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WO2018032574A1 - Anticorps à chaîne unique et son application - Google Patents

Anticorps à chaîne unique et son application Download PDF

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Publication number
WO2018032574A1
WO2018032574A1 PCT/CN2016/100210 CN2016100210W WO2018032574A1 WO 2018032574 A1 WO2018032574 A1 WO 2018032574A1 CN 2016100210 W CN2016100210 W CN 2016100210W WO 2018032574 A1 WO2018032574 A1 WO 2018032574A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
chain antibody
base sequence
region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2016/100210
Other languages
English (en)
Chinese (zh)
Inventor
林志国
殷一博
黄建萍
葛海涛
慕璐岩
暴洪博
杨昌霖
龙宇
金林春
胡力
王佳斌
夏松松
唐重阳
陶海鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO2018032574A1 publication Critical patent/WO2018032574A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention belongs to the field of genetic engineering technology, and relates to a single chain antibody and application thereof.
  • the object of the present invention is to provide a single-chain antibody and its use, which not only maintains the function and stability of the antibody, but also simplifies the preparation procedure and antibody structure, and improves the performance of the antibody.
  • a single-chain antibody which is composed of an IFNY secretion signal peptide, an antibody variable region, an IgG1 hinge fragment, and an IgG1 CH 2 CH 3 region, wherein: the amino acid sequence ⁇ ij of the IFNY secretion signal peptide is SEQ ID No.
  • the base sequence is shown in SEQ ID No. 2; the amino acid sequence of the IgG1 hinge fragment is shown in SEQ ID No. 3, and the base sequence ⁇ IJ is shown in SEQ ID No. 4.
  • the above single-chain antibodies are mainly used in the fields of medical and biotechnology, and the main uses are as follows: 1. It can be used as a coupled tracer for intraoperative tumor imaging; 2. As a targeted drug for targeted therapy; As a neutralizing antibody to which the target is bound, the function of inhibiting the bound target is achieved; 4. For detecting the expression of the antigen recognized by the variable region.
  • the funding sources for the research and development process of the present invention are: National International Science and Technology Cooperation Special No. 2014 DFA31630, Heilongjiangzhou Applied Technology Research and Development No. GA15C108, Harbin Medical University graduate Innovation Research Project No. YJSCX2015-20HYD.
  • the present invention uses an EGFR antibody linked to the variable region sequence of EGFR as an example.
  • the short peptide is used to directly link the variable regions of the heavy and light chains of the antibody, and the front end is linked to the IFN Y secretion signal peptide.
  • the IgGl hinge fragment was ligated to the IgG1 CH 2 CH 3 region, and this structure first guided the secretion of the antibody by the IFN Y secretion signal peptide, and the connection mode of the antibody was first proposed.
  • the variable region sequences of other antibodies can also be ligated into the antibody structures of the invention.
  • 1 is a linked form of a single-chain antibody.
  • the single-chain antibody provided by the present embodiment secretes a signal peptide from IFN Y.
  • the antibody variable region, the IgG1 hinge fragment and the IgG1 CH 2 CH 3 region are sequentially ligated, wherein: the IFN Y secretion signal peptide portion is used for secretion of the antibody synthesized into the cell structure, and the amino acid sequence thereof is represented by SEQ ID NO. , the base sequence is shown in SEQ ID No. 2; the antigen binding region can make the single-chain antibody play a phase The role of the intact antibody should be; the IgGl hinge fragment is used to ensure the function and stability of the single-chain antibody, the amino acid sequence ⁇ IJ is shown in SEQ ID No. 3, the base sequence ⁇ ij is shown as SEQ ID No.
  • CH 2 region and CH Region 3 is used to stabilize the antibody structure, and the purification of the antibody is facilitated.
  • the amino acid sequence CH ij of CH 2 is shown in SEQ ID No. 5, and the base sequence ⁇ IJ is shown in SEQ ID No. 6; CH 3
  • amino acid sequence of the region is shown in SEQ ID No. 7, and the nucleotide sequence is shown in SEQ ID No. 8.
  • This embodiment provides the use of a single-chain antibody which can be used as a coupled tracer for intraoperative tumor imaging, as follows:
  • the present embodiment provides the use of a single-chain antibody, which can be used for the targeted therapy of a drug as a drug, or as a neutralizing antibody to a target, to achieve inhibition.
  • the function of the combined target is not limited to the following abbreviations:
  • This embodiment provides a single-chain antibody, which can be used for detecting the expression of an antigen recognized by a variable region, and the single-chain antibody can be used for immunohistochemistry, Western blot and the like.
  • a qualitative and quantitative method for detecting the expression of an antigen recognized by a variable region which can be coupled to a fluorescent molecule, biotin or a labeled secondary antibody that targets a human IgG1Fc fragment for identification of a variable Expression of the antigen recognized by the region.
  • This embodiment uses an EGFR antibody linked to the variable region sequence of EGFR as an example, and uses a short peptide to directly link the variable regions of the antibody heavy and light chains, and the front end is linked to the IFN Y secretion signal. Peptide, back-end via IgGl hinge fragment with IgGlCH 2 CH 3
  • the preparation method is as follows:
  • an expression vector which may be a lentiviral vector, a retroviral vector, a sleeping beauty vector or a transient expression vector
  • transfecting or transforming a mammalian cell into the above vector the available cells include myeloma cells, C
  • HO cells monkey COS cells, human embryonic kidney HEK293 cells, etc.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un anticorps à chaîne unique et son application. L'anticorps à chaîne unique comprend : un peptide à signal de sécrétion IFNγ, une région variable d'anticorps, un fragment de charnière IgG1, et une région d'IgG1 CH2CH3 connectée dans cet ordre. Une séquence d'acides aminés du peptide signal sécrétoire IFNγ est présentée dans SEQ ID NO : 1, avec une séquence de base telle que représentée dans SEQ ID NO : 2. Une séquence d'acides aminés du fragment de charnière IgG1 est présentée dans SEQ ID NO : 3, avec une séquence de base telle que représentée dans SEQ ID NO : 4. L'anticorps à chaîne unique est utilisé principalement dans les domaines médical et biotechnologique, et les utilisations principales de celui-ci sont: 1, en tant que traceur de couplage pour l'imagerie tumorale pendant une chirurgie; 2, en tant que médicament de ciblage pour un traitement de ciblage; 3, en tant qu'anticorps neutralisant à une cible liée de celui-ci, inhibant une fonction de la cible liée; et 4, pour détecter l'expression d'un antigène reconnu par la région variable. La présente invention préserve les fonctions et la stabilité d'un anticorps, simplifie un processus de fabrication et une structure d'anticorps, et améliore la performance d'un anticorps.
PCT/CN2016/100210 2016-08-17 2016-09-26 Anticorps à chaîne unique et son application Ceased WO2018032574A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610680168.0 2016-08-17
CN201610680168.0A CN106220733A (zh) 2016-08-17 2016-08-17 一种单链抗体及其应用

