抗CD20嵌合抗体的构建、表达和功能研究以及CD20相互作用蛋白的初步研究
|
摘要
非霍奇金淋巴瘤(non-Hodgkin's lymphoma, NHL)是一组组织形态和生物学特性各异的淋巴系统恶性肿瘤,是威胁人类生命的十大恶性肿瘤之一,也是发病率增长最快的恶性肿瘤。美罗华(Rituximab)作为一种嵌合抗体彻底改变了非霍奇金淋巴瘤的治疗状况,并且成为了目前最成功的抗肿瘤药物。单独使用Rituximab治疗复发以及难治的NHL缓解率可达50%左右,若与化疗药物联用,在一些NHL亚型中治愈率可达100%,并且副作用很小。2008年美罗华的销售额达到近60亿美元,具有极大的市场价值。
美罗华取得成功的原因主要有两点:首先,美罗华是人鼠嵌合抗体,既保留了鼠源可变区的高亲和性,又具有人Fc段的多种免疫杀伤功能。在降低了人抗鼠抗体(HAMA)反应的发生率的同时,嵌合抗体作为完整抗体分子,在体内半寿期更长,并具有人Fc段的多种免疫杀伤功能。其次,NHL患者中约85-90%是B细胞淋巴瘤,而CD20是B淋巴细胞和B淋巴瘤细胞表面特有的膜表面蛋白,不表达于造血干细胞、祖细胞、正常浆细胞以及人体其它正常组织[1]。CD20在B细胞表面的表达非常稳定,在与抗CD20抗体结合以后不易脱落与内化[2]。抗体与CD20结合以后,通过诱导细胞凋亡以及ADCC、CDC作用发挥杀伤作用。
即便如此在临床应用中,病人还是会产生抗体抵抗。并且人们逐渐开始认识到美罗华的局限性。研制新型的抗CD20抗体成为目前抗肿瘤药物的新热点,并且以提高人源化水平和提高与Fc受体亲和力为目前的主流方法,尤其是人源化程度更高的抗体在临床实验中已经取得了较好的效果。还有研究人员试图从CD20抗原方面入手,解决抗CD20抗体的局限性问题。
但是CD20的功能一直都不能确定,甚至连其天然配体都难觅踪影。最初研究人员推测它有可能是一种钙离子通道,因为抗体和CD20结合以后,引起细胞膜内的钙离子流动。目前的研究显示,CD20有自身形成4聚体的趋势,这样推测CD20可能是一种膜表面受体,能起到传导细胞信号的作用[3]。但是其他研究显示了不同的结果:CD20本身并不是传导信号的主体,只是掺入到BCR介导的信号通路中,并抢夺一部分BCR的功能[4]。虽然这些肤浅的了解不足以揭开CD20功能之谜,但是这些研究都表明CD20功能与BCR关系最大。
本课题在利用现有条件的基础上,通过定点突变原有抗CD20抗体基因构建合成新的抗CD20嵌合抗体。在寻找比美罗华杀伤效果更好或接近的抗CD20嵌合抗体的同时,以CD20与BCR的作用作为研究CD20功能的一个突破口,寻找到CD20天然的作用蛋白,指导开发出新抗CD20抗体。本课题以抗体功能和寻找抗原相互作用蛋白这两方面入手,寻找构建抗CD20抗体的新方向。该课题使用方法和取得的结果的主要是:
1.获得了2种抗体基因,构建了哺乳动物细胞双基因表达载体。
通过重叠延伸PCR修改原有抗CD20抗体基因合成新抗CD20抗体轻链和重链可变区。上述序列均测序正确后,将轻链连入pBudCE4.1上游多克隆位点后,再将重链可变区和本室原有的CH1和Fc段序列连入该载体下游多克隆位点。构建完成哺乳动物细胞双基因表达载体。2种抗体分别命名为20R和20S。
2.获得了BCR各组分与EGFP融合表达载体。
以BCR各组分完整序列为模板,以NCBI上BCR各组分的结构域划分为依据,PCR得出Igα胞外区,Igβ胞外区和IgM重链序列。测序正确后构建与EGFP融合表达载体。其中IgM分成3段构建,包括CH1+CH2, CH2+CH3,CH3+CH4,这些片段与EGFP融合表达载体则分别命名为IgM1, IgM2, IgM3。Igα胞外区和Igβ胞外区与EGFP融合表达载体命名为Igα和Igβ。
3.在CHO-K1细胞中表达了抗体蛋白、纯化了表达产物并对其进行了初步鉴定。
将构建完成的2种含有抗体基因的双基因表达载体转染至CHO-K1细胞中进行表达并挑取单克隆,以直接ELISA筛选出高表达的细胞株。转瓶扩大培养阳性克隆,以蛋白A亲和层析柱纯化得到目的蛋白,说明其含有人Fc段;BCA法测定纯化后蛋白浓度。SDS-PAGE检测显示,纯化蛋白的相对分子量大小约为150kD,与标准抗体分子大小一致并在凝胶成像系统软件Gel-Pro Analyzer分析纯化蛋白中完整的抗体蛋白含量近70%。结合BCA法测定的蛋白浓度,最后得出20R和20S这2种纯化蛋白中完整抗体的浓度为:120 mg/L和77 mg/L。
4.两种方法验证抗体诱导细胞凋亡的能力
(1)细胞凋亡试剂盒检测抗体诱导细胞凋亡。
以CD20+细胞Raji、Ramous(均为Burkitt淋巴瘤细胞)检测表达抗体诱导CD20+细胞凋亡的能力;以人白血病细胞株HL60检测抗体与CD20-细胞凋亡的诱导能力。抗体蛋白以2μg/ml、5μg/ml、20μg/ml这3个浓度分别作用于Raji、Ramous细胞。最后结果显示这2种抗体均可诱导CD20 +细胞凋亡,并且有较明显的剂量依赖效应。而且对CD20-细胞的生长没有明显影响。说明这2种抗体具有特异诱导CD20+细胞凋亡的能力。
(2)流式细胞技术检测抗体诱导的细胞凋亡。
实验方法与细胞凋亡试剂盒检测抗体诱导细胞凋亡类似,但是细胞接种量扩大到3×106细胞/ml,并且在抗体作用的24小时后,只用PI单染细胞,并用流式细胞仪检测凋亡细胞的数量。
结果显示这2种细胞均可诱导细胞凋亡,并且20R比20S在同等剂量下诱导细胞凋亡的能力高1倍多。
5.嵌合抗体诱导的CDC效应的检测
美罗华、20R和20S分别以2μg/ml、5μg/ml、10μg/ml这3个浓度作用于2×106细胞/ml的Raji细胞。并设无抗体作用的阴性对照孔。其中实验组与阴性对照组各设3个副孔。在抗体与细胞孵育15分钟后,加入10%的未灭活的人血清,即引入补体。血清孵育30分钟后加入CCK8。当阴性对照组呈现橘红色后检测OD450nm吸光值。最后结果显示美罗华的杀伤效果最好,20R和20S次之。而且20R的杀伤效果大概为美罗华的66%左右,20S的杀伤效果不好。
