计算机辅助设计进行抗VEGF抗体体外亲和力成熟
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摘要
恶性肿瘤严重威胁人类健康,全球每年有700多万人死于恶性肿瘤,1000多万新增肿瘤患者。在我国,恶性肿瘤已成为城市人口的主要死亡原因,占死亡人口总数的21%。WHO预计,到2020年,全世界肿瘤发病率将是现在的两倍。对于肿瘤的治疗,早期主要采用手术及放、化疗的方法,但是这些方法存在着各自的缺点:手术治疗依赖于早期诊断,而放、化疗的副作用很大。因此迫切需要开发新型抗肿瘤药物,而抗血管生成的治疗性抗体的出现为肿瘤治疗开辟了新途径。新生血管的形成对肿瘤的生长和转移至关重要。血管内皮生长因子( vascular endothelial growth factor, VEGF)与其受体VEGFR相互作用,能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用。通过阻断VEGF/VEGFR,可以特异性阻断或抑制VEGF/VEGFR所介导的生物学活性,从而抑制肿瘤血管生成,达到治疗恶性肿瘤、抑制肿瘤进展的目的。因此针对VEGF开发治疗肿瘤的抗体成为人们关注热点。目前抗VEGF人源化抗体贝伐单抗(Bevacizumab,商品名Avastin )已成功上市,并产生了巨大的社会效益及经济价值,2009年年销售额达到57.7亿美元,并被认为是最有市场开发前景的治疗性抗体。但是国际上还没有成功应用于临床的全人源抗VEGF抗体。因此,开发以VEGF为靶标的全人源抗体具有广阔应用前景和重大意义。
本研究室通过对大容量全合成噬菌体人源抗体库的筛选,获得了近50株人源抗VEGF单链抗体,这为开发具有自主知识产权的抗VEGF治疗性抗体奠定了基础。为了便于研究,本研究将筛选到的两株抗VEGF单链抗体VA6、VK8改造为IgG1全抗体,并将这两株全抗体在FreeStyle~(TM)293细胞中进行了瞬时表达,表达量可达20mg/L,获得的全抗体纯品为以后的实验提供了充足稳定的实验材料。改造的两株全抗体保持了VEGF-A165特异性结合的活性,利用Bia-core方法测定了两株全抗体的亲和力,VA6、VK8亲和力分别为12.7nM和0.77nM。体外活性试验结果显示,两株全抗体都具有一定的体外活性,具有进一步开发为治疗性抗体的潜质。
由于VEGF/VEGFR2的亲和力在0.1nM水平,只有亲和力达到或超过这个水平的抗体才能较好地阻断VEGF和VEGFR结合,达到临床应用的要求。VA6、VK8两株抗体的亲和力尚不能达到此水平,因此需要进行体外亲和力成熟以提高这两株抗体的亲和力。本研究对这两株抗体进行了体外亲和力成熟,以期改善其体外、体内生物学活性,同时尝试建立可行的亲和力成熟方法,以便应用于其它抗体的开发研究,为治疗性抗体的研发提供技术支撑。我们以两株抗VEGF单克隆抗体为模版,采用计算机辅助设计的技术路线,根据构建的抗原-抗体复合物结构模型所提供的信息进行全抗体突变体设计,然后进行试验分析验证,寻找亲和力得以改善的突变体。在得到亲和力有所提高的突变体之后,再进行下一轮的突变体设计、分析与验证,如此反复进行,以期获得亲和力显著提高,功能有所改善的突变体。并在此基础上,通过进一步改进和完善,建立起高效的抗体亲和力成熟方法,为治疗性抗体的研发提供技术支持。
首先,我们通过同源模建的方法,构建了VA6和VEGF复合物的三维结构模型,通过对该模型的深入分析,发现该抗体与VEGF的结合表位与VEGF受体1、受体2的结合表位交错重叠。推测该抗体能够抑制VEGF与其受体的结合,从而抑制VEGF的生物学活性。VA6轻链CDR3区第90位赖氨酸、第92位天冬氨酸两个氨基酸残基由于所带电荷性质和对应的VEGF界面上的残基电荷性质相同,存在排斥作用,明显不利于抗原抗体的结合。根据结构模型所获信息,我们设计并构建了一系列突变体。利用FreeStyleTM293-F细胞双载体瞬时表达系统,对所构建的突变体进行了全抗体表达,用Protein A柱进行亲和层析纯化。在对抗体突变体亲和力分析过程中,通过反复尝试,我们建立了一种简便的定性比较抗体相对亲和力的ELISA方法。运用该方法初步分析了所构建突变体的亲和力,结果表明,突变体A6L-K90D_D92I和A6L-K90D_D92V亲和力分别有了不同程度的提高。Bia-core测定这两株突变体的亲和力,分别达到亲本抗体亲和力的1.5倍和2.3倍。这一结果证明所构建的模型准确,该亲和力成熟方法可行有效。
在此工作基础上,我们以A6L-K90D_D92V为目标抗体,构建了新的三维结构模型。通过对该结构的详细分析,确定抗体重链57位的丝氨酸与VEGF上86位组氨酸距离较近,将重链57位氨基酸改变为带负电荷的残基可以增强抗原-抗体之间的作用力。同时在结构模型中分析发现,重链98位氨基酸在CDR3区的loop区顶端,与抗原距离较远,因此决定在该区域引入3个氨基酸,以增加该区域与抗原的作用界面面积。按照第一轮突变体构建与分析策略,构建了突变体并进行了全抗体的表达与分析。结果显示,在将重链57位丝氨酸突变为谷氨酸后,抗体亲和力有所提高(A6H-S57E,与轻链A6L-K90D_D92V匹配),比A6L-K90D_D92V的亲和力高2倍,这样这一轮得到的突变体A6H-S57E亲和力达到亲本抗体的7倍。
随后,按照和前两轮相同的策略,对第二轮获得的亲和力最高的突变体进行了新一轮的三维结构模建和分析。结果显示,该突变体重链52位和54位的丝氨酸空间与VEGF表面相应区域空间结构契合不是十分严密,并且VEGF表面相应区域有氢键受体,所以可以改变这两个位置的氨基酸以增加契合面积,同时增加供体氢原子,以使该区域抗原抗体间形成氢键。根据这些信息设计了20株突变体并进行了亲和力分析,结果显示将52位和54位的丝氨酸都突变为苏氨酸之后抗体亲和力得到了进一步的提高(A6H-S52T_S54T_ S57E,与轻链A6L-K90D_D92V匹配),抗体亲和力达到初始亲本抗体的10倍以上。
