貓傳染性腹膜炎病毒單株抗體之重組抗體與單鏈可變片段之表現

Abstract

貓傳染性腹膜炎（feline infectious peritonitis, FIP）是一種由貓傳染性腹膜炎病毒（feline infectious peritonitis virus, FIPV）所引起之致死性疾病，好發於幼貓及多貓飼養環境。FIPV 能感染單核球及巨噬細胞，造成全身性發炎反應、血管炎及化膿性肉芽腫病變。由於抗體依賴性增強作用（antibody-dependent enhancement, ADE）可能促進病毒感染與疾病進展，FIP 疫苗及抗體治療之開發仍面臨重大挑戰。YB4D01 為一株針對第一型貓傳染性腹膜炎病毒（FIPV serotype I）UU4 株棘蛋白（spike protein）之中和單株抗體，具有作為抗體治療候選分子之潛力。本研究以融合瘤來源單株抗體 YB4D01 之重鏈與輕鏈可變區序列為基礎，建構全長重組單株抗體（recombinant monoclonal antibody）及單鏈可變片段抗體（single-chain variable fragment, scFv），分別利用 human embryonic kidney 293（HEK293）細胞及大腸桿菌表現系統進行表現。此外，亦評估不同表現條件對重組蛋白產量之影響，以建立後續蛋白表現最佳化之基礎。研究結果顯示，本研究已成功完成 YB4D01 重組單株抗體與 scFv 之基因建構與初步表現。重組單株抗體之表現受到重鏈與輕鏈質體比例及轉染條件影響；scFv 之表現則受到誘導溫度、IPTG 濃度及非離子界面活性劑添加條件影響。然而，目前尚未成功建立穩定且可大量生產之表現與純化流程，因此後續仍需進一步優化蛋白表現、純化及功能性分析，包括抗原結合能力與病毒中和活性之評估。綜合而言，本研究成功建立源自抗 FIPV 單株抗體 YB4D01 之重組單株抗體與 scFv 表現系統，為未來抗體工程改造、降低 ADE 風險之抗體設計，以及 FIP 抗體治療策略之發展提供初步基礎。

Feline infectious peritonitis (FIP) is a fatal and progressive disease of cats caused by feline infectious peritonitis virus (FIPV). The development of vaccines and antibody-based therapeutics against FIPV remains challenging because of the risk of antibody-dependent enhancement (ADE). YB4D01 is a neutralizing monoclonal antibody targeting the spike protein of the serotype I FIPV UU4 strain and represents a potential candidate for antibody-based therapeutic development. In this study, based on the variable region sequences of the heavy and light chains of YB4D01, a full-length recombinant monoclonal antibody and a single-chain variable fragment (scFv) were generated. The recombinant antibody and scFv were expressed using human embryonic kidney 293 (HEK293) cells and Escherichia coli expression systems, respectively. In addition, various expression conditions were evaluated to optimize recombinant protein production. The results demonstrated successful construction and expression of both the recombinant monoclonal antibody and scFv derived from YB4D01. Optimization studies revealed that recombinant antibody expression was affected by the heavy-chain to light-chain plasmid ratio and transfection conditions, whereas scFv expression was influenced by induction temperature, IPTG concentration, and surfactant supplementation. However, large-scale production and purification of the recombinant proteins remained unsuccessful, and further optimization is required before subsequent functional characterization, including antigen-binding and neutralization analyses, can be performed. In conclusion, this study successfully established recombinant antibody and scFv expression systems derived from the anti-FIPV monoclonal antibody YB4D01 and provides a foundation for future antibody engineering and the development of safer antibody-based therapeutics against FIPV.