过基因工程修饰，并将它们重新输回患者体内，以便攻击癌细胞。一旦T细胞被设计成CAR-T细胞，它就会成为患者体内的“活药物”[80]。目前CAR-T疗法在急性白血病和非霍奇金淋巴瘤的治疗上显示出良好的潜力并且即将上市，科济(CARsgen)公司开发的CAR-T疗法主要针对CLDN18.2过表达的晚期胃腺癌、胰腺癌，在上海长海医院(现为海军军区大学第一附属医院)进行临床招募，为针对claudins表达异常的肿瘤治疗提供了新的思路。
4 总结与展望Claudins在肿瘤代谢过程中的一些具体机理机制和关键步骤仍不清楚，但其作为肿瘤治疗靶标的潜力毋庸置疑。临床前实验和临床研究已充分证实了claudins作为肿瘤治疗靶标及相关应用的可能并且取得了一定进展：CPE及其衍生物用于肿瘤治疗和诊断的临床前研究效果显著；靶向CLDN18.2的抗体IMAB362和靶向CLDN4/6的抗体IMAB027已经进入临床研究阶段，IMAB027更是已获得FDA和欧盟授予的治疗胃癌和胰腺癌的孤儿药资格；科济生物医药(上海)有限公司所开发的针对CLDN18.2的CAR-T疗法也已进入临床研究阶段，用于癌症的免疫治疗将会有很光明的前景。
即便如此，研究过程中仍存在一些需要解决的问题和可以改进的方面，比如：是否能通过C端的蛋白修饰来调控claudins的功能；如何进一步避免CPE及其衍生物带来的毒副作用；如何确保治疗过程中claudins靶向的特异性。因此，还需更深入研究肿瘤细胞异常表达的claudins在肿瘤代谢中的作用机制，一些假说尚需更多实验去探索，临床取得的成果也需要进一步的验证。综合来看，将claudins作为靶标用于肿瘤治疗的前景光明，给目前的肿瘤治疗带来新的选择。
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