新型抗CD20人源化抗体的表征结构分析

发布时间：2018-10-23 18:26

【摘要】：研究目的： 非霍奇金淋巴瘤（NHL）是最常见的淋巴系统恶性肿瘤，好发于青壮年，其中绝大多数为B细胞来源，约占85%。CD20分子在95%以上的B细胞性NHL中均有表达，且抗原分子在膜上比较暴露，容易接近，与单抗结合后无显著内化和脱落，也不会因与抗体的结合而发生抗原调变，因此成为治疗B细胞淋巴瘤的理想靶点。目前已有人鼠嵌合抗CD20抗体上市（Rituximab-C2B8）。尽管Rituximab在临床治疗中已显示出较好的疗效，仍有部分的患者对Rituximab的治疗不产生反应，且单独使用该药治愈率仅为10％，大部分患者会产生复发和抵抗，并且具有产生人抗鼠抗体（HAMA）反应的潜在风险。基于此，本实验室重新构建并表达了全新的重组抗CD20人源化单克隆抗体，以期进一步提高抗体的功能，降低HAMA反应的风险。本研究首次通过超高压液相色谱串联四级杆飞行时间质谱（LC-ESI-Q-Tof）技术从不同水平对重组抗CD20人源化单克隆抗体的结构进行了详细的分析鉴定，并进行了体外生物活性的分析，为下一步的临床前和临床研究奠定了良好基础。 研究方法： SDS-PAGE和HPLC-SEC分析重组抗CD20人源化单克隆抗体的表观分子量和纯度。 LC/MS在完整蛋白水平上分析重组抗CD20人源化单克隆抗体的精确分子量及主要的翻译后修饰种类。 LC/MS在抗体轻、重链水平上分析重组抗CD20人源化单克隆抗体轻、重链的精确分子量及主要的翻译后修饰种类。 LC/MS分析重组抗CD20人源化单克隆抗体的质量肽图谱，对其一级结构进行匹配，标定糖基化位点，，并确认抗体的主要翻译后修饰类型。 LC/MS分析重组抗CD20人源化单克隆抗体的糖基化修饰类型，对游离寡糖进行鉴定，分析各糖形的比例分布。 体外测定重组抗CD20人源化抗体的ADCC和CDC效应，直接诱导细胞凋亡的作用以及抗体与CD20抗原的亲和力。 研究结果： SDS-PAGE及HPLC-SEC分析结果显示，重组抗CD20人源化单克隆抗体分子量与理论分子量一致；经过ProteinA亲和层析及疏水层析，HPLC纯度可达99%以上，有少量的多聚体存在。 PNGaseF酶切去除N糖基化修饰后的完整抗体的精确分子量为145424.92Da，与理论分子量一致。 还原状态下，PNGaseF酶切前后，重链的精确分子量分别为50671.28Da和49226.75Da，轻链分子量均为23489.18Da，证实轻链不存在N糖基化的翻译后修饰，重链则存在N糖基化修饰。 LC-MS质量肽图谱分析结果显示，一级覆盖率为100%，MSE二级覆盖率为96.7%（b，y≥3），一级结构与理论序列完全一致。糖基化位点位于重链第301位的Asn。 正相超高效液相色谱质谱（NP-UPLC-MS）分析共发现并确认的糖形为32种，以二天线复杂型糖形为主，有少量高甘露糖形，以及少量去岩藻糖化糖形，其中G0和G0F的比例分别为6.54%和45.96%。 在体外功能分析结果显示，重组抗CD20人源化单克隆抗体的ADCC、CDC、直接诱导细胞凋亡的作用和亲和力均优于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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