초록 ▼

◾ 다양한 재조합 단백질 자체 제조 및 평가: FlaB 등
◾ 항알레르기 항원 표지 conjugate 제조 및 평가
◾ 항알레르기 항원과 FlaB를 이용한 다양한 나노입자 제작 및 평가
◾ 마우스 모델을 이용한 천식 억제 효능 검증
◾ 마우스 모델을 이용한 천식 억제 작용 기전 규명
- 생체 내 투여 약물의 추적 및 면역세포에 미치는 영향 평가
- TLR5 KO 마우스를 이용한 증명
- 수지상 세포 활성에 미치는 영향 평가: In vitro 및 in vivo system에서 검증
- Pre

◾ 다양한 재조합 단백질 자체 제조 및 평가: FlaB 등
◾ 항알레르기 항원 표지 conjugate 제조 및 평가
◾ 항알레르기 항원과 FlaB를 이용한 다양한 나노입자 제작 및 평가
◾ 마우스 모델을 이용한 천식 억제 효능 검증
◾ 마우스 모델을 이용한 천식 억제 작용 기전 규명
- 생체 내 투여 약물의 추적 및 면역세포에 미치는 영향 평가
- TLR5 KO 마우스를 이용한 증명
- 수지상 세포 활성에 미치는 영향 평가: In vitro 및 in vivo system에서 검증
- Pre-immune status가 천식 면역치료에 미치는 영향 평가
◾ DC-기반 세포 치료법 최적화 연구
- 오르가노이드 이식법
- 면역미세환경 조절 면역치료법
◾ 항알레르기 융합 나노입자 도출 및 천식 억제 효능 평가: Derp2-FlaB 기반 나노입자
◾ 천식환자에서 플라젤린 기반 치료법의 효능 검증 및 작용 기전 규명
◾ 플라젤린 기반 천식 치료법이 NKT 세포에 미치는 효능 검증 및 작용 기전 규명


(출처 : 보고서 요약서 3P)

Abstract ▼

Purpose & Contents
□ Purpose
○ The 1^st year
- Synthesis of Ver. 1.0 FlaB-HDM conjugate
- Study for enhancing the efficiency of FlaB and better stability
- Decision of optimal administration route and dosage of conjugate
- Verification of antigen-specific effect of conjug

Purpose & Contents
□ Purpose
○ The 1^st year
- Synthesis of Ver. 1.0 FlaB-HDM conjugate
- Study for enhancing the efficiency of FlaB and better stability
- Decision of optimal administration route and dosage of conjugate
- Verification of antigen-specific effect of conjugate for asthma
○ The 2^nd year
- Development of advanced adjuvant system
- Mechanism study of FlaB conjugate (Ver. 2.0)
- Decision of optimal administration route and dosage of conjugate with optimal conjugate
- Verification of DC cell therapy efficiency with optimal conjugate and toxicity test of FlaB with mouse
○ The 3^rd year
- Development of advanced adjuvant system with controlling the mobility of DC
- Basic study for establishing optimal DC manufacture system to enhance the effect of serious asthma
- Confirmation of effect with DC controlled by conjugate
- Verification of DC immunotherapy efficiency to T cell and NKT cell with optimal conjugate and toxicity test of FlaB with human immune cells
□ Contents
○ The 1^st year:
- Establishment of asthma mouse model with house dust mite
- Synthesis of FlaB- HDM conjugate
- Decision of best route and optimal dosage with conjugate
- Synthesis of FlaB-allergen conjugate by confirming antigen-specific asthma suppressive effect
○ The 2^nd year:
- Verification of asthma treatment efficiency with the 1st year product
- Molecular mechanism study of FlaB-allergen conjugate
- Synthesis and activity test of APC-targeted nanoparticles loaded with stable FlaB-allergen
- Development of optimal FlaB-allergen delivery nanocarrier system to APC
- Verification of effect with DC cell therapy with asthma model mouse
○ The 3^rd year:
- Collection of PBDC and CD3 T cell from HDM asthma patient
- Confirmation of immune-controlling efficiency with T cell and NKT cell
- Development of advanced nanoparticle delivery system controlling the mobility in vivo
- Basic study for establishment to make the optimal DC manufacturing system to enhance the treatment effect for serious asthma patient