在获得了VA6高亲和力突变体的工作基础上,我们尝试用同样的方法对另外一株抗体VK8进行体外亲和力成熟,并选择界面残基相互作用能作为衡量突变体亲和力是否可能提高的参数,尝试将突变体设计的过程标准化,以便更高效地获得高亲和力突变体,但是却遇到了困难。按照构建的模型设计的所有突变体亲和力都未见提高。通过分析认为该模型与实际情况存在偏差。随后我们对模型构建策略进行了改进,通过“叠合优化”和“分子对接”两种方法分别获得了多个模型。通过分析这些模型,认为其中的模型5更有可能与真实的抗原-抗体复合物结构相符。通过对这些模型进行综合分析及实验验证,初步确定了正确的模型,并最终获得了亲和力有所提高的突变体。
通过上述研究,我们初步确定了构建抗原-抗体复合物结构模型的方法,建立了抗VEGF单克隆抗体VA6、VK8的三维结构模型,并以此为依据,通过计算机辅助设计构建了大量突变体,最终获得了这两株抗VEGF单克隆抗体的高亲和力突变体。高亲和力突变体的获得,为进一步将VA6、VK8开发成治疗性抗体提供了有效的技术手段,而计算机辅助设计进行抗体体外亲和力成熟方法的建立,为开发人源治疗性抗体提供了技术支持。
Therioma has been seriously threatened to human health. 7 millions people die due to therioma and 10 millions new cancer patients arise every year throughout the world. In China, cancer has become the predominant cause of death in urban, which accounts for 21% of the total number of death. As WHO has predicted, by 2020,the incidence of cancer around the world will nearly double. As for cancer treatment, it is mainly depends on early surgery and radiotherapy as well as chemotherapy, however, either of them has its disadvantage: surgical treatment relies on early diagnosis, and radiotherapy and chemotherapy have too many adverse side effects. Thus it is desired to develop novel anti-tumor drugs, Anti-angiogenesis therapeutic antibody is a new approach for tumor therapy. Angiogenesis is essential for tumor growth and metastasis. Vascular endothelial growth factor (VEGF), interacting with its receptors (VEGFRs), can specifically promote endothelial cell division, proliferation and migration, and plays a critical role in the process of angiogenesis. By specifically blocking or inhibiting the VEGF/VEGFR-mediated biological activity, the tumor angiogenesis is able to supress, which is necessary for tumor progression and metastasis. Thus it is a focus of the VEGF to develop antibody treatment of cancer development. The current anti-VEGF humanized antibody, bevacizumab (trade name Avastin) is considered as the therapeutic antibody drugs with most valuable market prospects as well as enormous social and economic value, the annual sales of it is as high as 5.77 billion U.S. dollars in 2009. But there has no fully human anti-VEGF antibody been successfully used in clinical. Therefore the development of VEGF-targeted human-derived antibody has broad application prospects and great significance.
