細(xì)胞治療的典范：嵌合抗原受體T細(xì)胞療法

錢麗玲，陳蔣慶，吳曉燕，荊瑞瑞，孫潔,2

生物工程與大健康

孫潔 博士，浙江大學(xué)醫(yī)學(xué)院研究員，博士生導(dǎo)師。目前研究方向?yàn)镃AR-T細(xì)胞殺傷、增殖及耗竭的分子機(jī)制；開(kāi)發(fā)新型生物傳感器與智能分子機(jī)器；優(yōu)化及探索新的細(xì)胞癌癥免疫療法。成果以第一作者或通訊作者發(fā)表在、、等國(guó)際專業(yè)期刊。

細(xì)胞治療的典范：嵌合抗原受體T細(xì)胞療法

錢麗玲1，陳蔣慶1，吳曉燕1，荊瑞瑞1，孫潔1,2

1 浙江大學(xué)醫(yī)學(xué)院附屬第一醫(yī)院骨髓移植中心 浙江大學(xué)醫(yī)學(xué)院細(xì)胞生物學(xué)系，浙江 杭州 310058 2 浙江大學(xué)血液學(xué)研究所 浙江省干細(xì)胞與免疫治療工程實(shí)驗(yàn)室，浙江 杭州 310058

嵌合抗原受體T (CAR-T) 細(xì)胞療法是一種利用合成受體特異性靶向抗原的過(guò)繼性細(xì)胞療法(ACT)，目前在血液腫瘤的治療中有極大的臨床應(yīng)用價(jià)值。雖然美國(guó)食品藥品監(jiān)督管理局 (FDA) 已經(jīng)批準(zhǔn)兩款CAR-T藥物上市，但CAR-T療法在治療過(guò)程中仍然存在一些副作用，如細(xì)胞因子釋放綜合征 (CRS)、神經(jīng)毒性、B細(xì)胞功能缺失等。同時(shí)，CAR-T療法在實(shí)體瘤治療中的效果甚微，主要原因是缺乏特異性靶點(diǎn)以及腫瘤微環(huán)境對(duì)CAR-T細(xì)胞功能的抑制等。文中將從CAR的結(jié)構(gòu)設(shè)計(jì)、臨床應(yīng)用、合成生物學(xué)對(duì)新型CAR的優(yōu)化來(lái)闡述應(yīng)用CAR-T細(xì)胞療法治療腫瘤所面臨的挑戰(zhàn)及廣闊前景。

細(xì)胞治療，免疫治療，嵌合抗原受體T細(xì)胞，合成生物學(xué)

20世紀(jì)80年代以前，藥物的研發(fā)主要依賴于天然化合物和小分子藥物的合成，開(kāi)發(fā)了許多有效的小分子藥物用于疾病治療。隨著單克隆抗體技術(shù)和蛋白質(zhì)工程技術(shù)的發(fā)展，生物大分子藥物如天然蛋白質(zhì)或具有特定功能的重組蛋白藥物應(yīng)運(yùn)而生，但仍然存在新的困難和挑戰(zhàn)，由于疾病的復(fù)雜性和多樣性，且存在個(gè)體差異和組織特異性，因此很難找到組織特異和個(gè)體特異的靶點(diǎn)。近年來(lái)，興起了第3種治療方法——將細(xì)胞作為藥物的細(xì)胞治療，該方法在疾病尤其是癌癥的治療方面具有劃時(shí)代的意義[1]。

