靶向ErbB2双特异性抗体在胃癌中的抗肿瘤作用及其机理
发布时间：2018-11-05 09:15
【摘要】：在全世界范围内,每年约有70万人死于胃癌,使得胃癌导致的死亡率在所有肿瘤中高居第二位,而以包括中国在内的大部分发展中国家更是胃癌的重灾区。并且由于大部分胃癌患者均确诊于进展期,使得以包括手术和化疗在内的常规治疗手段难以发挥良好的治疗效果,因而胃癌患者的整体预后相对较差。其中,6%至23%的胃癌患者高表达人表皮生长因子受体2(ErbB2),而该受体的高表达往往预示着更差的预后。近年来随着对肿瘤分子机制的进一步了解以及对大量基础及临床试验数据的分析,人们已经认识到了靶向治疗在进展期胃癌患者中的重要性。作为临床标准疗法的曲妥珠(Trastuzumab)是一种能特异性靶向ErbB2胞外IV区的单克隆抗体,且已被证明了能够延长ErbB2高表达进展期胃癌患者的总生存期。帕妥珠(Pertuzumab)是另一个靶向ErbB2的单克隆抗体,结合ErbB2胞外II区,其在ErbB2高表达进展期胃癌患者中也显示了积极的疗效。而Trastuzumab与Pertuzumab联合应用于ErbB2高表达进展期胃癌患者的临床试验也在进行中,并且已经初步显示出了可喜的结果。尽管ErbB2靶向疗法显示了一定的疗效,但Trastuzumab和Pertuzumab的疗效仍相对温和并且在不同胃癌患者中疗效差异明显,因此提高ErbB2靶向治疗在胃癌患者中的效果显得极为重要。近几十年来,基因工程的飞速发展给我们研发及优化抗体的结构和功能提供了多种多样的选择。在前期研究中,我们通过基因工程构建了一种以Trastuzumab和Pertuzumab为亲本的双特异性抗体TPL。在本研究中,我们进一步探讨此双特异抗体在胃癌中的抗肿瘤作用及机制。首先,通过MTS细胞活力检测实验,我们比较了Trastuzumab,Pertuzumab,Trastuzumab+Pertuzumab以及TPL在体外抑制ErbB2高表达胃癌细胞系N87增殖的能力。Trastuzumab+Pertuzumab联合应用比二者单用能更有效地抑制N87细胞的增殖。重要的是,双特异性抗体TPL显示出具有比Trastuzumab+Pertuzumab明显增强的抑制胃癌细胞增殖的能力。随后通过荷瘤小鼠模型,我们比较了Trastuzumab,Pertuzumab,Trastuzumab+Pertuzumab以及双特异性抗体TPL的体内抑制胃癌细胞的活性。与体外实验结果一致,即Trastuzumab+Pertuzumab联用能比二者单用更为有效地抑制肿瘤的生长,但TPL具有比Trastuzumab+Pertuzumab联用更显著的抗肿瘤作用。此前有研究表明,同时使用两种靶向ErbB2不同区域的单克隆抗体可通过诱导ErbB2内吞而增强靶向治疗的效果。考虑到这可能是双特异性抗体TPL发挥显著抗瘤作用的机制之一,我们比较了Trastuzumab,Pertuzumab,Trastuzumab+Pertuzumab以及双特异性抗体TPL诱导ErbB2内吞的能力。流式细胞术分析以及免疫荧光共聚焦实验表明,单独的Trastuzumab和Pertuzumab在8小时内均不能诱导ErbB2明显内吞,而Trastuzumab+Pertuzumab联用组在处理8小时后才能诱导ErbB2微弱的内吞。但TPL处理N87细胞2小时后就可导致ErbB2发生一定程度的内吞,处理8小时后可引起更为显著的ErbB2内吞。为了进一步阐明靶向ErbB2不同表位抗体组合与其诱导ErbB2内吞能力之间的关系,我们在随后的研究中通过标准杂交瘤技术获得了靶向ErbB2的5株特异性单克隆抗体。这5株单克隆抗体加上Trastuzumab和Pertuzumab可以两两组成21对抗体组合。通过ELISA竞争抑制试验,我们从这21对抗体组合中鉴定出了18对不竞争结合ErbB2的组合。但是免疫荧光共聚焦实验证明,这18对抗体组合中只有其中4对抗体组合能引起明显的ErbB2内吞。综上所述,我们的研究表明通过基因工程构建的双特异性抗体TPL能引起ErbB2的明显内吞并借此在体内外均展示出良好的抗肿瘤活性,该双特异性抗体也有可能发展成为ErbB2高表达进展期胃癌患者的一种新的有效靶向制剂。此外,对于靶向ErbB2不同表位的单克隆抗体组合而言,只有其中特定的抗体组合才能有效诱导ErbB2内吞,这可能与抗体所识别的具体表位有关。
[Abstract]:Around 700,000 people die in gastric cancer every year around the world, causing the death rate of gastric cancer to be second in all tumors, while most developing countries, including China, are the hardest hit areas of gastric cancer. and the overall prognosis of gastric cancer patients is relatively poor due to the fact that most gastric cancer patients are diagnosed with the progression phase, making it difficult to exert a good therapeutic effect in conventional treatment methods including surgery and chemotherapy. Of these, 6% to 23% of gastric cancer patients express high expression of human epidermal growth factor receptor 2 (ErbB2), and high expression of the receptor often indicates a worse prognosis. In recent years, with further understanding of tumor molecular mechanisms and analysis of a large number of basic and clinical trial data, it has been recognized that targeted therapy is of importance in patients with advanced gastric cancer. Trastuzumab as a clinical standard therapy is a monoclonal antibody that specifically targets the extracellular IV region of ErbB2 and has been demonstrated to be able to prolong the overall survival of patients with gastric cancer with high expression of ErbB2. Pertuzumab is another monoclonal antibody targeting ErbB2, binding to the ErbB2 extracellular II region, which also shows a positive therapeutic effect in patients with gastric cancer with high expression of ErbB2. The clinical trials of Trastuzumab in combination with Pertuzumab in patients with gastric cancer with high expression of ErbB2 are also ongoing and a preliminary indication has been shown. Although ErbB2 targeted therapy showed certain efficacy, the efficacy of Trastuzumab and Pertuzumab remained relatively mild and the efficacy difference was evident in patients with gastric cancer, thus increasing the effectiveness of ErbB2 targeted therapy in patients with gastric cancer. In recent decades, the rapid development of genetic engineering has provided a wide variety of choices for us to develop and optimize the structure and function of antibodies. In previous studies, we constructed a double-specific antibody TPL using Trastuzumab and Pertuzumab as the parent. In this study, we further explore the anti-tumor effect and mechanism of this bispecific antibody in gastric cancer. First, by MTS cell viability testing, we compared the ability of Trastuzumab, Pertuzumab, Trastuzumab + Pertuzumab, and TPL to inhibit the proliferation of ErbB2 high-expression gastric cancer cell line N87 in vitro. The combination of Trastuzumab and Pertuzumab can more effectively inhibit the proliferation