敏感分析聚乙二醇修飾藥物於體內的藥物動力學將能加速藥物的開發時程。本論文首先結合表現不同抗聚乙二醇抗體之捕捉細胞(293T/3.3 [binding PEG MW≥2000 Da]、293T/6.3 [binding PEG MW≥750 Da]及293T/15-2b [binding CH3O-end PEG]細胞)、四種含有不同抗聚乙二醇抗體之腹水(3.3、6.3、15-2b或7A4 [binding PEG MW≥750 Da])及辣根過氧化氫酶鏈結抗鼠源免疫球蛋白G固定區之二級抗體，開發出最佳化之抗聚乙二醇細胞基礎三明治酵素免疫吸附試驗來定量自由型聚乙二醇(mPEG2K-NH2及mPEG5K-NH2)或聚乙二醇鏈結之小分子(mPEG5K-Biotin及mPEG5K-NIR797)、蛋白(PegIntron及Pegasys)及奈米粒子(Liposomal-Doxorubicin; Lipo-Dox及Quantum-Dots; QDs)。結果指出293T/15-2b細胞能較293T/3.3及293T/6.3細胞於ng mL-1的層級更敏感的偵測自由型聚乙二醇、類聚乙二醇分子與聚乙二醇修飾蛋白。其中293T/15-2b細胞與7A4偵測抗體的組合具有最佳的偵測效率。另一方面，293T/3.3細胞結合15-2b抗體能偵測到2 ng mL-1的Lipo-Dox，擁有最高的敏感度。然而三種抗聚乙二醇抗體表現細胞結合15-2b抗體均能敏感偵測到7 pM的QD。這些結果指出結合293T/15-2b細胞及7A4抗體為敏感定量自由型聚乙二醇、類聚乙二醇分子與聚乙二醇修飾蛋白的最佳組合；而293T/3.3細胞與15-2b抗體較適合用於定量聚乙二醇修飾之奈米粒子。
即便最佳化的抗聚乙二醇細胞基礎三明治酵素免疫吸附試驗能敏感地定量自由型甲氧基聚乙二醇及甲氧基聚乙二醇修飾分子，但由於較少表位暴露的關係，抗聚乙二醇骨架之細胞基礎三明治酵素免疫吸附試驗偵測小於5000道爾吞之聚乙二醇的能力依舊很差。因此我們接著藉由表現不同立體空間的抗聚乙二醇骨架抗體(AGP3)於人類腎臟細胞表面(293T/S-αPEG；短型細胞、293T/L-αPEG；長型細胞及293T/SL-αPEG；混合型細胞)，開發出敏感的三明治酵素免疫吸附試驗來提升對自由型聚乙二醇及聚乙二醇修飾分子的負載力與偵測敏感度。結果顯示混合型細胞對類聚乙二醇分子(CH3-PEG5K-FITC及8-arm PEG20K-FITC)的負載力較表現單一長度抗聚乙二醇抗體之293T細胞高出10至80倍。混合型細胞基礎三明治酵素免疫吸附試驗能敏感偵測到14-137 ng ml-1的自由型聚乙二醇(OH-PEG3K-NH2及CH3-PEG5K-NH2)及類聚乙二醇分子(CH3-PEG5K-FITC, CH3-PEG5K-SHPP及CH3-PEG5K-NIR797)。293T/SL-αPEG細胞也能較293T/S-αPEG或293T/L-αPEG細胞乘載更多且更敏感地偵測到3.2-16 ng ml-1的聚乙二醇修飾蛋白(PegIntron)及多臂聚乙二醇大分子(8-arm PEG20K-NH2及8-arm PEG40K-NH2)。
由於聚乙二醇已經廣泛應用於我們的生活當中，內生性及誘導性抗聚乙二醇抗體於人體內越來越常見。這些抗聚乙二醇抗體可能會限制聚乙二醇修飾藥物的治療效率。因此研究抗聚乙二醇抗體對聚乙二醇修飾藥物之藥物動力學及療效的影響是相當重要的。論文的最後，我們將探討抗聚乙二醇抗體與聚乙二醇修飾之紅血球生成素(Mircera®)於腎性貧血患者體內的藥物動力學及療效之相關性。我們檢測以Mircera治療之腎性貧血患者體內抗聚乙二醇抗體的含量並將其與貧血指標進行相關性分析。結果顯示Mircera治療無效的族群體內抗聚乙二醇抗體含量較治療有效的族群要高出許多(P<0.05)。而抗聚乙二醇抗體的含量與貧血的治療指標(血紅素[Hb, P<0.001]、血比容[Hct, P<0.001]及紅血球數量[P<0.01])呈現顯著負相關的關係。於動物實驗也證實給予外生性抗聚乙二醇抗體(6.3)能明顯地加速聚乙二醇修飾之紅血球生成素於動物體內循環的清除。以上的結果說明了抗聚乙二醇的含量與Mircera的治療效果及貧血治療指標(血紅素、血比容及紅血球數量)均呈現負相關的關係。我們相信最佳化及不同長度的抗聚乙二醇抗體表現細胞基礎三明治酵素免疫吸附試驗能於藥物開發的過程中提供敏感、精確且方便的工具來定量各式各樣的聚乙二醇修飾分子。此外，抗聚乙二醇抗體與Mircera 療效的相關性研究成果也將協助臨床醫師為慢性腎臟病患者制定妥善的治療策略，達到個人化醫療之目的。

Sensitive analysis of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. In this thesis, we first developed an optimized anti-PEG cell-based sandwich enzyme-linked immunosorbent assay (ELISA) by combining different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3 [binding PEG MW≥2000 Da], 293T/6.3 [binding PEG MW≥750 Da] and 293T/15-2b [binding CH3O-end PEG] cells) with four ascites of anti-PEG antibodies (3.3, 6.3, 15-2b or 7A4 [binding PEG MW≥750 Da]) and HRP-conjugated anti-mouse IgG Fc secondary antibodies for quantification of free PEG (mPEG2K-NH2 and mPEG5K-NH2) or PEG-conjugated small molecules (mPEG5K-Biotin and mPEG5K-NIR797), proteins (PegIntron and Pegasys) and nanoparticles (Liposomal-Doxorubicin; Lipo-Dox and Quantum-Dots; QDs). Results indicated that 293T/15-2b had higher detection sensitivity of free PEG, PEG-like molecules and PEGylated proteins than 293T/3.3 and 293T/6.3 cells at ng mL-1 levels. This was particularly true of the combination of 293T/15-2b and 7A4 detection antibody, which had the best detection efficiency. On the other hand, 293T/3.3 combined with 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL-1. Notwithstanding, all three types of anti-PEG cells combined with 15-2b had high sensitivity for QD quantification at 7 pM. These results suggest that the combination of 293T/15-2b and 7A4 detection antibody is the optimal pair for sensitive quantification of free PEG, PEG-like molecules and PEGylated proteins; and the 293T/3.3 cells combined with 15-2b are more suitable for quantifying PEGylated nanoparticles.
