[보고서]허혈 망막질환 치료용 항 c-Met 작용 항체 후보물질 개발

김정훈

허혈 망막질환 치료용 항 c-Met 작용 항체 후보물질 개발
Lead Optimization of Anti-c-Met Agonistic Antibody for Treatment of Ischemic Retinal Diseases 원문보기

보고서 정보
주관연구기관	서울대학교 Seoul National University
연구책임자	김정훈
참여연구자	조동현 , 이석재 , 이기황 , 김수연 , 장하영 , 조창식 , 정준호 , 유덕균 , 김기현 , 김수현 , 김상일
보고서유형	1단계보고서
발행국가	대한민국
언어	한국어
발행년월	2020-07
과제시작연도	2019
주관부처	(범부처사업) NTIS
등록번호	TRKO202200010199
과제고유번호	9991006323
사업명	범부처전주기신약개발(R&D)
DB 구축일자	2022-09-24
키워드	허혈 망막질환.미숙아망막병증.당뇨망막병증.항체.간세포성장인자.Ischemic retinal vascular diseases.Retinopathy of prematurity.Diabetic retinopathy.c-Met.Antibody.Hepatocyte growth factor.

초록 ▼

Ⅳ. 연구개발결과
○ 작용기전 연구
◆ 항 c-Met 작용 항체인 m4A25를 통해 c-Met 및 하위 신호전달 경로의 활성화를 확인하였음
◆ 항 c-Met 작용 항체인 m4A25 및 RHM5를 안구 내로 주사하였을 때 망막의 혈관내피세포에 국한된 c-Met 활성화 확인하였음
◆ m4A25, RHM5를 처리하였을 때 혈관내피세포 이외의 다른 망막구성세포의 세포 증식이 촉진되지 않는 현상을 확인하였음
○ 항체 최적화
◆ Human과 mouse c-Met에 결합하여 활성화를 유도하는 다수의 클론을 확보하였음
○ 유효성 평가
◆ 유효성 평가모델을 확립하고 정량적 분석 기준을 설정함
◆ In vitro 모델에서 m4A25와 RHM5의 혈관신생 촉진 효과를 확인함
◆ In vivo 모델에서 m4A25와 RHM5의 혈관신생 촉진 효과를 확인함
○ 특허전략 수립
◆ 특허법인의 특허정보 분석보고서를 검토하고 특허전략을 수립함

(출처 : 요 약 문 4p)

Abstract ▼

Objective: This study aims to develop the candidate of anti-c-Met agonistic antibody to treat ischemic retinal diseases including retinopathy of prematurity and diabetic retinopathy, which put people of all ages at risk. Current treatment options for these diseases are restricted to therapeutic injections that refrain vascular endothelial growth factors from promoting pathological neovasculization or destruction of retinal ischemia using laser. On the contrary, anti-c-Met agonistic antibody will ultimately trigger physiological angiogenesis and thereby rise as a novel and effective treatment option.

Research content:
○ Investigation of mode of action
◆ Analysis of activation of c-Met through the lead anti-c-Met antibody
◆ Analysis of intraocular activation of c-Met
◆ Potential side effects
○ Optimization of the lead antibody
◆ Optimization of the lead antibody
◆ Preparation of backup antibodies
○ Efficacy test
◆ Establishment of efficacy test models and development of analysis protocols
◆ In vitro efficacy test
◆ In vivo efficacy test
○ Patent preparation
◆ Intellectual property analysis and preparation of the patent

Expected outcome:
○ Anti-c-Met antibody lead from this research can be developed to therapeutic agent for ischemic retinal diseases through pre-clinical and clinical studies in future research.
○ Upon successful development, the antibody promoting physiological angiogenesis is expected to be high in demand as a novel means of targeting ischemic retinal diseases

