陳思遠(yuǎn)，劉雪，羅文新

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緊密連接蛋白claudins應(yīng)用于腫瘤治療的進展

陳思遠(yuǎn)，劉雪，羅文新

廈門大學(xué) 生命科學(xué)學(xué)院 公共衛(wèi)生學(xué)院 國家傳染病診斷試劑與疫苗工程技術(shù)研究中心 分子疫苗學(xué)和分子診斷學(xué)國家重點實驗室，福建 廈門 361102

Claudins蛋白家族是組成緊密連接(Tight junctions，TJs) 必不可少的骨架蛋白，在維持上皮和內(nèi)皮細(xì)胞中的細(xì)胞極性、細(xì)胞間的粘附固定、細(xì)胞旁路的離子運輸?shù)劝l(fā)揮重要作用。近年來大量的研究結(jié)果證明，claudins在許多人類惡性腫瘤中異常表達。因此，claudins也被作為癌癥治療的潛在靶標(biāo)。文中就claudin蛋白家族在腫瘤中的表達情況及其相關(guān)藥物的研究進展進行闡述。

claudin，腫瘤，梭菌腸毒素，抗體，靶向治療

緊密連接 (Tight junctions，TJs)又叫occluding junctions或zonulae occludentes，是細(xì)胞黏附結(jié)構(gòu)的重要連接形式之一。緊密連接包括跨膜蛋白occludins和claudins (CLDNs) 以及膜外周蛋白如ZO-1[1-3]，其縫合了相鄰極化的上皮細(xì)胞間或內(nèi)皮細(xì)胞間的縫隙，使之具有屏障功能，同時調(diào)控著離子、水、大分子甚至癌細(xì)胞通過旁細(xì)胞途徑的轉(zhuǎn)運[4]。

Claudins的異常表達使得TJs功能受損，屏障功能降低，從而導(dǎo)致組織通透性提高，最終導(dǎo)致包括遺傳性、過敏性疾病，各個系統(tǒng)感染性疾病乃至腫瘤等多種疾病的發(fā)生[5]。Claudins表達水平的上調(diào)或下調(diào)對腫瘤的發(fā)生發(fā)展具有促進或抑制作用，在腫瘤的增殖、侵襲、遷移與轉(zhuǎn)移過程中扮演重要角色，其在腫瘤診斷和治療中具有潛在價值，可作為診斷標(biāo)志，又可作為免疫治療的靶點[6]。本文主要就claudin蛋白家族的結(jié)構(gòu)功能、在惡性腫瘤中的異常表達情況及其作為靶標(biāo)的腫瘤抗體的研究進展進行了綜述，為研究者進一步研究claudins提供參考。

1 Claudin蛋白家族及其結(jié)構(gòu)與功能

Claudin蛋白家族最早在1998年由Furuse Mikio從雞肝中克隆得到并為之命名[7]。從線蟲到人類，claudins的結(jié)構(gòu)十分相似，高度保守。迄今為止，claudins已經(jīng)發(fā)現(xiàn)有27個成員的蛋白家族，其中有24種在哺乳動物中表達，分子量從20–35 kDa不等，此外，人類和黑猩猩缺少CLDN13[4]。根據(jù)不同claudins的序列同源性的高低可以將其分為兩組，分別是經(jīng)典型claudins和非經(jīng)典型claudins (圖1)[8]。

Claudins成員之間具有相似的結(jié)構(gòu)(圖2)，包括胞內(nèi)一個短的N端(Amino-terminal)，四次穿膜形成一大一小的兩個胞外loop區(qū)，以及一個胞內(nèi)的C端(Carboxy-terminal)[8]。在較大的胞外loop區(qū)上含有帶電的氨基酸，可以調(diào)節(jié)細(xì)胞旁路對陰陽離子的選擇透過性。在這個loop上，claudins有兩個高度保守的半胱氨酸殘基(Cysteine)。較小的loop則和其他的細(xì)胞膜上的claudins通過芳香族殘基的疏水作用形成二聚體。除此之外，CLDN3、4、6、9的小loop是梭菌腸毒素enterotoxin (CPE) 的受體。C端是claudins中差異最大的部分。除了CLDN12之外，其他的claudins成員在C端都有PDZ結(jié)構(gòu)域(Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1) and Zonula occludens-1 protein (ZO-1))，這一區(qū)域使得claudins可以和包括TJs相關(guān)的ZO family的適配體在內(nèi)的細(xì)胞質(zhì)支架蛋白直接互相作用[8]。這一特點使其在細(xì)胞信號轉(zhuǎn)導(dǎo)中發(fā)揮重要作用，同時也使claudins能與肌動蛋白間接作用，對TJs間的穩(wěn)定性和選擇透過性起重要作用[9]。Claudins在TJs中的正確定位需要PDZ結(jié)合元件的上游的C端序列。這一區(qū)域含有的氨基酸殘基有利于翻譯后的各種修飾(圖2)，影響claudins的定位和功能[3, 10-14]。

