PD-1阻断或下调对CAR-T抗B-ALL效应影响与CAR-T治疗B-ALL临床研究
发布时间：2018-07-26 21:16
【摘要】：[目的]嵌合抗原受体修饰T细胞(CAR-T)治疗和程序性细胞死亡受体1(PD-1)/程序性细胞死亡配体1(PD-L1)免疫监测点抗体封闭手段已经成为肿瘤免疫治疗领域两大最具应用前景的治疗方法。虽然CAR-T细胞治疗在B细胞来源急性淋巴细胞白血病(B-ALL)治疗中已经取得了不错的治疗效果,但是回输患者体内的CAR-T细胞杀伤活性仍会被PD-1/PD-L1免疫抑制性途径所削弱。能否通过联合PD-1抗体封闭或PD-1下调来打破PD-1/PD-L1免疫抑制性途径对CAR-T细胞活性的限制作用,以进一步提高CAR-T细胞治疗效果?再者,这种联合会不会带来严重毒副作用?这方面研究尚未在B-ALL中有研究报道。因此,我们开展了PD-1抗体封闭或PD-1下调联合抗CD19 CAR-T细胞治疗B-ALL的临床前安全性和有效性研究。此外,考虑本课题为一项应用性课题,我们计划在临床前实验基础上开展一项有关抗CD19 CAR-T细胞治疗化疗复发或难治性B-ALL患者的I期临床试验,用于初步评估抗CD19 CAR-T细胞输注的安全性和有效性。[方法]我们基于microRNA 148a骨架结构利用前期已验证的PD-1 siRNA序列设计了PD-1的人工microRNA序列,在此基础上,采用合成的抗CD19CAR序列、PD-1人工microRNA序列和非靶向对照(NC)人工microRNA序列构建三种逆转录病毒主质粒,并进行相应的逆转录病毒包装和浓缩;原代人外周血单个核细胞病毒感染前先用可溶性CD3/CD28抗体活化3天,再采用离心法进行逆转录病毒感染;流式细胞术用于检测细胞表型如CAR、PD-1、PD-L1、CD3、CD19、CD25、CD45RA/RO、CD62L等表达;PD-1蛋白下调效果用蛋白印迹和流式细胞术进行检测;在PD-1抗体体外封闭实验中,PD-1封闭抗体或IgG同型对照抗体体外先与T细胞孵育24小时,再进行后续功能实验;T细胞体内外功能实验采用流式细胞术、流式微球分析技术和以NALM-6白血病建立的B-ALL NPG/Vst小鼠模型进行;在临床试验中,抗CD19 CAR-T治疗化疗复发或难治B-ALL患者的安全性和有效性采用NCI通用不良事件术语标准3.0版、流式细胞术、Q-PCR、流式微球分析技术和骨髓形态检测等来进行观察和评估,所有试验方案均经我院伦理委员会批准后进行。[结果]我们成功构建了三种抗CD19 CAR逆转录病毒载体(pRV-19-28ζ、pRV-iPD-1-19-28ζ和pRV-NC-19-28ζ),并经测序鉴定,蛋白印迹和流式细胞检测结果均证实PD-1人工microRNA能特异性和有效下调PD-1蛋白表达;我们对RV-19-28ζ感染原代人T细胞所制备的抗CD19 CAR-T细胞体外扩增和细胞表型变化特点进行了研究,并发现病毒感染不利于T细胞扩增和给予抗CD19 CAR-T细胞再刺激能明显增加CAR-T细胞比例,而且感染后T细胞表型在体外培养过程中处于动态变化的,但这种变化并不是由于逆转录病毒感染所致;基于靶细胞(NALM-6、OCI-Ly10和K562)的流式细胞细胞毒性检测技术显示抗CD19 CAR-T细胞具有CAR特异性和剂量依赖性杀伤靶细胞活性;然后,我们发现肿瘤细胞和T细胞体外共孵育后能上调总T细胞和抗CD19 CAR-T细胞表面PD-1蛋白表达,以及不同程度地上调B细胞肿瘤Raji、OCI-Ly10和NALM-6表面的PD-L1蛋白表达,其中NALM-6细胞表面PD-L1蛋白上调最为明显;在此基础上,我们体外研究首次证明PD-1抗体封闭能增强抗CD19CAR-T细胞对NALM-6、Raji和OCI-Ly10靶细胞的细胞毒性作用。进一步,我们又研究了PD-1下调对CAR-T细胞杀伤NALM-6靶细胞的杀伤效应的影响,结果同样表明:PD-1下调能增强抗CD19 CAR-T细胞杀伤活性;但我们发现PD-1抗体封闭并不能增强抗CD19 CAR-T细胞与NALM-6肿瘤细胞共孵育后IFN-γ的分泌,而PD-1下调能显著增强抗CD19 CAR-T细胞的IFN-γ分泌;更为重要的是,动物实验结果显示:PD-1抗体封闭或PD-1下调联合抗CD19 CAR-T细胞在B-ALL模型(NALM-6)中虽然没有导致严重毒副作用,但是无法延长小鼠生存时间和发病时间;在动物实验基础上,经我院伦理委员会批准我们开展了抗CD19 CAR-T治疗化疗复发或难治性B-ALL患者的I期临床试验,目前已经完成了3例患者的抗CD19 CAR-T细胞输注。初步治疗结果显示:2例患者对CAR-T治疗产生了反应,达到完全缓解,1例患者治疗失败。其中2例治疗有效患者中有1例患者治疗后产生了细胞因子释放综合症,该患者血清中IL-6和CRP治疗前后急剧变化,体温升高,后经2次托珠单抗和1次地塞米松联合治疗得以缓解。而且,这例患者接受的CAR-T细胞是半相合供者的外周血T细胞制备而成,输注后未产生GVHD反应。[结论]PD-1抗体封闭或PD-1下调联合抗CD19 CAR-T细胞输注在BALL模型中虽然安全性较好,但是无法进一步增强抗CD19 CAR-T细胞体内抗B-ALL效应;抗CD19 CAR-T细胞输注能在化疗复发或难治B-ALL患者中诱导产生临床反应,临床不良反应主要有细胞因子释放综合症,托珠单抗联合地塞米松能有效缓解细胞因子释放综合症,血清中200 mg/L浓度水平的CRP能用于细胞因子释放综合症发展进程的预测。
[Abstract]:[Objective] the immunotherapy of chimeric antigen receptor modified T cells (CAR-T) and programmed cell death receptor 1 (PD-1) / programmed cell death ligand 1 (PD-L1) antibody blocking method has become the two most promising treatment in the field of tumor immunotherapy. Although CAR-T cells are treated in B cells from acute lymphoblastic leukocyte origin A good therapeutic effect has been achieved in the treatment of disease (B-ALL), but the CAR-T cell killing activity in the transfusion patient is still weakened by the PD-1/PD-L1 immunosuppressive pathway. Can the restriction of the PD-1/PD-L1 immunosuppressive pathway to the activity of CAR-T cells be broken through the combination of PD-1 antibody closure or PD-1 downregulation to further improve CA R-T cell therapy effect? Again, does this association bring serious toxic and side effects? This research has not been reported in B-ALL. Therefore, we have developed PD-1 antibody blocking or PD-1 downregulation in combination with anti CD19 CAR-T cells for the treatment of B-ALL before and in clinical safety and effectiveness. In addition, we consider this subject as an application subject, We plan to develop a phase I clinical trial on anti CD19 CAR-T cell therapy for chemotherapy recurrent or refractory B-ALL patients on the basis of preclinical trials to evaluate the safety and effectiveness of anti CD19 CAR-T cell infusion. [Methods] we designed the PD-1 siRNA sequence that has been verified in the earlier period of use of the microRNA 148A skeleton structure. On the basis of the artificial microRNA sequence of PD-1, three kinds of retrovirus main plasmids were constructed