Anti-HER3抗體抑制Herceptin耐藥及功能優(yōu)化研究
發(fā)布時間：2019-03-15 08:57
【摘要】：隨著腫瘤發(fā)病分子機制的深入研究,針對關(guān)鍵基因、特異性細胞受體和調(diào)控分子的靶向性治療已成為腫瘤臨床治療的重要途徑�？贵w藥物以其特異性、靶向性、安全性以及臨床有效性等優(yōu)勢在腫瘤靶向性治療中發(fā)揮重要作用,目前比較熱門的腫瘤治療的靶標包括HER家族等一系列分子。HER2是HER家族成員之一,在乳腺癌、卵巢癌等多種癌癥中過表達。Herceptin、Perjeta、T-DM1等多個抗體、抗體-小分子偶聯(lián)藥物成功上市,并逐步成為HER2陽性腫瘤的臨床治療一線用藥。雖然HER2抗體在HER2陽性腫瘤的臨床治療中取得良好效果,但是HER2陽性乳腺癌的反應(yīng)率僅為20~30%,及Herceptin治療后期出現(xiàn)抗體耐藥的現(xiàn)象限制了抗體靶向性治療范圍和效率。因此,明確抗體的耐藥機制以及確定評估抗體療效的生物標志物,對于優(yōu)化治療方案、提高療效具有重要的意義。HER3是表皮生長因子受體HER家族成員,是PI3K/AKT信號通路中關(guān)鍵的激活因子。研究顯示,多種腫瘤在使用EGFR和HER2抑制劑治療時,HER3的表達上調(diào),磷酸化水平提高。因此,HER3在抗腫瘤中的作用逐步得到重視。各種靶向HER3的抑制劑中,單克隆抗體主要通過阻斷HER3與配體HRG結(jié)合或者阻斷HER3與其他HER家族成員形成異二聚體等分子機制,阻斷HER3下游信號通路激活,從而抑制腫瘤細胞生長。目前靶向HER3的單克隆抗體藥物Duligotuzumab、Patritumab均已進入臨床II期研究,Seribantuma、DL11、Seribantumab、LJM716、RG7116等多個抗體處于臨床前研究中。治療性抗體具有特異性高、半衰期長、副作用小等諸多優(yōu)勢,但因為其分子量大從而限制了其單獨用藥療效。近年來,抗體藥物偶聯(lián)(antibody-drug conjugate,ADC)技術(shù)發(fā)展迅速。ADC藥物結(jié)合了抗體藥物和細胞毒性藥物兩者的優(yōu)點,具有抗體藥物的特異性和細胞毒藥物的強細胞毒性,在治療腫瘤上取得了更好效果,是新一代靶向治療藥物的主要研究方向。Mylotarg TM、Adcetris TM、Kadcyla TM等ADC藥物成功上市,使得人們更為關(guān)注ADC藥物。本研究內(nèi)容基于前期獲得的對曲妥珠(Herceptin)具有耐藥性的卵巢癌細胞系(SKOV3/T),觀察比較了具有耐藥性的SKOV3/T細胞的增殖、信號通路和成瘤性的差異,并對SKOV3/T耐藥性中出現(xiàn)的HER3表達異常進行了深入探討;針對HER3靶點設(shè)計、優(yōu)化、篩選、表達了靶向HER3的全人單克隆抗體,并對HER3全人單克隆抗體和價靶向HER3的ADC藥物進行了體、內(nèi)外功能活性評價。本研究集中于以下三個方面:(一)曲妥珠耐藥細胞株SKOV3/T中HER3異常表達合理評價曲妥珠耐藥卵巢癌細胞系SKOV3/T的細胞增殖及成瘤能力。體外細胞增殖實驗以及體內(nèi)荷瘤動物實驗結(jié)果均表明,SKOV3/T細胞增殖及成瘤能力顯著高于正常卵巢癌細胞SKOV3。進一步通過流式細胞術(shù)和Western blot方法對SKOV3/T細胞和SKOV3細胞表面HER1、HER2以及HER3表達水平進行鑒定。實驗結(jié)果顯示,與SKOV3細胞株相比,SKOV3/T細胞株中HER1基因的表達未發(fā)生明顯改變,HER2基因表達顯著下調(diào),而HER3基因表達上調(diào)。(二)靶向HER3抗體抑制曲妥珠耐藥腫瘤細胞為了驗證HER3在曲妥珠耐藥中的作用,我們合成表達了特異性抗HER3抗體Lm Ab3,并對其生物學活性進行檢定。實驗結(jié)果顯示,Lm Ab3可以特異性識別重組表達的HER3抗原和天然的HER3分子,親和力Kon值為2.46E-10,并能特異性抑制HER家族分子磷酸化水平,并抑制HER3陽性的MCF7腫瘤細胞增殖。進一步利用特異性抗HER3抗體Lm Ab3為工具,探討HER3在曲妥珠耐藥中的作用。三維培養(yǎng)實驗結(jié)果顯示,特異性anti-HER3抗體阻斷后,SKOV3/T細胞增殖被顯著抑制。建立小鼠SKOV3/T耐藥細胞皮下荷瘤模型,使用特異性anti-HER3抗體治療后,腫瘤生長受到抑制,而生理鹽水對照組和Heceptin治療組則對SKOV3/T腫瘤未見明顯抑制作用。為了探討HER3抗體抑制曲妥珠耐藥細胞中的分子機制,我們進一步分析HER3激活的下游信號通路中HER1、HER2、HER3以及AKT等關(guān)鍵分子磷酸化水平,特異性anti-HER3抗體阻斷后,HER1、HER2、HER3以及AKT等分子磷酸化程度均受抑制。(三)靶向HER3抗體的優(yōu)化及生物學功能評價利用分子模擬技術(shù)對靶向HER3抗體進行合理優(yōu)化設(shè)計,結(jié)合生物學功能分析,獲得了一株高親和力、特異性識別HER3的新抗體FD001。進一步利用抗體偶聯(lián)技術(shù)將抗HER3抗體FD001與毒物小分子DM1進行偶聯(lián),獲得靶向HER3的ADC藥物FD001-DM1。體內(nèi)外生物學實驗結(jié)果顯示,FD001-DM1具有良好的抑制腫瘤增殖能力。本研究的創(chuàng)新之處:初步揭示了曲妥珠耐藥細胞株SKOV3/T中HER3異常表達,特異性Anti-HER3抗體可一定程度上抑制曲妥珠耐藥細胞增殖。進一步利用計算機分子模擬技術(shù)優(yōu)化設(shè)計并獲得了高親和力抗HER3新抗體FD001,并將該抗體偶聯(lián)小分子藥物DM1,獲得ADC分子FD001-DM1;FD001-DM1具有良好的體內(nèi)外抑制腫瘤增殖效應(yīng)。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R730.5
本文編號：2440479
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R730.5
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