圖1 Claudin蛋白家族成員

圖2 Claudins蛋白結(jié)構(gòu)及關(guān)鍵區(qū)段

2 Claudins在腫瘤中的表達及可能的機理

Claudins的異常表達可導(dǎo)致上皮細(xì)胞、內(nèi)皮細(xì)胞的結(jié)構(gòu)破壞及功能受損，其在多種上皮來源的腫瘤中異常表達，表明claudins可能在腫瘤的侵襲和轉(zhuǎn)移中發(fā)揮著重要的作用。細(xì)胞間的吸附消失被認(rèn)為和腫瘤的侵略性有關(guān)，因此有假說認(rèn)為，在腫瘤代謝中，claudins表達水平的下調(diào)加強了腫瘤的轉(zhuǎn)移和入侵。但另一方面，也有研究發(fā)現(xiàn)有些claudins的過量表達可能導(dǎo)致在某些腫瘤中蛋白的定位和功能異常，從而促進了腫瘤侵襲和轉(zhuǎn)移能力。因此，在上皮/內(nèi)皮相關(guān)腫瘤發(fā)生過程中，很可能看到claudins的異常表達[4]。

如表1所示，一些claudins在癌癥中表達下調(diào)，這被認(rèn)為是由于這些claudins在TJs形成和細(xì)胞黏附的屏障作用，從而抑制了腫瘤發(fā)生和發(fā)展。例如，在胸腺癌中，CLDN7在原位和浸潤性導(dǎo)管癌的表達降低，并且與腫瘤的組織化程度呈負(fù)相關(guān)，在高度病變腫瘤中CLDN7的表達明顯降低。在siRNA介導(dǎo)敲除CLDN7的TE食管鱗狀細(xì)胞系中，細(xì)胞的生長和轉(zhuǎn)移增加，伴隨鈣粘蛋白(E-cadherin) 的表達降低。而CLDN7的高表達逆轉(zhuǎn)了這些表型，使得細(xì)胞間黏附更強而入侵能力更弱，同時鈣粘蛋白表達增加[45]。類似的現(xiàn)象也有在CLDN1有關(guān)的一些癌癥中發(fā)現(xiàn)，在人胃癌細(xì)胞中敲除CLDN1促進了腫瘤的發(fā)生[46]，而在肺癌中CLDN1的過表達抑制腫瘤的分裂以及腫瘤細(xì)胞的遷移、入侵和代謝[15]。相關(guān)研究認(rèn)為，一種細(xì)胞膜蛋白激酶RON可能在胸腺癌中CLDN1的下調(diào)中有重要作用[47]。腫瘤發(fā)生過程中上/內(nèi)皮細(xì)胞向間充質(zhì)細(xì)胞轉(zhuǎn)化 (Epithelial/endothelialto mesenchymal transition，EMT) 途徑的激活被提出用于解釋這些現(xiàn)象。原發(fā)性腫瘤細(xì)胞的相關(guān)的claudins表達受到抑制從而降低，細(xì)胞極性被破壞同時細(xì)胞黏附性降低，腫瘤侵入性增強，透過基底膜入侵進入血流。接著這些循環(huán)的腫瘤細(xì)胞退出血液進行間充質(zhì)細(xì)胞向上/內(nèi)皮細(xì)胞轉(zhuǎn)化 (Mesenchymal to epithelial/endothelial transition，MET) 形成微轉(zhuǎn)移[48](圖3)。

