肝癌特異癌胚性抗原介導(dǎo)的免疫治療研究*

姚敏,姚登福

（南通大學(xué):1醫(yī)學(xué)院免疫學(xué)教研室；2附屬醫(yī)院臨床醫(yī)學(xué)研究中心，江蘇南通226001）

·肝病專欄·

肝癌特異癌胚性抗原介導(dǎo)的免疫治療研究*

姚敏1,姚登福2

（南通大學(xué):1醫(yī)學(xué)院免疫學(xué)教研室；2附屬醫(yī)院臨床醫(yī)學(xué)研究中心，江蘇南通226001）

肝細(xì)胞性肝癌(Hepatocellular carcinoma,HCC)的預(yù)防和有效治療仍是醫(yī)學(xué)界的難題[1-2]。人類HCC發(fā)生與肝炎病毒(HBV、HCV)慢性持續(xù)性感染、化學(xué)致癌物攝入等多病因作用相關(guān)。癌基因或癌相關(guān)基因激活、抗癌基因失活或胚胎期某些癌基因重新復(fù)活等諸多因素，引起肝細(xì)胞生長失控，經(jīng)啟動(dòng)、促進(jìn)、演變?yōu)槎嚯A段發(fā)病過程。因其惡性度高，起病隱匿，早期診斷率低，確診時(shí)往往已到中晚期，失去根治性切除機(jī)會(huì)，且術(shù)后復(fù)發(fā)機(jī)率很高，預(yù)后極差[3]。肝癌以手術(shù)切除為主的綜合治療，包括血管栓塞、射頻、放化療和生物治療等，雖可有效地治療局部病灶，但仍不能解決復(fù)發(fā)、轉(zhuǎn)移等問題[4-5]。肝癌特異性或相關(guān)抗原、某些通路的關(guān)鍵信號(hào)分子、生長因子及受體和癌基因產(chǎn)物等均為治療靶點(diǎn)，癌胚型抗原介導(dǎo)的治療靶點(diǎn)具有高度靶向特異性，在肝癌治療中可能具有良好的應(yīng)用前景。本文述評(píng)了近年對(duì)癌胚型抗原介導(dǎo)的肝癌分子免疫治療研究的新進(jìn)展。

1 癌胚性HBx抗原與肝癌免疫治療

肝癌為多基因變異的惡性腫瘤，伴隨DNA甲基化改變、MicroRNA、lncRNA表達(dá)及免疫應(yīng)答等[7-8]。我國肝癌患者中HBV感染者居多，在80%以上。HBV感染自然史分為免疫耐受期、免疫清除期、非活動(dòng)復(fù)制期和再活動(dòng)期，免疫力低下或勞累可誘導(dǎo)基因變異[9-10]。在HBV的A、B、C、D、E、F、G和H 8種基因型中，我國肝癌患者以C型和B型為主；C基因型占74.5%，尤其C2亞型比B基因型更易致癌，且術(shù)后又是復(fù)發(fā)的高危因素，值得重視[11-12]。HBV陽性肝癌中存在大量截短HBx蛋白為致癌基因，肝癌細(xì)胞基因組19q12、2q32.2、22q12是HBx整合位點(diǎn)。將HBx基因轉(zhuǎn)染至肝癌HepG2細(xì)胞，受轉(zhuǎn)染細(xì)胞第3、18和20號(hào)染色體發(fā)生改變，微核數(shù)量增加3倍；HBx蛋白調(diào)節(jié)胞內(nèi)基因、結(jié)合轉(zhuǎn)錄因子、激活相關(guān)信號(hào)通路與啟動(dòng)子共同作用，誘導(dǎo)肝細(xì)胞發(fā)生惡性轉(zhuǎn)化[13-14]。

HBxAg表達(dá)與HCC發(fā)生、發(fā)展密切相關(guān)[15]。在抗HBxAg單抗（188Re-anti-HBx Mab）治療HBV相關(guān)HCC研究中，以持續(xù)表達(dá)HBxAg的肝癌Hep3B2.1-7細(xì)胞制備裸鼠人肝癌移植瘤模型，以188Re-4H9 Mab治療，發(fā)現(xiàn)肝癌組織188Re-4H9 Mab特異性聚集，瘤體明顯縮小，呈劑量依賴性[16]。病理學(xué)檢查見瘤內(nèi)出現(xiàn)壞死、出血程度明顯高于對(duì)照組。因核素標(biāo)記的抗HBx Mab的靶點(diǎn)是癌細(xì)胞或病毒感染細(xì)胞的表面抗原，而非體內(nèi)自身抗原，與以往核素標(biāo)記單抗相比，特異性更高、對(duì)正常細(xì)胞毒性更低。提示該單抗與HBx抗原特異結(jié)合，不僅可用于HCC治療，且可在HBV持續(xù)感染而非癌變階段發(fā)揮治療作用[17-18]。

