초록 ▼

주관기관 ㈜뉴라클사이언스는 FAM19A5 조절을 통한 “신경교흉터 형성 억제 기전의 알츠하이머병 항체 신약 개발”의 최종 후보 물질 도출을 목적으로 하기의 세부 마일스톤 별 과업을 수행하였음.
1) 선도항체 스크리닝 및 항체 특성 분석 : 동물 면역화 및 인간 항체 라이브러리를 통하여 발굴한 다양한 항체의 물성 및 특성을 분석하고, 1차적으로 효능 평가 대상의 후보 항체군을 선정하였음.
2) 작용기전(MOA) 연구 : 선정된 후보 항체를 이용하여 FAM19A5의 뇌질환에서 작용기전 연구 및 in vitro potency

주관기관 ㈜뉴라클사이언스는 FAM19A5 조절을 통한 “신경교흉터 형성 억제 기전의 알츠하이머병 항체 신약 개발”의 최종 후보 물질 도출을 목적으로 하기의 세부 마일스톤 별 과업을 수행하였음.
1) 선도항체 스크리닝 및 항체 특성 분석 : 동물 면역화 및 인간 항체 라이브러리를 통하여 발굴한 다양한 항체의 물성 및 특성을 분석하고, 1차적으로 효능 평가 대상의 후보 항체군을 선정하였음.
2) 작용기전(MOA) 연구 : 선정된 후보 항체를 이용하여 FAM19A5의 뇌질환에서 작용기전 연구 및 in vitro potency 분석법 개발을 진행하였음.
3) 질환모델 약효 평가 : 대표적 알츠하이머병 동물 모델인 5xFAD 및 APP/Ps1 마우스 모델에서 FAM19A5 항체 투여 후 인지기능 개선 및 뇌조직 분석을 통하여 질환 개선 효과를 확인하였음.
4) 예비독성 평가 : 마우스에서 단회 및 반복 투여를 통한 후보 항체의 예비 독성을 확인하였으며, 인체 장기 조직 샘플에서의 Tissue Cross Reactivity assay를 수행하여 항체의 안전성과 특이성을 확인하였음.
이상의 결과를 바탕으로 주관기관은 본 과제를 통하여 최종적으로 FAM19A5항체 3-2 클론(Chicken origin)을 신약 후보 물질로 선정하였고, 본 결과를 바탕으로 항체 치료제 개발을 진행할 예정임.

(출처 : 요약서 3p)

Abstract ▼

IV. Research Results

1. FAM19A5 Antibody Discovery
(A) Panning was conducted on chicken immunization, rabbit immunization and human libraries and more than 20 FAM19A5-binding scFv were discovered.
(B) By examining the basic production capacity and stability of the antibodies converted

IV. Research Results

1. FAM19A5 Antibody Discovery
(A) Panning was conducted on chicken immunization, rabbit immunization and human libraries and more than 20 FAM19A5-binding scFv were discovered.
(B) By examining the basic production capacity and stability of the antibodies converted to IgG1 frame, 16 antibodies were selected and small scale production commenced.

2. Antibody Specificity Analysis
(A) The basic binding affinity of the antibodies were evaluated using the ELISA method antigen-antibody binding verification test and six top antibodies were selected.
(B) Six peptides covering FAM19A5 were synthesized, and the epitopes of each antibody clone were identified by comparing the binding of each antibody. The antibody having a specific epitope was screened and the efficacy of the antibody according to the binding site was investigated and candidates fulfilling the standard of therapeutic development was secured as a criterion for selection.
(C) Western blotting and immunocytochemistry tests were performed to select FAM19A5-specific antibodies. Finally, the 3-2 antibody and other antibodies of the chicken origin were selected as the antibodies of priority for efficacy evaluation.

3. In-vitro / in-vivo efficacy test
(A) FACS analysis
FACS analysis was performed on the 3-2 antibody and the chicken origin antibodies selected after antibody specificity analysis, and it confirmed the superior binding affinity of the 3-2 antibody over other antibodies.
(B) Glial scar inhibition in TBI animal model
TBI animal model, a representative model for nerve damage, was used to evaluate the efficacy 3-2 antibody and it was found to inhibit glial scar formation hypertrophic neuropathy.

