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Development of Anti-Cancer Drug using a Chimeric Antibody Targeting Nectin-2

  • 심윤희 (아주대학교 일반대학원)

초록/요약

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.

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초록/요약

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.

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목차

I. INTRODUCTION 1

II. MATERIALS AND METHODS 11
1. Generation of Chimeric 12G1 11
2. Cell Culture 12
3. RNA Extraction and Complementary DNA Synthesis 12
4. qRT-PCR 12
5. Western Blot Analysis 13
6. Flow Cytometry 14
7. Surface Plasmon Resonance Analysis 15
8. Enzyme-Linked Immunosorbent Assay (ELISA) 15
9. Knock-Down by siRNA 16
10. Epitope Mapping 16
11. Effector Function Assays 17
12. Internalization Assay 18
13. Generation of Antibody-Drug Conjugates (ADCs) 19
14. Drug-to-Antibody Ratio (DAR) Analysis 20
15. Cell Cycle Assay 21
16. In Vitro Cytotoxicity Assay 21
17. In Vivo Studies of ADCs 22
18. Statistical Analysis 22

III. RESULTS 27
1. The Expression of Nectin-2 in Cancer Cell Lines 27
2. Generation and Characterization of c12G1 30
3. Epitope Mapping of c12G1 on Nectin-2 31
4. Effector Function Assays of c12G1 31
5. Internalization of c12G1 41
6. Generation and Characterization of ADCs 41
7. In vitro Cytotoxicity Test of c12G1-ADCs 48
8. In vivo Studies of c12G1-DM1 52

IV. DISCUSSION 56

V. REFERENCES 60

VI. ABSTRACT IN KOREAN 66

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