【摘要】：嵌合性抗原受體(chimeric antigen receptor,CAR) T淋巴細胞(CAR-T)是通過將外源性人工設(shè)計的CAR基因?qū)隩淋巴細胞內(nèi)進行基因修飾改造,而后得到的表達CAR的特異性T細胞[1]。CAR主要由可特異性識別腫瘤抗原的膜外片段,連接片段,和含刺激信號的膜內(nèi)片段組成。膜外片段主要是由靶向抗原分子的單鏈抗體(Single-chain variable fragment, scFv)組成,膜內(nèi)效應(yīng)分子則是由共刺激分子和CD3ζ等T細胞活化基團組成[2]。主要的共刺激分子有CD28, CD27和CD137 (4-1BB)等。細胞外scFv與抗原結(jié)合后可以啟動胞內(nèi)段信號轉(zhuǎn)導(dǎo)并活化T淋巴細胞,最終活化的T淋巴細胞對腫瘤細胞產(chǎn)生殺傷。CAR-T細胞療法是通過對來源于患者自身的T淋巴細胞進行基因修飾而后將自體性CAR-T細胞回輸給病人治療腫瘤的方法,這是一種最前沿進展的腫瘤免疫治療方法。前期的臨床研究已經(jīng)顯示,特異性靶向CD19分子的CAR-T細胞能有效地治療B細胞性惡性腫瘤(表達有CD19分子),包括急性淋巴細胞白血病(ALL),慢性淋巴細胞白血病(CLL),和B細胞淋巴瘤。目前的數(shù)據(jù)表明其對晚期復(fù)發(fā)難治性ALL的治療有效率可達到80%以上[3,4],對CLL和部分B細胞淋巴瘤的有效率50%[5,6],相關(guān)成果已多次在新英格蘭醫(yī)學(xué)雜志等頂級期刊發(fā)表。CAR-T細胞療法已經(jīng)成為血液系統(tǒng)腫瘤治療的一個突破性進展。前期CAR-T在實體瘤的應(yīng)用較少,且療效并無其在血液腫瘤明顯,病人腫瘤緩解率低[7,8]。去年新英格蘭醫(yī)學(xué)雜志再次報道了該療法在實體瘤的重大突破,廣泛顱內(nèi)轉(zhuǎn)移的膠質(zhì)母細胞瘤病人在CAR-T細胞多次回輸后達到了完全緩解(Complete remission, CR),該結(jié)果極大肯定和明確了 CAR-T療法在實體瘤的應(yīng)用價值[9]。所以,開展CAR-T療法在實體瘤的應(yīng)用是十分必要的。癌胚抗原(Carcino-embryonic antigen, CEA)是經(jīng)典的腫瘤標志物,特別在結(jié)直腸癌病人中有80%以上陽性表達。正常組織細胞中只有消化道細胞有少量的CEA在細胞膜表達,而該CEA在生理條件下朝向胞腔內(nèi)表達從而避免被靶向CEA的CAR-T細胞識別[10]。所以CEA是CAR-T治療結(jié)直腸癌理想的靶點。針對CEA設(shè)計的CAR-T在國外已經(jīng)完成了動物實驗[11,12]以及Ⅰ期臨床試驗[7],現(xiàn)有的CAR-T細胞治療實體瘤的臨床結(jié)果都是在國外獲得,且目前大多處于臨床Ⅰ期階段,病例總數(shù)尚不多;而且在不同的治療機構(gòu)以及病人個體間,治療的輸注細胞數(shù)量都不等,其標準在不同的個體之間尚需進一步探索;更重要的是,我國的腫瘤在發(fā)病率,腫瘤類型,和死亡率等方面都和西方國家不盡相同,目前我們國內(nèi)還沒有開展CAR-T療法在實體腫瘤應(yīng)用。因此,在我們國家開展CAR-T對腫瘤的臨床治療研究是非常必要的。我們開展了 CAR-T靶向CEA的臨床研究,而在臨床實踐中也因為發(fā)現(xiàn)CAR-T細胞的不足而通過基礎(chǔ)研究來探究功能更強的CAR-T細胞。所以,我們的工作主要由臨床試驗和基礎(chǔ)研究兩部分組成:第一部分:CAR-T治療CEA陽性結(jié)直腸癌的臨床研究證實了其治療CEA陽性結(jié)直腸癌的安全性和有效性:為了開展靶向CEA的Ⅰ期CAR-T臨床試驗,我們前期完成了靶向CEA的CAR-T細胞的制備,也在體外和動物體內(nèi)驗證了其有效性和安全性。我們在國際臨床試驗機構(gòu)(clinicaltrial.gov)完成了注冊(編號:NCT 02349724),同時也通過了第三軍醫(yī)大學(xué)第一附屬醫(yī)院倫理委員會的批準。CEA在結(jié)直腸癌、胰腺癌、肺癌等腫瘤中都有廣泛的表達,尤其在結(jié)直腸癌病人中CEA陽性率超過80%。在前期的試驗過程中,我們將重心聚焦在結(jié)直腸癌病人的收治上。我們?nèi)虢M了符合納排標準的10例難治復(fù)發(fā)性CEA陽性的結(jié)直腸癌病人,這10例病人全部伴有腸外器官轉(zhuǎn)移(部分病人原發(fā)灶已切除),其中絕大部分為肝轉(zhuǎn)移(7例)。通過采集病人外周血并在GMP實驗室獲得T淋巴細胞,通過包含CAR序列的慢病毒(lentivirus)轉(zhuǎn)染T細胞獲得CAR-T細胞。在細胞回輸前,我們使用了淋巴清除(lymphodepletion)的化療方案(氟達拉濱+環(huán)磷酷胺,FC)對患者外周血的淋巴細胞進行清除,為CAR-T細胞的注入提供良好環(huán)境,提高CAR-T治療的療效。大劑量CAR-T細胞的回輸治療實體瘤可能存在致死風(fēng)險[13],為了確保治療的安全性,我們采用了爬坡階梯的輸注模式,在確認低劑量細胞回輸?shù)陌踩院?再對后續(xù)病人使用高劑量細胞回輸。通過試驗,我們驗證了 CAR-T治療CEA陽性結(jié)直腸癌的安全性。在10例病人中,治療劑量從107至1010逐步增加,最高細胞回輸劑量達到了5 1x10,所有病人均無嚴重不良反應(yīng)發(fā)生(除FC化療對三系的影響),大部分病人僅報告少量1-2級不良事件。7例在前期化療療效評價為疾病進展(Progressive disease, PD)的病人在接受CAR-T治療后腫瘤病灶大小保持穩(wěn)定,評價為病情穩(wěn)定(Stable disease, SD),部分病人維持SD的時間超過了 30周。雖然沒有達到部分緩解(Partial remission, PR)的診斷標準,但有兩例接受高劑量細胞回輸?shù)牟∪嗽谟跋駥W(xué)可見腫瘤有縮小。另外,大部分病人的血清CEA在治療后明顯下降,提示了 CAR-T細胞對CEA陽性腫瘤細胞的抑制及殺傷作用。CAR-T細胞在外周血和體內(nèi)的存續(xù)以及其在體內(nèi)的增殖一直是CAR-T細胞治療實體腫瘤的一個短板[8]。在我們的臨床試驗中,我們發(fā)現(xiàn)接受高劑量細胞回輸?shù)牟∪薈AR-T細胞可以在體內(nèi)存活一定時間,甚至發(fā)現(xiàn)了 CAR-T細胞在外周血的少量擴增�？偠灾�,我們課題組在國內(nèi)首個報道了 CAR-T在實體腫瘤應(yīng)用。