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Preclinical study on a novel DNA vaccine(HB-110) for the treatment of chronic Hepatitis B virus infection : B형 간염 치료용 DNA 백신 (HB-110)의 전임상 연구

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dc.contributor.advisor김병기l-
dc.contributor.author김채영-
dc.date.accessioned2017-07-27T16:31:08Z-
dc.date.available2017-07-27T16:31:08Z-
dc.date.issued2014-08-
dc.identifier.other000000021003-
dc.identifier.urihttps://hdl.handle.net/10371/134963-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 협동과정 생물화학공학전공, 2014. 8. 김병기l.-
dc.description.abstract현재까지 개발되어 사용되고 있는 Hepatitis B virus (HBV) 치료제들은 면역치료제인 Interferon-α및 화학요법제인 Lamivudine, Adefovir, Entecarvir 등이 있다. Interferon-α 경우 투여시 부작용이 심하며, 화학요법제들은 바이러스 증식억제의 장점을 가지고 있으나, 환자에 장기간 투여시 독성, 내성바이러스 출현이 발생되며, 투여 중단시 HBV의 recurrence가 발생되는 제한이 있다. 본 연구에서는 현재 사용하는 치료제보다 유효성 및 안전성이 우수한 HBV 치료제를 개발하기 위해HBV 감염자 대상으로 치료 DNA 백신 개발을 진행하고 있으며, 먼저 선도 물질인 HB-100을 개발하여 우크라이나 및 리투아니아에서 사전임상을 실시하여 안전성 및 유효성을 확인하였다. 그리고 HB-100보다 효능이 우수하며 경제적으로 생산이 가능한 HB-110을 개발하였다.
본 연구에서는 HB-110의 생산공정을 확립하였으며, 물리화학 및 생물학적 특성분석과 안정성, 독성, 역동력학 연구를 수행하여 전임상 연구를 완료하였다. HB-110은 HBV 항원유전자 및 변이체 IL-12 유전자를 발현하는 3종의 naked plasmid로 이루어진 DNA 백신으로, E. coli DH5에 각각의 plasmid를 형질전환시켜 생산세포주 bank를 제조하였다. 제조된 세포주 bank를 대상으로 세포주의 유전형, 표현형 및 오염여부 등에 대해 시험을 수행하였으며, 적합하게 제조된 것을 확인하였다. 제조된 세포주를 이용하여 생산공정을 확립하였으며, 배양공정에서 배양중 glycerol 첨가와 정제공정단계에 thiophilic/aromatic adsorption 크로마토그래피를 실시하여 supercoiled monomer 형태가 90% 이상 순도의 plasmid 시료를 확보 할 수 있었다. 확립된 생산공정을 이용하여 생산된 HB-110에 대해 유전자 치료제의 가이드를 적용하여 물리화학 및 생물학적 특성분석을 진행하였으며, 이를 기반으로 생산된 HB-110이 의약품으로 사용 적합성을 판단하는 품질기준 시험법을 수립하였다. 독성시험은 확립된 생산공정으로 생산한 HB-110을 대상으로 안전성 평가연구원 및 Ina(일본)에서 설치류 및 원숭이에서 평가를 실시하여, HB-110이 안전한 물질임을 (NOAEL ≥ 4mg/kg) 확인하였다. 약동력학 연구는 마우스에서 실시하였으며, 정맥으로 투여 후 평가결과 half-life가 1.9분이며 AUC가 103 ug min/ml로 나타났다. 투여 경로인 근육내 투여시 근육내에서 약 11일까지 HB-110이 잔류되는 개체가 존재하였으며, 타장기에서는 투여 후 8시간 이후에는 검출한계(0.01pg/mg tissue) 미만으로 나타났다. 이렇게 확보된 전임상 자료를 이용하여 제1상임상 시험계획서(IND)를 KFDA에 제출하여 승인을 득하였으며, 카톡릭의대 강남성모병원에서 HBV 감염자 대상으로 1상임상을 완료하였다.
또한 본 연구에서는 HB-110의 효능을 향상시키기 위해 전기천공법(electroporation) 적용연구를 실시하여, S항원 유전자의 발현을 대폭적으로 증가시켰으며, 세포성 면역반응이 항원에 따라 약 1.7∼3배 이상 향상되는 것을 mouse 시험을 통해 확인하였다. 향후 2상 임상시험에서는 전기천공법을 적용할 계획이며, HB-110의 효능증가를 기대 할 수 있을 것으로 예측된다.
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dc.description.abstractInterferon-a immunotherapy and chemotherapeutic agents including Lamivudine, Adefovir and Entecarvir have been developed so far for the treatment of Hepatitis B Virus (HBV) infection and are widely used. Interferon-a is associated with serious side effects, while chemotherapeutic agents, although being generally safe and effective at inhibiting virus proliferation, also show toxicities after long-term use. Importantly, chronic administration of chemotherapeutic agents is often associated with occurrence of resistant virus in patients. Also, recurrence of HBV is common once these agents are discontinued. With the purpose of providing an HBV therapy with efficacy and safety superior to existing therapies, in the present study a therapeutic DNA vaccine was developed for the treatment of patients with HBV infection. Safety and efficacy of the lead compound, HB-100, was demonstrated in a pilot clinical study conducted in Ukraine and Lithuania. Later, an optimized compound, HB-110, with higher efficacy and economic productivity compared to HB-100 was developed.
A production process for HB-110 was developed, and preclinical studies were conducted including physicochemical/biological characterization, stability, toxicity and pharmacokinetics (PK). HB-110 is a DNA vaccine comprised of 3 different naked plasmid vectors, which express HBV antigens and a mutant IL-12. Escherichia. coli DH5a cells were transfected with the plasmids to produce production cell lines for each plasmid. Production cell banks of each cell line were established and characterized for genotype, phenotype and adventitious contamination. Production processes using the cell lines were established for each plasmid, and plasmid material with purity higher than 90% supercoiled monomer could be obtained by a combination of a glycerol-based feeding strategy during the culture process and incorporation of TAA chromatography in the purification process. In accordance with regulatory guidelines for gene therapy products, physicochemical and biological characterizations were performed for HB-110 produced using the established production process, and quality test methods were established for the quality control and release testing of clinical-grade material. Toxicity evaluation of HB-110, conducted in rats and monkeys at KIT (Korea) and Ina (Japan), demonstrated that HB-110 was safe, with a NOAEL ≥ 4 mg/kg. A PK study conducted in a mouse model showed a half-life of 1.9 minutes and AUC of 103 ug min/ml after IV administration. Residual HB-110 was detected in the muscle at the site of administration until approximately 11 days after IM administration, whereas in other organs HB-110 levels fell below the limit of detection (0.01 pg/mg tissue) after 8 hours post-administration. Based on the data obtained from the preclinical studies, an investigational new drug application (IND) was submitted to and approved by the KFDA, and a phase 1 clinical study involving subjects with HBV infection was conducted and completed at The Catholic University of Korea Seoul St. Marys Hospital.
With the purpose of further enhancing the efficacy of HB-110, electroporation studies in mice were conducted which showed a sharp increase of S antigen gene expression and enhancement of cellular immune response by a factor of about 1.7 to 3 times depending on the antigen. The electroporation method is planned to be incorporated in future phase 2 clinical study protocols to further enhance the clinical efficacy of HB-110.
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dc.description.tableofcontentsABSTRACTS ............................................................................................................... i
CONTENTS .................................................................................................................iii
LIST OF TABLES .....................................................................................................viii
LIST OF FIGURES......................................................................................................x
LIST OF ABBREVIATIONS................................................................................... xiii