1 細(xì)胞治療的分類及發(fā)展

細(xì)胞治療是指將自體、同源異體或異種細(xì)胞經(jīng)體外工程化改造和擴(kuò)大培養(yǎng)后，輸注患者體內(nèi)治療疾病的療法。該治療方法與傳統(tǒng)的小分子藥物和蛋白藥物相比最大的特點(diǎn)是利用活細(xì)胞作為藥物來(lái)治療疾病，具有復(fù)雜性和可調(diào)節(jié)性等特 征[1]，具體來(lái)說(shuō)，將細(xì)胞作為藥物具有以下優(yōu)點(diǎn)：1) 選擇性高，細(xì)胞藥物能感知復(fù)雜的人體內(nèi)環(huán)境，只在特定的環(huán)境中激活，以發(fā)揮相應(yīng)功能，這意味著可以更大程度上限制藥物的副作用；2)局部濃度高，人體代謝、藥物效應(yīng)動(dòng)力學(xué)(Pharmacodynamics，PD) 和藥物代謝動(dòng)力學(xué)(Pharmacokinetics，PK) 決定了分子藥物靶向性較低，它不只在病變組織或細(xì)胞內(nèi)分布，而且分布于整個(gè)機(jī)體組織，這通常會(huì)造成嚴(yán)重的脫靶效應(yīng)，而細(xì)胞藥物的優(yōu)勢(shì)在于可主動(dòng)遷移到靶組織或靶細(xì)胞內(nèi)發(fā)揮作用；3) 更加個(gè)性化，由于個(gè)體差異，目前很難控制每個(gè)患者小分子藥物的使用劑量，但在細(xì)胞治療中，可應(yīng)用合成生物學(xué)設(shè)計(jì)基因開(kāi)關(guān)控制藥物的合成或釋放，也可以根據(jù)臨床需要設(shè)計(jì)不同細(xì)胞藥物以治療更多疾病[1]。但細(xì)胞藥物的開(kāi)發(fā)和合成生物學(xué)的應(yīng)用還需更多基礎(chǔ)研究的支撐，以解決細(xì)胞藥物在臨床上治療疾病種類少、副作用嚴(yán)重、費(fèi)用昂貴等問(wèn)題。

1.1 細(xì)胞治療的分類

細(xì)胞治療根據(jù)細(xì)胞類型可分為干細(xì)胞治療、免疫細(xì)胞治療和其他細(xì)胞治療。輸血是最早的細(xì)胞治療，現(xiàn)已經(jīng)發(fā)展到輸注特定血液成分進(jìn)行治療，這使得在提高血液利用率的同時(shí)減少副作用，目前研究較多的是干細(xì)胞治療和免疫細(xì)胞治療。干細(xì)胞治療是指把健康的干細(xì)胞輸注到患者體內(nèi)，從而修復(fù)或替換受損細(xì)胞或組織以治療疾病的方法。目前國(guó)內(nèi)外臨床試驗(yàn)應(yīng)用較廣泛的是利用間充質(zhì)干細(xì)胞治療神經(jīng)性疾病、糖尿病、慢性心臟疾病、腎臟病、肝臟疾病、艾滋病和癌癥等疾病[2-6]。免疫細(xì)胞治療的過(guò)程主要包括從患者或供體血液中提取免疫細(xì)胞，在體外進(jìn)行工程化改造和擴(kuò)增培養(yǎng)后，重新輸注入病人體內(nèi)或者直接注射到病灶處。目前用于臨床試驗(yàn)的免疫細(xì)胞療法主要有CAR-T、TCR-T和NK細(xì)胞療法等[7-9]。文中主要介紹CAR-T療法的發(fā)展、應(yīng)用和前景。

