Anti-HER3抗体抑制Herceptin耐药及功能优化研究

发布时间：2019-03-15 08:57

【摘要】：随着肿瘤发病分子机制的深入研究,针对关键基因、特异性细胞受体和调控分子的靶向性治疗已成为肿瘤临床治疗的重要途径。抗体药物以其特异性、靶向性、安全性以及临床有效性等优势在肿瘤靶向性治疗中发挥重要作用,目前比较热门的肿瘤治疗的靶标包括HER家族等一系列分子。HER2是HER家族成员之一,在乳腺癌、卵巢癌等多种癌症中过表达。Herceptin、Perjeta、T-DM1等多个抗体、抗体-小分子偶联药物成功上市,并逐步成为HER2阳性肿瘤的临床治疗一线用药。虽然HER2抗体在HER2阳性肿瘤的临床治疗中取得良好效果,但是HER2阳性乳腺癌的反应率仅为20~30%,及Herceptin治疗后期出现抗体耐药的现象限制了抗体靶向性治疗范围和效率。因此,明确抗体的耐药机制以及确定评估抗体疗效的生物标志物,对于优化治疗方案、提高疗效具有重要的意义。HER3是表皮生长因子受体HER家族成员,是PI3K/AKT信号通路中关键的激活因子。研究显示,多种肿瘤在使用EGFR和HER2抑制剂治疗时,HER3的表达上调,磷酸化水平提高。因此,HER3在抗肿瘤中的作用逐步得到重视。各种靶向HER3的抑制剂中,单克隆抗体主要通过阻断HER3与配体HRG结合或者阻断HER3与其他HER家族成员形成异二聚体等分子机制,阻断HER3下游信号通路激活,从而抑制肿瘤细胞生长。目前靶向HER3的单克隆抗体药物Duligotuzumab、Patritumab均已进入临床II期研究,Seribantuma、DL11、Seribantumab、LJM716、RG7116等多个抗体处于临床前研究中。治疗性抗体具有特异性高、半衰期长、副作用小等诸多优势,但因为其分子量大从而限制了其单独用药疗效。近年来,抗体药物偶联(antibody-drug conjugate,ADC)技术发展迅速。ADC药物结合了抗体药物和细胞毒性药物两者的优点,具有抗体药物的特异性和细胞毒药物的强细胞毒性,在治疗肿瘤上取得了更好效果,是新一代靶向治疗药物的主要研究方向。Mylotarg TM、Adcetris TM、Kadcyla TM等ADC药物成功上市,使得人们更为关注ADC药物。本研究内容基于前期获得的对曲妥珠(Herceptin)具有耐药性的卵巢癌细胞系(SKOV3/T),观察比较了具有耐药性的SKOV3/T细胞的增殖、信号通路和成瘤性的差异,并对SKOV3/T耐药性中出现的HER3表达异常进行了深入探讨;针对HER3靶点设计、优化、筛选、表达了靶向HER3的全人单克隆抗体,并对HER3全人单克隆抗体和价靶向HER3的ADC药物进行了体、内外功能活性评价。本研究集中于以下三个方面:(一)曲妥珠耐药细胞株SKOV3/T中HER3异常表达合理评价曲妥珠耐药卵巢癌细胞系SKOV3/T的细胞增殖及成瘤能力。体外细胞增殖实验以及体内荷瘤动物实验结果均表明,SKOV3/T细胞增殖及成瘤能力显著高于正常卵巢癌细胞SKOV3。进一步通过流式细胞术和Western blot方法对SKOV3/T细胞和SKOV3细胞表面HER1、HER2以及HER3表达水平进行鉴定。实验结果显示,与SKOV3细胞株相比,SKOV3/T细胞株中HER1基因的表达未发生明显改变,HER2基因表达显著下调,而HER3基因表达上调。(二)靶向HER3抗体抑制曲妥珠耐药肿瘤细胞为了验证HER3在曲妥珠耐药中的作用,我们合成表达了特异性抗HER3抗体Lm Ab3,并对其生物学活性进行检定。实验结果显示,Lm Ab3可以特异性识别重组表达的HER3抗原和天然的HER3分子,亲和力Kon值为2.46E-10,并能特异性抑制HER家族分子磷酸化水平,并抑制HER3阳性的MCF7肿瘤细胞增殖。进一步利用特异性抗HER3抗体Lm Ab3为工具,探讨HER3在曲妥珠耐药中的作用。三维培养实验结果显示,特异性anti-HER3抗体阻断后,SKOV3/T细胞增殖被显著抑制。建立小鼠SKOV3/T耐药细胞皮下荷瘤模型,使用特异性anti-HER3抗体治疗后,肿瘤生长受到抑制,而生理盐水对照组和Heceptin治疗组则对SKOV3/T肿瘤未见明显抑制作用。为了探讨HER3抗体抑制曲妥珠耐药细胞中的分子机制,我们进一步分析HER3激活的下游信号通路中HER1、HER2、HER3以及AKT等关键分子磷酸化水平,特异性anti-HER3抗体阻断后,HER1、HER2、HER3以及AKT等分子磷酸化程度均受抑制。(三)靶向HER3抗体的优化及生物学功能评价利用分子模拟技术对靶向HER3抗体进行合理优化设计,结合生物学功能分析,获得了一株高亲和力、特异性识别HER3的新抗体FD001。进一步利用抗体偶联技术将抗HER3抗体FD001与毒物小分子DM1进行偶联,获得靶向HER3的ADC药物FD001-DM1。体内外生物学实验结果显示,FD001-DM1具有良好的抑制肿瘤增殖能力。本研究的创新之处:初步揭示了曲妥珠耐药细胞株SKOV3/T中HER3异常表达,特异性Anti-HER3抗体可一定程度上抑制曲妥珠耐药细胞增殖。进一步利用计算机分子模拟技术优化设计并获得了高亲和力抗HER3新抗体FD001,并将该抗体偶联小分子药物DM1,获得ADC分子FD001-DM1;FD001-DM1具有良好的体内外抑制肿瘤增殖效应。
[Abstract]:With the further study of the molecular mechanism of the pathogenesis of the tumor, the targeted therapy for the key genes, the specific cell receptors and the regulatory molecules has become an important way of the clinical treatment of the tumor. Antibody drugs play an important role in the treatment of tumor targeting with the advantages of specificity, targeting, safety and clinical efficacy. HER2 is one of the HER family members and is overexpressed in a variety of cancers such as breast cancer, ovarian cancer and the like. Herceptin, Perjeta, T-DM1 and other antibodies, antibody-small-molecule coupled drugs have been successfully listed and become the first-line drugs for HER2-positive tumors. Although the HER2 antibody has a good effect in the clinical treatment of HER2-positive tumors, the reaction rate of the HER2-positive breast cancer is only 20-30%, and the phenomenon of antibody resistance in