靶向肝星状细胞的全人源化抗TGF-β1单链抗体药物设计及其抗肝纤维化研究

Liver fibrosis, a major cause of the morbidity and mortality of chronic liver disease, disrupts the normal architecture of the liver and leads to liver failure. Production of excessive transforming growth factor-β1 (TGF-β1) is considered as a major profibrogenic cytokine and a potent inducer of liver fibrosis. It is confirmed that attenuating the binding of TGF-β1 to transforming growth factor-beta receptor type II (TβRII) is effective to ameliorate TGF-β1-induced fibrosis. Studies have shown that platelet derived growth factor beta receptors (PDGFβR) are over-expressed on activated hepatic stellate cells (HSCs) in liver fibrosis. Therefore, the applicant proposes the hypothesis: novel humanized anti-TGF-β1 single-chain antibody (scFv) isolated by phage display, competes with TβRII for binding of TGF-β1 and suppresses elevated TGF-β1 activity. In the study, humanized anti-TGF-β1 scFv is genetically coupled with PDGFβR-binding peptide (BiPPB) via encoding Linker sequence (Gly4Ser)2, namely, BiPPB-Linker-scFv. Next, the effects of recombinant BiPPB-Linker-scFv on TGF-β1-induced LX-2 and HSC-T6 cells will be used to confirm whether novel scFv could compete with TβRII for binding of TGF-β1 and attenuate the function of excessive TGF-β1 in the process of fibrosis. We further to investigate the effects of recombinant BiPPB-Linker-scFv on carbon tetrachloride (CCl4)-induced acute liver fibrogenesis and advanced liver fibrosis animal model. Thus, it may represent a potential anti-fibrosis drug for liver fibrosis.

肝纤维化是导致慢性肝功能衰竭的主要原因，已成为危害人类健康的主要疾病。转化生长因子β1（TGF-β1）的过量产生是引起纤维化的根源。阻断TGF-β1与转化生长因子βII型受体（TβRII）结合，被证实能抑制过量TGF-β1所引发的纤维化。研究证实，血小板衍生生长因子β受体（PDGFβR）在纤维化的肝星状细胞表面过量表达。因此，本研究拟通过噬菌体展示技术筛选特异性结合TGF-β1的全人源化单链抗体（scFv），将其与PDGFβR的识别肽（BiPPB）通过Linker连接，即BiPPB-Linker-scFv；中试制备该药物来验证其是否能与胞内的TβRII竞争结合TGF-β1，并探查该药物对TGF-β1诱导的人肝星状细胞（LX-2）和大鼠肝星状细胞（HSC-T6）纤维化影响；并依托四氯化碳（CCl4）诱导的急性和慢性肝纤维化动物模型，验证该药物对其保护作用，为临床抗纤维化药物的开发提供可能。

肝纤维化是导致慢性肝功能衰竭的主要原因，已成为危害人类健康的主要疾病。转化生长因子β1（TGF-β1）的过量产生是引起纤维化的根源。阻断TGF-β1与转化生长因子βII型受体（TβRII）结合，被证实能抑制过量TGF-β1所引发的纤维化。本研究将血小板衍生生长因子β受体（PDGFβR）的识别肽（BiPPB）与可特异性结合TGF-β1的全人源化单链抗体（scFv）偶联BiPPB-scFv，结果表明，BiPPB-scFv特异性结合CCl4诱导的肝纤维化小鼠的纤维化肝组织，并抑制肝脏中纤维化相关基因mRNA和蛋白表达，同时缓解CCl4引起的肝脏病理结构和肝功能损害。因此，靶向药物BiPPB-scFv可为肝纤维化的靶向治疗提供新的策略。

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