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Title Page 2

Contents 5

Abstract 15

Literature Review 17

1. Research and Industrialization of Biological Products 18

2. Industrialization of Single Chain Variable Fragment (scFv) Therapies 20

3. Antibody-Based Antiviral Therapies 23

4. 3D8 scFv 25

5. Improving Stability and Solubility for the Industrialization of Protein Therapeutics 29

6. Discussion 31

References 34

Chapter 1. Stability Optimization of Recombinant 3D8 scFv Using Protein Engineering Approaches 44

1. Introduction 45

2. Materials & methods 49

A. Cell lines and Viruses 49

B. In silico Analysis of 3D8 scFv Sequence for Increased Stability of 3D8 scFv 49

C. Construction of 3D8 scFv mutants 50

D. 3D8 scFv protein production 52

E. Verification of Expression Levels of Produced 3D8 scFv Before Purification 54

F. Verification of Purity Post-Purification Using SE-HPLC 54

G. Abzyme Test to Confirm Nuclease Activity 55

H. Cell Toxicity Test Before Cell-Based Experiments 56

I. Post-Treatment Method for Confirming Antiviral Effect of 3D8 scFv 56

J. Analysis of Viral RNA Expression Using qRT-PCR 57

3. Results 59

A. In silico analysis of protein sequence for the stability enhancement of 3D8 scFv 59

B. Construction of 3D8 scFv Mutants 60

C. Verification of Expression of Constructed 3D8 scFv Mutants 63

D. Stability Verification of Expressed 3D8 scFv Mutants (SDS-PAGE & SE-HPLC) 68

E. Verification of Activity of Expressed 3D8 scFv Mutants (Nuclease Activity) 72

F. Verification of Cytotoxicity of Expressed 3D8 scFv Mutants 75

G. Verification of Antiviral Activity of Expressed 3D8 scFv Mutants 77

4. Discussion 80

References 84

Chapter 2. Process Optimization for High-Yield Production of Therapeutic scFv 3D8 88

1. Introduction 89

2. Materials & methods 93

A. Lonza expression system 93

B. Fermentation of 3D8 scFv 93

C. Purification of 3D8 scFv 94

D. Verification of 3D8 scFv Titer During Cultivation and Purification Yield Using Sandwich ELISA 97

E. SDS-PAGE Analysis for 3D8 scFv Purity Verification 99

F. Buffer Exchange and Concentration 99

G. SE-HPLC Analysis for 3D8 scFv Purity Verification 100

H. Endotoxin Analysis of 3D8 scFv 100

I. Verification of Nuclease Activity of 3D8 scFv 101

J. Analysis of Viral RNA Expression Using qRT-PCR 101

3. Results 103

A. Verification of ELISA Applicability for 3D8 Mutants 103

B. Application of 3D8 scFv to Lonza XS Expression Platform 105

C. Establishing Cultivation Process for High-Quality 3D8 scFv Production 108

D. Establishment of a purification process for high-quality 3D8 scFv production 114

E. Endotoxin Removal for Industrial Application 117

F. Verification of Purity After Improved Purification Methods (SDS-PAGE and SE-HPLC Analysis) 121

G. Verification of 3D8 scFv Activity After Process Improvement: Antiviral Effect 123

4. Discussion 125

References 128

Chapter 3. Evaluation of Antiviral Efficacy of Optimized 3D8 scFv Against Respiratory Viruses 131

1. Introduction 132

2. Materials & methods 135

A. Production of 3D8 scFv (Culture and Purification) 135

B. Purification of 3D8 scFv 135

C. Purity Analysis of 3D8 scFv 137

D. Endotoxin Analysis of 3D8 scFv 138

E. Host Cell Protein Analysis for Impurity Confirmation 139

F. Host Cell DNA Analysis for Impurity Confirmation 139

G. Nuclease Activity test of 3D8 scFv 139

H. Viral RNA Quantification by qRT-PCR 139

I. Antiviral Efficacy Against COVID-19: Immuno Fluorescence Assay 140

J. In Vivo Toxicity Evaluation 141

K. In Vivo Efficacy Evaluation 142

3. Results 143

A. Production and Purity Confirmation of 3D8 scFv through Established Process 143

B. Activity Confirmation of Expressed 3D8 scFv (WT, Y101P) (Nuclease Activity & Antiviral Effect) 145

C. Verification of Antiviral Efficacy of 3D8 scFv: Confirmation of Antiviral Efficacy Against SARS-CoV-2 148

D. In Vivo Toxicity Evaluation of 3D8 scFv Produced by the New Process 150

E. In Vivo Efficacy Evaluation of 3D8 scFv Produced by the New Process (hCoV OC43) 157

4. Discussion 161

References 165

논문요약 169

List of Tables 9

Literature Review 9

Table 1. Currently Approved Antiviral Therapeutic Antibodies 24

Table 2. Various Expression Systems Used to Study the Broad Antiviral Activity... 28

Chapter 1 10

Table 1. Primer sequences used for Mutagenesis 51

Table 2. Primers used in qRT-PCR for Antiviral Test 58

Table 3. Information of Plasmids of 3D8 scFv wild type & mutants 61

Chapter 2 10

Table 1. Affinity Chromatography step for 3D8 scFv purification 96

Table 2. Anion exchange chromatography step for 3D8 scFv purification 97

Table 3. Primers Used in qRT-PCR for Antiviral Test 102

Chapter 3 10

Table 1. Affinity Chromatography step for 3D8 scFv purification 136

Table 2. Anion exchange chromatography step for 3D8 scFv purification 137

Table 3. Primers Used in qRT-PCR for Antiviral Test 140

Table 4. In Vivo Two-Week Repeated Intravenous Toxicity Study Groups 141

Table 5. In vivo antiviral efficacy test of 3D8 WT and Y101P via intranasal... 142