1.2 CAR-T細(xì)胞治療的發(fā)展

早在20世紀(jì)80年代，得益于過(guò)繼性T細(xì)胞治療(Adoptive T-cell therapy，ACT) 的發(fā)展，臨床上已開(kāi)始利用輸注自體或異體供體淋巴細(xì)胞治療一些腫瘤，如轉(zhuǎn)移性黑色素瘤、復(fù)發(fā)性白血 病[10-11]等。但這種基于自然T細(xì)胞的ACT治療存在以下不足：首先，供體T細(xì)胞可能攻擊受體導(dǎo)致移植物抗宿主反應(yīng)(Graft-versus-host disease，GVHD)；同時(shí)，受體可能排斥輸注的T細(xì)胞，限制它們的持久性和療效；而且從體內(nèi)分離的自然T細(xì)胞數(shù)目少，靶向特異性低，抗腫瘤效率低。這些早期ACT的臨床結(jié)果表明，該療法需要增強(qiáng)靶向腫瘤的特異性并提高體外擴(kuò)增T細(xì)胞的數(shù)量，同時(shí)減少免疫排斥反應(yīng)[12-14]。T細(xì)胞工程的出現(xiàn)改變了傳統(tǒng)ACT的局限性，產(chǎn)生了特異性靶向腫瘤的TCR-T、CAR-T細(xì)胞[15]，其中通過(guò)在T細(xì)胞表達(dá)嵌合抗原受體(Chimeric antigen receptor，CAR) 而擺脫HLA限制性的治療方法即CAR-T療法，該方法已經(jīng)在血液瘤的治療中取得顯著的效果。本文首先簡(jiǎn)述了CAR的結(jié)構(gòu)設(shè)計(jì)以及CAR-T療法在血液瘤和實(shí)體瘤中的應(yīng)用現(xiàn)狀，然后總結(jié)了合成生物學(xué)為CAR-T細(xì)胞設(shè)計(jì)提供的新思路，最后展望CAR-T療法在腫瘤尤其是在實(shí)體瘤治療過(guò)程中的巨大應(yīng)用潛能。

2 CAR的設(shè)計(jì)及臨床應(yīng)用

2.1 CAR的設(shè)計(jì)

2.2 CAR-T療法的臨床應(yīng)用

臨床試驗(yàn)已證實(shí)CAR-T細(xì)胞療法在治療B細(xì)胞系血液腫瘤的療效顯著，其中包括非霍奇金淋巴瘤(Non-Hodgkinlymphoma, NHL)、慢性淋巴細(xì)胞白血病(Chronic lymphocytic leukemia，CLL)、急性淋巴細(xì)胞白血病(Acute lymphoblastic leukemia，ALL)，但CAR-T細(xì)胞在實(shí)體瘤治療中仍存在一定挑戰(zhàn)。

圖1 第一、二和三代CAR的結(jié)構(gòu)設(shè)計(jì)

