WO2014169494A1 - Anticorps monoclonal reconnaissant spécifiquement des protéines mutantes egfr, et son procédé de préparation et son utilisation - Google Patents

Anticorps monoclonal reconnaissant spécifiquement des protéines mutantes egfr, et son procédé de préparation et son utilisation Download PDF

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WO2014169494A1
WO2014169494A1 PCT/CN2013/074626 CN2013074626W WO2014169494A1 WO 2014169494 A1 WO2014169494 A1 WO 2014169494A1 CN 2013074626 W CN2013074626 W CN 2013074626W WO 2014169494 A1 WO2014169494 A1 WO 2014169494A1
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egfr
monoclonal antibody
cctcc
protein
cancer
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PCT/CN2013/074626
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Chinese (zh)
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吴正东
黄文俊
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武汉纽斯特生物技术有限公司
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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
    • C07K16/2863—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is in the field of biological sciences and pharmaceutical carriers, and in particular relates to monoclonal antibodies that specifically recognize EGFR mutant proteins, methods for their preparation, and their use as diagnostic and therapeutic agents. Background technique
  • the EGFR protein (Epidermal Growth Factor Receptor, also known as ErbB-1 or HER1) is a transmembrane receptor member of the epidermal growth factor receptor family on the cell surface. EGFR binds to extracellular epidermal growth factor (EGF), thereby transmitting extracellular signals into the cell. Mutations in EGFR proteins are present in a variety of human cancers, such as lung cancer, anal cancer, rectal cancer, and breast cancer (Paez JG et al., Science, 304 (5676): 1497-500 (2004)). These mutations result in sustained activation of the EGFR protein, which in turn promotes cell proliferation and cancer cell growth. Common EGFR muteins are G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D and V769L.
  • Inhibitors for EGFR muteins have been marketed, including IressaTM (Genf itinib), TarcevaTM (chemical name Erlotinib), etc. (Mayumi Onol and Michihiko Kuwano, Clin. Cancer Res 12: 7242 (2006) ). These inhibitor drugs are specific for the mutated EGFR protein, so before use, it is necessary to detect whether the patient has an EGFR mutation with an antibody that specifically binds to the mutated EGFR protein but does not bind to the wild-type EGFR protein (Pal lis AG et al., J. Cancer. 105 (1) : 1-8. (2011) ). This antibody can be used to rapidly detect small amounts of samples taken from patients for the diagnosis of cancer. Antibodies that specifically bind to EGFR muteins are also urgently needed to treat cancer. Summary of the invention
  • the object of the present invention is to provide specific recognition of EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D and respectively to solve the above problems.
  • the monoclonal antibodies prepared by the method of the present invention are capable of specifically recognizing G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D and V769L mutant proteins, respectively, without recognizing the wild type EGFR protein.
  • the monoclonal antibody or antigen binding site that specifically recognizes an EGFR mutein is characterized by a heavy chain and a light chain; CDR1, CDR2 and CDR3 of the heavy and light chains
  • the amino acid sequences are hybridomas from the China Type Culture Collection (CCTCC) under the accession numbers CCTCC C20130 K CCTCC C2012127, CCTCC C20121 18, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.
  • the amino acid sequences of the CDR1, CDR2 and CDR3 of the monoclonal antibody produced by the cell are determined.
  • the monoclonal antibody or antigen-binding site that specifically recognizes an EGFR mutein is characterized by comprising a heavy chain and a light chain of a monoclonal antibody produced by the hybridoma cell, respectively.
  • the antibody or antigen binding site comprises the amino acid sequences of the heavy and light chain variable regions of the monoclonal antibodies produced by the hybridoma cells described above.
  • the monoclonal antibody of the present invention may be a humanized or chimeric antibody.
  • the monoclonal antibody is IgG.
  • the invention includes preservation in the China Type Culture Collection (CCTCC) deposit number CCTCC C20130 K CCTCC C2012127, CCTCC C20121 18, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC Hybridoma cells of C201316.
  • CTCC China Type Culture Collection
  • the invention includes a medicament comprising the monoclonal antibody or antigen binding site or a carrier useful for the medicament.