6.得到了5种表达目的基因与EGFP融合表达的293细胞。
利用293细胞具有半贴壁和自动成团的趋势,在加压过程中反复挑选高表达的多克隆细胞团。用Westen Blotting检测表达状况。结果显示得到了表达Igα胞外区-EGFP、Igβ胞外区-EGFP、IgM1-EGFP、IgM2-EGFP、IgM3-EGFP这5种细胞。这些细胞表达的蛋白仍然用其载体的名字命名,分别为Igα、Igβ、IgM1、IgM2和IgM3。
7. GST-Pull Down验证CD20相互作用蛋白为Igβ和IgM。
将诱饵蛋白CD20-GST结合到GST Bind resin上,分别与Igα、Igβ、IgM1、IgM2、IgM3这5种细胞裂解液作用。离心、清洗数次并去掉上清后,用还原性Loading Buffer在100℃下洗脱resin,最后用Westen Blotting检测。最后结果显示CD20可以与Igβ和IgM的CH2区结合,与Igα和IgM3不结合。
本课题合成了2种抗CD20嵌合抗体,对CD20+细胞有一定杀伤作用。其中20R有成为药物应用于临床的潜力。但是这2种抗体与目前应用于临床中最好的抗CD20抗体具有一定差距。说明,修改的一系列位点可能为抗CD20抗体结合CD20的关键部位,或者为Fab’特殊的结构为必需。这样以后若合成抗CD20嵌合抗体可以避开这些区域,并结合一些抗CD20抗体的临床实验,设计合成新的抗CD20嵌合抗体。
在寻找CD20相互作用蛋白方面,本课题没有从大量的血液细胞膜表面蛋白中搜寻CD20相互作用蛋白。而是根据目前研究的状况,从BCR方面入手探讨CD20相互作用蛋白。发现了CD20可能与Igβ及IgM的CH2区作用。这样我们推测CD20所引起的细胞内钙离子流,极有可能就是BCR的部分功能。而且接下来可以利用Igβ和IgM的CH2区的序列特征设计新的抗CD20抗体,即用Igβ和IgM的CH2区这些人体内天然存在的蛋白构建抗体。这些抗体可能会达到更好的治疗效果和更小的副作用。本课题可能为抗CD20抗体的研制和CD20功能的继续探讨开辟了一个新的领域。
Non-hodgkin’s lymphoma is the most common hematologic cancer in adults, and one of top ten life-threatening malignancies. Some indolent or slow growing B-cell non-Hodgkin’s lymphomas are incurable. In contrast, others which are aggressive or very aggressive and may be rapidly fatal are often curable. Until presently, no chemotherapeutic regimen provided a survival benefit. The anti-CD20 monoclonal antibody rituximab with better curative effect and less side-effect, first approved for clinical use in 1997, has changed the standard of care for many patients with NHL. The overall response rate was 48%, with 6% of the patients having complete remissions; better response rates correlated with fewer previous treatments. The median duration of the response was about one year.
There are two reasons for the good therapeutic effect of rituximab listed as follow:
On one hand, Rituximab is a chimeric monoclonal antibody composed of murine variable regions from the anti-CD20 antibody 2B8 that are linked to a human Fc component directed against CD20 on B cells. Therefore, Rituximab has lower immunogenicity than murine monoclonal antibody. Furthermore, this results in lower incidence rate of HAMA than murine monoclonal antibody. And chimeric monoclonal antibodies have longer half life than other micromolecule recombination antibodies in human body. Moreover, it is obvious that human Fc fragment has diverse immunological functions which involve in kill lymphoma.
