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
  • C07K16/30—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.

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

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• 2012
  • 2012-09-28 CN CN201210367604.0A patent/CN103172744B/zh not_active Expired - Fee Related
  • 2012-10-11 WO PCT/CN2012/082763 patent/WO2014047973A1/fr not_active Ceased

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