【摘要】：[目的]嵌合抗原受體修飾T細(xì)胞(CAR-T)治療和程序性細(xì)胞死亡受體1(PD-1)/程序性細(xì)胞死亡配體1(PD-L1)免疫監(jiān)測點(diǎn)抗體封閉手段已經(jīng)成為腫瘤免疫治療領(lǐng)域兩大最具應(yīng)用前景的治療方法。雖然CAR-T細(xì)胞治療在B細(xì)胞來源急性淋巴細(xì)胞白血病(B-ALL)治療中已經(jīng)取得了不錯的治療效果,但是回輸患者體內(nèi)的CAR-T細(xì)胞殺傷活性仍會被PD-1/PD-L1免疫抑制性途徑所削弱。能否通過聯(lián)合PD-1抗體封閉或PD-1下調(diào)來打破PD-1/PD-L1免疫抑制性途徑對CAR-T細(xì)胞活性的限制作用,以進(jìn)一步提高CAR-T細(xì)胞治療效果?再者,這種聯(lián)合會不會帶來嚴(yán)重毒副作用?這方面研究尚未在B-ALL中有研究報道。因此,我們開展了PD-1抗體封閉或PD-1下調(diào)聯(lián)合抗CD19 CAR-T細(xì)胞治療B-ALL的臨床前安全性和有效性研究。此外,考慮本課題為一項應(yīng)用性課題,我們計劃在臨床前實(shí)驗基礎(chǔ)上開展一項有關(guān)抗CD19 CAR-T細(xì)胞治療化療復(fù)發(fā)或難治性B-ALL患者的I期臨床試驗,用于初步評估抗CD19 CAR-T細(xì)胞輸注的安全性和有效性。[方法]我們基于microRNA 148a骨架結(jié)構(gòu)利用前期已驗證的PD-1 siRNA序列設(shè)計了PD-1的人工microRNA序列,在此基礎(chǔ)上,采用合成的抗CD19CAR序列、PD-1人工microRNA序列和非靶向?qū)φ?NC)人工microRNA序列構(gòu)建三種逆轉(zhuǎn)錄病毒主質(zhì)粒,并進(jìn)行相應(yīng)的逆轉(zhuǎn)錄病毒包裝和濃縮;原代人外周血單個核細(xì)胞病毒感染前先用可溶性CD3/CD28抗體活化3天,再采用離心法進(jìn)行逆轉(zhuǎn)錄病毒感染;流式細(xì)胞術(shù)用于檢測細(xì)胞表型如CAR、PD-1、PD-L1、CD3、CD19、CD25、CD45RA/RO、CD62L等表達(dá);PD-1蛋白下調(diào)效果用蛋白印跡和流式細(xì)胞術(shù)進(jìn)行檢測;在PD-1抗體體外封閉實(shí)驗中,PD-1封閉抗體或IgG同型對照抗體體外先與T細(xì)胞孵育24小時,再進(jìn)行后續(xù)功能實(shí)驗;T細(xì)胞體內(nèi)外功能實(shí)驗采用流式細(xì)胞術(shù)、流式微球分析技術(shù)和以NALM-6白血病建立的B-ALL NPG/Vst小鼠模型進(jìn)行;在臨床試驗中,抗CD19 CAR-T治療化療復(fù)發(fā)或難治B-ALL患者的安全性和有效性采用NCI通用不良事件術(shù)語標(biāo)準(zhǔn)3.0版、流式細(xì)胞術(shù)、Q-PCR、流式微球分析技術(shù)和骨髓形態(tài)檢測等來進(jìn)行觀察和評估,所有試驗方案均經(jīng)我院倫理委員會批準(zhǔn)后進(jìn)行。[結(jié)果]我們成功構(gòu)建了三種抗CD19 CAR逆轉(zhuǎn)錄病毒載體(pRV-19-28ζ、pRV-iPD-1-19-28ζ和pRV-NC-19-28ζ),并經(jīng)測序鑒定,蛋白印跡和流式細(xì)胞檢測結(jié)果均證實(shí)PD-1人工microRNA能特異性和有效下調(diào)PD-1蛋白表達(dá);我們對RV-19-28ζ感染原代人T細(xì)胞所制備的抗CD19 CAR-T細(xì)胞體外擴(kuò)增和細(xì)胞表型變化特點(diǎn)進(jìn)行了研究,并發(fā)現(xiàn)病毒感染不利于T細(xì)胞擴(kuò)增和給予抗CD19 