- 學位論文
抗新型冠狀病毒Omicron變異株之單株抗體與雙專一性抗體研究
Study of Monoclonal and Bispecific Antibodies against SARS-CoV-2 Omicron Variants
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摘要
新型冠狀病毒 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) 已隨著全球大流行，迅速演化出不同的變異株。其中，Omicron (B.1.1.529) 變異株在S蛋白 (Spike) 受體結合區 (receptor-binding domain, RBD) 的大量突變，導致其能逃脫既有的疫苗與抗體保護力。Omicron的高傳播力也使帶有不同突變的Omicron亞型持續出現，因此，開發出廣效性中和抗體是對抗COVID-19疫情的長久之計。本研究以BA.2-RBD-hFc作為免疫小鼠之抗原，成功篩選出四株針對BA.2-RBD的單株抗體D1H5、D2G8、D1A4和D1B6。透過抗原決定位之分析以及RBD結合實驗，可知D1H5與D2G8的抗原決定位在RBD的高度保守區域，因而能廣泛地結合不同變異株的RBD。D1A4和D1B6則會因為來自BA.5與BQ.1變異株的L452R突變，喪失對RBD的結合能力。此外，除了D1H5具有對BA.5變異株的微弱中和力，其餘三株抗體皆無法中和SARS-CoV-2病毒。為了提升抗體的中和能力，將中和性抗體改造成雙專一性抗體 (bispecific antibodies, bsAbs) 是一種解方。本研究透過還原抗體鉸鏈區 (hinge region) 之雙硫鍵，再進行氧化，將中和性抗體S2-8D或S2-4A與無中和力的HR2單株抗體S2-8A重新組裝，成功製造出能結合兩種抗原決定位的雙專一性抗體bs8D8A與bs4A8A。此成果揭示了化學性改造對於開發雙專一性抗體之可行性。
並列摘要
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the global pandemic, has rapidly evolved into different variants. Among these variants, Omicron has garnered significant attention due to its extensive mutations on the receptor-binding domain (RBD) of Spike (S) protein, enabling it to evade vaccine and antibody therapies. The increased transmissibility of Omicron has led to the continuous emergence of subvariants with distinct mutations. Therefore, the development of broadly neutralizing antibodies (NAbs) is a long-term strategy to combat the COVID-19 pandemic. In this study, BA.2-RBD-hFc was employed as the antigen for mouse immunization, and four monoclonal antibodies (mAbs), D1H5, D2G8, D1A4, and D1B6, were successfully isolated and characterized. D1H5 and D2G8 were found to recognize highly conserved epitopes on RBDs of various SARS-CoV-2 variants. However, D1A4 and D1B6 lost their RBD-binding capabilities due to the L452R mutation found in BA.5 and BQ.1 subvariants. Furthermore, D1H5 showed weak neutralizing activity against BA.5 subvariant, but D2G8, D1A4, and D1B6 failed to neutralize SARS-CoV-2 authentic viruses. To enhance the neutralizing capacity of antibodies, engineering two mAbs into bispecific antibodies (bsAbs) is an effective approach. In this study, the SARS-CoV-2 NAbs S2-8D or S2-4A was re-assembled with the HR2-specific non-neutralizing mAb S2-8A by using a method which involves the reducing and re-oxidizing the disulfide bonds of antibody hinge regions to generate two bsAbs, bs8D8A and bs4A8A. This achievement highlights the feasibility of chemical modification in the development of bispecific antibodies.
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