KR101962483B1 - 혈액응고 제viii 인자의 기능을 대체하는 기능을 갖는 다중특이성 항원 결합 분자 - Google Patents
혈액응고 제viii 인자의 기능을 대체하는 기능을 갖는 다중특이성 항원 결합 분자 Download PDFInfo
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Abstract
Description
도 2는 스크리닝을 설명한 도면이다. 인간 F.IXa 및 인간 F.X에 대한 항체 유전자를 약 200종류씩 제작하여, 동물세포 발현 벡터에 삽입하였다. 항F.IXa 항체와 항F.X 항체의 조합으로서 4만 이상의 이중특이성 항체를 일과성 발현하였다. F.Xa 생산 촉진 활성 및 F.Xase 저해작용을 평가하여, F.Xa 생산 촉진 활성이 높고 F.Xase 저해작용이 낮은 이중특이성 항체를 스크리닝하였다. 추가로 필요에 따라 아미노산 치환을 행함으로써, 프로토타입 항체를 제작하였다.
도 3은 hA69-KQ/hB26-PF/hAL-AQ, Q1-G4k/J268-G4h/L45-k, Q1-G4k/J321-G4h/L45-k, Q31-z7/J326-z107/L2-k, Q64-z55/J344-z107/L45-k의 F.Xa 생산 촉진 활성을 나타내는 도면이다. 항체 용액의 농도는 300, 30, 3 ㎍/mL(Human Factor IXa, Novact(등록상표) M, Human Factor X와 항체 용액을 혼합한 후의 농도는 100, 10, 1 ㎍/mL), 효소반응 시간은 10분간, 발색시간은 50분간이다. 결과, 이들 항체는, WO 2006/109592에 기재된 hA69-KQ/hB26-PF/hAL-AQ와 비교하여, 높은 F.Xa 생산 촉진 활성을 나타내었다.
도 4는 hA69-KQ/hB26-PF/hAL-AQ, 프로토타입 항체, 및 아미노산 치환을 도입한 개변 항체의 F.Xa 생산 촉진 활성을 나타내는 도면이다. 항체 용액의 농도는 300, 30, 3 ㎍/mL(Human Factor IXa, Novact(등록상표) M, Human Factor X와 항체 용액을 혼합한 후의 농도는 100, 10, 1 ㎍/mL), 효소반응 시간은 2분간, 발색시간은 20분간이다. 결과, 이들 개변 항체는 프로토타입 항체보다도 높은 F.Xa 생산 촉진 활성을 나타내었다.
도 5는 hA69-KQ/hB26-PF/hAL-AQ, 프로토타입 항체, 및 아미노산 치환을 도입한 개변 항체의 F.Xase 저해작용을 나타내는 도면이다. hA69-KQ/hB26-PF/hAL-AQ, Q1-G4k/J268-G4h/L45-k, Q31-z7/J326-z107/L2-k, Q1-G4k/J321-G4h/L45-k, Q64-z55/J344-z107/L45-k, Q85-G4k/J268-G4h/L406-k, Q85-G4k/J321-G4h/L334-k, Q64-z7/J344-z107/L406-k, Q64-z7/J326-z107/L334-k, Q153-G4k/J142-G4h/L180-k, Q405-G4k/J232-G4h/L248-k, Q360-G4k/J232-G4h/L406-k, Q153-G4k/J232-G4h/L406-k, Q458-z106/J346-z107/L408-k, Q360-z118/J300-z107/L334-k, Q499-z118/J327-z107/L377-k, Q499-z121/J327-z119/L404-k, Q499-z121/J339-z119/L377-k, Q499-z118/J346-z107/L248-k, Q354-z106/J259-z107/L324-k, Q460-z121/J327-z119/L334-k, 및 Q499-z118/J327-z107/L334-k의 F.VIIIa 존재하에 있어서의 F.IXa에 의한 F.X 활성화에 대한 영향을 나타내는 도면이다. 항체의 F.Xase 저해작용은, 항체 첨가 반응액의 흡광도로부터 항체 무첨가 반응액의 흡광도를 뺀 값으로 나타내었다. 항체 용액의 농도는 300, 30 ㎍/mL(Human Factor IXa, F.VIIIa, Human Factor X와 항체 용액을 혼합한 후의 농도는 100, 10 ㎍/mL), 효소반응 시간은 6분간, 발색시간은 14분간이다. 가로축의 F.Xase 저해작용의 값이 플러스로 갈수록, F.Xase 저해작용이 약하다. 결과, WO 2006/109592에 기재된 hA69-KQ/hB26-PF/hAL-AQ는 강한 F.Xase 저해작용을 나타내었다. 본 발명의 항체 모두, hA69-KQ/hB26-PF/hAL-AQ와 비교하여, 약한 F.Xase 저해작용을 나타내거나, 또는 저해작용을 나타내지 않았다.
도 6a는 프로토타입 항체, 및 아미노산 치환을 도입한 개변 항체의 아미노산 서열을 나타낸 도면이다. Ref열에 서열명이 기재되어 있지 않은 경우는 Name열의 가변영역 서열을 기재하였다. Kabat 넘버링으로 해당하는 번호에 아미노산이 없는 개소는 「-(하이푼)」을 표시하였다. Name열과 Ref열의 가변영역을 비교하여, 동일한 아미노산의 개소를 「.(도트)」로서 표시하고, 상이한 개소는 Name열의 가변영역의 아미노산을 표시하였다. F.Xa 생산 촉진 활성 향상에 중요한 것이 명백해진 아미노산은 테두리를 둘러서 표시하였다.
