【摘要】： 腫瘤壞死因子α(TNF-α)是一種多功能的細(xì)胞因子，參與許多重要的生理功能，但TNF-α的過表達(dá)會(huì)破壞機(jī)體的免疫平衡，，與類風(fēng)濕性關(guān)節(jié)炎、Crohn's病等多種疾病密切相關(guān)�；A(chǔ)和臨床研究都證實(shí)，抑制TNF活性是治療這類疾病的一個(gè)有效措施。目前，已被FDA批準(zhǔn)的TNF-α拮抗劑有TNFRII-F_C融合蛋白和抗TNF-α單抗，但昂貴的生產(chǎn)成本或潛在的誘發(fā)人抗鼠抗體反應(yīng)(HAMA)的副作用，使其廣泛應(yīng)用受到一定限制。開發(fā)新型TNF-α拮抗分子必要而有意義，人源小分子抗體由于其獨(dú)特的治療價(jià)值而受到人們的矚目，但高親和力人源抗體的獲得有一定困難。 本實(shí)驗(yàn)室在TNF-α拮抗分子研究方面取得了一定進(jìn)展：自主研制的鼠抗TNF-α中和單抗Z12能特異性識(shí)別TNF-α的141～146位這一功能表位；根據(jù)理論模擬構(gòu)建的TNF／抗體相互作用的復(fù)合物模型，設(shè)計(jì)獲得功能性拮抗肽(PT2、PT3、PT4、PT7)；利用人抗體可變區(qū)重鏈框架(V_H5)展示拮抗肽(PT2、PT3、PT4)，設(shè)計(jì)并合成獲得單域抗體(PTVH5)，其活性明顯好于拮抗肽，證明抗體可變區(qū)框架適于展示拮抗肽。 基于此，借助已建立的“基于抗原—抗體相互作用結(jié)構(gòu)信息設(shè)計(jì)新型功能分子”的技術(shù)平臺(tái)，本論文提出以人抗體可變區(qū)(重鏈、輕鏈)框架展示拮抗肽(PT2、PT3、PT4、PT7)設(shè)計(jì)虛擬單鏈抗體構(gòu)象庫，借助TNF／抗體相互作用動(dòng)態(tài)模式，虛擬篩選新型TNF-α單鏈抗體分子，合成獲得單鏈抗體分子并通過生物學(xué)實(shí)驗(yàn)驗(yàn)證其功能。 研究結(jié)果如下： 1．在單域抗體PTVH5(用V_H5框架來展示拮抗肽PT2、PT3、PT4)的基礎(chǔ)上，按照“結(jié)構(gòu)匹配、靜電互補(bǔ)”的原則，從人的七類輕鏈可變區(qū)(V_L)通用框架中選擇與之匹配的V_κ1框架，用拮抗肽PT7替換V_κ1框架中的CDR3區(qū)，通過Linker連接，設(shè)計(jì)成新型分子TSA1。理論分析發(fā)現(xiàn)，TSA1穩(wěn)定性較好，識(shí)別TNF-α的141～146功能位點(diǎn)(即Z12識(shí)別的位點(diǎn))。生物學(xué)實(shí)驗(yàn)證明，TSA1能與TNF-α結(jié)合、抑制TNF-α與TNFR的結(jié)合、抑制TNF-α介導(dǎo)的細(xì)胞毒作用及NF-κB的激活，而且TSA1的活性比之前設(shè)計(jì)的單域抗體有顯著提高(中和TNF-α介導(dǎo)的細(xì)胞毒實(shí)驗(yàn)中，單域抗體PTVH5的IC_(50)值為56.5±3.2μmol／L；TSA1的IC_(50)值為0.0982±0.01μmol／L)。但與S-Remicade(上市抗體Remicade的單鏈形式)相比，TSA1的活性較弱。 2．從框架的合理選擇和拮抗肽的合理定位兩方面來優(yōu)化設(shè)計(jì)方案，設(shè)計(jì)虛擬單鏈抗體構(gòu)象庫，以期篩選得到活性更好的拮抗分子。TSA1與TNF的動(dòng)態(tài)作用模式表明，在與TNF的結(jié)合過程中，重鏈CDR3(HCDR3，即拮抗肽PT4)起著關(guān)鍵的作用。鑒于此，根據(jù)4種拮抗肽活性的差異(PT7＞PT4＞PT2≈PT3)，將其中活性最好的PT7安排在HCDR3的位置，其余三個(gè)拮抗肽PT2、PT3、PT4在HCDR1、HCDR2、LCDR3的位置上隨機(jī)組合。結(jié)合七類V_H和七類V_L框架產(chǎn)生的49種框架組合，利用計(jì)算機(jī)模建技術(shù)，設(shè)計(jì)人單鏈抗體構(gòu)象庫，虛擬篩選得到以V_λ1與V_H5為框架設(shè)計(jì)的新型單鏈抗體TSA2(拮抗肽PT2、PT3、PT7分別替換HCDR1、HCDR2、HCDR3，拮抗肽PT4替換LCDR3)。理論分析表明，TSA2在與TNF-α的相互作用區(qū)域、結(jié)合自由能、分子間氫鍵等方面均顯著優(yōu)于TSA1。生物學(xué)實(shí)驗(yàn)證明，與TSA1相比，TSA2的活性顯著提高，與理論預(yù)測結(jié)果一致。而NTSA2的活性在一定程度上與S-Remicade(上市抗體Remicade的單鏈形式)相接近，達(dá)到預(yù)期效果。 本論文初步驗(yàn)證了“借助計(jì)算機(jī)模建，基于人抗體可變區(qū)的框架結(jié)構(gòu)和拮抗肽，設(shè)計(jì)虛擬單鏈抗體構(gòu)象庫并從中篩選新型拮抗分子”的策略是可行的，從而為人源小分子抗體的制備提供了一條可供選擇的途徑。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 秦衛(wèi)松;基于抗原抗體相互作用的立體結(jié)構(gòu)信息設(shè)計(jì)新型TNF-α拮抗分子[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2005年

本文編號(hào)：2182635