  • the present invention also encompasses a diagnostic kit comprising the monoclonal antibody or antigen binding site thereof as referred to in the present invention.
  • the present invention can provide a method for cancer diagnosis (for example, lung cancer, non-small cell lung cancer, rectal cancer, colon cancer, papillary thyroid cancer, pancreatic cancer, esophageal cancer, Prostate cancer, ovarian cancer, glioma, brain cancer), the main steps thereof include: contacting a biological sample from a detection target with the monoclonal antibody of the present invention; detecting whether the antibody or a part thereof binds to the sample; Binding to the sample indicates that the subject is cancer or has a risk of developing cancer.
  • a method for cancer diagnosis for example, lung cancer, non-small cell lung cancer, rectal cancer, colon cancer, papillary thyroid cancer, pancreatic cancer, esophageal cancer, Prostate cancer, ovarian cancer, glioma, brain cancer
  • the main steps thereof include: contacting a biological sample from a detection target with the monoclonal antibody of the present invention; detecting whether the antibody or a part thereof binds to the sample; Binding to the sample
  • the invention provides a method for treating a tumor patient, comprising: detecting whether an EGFR protein of a G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutant form is present from In the patient's sample; if one of the mutations is present, an EGFR inhibitor (eg, Genf it inib and Erlotinib) is used for treatment.
  • an EGFR inhibitor eg, Genf it inib and Erlotinib
  • the present invention also provides a method of treating cancer patients (e.g., rectal cancer, papillary thyroid carcinoma, pancreatic cancer, esophageal cancer, prostate cancer, ovarian cancer, lung cancer).
  • the method of treatment comprises administering to a patient a drug comprising a monoclonal antibody of the present invention or an antigen-binding portion thereof in a composition.
  • the present invention further provides a method for treating melanoma, rectal cancer, thyroid cancer, pancreatic cancer, esophageal cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer, or hematopoiesis using the monoclonal antibody of the present invention or an antigen binding site thereof as an agent. Tissue cancer.
  • the present invention encompasses a purified nucleic acid molecule which is composed of a nucleic acid sequence capable of encoding a heavy chain or a light chain or an antigen-binding portion of a monoclonal antibody which specifically binds to a mutant EGFR protein of the present invention. Further the invention encompasses a vector comprising the nucleic acid sequence; the vector is optimized to comprise an expression control sequence comprising a nucleic acid molecule. In one embodiment, the invention provides a host cell comprising the nucleic acid molecule. In another application, the invention provides a cell line capable of producing said monoclonal antibody or antigen binding site.
  • the present invention also encompasses a method of producing a monoclonal antibody or antigen binding site that specifically binds to a mutant without binding to a wild type EGFR protein.
  • the steps include: culturing a host cell or a cell strain referred to in the present invention under appropriate conditions; and purifying the antibody.
  • the present invention is based on the discovery that monoclonal antibodies specifically bind to mutant EGFR proteins.
  • monoclonal antibodies specifically bind to mutant EGFR proteins.
  • these antibodies bind very well to mutant EGFR proteins (eg, EGFR proteins with G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D, or V769L mutations).
  • These antibodies are effective in diagnosing or treating diseases such as cancer.
  • the antibody is particularly effective for detecting trace amounts of EGFR protein in biological samples.
  • the EGFR described in this patent refers to a gene or protein of the Epidermal Growth Factor Receptor (EGFR) (Hunter T and Cooper JA, Annu Rev Biochem. 54:897-930 (1985)). This gene or protein is also known as ErbB-1 or HER1.
  • EGFR Epidermal Growth Factor Receptor
  • the wild type EGFR protein sequence includes SEQ ID NO: 7, and the wild type EGFR protein encodes the mRNA sequence including SEQ ID NO: 8.
  • EGFR is a transmembrane receptor member of the epidermal growth factor receptor family on the cell surface and is a receptor tyrosine kinase. EGFR binds to extracellular epidermal growth factor (EGF), dimerizes and activates its own kinase activity, phosphorylating a range of downstream proteins within the cell, thereby transmitting extracellular signals into the cell.
