CAR-T治疗CEA阳性结直肠癌的临床和基础研究
发布时间：2018-09-12 10:07
【摘要】：嵌合性抗原受体(chimeric antigen receptor,CAR) T淋巴细胞(CAR-T)是通过将外源性人工设计的CAR基因导入T淋巴细胞内进行基因修饰改造,而后得到的表达CAR的特异性T细胞[1]。CAR主要由可特异性识别肿瘤抗原的膜外片段,连接片段,和含刺激信号的膜内片段组成。膜外片段主要是由靶向抗原分子的单链抗体(Single-chain variable fragment, scFv)组成,膜内效应分子则是由共刺激分子和CD3ζ等T细胞活化基团组成[2]。主要的共刺激分子有CD28, CD27和CD137 (4-1BB)等。细胞外scFv与抗原结合后可以启动胞内段信号转导并活化T淋巴细胞,最终活化的T淋巴细胞对肿瘤细胞产生杀伤。CAR-T细胞疗法是通过对来源于患者自身的T淋巴细胞进行基因修饰而后将自体性CAR-T细胞回输给病人治疗肿瘤的方法,这是一种最前沿进展的肿瘤免疫治疗方法。前期的临床研究已经显示,特异性靶向CD19分子的CAR-T细胞能有效地治疗B细胞性恶性肿瘤(表达有CD19分子),包括急性淋巴细胞白血病(ALL),慢性淋巴细胞白血病(CLL),和B细胞淋巴瘤。目前的数据表明其对晚期复发难治性ALL的治疗有效率可达到80%以上[3,4],对CLL和部分B细胞淋巴瘤的有效率50%[5,6],相关成果已多次在新英格兰医学杂志等顶级期刊发表。CAR-T细胞疗法已经成为血液系统肿瘤治疗的一个突破性进展。前期CAR-T在实体瘤的应用较少,且疗效并无其在血液肿瘤明显,病人肿瘤缓解率低[7,8]。去年新英格兰医学杂志再次报道了该疗法在实体瘤的重大突破,广泛颅内转移的胶质母细胞瘤病人在CAR-T细胞多次回输后达到了完全缓解(Complete remission, CR),该结果极大肯定和明确了 CAR-T疗法在实体瘤的应用价值[9]。所以,开展CAR-T疗法在实体瘤的应用是十分必要的。癌胚抗原(Carcino-embryonic antigen, CEA)是经典的肿瘤标志物,特别在结直肠癌病人中有80%以上阳性表达。正常组织细胞中只有消化道细胞有少量的CEA在细胞膜表达,而该CEA在生理条件下朝向胞腔内表达从而避免被靶向CEA的CAR-T细胞识别[10]。所以CEA是CAR-T治疗结直肠癌理想的靶点。针对CEA设计的CAR-T在国外已经完成了动物实验[11,12]以及Ⅰ期临床试验[7],现有的CAR-T细胞治疗实体瘤的临床结果都是在国外获得,且目前大多处于临床Ⅰ期阶段,病例总数尚不多;而且在不同的治疗机构以及病人个体间,治疗的输注细胞数量都不等,其标准在不同的个体之间尚需进一步探索;更重要的是,我国的肿瘤在发病率,肿瘤类型,和死亡率等方面都和西方国家不尽相同,目前我们国内还没有开展CAR-T疗法在实体肿瘤应用。因此,在我们国家开展CAR-T对肿瘤的临床治疗研究是非常必要的。我们开展了 CAR-T靶向CEA的临床研究,而在临床实践中也因为发现CAR-T细胞的不足而通过基础研究来探究功能更强的CAR-T细胞。所以,我们的工作主要由临床试验和基础研究两部分组成:第一部分:CAR-T治疗CEA阳性结直肠癌的临床研究证实了其治疗CEA阳性结直肠癌的安全性和有效性:为了开展靶向CEA的Ⅰ期CAR-T临床试验,我们前期完成了靶向CEA的CAR-T细胞的制备,也在体外和动物体内验证了其有效性和安全性。我们在国际临床试验机构(clinicaltrial.gov)完成了注册(编号:NCT 02349724),同时也通过了第三军医大学第一附属医院伦理委员会的批准。CEA在结直肠癌、胰腺癌、肺癌等肿瘤中都有广泛的表达,尤其在结直肠癌病人中CEA阳性率超过80%。在前期的试验过程中,我们将重心聚焦在结直肠癌病人的收治上。我们入组了符合纳排标准的10例难治复发性CEA阳性的结直肠癌病人,这10例病人全部伴有肠外器官转移(部分病人原发灶已切除),其中绝大部分为肝转移(7例)。通过采集病人外周血并在GMP实验室获得T淋巴细胞,通过包含CAR序列的慢病毒(lentivirus)转染T细胞获得CAR-T细胞。在细胞回输前,我们使用了淋巴清除(lymphodepletion)的化疗方案(氟达拉滨+环磷酷胺,FC)对患者外周血的淋巴细胞进行清除,为CAR-T细胞的注入提供良好环境,提高CAR-T治疗的疗效。大剂量CAR-T细胞的回输治疗实体瘤可能存在致死风险[13],为了确保治疗的安全性,我们采用了爬坡阶梯的输注模式,在确认低剂量细胞回输的安全性后,再对后续病人使用高剂量细胞回输。通过试验,我们验证了 CAR-T治疗CEA阳性结直肠癌的安全性。在10例病人中,治疗剂量从107至1010逐步增加,最高细胞回输剂量达到了5 1x10,所有病人均无严重不良反应发生(除FC化疗对三系的影响),大部分病人仅报告少量1-2级不良事件。7例在前期化疗疗效评价为疾病进展(Progressive disease, PD)的病人在接受CAR-T治疗后肿瘤病灶大小保持稳定,评价为病情稳定(Stable disease, SD),部分病人维持SD的时间超过了 30周。虽然没有达到部分缓解(Partial remission, PR)的诊断标准,但有两例接受高剂量细胞回输的病人在影像学可见肿瘤有缩小。