On the other hand, CD20 plays an important role in the mechanism of action of Rituximab. Approximately 85% of non-Hodgkin’s lymphomas in adults are of B cell origin. CD20 is a 33-37 kD, non-glycosylated phosphoprotein expressed on the surface of normal B lymphocytes and about 95% of malignant B cells. This fact has lead that CD20-based immunotherapy have been successfully employed in the treatment, and that over one million patients accept Rituximab-based treatment world-wide with diseases such as Non-Hodgkins Lymphoma and various autoimmune disorders. Despite such clinical success and recent progress, researchers still remain largely ignorant of how anti-CD20 mAb operate in vivo. Expression of CD20 is restricted to the B-cell lineage from the pre-B-cell stage until terminal differentiation into plasma cells. Treatment with rituximab induces a pronounced, rapid and prolonged near-depletion of circulating B-cells. Circulating B-cells are replenished from bone marrow pro-B-cells within 4 to 12 months, sometimes longer. Interestingly, naive B-cells appear to recover faster than memory B-cells. Antibodies kill tumor cells through complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and induction of apoptosis. Despite the expression of antigens such as CD20, patients may not have a response to antibody therapy or resistance to the therapy may develop. Therefore, to clarity the foundation of CD20 is imperative to justify the mechanisms of resistance to anti-CD20 monoclonal antibodies potentially.
Some researchers claimed that CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. They provide direct evidence of CD20 homo-oligomerization into tetramers. After activation, BCR-CD20 complexes dissociated and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. So they deem that CD20 is a“store-operated”calcium entry (SOCE). Controversy, other researches data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and“hijack”the signaling potential of the BCR. But these researches did not provide not only specify of CD20 function but also determine of interaction protein of CD20.
This study means to construction, expression and characterization of two anti-CD20 chimeric monoclonal antibodies. The new genes of anti-CD20 chimeric monoclonal antibodies come from modification of another anti-CD20 chimeric antibody which has been preserved in our laboratory. At the moment, we try to find the native interaction protein of CD20 in order to explain some mechanisms of resistance to anti-CD20 monoclonal antibodies potentially, learn the foundation of CD20 and look for the new development way of anti-CD20 monoclonal antibodies.
Methods and Results
1. Construction of 2 anti-CD20 chimeric monoclonal antibody eukaryotic expression vector.
According to the patents of anti-CD20 antibodies, the synthesis of new anti-CD20 chimeric monoclonal antibodies light chain and VH gene is amplified by overlap extension PCR. The templates are the plasmids which were sroryed by our laboratory. Then, light chain, VH and Fc fragment were cloned into pBudCE4.1 different multiple clone sites. Each eukaryotic expression vector has two open reading frames, and regular two gene expressions.
2. Construction of various BCR various and EGFP fusion expression vectors.
BCR is composed of Igα, Igβand sIgM (or sIgD). The sIgM is the surface IgM monomer. The extracellular regions of Igαand Igβare synthesized by PCR and the templates are human cDNA. IgM heavy chain is spliced into 3 kinds of regions which are CH1CH2, CH2CH3 and CH3CH4. These regions gene is amplified by polymerase chain reaction (PCR) from the human cDNA. And then these gene and EGFP gene was linked and cloned into a mammalian expression vector pcDNA3.1. These vectors called after Igα, Igβ, IgM1, IgM2 and IgM3.
3. Purification of anti-CD20 antibody protein.
After anti-CD20 antibody eukaryotic expression vectors transfection into CHO-K1 cell, the positive clones were obtained by the screening of zeocin antibiotics. Then, the positive clones which are increased expression clones detected by ELISA inoculate into roller bottles. Supernatant of roller bottles culture with serum free medium load in Protein A affinity chromatography column. And then the proteins can be eluted with a high concentration of the carbohydrate. The collected protein can be detection by SDS-PAGE by compared molecular weights of antibody proteins. After BCA assay, the purity of proteins can be calculated. SDS-PAGE assay show that 20R and 20S protein molecular weight are about 150 kD. And their concentrations are 120 mg/L and 77 mg/L.
4. Anti-CD20-mediated apoptosis.
Anti-CD20-mediated apoptosis assays fall into two different methods.
(1) Apoptosis assay kits analysis of apoptosis.
Briefly, 5 x103 to 5 x104 Raji, Ramos, and HL60 cells were resuspended in 100μL culture medium and plated into 96-well flat-bottom microtiter plates. After incubating cells at 37°C for 24 hours with 20R 20S (density is 2μg/ml、5μg/ml、20μg/ml), the cells are added Annexin V-FITC and PI. The dyeing result can be observed with fluorescence microscope (wave length in 488nm). The result shows that 20R and 20S can both induct apoptosis of CD20+ cell and neither apoptosis of CD20- cells. And the effects of apoptosis present dose-respones relationship.
(2) Flow cytometric analysis of apoptosis.
Flow cytometric analysis of cellular DNA was performed following propidium iodide staining according to the method of Fried et al [5]. Briefly, after 3 x 106 Raji cells were incubated with 20R, 20S (density of 1μg/ml and 5μg/ml) 24 hours, the cells are washed in phosphate-buffered saline (PBS), and then are gently resuspended in 1mL PBS. Then PI is added into the cells. Samples were stored in the dark at 4°C until flow cytometric analysis of individual nuclei using a FACScan flow cytometer could be performed. The result show that 20R have better perform at induction of apoptosis of CD20+ cells.
5. Anti-CD20-mediated CDC.
First, 1 x105 Raji resuspended in 50μl culture medium and plated in 96-well flat-bottom microtiter plates. After incubating cells at 37°C for 15 minutes with 20R 20S (density is 2μg/ml、5μg/ml、10μg/ml), the cells are added 10μl human blood serum. Then, about 30 minutes later, the cells are added 10μl CCK8. When the colors of negative control become orange red (means to OD450nm exceed 1.0), the wells should be reading at OD450nm. The result shows that the ability of 20R-mediated CDC as 66% as that of Rituximab. And ability of 20S-mediated CDC is much worse than 20R.
