KR950700419A - 단량체 및 이량체 항체-단편 융합 단백질(monomeric and dimeric antibody-fragment fusion proteins) - Google Patents

단량체 및 이량체 항체-단편 융합 단백질(monomeric and dimeric antibody-fragment fusion proteins)

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KR950700419A
KR950700419A KR1019940702517A KR19940702517A KR950700419A KR 950700419 A KR950700419 A KR 950700419A KR 1019940702517 A KR1019940702517 A KR 1019940702517A KR 19940702517 A KR19940702517 A KR 19940702517A KR 950700419 A KR950700419 A KR 950700419A
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protein
fusion
fragment
peptide
antibody
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플뤼크툰 안드레아스
팍크 페터
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위르겐 오이만, 라인하르트 슈틀러
메르크 파텐트 게젤샤프트 미트 베쉬랭크터 하프퉁
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

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  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

본 발명은 항체의 Fv-단편을 포함하나 불변 항체 도메인을 이용하지 않는 새로운 부류의 항원 결합 분자를 제공한다. 또한 이들 분자는 다른 항체 단편 분자 또는 비-항체 단편 분자와 이량화되어 각각 이-또는 다-작용성 항체-단편 융합 단백질 및 소위 미니항체를 형성한다. 신규 융합 단백질은 진단 및 치료의학의 광범위한 분야에서 사용될 수 있다.

Description

단량체 및 이량체 항체-단편 융합 단백질(MONOMERIC AND DIMERIC ANTIBODY -FRAGMENT FUSION PROTEINS)
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제 1 도 scFv 단편을 함유하는 scFv-발현 벡터 pLISC-SE, 제 2 도 2시스트론 scFv-힌지-지퍼 발현 벡터 pACKxFyJ, 제 3 도 작용적 ELISA; OD280으로 측정한 친화성 정제 단백질의 농도(세로축)는 웰당 결합 부위의 몰 수(가로축)를 의미한다. ELISA 플레이트는 포스포콜린으로 피복되고 정제된 포스포콜린-특이적 미니항체-단백질이 결합되었으며 항-McPC603 항혈청으로 검출되었다. (a)다양한 미니항체의 비교, (b)ScFv 및 전체 IgA와 미니항체 scHLXc의 비교.

Claims (24)