the later stage of Herceptin treatment limits the therapeutic range and efficiency of the antibody. Therefore, the resistance mechanism of the antibody and the biomarkers for determining the therapeutic effect of the antibody have important significance for optimizing the treatment scheme and improving the curative effect. HE3 is a member of the HER family of the epidermal growth factor receptor, which is the key activation factor in the PI3K/ AKT signal pathway. The study showed that the expression of HE3 was up-regulated and the level of phosphorylation increased when multiple tumors were treated with EGFR and HER2 inhibitors. Therefore, the role of HE3 in anti-tumor has been paid more and more attention. In the various inhibitors of the targeting HE3, the monoclonal antibody can block the activation of the downstream signal path of the HER3 by blocking the binding of the HE3 with the ligand HRG or blocking the molecular mechanism of the HE3 and other HER family members, so as to inhibit the growth of the tumor cells. The current monoclonal antibody drug, Duliginozumab, targeting HE3, has entered clinical Phase II studies, and multiple antibodies such as Seribianuma, DL11, Serbiantumab, LJM716, RG7116, and the like are in preclinical studies. The therapeutic antibody has the advantages of high specificity, long half-life, small side effect and the like, but because of the large molecular weight, the curative effect of the therapeutic antibody is limited. In recent years, the technology of antibody drug coupling (ADC) has been developed rapidly. The drug combination of the ADC combines the advantages of both the antibody drug and the cytotoxic drug, has the specificity of the antibody drug and the strong cytotoxicity of the cytotoxic drug, has achieved a better effect on the treatment of the tumor, and is the main research direction of the next generation of targeted therapy medicaments. ADC drugs, such as Mylotarg (TM), Adcetoris (TM), Kadcylla (TM), have been successfully marketed, making it more interesting to focus on the ADC drug. The content of SKOV3/ T cell line (SKOV3/ T) with resistance of SKOV3/ T cells was observed, and the expression of HER3 in SKOV3/ T drug resistance was discussed. Aiming at the design, optimization and screening of the HER3 target, the whole human monoclonal antibody targeting the HER3 is expressed, and the human monoclonal antibody and the price of the HER3 human monoclonal antibody and the price target HER3 are used for carrying out the body, the internal and external functional activity evaluation. The study focused on the following three aspects: (1) the abnormal expression of HER3 in trastuzumab-resistant cell line SKOV3/ T can reasonably evaluate the cell proliferation and tumor-forming ability of trastuzumab-resistant ovarian cancer cell line SKOV3/ T. The results of in vitro cell proliferation and in vivo tumor-bearing animals showed that SKOV3/ T cell proliferation and tumor-forming ability were significantly higher than that of normal ovarian cancer cells SKOV3. The expression levels of HE1, HER2 and HER3 in SKOV3/ T cells and SKOV3 cells were further identified by flow cytometry and Western blot. The results of