6. Five kinds of Cells express the fusion protein.
After fusion expression vectors transfecting 293 cells, the positive clones were obtained by the screening of G418. The protein expressions of these cells can be analyzed by Western Blotting assay.
The results show that the lysis solutions of cells contain interest proteins.
7. GST-Pull Down
After CD20-GST co-incubated with Igα, Igβ, IgM1, IgM2, IgM3, negative control and positive control two hours at 4℃, the mixed liquor can be added GST Bind Resin. Two hours later, wash resins five times with GST Bing Buffer. Then, samples are heated toghter loading buffer to 100℃for 5 minutes. At least, analyze sample by Western Blotting. The result shows that the interaction protein of CD20 is Igβand IgM. And the region of interaction of IgM is CH2 region.
In sum, this study involves in construction two kind of anti-CD20 chimeric monoclonal antibodies. The two antibodies both can induct the apoptosis of CD20+ cells. And the effects of the assay and antibodies have dose-effect relationship. However, the effects of two antibodies mediated CDC are worse than Rituximab. And effect of 20R is about as 66% as Rituximab. This result illustrate that the region of mutation may be key to antibody affinity to CD20 or to maintain the constitution of anti-CD20 Fab’.
To surprised, this study finds the native interaction protein of CD20, as demonstrates that the foundation of CD20 is relevant to BCR. And the new discovery may result in constructions of new anti-CD20 antibody. The method of design of anti-CD20 would be changed.
美罗华取得成功的原因主要有两点:首先,美罗华是人鼠嵌合抗体,既保留了鼠源可变区的高亲和性,又具有人Fc段的多种免疫杀伤功能。在降低了人抗鼠抗体(HAMA)反应的发生率的同时,嵌合抗体作为完整抗体分子,在体内半寿期更长,并具有人Fc段的多种免疫杀伤功能。其次,NHL患者中约85-90%是B细胞淋巴瘤,而CD20是B淋巴细胞和B淋巴瘤细胞表面特有的膜表面蛋白,不表达于造血干细胞、祖细胞、正常浆细胞以及人体其它正常组织[1]。CD20在B细胞表面的表达非常稳定,在与抗CD20抗体结合以后不易脱落与内化[2]。抗体与CD20结合以后,通过诱导细胞凋亡以及ADCC、CDC作用发挥杀伤作用。
即便如此在临床应用中,病人还是会产生抗体抵抗。并且人们逐渐开始认识到美罗华的局限性。研制新型的抗CD20抗体成为目前抗肿瘤药物的新热点,并且以提高人源化水平和提高与Fc受体亲和力为目前的主流方法,尤其是人源化程度更高的抗体在临床实验中已经取得了较好的效果。还有研究人员试图从CD20抗原方面入手,解决抗CD20抗体的局限性问题。
但是CD20的功能一直都不能确定,甚至连其天然配体都难觅踪影。最初研究人员推测它有可能是一种钙离子通道,因为抗体和CD20结合以后,引起细胞膜内的钙离子流动。目前的研究显示,CD20有自身形成4聚体的趋势,这样推测CD20可能是一种膜表面受体,能起到传导细胞信号的作用[3]。但是其他研究显示了不同的结果:CD20本身并不是传导信号的主体,只是掺入到BCR介导的信号通路中,并抢夺一部分BCR的功能[4]。虽然这些肤浅的了解不足以揭开CD20功能之谜,但是这些研究都表明CD20功能与BCR关系最大。
本课题在利用现有条件的基础上,通过定点突变原有抗CD20抗体基因构建合成新的抗CD20嵌合抗体。在寻找比美罗华杀伤效果更好或接近的抗CD20嵌合抗体的同时,以CD20与BCR的作用作为研究CD20功能的一个突破口,寻找到CD20天然的作用蛋白,指导开发出新抗CD20抗体。本课题以抗体功能和寻找抗原相互作用蛋白这两方面入手,寻找构建抗CD20抗体的新方向。该课题使用方法和取得的结果的主要是:
1.获得了2种抗体基因,构建了哺乳动物细胞双基因表达载体。
通过重叠延伸PCR修改原有抗CD20抗体基因合成新抗CD20抗体轻链和重链可变区。上述序列均测序正确后,将轻链连入pBudCE4.1上游多克隆位点后,再将重链可变区和本室原有的CH1和Fc段序列连入该载体下游多克隆位点。构建完成哺乳动物细胞双基因表达载体。2种抗体分别命名为20R和20S。
2.获得了BCR各组分与EGFP融合表达载体。
