Affinity Maturation Of Anti-vegf Antibodies By Computor-aided Drug Design

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.