基于人抗体可变区框架结构和拮抗肽设计新型TNF-α人源小分子拮抗剂
发布时间：2018-08-14 10:57
【摘要】： 肿瘤坏死因子α(TNF-α)是一种多功能的细胞因子，参与许多重要的生理功能，但TNF-α的过表达会破坏机体的免疫平衡，，与类风湿性关节炎、Crohn's病等多种疾病密切相关。基础和临床研究都证实，抑制TNF活性是治疗这类疾病的一个有效措施。目前，已被FDA批准的TNF-α拮抗剂有TNFRII-F_C融合蛋白和抗TNF-α单抗，但昂贵的生产成本或潜在的诱发人抗鼠抗体反应(HAMA)的副作用，使其广泛应用受到一定限制。开发新型TNF-α拮抗分子必要而有意义，人源小分子抗体由于其独特的治疗价值而受到人们的瞩目，但高亲和力人源抗体的获得有一定困难。 本实验室在TNF-α拮抗分子研究方面取得了一定进展：自主研制的鼠抗TNF-α中和单抗Z12能特异性识别TNF-α的141～146位这一功能表位；根据理论模拟构建的TNF／抗体相互作用的复合物模型，设计获得功能性拮抗肽(PT2、PT3、PT4、PT7)；利用人抗体可变区重链框架(V_H5)展示拮抗肽(PT2、PT3、PT4)，设计并合成获得单域抗体(PTVH5)，其活性明显好于拮抗肽，证明抗体可变区框架适于展示拮抗肽。 基于此，借助已建立的“基于抗原—抗体相互作用结构信息设计新型功能分子”的技术平台，本论文提出以人抗体可变区(重链、轻链)框架展示拮抗肽(PT2、PT3、PT4、PT7)设计虚拟单链抗体构象库，借助TNF／抗体相互作用动态模式，虚拟筛选新型TNF-α单链抗体分子，合成获得单链抗体分子并通过生物学实验验证其功能。 研究结果如下： 1．在单域抗体PTVH5(用V_H5框架来展示拮抗肽PT2、PT3、PT4)的基础上，按照“结构匹配、静电互补”的原则，从人的七类轻链可变区(V_L)通用框架中选择与之匹配的V_κ1框架，用拮抗肽PT7替换V_κ1框架中的CDR3区，通过Linker连接，设计成新型分子TSA1。理论分析发现，TSA1稳定性较好，识别TNF-α的141～146功能位点(即Z12识别的位点)。生物学实验证明，TSA1能与TNF-α结合、抑制TNF-α与TNFR的结合、抑制TNF-α介导的细胞毒作用及NF-κB的激活，而且TSA1的活性比之前设计的单域抗体有显著提高(中和TNF-α介导的细胞毒实验中，单域抗体PTVH5的IC_(50)值为56.5±3.2μmol／L；TSA1的IC_(50)值为0.0982±0.01μmol／L)。但与S-Remicade(上市抗体Remicade的单链形式)相比，TSA1的活性较弱。 2．从框架的合理选择和拮抗肽的合理定位两方面来优化设计方案，设计虚拟单链抗体构象库，以期筛选得到活性更好的拮抗分子。TSA1与TNF的动态作用模式表明，在与TNF的结合过程中，重链CDR3(HCDR3，即拮抗肽PT4)起着关键的作用。鉴于此，根据4种拮抗肽活性的差异(PT7＞PT4＞PT2≈PT3)，将其中活性最好的PT7安排在HCDR3的位置，其余三个拮抗肽PT2、PT3、PT4在HCDR1、HCDR2、LCDR3的位置上随机组合。结合七类V_H和七类V_L框架产生的49种框架组合，利用计算机模建技术，设计人单链抗体构象库，虚拟筛选得到以V_λ1与V_H5为框架设计的新型单链抗体TSA2(拮抗肽PT2、PT3、PT7分别替换HCDR1、HCDR2、HCDR3，拮抗肽PT4替换LCDR3)。理论分析表明，TSA2在与TNF-α的相互作用区域、结合自由能、分子间氢键等方面均显著优于TSA1。生物学实验证明，与TSA1相比，TSA2的活性显著提高，与理论预测结果一致。而NTSA2的活性在一定程度上与S-Remicade(上市抗体Remicade的单链形式)相接近，达到预期效果。 本论文初步验证了“借助计算机模建，基于人抗体可变区的框架结构和拮抗肽，设计虚拟单链抗体构象库并从中筛选新型拮抗分子”的策略是可行的，从而为人源小分子抗体的制备提供了一条可供选择的途径。
[Abstract]:Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine involved in many important physiological functions. However, the overexpression of TNF-alpha can destroy the body's immune balance and is closely related to rheumatoid arthritis, Crohn's disease and other diseases. At present, the TNF-alpha antagonists approved by FDA include TNFRII-F_C fusion protein and anti-TNF-alpha monoclonal antibody, but the high cost of production or the potential side effects of inducing human anti-mouse antibody response (HAMA) restrict their wide application. Value has attracted the attention of people, but it is difficult to obtain high affinity human antibody.