以BCR各组分完整序列为模板,以NCBI上BCR各组分的结构域划分为依据,PCR得出Igα胞外区,Igβ胞外区和IgM重链序列。测序正确后构建与EGFP融合表达载体。其中IgM分成3段构建,包括CH1+CH2, CH2+CH3,CH3+CH4,这些片段与EGFP融合表达载体则分别命名为IgM1, IgM2, IgM3。Igα胞外区和Igβ胞外区与EGFP融合表达载体命名为Igα和Igβ。
3.在CHO-K1细胞中表达了抗体蛋白、纯化了表达产物并对其进行了初步鉴定。
将构建完成的2种含有抗体基因的双基因表达载体转染至CHO-K1细胞中进行表达并挑取单克隆,以直接ELISA筛选出高表达的细胞株。转瓶扩大培养阳性克隆,以蛋白A亲和层析柱纯化得到目的蛋白,说明其含有人Fc段;BCA法测定纯化后蛋白浓度。SDS-PAGE检测显示,纯化蛋白的相对分子量大小约为150kD,与标准抗体分子大小一致并在凝胶成像系统软件Gel-Pro Analyzer分析纯化蛋白中完整的抗体蛋白含量近70%。结合BCA法测定的蛋白浓度,最后得出20R和20S这2种纯化蛋白中完整抗体的浓度为:120 mg/L和77 mg/L。
4.两种方法验证抗体诱导细胞凋亡的能力
(1)细胞凋亡试剂盒检测抗体诱导细胞凋亡。
以CD20+细胞Raji、Ramous(均为Burkitt淋巴瘤细胞)检测表达抗体诱导CD20+细胞凋亡的能力;以人白血病细胞株HL60检测抗体与CD20-细胞凋亡的诱导能力。抗体蛋白以2μg/ml、5μg/ml、20μg/ml这3个浓度分别作用于Raji、Ramous细胞。最后结果显示这2种抗体均可诱导CD20 +细胞凋亡,并且有较明显的剂量依赖效应。而且对CD20-细胞的生长没有明显影响。说明这2种抗体具有特异诱导CD20+细胞凋亡的能力。
(2)流式细胞技术检测抗体诱导的细胞凋亡。
实验方法与细胞凋亡试剂盒检测抗体诱导细胞凋亡类似,但是细胞接种量扩大到3×106细胞/ml,并且在抗体作用的24小时后,只用PI单染细胞,并用流式细胞仪检测凋亡细胞的数量。
结果显示这2种细胞均可诱导细胞凋亡,并且20R比20S在同等剂量下诱导细胞凋亡的能力高1倍多。
5.嵌合抗体诱导的CDC效应的检测
美罗华、20R和20S分别以2μg/ml、5μg/ml、10μg/ml这3个浓度作用于2×106细胞/ml的Raji细胞。并设无抗体作用的阴性对照孔。其中实验组与阴性对照组各设3个副孔。在抗体与细胞孵育15分钟后,加入10%的未灭活的人血清,即引入补体。血清孵育30分钟后加入CCK8。当阴性对照组呈现橘红色后检测OD450nm吸光值。最后结果显示美罗华的杀伤效果最好,20R和20S次之。而且20R的杀伤效果大概为美罗华的66%左右,20S的杀伤效果不好。
6.得到了5种表达目的基因与EGFP融合表达的293细胞。
利用293细胞具有半贴壁和自动成团的趋势,在加压过程中反复挑选高表达的多克隆细胞团。用Westen Blotting检测表达状况。结果显示得到了表达Igα胞外区-EGFP、Igβ胞外区-EGFP、IgM1-EGFP、IgM2-EGFP、IgM3-EGFP这5种细胞。这些细胞表达的蛋白仍然用其载体的名字命名,分别为Igα、Igβ、IgM1、IgM2和IgM3。
7. GST-Pull Down验证CD20相互作用蛋白为Igβ和IgM。
将诱饵蛋白CD20-GST结合到GST Bind resin上,分别与Igα、Igβ、IgM1、IgM2、IgM3这5种细胞裂解液作用。离心、清洗数次并去掉上清后,用还原性Loading Buffer在100℃下洗脱resin,最后用Westen Blotting检测。最后结果显示CD20可以与Igβ和IgM的CH2区结合,与Igα和IgM3不结合。
本课题合成了2种抗CD20嵌合抗体,对CD20+细胞有一定杀伤作用。其中20R有成为药物应用于临床的潜力。但是这2种抗体与目前应用于临床中最好的抗CD20抗体具有一定差距。说明,修改的一系列位点可能为抗CD20抗体结合CD20的关键部位,或者为Fab’特殊的结构为必需。这样以后若合成抗CD20嵌合抗体可以避开这些区域,并结合一些抗CD20抗体的临床实验,设计合成新的抗CD20嵌合抗体。
在寻找CD20相互作用蛋白方面,本课题没有从大量的血液细胞膜表面蛋白中搜寻CD20相互作用蛋白。而是根据目前研究的状况,从BCR方面入手探讨CD20相互作用蛋白。发现了CD20可能与Igβ及IgM的CH2区作用。这样我们推测CD20所引起的细胞内钙离子流,极有可能就是BCR的部分功能。而且接下来可以利用Igβ和IgM的CH2区的序列特征设计新的抗CD20抗体,即用Igβ和IgM的CH2区这些人体内天然存在的蛋白构建抗体。这些抗体可能会达到更好的治疗效果和更小的副作用。本课题可能为抗CD20抗体的研制和CD20功能的继续探讨开辟了一个新的领域。
Non-hodgkin’s lymphoma is the most common hematologic cancer in adults, and one of top ten life-threatening malignancies. Some indolent or slow growing B-cell non-Hodgkin’s lymphomas are incurable. In contrast, others which are aggressive or very aggressive and may be rapidly fatal are often curable. Until presently, no chemotherapeutic regimen provided a survival benefit. The anti-CD20 monoclonal antibody rituximab with better curative effect and less side-effect, first approved for clinical use in 1997, has changed the standard of care for many patients with NHL. The overall response rate was 48%, with 6% of the patients having complete remissions; better response rates correlated with fewer previous treatments. The median duration of the response was about one year.
