新型人源化抗CD20抗体的设计及生物学功能研究
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摘要
非霍奇金淋巴瘤（Non-Hodgkin’s lymphoma，NHL）是最常见的血液系统恶性肿瘤，绝大多数NHL发生与B细胞相关，NHL是威胁人类生命的10大恶性肿瘤之一，许多传统的化疗药物对NHL的治疗效果并不好。利用单克隆抗体靶向治疗恶性肿瘤是人类长久以来的梦想。
1997年，世界上第一个抗CD20单克隆抗体——美罗华(Rituximab)上市应用于NHL的治疗，之后随着美罗华的广泛应用，NHL的死亡率得到控制并逐渐下降。在之后的10年时间里，人们摸索出了多种基于美罗华的治疗方案，旨在提高美罗华功效，取得了长足的进步，使得NHL患者的缓解率和完全缓解率有所提高，并且预后良好。美罗华的成功最重要的因素就是其选用了一个合适的治疗靶标—CD20。CD20是B淋巴细胞上的跨膜蛋白，由279个氨基酸组成，以非糖基化形式存在，其分子量因磷酸化程度不同而不同，范围在33～37kD。CD20分子有4个跨膜区，氨基端和羧基端都位于细胞质内侧，在第三跨膜区和第四跨膜区之间，有一个由43个氨基酸组成的环区（large loop），构成其主要的抗原表位。CD20抗原分子比较容易暴露，易于接近。CD20的生理作用至今尚不清晰，根据CD20与抗CD20的抗体结合后B细胞产生的一系列的生物学反应，推测CD20可能参与B细胞的增殖、分化、信号转导和钙离子的跨膜传递。
由于Rituximab是一种人鼠嵌合型的抗CD20抗体，在抗体分子中仍然包含约30%的鼠源序列，其在临床应用过程中约17-20%的病人产生了免疫原性，导致无法长期用药，严重制约了Rituximab的使用，尤其严重影响其在需要长期用药的自身免疫性疾病适应症治疗方面的应用。有鉴于此，对CD20抗体分子进行人源化改造，发现效果更好，免疫原性更低的新抗体分子一直是CD20抗体研究的热点。
在本课题研究中，采用独特的抗体人源化技术，在鼠单抗2B8（罗氏Rituximab的原始鼠源单抗）的序列基础之上，设计选择了与MHC II (HLA-DR)结合位点少的人胚系（germline）抗体框架序列进行鼠源单抗2B8的人源化改造，大大降低了其免疫原性。同时，在低免疫源性框架的前提下，通过分子对接的方法适当调整CDR区的序列结构，可保持或提高良好的抗原识别活性和体内体外的抗肿瘤活性。
将抗CD20抗体分子充分人源化，筛选得到了新的人源化CD20抗体分子，该抗体分子与Rituximab相比，鼠源序列由30%降低至7%，新抗体分子保留了与美罗华（Rituximab）相同的抗原抗体结合位点，即与CD20的large loop结合，作用机制与Rituximab完全一致，体外、体内药效明确，而免疫原性明显降低，据此预期该创新抗体分子不但在肿瘤治疗方面与Rituximab有类似的疗效和更低的副作用，而且对多种涉及B细胞异常激活的自身免疫疾病，也有更广泛的应用前景。
另外，本研究还设计了10种制剂处方用于人源化CD20抗体的工艺生产，通过SDS-PAGE和CE-SDS检测了这10种制剂处方样品在不同温度（-80℃，-20℃，4℃，25℃和40℃）下存放30天、60天、90天和120天的稳定性，拟初步采用3#制剂处方。同时，采用毛细管电泳技术对新型人源化CD20抗体的理化性质（包括纯度，等电点等）进行分析研究，发现不同批次样品的酸碱性组分含量有所差异，该差异在一定程度上影响了抗体分子的生物活性。
Non-Hodgkin’s lymphoma (NHL) is one of the most common cancer in the bloodsystem. The most of NHL is associated with B cell and the therapeutic efficiency using of many traditional chemotherapeutics for NHL is not very well. Use of monoclonal antibody to treat cancer targetly is the dream of human beings for a long time.
In1997, the first anti-CD20monoclonal antibody, named Rituximab, was appliedto the treatment of NHL in the world. With the using widely of Rituximab, the mortality rate of NHL was decreased gradually. In the following10years, many therapeuticregimens were developed based on Rituximab, to aim at an increasing on the effect ofRituximab and to obtain an obvious improvement. At the same time, the remission rate and complete remission rate of NHL patients were increased, and the prognosis iswell. The most important factor in the Rituximab is the choice of the proper target-CD20. CD20is the transmembrane protein on the B leukomonocyte and it contain279amino acids. As a non-glycosylation, the molecular weight is reported to be33-37kDdepending on the different levels of phosphorylation. The CD20has four transmembrane domains and both of the N-terminal and C-terminal are located on the inner side of cytoplasm, which is between the third and fourth transmembrane domain. It has a large loop, which contains43amino acids to form prime epitope. The CD20antigen is relatively easy to expose and also to approach. The physiological effect of CD20remains to be clarified, based on a series of biological response on B cell after the combination of the CD20antigen with CD20antibody, also infering that the CD20would join in the proliferation, differentiation, signal transduction and the transmembrane deliverof Ca~(2+) on B cell.