50 fully human anti-VEGF scFV antibodies has been obtained in our lab by screening a large fully synthetic human phage antibody library, which laid a good foundation for developing anti-VEGF therapeutic antibody with proprietary intellectual property rights. For further study, two anti-VEGF scFv monoclonal antibodies, named VA6 and VK8, were altered to IgG1 antibodies and transiently expressed in FreeStyleTM293 cells, the expression level of which is up to about 20mg/L, the purified whole antidody samples provide enough stable experimental materials for subsequent studies. The whole antibodies IgG1 of VA6 and VK8 retained specilly binding activities to antigen VEGF-A165. The affinities of these two whole antibodies to antigen VEGF165 were determined using Bia-core, the affinities of VA6 and VK8 were 12.7nM and 0.77nM,respectively. The results of in vitro activity study suggested that these two antibodies had some activity in vitro and potential of further development for the therapeutic antibody.
As the affinity of VEGF to VEGFR2 is at 0.1nM level, only antibodies whose affinity up to this level can block or inhibit the interaction between VEGF and its receptors efficiently, while the affinity of VA6 and VK8 could not achieve this level, therefore affinity improvement was needed. In this study, in vitro affinity maturation was carried out to improve the affinity of VA6 and VK8, and to improve their bioactivities in vitro and in vivo as well. And through this work, we also expect to consturct a genenral viable method of affinity maturation, which could be used to other antibodies’affinity maturation.
As parent antibody, VA6 and VK8 were used to construct antigen-antibody complex structure models by computer-aided design technology, and serials of antibody mutants were designed and constructed according to the information from the complex sturcture models. The mutants were expressed and the affinities were detected. After obtaining mutants with higher affinity, a new round of Models construction, mutants design and construction, affinity analysis were accomplished to get better mutants. This procedure was repeated based on the last round to look for Variants with significantly improved affinity, and on this basis, through further improvement and refinement, a highly efficient general method of antibody affinity maturation should be established to used for other antibody affinity maturation.
Firstly, we adopted the method of homology modeling to construct the VA6 and VEGF complex three-dimensional structure model. Analyzing results indicated that the antibody epitopes were overlapped with the epitopes of VEGFR 1 and VEGFR 2, So it was speculated that VA6 could inhibit VEGF binding to its receptors, thereby inhibit the biological activity of VEGF. In addition, Model indicated that electric charge of 90th lysine and 92nd aspartate from VA6 light chain CDR3 region countered with the corresponding interface residues on VEGF, then obstructed the binding between VA6 and VEGF. According to this information, a series of mutants were designed and built. These mutants were expressed in FreeStyleTM293-F cells dual-vector expression system, and purified with Protein A affinity chromatography columns. For affinity assay, a simple ELISA method to qualitatively compare the relative affinity of antibody had been established after repeated attempts. By using this method, a preliminary affinity analysis of the mutants was carried out and the results showed that the affinity of mutants A6L-K90D_D92I and A6L-K90D_D92V increased to some extent.. Affinity of these mutants was detected by BIA-core and the affinity of the Variants is 1.5 times and 2.3 times higher than that of the parent antibody respectively. The results implied that the constructed model was accurate and the affinity maturation method was effective.
Then, model of mutant A6L-K90D_D92V and VEGF complex was constructed, and another site with countered charge to VEGF, heavy chain 57th serine, was identified to be close with the 86th His, and the interactive effort could be improved by changing it to amino acids with negative charge residues. And the modeling showed that the 98th amino acid in HCDR3 was at the top of CDR3 loop and it was far away from antigen, so several variants with 3 amino acids insertion at this position were designed and constructed. All these mutants were constructed and analyzed as before. And the results showed that the mutant A6H-S57E had about 2-fold increase in affinity analysis. Up to here, a mutant, A6H-S57E (mating with light chain A6L-K90D_D92V), with 7 times higher affinity than parent antibody was identified.