2.2.1 CAR-T細(xì)胞在血液腫瘤中的應(yīng)用

目前，CAR-T療法在治療ALL[22-24]中的療效最好，應(yīng)用也最廣泛，其中靶向CD19的CAR在治療成人和兒童ALL中都取得顯著的療效[25-26]，文獻(xiàn)報(bào)道，CD19 CAR-T治療復(fù)發(fā)/難治性ALL的完全緩解率可高達(dá)90%[27]。目前FDA批準(zhǔn)的CD19 CAR-T細(xì)胞藥物分別以CD28或4-1BB為共刺激結(jié)構(gòu)域[28-29]的這兩種CAR-T細(xì)胞各有優(yōu)點(diǎn)，共刺激結(jié)構(gòu)域?yàn)镃D28的CAR-T細(xì)胞在反應(yīng)初期增殖更快，能介導(dǎo)更強(qiáng)的腫瘤殺傷能力，但同時(shí)副作用也更強(qiáng)，且在免疫反應(yīng)后期的持久性較差；而基于4-1BB的CAR-T細(xì)胞雖然在反應(yīng)初期增殖能力相比CD28 CAR較差，但持續(xù)性更好，在免疫反應(yīng)后期也能維持較高細(xì)胞數(shù)量[30-32]。另外，值得注意的是，CD19 CAR-T細(xì)胞在治療過(guò)程中也存在一些副作用，比如細(xì)胞因子釋放綜合征(Cytokine release syndrome，CRS)，神經(jīng)毒性，B細(xì)胞功能缺失，同時(shí)還存在腫瘤復(fù)發(fā)等問(wèn)題[21,33-35]。已有研究表明導(dǎo)致CRS的主要原因是單核細(xì)胞釋放的IL-1和IL-6[36]，目前臨床上主要采用耗竭單核細(xì)胞或使用IL-6拮抗劑(托珠單抗，Tocilizumab) 來(lái)降低CRS，也有文獻(xiàn)研究表明改良CAR的跨膜區(qū)和鉸鏈區(qū)結(jié)構(gòu)域可減少細(xì)胞因子釋放[36-40]；CAR-T療法造成的神經(jīng)毒性可能是由于巨噬細(xì)胞攻擊腦膜導(dǎo)致腦組織損傷，臨床上常用IL-1抑制劑阿那白滯素(Anakinr，別名Kineret) 來(lái)控制神經(jīng)毒性[36,41]；由于正常B細(xì)胞表面也表達(dá)CD19，故CD19 CAR-T細(xì)胞在殺傷腫瘤細(xì)胞的同時(shí)也會(huì)攻擊自身正常的B細(xì)胞，即“on-target，off-tumor”效應(yīng)，造成B細(xì)胞功能缺失，目前可通過(guò)定期向患者體內(nèi)輸注免疫球蛋白以彌補(bǔ)B細(xì)胞的功能缺失[42]。盡管針對(duì)CD19抗原的CAR-T療法療效突出，但仍有部分患者易復(fù)發(fā)，因此需要尋找新的腫瘤靶點(diǎn)進(jìn)行輔助治療。CD22在大多數(shù)急性淋巴細(xì)胞白血病患者B細(xì)胞較高表達(dá)，因此目前臨床上會(huì)對(duì)一些CD19 CAR-T治療后CD19陰性的患者，開(kāi)展針對(duì)CD22抗原的CAR-T治療的臨床試驗(yàn)，最新的結(jié)果表明在接受CD22 CAR-T細(xì)胞輔助治療后，73%的患者獲得了完全緩解(Complete remission，CR)[43]。此外，串聯(lián)CD19/CD22 CAR-T目前也已用于臨床試驗(yàn)[44]，它可同時(shí)識(shí)別CD19和CD22抗原從而減少抗原逃逸引起的復(fù)發(fā)。

同時(shí)，靶向BCMA抗原的CAR-T的初步臨床研究結(jié)果令人鼓舞。BCMA全稱為B細(xì)胞成熟抗原(B cell maturation antigen)，呈相對(duì)特異地高表達(dá)于骨髓瘤細(xì)胞表面，因此可作為多發(fā)性骨髓瘤免疫治療的理想靶點(diǎn)。臨床上靶向BCMA 的LCAR-B38M療法對(duì)復(fù)發(fā)/難治性多發(fā)性骨髓瘤晚期患者的療效較理想，總緩解率為88%，其中68%患者完全緩解(CR)，但安全性上仍存在以細(xì)胞因子釋放綜合征(CRS) 為主的副作用[45]。

