초록 ▼

· Ab417 항체 및 대조 항체 (hFc)를 대량 생산 및 정제하였음.
· SD rat에서 Ab417 항체의 pharmacokinetics 분석 결과, 반감기가 114 시간으로 나타났음.
· 정상 마우스에서 Ab417 항체의 단회 독성 시험 결과, 일반 독성 및 신경 독성이 나타나지 않았음.
· 간내 담도암 세포인 Choi-CK 이식 모델과 간외 담도암 세포인 TFK-1 이식 모델에서 Ab417 항체의 항암 효능을 확인하였음. 담낭암 모델로서 K-Ras 변이를 가진 JCRB1033 세포 이식 모델에서는 Ab417

· Ab417 항체 및 대조 항체 (hFc)를 대량 생산 및 정제하였음.
· SD rat에서 Ab417 항체의 pharmacokinetics 분석 결과, 반감기가 114 시간으로 나타났음.
· 정상 마우스에서 Ab417 항체의 단회 독성 시험 결과, 일반 독성 및 신경 독성이 나타나지 않았음.
· 간내 담도암 세포인 Choi-CK 이식 모델과 간외 담도암 세포인 TFK-1 이식 모델에서 Ab417 항체의 항암 효능을 확인하였음. 담낭암 모델로서 K-Ras 변이를 가진 JCRB1033 세포 이식 모델에서는 Ab417 항체의 효능이 미약하게 나타났음.
· Ab417 항체의 in vitro 작용 기전을 분석하였음.
· Ab417 항체의 생체 분포 분석 결과, L1CAM을 발현하고 있는 종양의 특이적인 섭취를 확인하였음.
· 간내 담도암에서 L1CAM의 발현 양상 및 임상적 의미를 연구하였음.
· Ab417 항체의 중쇄 및 경쇄 가변 영역의 아미노산 잔기 일부를 치환한 변이체를 제작하여 Ab417항체의 물성을 향상시켰음.
· Ab417 항체의 정제 공정 연구를 통해 항체의 생산성을 향상하였음.

(출처 : 보고서 요약서 3p)

Abstract ▼

Ⅳ. Results
1. Production and purification of the Ab417 antibody
· To obtain Ab417, we constructed CHO-DG44 stable cell line (#9-20 nM) and produced 1 g of Ab417 from the cell line. Purity of the purified antibodies was confirmed through SEC analysis and endotoxin detection assay (Kangwon Natio

Ⅳ. Results
1. Production and purification of the Ab417 antibody
· To obtain Ab417, we constructed CHO-DG44 stable cell line (#9-20 nM) and produced 1 g of Ab417 from the cell line. Purity of the purified antibodies was confirmed through SEC analysis and endotoxin detection assay (Kangwon National University).
· One gram of recombinant hFc was produced and its purity was confirmed through SEC analysis and endotoxin detection assay.
· The purified Ab417 and hFc were used for PK, toxicity, biodistribution, and efficacy studies.

2. Anaylsis of pharmacokinetics of the Ab417 antibody in rats
· ELISA methods to detect Ab417 in rat serum were established.
· For PK study, 3 or 10 mg/kg of Ab417 was injected into three SD rats and antibody levels quantified by ELISA. Then PK parameters were obtained. As a result, average half-life of Ab417 in rats was estimated to be 114 hours.

3. Single-dose toxicity study of Ab417 in normal mice
· Immunohistochemical analysis of mouse tissues using Ab417 indicated that expression profile of L1CAM in normal mouse tissues is similar to that in human tissues.
· Ab417 conjugated with Cy5.5 was distributed into whole body of mouse with the course of time after it was injected.
· Single dose of 10, 30, or 50 mg/kg of Ab417 was intravenously injected into normal ICR mouse. After 10 days of Ab417 treatment, autopsy was carried out and tissues were analyzed. No notable toxicity was observed.

4. In vivo efficacy studies of the Ab417 antibody in intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC) and gallbladder carcinoma (GBC) models
· We used Choi-CK (ICC), TFK-1 (ECC), and JCRB1033 (GBC) cell lines to evaluate anti-tumor activity of Ab417. Genomic sequence analysis showed that Choi-CK and TFK-1 cells have normal K-Ras gene, while JCRB1033 cell has a K-Ras activating mutation.
· L1CAM expression in Choi-CK, TFK-1, and JCRB1033 cell lines was confirmed.
· Biweekly injection of 10 mg/kg Ab417 inhibited the growth of tumor in Choi-CK xenograft nude mouse model. Combined treatment with Ab417 (10 mg/kg) and gemcitabine (10 mg/kg) or cisplatin (0.5 mg/kg) showed additive antitumor activity.
· In TFK-1 xenograft NOD/SCID mouse models, biweekly injection of 10 mg/kg Ab417 exhibited antitumor activity. Combination of Ab417 (6 mg/kg) with gemcitabine (5 mg/kg) increased antitumor activity compared to either agent.
· In JCRB1033 xenograft nude mouse models, injection of 10 mg/kg Ab417 thrice a week did not exhibit antitumor activity.