the experiment show that the expression of the HER1 gene in the SKOV3/ T cell line has not changed significantly compared with the SKOV3 cell line, and the expression of the HER2 gene is down-regulated, while the expression of the HER3 gene is up-regulated. And (2) targeting the HER3 antibody to inhibit the trastuzumab-resistant tumor cell in order to verify the role of the HER3 in the drug resistance of the trastuzumab, and the specific anti-HER3 antibody Lm Ab3 is synthesized and the biological activity is verified. The results of the experiment show that Lm Ab3 can specifically identify the HE3 antigen and the natural HER3 molecule of the recombinant expression, the affinity Kon value is 2.46E-10, and can specifically inhibit the phosphorylation level of the HER family, and inhibit the proliferation of the HR3-positive MCF7 tumor cells. The role of HER3 in the drug resistance of trastuzumab was also discussed by using the specific anti-HER3 antibody Lm Ab3 as a tool. The results of three-dimensional culture showed that the proliferation of SKOV3/ T cells was significantly inhibited after the specific anti-HER3 antibody was blocked. The mouse SKOV3/ T-resistant subdermal tumor-bearing model was established, and the tumor growth was inhibited after the treatment with the specific anti-HER3 antibody, while the normal saline control group and the Heceptin treatment group showed no significant inhibition on the SKOV3/ T tumor. In order to study the molecular mechanism of the HER3 antibody in the inhibition of trastuzumab-resistant cells, we further analyzed the phosphorylation levels of the key molecules such as HE1, HER2, HE3 and AKT in the downstream signal path activated by HER3, after the specific anti-HER3 antibody was blocked, HER1, HER2, The degree of phosphorylation of HE3 and AKT is inhibited. (3) The optimized and biological function evaluation of the targeted HE3 antibody is designed by using the molecular simulation technology to reasonably optimize the targeting HE3 antibody, and a high affinity is obtained in combination with the biological function analysis to obtain a new antibody FD001 with high affinity and specific identification of HE3. The anti-HER3 antibody FD001 is further coupled with the poison micromolecule DM1 by using the antibody coupling technology to obtain the ADC drug FD001-DM1 targeting the HER3. The results of in-vivo biological experiments show that FD001-DM1 has a good ability to inhibit the proliferation of tumor. The innovation of this study is that the abnormal expression of HE3 in the resistant cell line SKOV3/ T of the trastuzumab is preliminarily revealed, and the specific Anti-HER3 antibody can inhibit the proliferation of trastuzumab-resistant cells to a certain extent. The invention further utilizes the computer molecular simulation technology to optimize the design and obtain the high-affinity anti-HER3 new antibody FD001, and the antibody is coupled with the small-molecule drug DM1 to obtain the ADC molecular FD001-DM1; and the FD001-DM1 has a good in-vivo inhibition of the tumor proliferation effect.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.5

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