2.2.2 CAR-T療法在治療實(shí)體瘤中的應(yīng)用

CAR-T療法不僅在治療血液腫瘤方面取得了突破，在治療實(shí)體瘤方面也有一定進(jìn)展。目前，在實(shí)體瘤應(yīng)用方面，CAR-T療法靶向的腫瘤抗原大多是高表達(dá)的分化抗原，例如癌胚抗原(Carcinoembryonic antigen，CEA)、前列腺特異性膜抗原(Prostate specific membrane antigen, PSMA)、雙唾液酸神經(jīng)節(jié)苷脂(Disialoganglioside, GD2)、糖鏈抗原-125 (Carbohydrate antigen-125, CA-125)、人類表皮生長(zhǎng)因子受體2 (Human epidermal growth factor receptor-2，Her-2) 和間皮細(xì)胞抗原(Mesothelin) 等。雖然過(guò)表達(dá)的抗原種類多樣，但由于其表達(dá)特異性低，CAR-T細(xì)胞對(duì)低水平表達(dá)抗原的正常細(xì)胞也高度敏感，因此治療中存在on-target/off-tumor的副作用[21]。例如，使用高劑量的Her-2 CAR-T細(xì)胞已導(dǎo)致致命的副作用，其中部分原因是由于該抗原也在健康正常肺上皮和心血管細(xì)胞低表達(dá)[46]。因此，一個(gè)首選的實(shí)體腫瘤抗原靶點(diǎn)就要求其表達(dá)僅限于腫瘤細(xì)胞，或只發(fā)生在非常低水平的非重要正常組織。目前表皮生長(zhǎng)因子受體Ⅲ型突變體(Epidermal growth factor receptor variant type Ⅲ，EGFRvⅢ)、糖鏈抗原15-3 (Carbohydrate antigen 15-3，CA 15-3)和硫酸軟骨素蛋白聚糖4 (Chondroitin sulfate proteoglycan-4，CSPG-4) 分別被認(rèn)為是治療惡性膠質(zhì)瘤、胰腺癌、黑色素瘤較理想的CAR靶點(diǎn)[21]。此外，實(shí)體瘤的腫瘤微環(huán)境(Tumor microenvironment，TME) 也會(huì)對(duì)CAR-T細(xì)胞的功能產(chǎn)生抑制作用。首先，腫瘤細(xì)胞的無(wú)氧糖酵解途徑使其所處的TME呈現(xiàn)出缺氧、酸性、低營(yíng)養(yǎng)成分的特點(diǎn)而不利于T細(xì)胞存活[47-49]。其次腫瘤細(xì)胞表面表達(dá)PD-L1、MHCⅡ、Gal9等配體與T細(xì)胞上的PD1、LAG-3、TIM-3等受體結(jié)合，激活T細(xì)胞上的抑制性信號(hào)通路抑制T細(xì)胞功能[50]。腫瘤微環(huán)境中的腫瘤細(xì)胞和腫瘤相關(guān)細(xì)胞如腫瘤相關(guān)成纖維細(xì)胞(Cancer-associated fibroblast，CAF)、調(diào)節(jié)性T細(xì)胞(Regulatory T cell，Treg) 等則會(huì)分泌抑制性的細(xì)胞因子，如血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor，VEGF)、轉(zhuǎn)化生長(zhǎng)因子β (TGF-β)，或者通過(guò)產(chǎn)生活性氧(ROS)、前列腺素E2 (PGE2) 和乳酸等抑制T細(xì)胞的免疫應(yīng)答[51-53]。因此，CAR-T療法在實(shí)體瘤中的應(yīng)用還需要克服TME的抑制信號(hào)，增強(qiáng)CAR-T細(xì)胞的腫瘤識(shí)別能力、浸潤(rùn)能力和持久性并避免脫靶效應(yīng)[21]。目前已有一些應(yīng)對(duì)策略，可能會(huì)對(duì)CAR-T細(xì)胞在復(fù)雜的腫瘤微環(huán)境中發(fā)揮作用提供幫助，比如局部給藥，或使用分泌IL-12的“武裝CAR”、NK細(xì)胞受體CAR等[54-60]。

2.3 合成生物學(xué)方法優(yōu)化CAR的設(shè)計(jì)

為了克服免疫抑制(包括阻斷檢查點(diǎn)、抑制調(diào)節(jié)性T細(xì)胞和其他髓系細(xì)胞)，降低on-target/ off-tumor毒性，提高CAR-T細(xì)胞的抗腫瘤療效，目前正在探索促進(jìn)CAR-T細(xì)胞的浸潤(rùn)、增強(qiáng)CAR-T細(xì)胞的功能持久性的多種方法，其中CAR的設(shè)計(jì)與合成生物學(xué)技術(shù)的結(jié)合為設(shè)計(jì)安全 性更高、功能更強(qiáng)的CAR-T細(xì)胞提供了更多可能性。