There are two reasons for the good therapeutic effect of rituximab listed as follow:
On one hand, Rituximab is a chimeric monoclonal antibody composed of murine variable regions from the anti-CD20 antibody 2B8 that are linked to a human Fc component directed against CD20 on B cells. Therefore, Rituximab has lower immunogenicity than murine monoclonal antibody. Furthermore, this results in lower incidence rate of HAMA than murine monoclonal antibody. And chimeric monoclonal antibodies have longer half life than other micromolecule recombination antibodies in human body. Moreover, it is obvious that human Fc fragment has diverse immunological functions which involve in kill lymphoma.
On the other hand, CD20 plays an important role in the mechanism of action of Rituximab. Approximately 85% of non-Hodgkin’s lymphomas in adults are of B cell origin. CD20 is a 33-37 kD, non-glycosylated phosphoprotein expressed on the surface of normal B lymphocytes and about 95% of malignant B cells. This fact has lead that CD20-based immunotherapy have been successfully employed in the treatment, and that over one million patients accept Rituximab-based treatment world-wide with diseases such as Non-Hodgkins Lymphoma and various autoimmune disorders. Despite such clinical success and recent progress, researchers still remain largely ignorant of how anti-CD20 mAb operate in vivo. Expression of CD20 is restricted to the B-cell lineage from the pre-B-cell stage until terminal differentiation into plasma cells. Treatment with rituximab induces a pronounced, rapid and prolonged near-depletion of circulating B-cells. Circulating B-cells are replenished from bone marrow pro-B-cells within 4 to 12 months, sometimes longer. Interestingly, naive B-cells appear to recover faster than memory B-cells. Antibodies kill tumor cells through complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and induction of apoptosis. Despite the expression of antigens such as CD20, patients may not have a response to antibody therapy or resistance to the therapy may develop. Therefore, to clarity the foundation of CD20 is imperative to justify the mechanisms of resistance to anti-CD20 monoclonal antibodies potentially.
Some researchers claimed that CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. They provide direct evidence of CD20 homo-oligomerization into tetramers. After activation, BCR-CD20 complexes dissociated and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. So they deem that CD20 is a“store-operated”calcium entry (SOCE). Controversy, other researches data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and“hijack”the signaling potential of the BCR. But these researches did not provide not only specify of CD20 function but also determine of interaction protein of CD20.
This study means to construction, expression and characterization of two anti-CD20 chimeric monoclonal antibodies. The new genes of anti-CD20 chimeric monoclonal antibodies come from modification of another anti-CD20 chimeric antibody which has been preserved in our laboratory. At the moment, we try to find the native interaction protein of CD20 in order to explain some mechanisms of resistance to anti-CD20 monoclonal antibodies potentially, learn the foundation of CD20 and look for the new development way of anti-CD20 monoclonal antibodies.
Methods and Results
1. Construction of 2 anti-CD20 chimeric monoclonal antibody eukaryotic expression vector.
According to the patents of anti-CD20 antibodies, the synthesis of new anti-CD20 chimeric monoclonal antibodies light chain and VH gene is amplified by overlap extension PCR. The templates are the plasmids which were sroryed by our laboratory. Then, light chain, VH and Fc fragment were cloned into pBudCE4.1 different multiple clone sites. Each eukaryotic expression vector has two open reading frames, and regular two gene expressions.
2. Construction of various BCR various and EGFP fusion expression vectors.
BCR is composed of Igα, Igβand sIgM (or sIgD). The sIgM is the surface IgM monomer. The extracellular regions of Igαand Igβare synthesized by PCR and the templates are human cDNA. IgM heavy chain is spliced into 3 kinds of regions which are CH1CH2, CH2CH3 and CH3CH4. These regions gene is amplified by polymerase chain reaction (PCR) from the human cDNA. And then these gene and EGFP gene was linked and cloned into a mammalian expression vector pcDNA3.1. These vectors called after Igα, Igβ, IgM1, IgM2 and IgM3.
3. Purification of anti-CD20 antibody protein.
After anti-CD20 antibody eukaryotic expression vectors transfection into CHO-K1 cell, the positive clones were obtained by the screening of zeocin antibiotics. Then, the positive clones which are increased expression clones detected by ELISA inoculate into roller bottles. Supernatant of roller bottles culture with serum free medium load in Protein A affinity chromatography column. And then the proteins can be eluted with a high concentration of the carbohydrate. The collected protein can be detection by SDS-PAGE by compared molecular weights of antibody proteins. After BCA assay, the purity of proteins can be calculated. SDS-PAGE assay show that 20R and 20S protein molecular weight are about 150 kD. And their concentrations are 120 mg/L and 77 mg/L.
4. Anti-CD20-mediated apoptosis.
Anti-CD20-mediated apoptosis assays fall into two different methods.
(1) Apoptosis assay kits analysis of apoptosis.
Briefly, 5 x103 to 5 x104 Raji, Ramos, and HL60 cells were resuspended in 100μL culture medium and plated into 96-well flat-bottom microtiter plates. After incubating cells at 37°C for 24 hours with 20R 20S (density is 2μg/ml、5μg/ml、20μg/ml), the cells are added Annexin V-FITC and PI. The dyeing result can be observed with fluorescence microscope (wave length in 488nm). The result shows that 20R and 20S can both induct apoptosis of CD20+ cell and neither apoptosis of CD20- cells. And the effects of apoptosis present dose-respones relationship.
(2) Flow cytometric analysis of apoptosis.