Rituximab belongs to one of the human-mouse chimeric antibodies, and30%of its sequence comes from the original murine. During the clinical application, immunogenicity was reported in the17-20%patients. So it is unable to be used for a long time.The application of Rituximab was restrict especially for the treatment of autoimmunedisease. Keeping this in mind, to carry out the humanization of CD20, to find novel antibody with a better effect and a lower immunogenicity is the hot issue on the resear ch for anti-CD20antibody.
In this study, we use unique antibody humanization technology, based on the sequence of the murine monoclonal antibody2B8(the original murine monoclonal antibodyof Rituximab from Roche), designed and selected with germline antibody frame sequence which has less binding sites to the MHC II (HLA-DR), and the immunogenicityhad been decreased obviously. At the same time, we regulated to the sequence structure of CDR region through molecular dock, which can keep and increase the activity ofantigen recognition and anti-tumor effect in both vitro and vivo.
Screening the novel humanized anti-CD20antibody through full humanization, comparedwith Rituximab, the murine sequence of the novel humanized anti-CD20antibody wasdecreased by23%. The novel humanized anti-CD20antibody retained the same antigenbinding sites on the large loop of Rituximab. The mechanism of action is same to Rituximaband the activity is clear in vitro and in vivo, while the immunogenicity is decreased obviously.On these grounds, compared to Rituximab, the novel humanized anti-CD20antibody has thesimilar effect on anti-tumor and lower toxicity. On the other hand, it also has an extensiveapplication prospect on the treatment of autoimmune diseases, which involves many kinds ofB cell abnormal.
In this study, we designed ten kinds of formulations for the process of humanizedanti-CD20antibody and investigated the stability of the antibody at different temperature(-80℃，-20℃，4℃，25℃and40℃) for30days、60days、90days and120days. The third one wasplaned to be using as the formulation process of the anti-CD20antibody. We also analyzed thephysicochemical property (such as purity and isoelectric point) of the novel humanizedanti-CD20antibody through capillary electrophoresis, which presented that the samples withdifferent lots had different percentage of basic and acid components. The different influenceon the biological activity of the antibody.
1997年，世界上第一个抗CD20单克隆抗体——美罗华(Rituximab)上市应用于NHL的治疗，之后随着美罗华的广泛应用，NHL的死亡率得到控制并逐渐下降。在之后的10年时间里，人们摸索出了多种基于美罗华的治疗方案，旨在提高美罗华功效，取得了长足的进步，使得NHL患者的缓解率和完全缓解率有所提高，并且预后良好。美罗华的成功最重要的因素就是其选用了一个合适的治疗靶标—CD20。CD20是B淋巴细胞上的跨膜蛋白，由279个氨基酸组成，以非糖基化形式存在，其分子量因磷酸化程度不同而不同，范围在33～37kD。CD20分子有4个跨膜区，氨基端和羧基端都位于细胞质内侧，在第三跨膜区和第四跨膜区之间，有一个由43个氨基酸组成的环区（large loop），构成其主要的抗原表位。CD20抗原分子比较容易暴露，易于接近。CD20的生理作用至今尚不清晰，根据CD20与抗CD20的抗体结合后B细胞产生的一系列的生物学反应，推测CD20可能参与B细胞的增殖、分化、信号转导和钙离子的跨膜传递。
由于Rituximab是一种人鼠嵌合型的抗CD20抗体，在抗体分子中仍然包含约30%的鼠源序列，其在临床应用过程中约17-20%的病人产生了免疫原性，导致无法长期用药，严重制约了Rituximab的使用，尤其严重影响其在需要长期用药的自身免疫性疾病适应症治疗方面的应用。有鉴于此，对CD20抗体分子进行人源化改造，发现效果更好，免疫原性更低的新抗体分子一直是CD20抗体研究的热点。
在本课题研究中，采用独特的抗体人源化技术，在鼠单抗2B8（罗氏Rituximab的原始鼠源单抗）的序列基础之上，设计选择了与MHC II (HLA-DR)结合位点少的人胚系（germline）抗体框架序列进行鼠源单抗2B8的人源化改造，大大降低了其免疫原性。同时，在低免疫源性框架的前提下，通过分子对接的方法适当调整CDR区的序列结构，可保持或提高良好的抗原识别活性和体内体外的抗肿瘤活性。
将抗CD20抗体分子充分人源化，筛选得到了新的人源化CD20抗体分子，该抗体分子与Rituximab相比，鼠源序列由30%降低至7%，新抗体分子保留了与美罗华（Rituximab）相同的抗原抗体结合位点，即与CD20的large loop结合，作用机制与Rituximab完全一致，体外、体内药效明确，而免疫原性明显降低，据此预期该创新抗体分子不但在肿瘤治疗方面与Rituximab有类似的疗效和更低的副作用，而且对多种涉及B细胞异常激活的自身免疫疾病，也有更广泛的应用前景。
另外，本研究还设计了10种制剂处方用于人源化CD20抗体的工艺生产，通过SDS-PAGE和CE-SDS检测了这10种制剂处方样品在不同温度（-80℃，-20℃，4℃，25℃和40℃）下存放30天、60天、90天和120天的稳定性，拟初步采用3#制剂处方。同时，采用毛细管电泳技术对新型人源化CD20抗体的理化性质（包括纯度，等电点等）进行分析研究，发现不同批次样品的酸碱性组分含量有所差异，该差异在一定程度上影响了抗体分子的生物活性。
Non-Hodgkin’s lymphoma (NHL) is one of the most common cancer in the bloodsystem. The most of NHL is associated with B cell and the therapeutic efficiency using of many traditional chemotherapeutics for NHL is not very well. Use of monoclonal antibody to treat cancer targetly is the dream of human beings for a long time.