Subsequently, model of the highest affinity mutant and VEGF complex was constructed. The results showed that heavy chain 52th serine and 54th serine of the mutant is not very tight fit with VEGF surface and there is a hydrogen bond acceptor on the corresponding VEGF interaction surface, so hydrogen donor could be taken in the region around 52 serine and 54 serine to enhance antibody-antigen interaction force. Then based on this information, 20 new mutants were designed and tested, then a new mutant named A6H-S52T_S54T_ S57E (mating with light chain A6L-K90D_D92V) was obtained, whose affinity was about 10-fold higher than affinity of parent antibody, reached the level of nanomolar.
Based on the work of affinity maturation of VA6, the similar method were used in the study of in vitro affinity maturation of another antibody VK8, and the interface residue interaction energy was introduced as a measurable parameter for predicting whether the mutants might increase the affinity, and tried to standardize, the process of mutant design. But unfortunately, the first try of maturation on VK8 failed and the reason was summarized as inaccurate modeling. Then several models were built by structural superposition and molecular docking. By analyzing these models, model 5 were more likely to mimic the real structure of antibody-antigen complex. Based on the model 5, a series of mutants were designed and tested, and finally some mutants with improved affinity were obtained.
In conclusion, Method was primarily established to build antibody-antigen complex structural model, and three-dimensional structure models of anti-VEGF monoclonal antibodies VA6 and VK8 were constructed. On this basis, a large number of mutants were designed and tested, from which two anti-VEGF monoclonal antibodies with higher affinity were identified. Thus, these high-affinity mutants opened a new situation for the further development of VA6 and VK8, and in vitro antibody affinity maturation with computer-aided design provided effective technical support for development of human therapeutic antibodies.
本研究室通过对大容量全合成噬菌体人源抗体库的筛选,获得了近50株人源抗VEGF单链抗体,这为开发具有自主知识产权的抗VEGF治疗性抗体奠定了基础。为了便于研究,本研究将筛选到的两株抗VEGF单链抗体VA6、VK8改造为IgG1全抗体,并将这两株全抗体在FreeStyle~(TM)293细胞中进行了瞬时表达,表达量可达20mg/L,获得的全抗体纯品为以后的实验提供了充足稳定的实验材料。改造的两株全抗体保持了VEGF-A165特异性结合的活性,利用Bia-core方法测定了两株全抗体的亲和力,VA6、VK8亲和力分别为12.7nM和0.77nM。体外活性试验结果显示,两株全抗体都具有一定的体外活性,具有进一步开发为治疗性抗体的潜质。
由于VEGF/VEGFR2的亲和力在0.1nM水平,只有亲和力达到或超过这个水平的抗体才能较好地阻断VEGF和VEGFR结合,达到临床应用的要求。VA6、VK8两株抗体的亲和力尚不能达到此水平,因此需要进行体外亲和力成熟以提高这两株抗体的亲和力。本研究对这两株抗体进行了体外亲和力成熟,以期改善其体外、体内生物学活性,同时尝试建立可行的亲和力成熟方法,以便应用于其它抗体的开发研究,为治疗性抗体的研发提供技术支撑。我们以两株抗VEGF单克隆抗体为模版,采用计算机辅助设计的技术路线,根据构建的抗原-抗体复合物结构模型所提供的信息进行全抗体突变体设计,然后进行试验分析验证,寻找亲和力得以改善的突变体。在得到亲和力有所提高的突变体之后,再进行下一轮的突变体设计、分析与验证,如此反复进行,以期获得亲和力显著提高,功能有所改善的突变体。并在此基础上,通过进一步改进和完善,建立起高效的抗体亲和力成熟方法,为治疗性抗体的研发提供技术支持。
首先,我们通过同源模建的方法,构建了VA6和VEGF复合物的三维结构模型,通过对该模型的深入分析,发现该抗体与VEGF的结合表位与VEGF受体1、受体2的结合表位交错重叠。推测该抗体能够抑制VEGF与其受体的结合,从而抑制VEGF的生物学活性。VA6轻链CDR3区第90位赖氨酸、第92位天冬氨酸两个氨基酸残基由于所带电荷性质和对应的VEGF界面上的残基电荷性质相同,存在排斥作用,明显不利于抗原抗体的结合。根据结构模型所获信息,我们设计并构建了一系列突变体。利用FreeStyleTM293-F细胞双载体瞬时表达系统,对所构建的突变体进行了全抗体表达,用Protein A柱进行亲和层析纯化。在对抗体突变体亲和力分析过程中,通过反复尝试,我们建立了一种简便的定性比较抗体相对亲和力的ELISA方法。运用该方法初步分析了所构建突变体的亲和力,结果表明,突变体A6L-K90D_D92I和A6L-K90D_D92V亲和力分别有了不同程度的提高。Bia-core测定这两株突变体的亲和力,分别达到亲本抗体亲和力的1.5倍和2.3倍。这一结果证明所构建的模型准确,该亲和力成熟方法可行有效。
