共刺激分子增强双功能抗体介导的T细胞杀伤

发布时间：2018-01-12 08:21

本文关键词：共刺激分子增强双功能抗体介导的T细胞杀伤　出处：《北京协和医学院》2011年硕士论文　论文类型：学位论文

【摘要】：背景与目的T淋巴细胞是细胞免疫的主要效应细胞,其活化程度直接影响抗肿瘤治疗效果。调控T细胞增殖成为效应细胞需要抗原肽-MHC分子复合物介导的特异性刺激信号和B7、4-1BBL等分子提供的共刺激信号。如果在增殖分化过程中未能获得共刺激信号,T细胞可能会出现活化诱导的细胞死亡(activation-induced cell death, AICD)或者无能状态,进而导致抗肿瘤免疫功能低下。而通过低表达共刺激分子降低免疫原性,使得识别它的抗原提呈细胞或淋巴细胞得不到充分的活化信号,是肿瘤细胞免疫逃逸的重要机制之一。本课题通过克隆人4-1BBL胞膜外区及构建4-1BBL/anti-CD20融合蛋白来激活T细胞表面4-1BB信号通路、通过联合应用阿糖胞苷提高靶细胞表面B7分子的表达水平达到提高共刺激信号的目的,以促进淋巴细胞的活化,增强抗肿瘤免疫反应,更好地抑制肿瘤甚至彻底清除肿瘤。方法与结果第一部分实验中,体外用CytoTox96Nonradioactived Assay kit检测了4-1BBL胞膜外区蛋白联合anti-CD3/anti-Pgp微型双特异抗体对靶向的K562/A02细胞的细胞毒作用,用Calcein-AM法检测了4-1BBL胞膜外区蛋白及4-1BBL/anti-CD20融合蛋白联合anti-CD3/anti-CD20微型双特异抗体对靶向的Raji细胞的细胞毒作用,证明增强4-1BBL信号确实能够提高T细胞杀伤效果,而具有CD20和4-1BBL双靶向的融合蛋白激活信号能力较单一的4-1BBL分子更为理想,联合杀伤效果更为突出。在体内联合应用4-1BBL胞膜外区蛋白和anti-CD3/anti-Pgp治疗裸鼠K562/A02移植瘤,联合应用4-1BBL/anti-CD20和anti-CD3/anti-CD20治疗裸鼠Raji移植瘤,显著控制了肿瘤的生长并清除肿瘤,观察至100天未见肿瘤复发。第二部分实验中,取不同终浓度的抗代谢类化疗药阿糖胞苷(Ara-C),作用靶细胞72h,FACS测定B7分子(CD80和CD86)表达。取不同终浓度Ara-C作用靶细胞72h,MTT法检测Ara-C对靶细胞的抑制率。确定使靶细胞CD80和CD86表达升高但又不产生抑制效果的Ara-C终浓度0.251μmol/l。Calcein-AM法测定双功能抗体联合Ara-C的体外杀伤活性,杀伤效果比未经Ara-C处理的效果明显(p0.05)。体内采用K562/A02细胞移植瘤模型,联合阿糖胞苷和anti-CD3/anti-PGP Diabody治疗,效果优于双功能抗体单用,并且对防止肿瘤二次复发有很好效果。结论4-1BBL、4-1BBL/anti-CD20和anti-CD3/anti-Pgp、anti-CD3/anti-CD20联合使用体内外均有效激活T细胞,增强其杀伤作用。非抑制剂量化疗药阿糖胞苷使靶细胞CD80表达升高,再联合双功能抗体使体外介导T细胞杀伤靶细胞的效果提高近20%。体内K562/A02移植瘤模型也证实阿糖胞苷在双功能抗体的治疗中起到重要辅助作用,可以有效提升疗效。以上结果均说明共刺激途径的激活对于维持和提高T细胞杀伤能力是至关重要的。
[Abstract]:Background and objective T lymphocyte is the main effector cell immunity, its activation degree directly affect the anti tumor therapy effect. The proliferation of T cells into costmulatory signal effect cells require specific stimulation signal and B7,4-1BBL antigen -MHC molecular complex mediated offer. If failed in the proliferation and differentiation in the process of CO the stimulation signal, T cells may lead to activation induced cell death (activation-induced cell, death, AICD) or incompetent state, resulting in antitumor immunity. The low expression of costimulatory molecules to reduce immunogenicity, it makes the recognition of antigen presenting cells or lymphocytes are not fully activated signal is. One of the important mechanisms of tumor immune escape. The cloning of human 4-1BBL extracellular domain and construct 4-1BBL/anti-CD20 fusion protein to activate T cell surface 4 -1BB signaling pathway can enhance the expression level of B7 molecules on target cells by enhancing the expression level of target molecules on the surface of target cells, so as to enhance the activation of costimulatory signals, promote the activation of lymphocytes, enhance the anti-tumor immune response, and further inhibit the tumor and even completely remove the tumor.
Methods and results
The first part of the experiment, the in vitro detection of extracellular domain of human 4-1BBL and anti-CD3/anti-Pgp micro bispecific antibody targeting on the cytotoxicity of K562/A02 cells by CytoTox96Nonradioactived Assay kit, the extracellular domain of human 4-1BBL and 4-1BBL/ anti-CD20 fusion protein and anti-CD3/anti-CD20 Mini bispecific antibody targeting on the cytotoxicity of Raji cells detected by Calcein-AM method that enhanced 4-1BBL signal can indeed improve the killing effect of T cells, with CD20 and 4-1BBL dual targeting fusion protein activation signal ability than a single 4-1BBL molecule is more ideal, and the killing effect is more outstanding. In the combined application of in vivo extracellular domain of human 4-1BBL and anti-CD3/anti-Pgp treatment of K562/A02 xenografts in nude mice. The combined application of 4-1BBL/anti-CD20 and anti-CD3/anti-CD20 treatment of Raji xenografts in nude mice, significantly control tumor growth The tumor was removed and no recurrence of the tumor was observed for 100 days.
The second part of the experiment, cytarabine from different concentrations of anti metabolism drugs (Ara-C), the target cell 72h, FACS of B7 molecules (CD80 and CD86). The expression of different concentrations of Ara-C target cells 72h, inhibition of target cell detection rate of Ara-C MTT method. To determine the target cells of CD80 and the expression of CD86 increased but does not produce the inhibitory effect of Ara-C determination of cytotoxicity of bispecific antibody combined with Ara-C in vitro at concentration of 0.251 mol/l.Calcein-AM, the killing effect than the untreated Ara-C (P0.05). The effect is obvious in vivo by using K562/A02 cell xenograft model combined with cytarabine and anti-CD3/anti-PGP Diabody treatment is better than the double function antibody alone, and to prevent the tumor recurred two times has a very good effect.
Conclusion 4-1BBL, 4-1BBL/anti-CD20 and anti-CD3/anti-Pgp, anti-CD3/anti-CD20 combined with in vitro and in vivo effectively activate T cells and enhance its cytotoxic effect. Non inhibitor chemotherapy cytarabine target cells increase the expression of CD80, and combined with the bifunctional antibody in vitro mediated T cell target cell killing effect increased by nearly 20%. in K562/A02 transplanted tumor model also confirmed Ara plays an important role in the treatment of auxiliary bifunctional antibody, can effectively improve the curative effect. The above results suggest that the activation of costimulatory pathway is crucial for maintaining and improving the ability of T cells.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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