Despite the fact that the optimized anti-PEG cell-based sandwich ELISA can efficiently quantify free mPEG and mPEGylated molecules, the detection sensitivity remains relatively poor for PEG molecules smaller than 5000 Da in anti-PEG backbone cell-based sandwich ELISA because of fewer epitopes for anti-PEG detection antibodies. Thus, we next developed a sensitive sandwich ELISA for PEG by tethering anti-PEG antibodies (AGP3) via tethers with different dimensions on the surface of 293T cells (293T/S-αPEG, short-type cells; 293T/L-αPEG, long-type cells and 293T/SL-αPEG, hybrid-type cells) to improve the binding capacity and detection sensitivity for free PEG and PEGylated molecules. The binding capacity of hybrid-type cells for PEG-like molecules (CH3-PEG5K-FITC and 8-arm PEG20K-FITC) was at least 10- to 80-fold greater than for 293T cells expressing anti-PEG antibodies with uniform tether lengths. The detection sensitivity of free PEG (OH-PEG3K-NH2 and CH3-PEG5K-NH2) and PEG-like molecules (CH3-PEG5K-FITC, CH3-PEG5K-SHPP and CH3-PEG5K-NIR797) was 14-137 ng ml-1 in the hybrid-type cell-based sandwich ELISA. 293T/SL-αPEG cells also had significantly higher sensitivity for quantification of a PEGylated protein (PegIntron) and multi-arm PEG macromolecules (8-arm PEG20K-NH2 and 8-arm PEG40K-NH2) at 3.2, 16 and 16 ng ml-1, respectively. Additionally, the overall binding capacity of 293T/SL-αPEG cells for PEGylated macromolecules was higher than 293T/S-αPEG or 293T/L-αPEG cells.
Naturally occurring or induced anti-PEG antibodies have been commonly observed in humans and PEG is widely encountered in our daily lives. The associated anti-PEG antibodies may restrict the therapeutic efficacy of PEGlyated drugs. Therefore, it is important to study the effect of anti-PEG antibodies on the pharmacokinetics and therapeutic efficacy of PEGylated drugs before therapeutic performance. Finally, we focus on the study of correlation between anti-PEG antibodies with the pharmacokinetics and therapeutic efficacy of PEG-EPO (Mircera) in renal anemia patients. We analyzed the level of anti-PEG antibodies and correlated it with anemia indexes in Mircera-treated renal anemia patients. Results show that the level of anti-PEG antibodies in the Mircera insensitive population was significantly (P < 0.05) higher than in the Mircera sensitive population. The level of anti-PEG antibodies was negatively correlated with treatment indexes (hemoglobin [Hb, P < 0.001], hematocrit [Hct, P < 0.001] and RBC count [P < 0.01]) of anemia. The exogenous anti-PEG antibody (6.3) dramatically accelerated the clearance of PEG-EPO from the circulation in an animal model as compared with control mice. Together, the level of anti-PEG antibodies was negatively correlated with therapeutic efficacy of Mircera and the treatment indexes of anemia (Hb, Hct and RBC count). We believe that the optimized and variable length anti-PEG Ab tethered cell-based sandwich ELISA can provide a sensitive, precise and convenient tool for the quantification of each type of PEGylated molecule during drug development. This observational study of correlation between anti-PEG antibody and therapeutic efficacy of Mircera provides strong support for clinicians to decide therapeutic strategies and carry out personal medicine in chronic kidney disease.