  • EGF extracellular epidermal growth factor
  • Mutations of the EGFR protein include insertions, deletions, substitutions, and the like of one or more amino acids.
  • the invention encompasses monoclonal antibodies that specifically bind to a mutant EGFR protein but do not bind to a wild-type EGFR protein.
  • Antibody or "immunoglobulin Ig” refers to a tetramer that is linked by a disulfide bond by a heavy chain (H, about 50-70 kDa) and a light chain (L, about 25 kDa).
  • Light chain It can be a lambda light chain or a kappa light chain.
  • Heavy chains can be divided into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and the combination of different heavy and light chains determines that the type of antibody is IgM, IgD, IgG, IgA or IgE, respectively.
  • Each heavy chain (sometimes referred to as the H chain, or HC) contains a heavy chain variable region (also known as v H ) and a heavy chain constant region (also known as C H ).
  • the heavy chain constant region is comprised of three structures, namely the C H 1, C H 2 and C H 3.
  • Each light chain (sometimes also an L chain, or LC) also has a light chain variable region (also called V) and a light chain constant region (CL).
  • the light chain constant region contains only one structure and CL.
  • the variable and constant regions are linked by a "J" shaped structure of approximately 12 amino acids. There is also a "D" shaped area of about 3 amino acids inside the heavy chain.
  • the heavy chain variable region and the light chain variable region can be further divided into a hypervariable region, also called a complementarity determining region (CDR), and a slightly conserved framework region (FR) interspersed in the middle. These regions are arranged from the N terminal to the C terminal as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDR complementarity determining region
  • FR slightly conserved framework region
  • the amino acid sequence of each structural region is ranked according to Kabat (Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD (1987 and 1991)) and Chothia & Lesk (Chothia & Lesk, J. Mol. Biol. 196:901 -917 (1987); Chothia et al., Nature 342: 878-883 (1989)).
  • Antigen binding site refers to a stretch of antibody fragments on an antibody that specifically recognizes and binds to an antigen, such as a polypeptide fragment containing a particular mutation on an EGFR protein. It is currently believed that the antigen binding function of an antibody can be achieved by a partial fragment of a full length antibody.
  • the "antigen binding site" of an antibody is characterized by comprising: (1) a Fab fragment, a monovalent fragment comprising a VL, VH , CL, Ch1 domain; (2) an F(ab') 2 fragment, 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).
  • a Fab fragment a monovalent fragment comprising a VL, VH , CL, Ch1 domain
  • an F(ab') 2 fragment a bivalent fragment comprising connected via a disulfide bond of the hinge region Fab fragments
  • V H and ⁇ of the F v fragment are encoded by different independent genes, but they can be recombined by synthesizing a link region to form a pair of V H and VL pairs inside the single-stranded protein.
  • Monovalent molecule also known as single-chain F v (scF v ) (Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988) )).
  • scF v single-chain F v
  • Such single chain antibodies are also included within the scope of "antigen binding sites”.
  • a portion of antibodies can be obtained from full length antibodies by conventional techniques, such as papain or pepsin enzymatic digestion. They can also be obtained by standard DNA recombination techniques as described below.
  • the antibody of the present invention can be produced by immunizing a non-human animal (e.g., mouse, rabbit, rat or hamster) with an antigen derived from a human EGFR mutein. These antibodies can also be obtained by phage display technology or other known antibody preparation techniques.
  • the antigen may be a purified polypeptide or polypeptide, or may be a polypeptide or polypeptide expressed in a cell.
  • An antibody that specifically binds to the EGFR G719A mutein of the present invention can be passed through a polypeptide
  • ETEFKKIKVLASGAFGTV (SEQ ID NO: 1) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization of the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C201301, and the name is hybridoma cell line M116.
  • An antibody that specifically binds to the EGFR G719C mutein of the present invention can be passed through a polypeptide
  • ETEFKKIKVLCSGAFGTV (SEQ ID NO: 2) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization of the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C2012127, and the name is hybridoma cell line M32_8.