Results
○ FlaB-HDM production
- Improvement of protocol for FlaB-HDM production: sequential cross link -> step-by step cross link
○ Establishment of optimal FlaB production system
- Optimal plasmid construct / optimal protein expression condition
○ Optimization of FlaB protein purification
- Optimal condition for the plasmid construct
- Validation: SDS-PAGE / W-blotting / native gel electrophoresis / LPS removal / endotoxin test etc.
○ Validation of biological function of the FlaB
- TLR5-dependent NFkB-luciferase reporter assay
- FlaB stability test: 6 months storage / W-blotting
○ Production of allergen-conjugated particle
- Production of bio-compatible & APC-targeting nanoparticle (ex> micelle)
- Production of FlaB-conjugated nanoparticle platform
- Production of HDM allergen-conjugated nanoparticle platform
- Production of FlaB-HDM-conjugated nanoparticle platform
○ Efficacy test and mechanism of action: anti-asthma
- Establishment of asthma animal model: 예> OVA, HDM etc
- Efficacy test: AHR / BAL cell count / immune cell infiltration in a lung / BALF cytokine / allergen-specific cytokine production in the draining lymph nodes / allergen-specific nserum IgE -> treatment of FlaB and allergen induced anti-asthma effects
- Optomization of treatment model
- Efficacy test for HDM-FlaB conjugate(prototype): 3.5μg HDM-FlaB conjugate -> effective
- Comparison of administration routes: eg> intranasal, sublingual etc.
- Determination of allergen-specific effect by using non-specific allergens
○ Production of various nanoparticles and validation
- 4arm-PEG-based FlaB-HDM conjugate
- SH-PEI-based FlaB-HDM conjugate
- HA-based FlaB-HDM/OVA conjugate
- Gold particle-based FlaB-HDM conjugate
- Pluronics-based FlaB-OVA conjugate
- Liposome-based FlaB-OVA conjugate -> perform efficacy test
- FlaB fusion protein-based FlaB-HDM/OVA conjugate
○ In vivo tracing of FlaB and determination interaction of FlaB with immune cells
- Preparation of nfluorochrome-tagged FlaB -> detemination of interaction with DCs and/or T cells in the draining lymph nodes
○ Determination of TLR5-mediated anti-asthma effect using TLR5 knock out mice K
- Efficacy test: AHR / BAL cell count / immune cell infiltration in a lung / BALF cytokine / allergen-specific cytokine production in the draining lymph nodes / allergen-specific nserum IgE
- Anti-asthmatic effects were abrogated in the TLR5 KO mice -> Confirmation of FlaB dependent effect
○ Investigation of underlying mechanism of the flagellin-based specific immunotherapy
- Effect of experimental condition on the suppression of asthma: eg> dosages, routes, LPS contamination in the reagents
- Systems biological analysis of signaling and regulatory network of in vivo treated asthma immunomodulatory: Investigation of immune response induction mechanism through different dosage flagellin treatment by applying systems biological approaches
○ Evaluation of therapeutic effect of liposome based FlaB-OVA conjugate
- Efficacy test: AHR / BAL cell count / immune cell infiltration in a lung / BALF cytokine / allergen-specific cytokine production in the draining lymph nodes / allergen-specific nserum IgE
- Collaboration-based investigation
○ Investigation of DC (dendritic cell) based immunotherapy
- Evaluation of therapeutic effect of adoptive transfer based DC immunotherapy against asthma
- Investigation of characteristics of in vivo immune responses by different doses of flagellin treatment
- Molecular immunological investigation of flagellin-treated DC
- Evaluation of DC adoptive transfer by using TLR5 KO mice
- Determination of optimal flagellin dosage and measurement of antigen specific immunologic parameters which induces optimal asthma therapeutic effect
- Investigation of Treg related mechanism by treatment of flagellin
○ Investigation of effects of pre-immune status on flagellin based asthma immunotherapy
- Asthma vs normal 상태: immune cell function -> different status
- Basic research for clinical application
○ Optimization of DC-based cell therapy
- Production and evaluation of HA-based and adhesion molecule targeting nanoparticles
○ Development of allergen and FlaB fusion protein-based nanoparticle
- Development of allergen-FlaB fusion protein: Derp2-FlaB chimeric fusion protein
• Derp2-FlaB chimeric fusion protein: purification
• Derp2-FlaB chimeric fusion protein: determination of in vitro biologicval activity
• Derp2-FlaB chimeric fusion protein: efficacy test
• Collaboration-based investigation
- Development/validation of allergen-FlaB fusion protein-based nanoparticle
• Development of nanoparticle complex carring Derp2-FlaB using cationic nanoparticle (DOTAP/Cholesterol)
• Validation: determination of light scattering pattern / Native gel electrophoresis
- determination of anti-asthmatic effect of the Derp2-FlaB-NP
• Efficacy test: AHR / BAL count / Derp2-specific serum IgE / Derp2-specific cytokine production in bronchial LNs
==> significant suppression of Der2-induced asthma
• Collaboration-based investigation
○ Optimization of DC-based cell therapy: Organoid transplantation
- Application of Tertiary Lymphoid Organ (enTLO) on DC-based immunotherapy
○ Potentiation of therapeutic afficacy of immmunotherapy by modulating the in vivo immune microenvironment
- Development of novel strategy for potentiation of specific immunotherapy to asthma
○ Efficacy of flagellin-based specific immunotherapy in human-originated specimens and investigation of underlying mechanism
- Evaluation of Immune Cells Derived from Asthmatic Patients
- Isolation of PBMC from asthmatic patients: MNC / T cell
- Differentation of DC: GM-CSF / IL4 treatment -> CD11c DC
- Co-culture system: determination of T cell function
○ Effect of flagellin- based SIP on NKT: efficacy test / mechanism study
- Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells
->new therapeutic modality