表1 部分claudins在一些癌癥組織中的表達變化

另一方面，也可以在人卵巢癌表面上皮細(xì)胞中過量表達CLDN3與CLDN4，使其在轉(zhuǎn)移、入侵和生存能力上都有提升[49]。CLDN4的過表達促進腫瘤細(xì)胞的入侵能力，這在腸道癌細(xì)胞系Caco-2中也有被發(fā)現(xiàn)，而這被認(rèn)為可能與基質(zhì)金屬蛋白酶 (Matrix metalloproteinase，MMP) 中的MMP-2和MMP-9的激活有關(guān)[50]，這二者有能力降解基底膜的三重螺旋的Ⅳ和Ⅴ型膠原蛋白，從而導(dǎo)致惡性腫瘤的侵襲和轉(zhuǎn)移[51]。在人腎上皮細(xì)胞293T細(xì)胞上的一些實驗表明包括CLDN1、3、4、5在內(nèi)的許多種claudins可以介導(dǎo)pro-MMP2的激活，從而促進腫瘤的侵襲和轉(zhuǎn)移。同時另有研究表明胃癌中CLDN4在MMP-9的表達中或許十分重要，并且可能決定了彌漫性胃癌中腸型的發(fā)展[52-53]。在肝癌細(xì)胞中，CLDN10的表達促進了細(xì)胞的存活、運動和入侵，同時MMP-2表達上調(diào)[54]。除此之外，也有研究發(fā)現(xiàn)，肝轉(zhuǎn)移中的CLDN2表達水平升高可以促進腫瘤細(xì)胞/細(xì)胞外基質(zhì)粘附或促進腫瘤細(xì)胞與駐留肝細(xì)胞之間的相互作用，從而增強乳腺癌細(xì)胞的存活[55]。

圖3 Claudins有關(guān)的腫瘤細(xì)胞轉(zhuǎn)移

3 以claudins為腫瘤治療靶點的相關(guān)研究

在許多癌癥中claudins的異常表達和其在質(zhì)膜上的定位，使他們成為癌癥治療的潛在靶標(biāo)候選。其中，CLDN3、4、6、18.2在許多癌癥中表達量都有顯著增高，是腫瘤靶向治療的熱門靶標(biāo)(表2)。人們越來越關(guān)注在癌癥惡化過程中針對claudins制定策略，有很多種針對腫瘤細(xì)胞表達claudins的策略值得期待，包括：1) CPE結(jié)合介導(dǎo)腫瘤細(xì)胞溶解；2) C-CPE偶聯(lián)藥物遞送細(xì)胞毒性藥物或小分子抑制劑，或作為示蹤試劑；3) 以claudins為靶點的抗體或抗體偶聯(lián)藥物 (Antibody-drug conjugates，ADC)作為遞送細(xì)胞毒性藥物或小分子抑制劑的載體，或連接熒光基團作為示蹤試劑；4) 以claudins為靶點的嵌合抗原受體T細(xì)胞免疫療法 (Chimeric antigen receptor T-cell immunotherapy，CAR-T) (圖4)。

3.1 以claudins為腫瘤治療靶點的CPE相關(guān)研究

CPE是一個35 kDa的蛋白，其C端在與受體claudins第二個loop結(jié)合的過程中誘導(dǎo)穿孔的形成，可導(dǎo)致細(xì)胞膜的通透和上皮細(xì)胞的溶解，繼而引導(dǎo)caspase-3途徑的細(xì)胞凋亡或細(xì)胞脹裂[56]。CPE蛋白可以被分為兩部分，N端為細(xì)胞毒性域，和寡聚化及穿孔形成有關(guān)，C端為受體結(jié)合域，即C-CPE[56-57](圖5)。CPE特異地與游離的CLDN結(jié)合而極少與整合入TJs的claudins結(jié)合。許多研究發(fā)現(xiàn)，CLDN3、4、6、7、9是具有高親和力CPE/C-CPE受體，CLDN1、2、8、19是低親和力CPE/C-CPE受體[58-66]。

表2 Claudins相關(guān)臨床治療試驗進展

fAll the experimental information above was obtained from the ClinicalTrials.gov website and updated until October 10, 2018.

研究顯示，CLDN3和CLDN4在幾個卵巢癌病例中的表達量是正常上皮細(xì)胞的83–109倍[66]。此外，DNA微陣列分析顯示，CLDN3和CLDN4是卵巢癌中表達差異最大的5個基因中的2個[67]。CLDN6和CLDN7在卵巢癌中過表達[39, 68]，意味著CPE敏感的幾個claudins在卵巢癌中的表達往往是增多的[69]。此外，有研究報道化療耐藥或復(fù)發(fā)性卵巢癌的CLDN3和CLDN4的表達水平高于化療敏感性卵巢癌[69-70]，這為CPE用于卵巢癌的靶向治療提供了希望。