  • An antibody that specifically binds to the EGFR 719S mutein of the present invention can be passed through a polypeptide
  • ETEFKKIKVLSSGAFGTV (SEQ ID NO: 3) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization of the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C2012118, and the name is hybridoma cell line M20_18.
  • An antibody that specifically binds to an EGFR D761Y mutein in the present invention can be passed through a polypeptide
  • EATSPKANKEILYEAYVM (SEQ ID NO: 4) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization of the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C201303, and the name is hybridoma cell line M121ft l.
  • An antibody that specifically binds to the EGFR L858R mutein of the present invention can be passed through a polypeptide
  • KTPQHVKITDFGRAKLLG (SEQ ID NO: 5) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization of the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C2012125, and the name is hybridoma cell line M85_13.
  • An antibody that specifically binds to the EGFR L861Q mutein of the present invention can be passed through a polypeptide
  • ITDFGLAKQLGAEEKE (SEQ ID NO: 6) was immunized to obtain mice.
  • the hybridoma cells obtained by immunization with the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C201315, and the name is hybridoma cell line M80_14.
  • An antibody that specifically binds to the EGFR A839T mutein of the present invention can be passed through a polypeptide
  • DRRLVHRDLTARNVLVKTP (SEQ ID NO: 7) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization of the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C201302, and the name is hybridoma cell line M1 18ft7.
  • An antibody that specifically binds to the EGFR N826S mutein of the present invention can be passed through a polypeptide
  • IAKGMSYLEDRRLVHR (SEQ ID NO: 8) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization with the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C201320, and the name is hybridoma cell line M1 17_10.
  • An antibody that specifically binds to the EGFR G863D mutein of the present invention can be passed through a polypeptide
  • ITDFGLAKLLDAEEKE (SEQ ID NO: 9) was immunized to obtain mice.
  • the hybridoma cells obtained by immunization with the polypeptide in the present invention have been deposited in the China Center for Type Culture Collection in accordance with the Budapest Agreement, and the accession number is CCTCC C201304, and the name is hybridoma cell line M120.
  • An antibody that specifically binds to the EGFR V769L mutein of the present invention can be passed through a polypeptide
  • EAYVMASLDNPHV (SEQ ID NO: 10) was obtained by immunizing mice.
  • the hybridoma cells obtained by immunization of the polypeptide in the present invention have been preserved in a typical Chinese culture in accordance with the Budapest Agreement. (2015) Center, the deposit number is CCTCC C201316, the name is hybridoma cell line M122ftl.
  • the invention further comprises a monoclonal antibody or antigen binding site thereof, which includes the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.
  • the monoclonal antibodies of the present invention may only comprise hybrids with the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the heavy chain amino acid sequence or light chain amino acid sequence of the monoclonal antibody produced by the tumor cell may only comprise hybrids with the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the anti-EGFR antibody or antigen binding site of the invention comprises one or more of the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC The amino acid sequence of the heavy or light chain or CDR of the monoclonal antibody produced by the hybridoma cells of C201304 and CCTCC C201316.
  • the antibody or antigen binding site includes hybridoma cells from the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the amino acid sequences of all CDR1, CDR2 and CDR3 of the heavy and light chains of the produced monoclonal antibodies are included in the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the anti-EGFR antibodies or antigen binding sites of the invention are comprised by the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the amino acid sequence of the heavy or light chain variable region of the monoclonal antibody produced by the hybridoma cell are comprised by the accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the antibody or antigen binding site also contains hybridomas with accession numbers C CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.
  • Cell produced The amino acid sequence of the heavy or light chain variable region of a monoclonal antibody.
  • the antibody or antigen binding site of the present invention is capable of specifically binding to the mutant EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L proteins, respectively, but not Binding to the wild-type EGFR protein (not bound at all or not bound to some extent).
  • the antibody or antigen binding site is capable of distinguishing between mutant and wild type human EGFR proteins. The specificity of this binding can be confirmed by known common detection methods, such as immunoblotting experiments.
  • the antibody is capable of specifically preferentially binding to a mutant EGFR protein, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 250 fold, or at least for a mutant EGFR protein. It is 500 times stronger than its ability to bind to wild-type EGFR protein.