通過入組10例符合倫理納排標準的晚期結(jié)直腸癌病人,結(jié)合爬坡劑量的回輸模式,我們確認了靶向CEA的CAR-T細胞在治療復(fù)發(fā)難治性結(jié)直腸癌的安全性�？紤]到入組的病人均為臨床一線二線化療無效的難治性腫瘤,通過CAR-T治療后,大部分病人(7例/10例)的腫瘤得到了控制,一些病人的腫瘤病灶甚至在影像學(xué)上可以看到縮小。另外,CAR-T細胞的存活與擴增也在我們的試驗得到了驗證。本臨床試驗證明了 CAR-T治療實體瘤的安全性以及其功能,為后續(xù)CAR-T的細胞優(yōu)化及臨床應(yīng)用提供了良好依據(jù)。第二部分:CAR-T治療CEA陽性結(jié)直腸癌的基礎(chǔ)研究通過篩選和對比,我們找到了 CAR-T最適、功能最佳的單鏈抗體scFv在靶向CEA陽性腫瘤的CAR-T臨床試驗中我們也發(fā)現(xiàn)了目前CAR-T細胞所需要優(yōu)化和改良的地方。CAR-T可以控制和抑制實體腫瘤的生長,但未能完成對腫瘤的清除,這提示我們需要功能更強的CAR-T細胞。目前CAR-T細胞聯(lián)用PD-1,趨化因子受體(Cxcchemokine receptor, CXCR)均已被證實可以提高CAR-T細胞療效[14,15]。CAR自身結(jié)構(gòu)中的scFv和共刺激信號也會對CAR-T細胞的功能造成不同的影響,不同scFv與共刺激信號會產(chǎn)生不同功能的CAR[16,17]。前期報道提示低親和力的scFv相比于高親和力的scFv (兩個scFv為源于同一個單抗的不同突變體)甚至表現(xiàn)出對CEA陽性腫瘤更好的殺傷以及對CEA陰性或弱陽性細胞更好的安全性[18]。而在不同雜交瘤來源的scFv (靶向不同表位)之間對比發(fā)現(xiàn),高親和力利于提高CAR-T的功能[19,20]。所以,scFv的親和力與CAR-T細胞功能是否呈正相關(guān)尚無定論。另外,在培養(yǎng)病人的CAR-T細胞過程中,我們也發(fā)現(xiàn)CAR-T細胞表面的CAR表達陽性率CAR+%在培養(yǎng)過程中呈逐漸下降的趨勢,部分病人的CAR+%在細胞回輸前降至20%以下,個別病人甚至因為低于10%的CAR+%而取消細胞回輸。我們檢測了靶向HER2和CD19的CAR-T細胞(僅僅替換了 CEA CAR結(jié)構(gòu)的scFv)的CAR變化,上述兩種CAR-T細胞的CAR+%在培養(yǎng)過程中保持穩(wěn)定。因此,我們推測CEA CAR-T的CAR+%下降的原因可能與scFv有關(guān)。因此,為探究scFv與CAR+%下降的問題并篩選功能最佳的scFv,我們篩選了幾種已知的CEA單抗。我們用于CEA CAR-T的scFv序列是從靶向CEA的單克隆抗體(Monoclonal antibody, mAb) BW431/26中獲得[21],同時我們也找到了其他在目前廣泛應(yīng)用的CEA單克隆抗體M5A,hMN-14和C2-45。我們找到上述四種單抗的序列并構(gòu)建出相應(yīng)的scFv,通過真核細胞表達和蛋白純化得到4種scFv蛋白,我們檢測了 4種scFv的親和力,發(fā)現(xiàn)M5A和hMN-14的親和力最高,而BW431/26的親和力最低。同時,我們把各scFv的序列裝載到相同的3代CAR骨架并構(gòu)建出對應(yīng)的病毒且完成了對T細胞的感染,獲得了相應(yīng)的CAR-T。通過比較4個CAR的CAR+%,我們發(fā)現(xiàn)M5A和hMN-14的CAR-T細胞的CAR+%在培養(yǎng)過程中可以穩(wěn)定表達,而BW431/26的CAR+%仍呈下降趨勢,C2-45的CAR+%則持續(xù)檢測不到。為了驗證CAR-T表面的CAR功能,我們用重組CEA蛋白刺激各CAR-T后檢測凋亡和表面凋亡分子(Tim-3)發(fā)現(xiàn)M5A,hMN-14和BW431/26 CAR-T的相應(yīng)指標有明顯升高,提示上述CAR-T對CEA刺激的反應(yīng),這也證明了上述CAR-T細胞表面的CAR是有功能的。另外,我們也通過細胞殺傷試驗比較了各CAR-T細胞的功能,發(fā)現(xiàn)M5A CAR-T對CEA陽性腫瘤細胞的體外殺傷效果優(yōu)于hMN-14和BW431/26,而C2-45 CAR-T則對腫瘤細胞無殺傷作用。我們進一步在NOD-SCIDy-/- (NSG)小鼠體內(nèi)驗證和比較了各個CAR-T的功能。與體外殺傷實驗一致,M5ACAR-T對腫瘤的抑制和殺傷效果優(yōu)于hMN-14和BW431/26。綜合scFv親和力,CAR+%的表達和腫瘤的殺傷抑制作用,我們認為M5A優(yōu)于其他靶向CEA的scFv并可以成為后續(xù)CEA CAR-T臨床試驗的最佳scFv。
[Abstract]:Chimeric antigen receptor (CAR) T lymphocyte (CAR-T) is a specific T cell expressing CAR by introducing exogenous and artificially designed CAR gene into T lymphocyte for gene modification. The extracellular fragments are mainly composed of single-chain variable fragments (scFv) targeting antigen molecules, while the intramembrane effectors are composed of co-stimulatory molecules and CD3 T cell activating groups [2]. The main costimulatory molecules are CD28, CD27 and CD137 (4-1BB). The extracellular scFv binds to antigen. Car-T cell therapy is the most advanced approach to cancer treatment by genetically modifying T lymphocytes derived from the patient's own and then transfusing autologous CAR-T cells back to the patient. Previous clinical studies have shown that specifically