I. INTRODUCTION……………………………………………………………1
1.1 Hepatitis B Virus……………………………………………………………1
1.1.1 HBV Structure………………………………………………………….1
1.1.2 Life cycle……………………………………………………………….3
1.1.3 Transmission……………………………………………………………3
1.1.4 Epidemiology…………………………………………………………...4
1.1.5 Disease progression……………………………………………………..7
1.2 Therapeutic DNA Vaccine ...………………………………………………...7
1.2.1 Why is Therapeutic DNA Vaccine Needed ?.……………………... …….7
1.2.2 Possibility for the Development of Therapeutic Vaccine……………........9
1.2.3 Antigen Gene Selection ……………………………………………….11
1.3 Current Development Status of Therapeutic Vaccine ....……………………12
1.4 Preliminary Study: HB-100 (Lead) Study………………………………......12
1.5 Selection and Development System of Therapeutic DNA Vaccine Candidate (HB-110)……………………………………………………………………16
1.5.1 Candidate (HB-110) Selection…………………………………………16
1.5.2 Development System………………………………………………….20

II. MATERIALS AND METHODS .....................................................................22
2.1 Establishment of the Production Process for HB-110 Sample ……….……22
2.1.1 Selection of Host Cell and Cell Line Manufacturing…………………22
2.1.2 Fermentation Process ………………………………………………..23
2.1.3 Purification Process………………………………………………….23
2. 2 CMC (chemistry, manufacturing & control) Study………………………....24
2.2.1 Physicochemical Characterization……………………………………24
2.2.2 Biological Characterization…………………………………………..24
2. 3 Specifications and Test Procedures………………………………………...25
2.3.1 Quality Control Items and Specifications for Plasmid DNA…………..25
2.3.2 Establishment and Validation of HBcAg Quantification………………25
2.3.3 Identification of the Expression of L and Polymerase Proteins. ………25
2.4 Selection of Dosage Form and Stability Test……………………………….26
2.4.1 Dosage Form Design and Long-term Test……………………………26
2.5 Safety Evaluation…………………………………………………………..29
2.5.1 HB-110 Sample Production for Safety Evaluation……………………29
2.5.2 Safety Assessment in Small Animals…………………………………29
2.5.2.1 Acute Toxicity Test for Single Intramuscular Injection in Rats……30
2.5.2.2 Acute Toxicity Test for Single Intravenous Injection in Rats ……..30
2.5.2.3 26-Week Chronic Toxicity Test for intravenous Injection in Rats…30
2.5.2.4 Genetic and Reproductive Toxicity Test for HB-110 in Rats...……30
2.4.2.5 Immunotoxicity Test for HB-110 in Mice……………………..…30
2.4.3 Safety Evaluation in Monkeys……………………………………30
2.6 Pharmacokinetic Study……………………………………………………31
2.6.1 HB-110 Preparation…………………………………………………31
2.6.2 Intravenous Administration of HB-110………………………………31
2.6.3 Intramuscular Administration of HB-110…………………………….33
2.6.4 Analysis of HB-110 in Blood Samples by Polymerase Chain Reaction
(PCR) ………………………………………………………………..33
2.6.5 Analysis of HB-110 in Tissue Samples by Polymerase Chain Reaction
(PCR) ………………………………………………………………..34
2.6.6 Evaluation of Biodistribution using RT-PCR…………………………34
2.7 HB-110 Manufacturing for Phase 1 Clinical Trial………………………….35
2.8 Phase 1 Clinical Trial on HB-110…………………………………………..35
2.9 Efficacy Enhancement Study………………………………………………35
2.9.1 HB-110 Preparation………………………………………………….37
2.9.2 Experimental Animals and Immunization ……………………………37
2.9.3 Measurement of Expressed S Antigen ……………………………….37
2.9.4 Measurement of Antibody Responses ………………………………..38
2.9.5 IFN-r ELISPOT Assay ………………………………………………38
2.9.6 HBsAg Seroconversion Analysis ……………………………………38