(source: SUMMARY 6p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
요 약 문 ... 4
SUMMARY ... 6
CONTENTS ... 7
목차 ... 8
제1장 연구개발과제의 개요 ... 9
제1절 타겟 환자군(Target Patient Population) ... 9
제2절 미충족 의학적 수요(Unmet Medical Needs) ... 10
제3절 시장규모와 성장세(Size and Growth) ... 12
제4절 연구개발 목표 ... 13
제2장 국내외 기술개발 현황 ... 14
제3장 연구개발수행 내용 및 결과 ... 17
제1절 작용기전 연구 ... 17
제2절 항체 최적화 ... 20
제3절 유효성 평가 ... 31
제4절 특허전략 수립 ... 34
제4장 목표달성도 및 관련분야에의 기여도 ... 35
제1절 작용기전 연구 ... 35
제2절 항체 최적화 ... 35
제3절 유효성 평가 ... 36
제4절 특허전략 수립 ... 36
제5장 연구개발결과의 활용계획 ... 37
제1절 연구개발 결과물 활용 방안 ... 37
제2절 사업화 전략 및 방법(Commercialization) ... 37
제3절 기대성과(일자리 창출, 특허, 논문, 기술이전) ... 37
제6장 연구개발과정에서 수집한 해외과학기술정보 ... 38
제7장 연구시설ᆞ장비 현황 ... 39
제8장 참고문헌 ... 40
끝페이지 ... 42

표/그림 (21)