by synthetic anti CD19CAR sequence, PD-1 artificial microRNA sequence and non target control (NC) artificial microRNA sequence, and corresponding retrovirus packaging and concentration were carried out. The original human peripheral blood mononuclear cell virus infection first used soluble CD3/CD28. The antibody was activated for 3 days and then centrifugation was used for retroviral infection. Flow cytometry was used to detect the expression of cell phenotypes such as CAR, PD-1, PD-L1, CD3, CD19, CD25, CD45RA/RO, CD62L and so on. The down-regulation effect of PD-1 protein was detected by Western blot and flow cytometry; PD-1 closed antibody or IgG homotype resistance was used in the closed test of PD-1 antibody in vitro. The body was incubated with T cells for 24 hours in vitro and followed by subsequent functional experiments. Flow cytometry, flow microsphere analysis and B-ALL NPG/Vst mice model established with NALM-6 leukemia were performed in the T cells in vivo and in vitro. In clinical trials, anti CD19 CAR-T was used to treat the safety and effectiveness of chemotherapy for recurrent or refractory B-ALL patients. The NCI general adverse event terminology standard 3, flow cytometry, Q-PCR, flow microsphere analysis and bone marrow morphometry were observed and evaluated. All the test programs were approved by our Institute of ethics. [results] we successfully constructed three kinds of anti CD19 CAR retroviral vectors (pRV-19-28 zeta, pRV-iPD-1-19-28 zeta and) PRV-NC-19-28 zeta), and by sequencing, the results of Western blot and flow cytometry showed that PD-1 artificial microRNA could specifically and effectively reduce the expression of PD-1 protein. We studied the amplification of anti CD19 CAR-T cells in vitro and the phenotypic variation of CD19 CAR-T cells from the original T cells of RV-19-28 zeta infection, and found that the virus infection was unfavourable. The amplification of T cells and the stimulation of anti CD19 CAR-T cells can significantly increase the proportion of CAR-T cells, and the phenotype of T cells after infection is dynamically changed in the process of culture in vitro, but this change is not due to retroviral infection; flow cytometric detection techniques based on target cells (NALM-6, OCI-Ly10 and K562) The anti CD19 CAR-T cells showed CAR specific and dose dependent killer cell activity. Then, we found that the expression of PD-1 protein on the surface of the total T cells and the anti CD19 CAR-T cells was up-regulated after the tumor cells and T cells were reincubated in vitro, and the expression of the protein expression of Raji, OCI-Ly10 and NALM-6 surfaces of B cell tumors was up-regulated to varying degrees. The most obvious up regulation of PD-L1 protein on the surface of ALM-6 cells; on this basis, our in vitro study showed that PD-1 antibody blocking could enhance the cytotoxic effect of anti CD19CAR-T cells on NALM-6, Raji and OCI-Ly10 target cells. Further, we also studied the effect of PD-1 down regulation on the killing effect of CAR-T cells on the killing of NALM-6 target cells. The results showed that the down regulation of PD-1 could enhance the anti CD19 CAR-T cell killing activity, but we found that the blocking of PD-1 antibody did not enhance the secretion of IFN- gamma after the reincubation of CD19 CAR-T cells and NALM-6 tumor cells, and the downregulation of PD-1 could significantly enhance the IFN- gamma secretion of the anti CD19 CAR-T cells; the more important thing is that the animal experiment results show that the antibody is closed. Or PD-1 down-regulation of CD19 CAR-T cells in B-ALL model (NALM-6) did not lead to severe toxic and side effects, but could not prolong the survival time and time of onset of mice. On the basis of animal experiments, our hospital ethics committee approved the trial of I stage clinical trials of anti CD19 CAR-T therapy for recurrent or refractory B-ALL patients. 3 cases of anti CD19 CAR-T cell infusion have been completed. Preliminary results showed that 2 patients responded to CAR-T treatment, achieved complete remission, and 1 patients failed. 