靶向ErbB2雙特異性抗體在胃癌中的抗腫瘤作用及其機(jī)理
發(fā)布時間：2018-11-05 09:15
【摘要】：在全世界范圍內(nèi),每年約有70萬人死于胃癌,使得胃癌導(dǎo)致的死亡率在所有腫瘤中高居第二位,而以包括中國在內(nèi)的大部分發(fā)展中國家更是胃癌的重災(zāi)區(qū)。并且由于大部分胃癌患者均確診于進(jìn)展期,使得以包括手術(shù)和化療在內(nèi)的常規(guī)治療手段難以發(fā)揮良好的治療效果,因而胃癌患者的整體預(yù)后相對較差。其中,6%至23%的胃癌患者高表達(dá)人表皮生長因子受體2(ErbB2),而該受體的高表達(dá)往往預(yù)示著更差的預(yù)后。近年來隨著對腫瘤分子機(jī)制的進(jìn)一步了解以及對大量基礎(chǔ)及臨床試驗(yàn)數(shù)據(jù)的分析,人們已經(jīng)認(rèn)識到了靶向治療在進(jìn)展期胃癌患者中的重要性。作為臨床標(biāo)準(zhǔn)療法的曲妥珠(Trastuzumab)是一種能特異性靶向ErbB2胞外IV區(qū)的單克隆抗體,且已被證明了能夠延長ErbB2高表達(dá)進(jìn)展期胃癌患者的總生存期。帕妥珠(Pertuzumab)是另一個靶向ErbB2的單克隆抗體,結(jié)合ErbB2胞外II區(qū),其在ErbB2高表達(dá)進(jìn)展期胃癌患者中也顯示了積極的療效。而Trastuzumab與Pertuzumab聯(lián)合應(yīng)用于ErbB2高表達(dá)進(jìn)展期胃癌患者的臨床試驗(yàn)也在進(jìn)行中,并且已經(jīng)初步顯示出了可喜的結(jié)果。盡管ErbB2靶向療法顯示了一定的療效,但Trastuzumab和Pertuzumab的療效仍相對溫和并且在不同胃癌患者中療效差異明顯,因此提高ErbB2靶向治療在胃癌患者中的效果顯得極為重要。近幾十年來,基因工程的飛速發(fā)展給我們研發(fā)及優(yōu)化抗體的結(jié)構(gòu)和功能提供了多種多樣的選擇。在前期研究中,我們通過基因工程構(gòu)建了一種以Trastuzumab和Pertuzumab為親本的雙特異性抗體TPL。在本研究中,我們進(jìn)一步探討此雙特異抗體在胃癌中的抗腫瘤作用及機(jī)制。首先,通過MTS細(xì)胞活力檢測實(shí)驗(yàn),我們比較了Trastuzumab,Pertuzumab,Trastuzumab+Pertuzumab以及TPL在體外抑制ErbB2高表達(dá)胃癌細(xì)胞系N87增殖的能力。Trastuzumab+Pertuzumab聯(lián)合應(yīng)用比二者單用能更有效地抑制N87細(xì)胞的增殖。重要的是,雙特異性抗體TPL顯示出具有比Trastuzumab+Pertuzumab明顯增強(qiáng)的抑制胃癌細(xì)胞增殖的能力。隨后通過荷瘤小鼠模型,我們比較了Trastuzumab,Pertuzumab,Trastuzumab+Pertuzumab以及雙特異性抗體TPL的體內(nèi)抑制胃癌細(xì)胞的活性。與體外實(shí)驗(yàn)結(jié)果一致,即Trastuzumab+Pertuzumab聯(lián)用能比二者單用更為有效地抑制腫瘤的生長,但TPL具有比Trastuzumab+Pertuzumab聯(lián)用更顯著的抗腫瘤作用。此前有研究表明,同時使用兩種靶向ErbB2不同區(qū)域的單克隆抗體可通過誘導(dǎo)ErbB2內(nèi)吞而增強(qiáng)靶向治療的效果�？紤]到這可能是雙特異性抗體TPL發(fā)揮顯著抗瘤作用的機(jī)制之一,我們比較了Trastuzumab,Pertuzumab,Trastuzumab+Pertuzumab以及雙特異性抗體TPL誘導(dǎo)ErbB2內(nèi)吞的能力。流式細(xì)胞術(shù)分析以及免疫熒光共聚焦實(shí)驗(yàn)表明,單獨(dú)的Trastuzumab和Pertuzumab在8小時內(nèi)均不能誘導(dǎo)ErbB2明顯內(nèi)吞,而Trastuzumab+Pertuzumab聯(lián)用組在處理8小時后才能誘導(dǎo)ErbB2微弱的內(nèi)吞。但TPL處理N87細(xì)胞2小時后就可導(dǎo)致ErbB2發(fā)生一定程度的內(nèi)吞,處理8小時后可引起更為顯著的ErbB2內(nèi)吞。為了進(jìn)一步闡明靶向ErbB2不同表位抗體組合與其誘導(dǎo)ErbB2內(nèi)吞能力之間的關(guān)系,我們在隨后的研究中通過標(biāo)準(zhǔn)雜交瘤技術(shù)獲得了靶向ErbB2的5株特異性單克隆抗體。這5株單克隆抗體加上Trastuzumab和Pertuzumab可以兩兩組成21對抗體組合。通過ELISA競爭抑制試驗(yàn),我們從這21對抗體組合中鑒定出了18對不競爭結(jié)合ErbB2的組合。但是免疫熒光共聚焦實(shí)驗(yàn)證明,這18對抗體組合中只有其中4對抗體組合能引起明顯的ErbB2內(nèi)吞。綜上所述,我們的研究表明通過基因工程構(gòu)建的雙特異性抗體TPL能引起ErbB2的明顯內(nèi)吞并借此在體內(nèi)外均展示出良好的抗腫瘤活性,該雙特異性抗體也有可能發(fā)展成為ErbB2高表達(dá)進(jìn)展期胃癌患者的一種新的有效靶向制劑。此外,對于靶向ErbB2不同表位的單克隆抗體組合而言,只有其中特定的抗體組合才能有效誘導(dǎo)ErbB2內(nèi)吞,這可能與抗體所識別的具體表位有關(guān)。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
本文編號：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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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
，本文編號：2311611
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