圖4 靶向claudins的治療策略

圖5 CPE的結(jié)構(gòu)功能區(qū)域和細(xì)胞裂解作用(改自Hashimoto等[56])

有學(xué)者嘗試直接使用CPE治療腫瘤。腹腔注射CPE的抗化療原發(fā)性卵巢癌細(xì)胞的小鼠移植模型腫瘤生長得到抑制，且呈劑量依賴，并且并未引發(fā)明顯的副作用[69, 71]。在胰腺癌細(xì)胞Panc-1移植的小鼠體內(nèi)注射CPE則完全抑制腫瘤生長，導(dǎo)致腫瘤生長明顯受抑和壞死，同時未造成任何不良影響[27]。

由于CLDN3/4在肺、胃等正常組織中也有表達，因此也有研究者為了減少CPE潛在的毒副風(fēng)險，同時也為了使其具有更小的抗原特性，嘗試使用C-CPE作為抗腫瘤藥物穿透的增強劑并應(yīng)用于癌癥診斷。反復(fù)腹腔注射紫杉醇聯(lián)合C-CPE，在植入皮下腫瘤的小鼠產(chǎn)生了顯著的協(xié)同抗腫瘤作用，同時發(fā)現(xiàn)C-CPE誘導(dǎo)了卵巢癌細(xì)胞的形態(tài)學(xué)改變[72]。對腹腔移植了腹腔轉(zhuǎn)移卵巢癌細(xì)胞OSPC-ARK-1的小鼠靜脈注射C-CPE聯(lián)合熒光染料，能夠檢測到以往視覺觀察難以發(fā)現(xiàn)的小至1 mm2區(qū)域的腫瘤組織[73]。用111共軛谷胱甘肽S-轉(zhuǎn)移酶和C-CPE結(jié)合的斷層顯像研究顯示，CLDN4陽性的乳腺癌細(xì)胞MDA-MB-468移植小鼠的腫瘤組織中，放射性示蹤物的累積與CLDN4陰性移植小鼠對比明顯增加[74]。C-CPE聯(lián)合熒光素在CLDN4陽性的胰腺癌細(xì)胞Capan-1小鼠中的累積與CLDN4陰性移植小鼠相比更高[75]。

3.2 以claudins為腫瘤治療靶點的抗體及CAR-T研究

目前已有許多以claudins為抗原篩選得到的抗體，抗原包含了claudins的短肽、細(xì)胞、DNA或是病毒顆粒類似物。另一種靶向腫瘤claudins的治療策略是特異性抗體的應(yīng)用，CLDN3、4、6、18.2是該研究中的熱門靶標(biāo)，有許多研究團隊已經(jīng)篩選了特異性針對腫瘤細(xì)胞高表達的claudins胞外區(qū)的抗體。

如前文所述，CLDN3和CLDN4在許多卵巢癌病例中的表達量異常增高，因此靶向CLDN3/4抑或二者的抗體有作為治療手段的潛力，一些抗體也被證明在臨床前動物模型上有抗腫瘤作用，更是有抗體已經(jīng)進入了臨床階段(表2)。篩選得到能夠識別并結(jié)合CLDN4胞外第2個loop的鼠抗KM3900并人源化后得到的抗體KM3934，在體外實驗中表現(xiàn)出抗體依賴性的細(xì)胞毒性 (Antibody dependent cytotoxicity，ADCC) 和補充依賴性細(xì)胞毒性 (Complement dependent cytotoxicity，CDC)，并在移植人卵巢癌細(xì)胞系MCAS或人胰腺癌細(xì)胞系CFPAC-1的免疫缺陷小鼠體內(nèi)能夠顯著抑制腫瘤的生長[76]。CLDN6在一定比例的晚期卵巢癌中高水平表達，在正常成人組織中沒有發(fā)現(xiàn)。IMAB027是一株特異性結(jié)合CLDN6的單克隆抗體。臨床前實驗證明這種抗體有抑制腫瘤生長和殺死癌細(xì)胞的ADCC和CDC效應(yīng)，目前正在進行復(fù)發(fā)性晚期卵巢癌患者的臨床Ⅰ期試驗(NCT02054351)。早期的數(shù)據(jù)表明，IMAB027具有良好的耐受性[77]。CLDN18.2參與腫瘤的發(fā)展和進展，暴露在細(xì)胞外的loop可用于單克隆抗體結(jié)合。這些生物學(xué)特性表明它是靶向治療的理想分子，是目前claudins相關(guān)腫瘤臨床治療研究中最熱門的靶標(biāo)。除了胃內(nèi)層的正常組織中，CLDN18.2在70%–90%的胃、胰、膽管癌中高表達。IMAB362就是一株抗CLDN18.2的單克隆抗體[78]，能夠誘導(dǎo)ADCC和CDC效應(yīng)，及介導(dǎo)腫瘤被破壞。來自二期臨床研究的發(fā)現(xiàn)表明，單純化療相比，將實驗性抗體IMAB362 (Ganymed藥物) 添加到標(biāo)準(zhǔn)化療中，可以提高以前未經(jīng)治療的胃癌患者的整體生存3–5個月，而在CLDN18.2表達超過70%的腫瘤上，甚至能由9個月提高至16.7個月[79]。

