WO2014047973A1 - Anticorps monoclonaux reconnaissant, de façon spécifique, les protéines mutantes b-raf, leur procédé de préparation et leur utilisation - Google Patents

Anticorps monoclonaux reconnaissant, de façon spécifique, les protéines mutantes b-raf, leur procédé de préparation et leur utilisation Download PDF

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Publication number
WO2014047973A1
WO2014047973A1 PCT/CN2012/082763 CN2012082763W WO2014047973A1 WO 2014047973 A1 WO2014047973 A1 WO 2014047973A1 CN 2012082763 W CN2012082763 W CN 2012082763W WO 2014047973 A1 WO2014047973 A1 WO 2014047973A1
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WO
WIPO (PCT)
Prior art keywords
raf
monoclonal antibody
cancer
cctcc
antibody
Prior art date
Application number
PCT/CN2012/082763
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English (en)
Chinese (zh)
Inventor
黄文俊
Original Assignee
武汉纽斯特生物技术有限公司
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Application filed by 武汉纽斯特生物技术有限公司 filed Critical 武汉纽斯特生物技术有限公司
Publication of WO2014047973A1 publication Critical patent/WO2014047973A1/fr

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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • the present invention provides a method of treating a tumor patient, comprising: detecting whether a V600E, N581S or V600K mutant form of a B-Raf protein is present in a sample from a patient; if one of the mutations is present, Treatment with a B-Raf inhibitor (eg GDC-0879).
  • the invention also provides a method for treating cancer patients (for example, melanoma, thyroid cancer, Methods for pancreatic cancer, esophageal cancer, colon cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer, and liver cancer patients).
  • the method of treatment comprises administering to the patient a drug comprising a monoclonal antibody of the invention or an antigen binding portion thereof in a composition.
  • the present invention is based on the discovery that a monoclonal antibody specifically binds to a mutant B-Raf protein. We have found that these antibodies bind very well to mutant B-Raf proteins (such as B-Raf proteins with N581S, V600E or V600K mutations). These antibodies are effective in diagnosing or treating diseases such as cancer. These antibodies are particularly effective in detecting trace amounts of B-Raf protein in biological samples.
  • B-Raf binds to a mutant B-Raf protein.
  • B-Raf N581S mutation described in this patent refers to a mutation in which the aspartic acid (N) at position 581 is replaced by serine (S); B-Raf V600E refers to the proline at position 600 (V) A mutation replaced by glutamic acid (E); B-Raf V600K refers to a mutation in which the proline (V) at position 600 is replaced by lysine (K).
  • S aspartic acid
  • B-Raf V600E refers to the proline at position 600
  • E glutamic acid
  • K lysine
  • the present invention encompasses monoclonal antibodies that specifically bind to a mutant B-Raf protein but do not bind to a wild-type B-Raf protein.
  • Antibody or "immunoprotein Ig” refers to a tetramer linked by a heavy chain (H, about 50-70 kDa) and a light chain (L, about 25 kDa) through a disulfide bond.
  • the light chain can be either a light chain or a kappa light chain.
  • antigen binding site of an antibody that comprises: (1) a Fab fragment, containing the VL, a monovalent fragment V H, C, C H 1 domain; (2) - of F (ab ') 2 fragments, a bivalent fragment comprising connected via a disulfide bond of the hinge region Fab fragments; (3) - comprising a V H and C H 1 domain Fd fragments; (4) comprises a single arm of an antibody VL, V H Fv fragment of the domain; (5) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (6) an independent complementarity determining region (CDR).
  • the anti-B-Raf antibody or antigen binding site of the invention comprises the weight of a monoclonal antibody produced by a hybridoma cell having the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, CCTCC C2012112.
  • the antibody or antigen-binding site contains the heavy or light chain variable of the monoclonal antibody produced by the hybridoma cells of the CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, CCTCC C2012112.
  • the amino acid sequence of the region is provided.
  • the antibody or antigen-binding site of the present invention may only bind to one B-Raf mutein and not to other B-Raf muteins (eg, one of B-Raf V600E, B-Raf V600K, B-Raf N581S) .