of N87 cells than both. Importantly, the bispecific antibody TPL shows the ability to inhibit the proliferation of gastric cancer cells significantly enhanced than Trastuzumab + Pertuzumab. The activity of Trastuzumab, Pertuzumab, Trastuzumab + Pertuzumab, and dual-specific antibody TPL in vivo inhibition of gastric cancer cells was then compared through a tumor-bearing mouse model. Consistent with the in vitro experimental results, Trastuzumab + Pertuzumab can more effectively inhibit tumor growth than both, but TPL has a more significant anti-tumor effect than Trastuzumab plus Pertuzumab. Previous studies have shown that simultaneous use of two monoclonal antibodies targeting different regions of ErbB2 can enhance the effect of targeted therapy by inducing ErbB2 internalization. Considering that this may be one of the mechanisms for a significant anti-tumor effect of the double-specific antibody TPL, we compared the ability of Trastuzumab, Pertuzumab, Trastuzumab + Pertuzumab, and dual-specific antibody TPL to induce ErbB2 internalization. Flow cytometry analysis, as well as immunofluorescence confocal experiments, demonstrated that the individual Trastuzumab and Pertuzumab could not induce ErbB2 significantly within 8 hours, while the Trastuzumab + Pertuzumab combination group could induce an ErbB2 weak endocytic after 8 hours of treatment. However, after 2 hours of N87 cells treated with TPL, ErbB2 could be swallowed up to some extent, and more pronounced ErbB2 could be caused after 8 hours of treatment. In order to further clarify the relationship between the combination of different table-position antibodies targeting ErbB2 and its ability to induce ErbB2, we obtained five specific monoclonal antibodies targeting ErbB2 in subsequent studies by standard hybridoma techniques. These five monoclonal antibodies plus Trastuzumab and Pertuzumab can be combined with two to 21 pairs of antibodies. Through an ELISA competitive inhibition assay, we identified 18 combinations of non-competitive binding ErbB2 from this 21 pair of antibody combinations. However, immunofluorescence confocal experiments demonstrated that only 4 of these 18 antibody combinations could cause significant ErbB2 internalization. In conclusion, our research shows that the double-specific antibody TPL constructed by genetic engineering can cause the obvious internal annexation of ErbB2, thereby demonstrating good anti-tumor activity outside the body. The bispecific antibody may also develop a novel effective targeting formulation for patients with gastric cancer with high expression of ErbB2. In addition, for a monoclonal antibody combination targeting ErbB2 different table bits, only specific antibody combinations can effectively induce ErbB2 internalization, which may be associated with specific table bits identified by the antibody.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2311611
[Abstract]:Around 700,000 people die in gastric cancer every year around the world, causing the death rate of gastric cancer to be second in all tumors, while most developing countries, including China, are the hardest hit areas of gastric cancer. and the overall prognosis of gastric cancer patients is relatively poor due to the fact that most gastric cancer patients are diagnosed with the progression phase, making it difficult to exert a good therapeutic effect in conventional treatment methods including surgery and chemotherapy. Of these, 6% to 23% of gastric cancer patients express high expression of human epidermal growth factor receptor 2 (ErbB2), and high expression of the receptor often indicates a worse prognosis. In recent years, with further understanding of tumor molecular mechanisms and analysis of a large number of basic and clinical trial data, it has been recognized that targeted therapy is of importance in patients with advanced gastric cancer. Trastuzumab as a clinical standard therapy is a monoclonal antibody that specifically targets the extracellular IV region of ErbB2 and has been demonstrated to be able to prolong the overall survival of patients with gastric cancer with high expression of ErbB2. Pertuzumab is another monoclonal antibody targeting ErbB2, binding to the ErbB2 extracellular II region, which also shows a positive therapeutic effect in patients with gastric cancer with high expression of ErbB2. The clinical trials of Trastuzumab in combination with Pertuzumab in patients with gastric cancer with high expression of ErbB2 are also ongoing