2 炎癥介質(zhì)NF-κB與肝癌

臨床證實(shí)HCC為炎癥相關(guān)性惡性腫瘤，核轉(zhuǎn)錄因子-κB（Nuclear factor-κB，NF-κB）活化在炎癥和癌變間充當(dāng)橋梁角色并與多藥耐藥的發(fā)生相關(guān)[19]。肝細(xì)胞癌變過程中NF-κB異常表達(dá)，與其受體結(jié)合經(jīng)信號(hào)轉(zhuǎn)導(dǎo)途徑誘導(dǎo)病變細(xì)胞凋亡，以建立細(xì)胞快速防御機(jī)制，發(fā)揮抗凋亡作用?；罨疦F-κB促腫瘤壞死因子(TNF)-α轉(zhuǎn)錄產(chǎn)生反饋調(diào)節(jié)，使NF-κB抗凋亡效應(yīng)增強(qiáng)從而促進(jìn)癌變，以蛋白酶體抑制劑阻斷NF-κB活化可逆轉(zhuǎn)癌細(xì)胞對(duì)TRAIL(TNF-related apoptosis-inducing ligand)耐受性，增強(qiáng)TRAIL所誘導(dǎo)癌細(xì)胞凋亡[20]。肝癌HepG2、SMMC 7721細(xì)胞死亡受體-5 (DR5)表達(dá)水平明顯升高，正常肝細(xì)胞卻很少。抗DR5單抗干預(yù)TRAIL介導(dǎo)的NF-κB活化，可選擇性誘導(dǎo)癌細(xì)胞凋亡，抑制P38絲裂原活化蛋白激酶磷酸化和NF-κB活化，下調(diào)MMP-9和細(xì)胞間粘附分子1表達(dá)，進(jìn)而抑制癌細(xì)胞增殖、侵襲和血管生成，改變肝癌細(xì)胞的生物學(xué)特性[21-16]。

NF-κB是炎癥相關(guān)多中心、多基因和多步驟肝癌的重要介質(zhì)，從慢性肝炎到肝癌過程中癌組織NF-κB表達(dá)，顯著高于肝硬化或慢性肝炎的癌周組織（伴不典型增生），并伴肝免疫炎癥反應(yīng)相關(guān)基因、肝炎病毒相關(guān)基因和原癌基因轉(zhuǎn)錄表達(dá)[22]。NF-κB為誘導(dǎo)DNA結(jié)合型轉(zhuǎn)錄因子，兩關(guān)鍵分子NF-κB1和NF-κBIα基因具有啟動(dòng)子功能的單核苷酸多態(tài)性，為HBV C基因型HCC的獨(dú)立危險(xiǎn)因素。肝癌中NF-κB 1-94ATTG2和NF-κBIA 3’非翻譯區(qū)GG等位基因頻率，顯著高于對(duì)照和慢性乙肝患者；NF-κBIA-826CT和NF-κBIA-881AG等位基因頻率顯著高于非HCC感染者，且這種關(guān)聯(lián)性在C型HBV感染致癌中顯著，提示C基因型HBV致癌能力高于B基因型，且與NF-κB遺傳易感性有關(guān)[23-24]。