1 of the 2 patients were treated with cytokine release after treatment, and the patient's serum IL-6 and CRP were urgent. The play changes, the body temperature rises, after 2 times of the combined treatment of the CAR-T and 1 dexamethasone. Moreover, this patient receives the T cells from the peripheral blood of the half of the donor, and does not produce the GVHD reaction. [conclusion]PD-1 antibody closed or PD-1 down-regulation against CD19 CAR-T cell infusion in BALL model. Safety is good, but it can not further enhance the anti B-ALL effect of anti CD19 CAR-T cells in vivo; anti CD19 CAR-T cell infusion can induce clinical response in patients with relapse of chemotherapy or refractory B-ALL patients. The main clinical adverse reactions are cytokine release syndrome, and the combination of tubeumab combined with dexamethasone can effectively alleviate cytokine release synthesis. The CRP of 200 mg/L in serum can be used to predict the development process of cytokine release syndrome.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.51
[Abstract]:[Objective] the immunotherapy of chimeric antigen receptor modified T cells (CAR-T) and programmed cell death receptor 1 (PD-1) / programmed cell death ligand 1 (PD-L1) antibody blocking method has become the two most promising treatment in the field of tumor immunotherapy. Although CAR-T cells are treated in B cells from acute lymphoblastic leukocyte origin A good therapeutic effect has been achieved in the treatment of disease (B-ALL), but the CAR-T cell killing activity in the transfusion patient is still weakened by the PD-1/PD-L1 immunosuppressive pathway. Can the restriction of the PD-1/PD-L1 immunosuppressive pathway to the activity of CAR-T cells be broken through the combination of PD-1 antibody closure or PD-1 downregulation to further improve CA R-T cell therapy effect? Again, does this association bring serious toxic and side effects? This research has not been reported in B-ALL. Therefore, we have developed PD-1 antibody blocking or PD-1 downregulation in combination with anti CD19 CAR-T cells for the treatment of B-ALL before and in clinical safety and effectiveness. In addition, we consider this subject as an application subject, We plan to develop a phase I clinical trial on anti CD19 CAR-T cell therapy for chemotherapy recurrent or refractory B-ALL patients on the basis of preclinical trials to evaluate the safety and effectiveness of anti CD19 CAR-T cell infusion. [Methods] we designed the PD-1 siRNA sequence that has been verified in the earlier period of use of the microRNA 148A skeleton structure. On the basis of the artificial microRNA sequence of PD-1, three kinds of retrovirus main plasmids were constructed by synthetic anti CD19CAR sequence, PD-1 artificial microRNA sequence and non target control (NC) artificial microRNA sequence, and corresponding retrovirus packaging and concentration were carried out. The original human peripheral blood mononuclear cell virus infection first used soluble CD3/CD28. The antibody was activated for 3 days and then centrifugation was used for retroviral infection. Flow cytometry was used to detect the expression of cell phenotypes such as CAR, PD-1, PD-L1, CD3, CD19, CD25, CD45RA/RO, CD62L and so on. The down-regulation effect of PD-1 protein was detected by Western blot and flow cytometry; PD-1 closed antibody or IgG homotype resistance was used in the closed test of PD-1 antibody in vitro. The body was incubated with T cells for 24 hours in vitro and followed by subsequent functional experiments. Flow cytometry, flow microsphere analysis and B-ALL NPG/Vst mice model established with NALM-6 leukemia were performed in the T cells in vivo and in vitro. In clinical trials, anti CD19 CAR-T was used to treat the safety and effectiveness of chemotherapy for recurrent or refractory B-ALL patients. The NCI general adverse event terminology standard 3, flow