Flow cytometric analysis of cellular DNA was performed following propidium iodide staining according to the method of Fried et al [5]. Briefly, after 3 x 106 Raji cells were incubated with 20R, 20S (density of 1μg/ml and 5μg/ml) 24 hours, the cells are washed in phosphate-buffered saline (PBS), and then are gently resuspended in 1mL PBS. Then PI is added into the cells. Samples were stored in the dark at 4°C until flow cytometric analysis of individual nuclei using a FACScan flow cytometer could be performed. The result show that 20R have better perform at induction of apoptosis of CD20+ cells.
5. Anti-CD20-mediated CDC.
First, 1 x105 Raji resuspended in 50μl culture medium and plated in 96-well flat-bottom microtiter plates. After incubating cells at 37°C for 15 minutes with 20R 20S (density is 2μg/ml、5μg/ml、10μg/ml), the cells are added 10μl human blood serum. Then, about 30 minutes later, the cells are added 10μl CCK8. When the colors of negative control become orange red (means to OD450nm exceed 1.0), the wells should be reading at OD450nm. The result shows that the ability of 20R-mediated CDC as 66% as that of Rituximab. And ability of 20S-mediated CDC is much worse than 20R.
6. Five kinds of Cells express the fusion protein.
After fusion expression vectors transfecting 293 cells, the positive clones were obtained by the screening of G418. The protein expressions of these cells can be analyzed by Western Blotting assay.
The results show that the lysis solutions of cells contain interest proteins.
7. GST-Pull Down
After CD20-GST co-incubated with Igα, Igβ, IgM1, IgM2, IgM3, negative control and positive control two hours at 4℃, the mixed liquor can be added GST Bind Resin. Two hours later, wash resins five times with GST Bing Buffer. Then, samples are heated toghter loading buffer to 100℃for 5 minutes. At least, analyze sample by Western Blotting. The result shows that the interaction protein of CD20 is Igβand IgM. And the region of interaction of IgM is CH2 region.
In sum, this study involves in construction two kind of anti-CD20 chimeric monoclonal antibodies. The two antibodies both can induct the apoptosis of CD20+ cells. And the effects of the assay and antibodies have dose-effect relationship. However, the effects of two antibodies mediated CDC are worse than Rituximab. And effect of 20R is about as 66% as Rituximab. This result illustrate that the region of mutation may be key to antibody affinity to CD20 or to maintain the constitution of anti-CD20 Fab’.
To surprised, this study finds the native interaction protein of CD20, as demonstrates that the foundation of CD20 is relevant to BCR. And the new discovery may result in constructions of new anti-CD20 antibody. The method of design of anti-CD20 would be changed.
引文
1. Bubien JK,Zhou LJ,Bell PD,et al. Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes.J Cell Biol ,1993,121:1121
2. Waubant E. Spotlight on anti-CD20. Int MS J, 2008, 15(1):19-25.
3. Polyak M J, Li H, Shariat N, et al. CD20 homo-oligomers physically associate with the B cell antigen receptor. J Biol Chem, 2008,283(27): 18545-18552.
4. Walshe C A, Beers S A, French R R, et al. Induction of cytosolic calcium flux by CD20 is dependent upon B Cell Antigen Receptor Signaling. J Biol Chem, 2008,283(25): 16971-16984.
5. Fried J, Perez AG, Clarkson BD: Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures: Some pitfalls in staining and data analysis. J Histochem Cytochem 26:921, 1978
6. National Cancer Institute. Adult non-Hodgkin’s lymphoma (PDQ.): treatment [EB/OL]. http://www.cancer.gov/ cancerinfo/pdq/treatment/adult-non-hodgkins/ healthprofessional/, 2002-09-20.
7. Bubien JK,Zhou LJ,Bell PD,et al. Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes.J Cell Biol ,1993,121:1121
8. Press et al. Monoclonal antibody 1F5 (Anti-CD20) Serotherapy of Human B-Cell lymphomas.Blood,1987,69/2:584-591
9. Grillo-L′opez AJ, Hedrick E, Rashford M, et al. Rituximab: ongoing and future clinical development. Semin Oncol ,2002 Feb,29 (1 Suppl. 2): 105-12
10. Maloney DG, Smith B, Appelbaum FR. The anti-tumor effect of monoclonal anti-CD20 antibody (mAb) therapy includes direct anti-proliferative activity and induction of apoptosis in CD20 positive non-Hodgkin’s lymphoma (NHL) cell lines. Blood,1996,84:2535a
11. Anderson,et al. Chimeric and radiolabelled antibodies specific to human CD20 antigen and use thereof for treatment of B-cell lymphoma.United States Patent,6399061, 2002-06-04
12. Robinson et al. Chimeric antibody with specificity to human B-cell surface antigen. United States Patent, 5500362,1996-03-19
13. Dieffenbach CW,Dveksler GS.PCR Primer:A laboratory manual.2nd ed. New York: Cold Spring Harbor Laboratory Press,1995
14. Sambrook J, Ruussel DW. Molecular Cloning: A Laboratory Manual. 3rd ed, New York: Cold Spring Harbor Laboratory Press,2001
15. Fletcher S P, Jackson R J. Pestivirus internal ribosome entry site ( IRES) structure and function: elements in the 5′untranslated region important for IRES function. J Virol, 2002, 76 ( 10) : 5024 - 5033.