In1997, the first anti-CD20monoclonal antibody, named Rituximab, was appliedto the treatment of NHL in the world. With the using widely of Rituximab, the mortality rate of NHL was decreased gradually. In the following10years, many therapeuticregimens were developed based on Rituximab, to aim at an increasing on the effect ofRituximab and to obtain an obvious improvement. At the same time, the remission rate and complete remission rate of NHL patients were increased, and the prognosis iswell. The most important factor in the Rituximab is the choice of the proper target-CD20. CD20is the transmembrane protein on the B leukomonocyte and it contain279amino acids. As a non-glycosylation, the molecular weight is reported to be33-37kDdepending on the different levels of phosphorylation. The CD20has four transmembrane domains and both of the N-terminal and C-terminal are located on the inner side of cytoplasm, which is between the third and fourth transmembrane domain. It has a large loop, which contains43amino acids to form prime epitope. The CD20antigen is relatively easy to expose and also to approach. The physiological effect of CD20remains to be clarified, based on a series of biological response on B cell after the combination of the CD20antigen with CD20antibody, also infering that the CD20would join in the proliferation, differentiation, signal transduction and the transmembrane deliverof Ca~(2+) on B cell.
Rituximab belongs to one of the human-mouse chimeric antibodies, and30%of its sequence comes from the original murine. During the clinical application, immunogenicity was reported in the17-20%patients. So it is unable to be used for a long time.The application of Rituximab was restrict especially for the treatment of autoimmunedisease. Keeping this in mind, to carry out the humanization of CD20, to find novel antibody with a better effect and a lower immunogenicity is the hot issue on the resear ch for anti-CD20antibody.
In this study, we use unique antibody humanization technology, based on the sequence of the murine monoclonal antibody2B8(the original murine monoclonal antibodyof Rituximab from Roche), designed and selected with germline antibody frame sequence which has less binding sites to the MHC II (HLA-DR), and the immunogenicityhad been decreased obviously. At the same time, we regulated to the sequence structure of CDR region through molecular dock, which can keep and increase the activity ofantigen recognition and anti-tumor effect in both vitro and vivo.
Screening the novel humanized anti-CD20antibody through full humanization, comparedwith Rituximab, the murine sequence of the novel humanized anti-CD20antibody wasdecreased by23%. The novel humanized anti-CD20antibody retained the same antigenbinding sites on the large loop of Rituximab. The mechanism of action is same to Rituximaband the activity is clear in vitro and in vivo, while the immunogenicity is decreased obviously.On these grounds, compared to Rituximab, the novel humanized anti-CD20antibody has thesimilar effect on anti-tumor and lower toxicity. On the other hand, it also has an extensiveapplication prospect on the treatment of autoimmune diseases, which involves many kinds ofB cell abnormal.
In this study, we designed ten kinds of formulations for the process of humanizedanti-CD20antibody and investigated the stability of the antibody at different temperature(-80℃，-20℃，4℃，25℃and40℃) for30days、60days、90days and120days. The third one wasplaned to be using as the formulation process of the anti-CD20antibody. We also analyzed thephysicochemical property (such as purity and isoelectric point) of the novel humanizedanti-CD20antibody through capillary electrophoresis, which presented that the samples withdifferent lots had different percentage of basic and acid components. The different influenceon the biological activity of the antibody.
引文
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