如表2所示，目前也有針對CLDN18.2的CAR-T細(xì)胞療法的研究在一期臨床招募階段。CAR-T細(xì)胞設(shè)計的基本原理涉及結(jié)合抗原結(jié)合和T細(xì)胞激活功能的重組受體，從人身上取出T細(xì)胞，通過基因工程修飾，并將它們重新輸回患者體內(nèi)，以便攻擊癌細(xì)胞。一旦T細(xì)胞被設(shè)計成CAR-T細(xì)胞，它就會成為患者體內(nèi)的“活藥物”[80]。目前CAR-T療法在急性白血病和非霍奇金淋巴瘤的治療上顯示出良好的潛力并且即將上市，科濟(CARsgen) 公司開發(fā)的CAR-T療法主要針對CLDN18.2過表達的晚期胃腺癌、胰腺癌，在上海長海醫(yī)院(現(xiàn)為海軍軍區(qū)大學(xué)第一附屬醫(yī)院)進行臨床招募，為針對claudins表達異常的腫瘤治療提供了新的思路。

4 總結(jié)與展望

Claudins在腫瘤代謝過程中的一些具體機理機制和關(guān)鍵步驟仍不清楚，但其作為腫瘤治療靶標(biāo)的潛力毋庸置疑。臨床前實驗和臨床研究已充分證實了claudins作為腫瘤治療靶標(biāo)及相關(guān)應(yīng)用的可能并且取得了一定進展：CPE及其衍生物用于腫瘤治療和診斷的臨床前研究效果顯著；靶向CLDN18.2的抗體IMAB362和靶向CLDN4/6的抗體IMAB027已經(jīng)進入臨床研究階段，IMAB027更是已獲得FDA和歐盟授予的治療胃癌和胰腺癌的孤兒藥資格；科濟生物醫(yī)藥(上海) 有限公司所開發(fā)的針對CLDN18.2的CAR-T療法也已進入臨床研究階段，用于癌癥的免疫治療將會有很光明的前景。

即便如此，研究過程中仍存在一些需要解決的問題和可以改進的方面，比如：是否能通過C端的蛋白修飾來調(diào)控claudins的功能；如何進一步避免CPE及其衍生物帶來的毒副作用；如何確保治療過程中claudins靶向的特異性。因此，還需更深入研究腫瘤細(xì)胞異常表達的claudins在腫瘤代謝中的作用機制，一些假說尚需更多實驗去探索，臨床取得的成果也需要進一步的驗證。綜合來看，將claudins作為靶標(biāo)用于腫瘤治療的前景光明，給目前的腫瘤治療帶來新的選擇。

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Advances in the application of claudins to tumor therapy

Siyuan Chen, Xue Liu, and 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

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.

claudin, tumor,enterotoxin, antibody, targeted therapy

October 25, 2018;

December 28, 2018

National Natural Science Foundation of China (No. 31870925), Major Projects of Infectious Diseases (No. 2017ZX10202203-001).

Wenxin Luo. Tel: +86-592-2188657; Fax: +86-592-2181258; E-mail: wxluo@xmu.edu.cn

國家自然科學(xué)基金 (No. 31870925)，傳染病重大專項 (No. 2017ZX10202203-001) 資助。

10.13345/j.cjb.180435

陳思遠(yuǎn), 劉雪, 羅文新. 緊密連接蛋白claudins應(yīng)用于腫瘤治療的進展. 生物工程學(xué)報, 2019, 35(6): 931–941.

Chen SY, Liu X, Luo WX. Advances in the application of claudins to tumor therapy. Chin J Biotech, 2019, 35(6): 931–941.

(本文責(zé)編 陳宏宇)