cytometry, Q-PCR, flow microsphere analysis and bone marrow morphometry were observed and evaluated. All the test programs were approved by our Institute of ethics. [results] we successfully constructed three kinds of anti CD19 CAR retroviral vectors (pRV-19-28 zeta, pRV-iPD-1-19-28 zeta and) PRV-NC-19-28 zeta), and by sequencing, the results of Western blot and flow cytometry showed that PD-1 artificial microRNA could specifically and effectively reduce the expression of PD-1 protein. We studied the amplification of anti CD19 CAR-T cells in vitro and the phenotypic variation of CD19 CAR-T cells from the original T cells of RV-19-28 zeta infection, and found that the virus infection was unfavourable. The amplification of T cells and the stimulation of anti CD19 CAR-T cells can significantly increase the proportion of CAR-T cells, and the phenotype of T cells after infection is dynamically changed in the process of culture in vitro, but this change is not due to retroviral infection; flow cytometric detection techniques based on target cells (NALM-6, OCI-Ly10 and K562) The anti CD19 CAR-T cells showed CAR specific and dose dependent killer cell activity. Then, we found that the expression of PD-1 protein on the surface of the total T cells and the anti CD19 CAR-T cells was up-regulated after the tumor cells and T cells were reincubated in vitro, and the expression of the protein expression of Raji, OCI-Ly10 and NALM-6 surfaces of B cell tumors was up-regulated to varying degrees. The most obvious up regulation of PD-L1 protein on the surface of ALM-6 cells; on this basis, our in vitro study showed that PD-1 antibody blocking could enhance the cytotoxic effect of anti CD19CAR-T cells on NALM-6, Raji and OCI-Ly10 target cells. Further, we also studied the effect of PD-1 down regulation on the killing effect of CAR-T cells on the killing of NALM-6 target cells. The results showed that the down regulation of PD-1 could enhance the anti CD19 CAR-T cell killing activity, but we found that the blocking of PD-1 antibody did not enhance the secretion of IFN- gamma after the reincubation of CD19 CAR-T cells and NALM-6 tumor cells, and the downregulation of PD-1 could significantly enhance the IFN- gamma secretion of the anti CD19 CAR-T cells; the more important thing is that the animal experiment results show that the antibody is closed. Or PD-1 down-regulation of CD19 CAR-T cells in B-ALL model (NALM-6) did not lead to severe toxic and side effects, but could not prolong the survival time and time of onset of mice. On the basis of animal experiments, our hospital ethics committee approved the trial of I stage clinical trials of anti CD19 CAR-T therapy for recurrent or refractory B-ALL patients. 3 cases of anti CD19 CAR-T cell infusion have been completed. Preliminary results showed that 2 patients responded to CAR-T treatment, achieved complete remission, and 1 patients failed. 1 of the 2 patients were treated with cytokine release after treatment, and the patient's serum IL-6 and CRP were urgent. The play changes, the body temperature rises, after 2 times of the combined treatment of the CAR-T and 1 dexamethasone. Moreover, this patient receives the T cells from the peripheral blood of the half of the donor, and does not produce the GVHD reaction. [conclusion]PD-1 antibody closed or PD-1 down-regulation against CD19 CAR-T cell infusion in BALL model. Safety is good, but it can not further enhance the anti B-ALL effect of anti CD19 CAR-T cells in vivo; anti CD19 CAR-T cell infusion can induce clinical response in patients with relapse of chemotherapy or refractory B-ALL patients. The main clinical adverse reactions are cytokine release syndrome, and the combination of tubeumab combined with dexamethasone can effectively alleviate cytokine release synthesis. The CRP of 200 mg/L in serum can be used to predict the development process of cytokine release syndrome.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.51
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