靶向PSCA和MUC1的嵌合抗原受体T细胞治疗非小细胞肺癌的研究

发布时间：2018-09-06 18:57

【摘要】：全球范围来说,肺癌是最常见的恶性肿瘤之一。在肺癌类型中,大约85%左右的都是非小细胞肺癌(NSCLC)。目前,肺癌的治疗方式一般有手术切除,放疗,化疗,但是这些常见的治疗手段并没有显著性地延长肺癌病人的生存周期。分子靶向治疗是近年肺癌治疗上的重大突破。针对NSCLC有效的药物主要是表皮生长因子受体(EGFR)酪氨酸激酶抑制剂(Tyrosine kinase inhibitors,TKI),还有针对棘皮动物微管相关蛋白样4 (EML4) /间变淋巴瘤激酶(ALK)融合基因EML4-ALK突变的药物,还有其他分子靶向药物。分子靶向药物的治疗效果很好,但是肺癌病人在用药1年左右往往会出现耐药现象。而且最近引入的肿瘤免疫疗法,例如免疫检查点抑制剂CTLA-4抗体,PD-1抗体,PD-L1抗体对部分病人有效,对另外一部分病人则无应答。所以免疫检查点抑制剂的疗效还有待进一步的提高。因此,肺癌治疗仍然需要新的治疗方案。嵌合型抗原受体基因修饰的T细胞(CART)作为肿瘤靶向免疫治疗,在体外和临床实验中都表现出良好的靶向性和有效的杀伤性,其中以靶向CD19分子的CAR-CD19T在治疗白血病和淋巴瘤中获得了令人兴奋的结果,取得了巨大的成功。但是,CART细胞在实体瘤中的应用并没有取得很好的疗效,主要是因为实体瘤缺少真正的肿瘤特异性的抗原,还有其表面的肿瘤相关性抗原的异质性。简而言之,就是选取不同的肿瘤相关性抗原作为CART细胞的靶点,在不同的实体瘤取得的治疗效果不同,即使是同一个肿瘤也有可能治疗效果不一样。因此,选择肿瘤相关性抗原作为CART的靶点来治疗实体瘤是很重要的。只有很少的肿瘤相关性抗原已经被筛选出来作为治疗NSCLC的CART细胞的靶点。最近有文献报道,聚糖蛋白-3 (glypican-3)作为治疗非小细胞肺癌中-肺鳞状细胞癌,是非常有希望的CART的靶点。然而,在针对表皮生长因子受体(EGFR)的CART细胞治疗,在临床一期的数据表明,在11个病人中只有2个病人达到了部分缓解。黏蛋白MUC1,是一种跨膜的糖蛋白,在很多类型的肿瘤,包括非小细胞肺癌都会有异常的高表达。以黏蛋白MUC1为CART细胞治疗靶点的临床试验正在招募四种类型的实体瘤病人,其中就包括非小细胞肺癌的病人(临床试验编号:NCT02587689)。因此,基于以上研究进展,黏蛋白MUC1可以是作为治疗非小细胞肺癌的一个很有希望的CAR T细胞的靶点。前列腺干细胞抗原(PSCA)是以糖基磷脂酰肌醇(GPI)方式锚定在细胞表面的肿瘤相关性抗原,主要是在前列腺癌表面异常高表达,也有报道说PSCA也在其他肿瘤表面,例如胆囊腺癌和胃癌。有意思的是,也有报道证明PSCA经常在非小细胞肺癌肺癌中过表达。当然,PSCA是否在非小细胞肺癌中具有普遍性的过表达还需要进一步的验证。但是基于PSCA抗原的治疗方案到目前来说已经比较成熟。有很多报道表明,基于抗PSCA抗体的,以PSCA作为靶点的治疗方式,以及多肽疫苗已经用来治疗前列腺癌。进一步来说,以PSCA为靶点的CART细胞疗法已经用来在人源化小鼠中来治疗胰腺癌。而且,靶向PSCA抗原的CART细胞治疗前列腺癌,膀胱癌和胰腺癌的临床试验已经在进行中(临床试验编号:NCT02092948; NCT02744287)。以上的临床前实验以及在进行的临床试验已经充分说明PSCA是一个很理想的CART细胞治疗靶点。但是,关于PSCA是否也可以作为治疗非小细胞肺癌的CART细胞的靶点,还没有相关的文献报道。这也是本文中需要研究证明的。病人来源的异种移植模型(PDX model)已经被广泛应用到转化医学研究中,特别是人类癌症的研究。在PDX模型中,来自病人的原代标本可以在免疫缺陷的小鼠中不断传代,而且在不同的代数之间保持了与原代标本基本上相同的特征。在本论文的第一部分,我们首先建立了病人来源的非小细胞肺癌的PDX模型,并且验证了在小鼠体内的非小细胞肺癌仍然维持着与原代病人标本相似的形态、免疫表型、分子标记、基因表达水平等特点。紧接着的下一步实验,我们用免疫组化的方式证明了在非小细胞肺癌PDX模型中确实有MUC1和PSCA过表达的现象。然后,我们分别成功构建了靶向MUC1和PSCA的嵌合抗原受体(MUC1.CAR和PSCA.CAR)的慢病毒载体,并在体外用肺癌细胞系验证了靶向MUC1和PSCA的嵌合抗原受体T细胞杀伤的特异性以及有效性。最后,在PDX模型中,靶向PSCA的嵌合抗原受体T细胞可以抑制异常高表达PSCA的非小细胞肺癌在小鼠体内的生长。更重要的是,靶向PSCA的嵌合抗原受体T细胞联合靶向MUC1的嵌合抗原受体T细胞,在治疗PDX模型中表达PSCA和MUC1双阳性的非小细胞肺癌的效果更加显著。综上所述,在本论文中,我们的研究表明肿瘤相关性抗原PSCA和MUC1都可以作为治疗非小细胞肺癌的CAR T细胞的很好的靶点,而且两种CAR T细胞的联合治疗会进一步加强抗肿瘤的疗效。
[Abstract]:Lung cancer is one of the most common malignancies worldwide. About 85% of lung cancer types are non-small cell lung cancer (NSCLC). Currently, lung cancer is usually treated by surgery, radiotherapy, chemotherapy, but these common treatments do not significantly prolong the survival cycle of lung cancer patients. Effective drugs for NSCLC include epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), echinoderm microtubule-associated protein-4 (EML4) / anaplastic lymphoma kinase (ALK) fusion gene EML4-ALK mutations, and other molecules. Targeted drugs. Molecular targeted drugs have a good therapeutic effect, but lung cancer patients are often resistant to drugs for about a year. And recently introduced tumor immunotherapy, such as immunocheckpoint inhibitor CTLA-4 antibody, PD-1 antibody, PD-L1 antibody is effective in some patients, but not in others. So immunoassay The efficacy of point-checking inhibitors needs to be further improved. Therefore, new treatments are still needed for lung cancer. Chimeric antigen receptor gene-modified T cells (CART), as tumor-targeted immunotherapy, have shown good targeting and effective killing effects in vitro and clinical trials, including CAR-CD19T, which targets CD19 molecules. The exciting results have been achieved in the treatment of leukemia