Publications (1)

Publication Number Publication Date
WO2018032574A1 true WO2018032574A1 (fr) 2018-02-22

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PCT/CN2016/100210 Ceased WO2018032574A1 (fr) 2016-08-17 2016-09-26 Anticorps à chaîne unique et son application

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CN (1) CN106220733A (fr)
WO (1) WO2018032574A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060204496A1 (en) * 2003-11-28 2006-09-14 Tetsuo Kojima Agonist antibody against heteroreceptor
WO2007122511A2 (fr) * 2006-04-21 2007-11-01 Mab-Factory Gmbh Conjugué anticorps-rnase
CN101240024A (zh) * 2008-02-01 2008-08-13 南京师范大学 hBAFF的单链抗体scFv及其在生产BAFF人源性单克隆抗体中的应用
CN102241774A (zh) * 2010-05-27 2011-11-16 四川大学 重组IgE-Fc-抗EGFR单链抗体融合蛋白及其制备方法和用途
CN103981191A (zh) * 2014-04-10 2014-08-13 吉林农业大学 一种抗cd20单链抗体的植物种子表达系统

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199218A (zh) * 2011-04-29 2011-09-28 中国人民解放军军事医学科学院基础医学研究所 一种抗Her2抗体-白细胞介素2融合蛋白及其用途
AU2015273353B2 (en) * 2014-06-13 2020-09-10 Tenboron Oy Conjugates comprising an anti-EGFR1 antibody

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060204496A1 (en) * 2003-11-28 2006-09-14 Tetsuo Kojima Agonist antibody against heteroreceptor
WO2007122511A2 (fr) * 2006-04-21 2007-11-01 Mab-Factory Gmbh Conjugué anticorps-rnase
CN101240024A (zh) * 2008-02-01 2008-08-13 南京师范大学 hBAFF的单链抗体scFv及其在生产BAFF人源性单克隆抗体中的应用
CN102241774A (zh) * 2010-05-27 2011-11-16 四川大学 重组IgE-Fc-抗EGFR单链抗体融合蛋白及其制备方法和用途
CN103981191A (zh) * 2014-04-10 2014-08-13 吉林农业大学 一种抗cd20单链抗体的植物种子表达系统

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Construction of Anti-VEGFR-2 scFV-Fc Fusion Antibody and Stable Expression in CHO-k Cells", CHINESE JOURNAL OF PHARMACEUTICAL BIOTECHNOLOGY, vol. 18, no. 3, 31 December 2011 (2011-12-31), pages 206 - 210 *
DATABASE GenBank [O] 11 November 2015 (2015-11-11), HERAUD, J.M. ET AL.: "RecName: Full-Interferon gamma; Short-IFN-gamma; Flags: Precursor", XP055465193, Database accession no. Q8MKF5.1 *
DATABASE GenBank [O] 25 July 2016 (2016-07-25), NIRANJANA, K.R.P. ET AL.: "Fc IgGl heavy chain constant region, partial [Homo sapiens", XP055465202, Database accession no. AEV43323.1 *

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