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Development of Anti-Cancer Drug using a Chimeric Antibody Targeting Nectin-2
- Author(s)
- 심윤희
- Advisor
- 박상규
- Department
- 일반대학원 약학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2022-08
- Language
- eng
- Abstract
- Nectin-2는 칼슘 독립적인 세포 부착 분자로 알려져 있다. Nectin-2는 T cell 표면에 존재하는 면역 체크포인트인 PVRIG의 리간드이며, 다양한 암세포들, 특히 난소암에 과발현 되어 있다. 이 연구에서는, CHO 세포 발현 시스템을 통해 Nectin-2에 대한 키메라 항체인 c12G1을 생산하였으며 항체 특성화를 위해 간접 효소면역측정법과 표면 플라즈몬 공명 분석 및 유세포 분석을 진행했다. Nectin-2에 대한 c12G1의 특이성은 siRNA의 형질도입을 통한 Nectin-2의 녹다운에 의해 확인되었다. 또한, 면역침전법을 통해 항체의 결합 부위를 확인하기 위한 항원 결정기 맵핑을 수행하였다. c12G1은 Nectin-2의 첫 번째 C2 도메인에 결합하였는데, 이는 c12G1이 Nectin-2와 PVRIG와의 상호작용을 차단할 수 없음을 의미한다. 따라서 DM1 또는 MMAE를 c12G1에 접합하여 두 가지 유형의 항체-약물 접합체를 제작하였고 이들의 항암 효과를 평가하였다. 제작한 항체-약물 접합체가 Nectin-2 양성 세포의 세포 주기 정지를 유도하는 것을 확인하였고, 시험관 내 연구를 통해 반수 최대 억제 농도를 측정하였다. 또한, c12G1-DM1은 면역결핍 마우스 이종이식 모델에 투여하였고, c12G1-DM1은 종양 성장을 억제하였다. 종합해보면, 이러한 연구들은 항-Nectin-2 항체가 난소암 치료를 위한 항암제로 적용될 수 있음을 시사한다.
- Alternative Abstract
- Nectin-2 is known as a calcium-independent cell adhesion molecule. It is overexpressed in various cancer cells, especially ovarian cancer, and serves as a ligand for interaction with poliovirus receptor-related immunoglobulin domain-containing (PVRIG) known as the immune checkpoint on T cells. In this study, c12G1, a chimeric antibody against Nectin-2 was generated by the CHO cell expression system and characterized by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) analysis, and flow cytometry. The specificity of c12G1 to Nectin-2 was confirmed by the knock-down of Nectin-2 using siRNA. In addition, epitope mapping was performed through immunoprecipitation (IP) to identify the binding site of the antibody. c12G1 was bound to the first C2 domain of Nectin-2, which means that this antibody cannot block the interaction with PVRIG. Therefore, by conjugating DM1 or MMAE to c12G1, two types of antibody-drug conjugates (ADCs) were generated, and their anti-cancer effects were investigated. The ability of ADCs to induce cell cycle arrest was assessed and half-maximal inhibitory concentration (IC50) values were deduced through in vitro studies. Furthermore, c12G1-DM1 was administered to an immunodeficient mouse xenograft model to evaluate the efficacy, and c12G1-DM1 inhibited tumor growth. Taken together, these studies suggest that the anti-Nectin-2 antibody could be applied as an anti-cancer drug to treat ovarian cancer.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Pharmacy > 3. Theses(Master)
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