ΙΙΙ. RESULTS .. ...........................................................................................................40
3.1. Establishment of HB-110 Production Process……………………..………40
3.1.1 Cell Line Preparation………………………………………………...40
3.1.2 Fermentation Process………………………………………………...40
3.1.3 Purification Process…………………………………………………..45
3.1.3.1 Thiophilic/aromatic adsorption (TAA) chromatography…………45
3.1.3.2 Flowchart of Overall Manufacturing Process……………………45
3.1.3.3 Yield per Purification Step and In-process Test…………………..48
3.2 CMC Study……………………………………………………………….48
3.2.1 Physicochemical Characterization…………………………….……..48
3.2.2 Biological Characterization………………………………………….51
3.3. Quality Assessment Specification………………………………………....51
3.3.1 Quality Assessment Items and Specifications for HB-110 plasmid DNA
……………………………………………………………………………51
3.3.2 Establishment and Validation of HBcAg Quantification……………...56
3.3.3 Identification of the Expression of L and Polymerase Proteins………..56
3.4 Stability Tests……………………………………………………………...56
3.4.1 HB-110 Long-term Stability Test ………………………………….....56
3.5 Safety Evaluation………………………………………………………......60
3.5.1 Material for Safety Evaluation………………………………………..60
3.5.2 Safety Assessment in Small Animals and Monkeys…………………..60
3.6 Pharmacokinetic Study…………………………………………………….62
3.6.1 In Vivo Kinetics of HB-110 after Intravenous Administration………...62
3.6.2 In Vivo Kinetics of HB-110 after Intramuscular Administration……....62
3.6.3 Tissue Distribution of HB-110 after Intramuscular Administration…....67
3.6.4 Evaluation of Biodistribution using RT-PCR…………………………67
3.6.4.1 Pharmacokinetics after a Single Dose…………………………....67
3.6.4.2 Pharmacokinetics after a Repeated Dose…………………………70
3.7 HB-110 manufacturing for Phase 1 Clinical Trial…………………………..72
3.8 Phase 1 Clinical Trial………………………………………………………74
3.9 Study of Electroporation Application……………………………………..74
3.9.1 HBs Antigen Expression……………………………………………..74
3.9.2 Antibody Responses……………………………………………….....77
3.9.3 Cellular Immune Response…………………………………………..81
3.9.4 HBsAg Seroconversion Analysis…………………………………….85
3.9.5 Conclusion of Electroporation Study………………………………....85

ΙV. DISCUSSION ......................................................................................................87

V. REFERENCES ......................................................................................................91

국문초록..................................................................................................... ……….96
APPENDIX .............................................................................................................. 98
Appendix 1. Safety assessment in small animals……………………………98
Appendix 2. Identification of anti-HBs Ab after 6 month repeated HB-110 dose
in monkeys……………………………………………………99
Appendix 3. Safety results of HB-110 phase 1 clinical trial………………..100
Appendix 4. HBe Ag seroconversion rate of HB-110 phase 1 clinical trial....101
Appendix 5. Publications……………………………………………….....102
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dc.formatapplication/pdf-
dc.format.extent1992928 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectHBV-
dc.subjectTherapeutic DNA vaccine-
dc.subject.ddc660-
dc.titlePreclinical study on a novel DNA vaccine(HB-110) for the treatment of chronic Hepatitis B virus infection-
dc.title.alternativeB형 간염 치료용 DNA 백신 (HB-110)의 전임상 연구-
dc.typeThesis-
dc.contributor.AlternativeAuthorChae-Young Kim-
dc.description.degreeDoctor-
dc.citation.pages118-
dc.contributor.affiliation공과대학 협동과정-
dc.date.awarded2014-08-
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