  1. 비공유 상호작용에 이해 다른 펩티드와 이량체를 형성할 수 있는 펩티드 및 항체의 Fv-단편으로 필수적으로 구성되는 항체-단편 융합 단량체 단백질.
  2. 제 1 항에 있어서, 상기 Fv-단편이 단일 사슬 단편임을 특징으로 하는 단량체 단백질.
  3. 제 1 항 또는 제 2 항에 있어서, 상기 상호작용 펩트드가 10 내지 50개, 바람직하게는 10 내지 30개의 아미노산으로 구성됨을 특징으로 하는 단량체 단백질.
  4. 제 1 항 내지 제 3 항중 어느 한 항에 있어서, 상기 펩티드가 적어도 하나의 나선으로 구성됨을 특징으로 하는 단량체 단백질.
  5. 제 4 항에 있어서, 상기 나선 펩티드가 나선, 돌기 및 다른 나선으로 구성됨을 특징으로 하는 단량체 단백질.
  6. 제 4 랑에 있어서, 상기 펩티드가 매 7 번째 아미노산이 류신인 몇개의 반복 아미노산을 가지는 류신지퍼 분자를 포함함을 특징으로 하는 단량체 단백질.
  7. 제 4 항에 있어서, 상기 펩티드가 전하를 띤 잔기임을 특징으로 하는 단량체 단백질.
  8. 제 1 항 내지 제 7 항중 어느 한항에 있어서, 상기 결합 펩티드가 Fv-단편과 펩티드사이에 존재함을 특징으로 하는 단량체 단백질.
  9. 제 8 항에 있어서, 상기 결합 펩티드가 항체의 힌지 영역 서열 또는 그의 단편임을 특징으로 하는 단량체 단백질.
  10. Fv-단편, 상호작용 펩티드 및 필요하다면 결합 펩티드를 암호화하는 유전자를 하나의 발현 플라스미드를 클로닝시키고, 이 발현 플라스미드로 숙주 세포를 형질전환시키고, 자양액중에서 숙주 세포를 배양하고, 융합 단량체 단백질을 세포내에서 발현시키거나 또는 배지로 분비시킴을 특징으로 하는, 제 1 항 내지 제 9 항중 어느 한 항에 정의된 바와 같은 항체 융합 단량체 단백질의 제조방법.
  11. 제10항에 있어서, 상기 숙주 세포가 대장균임을 특징으로 하는 방법.
  12. 적어도 하나의 단량체 단위가 제 1 항내지 제 9 항중 어느 한 항에서 정의된 바와 같은 항체-단편 융합 단백질임을 특징으로 하는, 단량체 단위의 결합이 같거나 다른 펩티드의 비공유 상호 작용에 기초하는 두개의 융합 단량체 단백질로 필수적으로 구성되는 융합 이량체 단백질.
  13. 제12항에 있어서, 상기 상호작용 펩티드가 동일한 융합 이량체 단백질.
  14. 제12항 또는 제13항에 있어서, 상기 제 2 단량체 단위가 다른 특이성을 가지는, 제 1 항 내지 제 9 항에서 정의된 바와 같은 항체-단편 융합 단백질임을 특징으로 하는 이량체 단백질.
  15. 제12항 또는 제13항에 있어서, 상기 제 2 단량체가, 항체-단편(Fv)이 비-항체 단백질 또는 펩티드로 대체된 제 1 항 내지 제 9 항중 어느 한 항에서 정의된 바와 같은 융합 단백질임을 특징으로 하는 이량체 단백질.
  16. 제15항에 있어서, 상기 단백질 또는 펩티드가 독소, 펩티드 킬레이트화제, 금속 결합 단백질 또는 효소이거나, 또는 대응하는 특이적 결합 부위를 가짐을 특징으로 하는 이량체 단백질.
  17. 제15항에 있어서, 상기 단백질 또는 펩티드가 T-세포- 또는 T-세포 단편 특이적 결합 부위임을 특징으로 하는 이량체 단백질.
  18. 제12항 내지 제17항중 어느 한 항에 있어서, 상기 다른 단백질이 개재 펩티드의 하나 또는 둘다의 C-말단에 융합함을 특징으로 하는 이량체 단백질.
  19. 제18항에 있어서, 상기 융합 단백질이 독소, 펩티드 킬레이트화제, 금속 결합 단백질 또는 효소이거나, 또는 대응하는 특이적 결합 부위를 가지거나, 또는 T-세포(단편) 특이적 결합 부위를 가짐을 특징으로 하는 이량체 단백질.
  20. 완전한 융합 단량체 단백질 또는 그의 일부분을 암호화하는 유전자를 최소한 하나의 발현 플라스미드에 클로닝시키고, 이 발현 플라스미드(들)로 숙주 세포를 형질전환시켜 자양액중에서 배양하고, 완전한 융합 이량체 단백질을 세포내에서 발현시키거나 배지로 발현시키거나, 또는 융합 단량체 단백질을 별도로 발현시키고, 두개의 단량체 단위 사이의 비공유 결합을 배지 또는 실험관내에서 실시하고, 융합 단백질의 일부만이 클로닝될 경우에는 단백질 공학 단계를 부가적으로 실시함을 특징으로 하는, 제12항 내지 제19항중 어느 한 항에서 정의된 바와 같은 융합 이량체 단백질의 제조 방법.
  21. 제20항에 있어서, 제 1 융합 단량체 단백질을 암호화하는 유전자를 제 1 발현 플라스미드에 클로닝하고, 제 2 융합 단량체 단백질을 암호화하는 유전자를 제 2 발현 플라스미드에 클로닝함을 특징으로 하는 방법.
  22. 제20항에 있어서, 융합 이량체 단백질을 형성하는 단량체 단위사이의 비공유 결합을 실험관내에서 실시함을 특징으로 하는 방법.
  23. 제20항 내지 제22항중 어느 한 항에 있어서, 숙주 세포가 대장균임을 특징으로 하는 방법.
  24. (a)제 1 항 내지 제 8 항중 어느 한 항에서 정의된 바와 같은 항체-단편 융합 단량체 단백질 및 (b)(a)에서 정의된 바와 같은 제 2 융합 단량체 단백질을 포함하는 제12항 내지 제19항중 어느 한 항에서 정의된 바와 같은 항체-단편 융합 단백질(항체-단편은 같거나 다른 항원 특이성을 가지거나 또는 항체 단편 단위는 비-항체 단백질/펩티드로 대체된다)의 선별적인 이량체를 제조하기 위한 제조 킷트.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019940702517A 1992-01-23 1993-01-15 단량체 및 이량체 항체-단편 융합 단백질 KR100254759B1 (ko)

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EP92101069 1992-01-23
EP92101069.0 1992-01-23
DE92101069.0 1992-01-23
PCT/EP1993/000082 WO1993015210A1 (en) 1992-01-23 1993-01-15 Monomeric and dimeric antibody-fragment fusion proteins

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KR100254759B1 KR100254759B1 (ko) 2000-05-01

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US (1) US5910573A (ko)
EP (1) EP0654085B1 (ko)
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AT (1) ATE151113T1 (ko)
AU (1) AU676150B2 (ko)
CA (1) CA2128511C (ko)
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WO (1) WO1993015210A1 (ko)

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EP0654085B1 (en) 1997-04-02
HU215180B (hu) 1998-10-28
RU2128709C1 (ru) 1999-04-10
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HUT68798A (en) 1995-07-28
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KR100254759B1 (ko) 2000-05-01
ES2102007T3 (es) 1997-07-16
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CA2128511C (en) 2006-11-07
DE69309472T2 (de) 1997-10-23
DK0654085T3 (da) 1997-09-22
WO1993015210A1 (en) 1993-08-05
NO942750D0 (no) 1994-07-22
JP3490437B2 (ja) 2004-01-26
EP0654085A1 (en) 1995-05-24
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US5910573A (en) 1999-06-08
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AU676150B2 (en) 1997-03-06
ATE151113T1 (de) 1997-04-15
GR3023860T3 (en) 1997-09-30
RU94045249A (ru) 1996-05-27
JP2004041221A (ja) 2004-02-12
CZ175794A3 (en) 1994-12-15

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