2.3.1 控制CAR-T細(xì)胞的毒性和活性

目前臨床上使用的CAR-T療法所帶來(lái)的器官毒性、脫靶毒性等問(wèn)題亟待解決，已有報(bào)道表明嚴(yán)重CRS和腦水腫可引起治療相關(guān)死亡。合成生物學(xué)的發(fā)展為更好地調(diào)控人體內(nèi)的CAR-T細(xì)胞提供了新的思路。其中一種策略是通過(guò)小分子藥物來(lái)控制CAR-T細(xì)胞自凋亡或抗原抗體結(jié)合來(lái)降低CAR-T細(xì)胞治療過(guò)程中的毒性反應(yīng)。其具體設(shè)計(jì)有以下幾種方法：其一，在CAR-T細(xì)胞內(nèi)加入相應(yīng)的信號(hào)蛋白作為分子開(kāi)關(guān)，實(shí)現(xiàn)對(duì)T細(xì)胞可逆調(diào)控[61]或“自殺”基因如單純皰疹病毒胸苷激酶(HSV-TK) 基因，一旦CAR-T細(xì)胞在患者體內(nèi)發(fā)生不良反應(yīng)，通過(guò)施加藥物介導(dǎo)T細(xì)胞的自殺基因激活誘導(dǎo)CAR-T細(xì)胞凋亡[62-63]。其二，利用小分子藥物，如AP1903二聚化誘導(dǎo)型Caspase 9 (iCasp9) 激活T細(xì)胞的自殺開(kāi)關(guān)，誘導(dǎo)其發(fā)生凋亡終止CAR-T細(xì)胞發(fā)揮作用。其三，某些小分子藥物能夠通過(guò)介導(dǎo)腫瘤抗原和CAR之間的結(jié)合來(lái)調(diào)節(jié)CAR-T細(xì)胞識(shí)別腫瘤抗原的能力。最后，將CAR的scFv與信號(hào)轉(zhuǎn)導(dǎo)結(jié)構(gòu)域分隔開(kāi)，兩者之間依靠小分子二聚化的可逆結(jié)合得以更靈活地調(diào)控CAR-T細(xì)胞的功能[64-65]。