4. MoA Study
(A) Receptor Study
FAM19A5 receptor research was carried out by investigating protein phosphorylation, and intracellular signal transduction. Additional studies on the candidate receptor protein are being currently carried out by the institution, a consigned research institute (Korea University Medical School), and external research service organizations.
(B) Confirmation of Vessel Normalization and Phagocytosis mechanism
To investigate the efficacy of the final candidate antibody the brain of the TBI mouse model using the clarity method was used and nerve regeneration was observed. In addition, vessel normalization was observed in the brain tissue suggesting that the antibody not only causes nerve and immune system recovery but also allows for neurovascular coupling. Also based on the close interrelationship between neuronal damage and phagocytosis of microglia, the effect of 3-2 antibody in improving phagocytosis of BV-2 cells through FAM19A5 regulation was observed.
(C) Confirmation of Neuronal Regeneration Effect
Improvements to the behavioral measures were observed in rat and mouse spinal cord injury mouse models following 3-2 antibody administration and study is continuing into nerve regeneration effect in tissue.
(D) Establishment and Verification of Patient Serum Analysis
Based on the evidence of increased FAM19A5 expression in neuronal damage, degenerative brain disease and its increased concentration in patient serum, we established ELISA assay to detect FAM19A5 with patient blood and obtained blood samples of 16 patients and confirmed that the FAM19A5 level increases in patients compared to the normal group.

5. Efficacy Evaluation in Disease model
(A) 5xFAD Model
In the 5xFAD model known for its rapid Alzheimer`s disease progression, inhibition of reactive gliosis and neuroinflammation efficacy was confirmed after the administration of the antibody. In addition, inhibitory effect of amyloid beta protein accumulation and cognitive defects. Moreover, we confirmed the expression of FAM19A5 in the demented lesion tissues and the blood of patients. With these findings study investigating the possible correlation between Alzheimer’s disease and FAM19A5 regulation was performed concurrently.
(B) APP/Ps1 Mouse Model
The effect of FAM19A5 antibody administration on the cognitive function was tested through a test of object cognitive ability (water maze test) and spatial cognitive ability (passive avoidance test) using a Alzheimer's disease mouse model. Also, through brain tissue staining analysis it was confirmed that the antibody effectively inhibits the formation of amyloid beta plaques.

6. Toxicity Study
(A) Preliminary toxicity Study
Single / repeated doses of the FAM19A5 (3-2) antibody was administered to test for toxicity. Results demonstrate no toxicity of the administered antibodies.
(B) Tissue cross reactivity
Cross-reactivity of antibodies in human tissue samples (brain tumor and Alzheimer's disease patients) to confirm the strong positive reaction in Alzheimer's disease and brain tumor lesions. However, we confirmed that there is no binding of antibody clones to organs order than the desired.

7. Conclusion
Based on the above results, the company selected FAM19A5 antibody 3-2 clone(chicken origin) as the lead substance for the new drug, and will develop the next stage for the antibody therapeutic of Alzheimer`s disease. And, company selected 1-65 and 2-13 clones for backup substances.

(출처 : SUMMARY 10p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보 고 서 요 약 서 ... 3
• 요 약 문 ... 4
• SUMMARY ... 8
• CONTENTS ... 14
• 목차 ... 15
• 제1장 연구개발과제의 개요 ... 16
• 제1절 연구개발의 필요성 ... 16
• 제2절 연구개발의 목적 및 범위 ... 20
• 제2장 국내외 기술개발 현황 ... 29
• 제1절 기존 치료제 현황 ... 29
• 제2절 최근 개발 동향 ... 30
• 제3장 연구개발수행 내용 및 결과 ... 32
• 제1절 항체 스크리닝 및 항체 특성분석 ... 33
• 제2절 MOA Study ... 50
• 제3절 시료 생산 ... 60
• 제4절 질환모델 약효 평가(알츠하이머 모델) ... 60
• 제5절 Toxicity study ... 68
• 제6절 결론 ... 71
• 제4장 목표달성도 및 관련분야에의 기여도 ... 73
• 제1절 마일스톤 기반의 목표 달성도 ... 73
• 제2절 관련분야 기여도 ... 75
• 제5장 연구개발결과의 활용계획 ... 76
• 제1절 후속 연구 계획 ... 76
• 제2절 적응증 확장 계획 ... 77
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 78
• 제1절 과제 진행 기간 출간된 신규 논문(SCI) ... 78
• Reference ... 78
• 끝페이지 ... 79