and a preliminary indication has been shown. Although ErbB2 targeted therapy showed certain efficacy, the efficacy of Trastuzumab and Pertuzumab remained relatively mild and the efficacy difference was evident in patients with gastric cancer, thus increasing the effectiveness of ErbB2 targeted therapy in patients with gastric cancer. In recent decades, the rapid development of genetic engineering has provided a wide variety of choices for us to develop and optimize the structure and function of antibodies. In previous studies, we constructed a double-specific antibody TPL using Trastuzumab and Pertuzumab as the parent. In this study, we further explore the anti-tumor effect and mechanism of this bispecific antibody in gastric cancer. First, by MTS cell viability testing, we compared the ability of Trastuzumab, Pertuzumab, Trastuzumab + Pertuzumab, and TPL to inhibit the proliferation of ErbB2 high-expression gastric cancer cell line N87 in vitro. The combination of Trastuzumab and Pertuzumab can more effectively inhibit the proliferation of N87 cells than both. Importantly, the bispecific antibody TPL shows the ability to inhibit the proliferation of gastric cancer cells significantly enhanced than Trastuzumab + Pertuzumab. The activity of Trastuzumab, Pertuzumab, Trastuzumab + Pertuzumab, and dual-specific antibody TPL in vivo inhibition of gastric cancer cells was then compared through a tumor-bearing mouse model. Consistent with the in vitro experimental results, Trastuzumab + Pertuzumab can more effectively inhibit tumor growth than both, but TPL has a more significant anti-tumor effect than Trastuzumab plus Pertuzumab. Previous studies have shown that simultaneous use of two monoclonal antibodies targeting different regions of ErbB2 can enhance the effect of targeted therapy by inducing ErbB2 internalization. Considering that this may be one of the mechanisms for a significant anti-tumor effect of the double-specific antibody TPL, we compared the ability of Trastuzumab, Pertuzumab, Trastuzumab + Pertuzumab, and dual-specific antibody TPL to induce ErbB2 internalization. Flow cytometry analysis, as well as immunofluorescence confocal experiments, demonstrated that the individual Trastuzumab and Pertuzumab could not induce ErbB2 significantly within 8 hours, while the Trastuzumab + Pertuzumab combination group could induce an ErbB2 weak endocytic after 8 hours of treatment. However, after 2 hours of N87 cells treated with TPL, ErbB2 could be swallowed up to some extent, and more pronounced ErbB2 could be caused after 8 hours of treatment. In order to further clarify the relationship between the combination of different table-position antibodies targeting ErbB2 and its ability to induce ErbB2, we obtained five specific monoclonal antibodies targeting ErbB2 in subsequent studies by standard hybridoma techniques. These five monoclonal antibodies plus Trastuzumab and Pertuzumab can be combined with two to 21 pairs of antibodies. Through an ELISA competitive inhibition assay, we identified 18 combinations of non-competitive binding ErbB2 from this 21 pair of antibody combinations. However, immunofluorescence confocal experiments demonstrated that only 4 of these 18 antibody combinations could cause significant ErbB2 internalization. In conclusion, our research shows that the double-specific antibody TPL constructed by genetic engineering can cause the obvious internal annexation of ErbB2, thereby demonstrating good anti-tumor activity outside the body. The bispecific antibody may also develop a novel effective targeting formulation for patients with gastric cancer with high expression of ErbB2. In addition, for a monoclonal antibody combination targeting ErbB2 different table bits, only specific antibody combinations can effectively induce ErbB2 internalization, which may be associated with specific table bits identified by the antibody.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
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相关期刊论文 前3条
1 Wanqing Chen;Rongshou Zheng;Hongmei Zeng;Siwei Zhang;;The updated incidences and mortalities of major cancers in China, 2011[J];Chinese Journal of Cancer;2015年11期
2 Tasuku Matsuoka;Masakazu Yashiro;;Recent advances in the HER2 targeted therapy of gastric cancer[J];World Journal of Clinical Cases;2015年01期
3 JonathanSMARVIN;ZhenpingZHU;;Recombinant approaches to IgG-like bispecific antibodies[J];Acta Pharmacologica Sinica;2005年06期
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