Table 6. In vivo antiviral efficacy test of 3D8 Y101P via intranasal... 142

Table 7. Purity/Impurity Standards and Analysis Results for 3D8 scFv to be... 145

Table 8. Hematology values for male and female mouse administered 3D8 WT... 153

Table 9. Blood chemistry values for male and female mouse administered 3D8... 154

Table 10. Organ weight (g) for male and female mouse administered 3D8 WT... 154

Table 11. Hematology values for male and female mouse administered 3D8... 155

Table 12. Blood chemistry values for male and female mouse administered 3D8... 156

Table 13. Organ weight for male and female mouse administered 3D8 Y101P... 156

List of Figures 11

Literature Review 11

Figure 1. Stages of drug development: target identification, clinical trials,... 19

Chapter 1 11

Figure 1. Purification Process of 3D8 scFv Using FPLC Equipment 53

Figure 2. 3D Structure of 3D8 scFv wild type and mutants represented using... 60

Figure 3. Sequencing Confirmation Results of 3D8 Mutants: Modified Sequence... 62

Figure 4. Growth Curve of Cultured Mutants 64

Figure 5. Verification of Expression of Constructed 3D8 scFv Mutants 67

Figure 6. SDS-PAGE Results of Purified 3D8 Wildtype and 13 Mutants 69

Figure 7. Verification of Stability of 3D8 scFv Mutants Using SE-HPLC 71

Figure 8. Verification of Nuclease Activity Retention in Constructed 3D8 Mutants 74

Figure 9. MTT Assay Results of 3D8 scFv Wildtype and Mutants on Vero E6 Cells 76

Figure 10. Antiviral Efficacy Test Results of 3D8 scFv Wildtype and Mutants Against hCoV OC43 Infection Using... 79

Chapter 2 12

Figure 1. Verification of ELISA Applicability for 3D8 scFv Wildtype and... 104

Figure 2. Schematic Diagram of Plasmid DNA Used in 3D8 Expression Process 107

Figure 3. Parameter Trends and Growth Curves During the Cultivation Process... 110

Figure 4. SDS-PAGE Results of 3D8 scFv Expression Levels Over Cultivation... 111

Figure 5. Verification of 3D8 scFv Expression Levels in Culture Media... 113

Figure 6. Verification of Protein State and Purity at Each Step of the Purification Process 116

Figure 7. Maintenance of Nuclease Activity and Endotoxin Levels at Additional... 120

Figure 8. Comparison of Purity Between 3D8 scFv WT and Y101P After... 122

Figure 9. In Vitro Testing for Efficacy Verification of 3D8 scFv WT and 3D8... 124

Chapter 3 13

Figure 1. Purity Analysis Results of 3D8 WT and 3D8 Y101P 144

Figure 2. Activity Confirmation of Expressed 3D8 scFv (WT, Y101P) 147

Figure 3. Results of the SARS-CoV-2 Antiviral Assay Using... 149

Figure 4. Changes in Body Weight of Mice During the 2-Week Repeated... 152

Figure 5. In Vivo Antiviral Efficacy Test of 3D8 WT and Y101P Against hCoV... 159

Figure 6. Reconfirmation In Vivo Test of Antiviral Efficacy of 3D8 Y101P... 160

초록보기

 생물학적 제제의 개발 공정은 많은 발전을 거치며 바이러스 감염 등의 치료에 가능성을 보여주지만 산업적 개발 과정 중 심각한 어려움에 직면할 수 있습니다. 높은 바이러스 돌연변이율, 복잡한 생산 공정, 규모 확장의 어려움으로 인해 실험실 연구에서 대량 생산으로 전환하는 데 방해가 되는 경우가 많습니다.

이 연구는 치료 옵션으로서 핵산 가수분해 활성을 갖는 광범위한 항바이러스제 후보인 3D8 단일 사슬 가변 단편(scF v)의 개발에 중점을 두고 있습니다. 이 연구에서는 산업 응용에 필수적인 3D8 scF v의 안정성에 대한 중요한 문제를 해결하는 것으로 시작됩니다. 먼저, in silico 분석을 기반으로 안정성을 향상시키고 3D8의 응집을 감소시켜 안정적이고 효과적인 돌연변이 후보를 도출하기 위한 및 돌연변이 유발 방법 및 안정성과 효능 평가에 대해 자세히 설명합니다. 다음으로 상업적 적용을 목적으로 대량 생산을 하기 위한 특정 재조합 단백질 발현 시스템을 사용하여 이러한 3D8 야생형과 돌연변이의 생산성을 최적화하여 생산 수율을 크게 높였습니다. 또한 전임상 및 임상 적용에 필수적인 높은 순도와 확장성을 보장하기 위해 고급 발효 및 정제 공정이 개발되었습니다. 마지막으로 앞에서 선택된 3D8 scFv 후보, 특히 Y 101P의 hCoV OC43 및 SARS- CoV - 2를 포함한 호흡기 바이러스에 대한 항바이러스 효능을 비교합니다. 시험관 내 및 생체 내 테스트 모두 강력한 항바이러스 활성과 안전성 및 최소 독성을 입증하여 3D8을 유망한 항바이러스 치료 후보로 자리매김했습니다.

이 연구는 생물학적 항바이러스제 개발에서 안정성, 생산성 및 효능 문제를 해결하기 위한 포괄적인 접근 방식을 제시하여 3D8 scF v의 향후 치료 적용을 위한 길을 제안합니다.

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