以大鼠制備肝癌發(fā)生的動(dòng)態(tài)模型觀察，在肝細(xì)胞變性、癌前病變和癌變的過程中，肝組織總RNA呈梯度進(jìn)行性增加，DNA合成及核酸代謝旺盛。全基因組（鼠28 000個(gè)基因）表達(dá)譜分析，發(fā)現(xiàn)與對(duì)照組相比早期HCC有2 898個(gè)基因上調(diào)，其中1 179個(gè)基因上調(diào)幅度大于2倍，268個(gè)基因上調(diào)幅度大于8倍；晚期HCC有3 208個(gè)基因上調(diào)；晚期HCC與早期HCC相比2 416基因上調(diào)，涉及細(xì)胞周期相關(guān)基因、與信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因、腫瘤轉(zhuǎn)移相關(guān)基因、凋亡抑制基因、抑癌基因及黏附分子等，可見癌變過程復(fù)雜，決定了肝癌靶向治療極其艱難。然而干預(yù)NF-κB活化或中和TNF-α所介導(dǎo)的信號(hào)傳導(dǎo)可延緩肝癌發(fā)生，或者干預(yù)肝癌細(xì)胞中NF-κB的轉(zhuǎn)錄，可明顯抑制肝癌細(xì)胞增殖和生物學(xué)特性的改變[25-26]。

3 癌胚性甲胎蛋白(AFP)抗原與肝癌免疫治療

癌胚性AFP是胚胎期肝細(xì)胞合成的由609個(gè)氨基酸單鏈多肽組成的糖蛋白，3個(gè)結(jié)構(gòu)域N端含19個(gè)氨基酸殘基的前導(dǎo)信號(hào)點(diǎn)[27-28]，可被主要組織相容性復(fù)合體（Major histocompatibility complex,MHC）I或II類分子識(shí)別，遞呈CD4+和CD8+T細(xì)胞，活化T細(xì)胞可識(shí)別免疫顯性抗原（Immuno dominant）和亞免疫顯性抗原（Sub-dominant）表位，表1。發(fā)揮免疫調(diào)節(jié)作用，其多肽疫苗作用和策略是誘導(dǎo)和激發(fā)機(jī)體抗AFP特異免疫反應(yīng)[29-30]。

表1 AFP免疫和亞免疫顯性抗原表位的分子結(jié)構(gòu)

免疫顯性及亞顯性表位均能被MHCⅠ類分子所識(shí)別，都有特異性誘導(dǎo)T淋巴細(xì)胞活化的能力，且人類T細(xì)胞庫在MHCⅠ類免疫反應(yīng)中可識(shí)別AFP抗原表位。HLA-A24陽性HCC患者外周血單核細(xì)胞，發(fā)現(xiàn)5種HLA-A*2402限制性T細(xì)胞表位來源于AFP衍生肽，可誘導(dǎo)CTL產(chǎn)生γ干擾素（INF-γ），且可殺死AFP陽性肝癌細(xì)胞。以AFP衍生肽作為抗原表位殺傷肝癌細(xì)胞研究已進(jìn)入臨床試驗(yàn)。即使AFP衍生肽能有效地作為肝癌特異性靶點(diǎn)但問題很多，如DC功能、特異CTL、CD8+T細(xì)胞反應(yīng)和AFP陰性HCC靶向治療等有待研究[31-32]。

4 癌胚性磷脂酰肌醇蛋白多糖-3 與肝癌免疫治療

磷脂酰肌醇蛋白多糖-3（Glypican-3，GPC-3）為胞膜表面糖蛋白，正常肝無表達(dá)，HCC組織過表達(dá)并位于胞質(zhì)和胞膜，高于癌旁組織與HBV感染相關(guān)，遠(yuǎn)癌未見表達(dá)[33-34]。構(gòu)建位于HepG2胞膜的錨定蛋白(GPC3+α+EGFP)與淋巴細(xì)胞共培養(yǎng)，經(jīng)Fas-Fas L通路抑制癌細(xì)胞增值，促進(jìn)凋亡[35-36]。GPC-3過表達(dá)與基質(zhì)金屬蛋白酶(MMP)-2、纖維母細(xì)胞生長因子(FGF)-2、FGF受體-1、FGF受體-2、硫酸酯酶-1（SULF-1)和SULF-2顯著相關(guān)，抑制GPC-3可下調(diào)MMP；GPC-3表達(dá)，胞核/胞質(zhì)β-catenin顯著增高；與MMP及增殖信號(hào)結(jié)合，促進(jìn)HCC進(jìn)展。SULF-2增強(qiáng)細(xì)胞表面GPC-3和Wnt3a表達(dá)，穩(wěn)定β-catenin激活T細(xì)胞轉(zhuǎn)錄因子，Wnt/β-catenin靶基因cyclinD1，SULF-2致癌作用，經(jīng)上調(diào)GPC-3刺激Wnt/β-catenin通路，誘導(dǎo)癌細(xì)胞增殖；與高親和Hh競爭性抑制相應(yīng)受體結(jié)合，調(diào)控增殖和分化等[37-38]。

