人CD38分子的克隆表达、单克隆抗体的制备及其功能的初步研究

发布时间：2018-02-16 20:03

本文关键词： CD38 基因克隆基因表达载体单克隆抗体抗体识别表位　出处：《第二军医大学》2005年博士论文　论文类型：学位论文

【摘要】：淋巴瘤、骨髓瘤是严重威胁人类生命的恶性肿瘤,对于这类疾病的治疗,传统的化疗、放疗、免疫调节等治疗方法,难以取得令人满意的疗效。近年来,针对细胞表面分子的抗原靶向性治疗已取得了巨大进展,并成为一种非常有发展前途的治疗方法。CD38分子是一个功能性的膜分子,最初被认为是一种活化抗原,主要用于白细胞分类及表型鉴定。但近年来,人们发现CD38分子表达与分布相当广泛,无细胞系限制性,而与细胞的分化和活化状态有关。CD38分子具有许多复杂而又独特的生物学特性及功能:1)受体特性,与其配体或相应单克隆抗体结合对不同细胞可产生多种复杂的生物学效应;2)双功能酶活性:CD38分子的胞外结构域与核糖核苷二磷酸(ADPR)酶家族同源,能催化NAD+产生cADPR,cADPR被认为是一种第二信使,可不依赖于IP_3调节胞内Ca~(2+)浓度。3)粘附分子特性,参与调节淋巴细胞再循环及淋巴细胞归巢。4)细胞活化及细胞因子产生因此,对CD38分子的研究越来越受到广泛的重视。有文献报道,CD38分子的单克隆抗体可用于人骨髓瘤、淋巴瘤的临床治疗。研究结果显示,由抗CD38的单克隆抗体与毒素相偶联所形成的免疫毒素,对表达CD38的肿瘤细胞有较强的毒性,而对正常外周血细胞的毒性很小,几可忽略。说明,这种方法对骨髓瘤、淋巴瘤、白血病的治疗会非常有效。目前已报道的CD38分子的单克隆抗体根据其识别的抗原表位不同,大致可分为两组,分别识别胞外段近羧基端220—241位氨基酸(OKT10、AT1和SUN-487)和273—285位氨基酸(IB4、AT2和IB6),C.M.Ausiello等研究表明,已报道的CD38单抗IB4和OKT10是功能性抗体,它们均可诱导外周血淋巴细胞增殖和细胞因子释放,只有IB4还可诱导胞内蛋白酪氨酸磷酸化和胞内Ca~(2+)浓度增加。说明抗CD38单克隆抗体的功能与其所识别的抗原位点有关。尽管CD38—McAb目前国外已有临床应用,且有较好的发展前景,但国内尚未有功能性抗体的研究报道,而且单抗与相应的分子结合后,产生何种效应机制,单抗识别的抗原表位与其生物学功能之间的关系等都有待研究阐明。基于上述的研究背景和实际的应用情况,本课题旨在制备出有功能活性的CD38-McAb,研究其生物学功能,探讨其可能的效应机制,并对其所识别的抗
[Abstract]:Lymphoma and myeloma are malignant tumors that threaten human life seriously. In recent years, it is difficult to obtain satisfactory results for the treatment of these diseases, traditional chemotherapy, radiotherapy, immunomodulation and so on. Antigen-targeted therapy for cell surface molecules has made great progress and has become a promising therapeutic method. CD38 molecule is a functional membrane molecule, which was initially considered as an active antigen. In recent years, however, it has been found that the expression and distribution of CD38 molecules are quite extensive, and there is no cell line restriction. However, the CD38 molecule has many complex and unique biological characteristics and function: 1) receptor properties related to the differentiation and activation of cells. Binding with its ligands or corresponding monoclonal antibodies can produce many complex biological effects on different cells. 2) the extracellular domain of the bifunctional CD38 molecule is homologous to the ribonucleoside diphosphate ADPRase family. CADPRN cADPR can catalyze the production of NAD and is considered to be a second messenger, independent of IP_3 regulating intracellular Ca~(2 concentration. 3) Adhesion molecule, involved in the regulation of lymphocyte recirculation and lymphocyte homing. 4) Cell activation and cytokine production. More and more attention has been paid to the study of CD38 molecule. It has been reported that the monoclonal antibody of CD38 molecule can be used in the clinical treatment of human myeloma and lymphoma. The immunotoxin formed by the conjugation of monoclonal antibody against CD38 with toxin has strong toxicity to tumor cells expressing CD38, but little toxicity to normal peripheral blood cells, which can be neglected. The treatment of leukemia can be very effective. The reported monoclonal antibodies of CD38 molecules can be divided into two groups according to the antigenic epitopes they recognize. They recognize OKT10 AT1 and SUN-487 at the extracellular region near the carboxyl terminal of the 220-241 amino acids, and the studies on the amino acids IB4, AT2 at the 273-285 sites and C.M. Ausiello at the 273-285 positions show that, The reported CD38 monoclonal antibodies IB4 and OKT10 are functional antibodies that can induce lymphocyte proliferation and cytokine release in peripheral blood. Only IB4 could induce the intracellular protein tyrosine phosphorylation and the increase of intracellular Ca~(2 concentration, which indicated that the function of anti CD38 monoclonal antibody was related to the antigenic site it recognized. Although CD38-McAb has been used in clinic abroad and has a good prospect of development, there are no reports on functional antibodies in China, and what kind of effect mechanism does the monoclonal antibody produce when it binds to the corresponding molecule? The relationship between antigen epitopes recognized by McAbs and their biological functions needs to be elucidated. Based on the above research background and practical application, the purpose of this paper is to prepare CD38-McAbs with functional activity, to study their biological functions, to explore the possible mechanism of their effects, and to study the resistance of CD38-McAbs recognized by CD38-McAbs.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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