targeted CD19 CAR-T cells can effectively treat B-cell malignancies (with CD19 expression), including acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma. Current data indicate that they are effective against advanced relapsed and refractory ALL. CAR-T cell therapy has become a breakthrough in the treatment of hematological malignancies. Previous CAR-T has been less used in solid tumors and its efficacy has not been achieved. The significant breakthrough in solid tumors was reported in the New England Journal of Medicine last year. Patients with extensively intracranial metastatic glioblastoma achieved complete remission (CR) after repeated transfusion of CAR-T cells. The results strongly confirm and confirm CAR-T therapy. Carcino-embryonic antigen (CEA) is a classical tumor marker, especially in colorectal cancer patients, with over 80% positive expression. So CEA is an ideal target for the treatment of colorectal cancer. Car-T designed for CEA has been completed in animal experiments [11,12] and phase I clinical trials [7]. The number of transfused cells in different treatment institutions and individual patients is different, and the criteria for transfusing cells in different individuals need to be further explored. More importantly, the incidence, types and mortality of tumors in China are still not very high. There is no CAR-T therapy for solid tumors in our country. Therefore, it is very necessary to carry out clinical research on CAR-T therapy for tumors in our country. We have carried out clinical research on CAR-T targeted CEA, and in clinical practice, we have passed the foundation because of the lack of CAR-T cells. Our work consists of two parts: clinical trials and basic research. Part I: Clinical studies of CAR-T for CEA-positive colorectal cancer confirm the safety and efficacy of CAR-T for CEA-positive colorectal cancer. In order to carry out phase I CAR-T clinical trials targeting CEA, we have done the following We have completed the preparation of CAR-T cells targeting CEA in vitro and in vivo. We have registered with the International Clinical Trial Institute (NCT 02349724) and approved by the Ethics Committee of the First Affiliated Hospital of the Third Military Medical University. Cancer, lung cancer and other tumors are widely expressed, especially in colorectal cancer patients with a positive rate of more than 80%. In the previous trial, we focused on the treatment of colorectal cancer patients. Organ metastases (some patients had primary lesions removed), most of which were liver metastases (7 cases). T lymphocytes were obtained by collecting peripheral blood from patients and transfecting T cells with lentivirus containing CAR sequence into CAR-T cells. We used lymphodepletion chemotherapy before cell transfusion. Case (fludarabine + cyclophosphatidyl, FC) clears the peripheral blood lymphocytes of