2.3.2 增強(qiáng)CAR-T細(xì)胞腫瘤識(shí)別的特異性

利用合成生物學(xué)制備多靶點(diǎn)CAR或Boolean logic-gated CAR，用于識(shí)別一種或兩種腫瘤抗原的方式來(lái)增強(qiáng)工程化的CAR-T細(xì)胞識(shí)別腫瘤抗原的能力，比如合成Notch (synNotch) 受體、嵌合共刺激受體(CCR)、抑制性嵌合抗原受體(iCAR) 等。其中SynNotch受體是一種精確識(shí)別腫瘤抗原的設(shè)計(jì)，該策略利用Notch受體獨(dú)特的信號(hào)傳導(dǎo)機(jī)制，即抗原A特異性的synNotch在結(jié)合了腫瘤抗原A之后，轉(zhuǎn)錄因子被切割激活，進(jìn)而轉(zhuǎn)運(yùn)到細(xì)胞核引起了抗原B特異性的CAR的表達(dá)，最終與腫瘤表面的抗原B結(jié)合，T細(xì)胞被激活[66-68](圖2A)。此外，synNotch受體系統(tǒng)還具有整合多個(gè)細(xì)胞外信號(hào)的潛能[69]。CCR是將用于T細(xì)胞活化的一代CAR和用于共刺激的CCR組合到一起，只有當(dāng)兩種抗原同時(shí)存在于腫瘤細(xì)胞表面時(shí)，CAR-T細(xì)胞才能激活并介導(dǎo)腫瘤殺傷(圖2B)。這樣的設(shè)計(jì)增強(qiáng)了CAR-T細(xì)胞識(shí)別腫瘤細(xì)胞抗原的特異性，減輕副作用[70]。SynNotch受體和CCR的識(shí)別邏輯都是“AND gate”。另一種策略則是將識(shí)別不同腫瘤抗原的scFv串聯(lián)在一起，只要腫瘤細(xì)胞表達(dá)其中一個(gè)抗原[71]，就能活化T細(xì)胞介導(dǎo)腫瘤殺傷(圖2C)，即“OR gate”?；贑TLA-4和PD-1的iCAR通過(guò)識(shí)別正常細(xì)胞表面的抗原來(lái)發(fā)揮作用[72]，是一種“AND OR NOT gate”，CAR-T細(xì)胞一旦識(shí)別在正常細(xì)胞表面的抗原，就會(huì)傳遞抑制信號(hào)抑制T細(xì)胞的活化，因此可有效預(yù)防CAR-T細(xì)胞對(duì)正常細(xì)胞的不良反應(yīng)(圖2D)。SUPRA (Split，universal and programmable) CARs則整合了以上3種腫瘤識(shí)別邏輯[71,73-75]，這是一種通用型的CAR，該策略是將CAR分成兩個(gè)部分Zip CAR (不含scFv) 和Zip Fv (不含信號(hào)轉(zhuǎn)導(dǎo)結(jié)構(gòu)域)，二者能通過(guò)匹配的亮氨酸拉鏈結(jié)合以形成完整的CAR，且可通過(guò)使用不同組合的抗原靶向部分控制對(duì)腫瘤抗原的識(shí)別能力[74]，SUPRA CAR的開(kāi)發(fā)表明多個(gè)高級(jí)邏輯識(shí)別抗原功能可在單個(gè)集成系統(tǒng)中實(shí)現(xiàn)(圖2E)。

