WO2021190441A1 - Anticorps dirigé contre cd47/cd47 humanisé ou fragment de liaison à l'antigène ou fragment immunologiquement actif de celui-ci et utilisation associée - Google Patents

Anticorps dirigé contre cd47/cd47 humanisé ou fragment de liaison à l'antigène ou fragment immunologiquement actif de celui-ci et utilisation associée Download PDF

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Publication number
WO2021190441A1
WO2021190441A1 PCT/CN2021/082047 CN2021082047W WO2021190441A1 WO 2021190441 A1 WO2021190441 A1 WO 2021190441A1 CN 2021082047 W CN2021082047 W CN 2021082047W WO 2021190441 A1 WO2021190441 A1 WO 2021190441A1
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WIPO (PCT)
Prior art keywords
seq
antibody
humanized
cells
sequence
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PCT/CN2021/082047
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English (en)
Chinese (zh)
Inventor
张崇骞
张楠
李虹侠
宗超
王雷
黄苏萍
汤春
张晓霞
彭继荣
张晓东
Original Assignee
倍而达药业（苏州）有限公司
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Priority claimed from CN202010209465.3A external-priority patent/CN111454359B/zh
Priority claimed from CN202110061157.5A external-priority patent/CN112679611B/zh
Application filed by 倍而达药业（苏州）有限公司 filed Critical 倍而达药业（苏州）有限公司
Publication of WO2021190441A1 publication Critical patent/WO2021190441A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine, specifically to the field of antibody technology and immunity, and more specifically to the CD47/humanized CD47 antibody or its antigen binding fragments, immunologically active fragments and applications.
  • CD47 is a "self” mark, which represents a "don't eat me” signal.
  • the human body needs 20-30 trillion red blood cells in order to maintain the effective transport of oxygen throughout the body.
  • the life cycle of red blood cells is short, only 120 days, 10 billion red blood cells are produced every hour, and countless senescent red blood cells are swallowed and cleared by macrophages.
  • macrophages distinguish young and senescent red blood cells, and the mechanism of only attacking senescent red blood cells is unknown.
  • Oldenborg et al. confirmed that CD47 is an important "self” marker on the cell surface and an important signal that regulates the phagocytosis of macrophages.
  • the present invention provides a novel CD47/humanized CD47 antibody or antigen-binding fragment or immunologically active fragment thereof.
  • the CD47/humanized CD47 antibody can specifically block the interaction between CD47 protein and SIRP ⁇ , and has corresponding biological functions , Can activate macrophages at different levels to mediate the phagocytosis of CD47+ cells.
  • the CD47/humanized CD47 antibody exhibits extremely weak levels of low or no binding to platelets and red blood cells, showing a more specific targeting specificity for CD47+ tumor cells.
  • the CD47/humanized CD47 antibody can specifically target tumor cells in a mixed cell population of tumor cells and human normal red blood cells or human peripheral blood PBMC or other normal cells that may express CD47 molecules.
  • Figure 5A shows the dose effect of CD47 antibody on the blocking of Jurkat and SIRP ⁇ binding
  • Figure 5B shows the dose effect of CD47 antibody on the blocking of CCRF and SIRP ⁇ binding.
  • Figure 15A Binding activity of humanized 6E8 antibody to CD47 protein
  • Figure 15B Binding activity of humanized 100 and 99B5 antibodies to CD47 protein
  • Figure 15C Binding activity of humanized 81C1 antibody to CD47 protein
  • Figure 15D The binding activity of humanized 99B5 antibody to CD47 protein.
  • the transfection reagent used is the ExpiFectamine 293 Transfection Kit (Theromfisher, Lot#: A14524), the cell density during transfection is 25*10 5 cells/ml, and the expression enhancers Enhancer1 and Enhancer2 are added 16-18h after transfection, and 5 after transfection.
  • the cell supernatant was collected on the same day. After purification by Protein A, the supernatant was centrifuged at 10000rpm/min for 30min at 4°C to remove cell debris.
  • the binding activity of the CD47 antibodies 99B5 and 100E9 of the present invention to red blood cells is also significantly weaker than the binding activity of Hu5F9-G4 to red blood cells.
  • the detection method is the same as that of Experimental Example 10, replacing tumor cells with red blood cells as target cells, and detecting whether the CD47 antibody of the present invention has an activating effect on macrophages phagocytosing red blood cells.
  • the NAMD open source program minimizes the energy of the transplanted Grafted 3D model, and mutates a small number of residues back to mouse-derived residues according to the three-dimensional structure, physical and chemical properties and energy minimization rules.
  • the sequence of the above model was optimized according to isoelectric point, PTMs, aggregation, developability, immunogenicity, etc. Finally, the following humanized sequence was obtained as a candidate sequence, in which the code number 100E9 and the code number 100 correspond.
  • Figure 15A shows the better binding activity of humanized 6E8 antibody to CD47 protein
  • Figure 15B shows the better binding activity of humanized 100 and 99B5 antibodies to CD47 protein
  • Figure 15C shows the humanized 81C1 antibody and CD47 protein has better binding activity
  • Figure 15D shows the better binding activity of humanized 99B5 antibody to CD47 protein.
  • the results show that the exemplary humanized CD47 antibodies of the present invention have strong binding ability to CD47 fusion proteins.