patients, provides a good environment for the injection of CAR-T cells, and improves the efficacy of CAR-T therapy. After confirming the safety of low-dose cell reinfusion, high-dose cell reinfusion was given to follow-up patients. The safety of CAR-T in the treatment of CEA-positive colorectal cancer was validated by experiments. In addition to the effects of FC chemotherapy on the tertiary system, most patients reported only a small number of grade 1-2 adverse events. Seven patients with advanced disease (PD) were assessed as having stable tumor size after CAR-T treatment, and some patients were assessed as having stable disease (SD). Thirty weeks. Although the diagnostic criteria for partial remission (PR) were not met, two patients receiving high-dose cell reinfusion showed tumor shrinkage on imaging. In addition, the serum CEA of most patients decreased significantly after treatment, suggesting that CAR-T cells inhibited and killed CEA-positive tumor cells. The survival and proliferation of CAR-T cells in the peripheral blood and in vivo have always been a short board for the treatment of solid tumors [8].In our clinical trials, we found that CAR-T cells in patients receiving high-dose cell transfusion could survive for a certain period of time in vivo, and even found a small expansion of CAR-T cells in the peripheral blood. Our research group first reported the application of CAR-T in solid tumors in China. We confirmed the safety of CEA-targeted CAR-T cells in the treatment of relapsed and refractory colorectal cancer by combining the slope-climbing dose-reinfusion model in 10 patients with advanced colorectal cancer who met the ethical admission criteria. Most of the patients (7/10) had tumors under control after CAR-T therapy, and some of the tumors were even reduced in imaging. In addition, the survival and amplification of CAR-T cells were also verified in our experiments. This clinical trial proved that CAR-T therapy was effective. The safety and function of carcinomas provide a good basis for the subsequent optimization of CAR-T cells and clinical application. Part II: Basic research on CAR-T for CEA-positive colorectal cancer through screening and comparison, we found the best single-chain antibody scFv CAR-T, the best function in targeting CEA-positive tumors in the clinical trials of CAR-T. CAR-T cells can control and inhibit the growth of solid tumors, but can not complete the clearance of tumors, which suggests that we need more powerful CAR-T cells. At present, CAR-T cells combined with PD-1, chemokine receptor (CXCR) has been proved to be able to improve CAR-T fineness. Cytotherapy [14,15]. ScFv and costimulatory signals in CAR's own structure also have different effects on the function of CAR-T cells. Different scFv and costimulatory signals produce different functions of CAR [16,17]. Compared with different