표 실명을 유발하는 허혈 망막질환
표 허혈 망막질환에 대한 새로운 치료제로서 항 c-Met 항체 치료제
표 당뇨망막병증 시장규모 예상치 (Global Market Insights, 2018; Global View Research, 2018; Market Research, 2018; Market Watch, 2018; Research Markets, 2018)
표 미숙아망막병증 임상시험 현황
표 HGF 관련 약물의 임상시험 현황
표 Generation and characterization of recombinant anti-human c-Met scFv human IgG4 Fc fusion protein, m4A25-hFc4. (A) Nucleotide and amino acid sequences of m4A25 scFv gene. (B) affinity purified m4A25-hFc4 fusion protein is quantified and production yield is calculated as 6.165 mg/L culture. (C) SDS-PAGE gel results of 500 ng and 1 μg of m4A25-hFc4 and bovine serum albumin under reducing and non reducing condition respectively
표 Agonistic effiacy of anti-c-Met antibody m4A25-hFc4. (A) Phosphorylation of Met, FAK, Akt, Erk1/2 is induced by treatment of HGF (1 nM), m4A25-hFc4 (100 nM), isotype control antibody (100 nM) for 5 minutes. (B) Wound healing assay was made in bEnd.3 with 24 hours of recovery. The groups anlyzed were : rmHGF (1 nM), m4A5-hFc4 (100 nM), isotype control antibody (100 nM). (C) The graphics present cell free area of bEnd.3 cell after treatment. rmHGF and m4A25-hFc4 treated cells recover the wound faster than control antibody treated cells. (D) bEnd.3 cells were treated with rmHGF (1 nM), m4A5-hFc4 (100 nM), isotype control antibody (100 nM) and cell proliferation is measured immediately, 24 hours, 48 hours after treatment but did not show significant differences in cell proliferation
표 Expression and activation of c-Met in ocular tissues. (A) Expression of c-Met in the superficial layer of the retina. (B) Expression of the phosphorylated form of c-Met in the retinal vasculature at 6 hours after intravitreal injection of m4A25 (100 nM). (C) Expression of the phosphorylated form of c-Met in the retinal vasculature at 6 hours after intravitreal injection of RHM5 (100 nM)
표 Differential effects of HGF, RHM5, and m4A25 on the proliferation of retinal constituent cells. (A) HUVECs. (B) HRMECs. (C) ARPE-19. (D) Human retinal pericytes. (E) bEnd.3 cells. (F) Mouse brain pericytes
표 Serum antibody titer before and after immunization of the chicken with human c-Met antigen
표 Screening of c-Met reactive phage clones from the last round of the biopanning. (A) Phage ELISA results. Phage expression represent the signal of the HA specific antibody which binds to phage coat protein and enbrel is coated as negative control for c-Met antigen. (B) 6 different HCDR3s were identified from Sanger sequencing
표 Characterization of anti-human c-Met antibody. (A) Binding activity of anti-human c-Met antibody (RM001 – RM006) was evaluated with ELISA against recombinant human c-Met-HIS conjugated protein. (B) Binding activity against cell surface human c-Met was evaluated with PC-3 human prostate cancer cell line by flow cytometry analysis. (C) Agonistic efficacy against c-Met was evaluated with detection of phosphorylated c-Met after treatment of antibody to the human c-Met expressing cell line PC-3
표 Estimates of the sample coverage and clonal diversity of functional reads from singleplex-prepared and multi-plex-prepared phage libraries. (A) Sample coverage estimations of each libraries. Results are shown by dash and dotted line of the curve, which means the coverage val-ues from rarefaction curve and extrapolation curve respectively. (B) Enlarged figure of rectangular region of (a) and sample coverage and whole readcount of the acquired data. Dots represented the data that were achieved from libraries (singleplex-prepared and multiplex-prepared). (c - e) R/E curve (Rarefaction and extrapolation curve) of each libraries with diversity order number (c) q = 0, (d) q = 1, and (e) q = 2. R/E curves were obtained considering clonal frequencies of each functional clones
표 (A) The number of unique functional reads from each library. (B) The clonal distribution of co-existing functional reads in both libraries. (C) V gene usage of functional reads from each library. (D) Inequality (Gini index) of functional reads from each library with respect to V genes
표 Construction of rabbit immune library using in-house designed immunoglobulinl gene specific primer. (A) Titration of serum immunoglobulin from 3 human c-Met immunized rabbit. (B) Extraction of total RNA from 2 rabbits with high serum antibody titer. SP, BM, PB represent spleen, bone marrow, peripheral blood respectively. 500 ng of total RNA were loaded. (C) Minimum-path tracking algorithm derived primer design logic. Central single mismatch and terminal double mismatch was allowed along with 3’ perfect match for 6 nucleotide molecules
표 Biopanning titer: rabbit immune library
표 Screening of c-Met reactive phage clones from the last round of the biopanning. (A-B) Phage ELISA results. Phage expression represent the signal of the HA specific antibody which binds to phage coat protein and enbrel is coated as negative control for c-Met antigen. (C) 10 different HCDR3s were identified from Sanger sequencing
표 Characterization of anti-human c-Met antibody identified from rabbit immune library. (A) Binding activity of anti-human c-Met antibody (RHM001 – RHM006, scFv-human IgG1 Fc conjugated format) was evaluated with ELISA against recombinant human c-Met-HIS conjugated protein. (B) RHM5 clone was produced and tested for cross-reactivity against human, mouse and monkey c-Met. (C) Agonistic efficacy against c-Met was evaluated with detection of phosphorylated c-Met after treatment of antibody to the human c-Met expressing cell line PC-3 and mouse c-Met expressing cell line CT-26
표 Effects of m4A25 and RHM5 on in vitro angiogenesis assays. (A) The effects of HGF and m4A25 on the proliferation of bEnd.3 cells. (B) The effects of HGF and RHM5 on the proliferation of HUVECs. (C) The effects of HGF and RHM5 on the migration of HUVECs. (D) The effects of HGF and RHM5 on the tube formation of HUVECs
표 Effects of m4A25 and RHM5 on in vivo oxygen-induced retinopathy model. (A) The effects of m4A25 on in vivo oxygen-induced retinopathy model. (B) The effects of RHM5 on in vivo oxygen-induced retinopathy model
표 당뇨망막병증 임상시험 현황(2019년 7월 1일 이후 게시)

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연합인증

허혈 망막질환 치료용 항 c-Met 작용 항체 후보물질 개발
Lead Optimization of Anti-c-Met Agonistic Antibody for Treatment of Ischemic Retinal Diseases 원문보기