16. Spiker S, Thompson W F, Plant Phsiol, 1996; 110:15-21
17. Dopf J ,Horiagon TM. Gene ,1996 ;173 :39～44
18. Guohong Z ,Vanessa G,Steven RK.Biochem Biophys Res Commun ,1996 ;227 :707～711
19. Freshney R I. Animal Cell Culture: A Practical Approach. 2nd edition, Oxford: Oxford University Press, 1992
20.汪家政,范明。蛋白质技术手册。北京:科学出版社,2000
21. Sambrook J, Ruussel DW. Molecular Cloning: A Laboratory Manual. 3rd ed, New York: Cold Spring Harbor Laboratory Press,2001
22.李建武。生物化学实验原理和方法。北京大学出版社,1994
23. Flieger D, Renoth S, Beier I, et al. Mechanism of cytotoxicity induced by chimeric mouse human monoclonal antibody IDEC-C2B8 in CD20-expressing lymphoma cell lines.Cell Immunol, 2000, 204: 55–63
24. Manches O, Lui G, Chaperot L, et al. In vitro mechanisms of action of rituximab on primary non- Hodgkin lymphomas. Blood, 2003, 101 (3): 949-54
25. Bruce D, Cheson, and John P. Leonard. Monoclonal Antibody Therapy for B-Cell Non-Hodgkin’s Lymphoma. N Engl J Med 2008;359:613-26.
26. Bello C, Sotomayor EM. Monoclonal antibodies for B-cell lymphomas: rituximab and beyond. Hematology Am Soc Hematol Educ Program, 2007:233-242
2. Waubant E. Spotlight on anti-CD20. Int MS J, 2008, 15(1):19-25.
3. Polyak M J, Li H, Shariat N, et al. CD20 homo-oligomers physically associate with the B cell antigen receptor. J Biol Chem, 2008,283(27): 18545-18552.
4. Walshe C A, Beers S A, French R R, et al. Induction of cytosolic calcium flux by CD20 is dependent upon B Cell Antigen Receptor Signaling. J Biol Chem, 2008,283(25): 16971-16984.
5. Fried J, Perez AG, Clarkson BD: Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures: Some pitfalls in staining and data analysis. J Histochem Cytochem 26:921, 1978
6. National Cancer Institute. Adult non-Hodgkin’s lymphoma (PDQ.): treatment [EB/OL]. http://www.cancer.gov/ cancerinfo/pdq/treatment/adult-non-hodgkins/ healthprofessional/, 2002-09-20.
7. Bubien JK,Zhou LJ,Bell PD,et al. Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes.J Cell Biol ,1993,121:1121
8. Press et al. Monoclonal antibody 1F5 (Anti-CD20) Serotherapy of Human B-Cell lymphomas.Blood,1987,69/2:584-591
9. Grillo-L′opez AJ, Hedrick E, Rashford M, et al. Rituximab: ongoing and future clinical development. Semin Oncol ,2002 Feb,29 (1 Suppl. 2): 105-12
10. Maloney DG, Smith B, Appelbaum FR. The anti-tumor effect of monoclonal anti-CD20 antibody (mAb) therapy includes direct anti-proliferative activity and induction of apoptosis in CD20 positive non-Hodgkin’s lymphoma (NHL) cell lines. Blood,1996,84:2535a
11. Anderson,et al. Chimeric and radiolabelled antibodies specific to human CD20 antigen and use thereof for treatment of B-cell lymphoma.United States Patent,6399061, 2002-06-04
12. Robinson et al. Chimeric antibody with specificity to human B-cell surface antigen. United States Patent, 5500362,1996-03-19
13. Dieffenbach CW,Dveksler GS.PCR Primer:A laboratory manual.2nd ed. New York: Cold Spring Harbor Laboratory Press,1995
14. Sambrook J, Ruussel DW. Molecular Cloning: A Laboratory Manual. 3rd ed, New York: Cold Spring Harbor Laboratory Press,2001
15. Fletcher S P, Jackson R J. Pestivirus internal ribosome entry site ( IRES) structure and function: elements in the 5′untranslated region important for IRES function. J Virol, 2002, 76 ( 10) : 5024 - 5033.
16. Spiker S, Thompson W F, Plant Phsiol, 1996; 110:15-21
17. Dopf J ,Horiagon TM. Gene ,1996 ;173 :39～44
18. Guohong Z ,Vanessa G,Steven RK.Biochem Biophys Res Commun ,1996 ;227 :707～711
19. Freshney R I. Animal Cell Culture: A Practical Approach. 2nd edition, Oxford: Oxford University Press, 1992
20.汪家政,范明。蛋白质技术手册。北京:科学出版社,2000
21. Sambrook J, Ruussel DW. Molecular Cloning: A Laboratory Manual. 3rd ed, New York: Cold Spring Harbor Laboratory Press,2001
22.李建武。生物化学实验原理和方法。北京大学出版社,1994
23. Flieger D, Renoth S, Beier I, et al. Mechanism of cytotoxicity induced by chimeric mouse human monoclonal antibody IDEC-C2B8 in CD20-expressing lymphoma cell lines.Cell Immunol, 2000, 204: 55–63
24. Manches O, Lui G, Chaperot L, et al. In vitro mechanisms of action of rituximab on primary non- Hodgkin lymphomas. Blood, 2003, 101 (3): 949-54
25. Bruce D, Cheson, and John P. Leonard. Monoclonal Antibody Therapy for B-Cell Non-Hodgkin’s Lymphoma. N Engl J Med 2008;359:613-26.
26. Bello C, Sotomayor EM. Monoclonal antibodies for B-cell lymphomas: rituximab and beyond. Hematology Am Soc Hematol Educ Program, 2007:233-242