hybridoma-derived scFv (targeting different epitopes), it was found that high affinity could improve the function of CAR-T. Therefore, whether the affinity of scFv was positively correlated with the function of CAR-T cells is still uncertain. In addition, we also found that the positive rate of CAR +% on the surface of CAR-T cells decreased gradually during culture. In some patients, the CAR +% decreased to below 20% before cell transfusion. In some patients, cell transfusion was cancelled even because the CAR +% was below 10%. CAR+% of the above two kinds of CAR-T cells remained stable during the culture process. Therefore, we speculated that the reason for the decrease of CAR+% of CEA CAR-T might be related to scFv. Therefore, in order to explore the problem of the decrease of SCFv and CAR+% and to screen the best functioning scFv, we screened several known CEA CAR-T cells. McAbs A. The scFv sequences we used for CEA CAR-T were obtained from monoclonal antibody (mAb) BW431/26 targeting CEA, and we also found other CEA monoclonal antibodies M5A, hMN-14 and C2-45 which are widely used at present. We found the sequences of these four McAbs and constructed the corresponding scFv through eukaryotic cells. Four scFv proteins were expressed and purified. We detected the affinity of four scFv proteins and found that M5A and hMN-14 had the highest affinity, while BW431/26 had the lowest affinity. Comparing the CAR+% of four CARs, we found that CAR+% of the CAR-T cells of M5A and hMN-14 could be stably expressed in the culture process, while the CAR+% of BW431/26 still showed a downward trend, and the CAR+% of C2-45 could not be detected continuously. At present, the corresponding indexes of M5A, hMN-14 and BW431/26 CAR-T were significantly increased, suggesting that the above-mentioned CAR-T reacted to CEA stimulation, which also proved that the CAR-T cell surface is functional. In addition, we also compared the function of each CAR-T cell through the cytotoxicity test, and found that M5A CAR-T is superior to CEA-positive tumor cells in vitro killing effect. HMN-14 and BW431/26, while C2-45 CAR-T had no killing effect on tumor cells. We further validated and compared the functions of CAR-T in NOD-SCIDy-/(NSG) mice in vivo. The inhibitory and killing effects of M5ACAR-T on tumor were better than those of hMN-14 and BW431/26. The combined scFv affinity, CAR+% expression and tumor killing effect of M5ACAR-T were better than those of hMN-14 and BW431/26. We believe that M5A is superior to other scFv targeting CEA and may be the best scFv for subsequent CEA CAR-T clinical trials.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.34

【參考文獻】

相關(guān)期刊論文 前1條

1 Shengmeng Di;Zonghai Li;;Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects[J];Science China(Life Sciences);2016年04期

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本文編號：2238681