도 6b는 도 6a의 계속되는 도면이다.
도 6c는 도 6b의 계속되는 도면이다.
도 6d는 도 6c의 계속되는 도면이다.
Claims (29)
- 혈액응고 제IX 인자 및/또는 활성화 혈액응고 제IX 인자를 인식하는 제1 항원 결합 부위, 및 혈액응고 제X 인자를 인식하는 제2 항원 결합 부위를 포함하는 다중특이성 항원 결합 분자로서, 상기 제1 항원 결합 부위가 이하의 (a)~(n) 및 (u) 중 어느 하나에 기재된 이중특이성 항체에 결합하는 혈액응고 제IX 인자 및/또는 활성화 혈액응고 제IX 인자상의 에피토프와 중복되는 에피토프에 결합하고, 상기 제2 항원 결합 부위가 상기 이중특이성 항체에 결합하는 혈액응고 제X 인자상의 에피토프와 중복되는 에피토프에 결합하는, 다중특이성 항원 결합 분자:
(a) 제1 폴리펩티드가 서열번호:1에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:4에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:9에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q1-G4k/J268-G4h/L45-k),
(b) 제1 폴리펩티드가 서열번호:1에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:5에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:9에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q1-G4k/J321-G4h/L45-k),
(c) 제1 폴리펩티드가 서열번호:2에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:6에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:8에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q31-z7/J326-z107/L2-k),
(d) 제1 폴리펩티드가 서열번호:3에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:7에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:9에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q64-z55/J344-z107/L45-k),
(e) 제1 폴리펩티드가 서열번호:10에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:6에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:30에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q64-z7/J326-z107/L334-k),
(f) 제1 폴리펩티드가 서열번호:10에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:7에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:33에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q64-z7/J344-z107/L406-k),
(g) 제1 폴리펩티드가 서열번호:11에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:4에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:33에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q85-G4k/J268-G4h/L406-k),
(h) 제1 폴리펩티드가 서열번호:11에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:5에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:30에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q85-G4k/J321-G4h/L334-k),
(i) 제1 폴리펩티드가 서열번호:12에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:21에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:33에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q153-G4k/J232-G4h/L406-k),
(j) 제1 폴리펩티드가 서열번호:13에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:22에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:29에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q354-z106/J259-z107/L324-k),
(k) 제1 폴리펩티드가 서열번호:14에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:21에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:33에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q360-G4k/J232-G4h/L406-k),
(l) 제1 폴리펩티드가 서열번호:15에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:23에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:30에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q360-z118/J300-z107/L334-k),
(m) 제1 폴리펩티드가 서열번호:16에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:21에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:28에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q405-G4k/J232-G4h/L248-k),
(n) 제1 폴리펩티드가 서열번호:17에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:27에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:34에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q458-z106/J346-z107/L408-k),
(u) 제1 폴리펩티드가 서열번호:12에 기재된 아미노산 서열로 이루어지는 H쇄, 제2 폴리펩티드가 서열번호:170에 기재된 아미노산 서열로 이루어지는 H쇄 및 제3 폴리펩티드와 제4 폴리펩티드가 서열번호:171에 기재된 공통 L쇄로 이루어지는 이중특이성 항체(Q153-G4k/J142-G4h/L180-k). - 제 1 항에 있어서,
혈액응고 제VIII 인자의 기능을 대체하는, 다중특이성 항원 결합 분자. - 제 2 항에 있어서,
혈액응고 제VIII 인자의 기능을 대체하는 기능이, 서열번호: 165와 166으로 이루어지는 H쇄 및 서열번호: 167로 이루어지는 공통 L쇄를 갖는 이중특이성 항체(hA69-KQ/hB26-PF/hAL-AQ)와 비교하여 높은 활성화 혈액응고 제X 인자(F, Xa) 생산 촉진 활성에 기인하는 기능인, 다중특이성 항원 결합 분자. - 제 1 항 내지 제 3 항 중 어느 한 항에 있어서,
다중특이성 항원 결합 분자가 다중특이성 항체인, 다중특이성 항원 결합 분자. - 제 1 항 내지 제 3 항 중 어느 한 항에 기재된 다중특이성 항원 결합 분자를 코드하는 핵산.
- 제 5 항에 기재된 핵산이 삽입된 벡터.
- 제 5 항에 기재된 핵산 또는 상기 핵산이 삽입된 벡터를 포함하는 숙주세포.
- 제 7 항에 기재된 숙주세포를 배양함으로써, 다중특이성 항원 결합 분자 또는 이중특이성 항체를 제조하는 방법.
- 제 1 항 내지 제 3 항 중 어느 한 항에 기재된 다중특이성 항원 결합 분자, 및 약학적으로 허용되는 담체를 포함하는, 혈우병 A, 후천성 혈우병 또는 폰 빌레브란트병의 예방 및/또는 치료를 위한 의약 조성물.
- 제 1 항 내지 제 3 항 중 어느 한 항에 기재된 다중특이성 항원 결합 분자를 포함하는, 혈우병 A, 후천성 혈우병 또는 폰 빌레브란트병을 예방 및/또는 치료하는 방법에 사용하기 위한 키트.
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