CD133表位联合gp96佐剂疫苗引发抗淋巴细胞性白血病的免疫应答

发布时间：2018-07-05 09:39

本文选题：CD133 + 表位　；参考：《河北大学》2017年硕士论文

【摘要】：白血病是青少年常见的恶性肿瘤之一,由于化疗、骨髓移植和嵌合抗原受体T细胞(CAR T)治疗技术的逐渐提高,使得白血病的5年存活率达62%以上。然而,白血病的复发和难治疗性的问题仍然比较严峻。虽然临床试验已经有药物用于靶向治疗白血病,但是耐药性和毒副作用等问题比较显著。由于肿瘤特异性T细胞,特别是CD8+T细胞(CTL)在杀伤和清除肿瘤中发挥重要作用,因此研究新型特异性高、安全性好的肿瘤免疫治疗性疫苗用于活化特异性CTL,是应对白血病的有效手段之一。肿瘤干细胞因为具备自我更新、无限增殖和分化形成肿瘤的能力,并且对化疗、放疗具有抗性,被认为是肿瘤复发的根源所在。多种肿瘤中都发现了CD133~+肿瘤干细胞,提示CD133抗原可以作为CTL识别的靶点,这也为设计新型白血病治疗性疫苗提供了线索。热休克蛋白gp96作为分子伴侣不仅可以激活T细胞免疫,同时也可以激活先天免疫。本研究首先利用昆虫细胞表达系统,表达有免疫学功能的重组gp96蛋白。通过表位预测,发现CD133含有6个潜在H2-Kd限制性的表位,分别是CD133272-281(QNMSSSLKSL)、CD133419-428(RYLNQELPKL)、CD133452-461(FFFLGLLCGV)、CD133601-609(NLNVNIDSI)、CD133702-710(TLRQSVWTL)和CD133760-769(HYLHWVFYAI)。进一步将重组gp96作为T细胞疫苗佐剂,通过小鼠免疫实验鉴定出3个可活化特异性的CTL表位,分别是CD133419-428、CD133702-710和CD133760-769。在此基础上,将这三个表位联合gp96佐剂制备新型表位疫苗,在小鼠CD133~+淋巴细胞性白血病的模型中,CD133表位疫苗引发抗肿瘤特异性T细胞免疫应答。在预防模型中,免疫表位疫苗后小鼠肿瘤体积和重量与对照组相比都下降了43%(P0.05);ELISPOT实验证明表位疫苗免疫后CD133419-428、CD133702-710和CD133760-769表位特异的T细胞比对照组分别增加了4.6、0.6和1.2倍;分离免疫后小鼠的脾脏T细胞进行细胞毒性实验,发现当效应细胞与靶细胞比为20:1时,表位疫苗组对靶细胞的特异性杀伤作用明显地高于对照组(P0.05)。在治疗模型中,免疫表位疫苗后小鼠肿瘤体积和重量与对照相比分别下降了31%(P0.05)和30%(P0.05);分离免疫后小鼠的脾脏T细胞进行细胞毒性实验,发现当效应细胞与靶细胞比为20:1时,表位疫苗组对靶细胞的特异性杀伤作用极显著地高于对照组(P0.01)。最后,在荷瘤小鼠体内转输CD133表位疫苗激活的特异T细胞,能显著地抑制肿瘤生长。综上所述,本研究以小鼠淋巴细胞性白血病为模型,设计了以gp96为佐剂的、新型的抗CD133~+肿瘤的治疗性表位疫苗,通过小鼠实验验证该疫苗能够活化CD133特异性CTL应答,显著抑制小鼠淋巴瘤的生长,这为进一步研发靶向CD133~+的白血病等肿瘤治疗性疫苗提供了依据。
[Abstract]:Leukemia is one of the most common malignant tumors in adolescents. Because of chemotherapy, bone marrow transplantation and treatment of chimeric antigen receptor T cells (car T), the 5-year survival rate of leukemia is over 62%. However, the recurrence of leukemia and difficult to treat the problem is still relatively serious. Although drugs have been used to target leukemia in clinical trials, drug resistance and side effects are significant. Because tumor specific T cells, especially CD8 T cells (CTL), play an important role in killing and clearing tumors, new types of tumor specific T cells are studied with high specificity. Tumor immunotherapy vaccine with good safety is one of the effective methods for activating specific CTLs. Tumor stem cells have the ability of self-renewal, infinite proliferation and differentiation to form tumors, and are resistant to chemotherapy and radiotherapy, which is considered to be the root cause of tumor recurrence. CD133- tumor stem cells have been found in many kinds of tumors, suggesting that CD133 antigen can be used as a target for CTL recognition, which provides clues for the design of a new therapeutic vaccine for leukemia. Heat shock protein (gp96), as a molecular chaperone, not only activates T cell immunity, but also activates innate immunity. In this study, the recombinant gp96 protein with immunological function was expressed by insect cell expression system. By epitope prediction, it was found that CD133 contained six potential H2-Kd restricted epitopes, CD133272-281 (QNMSSSLKSL), CD133419-428 (RYLNQELPKL) CD133452-461 (FFLGLLCGV) CD133601-609 (NLNVNIDSI) CD133702-710 (TLRQSVWTL) and CD133760-769 (HYLHWVFYAI). The recombinant gp96 was further used as the adjuvant of T cell vaccine. Three specific CTL epitopes, CD133419-428, CD133702-710 and CD133760-769, were identified by immunological assay. On this basis, the three epitopes combined with gp96 adjuvant were used to prepare the novel epitope vaccine, and CD133 epitope vaccine induced anti-tumor specific T cell immune response in mouse CD133- lymphocytic leukemia model. In the preventive model, the tumor volume and weight of mice after immunization with epitope vaccine decreased by 43% (P0.05) compared with the control group (P0.05). The results of Elisa showed that the epitope specific T cells of CD133419-428 and CD133702-710 and CD133760-769 increased 4.60.6 and 1.2 times, respectively, compared with the control group. When the ratio of effector cells to target cells was 20:1, the specific killing effect of epitope vaccine group on target cells was significantly higher than that of control group (P0.05). In the treatment model, the tumor volume and weight of mice decreased by 31% (P0.05) and 30% (P0.05), respectively, compared with the control group, and the cytotoxicity test of spleen T cells of the immunized mice showed that when the ratio of effector cells to target cells was 20:1, the tumor size and weight of mice decreased by 31% (P0.05) and 30% (P0.05) respectively after immunization, and the ratio of effector cells to target cells was 20:1. The specific killing effect of epitope vaccine group on target cells was significantly higher than that of control group (P0.01). Finally, transfusions of specific T cells activated by CD133 epitope vaccine in tumor-bearing mice significantly inhibited tumor growth. To sum up, a novel therapeutic epitope vaccine against CD133~ tumor was designed using gp96 as adjuvant in murine lymphoblastic leukemia model. The results of mouse experiments showed that the vaccine could activate CD133-specific CTL response. The growth of murine lymphoma was significantly inhibited, which provided a basis for the further development of tumor therapeutic vaccines targeting CD133- to leukemia.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7

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