and lymphoma, and great success has been achieved. However, the use of CART cells in solid tumors has not been very effective, mainly because solid tumors lack real tumor-specific antigens, and the heterogeneity of tumor-associated antigens on their surfaces. Choosing different tumor-associated antigens as the targets of CART cells has different therapeutic effects in different solid tumors, and even the same tumor may have different therapeutic effects. Therefore, it is important to select tumor-associated antigens as the targets of CART to treat solid tumors. Recently, it has been reported that polyglycoprotein-3 (glypican-3) is a promising target for CART in non-small cell lung cancer (NSCLC). Mucoprotein MUC1, a transmembrane glycoprotein, is abnormally high in many types of tumors, including non-small cell lung cancer. Clinical trials targeting mucin MUC1 as a CART cell therapy target are recruiting four types of solid tumor patients, including non-small cell lung cancer. Patient (Clinical Trial Number: NCT02587689). Therefore, based on these advances, mucin MUC1 may be a promising target for the treatment of non-small cell lung cancer (NSCLC). Prostate stem cell antigen (PSCA) is a tumor-associated antigen anchored on the cell surface by glycosylphosphatidylinositol (GPI), predominantly preceding. It is interesting to note that PSCA is frequently overexpressed in non-small cell lung cancer. Of course, whether PSCA is ubiquitous in non-small cell lung cancer needs further validation. Antigen-based therapies have been well established. Many reports have shown that PSCA-targeted therapies based on anti-PSCA antibodies, and polypeptide vaccines have been used to treat prostate cancer. Furthermore, PSCA-targeted CART cell therapies have been used to treat pancreatic cancer in humanized mice. Pre-clinical trials and clinical trials have shown that PSCA is an ideal target for the treatment of prostate cancer, bladder cancer and pancreatic cancer (clinical trial number: NCT02092948; NCT02744287). Patient-derived xenotransplantation (PDX) models have been widely used in translational medicine, especially in the study of human cancers. In PDX models, primary specimens from patients can be used in the treatment of non-small cell lung cancer. In the first part of this paper, we first established a PDX model of patient-derived non-small cell lung cancer (NSCLC) and verified that NSCLC in mice still maintained the same characteristics as the original specimen. Similar morphology, immunophenotype, molecular markers, and gene expression levels were observed. Next step, we demonstrated that MUC1 and PSCA were overexpressed in the PDX model of non-small cell lung cancer by immunohistochemistry. Then, we successfully constructed chimeric antigen receptors (MUC1.CAR and PSCA.C) targeting MUC1 and PSCA, respectively. Finally, in PDX model, chimeric antigen receptor T cells targeting PSCA can inhibit the growth of non-small cell lung cancer cells with abnormally high expression of PSCA. Chimeric antigen receptor T cells targeting MUC1 combined with chimeric antigen receptor T cells targeting PSCA are more effective in the treatment of non-small cell lung cancer with positive expression of PSCA and MUC1 in PDX models. The combination of two CAR T cells will further enhance the antitumor efficacy.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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