圖2 運(yùn)用合成生物學(xué)設(shè)計(jì)的新型CARs

目前，對(duì)這些CAR的研究還處在初級(jí)階段，在向臨床研究轉(zhuǎn)化的過(guò)程中，還存在許多困難和挑戰(zhàn)等待研究者去克服。

3 CAR-T治療的前景

腫瘤免疫治療于2013年被雜志評(píng)為十大科技突破之首，其中CAR-T作為具有抗原靶向性且有一定持久性的“活細(xì)胞藥物”，可重編程T 細(xì)胞的效應(yīng)和分化等功能，在接觸抗原后增殖并發(fā)揮抗腫瘤作用，尤其是CAR-T療法在B細(xì)胞血液瘤中取得了突破，給復(fù)發(fā)/難治性白血病及淋巴瘤患者帶來(lái)了希望[26-27]。但腫瘤微環(huán)境的免疫抑制[76]、抗原表達(dá)缺失或下調(diào)以及CAR-T細(xì)胞持久性不足等導(dǎo)致仍有患者復(fù)發(fā)。此外，初步臨床結(jié)果表明CAR-T療法在對(duì)標(biāo)準(zhǔn)治療有耐藥性的多發(fā)性骨髓瘤治療中取得重大進(jìn)展[45]。雖然CAR-T在實(shí)體瘤治療上仍缺乏重大突破，但綜合目前CAR-T治療在臨床以及以下各個(gè)相關(guān)領(lǐng)域的進(jìn)展，不可否認(rèn)的是CAR-T療法具有廣闊前景：1) 隨著基因編輯技術(shù)和合成生物學(xué)的發(fā)展，CAR結(jié)構(gòu)的設(shè)計(jì)更具靈活性和多元化。2) 已有研究表明敲除T細(xì)胞受體的同時(shí)將CAR插入T細(xì)胞受體位點(diǎn)可延遲T細(xì)胞耗竭從而增強(qiáng)抗癌作用，這為未來(lái)CAR-T設(shè)計(jì)提供重要思路[44]。3) 選擇最優(yōu)的T細(xì)胞亞群，如調(diào)整CD4/CD8 T細(xì)胞比率和初始T細(xì)胞/效應(yīng)T細(xì)胞比率用于CAR治療，將進(jìn)一步提高CAR治療的療效和安全性[21]。4) 此外，初步臨床結(jié)果表明CAR-T治療與PD-1/PD-L1抑制劑聯(lián)用，或兩個(gè)不同靶點(diǎn)的CAR-T聯(lián)用在淋巴瘤治療上有較好成效[21,77-78]。5) 為了使CAR-T細(xì)胞治療成為更普遍的治療手段，已經(jīng)出現(xiàn)了“通用型”CAR-T細(xì)胞，并在機(jī)體表現(xiàn)出很強(qiáng)免疫抑制的B-ALL患兒中取得了成功[79]。開(kāi)發(fā)出具有更優(yōu)性能的“通用型”CAR-T細(xì)胞，重點(diǎn)是避免機(jī)體和T細(xì)胞的相互排斥，因此通過(guò)使用基因編輯技術(shù)沉默基因、Ⅰ類基因的同種異體T細(xì)胞，不僅能有效消除可能造成的GVHD[76]，經(jīng)過(guò)改造后，在患者急需治療時(shí)能提供大量可用的現(xiàn)成CAR-T細(xì)胞，這對(duì)臨床治療具有重要意義。6) 對(duì)腫瘤發(fā)病、復(fù)發(fā)機(jī)制以及對(duì)T細(xì)胞和CAR-T細(xì)胞功能機(jī)制的知識(shí)體系的擴(kuò)展和更新也為CAR-T治療提供深厚的理論基礎(chǔ)。隨著CAR-T基礎(chǔ)研究與臨床實(shí)踐的不斷深入，CAR-T細(xì)胞必將在腫瘤生物細(xì)胞免疫治療中發(fā)揮越來(lái)越重要的作用。

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Cell therapy’s poster child: Chimeric antigen receptor T cell therapy

Liling Qian1, Jiangqing Chen1, Xiaoyan Wu1, Ruirui Jing1, and Jie Sun1,2

1 Bone Marrow Transplantation Center of the First Affiliated Hospital, and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang, China 2 Institute of Hematology, Zhejiang University& Laboratory of Stem Cell and Immunotherapy Engineering, Hangzhou 310058, Zhejiang, China

Chimeric antigen receptor T (CAR-T) cell therapy, which adoptively transfers engineered T cells expressing synthetic receptors to target specific antigens, has achieved great clinical success in treating hematological malignancies. Though FDA has approved two CAR-T products, CAR-T therapy can cause some side effects, such as cytokine release syndrome (CRS), neurotoxicity and B cell aplasia. Meanwhile, lacking tumor specific antigen and the suppressive tumor environment limit the efficacy of CAR-T therapy in solid tumor. This review focuses on the structural components, clinical applications and synthetic biology approaches on CAR-T cell design, and summarizes the challenges and perspectives of CAR-T therapy as a revolutionary cancer immunotherapy.

cell therapy, immunotherapy, chimeric antigen receptor T cell, synthetic biology

July 1, 2019;

September 4, 2019

s:Jie Sun. Tel: +86-571-88208509; Fax: +86-571-88208094; E-mail: sunj4@zju.edu.cn

2019-10-10

http://kns.cnki.net/kcms/detail/11.1998.Q.20191010.0940.003.html

錢麗玲, 陳蔣慶, 吳曉燕, 等. 細(xì)胞治療的典范：嵌合抗原受體T細(xì)胞療法. 生物工程學(xué)報(bào), 2019, 35(12): 2339–2349.

Qian LL, Chen JQ, Wu XY, et al. Cell therapy’s poster child: Chimeric antigen receptor T cell therapy. Chin J Biotech, 2019, 35(12): 2339–2349.

(本文責(zé)編 陳宏宇)