  • the ability of the antibody to specifically bind to the B-Raf V600K protein may be at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 250-fold, or at least 500-fold stronger than any of the other Mutant ability to mutate B-Raf protein.
  • the present invention encompasses a nucleic acid sequence encoding a monoclonal antibody capable of specifically binding to a human mutant B-Raf protein but not a wild-type B-Raf protein and an antigen binding site thereof.
  • Human mutant B-Raf proteins include: V600E, V600K and N581S.
  • the nucleic acid molecule encodes only the heavy or light chain of the monoclonal antibody or antigen binding site. In other operational examples, the nucleic acid molecule simultaneously encodes the heavy and light chains of the monoclonal antibody and its antigen binding site.
  • the present invention also encompasses an antibody against which the anti-B-Raf monoclonal antibody or antigen-binding site thereof described above is modified by at least one additional molecule or group.
  • the modification can be for purifying or detecting the antibody, and/or enhancing its therapeutic effect.
  • an antibody or antigen binding site can be linked to a detection reagent, tag, cytotoxic agent, drug molecule, and/or protein or polypeptide for attachment to the antibody or antigen binding site thereof to another molecule (eg, avidin) A combination of a core region, or a polyhistidine tag).
  • a medical professional is administered an antibody or moiety of the invention that is capable of achieving an effective dose for a particular disease, typically a chimeric or humanized antibody or moiety, thereby slowing or treating the disease, preventing cancer. Transfer or further development.
  • the mode of administration of the antibody can be, for example, injection or infusion.
  • the dose of the antibody or part can be determined by the medical staff, roughly The range is from 0.1 to 100 mg/kg body weight, more preferably from 0.5 to 50 mg/kg body weight, more preferably from 1 to 20 mg/kg body weight, more preferably from 1 to 10 mg/kg body weight.
  • the effect of the treatment can be illustrated by monitoring, for example, the extent to which the tumor is reduced in size.
  • Figure 5 shows the immunofluorescence assay map of the antibody produced by the cell line with the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285 specifically binding to the B-Raf V600E protein but not the wild-type B-Raf protein.
  • the green fluorescent protein (GFP) signal indicates the expression of the wild-type B-Raf (top) and the mutant B-Raf V600E protein (below), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the B-Raf protein and appears only in cells expressing the B-Raf V600E protein (below).
  • the V600K protein does not bind to the immunofluorescence assay of wild-type B-Raf protein.
  • the green fluorescent protein (GFP) signal indicates the expression of wild-type B-Raf (top) and mutant B-Raf V600K protein (lower), respectively.
  • the red fluorescent signal is indicative of binding of the antibody to the B-Raf protein and appears only in cells expressing the B-Raf V600K protein (below).
  • the N581S protein does not bind to the immunohistochemical experimental map of wild-type B-Raf protein.
  • the brown signal indicates that the antibody binds to the B-Raf N581S mutein in cancer cells.
  • the blue signal indicates the nucleus.
  • Tumor tissue obtained from a patient who has been diagnosed with melanoma is made into a formaldehyde-fixed paraffin section. These cancer cases are confirmed by DNA sequencing methods containing specific B-Raf mutations.
  • the sections were dried at 60 Torr for 1 hour and then soaked twice in xylene for 10 minutes each time. The sections were then placed in 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol for 5 minutes. The sections were rinsed three times with distilled water and soaked in 30% hydrogen peroxide (H 2 0 2 ) for 10 minutes. The sections were rinsed three times with distilled water and boiled in citrate buffer for 90 seconds. After rinsing three times with PBS, the sections were placed in mouse serum for 30 minutes.
  • the sections were then placed in hematoxylin and soaked for 30 seconds at room temperature. Then remove the hematoxylin and place the slices in 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, soak each alcohol 2 minute. The sections were then placed in xylene for 10 minutes. Finally, the sections were closed with a neutral resin. The sections were placed under a visible light microscope and photographed.