Abstract

L'invention concerne un anticorps dirigé contre CD47/CD47 humanisé ou un fragment de liaison à l'antigène ou un fragment immunologiquement actif de celui-ci, qui est capable de bloquer la liaison de CD47 et de SIRPα, et d'activer et de favoriser l'activité phagocytaire des macrophages sur des cellules tumorales. L'invention concerne en outre un acide nucléique correspondant, un vecteur d'expression, une cellule hôte et une composition pharmaceutique qui peut être utilisée dans la préparation d'un médicament pour le traitement de maladies à médiation par CD47.
PCT/CN2021/082047 2020-03-23 2021-03-22 Anticorps dirigé contre cd47/cd47 humanisé ou fragment de liaison à l'antigène ou fragment immunologiquement actif de celui-ci et utilisation associée WO2021190441A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202010209465.3 2020-03-23
CN202010209465.3A CN111454359B (zh) 2020-03-23 2020-03-23 Cd47抗体或其免疫活性片段及应用
CN202110061157.5 2021-01-18
CN202110061157.5A CN112679611B (zh) 2021-01-18 2021-01-18 人源化cd47抗体或其抗原结合片段及应用

Publications (1)

Publication Number Publication Date
WO2021190441A1 true WO2021190441A1 (fr) 2021-09-30

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WO (1) WO2021190441A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116813774A (zh) * 2023-06-02 2023-09-29 生工生物工程（上海）股份有限公司 一种抗人cd47蛋白的抗体、核酸分子及其应用
CN116836241A (zh) * 2023-06-09 2023-10-03 湖南中晟全肽生化有限公司 抑制CD47与SIRPα结合的多肽及其应用
WO2024061350A1 (fr) * 2022-09-23 2024-03-28 广州凌腾生物医药有限公司 Protéine de fusion et son utilisation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105102479A (zh) * 2012-12-12 2015-11-25 瓦斯库劳克斯有限公司 治疗性cd47抗体
CN108779179A (zh) * 2016-11-28 2018-11-09 江苏恒瑞医药股份有限公司 Cd47抗体、其抗原结合片段及其医药用途
CN110066336A (zh) * 2019-05-12 2019-07-30 杭州科兴生物科技有限公司 抗cd47单克隆抗体、片段及其医药用途
CN110872350A (zh) * 2018-08-31 2020-03-10 南京圣和药业股份有限公司 抗cd47抗体及其应用
CN111454359A (zh) * 2020-03-23 2020-07-28 倍而达药业（苏州）有限公司 Cd47抗体或其免疫活性片段及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105102479A (zh) * 2012-12-12 2015-11-25 瓦斯库劳克斯有限公司 治疗性cd47抗体
CN108779179A (zh) * 2016-11-28 2018-11-09 江苏恒瑞医药股份有限公司 Cd47抗体、其抗原结合片段及其医药用途
CN110872350A (zh) * 2018-08-31 2020-03-10 南京圣和药业股份有限公司 抗cd47抗体及其应用
CN110066336A (zh) * 2019-05-12 2019-07-30 杭州科兴生物科技有限公司 抗cd47单克隆抗体、片段及其医药用途
CN111454359A (zh) * 2020-03-23 2020-07-28 倍而达药业（苏州）有限公司 Cd47抗体或其免疫活性片段及应用

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024061350A1 (fr) * 2022-09-23 2024-03-28 广州凌腾生物医药有限公司 Protéine de fusion et son utilisation
CN116813774A (zh) * 2023-06-02 2023-09-29 生工生物工程（上海）股份有限公司 一种抗人cd47蛋白的抗体、核酸分子及其应用
CN116813774B (zh) * 2023-06-02 2024-04-30 生工生物工程（上海）股份有限公司 一种抗人cd47蛋白的抗体、核酸分子及其应用
CN116836241A (zh) * 2023-06-09 2023-10-03 湖南中晟全肽生化有限公司 抑制CD47与SIRPα结合的多肽及其应用
CN116836241B (zh) * 2023-06-09 2024-04-19 湖南中晟全肽生物科技股份有限公司 抑制CD47与SIRPα结合的多肽及其应用

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