在此工作基础上,我们以A6L-K90D_D92V为目标抗体,构建了新的三维结构模型。通过对该结构的详细分析,确定抗体重链57位的丝氨酸与VEGF上86位组氨酸距离较近,将重链57位氨基酸改变为带负电荷的残基可以增强抗原-抗体之间的作用力。同时在结构模型中分析发现,重链98位氨基酸在CDR3区的loop区顶端,与抗原距离较远,因此决定在该区域引入3个氨基酸,以增加该区域与抗原的作用界面面积。按照第一轮突变体构建与分析策略,构建了突变体并进行了全抗体的表达与分析。结果显示,在将重链57位丝氨酸突变为谷氨酸后,抗体亲和力有所提高(A6H-S57E,与轻链A6L-K90D_D92V匹配),比A6L-K90D_D92V的亲和力高2倍,这样这一轮得到的突变体A6H-S57E亲和力达到亲本抗体的7倍。
随后,按照和前两轮相同的策略,对第二轮获得的亲和力最高的突变体进行了新一轮的三维结构模建和分析。结果显示,该突变体重链52位和54位的丝氨酸空间与VEGF表面相应区域空间结构契合不是十分严密,并且VEGF表面相应区域有氢键受体,所以可以改变这两个位置的氨基酸以增加契合面积,同时增加供体氢原子,以使该区域抗原抗体间形成氢键。根据这些信息设计了20株突变体并进行了亲和力分析,结果显示将52位和54位的丝氨酸都突变为苏氨酸之后抗体亲和力得到了进一步的提高(A6H-S52T_S54T_ S57E,与轻链A6L-K90D_D92V匹配),抗体亲和力达到初始亲本抗体的10倍以上。
在获得了VA6高亲和力突变体的工作基础上,我们尝试用同样的方法对另外一株抗体VK8进行体外亲和力成熟,并选择界面残基相互作用能作为衡量突变体亲和力是否可能提高的参数,尝试将突变体设计的过程标准化,以便更高效地获得高亲和力突变体,但是却遇到了困难。按照构建的模型设计的所有突变体亲和力都未见提高。通过分析认为该模型与实际情况存在偏差。随后我们对模型构建策略进行了改进,通过“叠合优化”和“分子对接”两种方法分别获得了多个模型。通过分析这些模型,认为其中的模型5更有可能与真实的抗原-抗体复合物结构相符。通过对这些模型进行综合分析及实验验证,初步确定了正确的模型,并最终获得了亲和力有所提高的突变体。
通过上述研究,我们初步确定了构建抗原-抗体复合物结构模型的方法,建立了抗VEGF单克隆抗体VA6、VK8的三维结构模型,并以此为依据,通过计算机辅助设计构建了大量突变体,最终获得了这两株抗VEGF单克隆抗体的高亲和力突变体。高亲和力突变体的获得,为进一步将VA6、VK8开发成治疗性抗体提供了有效的技术手段,而计算机辅助设计进行抗体体外亲和力成熟方法的建立,为开发人源治疗性抗体提供了技术支持。
Therioma has been seriously threatened to human health. 7 millions people die due to therioma and 10 millions new cancer patients arise every year throughout the world. In China, cancer has become the predominant cause of death in urban, which accounts for 21% of the total number of death. As WHO has predicted, by 2020,the incidence of cancer around the world will nearly double. As for cancer treatment, it is mainly depends on early surgery and radiotherapy as well as chemotherapy, however, either of them has its disadvantage: surgical treatment relies on early diagnosis, and radiotherapy and chemotherapy have too many adverse side effects. Thus it is desired to develop novel anti-tumor drugs, Anti-angiogenesis therapeutic antibody is a new approach for tumor therapy. Angiogenesis is essential for tumor growth and metastasis. Vascular endothelial growth factor (VEGF), interacting with its receptors (VEGFRs), can specifically promote endothelial cell division, proliferation and migration, and plays a critical role in the process of angiogenesis. By specifically blocking or inhibiting the VEGF/VEGFR-mediated biological activity, the tumor angiogenesis is able to supress, which is necessary for tumor progression and metastasis. Thus it is a focus of the VEGF to develop antibody treatment of cancer development. The current anti-VEGF humanized antibody, bevacizumab (trade name Avastin) is considered as the therapeutic antibody drugs with most valuable market prospects as well as enormous social and economic value, the annual sales of it is as high as 5.77 billion U.S. dollars in 2009. But there has no fully human anti-VEGF antibody been successfully used in clinical. Therefore the development of VEGF-targeted human-derived antibody has broad application prospects and great significance.