Expected Contribution
□ Application plan
○ Try to apply to clinical studies with the results from this project
- Proceed to preclinical (GLP) and clinical studies
- Transfer the origin technology or the patent to hospitals or company
- Clinical trial
- Get the approval for new treatment
- Share the benefits with the asthma patients
□ Expected effect
○ Technical aspect
- Possibility to develop drugs for other immune diseases by using dendritic cell therapy
- Development of highly effective immunotherapy platform for allergic diseases
- Innovation in allergy treatment modalities with less side effects
- New original technology combining with NT and BT
○ Economic and industrial aspect
- Decrease of cost related to asthma treatment
- Develop new market in the immunotherapy field
- Contribution to international competitiveness of Korean medical industry with new technology


(출처 : SUMMARY 7P)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 국문 요약문 ... 4
• SUMMARY ... 7
• Contents ... 10
• 목차 ... 11
• 1. 연구개발과제의 개요 ... 12
• 1-1. 연구개발 목적 ... 12
• 1-2. 연구개발의 필요성 ... 12
• 1-3. 연구개발 범위 ... 12
• 2. 국내외 기술개발 현황 ... 14
• 3. 연구수행 내용 및 결과 ... 19
• 3-1. 연구 내용 및 결과 ... 19
• 3-2. 연구개발 추진전략 및 방법 ... 62
• 3-3. 연구개발 추진체계 ... 63
• 3-4. 연구개발 추진일정 ... 64
• 4. 목표달성도 및 관련분야 기여도 ... 65
• 4-1. 목표달성도 ... 65
• 4-2. 관련분야 기여도 ... 68
• 5. 연구결과의 활용계획 ... 69
• 6. 연구과정에서 수집한 해외과학기술정보 ... 70
• 7. 연구개발성과의 보안등급 ... 74
• 8. 국가과학기술종합정보시스템에 등록한 연구시설·장비 현황 ... 75
• 9. 연구개발과제 수행에 따른 연구실 등의 안전조치 이행실적 ... 76
• 10. 연구개발과제의 대표적 연구실적 ... 79
• 11. 기타사항 ... 80
• 12. 참고문헌 ... 81
• 별첨4. 실적 증빙자료 ... 84
• 별첨5. 기타 첨부서류 ... 133
• 끝페이지 ... 148