CAR-T細(xì)胞再刺激能明顯增加CAR-T細(xì)胞比例,而且感染后T細(xì)胞表型在體外培養(yǎng)過程中處于動態(tài)變化的,但這種變化并不是由于逆轉(zhuǎn)錄病毒感染所致;基于靶細(xì)胞(NALM-6、OCI-Ly10和K562)的流式細(xì)胞細(xì)胞毒性檢測技術(shù)顯示抗CD19 CAR-T細(xì)胞具有CAR特異性和劑量依賴性殺傷靶細(xì)胞活性;然后,我們發(fā)現(xiàn)腫瘤細(xì)胞和T細(xì)胞體外共孵育后能上調(diào)總T細(xì)胞和抗CD19 CAR-T細(xì)胞表面PD-1蛋白表達(dá),以及不同程度地上調(diào)B細(xì)胞腫瘤Raji、OCI-Ly10和NALM-6表面的PD-L1蛋白表達(dá),其中NALM-6細(xì)胞表面PD-L1蛋白上調(diào)最為明顯;在此基礎(chǔ)上,我們體外研究首次證明PD-1抗體封閉能增強(qiáng)抗CD19CAR-T細(xì)胞對NALM-6、Raji和OCI-Ly10靶細(xì)胞的細(xì)胞毒性作用。進(jìn)一步,我們又研究了PD-1下調(diào)對CAR-T細(xì)胞殺傷NALM-6靶細(xì)胞的殺傷效應(yīng)的影響,結(jié)果同樣表明:PD-1下調(diào)能增強(qiáng)抗CD19 CAR-T細(xì)胞殺傷活性;但我們發(fā)現(xiàn)PD-1抗體封閉并不能增強(qiáng)抗CD19 CAR-T細(xì)胞與NALM-6腫瘤細(xì)胞共孵育后IFN-γ的分泌,而PD-1下調(diào)能顯著增強(qiáng)抗CD19 CAR-T細(xì)胞的IFN-γ分泌;更為重要的是,動物實(shí)驗結(jié)果顯示:PD-1抗體封閉或PD-1下調(diào)聯(lián)合抗CD19 CAR-T細(xì)胞在B-ALL模型(NALM-6)中雖然沒有導(dǎo)致嚴(yán)重毒副作用,但是無法延長小鼠生存時間和發(fā)病時間;在動物實(shí)驗基礎(chǔ)上,經(jīng)我院倫理委員會批準(zhǔn)我們開展了抗CD19 CAR-T治療化療復(fù)發(fā)或難治性B-ALL患者的I期臨床試驗,目前已經(jīng)完成了3例患者的抗CD19 CAR-T細(xì)胞輸注。初步治療結(jié)果顯示:2例患者對CAR-T治療產(chǎn)生了反應(yīng),達(dá)到完全緩解,1例患者治療失敗。其中2例治療有效患者中有1例患者治療后產(chǎn)生了細(xì)胞因子釋放綜合癥,該患者血清中IL-6和CRP治療前后急劇變化,體溫升高,后經(jīng)2次托珠單抗和1次地塞米松聯(lián)合治療得以緩解。而且,這例患者接受的CAR-T細(xì)胞是半相合供者的外周血T細(xì)胞制備而成,輸注后未產(chǎn)生GVHD反應(yīng)。[結(jié)論]PD-1抗體封閉或PD-1下調(diào)聯(lián)合抗CD19 CAR-T細(xì)胞輸注在BALL模型中雖然安全性較好,但是無法進(jìn)一步增強(qiáng)抗CD19 CAR-T細(xì)胞體內(nèi)抗B-ALL效應(yīng);抗CD19 CAR-T細(xì)胞輸注能在化療復(fù)發(fā)或難治B-ALL患者中誘導(dǎo)產(chǎn)生臨床反應(yīng),臨床不良反應(yīng)主要有細(xì)胞因子釋放綜合癥,托珠單抗聯(lián)合地塞米松能有效緩解細(xì)胞因子釋放綜合癥,血清中200 mg/L濃度水平的CRP能用于細(xì)胞因子釋放綜合癥發(fā)展進(jìn)程的預(yù)測。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R730.51

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