Our laboratory has made some progress in the study of TNF-alpha antagonist molecules: mouse anti-TNF-alpha neutralizing antibody Z12 and mouse anti-TNF-alpha neutralizing antibody Z12 can specifically recognize the 141-146 functional epitopes of TNF-alpha; functional antagonist peptides (PT2, PT3, PT4, PT7) were designed and obtained by theoretical modeling of TNF/antibody interaction complex model. Single domain antibody (PTVH5) was designed and synthesized by displaying antagonistic peptides (PT2, PT3, PT4) on variable region heavy chain frame of human antibody. The activity of the antibody variable region framework was significantly better than that of the antagonistic peptides.
Based on this, a virtual single-chain antibody conformation library is designed with the help of the established "new functional molecule design platform based on the structure information of antigen-antibody interaction". In this paper, we propose to display antagonistic peptides (PT2, PT3, PT4, PT7) in the framework of human antibody variable region (heavy chain, light chain) to screen the virtual single-chain antibody conformation library. A novel TNF-a single chain antibody molecule was synthesized and its function was verified by biological experiments.
The results are as follows:
1. On the basis of single-domain antibody PTVH5 (using V_H5 framework to display antagonistic peptides PT2, PT3, PT4), according to the principle of "structure matching, electrostatic complementarity", the matching V_kappa 1 frame was selected from the seven light chain variable region (V_L) general frame, and the CDR3 region in V_kappa 1 frame was replaced by antagonistic peptide PT7. A new molecular TS was designed by linker connection. A1. Theoretical analysis showed that TSA1 was stable and could identify 141-146 functional sites of TNF-a (i.e. Z12-recognized sites). Biological experiments showed that TSA1 could bind to TNF-a, inhibit the binding of TNF-a to TNFR, inhibit the cytotoxicity mediated by TNF-a and the activation of NF-kappa B, and the activity of TSA1 was significantly higher than that of the previously designed monoclonal antibody (medium) In the cytotoxicity test mediated by TNF-a, the IC_ (50) value of monoclonal antibody PTVH 5 was 56.5 (+ 3.2) micromol/L, and the IC_ (50) value of TSA1 was 0.0982 (+ 0.01) micromol/L. However, compared with S-Remicade, the activity of TSA1 was weaker.
2. To optimize the design scheme from two aspects: the reasonable choice of framework and the rational localization of antagonist peptides, a virtual single-chain antibody conformation library was designed in order to screen the more active antagonist molecules. The dynamic interaction model between TSA1 and TNF showed that heavy-chain CDR3 (HCDR3, antagonist peptide PT4) played a key role in the binding process with TNF. According to the activity difference of four antagonistic peptides (PT7 > PT4 > PT2 PT3), PT7 with the best activity was arranged at the position of HCDR3, and the other three antagonistic peptides PT2, PT3, PT4 were randomly combined at the position of HCDR1, HCDR2, LCDR3. A novel single-chain antibody TSA2 (antagonist peptide PT2, PT3, PT7 replacing HCDR1, HCDR2, HCDR3, antagonist peptide PT4 replacing LCDR3) was obtained by virtual screening. Theoretical analysis showed that TSA2 was superior to TSA1 in the interaction region with TNF-a, binding free energy, intermolecular hydrogen bonding and so on. Compared with SA1, the activity of TSA2 was significantly increased, which was consistent with the theoretical predictions. However, the activity of NTSA2 was similar to S-Remicade to a certain extent, and achieved the desired results.