癌胚性GPC-3除診斷與鑒別HCC外，極可能為HCC治療的新靶點(diǎn)，表2?？笹PC-3單抗(GC33)誘導(dǎo)GPC-3陽性肝癌細(xì)胞產(chǎn)生明顯抗體依賴的細(xì)胞介導(dǎo)的細(xì)胞毒(ADCC)作用，對(duì)肝癌Huh-7和HepG2細(xì)胞移植瘤具有明顯生長抑制作用。肝癌患者GPC-3高表達(dá)，其免疫原性在鼠模型和細(xì)胞培養(yǎng)物得以證明。因GPC-3特異性T細(xì)胞在肝癌患者血中表達(dá)較低，但在優(yōu)化共刺激下體外擴(kuò)增會(huì)增高。GPC-3對(duì)肝再生和肝細(xì)胞增殖起負(fù)調(diào)節(jié)作用[39-40]。以RNAi沉默GPC-3，可顯著抑制肝癌MHCC-97H細(xì)胞的增殖和侵襲，瘤體衰減。

表2 GPC-3作為肝癌免疫治療的分子靶點(diǎn)

GPC-3位于Wnt/β-catenin信號(hào)通路的上游，下調(diào)SULF2或干預(yù)其基因轉(zhuǎn)錄后，通過該通路可抑制HCC生長，其可作為治療新方案，具有開發(fā)應(yīng)用前景[43-44]?？笹PC-3單抗(GC33)治療HCC，抑制HepG2和Huh7增殖，使HepG2細(xì)胞合成AFP下降。將GC33與化療藥物索拉非尼聯(lián)合能有效抑制腫瘤。以GPC-3轉(zhuǎn)染修飾樹突狀細(xì)胞(DCs)激活人T細(xì)胞并引發(fā)細(xì)胞介導(dǎo)免疫反應(yīng)，對(duì)抗HepG2細(xì)胞增殖和轉(zhuǎn)移，GPC-3轉(zhuǎn)染DCs細(xì)胞對(duì)HepG2細(xì)胞產(chǎn)生高特異細(xì)胞毒性，用pEF-hGPC3質(zhì)粒轉(zhuǎn)染DCs刺激效應(yīng)細(xì)胞可有效溶解HepG2細(xì)胞，有望成為HCC新疫苗，建立缺GPI錨定區(qū)的突變GPC-3(sGPC3)載體，轉(zhuǎn)染HCC細(xì)胞，sGPC3通過Wnt信號(hào)顯著抑制癌細(xì)胞增殖，抑制HCC生長，是有價(jià)值的治療工具[41-45]。

5 展望

綜上所述，肝癌與病毒慢性感染，癌基因及癌相關(guān)基因激活及抑癌基因失活等相關(guān)，癌變機(jī)制復(fù)雜，有效治療仍是世界性難題[1,46]。手術(shù)治療仍是目前首選方法，療效的關(guān)鍵取決于早診診斷；晚期肝癌缺乏有效治療手段，免疫治療與手術(shù)、放化療、介入等綜合治療，可望改善療效，延長存活時(shí)間[47-48]。隨著基因組學(xué)、蛋白質(zhì)組學(xué)飛速發(fā)展，分子病理學(xué)、分子藥理學(xué)及基因工程技術(shù)的不斷進(jìn)步，分子剪接、基因沉默、干預(yù)轉(zhuǎn)錄和單抗技術(shù)可直接封閉癌細(xì)胞生長相關(guān)通路的信號(hào)分子或作為放射性核素、藥物和毒素等載體，進(jìn)行基因置換、基因增補(bǔ)、基因矯正和免疫治療等分子靶向研究，將會(huì)有更多、特異性更強(qiáng)、副作用更小的方法研制成功并在肝癌靶向治療中發(fā)揮作用。

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（2016-7-30收稿）

R392.1

A

10.3969/j.issn.1000-2669.2016.05.001

*國家國際科技合作專項(xiàng)(2013DFA32150)，江蘇省“六大人才高峰”項(xiàng)目(2014-YY-028)

姚登福，男，教授，博士生導(dǎo)師。E-mail:yaodf@ahnmc.com