開發可辨認 H7N9 HA1 及 HA2 之雙特異性抗體

Abstract

目前對於新型H7N9流感仍無有效的疫苗，且病毒株已對克流感產生抗藥性，造成治療的難度增加，因此本研究擬將可分別辨認H7N9 HA1及HA2的單株抗體F3-2及1C6B，製備成可同時結合上HA1及HA2之雙特異性抗體 (Bispecific antibody, BsAb)，藉此形成類似”分子鉗”的結構配置，達到抑制血球凝集素的構形改變，進而抑制H7N9病毒感染宿主細胞的功效。實驗首先擷取F3-2或1C6B之變異區 (Variable region) 以組成單鏈抗體 (scFv)，再利用不同長度的linker (例如G、(G4S)x2、(G4S)x4、(G4S)x6 或 (G4S)x8) 來連結F3-2 scFv及1C6B scFv，以增加結構的延展性與靈活性。於大腸桿菌中進行蛋白質表現時，此BsAb大部分會形成不可溶之包涵體，僅有極少量的BsAb為可溶狀態，但是此可溶狀態之BsAb具有可同時結合HA1及HA2之能力。然而，用尿素緩衝溶液所純化出之BsAb，則會喪失其抗原結合能力。因此，本論文繼續嘗試不同的表現條件以解決BsAb不可溶且產量低的問題。最後以GST-BsAb融合蛋白質的方式進行表現，可以製備出較多可溶之GST-BsAb，但是此融合蛋白質雖然仍可辨認HA1，但是卻無法辨認HA2。進一步利用TEV protease將GST-BsAb截切成free form BsAb後，仍無法使其恢復辨認HA2的能力，顯示此BsAb可能仍須直接表現為可溶形式的單體，才具有能同時結合上HA1及HA2之功能。

There is currently no effective vaccine for prevention of the novel H7N9 influenza pandemic, and the virus strain has developed resistance to Tamiflu, resulting in increased treatment difficulties. Thus, the present study aimed to develpe bispecific antibodies (BsAbs), composed of the single-chain variable fragments (scFvs) derived from the monoclonal antibody F3-2 and 1C6B which can respectively bind to H7N9 HA1 and HA2, to form a "molecular clamp" structural configuration for increasing efficacy of inhibiting the conformational change of hemagglutinin and the H7N9 infection in host cells. In addition, a linker with different length (i.e., G, (G4S)x2, (G4S)x4, (G4S)x6, or (G4S)x8) was also introduced in the BsAb to connect the F3-2 scFv with the 1C6B scFv for increasing the extensibility and flexibility. When performing protein expression in Escherichia coli, most of the BsAbs formed insoluble inclusion bodies, and only a very small amount of BsAb was soluble, but this soluble BsAb contained the ability to specifically bind to HA1 and HA2. However, the BsAb purified with a urea buffer lost its antigen binding ability. To reach the goal of getting large amount of soluble BsAbs, different protein expression protocols and plasmids were also applied in the experiments. The results showed that more soluble GST-BsAb can be produced by expressing it as the GST fusion protein. This GST-BsAb fusion protein can still recognize HA1, but it can not recognize HA2. Further use of TEV protease to cut the GST-BsAb into a free form BsAb still failed to restore its ability to recognize HA2, indicating that this BsAb may still need to be directly expressed as a soluble form of monomer to retain the function of simultaneously binding to HA1 and HA2.