Abstract

La présente invention concerne des anticorps monoclonaux reconnaissant, de façon spécifique, les protéines mutantes B-Raf, leur procédé de préparation et leur utilisation. Les trois anticorps monoclonaux préparés par le procédé de la présente invention peuvent reconnaître, de façon spécifique, trois protéines mutantes, à savoir, respectivement, les protéines mutantes B-Raf N581S, B-Raf V600E et B-Raf V600K, et ils peuvent être utilisés pour détecter la présence de mutations N581S, V600E et V600K dans les protéines B-Raf des tissus d'un animal ou d'un être humain, à des fins de diagnostic et de traitement du cancer de la peau et de divers autres cancers en clinique.
PCT/CN2012/082763 2012-09-28 2012-10-11 Anticorps monoclonaux reconnaissant, de façon spécifique, les protéines mutantes b-raf, leur procédé de préparation et leur utilisation WO2014047973A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210367604.0A CN103172744B (zh) 2012-09-28 2012-09-28 特异性识别B-Raf突变蛋白的单克隆抗体、制备方法及其应用
CN201210367604.0 2012-09-28

Publications (1)

Publication Number Publication Date
WO2014047973A1 true WO2014047973A1 (fr) 2014-04-03

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CN (1) CN103172744B (fr)
WO (1) WO2014047973A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018098352A2 (fr) 2016-11-22 2018-05-31 Jun Oishi Ciblage d'expression du point de contrôle immunitaire induit par kras
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111285935B (zh) * 2020-05-13 2020-09-11 方达医药技术(上海)有限公司 特异性检测braf基因突变的抗体及其在制备癌症检测试剂盒中的用途

Citations (3)

* Cited by examiner, † Cited by third party
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WO2005047542A1 (fr) * 2003-10-16 2005-05-26 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Traitements inhibant le developpement et la progression de naevi et de melanomes presentant des mutations de braf
WO2012042009A1 (fr) * 2010-09-30 2012-04-05 Deutsches Krebsforschungszentrum Moyens et procédés de diagnostic du cancer en utilisant un anticorps se liant spécifiquement à braf v600e
WO2012075324A1 (fr) * 2010-12-02 2012-06-07 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Procédés de traitement d'une tumeur utilisant un anticorps se liant spécifiquement au hmw-maa

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WO2007002811A2 (fr) * 2005-06-29 2007-01-04 The Wistar Institute Of Anatomy And Biology Methodes et compositions permettant de traiter le melanome
CN102586401A (zh) * 2011-01-05 2012-07-18 苏州科贝生物技术有限公司 一种检测人结直肠癌braf基因突变的方法和试剂盒
CN102242207B (zh) * 2011-06-29 2013-06-05 浙江大学 癌基因brafv600e突变检测的引物和探针

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005047542A1 (fr) * 2003-10-16 2005-05-26 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Traitements inhibant le developpement et la progression de naevi et de melanomes presentant des mutations de braf
WO2012042009A1 (fr) * 2010-09-30 2012-04-05 Deutsches Krebsforschungszentrum Moyens et procédés de diagnostic du cancer en utilisant un anticorps se liant spécifiquement à braf v600e
WO2012075324A1 (fr) * 2010-12-02 2012-06-07 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Procédés de traitement d'une tumeur utilisant un anticorps se liant spécifiquement au hmw-maa

Non-Patent Citations (1)

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Title
CAPPER, D. ET AL.: "Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody.", ACTA NEUROPATHOLOGICA., vol. 122, no. 1, 3 June 2011 (2011-06-03), pages 11 - 19, XP055017442, DOI: doi:10.1007/s00401-011-0841-z *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018098352A2 (fr) 2016-11-22 2018-05-31 Jun Oishi Ciblage d'expression du point de contrôle immunitaire induit par kras
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death

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Publication number Publication date
CN103172744A (zh) 2013-06-26
CN103172744B (zh) 2016-01-13

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