5. Analysis of the mechanism of action of Ab417
· Fine epitope of Ab417 in the Ig5 domain of L1CAM was determined through HDX-MS analysis.
· Ab417 did not inhibit proliferation of Choi-CK, TFK-1, or JCRB1033 cells in vitro. Considering that Ab417 inhibited the tumor growth in vivo, Ab417 may act in tumor microenvironment of cholangiocarcinoma to inhibit tumor growth.
· Ab417 exhibited weak ADCC activity in vitro.
· L1CAM was not shedded from Choi-CK and TFK-1 cells.

6. Biodistribution study of Ab417 antibody in mice models
· 64Cu-NOTA-Ab417 was prepared and its antigen-binding activity was confirmed by flow cytometric analysis using cholangiocarcinoma cell lines.
· Biodistribution study of 64Cu-NOTA-Ab417 in Choi-CK, SCK-L1 (SCK cells over-expressing L1CAM) and JCRB1033 xenograft models showed tumor uptake rate of 13 %ID/g, I5 %ID/g, and 13 %ID/g, respectively.
· 64Cu-NOTA-Ab417 was highly uptaken by the spleen.
· 64Cu-NOTA-Ab417 was rapidly disappeared in the blood and other tissues as well.

7. Clinicopathological study on L1CAM expression in intrahepatic cholangiocarcinoma (ICC)
· Tumors were collected from 119 ICC patients and database was constructed. Tissue optimization was carried out for immunohistochemistry.
· L1CAM was expressed in 32% of ICC tumors. There was no statistical significance between L1CAM expression and age, gender, pathologic T stage, nodal metastasis, grade, lymphatic invasion, or perineural invasion.
· There was no correlation between β-catenin and EGFR or L1CAM expression. However, most of L1CAM-positive tumors were EGFR-positive, although this was not statistically significant. In the case of extrahepatic cholangiocarcinoma, L1CAM expression correlated with EGFR expression.

8. Modification of the Ab417 antibody for the enhancement of productivity
· To improve the stability of Ab417, AggreSolveTM service was conducted from Lonza. Two variants (Ab417-H6L2 and Ab417-H6L6) of Ab417 were constructed and characterized. The variants showed lower pI and increased productivity, while their antigen-binding affinity and PK were not significantly different from Ab417.
· Purification process of Ab417 was optimized to solve an aggregation problem and thus stability of purified Ab417 was improved.

(출처 : Summary 12p)

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 4
• SUMMARY ... 10
• CONTENTS ... 16
• 목차 ... 17
• 제 1 장. 연구개발과제의 개요 ... 18
• 제 1절 연구개발 목적 ... 18
• 제 2절 연구개발 필요성 ... 18
• 제 3절 연구개발 범위 ... 29
• 제2장 국내외 기술개발 현황 ... 30
• 제 1절 항암 항체치료제의 국외 기술 개발 현황 ... 30
• 제 2절 항체치료제의 국내 기술 개발 현황 ... 33
• 제 3절 연구결과가 국내외 기술개발현황에서 차지하는 위치 ... 35
• 제 3장 연구개발수행 내용 및 결과 ... 36
• 제 1절 Ab417 항체, 대조(hFc) 항체의 대량 생산 및 정제 ... 36
• 제 2절 Ab417 항체의 PK 분석 ... 44
• 제 3절 Ab417 항체의 정상 마우스에서의 일반 독성 시험 (신경 독성 포함) ... 51
• 제 4절 Ab417 항체의 항암 효능 시험 (ICC, ECC, GBC) ... 58
• 제 5절 Ab417 항체의 작용 기전 분석 ... 87
• 제 6절 Ab417 항체의 생체분포 및 종양 표적능 평가 연구 ... 100
• 제 7절 간내 담도암에서 L1CAM과 암진행/전이조절 인자의 발현 양상과 치료 타겟으로서의 임상적 유용성 검증 ... 125
• 제 8절 Ab417 항체의 생산성 향상을 위한 항체 개량 연구 ... 132
• 제4장 목표달성도 및 관련분야에의 기여도 ... 144
• 제 1절 연구목표 및 연구내용 ... 144
• 제 2절 연구목표의 달성도 및 관련 분야에의 기여도 ... 145
• 제5장 연구개발결과의 활용계획 ... 151
• 제 1절 추가연구의 필요성 ... 151
• 제 2절 연구개발결과의 활용방안 ... 152
• 제 3절 기업화 추진방안 ... 152
• 제6장 연구개발과정에서 수집한 해외과학기술 정보 ... 153
• 제7장 연구시설 · 장비 현황 ... 154
• 제8장 참고문헌 ... 156
• 끝페이지 ... 163