This paper preliminarily validates the feasibility of the strategy of "designing a virtual single chain antibody conformation library and screening new antagonistic molecules based on the frame structure of variable region of human antibody and antagonistic peptides by computer", thus providing an alternative way for the preparation of human small molecular antibodies.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2182635
[Abstract]:Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine involved in many important physiological functions. However, the overexpression of TNF-alpha can destroy the body's immune balance and is closely related to rheumatoid arthritis, Crohn's disease and other diseases. At present, the TNF-alpha antagonists approved by FDA include TNFRII-F_C fusion protein and anti-TNF-alpha monoclonal antibody, but the high cost of production or the potential side effects of inducing human anti-mouse antibody response (HAMA) restrict their wide application. Value has attracted the attention of people, but it is difficult to obtain high affinity human antibody.
Our laboratory has made some progress in the study of TNF-alpha antagonist molecules: mouse anti-TNF-alpha neutralizing antibody Z12 and mouse anti-TNF-alpha neutralizing antibody Z12 can specifically recognize the 141-146 functional epitopes of TNF-alpha; functional antagonist peptides (PT2, PT3, PT4, PT7) were designed and obtained by theoretical modeling of TNF/antibody interaction complex model. Single domain antibody (PTVH5) was designed and synthesized by displaying antagonistic peptides (PT2, PT3, PT4) on variable region heavy chain frame of human antibody. The activity of the antibody variable region framework was significantly better than that of the antagonistic peptides.
Based on this, a virtual single-chain antibody conformation library is designed with the help of the established "new functional molecule design platform based on the structure information of antigen-antibody interaction". In this paper, we propose to display antagonistic peptides (PT2, PT3, PT4, PT7) in the framework of human antibody variable region (heavy chain, light chain) to screen the virtual single-chain antibody conformation library. A novel TNF-a single chain antibody molecule was synthesized and its function was verified by biological experiments.
The results are as follows:
1. On the basis of single-domain antibody PTVH5 (using V_H5 framework to display antagonistic peptides PT2, PT3, PT4), according to the principle of "structure matching, electrostatic complementarity", the matching V_kappa 1 frame was selected from the seven light chain variable region (V_L) general frame, and the CDR3 region in V_kappa 1 frame was replaced by antagonistic peptide PT7. A new molecular TS was designed by linker connection. A1. Theoretical analysis showed that TSA1 was stable and could identify 141-146 functional sites of TNF-a (i.e. Z12-recognized sites). Biological experiments showed that TSA1 could bind to TNF-a, inhibit the binding of TNF-a to TNFR, inhibit the cytotoxicity mediated by TNF-a and the activation of NF-kappa B, and the activity of TSA1 was significantly higher than that of the previously designed monoclonal antibody (medium) In the cytotoxicity test mediated by TNF-a, the IC_ (50) value of monoclonal antibody PTVH 5 was 56.5 (+ 3.2) micromol/L, and the IC_ (50) value of TSA1 was 0.0982 (+ 0.01) micromol/L. However, compared with S-Remicade, the activity of TSA1 was weaker.
2. To optimize the design scheme from two aspects: the reasonable choice of framework and the rational localization of antagonist peptides, a virtual single-chain antibody conformation library was designed in order to screen the more active antagonist molecules. The dynamic interaction model between TSA1 and TNF showed that heavy-chain CDR3 (HCDR3, antagonist peptide PT4) played a key role in the binding process with TNF. According to the activity difference of four antagonistic peptides (PT7 > PT4 > PT2 PT3), PT7 with the best activity was arranged at the position of HCDR3, and the other three antagonistic peptides PT2, PT3, PT4 were randomly combined at the position of HCDR1, HCDR2, LCDR3. A novel single-chain antibody TSA2 (antagonist peptide PT2, PT3, PT7 replacing HCDR1, HCDR2, HCDR3, antagonist peptide PT4 replacing LCDR3) was obtained by virtual screening. Theoretical analysis showed that TSA2 was superior to TSA1 in the interaction region with TNF-a, binding free energy, intermolecular hydrogen bonding and so on. Compared with SA1, the activity of TSA2 was significantly increased, which was consistent with the theoretical predictions. However, the activity of NTSA2 was similar to S-Remicade to a certain extent, and achieved the desired results.
This paper preliminarily validates the feasibility of the strategy of "designing a virtual single chain antibody conformation library and screening new antagonistic molecules based on the frame structure of variable region of human antibody and antagonistic peptides by computer", thus providing an alternative way for the preparation of human small molecular antibodies.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【共引文献】
相关博士学位论文 前1条
1 秦卫松;基于抗原抗体相互作用的立体结构信息设计新型TNF-α拮抗分子[D];中国人民解放军军事医学科学院;2005年
本文编号：2182635
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