50 fully human anti-VEGF scFV antibodies has been obtained in our lab by screening a large fully synthetic human phage antibody library, which laid a good foundation for developing anti-VEGF therapeutic antibody with proprietary intellectual property rights. For further study, two anti-VEGF scFv monoclonal antibodies, named VA6 and VK8, were altered to IgG1 antibodies and transiently expressed in FreeStyleTM293 cells, the expression level of which is up to about 20mg/L, the purified whole antidody samples provide enough stable experimental materials for subsequent studies. The whole antibodies IgG1 of VA6 and VK8 retained specilly binding activities to antigen VEGF-A165. The affinities of these two whole antibodies to antigen VEGF165 were determined using Bia-core, the affinities of VA6 and VK8 were 12.7nM and 0.77nM,respectively. The results of in vitro activity study suggested that these two antibodies had some activity in vitro and potential of further development for the therapeutic antibody.
As the affinity of VEGF to VEGFR2 is at 0.1nM level, only antibodies whose affinity up to this level can block or inhibit the interaction between VEGF and its receptors efficiently, while the affinity of VA6 and VK8 could not achieve this level, therefore affinity improvement was needed. In this study, in vitro affinity maturation was carried out to improve the affinity of VA6 and VK8, and to improve their bioactivities in vitro and in vivo as well. And through this work, we also expect to consturct a genenral viable method of affinity maturation, which could be used to other antibodies’affinity maturation.
As parent antibody, VA6 and VK8 were used to construct antigen-antibody complex structure models by computer-aided design technology, and serials of antibody mutants were designed and constructed according to the information from the complex sturcture models. The mutants were expressed and the affinities were detected. After obtaining mutants with higher affinity, a new round of Models construction, mutants design and construction, affinity analysis were accomplished to get better mutants. This procedure was repeated based on the last round to look for Variants with significantly improved affinity, and on this basis, through further improvement and refinement, a highly efficient general method of antibody affinity maturation should be established to used for other antibody affinity maturation.
Firstly, we adopted the method of homology modeling to construct the VA6 and VEGF complex three-dimensional structure model. Analyzing results indicated that the antibody epitopes were overlapped with the epitopes of VEGFR 1 and VEGFR 2, So it was speculated that VA6 could inhibit VEGF binding to its receptors, thereby inhibit the biological activity of VEGF. In addition, Model indicated that electric charge of 90th lysine and 92nd aspartate from VA6 light chain CDR3 region countered with the corresponding interface residues on VEGF, then obstructed the binding between VA6 and VEGF. According to this information, a series of mutants were designed and built. These mutants were expressed in FreeStyleTM293-F cells dual-vector expression system, and purified with Protein A affinity chromatography columns. For affinity assay, a simple ELISA method to qualitatively compare the relative affinity of antibody had been established after repeated attempts. By using this method, a preliminary affinity analysis of the mutants was carried out and the results showed that the affinity of mutants A6L-K90D_D92I and A6L-K90D_D92V increased to some extent.. Affinity of these mutants was detected by BIA-core and the affinity of the Variants is 1.5 times and 2.3 times higher than that of the parent antibody respectively. The results implied that the constructed model was accurate and the affinity maturation method was effective.
Then, model of mutant A6L-K90D_D92V and VEGF complex was constructed, and another site with countered charge to VEGF, heavy chain 57th serine, was identified to be close with the 86th His, and the interactive effort could be improved by changing it to amino acids with negative charge residues. And the modeling showed that the 98th amino acid in HCDR3 was at the top of CDR3 loop and it was far away from antigen, so several variants with 3 amino acids insertion at this position were designed and constructed. All these mutants were constructed and analyzed as before. And the results showed that the mutant A6H-S57E had about 2-fold increase in affinity analysis. Up to here, a mutant, A6H-S57E (mating with light chain A6L-K90D_D92V), with 7 times higher affinity than parent antibody was identified.