CONTENT
誌謝 ii
中文摘要 iii
ABSTRACT v
LIST OF FIGURES x
TABLE OF CONTENTS xii
APPENDICES xiii
ABBREVIATIONS xiv
INTRODUCTION 1
Chapter 1: Optimization of an anti-poly(ethylene glycol) (anti-PEG) cell-based capture system to quantify PEG and PEGylated molecules 10
1. Introduction 11
2. Materials and Methods 14
2-1. Reagents 14
2-2. Cells and Animals 14
2-3. Antibodies 15
2-4. Characterization of the Anti-PEG Antibody 16
2-5. Plasmid Construction 17
2-6. Generation of anti-PEG Expressing Cells by Lentiviral Transduction 17
2-7. Fluorescence-Activated Cell Sorting Analysis of the anti-PEG Expressing Cells 18
2-8. Anti-PEG Cell-Based Sandwich ELISA 18
2-9. ELISA Data Analysis 19
3. Results 21
3-1. Characterization of Anti-PEG Antibodies 21
3-2. Surface Display of Functional Anti-PEG Antibodies on 293T Cells 22
3-3. Quantification of free PEG and PEG-like Molecules by anti-PEG Cell-Based Sandwich ELISA 22
3-4. Quantification of PEGylated Proteins by anti-PEG Cell-Based Sandwich ELISA 24
3-5. Quantification of PEGylated Nanoparticles by anti-PEG Cell-Based Sandwich ELISA 25
4. Figures and Tables 26
5. Discussion 34
6. Conclusion 39
Chapter 2: Steric membrane anti-poly(ethylene glycol) antibody enhances sensitivity for quantifying PEG and PEGylated molecules 40
1. Introduction 41
2. Materials and Methods 45
2-1. Cells and Reagents 45
2-2. Plasmid Construction 45
2-3. Generation of Short-, Long- and Hybrid-Type Anti-PEG Expressing Cells by Lentiviral Transduction 46
2-4. Western Blot Analysis of the anti-PEG Expressing Cells 47
2-5. Fluorescence-Activated Cell Sorting Analysis of the anti-PEG Expressing Cells 47
2-6. Anti-PEG Cell-Based Sandwich ELISA 48
2-7. Modeling the structures and calculating the length of short- (AGP3-eB7) and long-type (AGP3-PTK7) anti-PEG Fab 49
2-8. Statistical Analysis 50
3. Results 51
3-1. Characterization of Short-, Long- or Hybrid-type Anti-PEG Antibodies-Expressing 293T cells 51
3-2. Binding Capacity of PEGylated Molecules in Short-, Long- or Hybrid-type Anti-PEG Antibodies-Expressing 293T cell 52
3-3. Quantification of Free PEG and PEG-like Molecules by anti-PEG Cell-Based Sandwich ELISA 53
3-4. Quantification of PEGylated protein and multi-arm PEG macromolecules by anti-PEG Cell-Based Sandwich ELISA 54
3-5. Speculative Model for Higher Capacity and Detective Sensitivity of PEG molecules in Hybrid-tethered Anti-PEG Antibody-Expressing 293T cells 55
4. Figures 57
5. Discussion 64
6. Conclusion 69
Chapter 3: The effect of endogenous anti-poly(ethylene glycol) antibody on the therapeutic efficiency of long-acting erythropoietin in renal anemia triggered by peritoneal dialysis in CKD patients Introduction 71
1. Introduction 72
2. Materials and Methods 76
2-1. Patients 76
2-2. Animals 76
2-3. Antibodies and Reagents 76
2-4. Analysis of anti-PEG antibodies in plasma of CKD patients 77
2-5. Clearance of Mircera in vivo 78
2-6. Statistical Analysis 78
3. Results 79
3-1. Hemoglobin level in Mircera-treated chronic kidney disease patients 79
3-2. Detection of anti-PEG antibodies in plasma samples of CKD patients by direct PEG-based ELISA 79
3-3. Correlation of the level of anti-PEG antibodies and the effectiveness of Mircera-treated CKD patients 79
3-4. Relationship between the level of anti-PEG antibodies and anemia indexes of Mircera-treated CKD patients 80
3-5. In vivo clearance of PEG-EPO 81
4. Figures and Tables 83
5. Discussion 100
6. Conclusions 108
CONCLUSIONS 109
REFERENCES 115

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