另外,大部分病人的血清CEA在治疗后明显下降,提示了 CAR-T细胞对CEA阳性肿瘤细胞的抑制及杀伤作用。CAR-T细胞在外周血和体内的存续以及其在体内的增殖一直是CAR-T细胞治疗实体肿瘤的一个短板[8]。在我们的临床试验中,我们发现接受高剂量细胞回输的病人CAR-T细胞可以在体内存活一定时间,甚至发现了 CAR-T细胞在外周血的少量扩增。总而言之,我们课题组在国内首个报道了 CAR-T在实体肿瘤应用。通过入组10例符合伦理纳排标准的晚期结直肠癌病人,结合爬坡剂量的回输模式,我们确认了靶向CEA的CAR-T细胞在治疗复发难治性结直肠癌的安全性。考虑到入组的病人均为临床一线二线化疗无效的难治性肿瘤,通过CAR-T治疗后,大部分病人(7例/10例)的肿瘤得到了控制,一些病人的肿瘤病灶甚至在影像学上可以看到缩小。另外,CAR-T细胞的存活与扩增也在我们的试验得到了验证。本临床试验证明了 CAR-T治疗实体瘤的安全性以及其功能,为后续CAR-T的细胞优化及临床应用提供了良好依据。第二部分:CAR-T治疗CEA阳性结直肠癌的基础研究通过筛选和对比,我们找到了 CAR-T最适、功能最佳的单链抗体scFv在靶向CEA阳性肿瘤的CAR-T临床试验中我们也发现了目前CAR-T细胞所需要优化和改良的地方。CAR-T可以控制和抑制实体肿瘤的生长,但未能完成对肿瘤的清除,这提示我们需要功能更强的CAR-T细胞。目前CAR-T细胞联用PD-1,趋化因子受体(Cxcchemokine receptor, CXCR)均已被证实可以提高CAR-T细胞疗效[14,15]。CAR自身结构中的scFv和共刺激信号也会对CAR-T细胞的功能造成不同的影响,不同scFv与共刺激信号会产生不同功能的CAR[16,17]。前期报道提示低亲和力的scFv相比于高亲和力的scFv (两个scFv为源于同一个单抗的不同突变体)甚至表现出对CEA阳性肿瘤更好的杀伤以及对CEA阴性或弱阳性细胞更好的安全性[18]。而在不同杂交瘤来源的scFv (靶向不同表位)之间对比发现,高亲和力利于提高CAR-T的功能[19,20]。所以,scFv的亲和力与CAR-T细胞功能是否呈正相关尚无定论。另外,在培养病人的CAR-T细胞过程中,我们也发现CAR-T细胞表面的CAR表达阳性率CAR+%在培养过程中呈逐渐下降的趋势,部分病人的CAR+%在细胞回输前降至20%以下,个别病人甚至因为低于10%的CAR+%而取消细胞回输。我们检测了靶向HER2和CD19的CAR-T细胞(仅仅替换了 CEA CAR结构的scFv)的CAR变化,上述两种CAR-T细胞的CAR+%在培养过程中保持稳定。因此,我们推测CEA CAR-T的CAR+%下降的原因可能与scFv有关。因此,为探究scFv与CAR+%下降的问题并筛选功能最佳的scFv,我们筛选了几种已知的CEA单抗。我们用于CEA CAR-T的scFv序列是从靶向CEA的单克隆抗体(Monoclonal antibody, mAb) BW431/26中获得[21],同时我们也找到了其他在目前广泛应用的CEA单克隆抗体M5A,hMN-14和C2-45。我们找到上述四种单抗的序列并构建出相应的scFv,通过真核细胞表达和蛋白纯化得到4种scFv蛋白,我们检测了 4种scFv的亲和力,发现M5A和hMN-14的亲和力最高,而BW431/26的亲和力最低。同时,我们把各scFv的序列装载到相同的3代CAR骨架并构建出对应的病毒且完成了对T细胞的感染,获得了相应的CAR-T。通过比较4个CAR的CAR+%,我们发现M5A和hMN-14的CAR-T细胞的CAR+%在培养过程中可以稳定表达,而BW431/26的CAR+%仍呈下降趋势,C2-45的CAR+%则持续检测不到。为了验证CAR-T表面的CAR功能,我们用重组CEA蛋白刺激各CAR-T后检测凋亡和表面凋亡分子(Tim-3)发现M5A,hMN-14和BW431/26 CAR-T的相应指标有明显升高,提示上述CAR-T对CEA刺激的反应,这也证明了上述CAR-T细胞表面的CAR是有功能的。另外,我们也通过细胞杀伤试验比较了各CAR-T细胞的功能,发现M5A CAR-T对CEA阳性肿瘤细胞的体外杀伤效果优于hMN-14和BW431/26,而C2-45 CAR-T则对肿瘤细胞无杀伤作用。我们进一步在NOD-SCIDy-/- (NSG)小鼠体内验证和比较了各个CAR-T的功能。与体外杀伤实验一致,M5ACAR-T对肿瘤的抑制和杀伤效果优于hMN-14和BW431/26。综合scFv亲和力,CAR+%的表达和肿瘤的杀伤抑制作用,我们认为M5A优于其他靶向CEA的scFv并可以成为后续CEA CAR-T临床试验的最佳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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2238681
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34
【参考文献】
相关期刊论文 前1条
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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