Subsequently, model of the highest affinity mutant and VEGF complex was constructed. The results showed that heavy chain 52th serine and 54th serine of the mutant is not very tight fit with VEGF surface and there is a hydrogen bond acceptor on the corresponding VEGF interaction surface, so hydrogen donor could be taken in the region around 52 serine and 54 serine to enhance antibody-antigen interaction force. Then based on this information, 20 new mutants were designed and tested, then a new mutant named A6H-S52T_S54T_ S57E (mating with light chain A6L-K90D_D92V) was obtained, whose affinity was about 10-fold higher than affinity of parent antibody, reached the level of nanomolar.
Based on the work of affinity maturation of VA6, the similar method were used in the study of in vitro affinity maturation of another antibody VK8, and the interface residue interaction energy was introduced as a measurable parameter for predicting whether the mutants might increase the affinity, and tried to standardize, the process of mutant design. But unfortunately, the first try of maturation on VK8 failed and the reason was summarized as inaccurate modeling. Then several models were built by structural superposition and molecular docking. By analyzing these models, model 5 were more likely to mimic the real structure of antibody-antigen complex. Based on the model 5, a series of mutants were designed and tested, and finally some mutants with improved affinity were obtained.
In conclusion, Method was primarily established to build antibody-antigen complex structural model, and three-dimensional structure models of anti-VEGF monoclonal antibodies VA6 and VK8 were constructed. On this basis, a large number of mutants were designed and tested, from which two anti-VEGF monoclonal antibodies with higher affinity were identified. Thus, these high-affinity mutants opened a new situation for the further development of VA6 and VK8, and in vitro antibody affinity maturation with computer-aided design provided effective technical support for development of human therapeutic antibodies.
引文
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[14].Iwai, H., et al. , Antibody affinity maturation in vitro using unconjugated peptide antigen. Protein Eng Des Sel, 2010. 23(4): p. 185-93.
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[21].Fermer, C., et al. , Specificity rescue and affinity maturation of a low-affinity IgM antibody against pro-gastrin-releasing peptide using phage display and DNA shuffling. Tumour Biol, 2004. 25(1-2): p. 7-13.
[22].Chodorge, M., et al. , In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants. Protein Eng Des Sel, 2008. 21(5): p. 343-51.
[23].Yoshinaga, K., et al. , Ig L-chain shuffling for affinity maturation of phage library-derived human anti-human MCP-1 antibody blocking its chemotactic activity. J Biochem, 2008. 143(5): p. 593-601.
[24].Luginbuhl, B., et al. , Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation. J Mol Biol, 2006. 363(1): p. 75-97.
[25].Yau, K.Y., et al. , Affinity maturation of a V(H)H by mutational hotspot randomization. J Immunol Methods, 2005. 297(1-2): p. 213-24.
[26].Chowdhury, P.S. and I. Pastan, Analysis of cloned Fvs from a phage display library indicates that DNA immunization can mimic antibody response generated by cell immunizations. J Immunol Methods, 1999. 231(1-2): p. 83-91.
[27].Yau, K.Y., et al. , Affinity maturation of a V(H)H by mutational hotspot randomization. J Immunol Methods, 2005. 297(1-2): p. 213-24.
[28].Montgomery, D.L., et al. , Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41. MAbs, 2009. 1(5): p. 462-74.
[29]. Steidl S, Ratsch O, Brocks B, Dürr M, Thomassen-Wolf E. In vitro affinity maturation of human GM-CSF antibodies by targeted CDR-diversification.Mol Immunol. 2008 Nov;46(1):135-44.
[30].Clark, L.A., et al. , Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design. Protein Sci, 2006. 15(5): p. 949-60.
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[32].Barderas, R., et al. , Affinity maturation of antibodies assisted by in silico modeling. Proc Natl Acad Sci U S A, 2008. 105(26): p. 9029-34.
[33].Fermer, C., et al. , Specificity rescue and affinity maturation of a low-affinity IgM antibody against pro-gastrin-releasing peptide using phage display and DNA shuffling. Tumour Biol, 2004. 25(1-2): p. 7-13.
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