【摘要】：研究目的： 非霍奇金淋巴瘤（NHL）是最常見的淋巴系統(tǒng)惡性腫瘤，好發(fā)于青壯年，其中絕大多數(shù)為B細(xì)胞來源，約占85%。CD20分子在95%以上的B細(xì)胞性NHL中均有表達，且抗原分子在膜上比較暴露，容易接近，與單抗結(jié)合后無顯著內(nèi)化和脫落，也不會因與抗體的結(jié)合而發(fā)生抗原調(diào)變，因此成為治療B細(xì)胞淋巴瘤的理想靶點。目前已有人鼠嵌合抗CD20抗體上市（Rituximab-C2B8）。盡管Rituximab在臨床治療中已顯示出較好的療效，仍有部分的患者對Rituximab的治療不產(chǎn)生反應(yīng)，且單獨使用該藥治愈率僅為10％，大部分患者會產(chǎn)生復(fù)發(fā)和抵抗，并且具有產(chǎn)生人抗鼠抗體（HAMA）反應(yīng)的潛在風(fēng)險�；诖�，本實驗室重新構(gòu)建并表達了全新的重組抗CD20人源化單克隆抗體，以期進一步提高抗體的功能，降低HAMA反應(yīng)的風(fēng)險。本研究首次通過超高壓液相色譜串聯(lián)四級桿飛行時間質(zhì)譜（LC-ESI-Q-Tof）技術(shù)從不同水平對重組抗CD20人源化單克隆抗體的結(jié)構(gòu)進行了詳細(xì)的分析鑒定，并進行了體外生物活性的分析，為下一步的臨床前和臨床研究奠定了良好基礎(chǔ)。 研究方法： SDS-PAGE和HPLC-SEC分析重組抗CD20人源化單克隆抗體的表觀分子量和純度。 LC/MS在完整蛋白水平上分析重組抗CD20人源化單克隆抗體的精確分子量及主要的翻譯后修飾種類。 LC/MS在抗體輕、重鏈水平上分析重組抗CD20人源化單克隆抗體輕、重鏈的精確分子量及主要的翻譯后修飾種類。 LC/MS分析重組抗CD20人源化單克隆抗體的質(zhì)量肽圖譜，對其一級結(jié)構(gòu)進行匹配，標(biāo)定糖基化位點，，并確認(rèn)抗體的主要翻譯后修飾類型。 LC/MS分析重組抗CD20人源化單克隆抗體的糖基化修飾類型，對游離寡糖進行鑒定，分析各糖形的比例分布。 體外測定重組抗CD20人源化抗體的ADCC和CDC效應(yīng)，直接誘導(dǎo)細(xì)胞凋亡的作用以及抗體與CD20抗原的親和力。 研究結(jié)果： SDS-PAGE及HPLC-SEC分析結(jié)果顯示，重組抗CD20人源化單克隆抗體分子量與理論分子量一致；經(jīng)過ProteinA親和層析及疏水層析，HPLC純度可達99%以上，有少量的多聚體存在。 PNGaseF酶切去除N糖基化修飾后的完整抗體的精確分子量為145424.92Da，與理論分子量一致。 還原狀態(tài)下，PNGaseF酶切前后，重鏈的精確分子量分別為50671.28Da和49226.75Da，輕鏈分子量均為23489.18Da，證實輕鏈不存在N糖基化的翻譯后修飾，重鏈則存在N糖基化修飾。 LC-MS質(zhì)量肽圖譜分析結(jié)果顯示，一級覆蓋率為100%，MSE二級覆蓋率為96.7%（b，y≥3），一級結(jié)構(gòu)與理論序列完全一致。糖基化位點位于重鏈第301位的Asn。 正相超高效液相色譜質(zhì)譜（NP-UPLC-MS）分析共發(fā)現(xiàn)并確認(rèn)的糖形為32種，以二天線復(fù)雜型糖形為主，有少量高甘露糖形，以及少量去巖藻糖化糖形，其中G0和G0F的比例分別為6.54%和45.96%。 在體外功能分析結(jié)果顯示，重組抗CD20人源化單克隆抗體的ADCC、CDC、直接誘導(dǎo)細(xì)胞凋亡的作用和親和力均優(yōu)于Rituximab。
[Abstract]:Objective: non Hodgkin's lymphoma (NHL) is the most common malignant tumor of the lymphatic system, which occurs in young adults, most of which are from B cells, accounting for about 95% of 85%.CD20 molecules expressed in B cell NHL. Moreover, antigen molecules are exposed on the membrane, easy to approach, no significant internalization and exfoliation after binding with monoclonal antibodies, and no antigenic modulation due to the binding with antibodies, so it is an ideal target for the treatment of B-cell lymphoma. Mouse chimeric antibody against CD20 (Rituximab-C2B8) has been published. Although Rituximab has shown good effect in clinical treatment, there are still some patients who do not respond to the treatment of Rituximab, and the cure rate of using the drug alone is only 10. The majority of patients will have relapse and resistance. It also has the potential risk of producing human anti-mouse antibody (HAMA) reaction. Based on this, a novel recombinant monoclonal antibody against CD20 was constructed and expressed in order to further improve the function of the antibody and reduce the risk of HAMA reaction. In this study, the structure of recombinant human monoclonal antibody against CD20 was analyzed and identified at different levels by ultrahigh pressure liquid chromatography tandem four-pole time-of-flight mass spectrometry (LC-ESI-Q-Tof) for the first time, and the bioactivity of recombinant monoclonal antibody against CD20 was analyzed in vitro. For the next clinical and clinical research laid a good foundation. Methods: SDS-PAGE and HPLC-SEC were used to analyze the apparent molecular weight and purity of recombinant anti-CD20 humanized monoclonal antibody. LC/MS was used to analyze the exact molecular weight of recombinant anti-CD20 humanized monoclonal antibody at the intact protein level. LC/MS in antibody light, Heavy chain level analysis of recombinant anti-human monoclonal antibody against CD20 light, heavy chain precise molecular weight and the main post-translational modification. LC/MS analysis of the recombinant anti-CD20 humanized monoclonal antibody mass peptide map, The primary structure was matched, the glycosylation sites were calibrated, and the main posttranslational modification types of antibodies were confirmed. LC/MS was used to analyze the glycosylation modification types of recombinant monoclonal antibodies against CD20 humanization, and free oligosaccharides were identified. The proportion distribution of each sugar form was analyzed. In vitro, the ADCC and CDC effects of recombinant anti CD20 humanized antibodies were determined, and the effect of direct induction of apoptosis and the affinity of the antibodies to CD20 antigens were also investigated. The results of SDS-PAGE and HPLC-SEC analysis showed that the molecular weight of the recombinant anti-CD20 humanized monoclonal antibody was the same as the theoretical molecular weight, and the purity of HPLC was over 99% by ProteinA affinity chromatography and hydrophobic chromatography. There was a small amount of polymer. The exact molecular weight of the complete antibody after removal of N-glycosylation modified by PNGaseF was 145424.92 Daa, which was consistent with the theoretical molecular weight. The exact molecular weights of heavy chain were 50671.28Da and 49226.75Da. the molecular weight of light chain was 23489.18Da. it was confirmed that there was no post-translational modification of N-glycosylation in the light chain. The heavy chain was modified by N-glycosylation. The results of LC-MS mass peptide analysis showed that the primary coverage rate was 96.7% (By 鈮

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