  • the antibody or antigen binding site of the invention specifically binds to the mutant EGFR protein at a dissociation equilibrium constant K D of at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 250 fold, At least 500 times, or at least 1000 times stronger than the dissociation equilibrium constant (K D ) for binding to the wild-type EGFR protein.
  • the dissociation equilibrium constant (K D ) can be measured by surface ion resonance or flow cytometry.
  • the antibody or antigen-binding site of the present invention may only bind to one EGFR mutein and not to other EGFR muteins (eg, EGFR G719A, EGFR G719C, EGFR 719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S) , EGFR G863D and one of EGFR V769L).
  • the ability of the antibody to specifically bind to the EGFR G719A 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 other mutant EGFR. Protein binding ability.
  • the antibody may only bind to the EGFR G719A protein without binding.
  • Other mutant EGFR proteins eg, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L.
  • the ability of the antibody to specifically bind to the EGFR G719A 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 other mutant EGFR.
  • Binding ability of proteins such as EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L.
  • the antibody may only bind to the EGFR G719C protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L).
  • the ability of the antibody to specifically bind to the EGFR G719C 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 other mutant EGFR.
  • Binding ability of proteins such as EGFR G719A, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L.
  • the antibody may only bind to the EGFR G719S protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719C, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L).
  • the ability of the antibody to specifically bind to the EGFR G719S 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 other mutant EGFR.
  • Binding ability of proteins such as EGFR G719A, EGFR G719C, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L.
  • the antibody may only bind to the EGFR D761Y protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719C, EGFR G719S, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L).
  • mutant EGFR proteins eg, EGFR G719A, EGFR G719C, EGFR G719S, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L.
  • the ability of the antibody to specifically bind to the EGFR D761Y protein may be 10 times less, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times stronger than any other mutant EGFR protein (eg EGFR G719A, EGFR G719C, EGFR G719S, EGFR L858R, EGFR) Binding ability of L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L).
  • any other mutant EGFR protein eg EGFR G719A, EGFR G719C, EGFR G719S, EGFR L858R, EGFR
  • the antibody may only bind to the EGFR L861Q protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L) .
  • the ability of the antibody to specifically bind to the EGFR L861Q 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 other mutant EGFR.
  • Binding ability of proteins such as EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR A839T, EGFR N826S, EGFR G863D, and EGFR V769L.
  • the antibody may only bind to the EGFR A839T protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR N826S, EGFR G863D, and EGFR V769L).
  • the ability of the antibody to specifically bind to the EGFR A839T 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 other mutant EGFR.
  • EGFR G719A The binding capacity of proteins such as EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR N826S, EGFR G863D and EGFR V769L.
  • proteins such as EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR N826S, EGFR G863D and EGFR V769L.
  • the antibody may only bind to the EGFR N826S protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR G863D and EGFR V769L).
  • the ability of the antibody to specifically bind to the EGFR N826S 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 other mutant EGFR.
  • Proteins eg EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR G863D And the binding ability of EGFR V769L).
  • the antibody may only bind to the EGFR G863D protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, and EGFR V769L).
  • the ability of the antibody to specifically bind to the EGFR G863D 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 other mutant EGFR.
  • Binding ability of proteins such as EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, and EGFR V769L.
  • the antibody may only bind to the EGFR V769L protein but not to other mutant EGFR proteins (eg, EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, and EGFR G863D).
  • the ability of the antibody to specifically bind to the EGFR V769L 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 other mutant EGFR.
  • Binding ability of proteins such as EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, and EGFR G863D.
  • the antibody or antigen binding site of the invention is capable of specifically binding to an EGFR polypeptide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the antibody or antigen binding site of the present invention specifically binds to the CCTCC C20130 K CCTCC C2012127, CCTCC C20121 18, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 or CCTCC C201316.
  • the present invention encompasses a nucleic acid sequence encoding a monoclonal antibody capable of specifically binding to a human mutant EGFR protein but not a wild type EGFR protein and an antigen binding site thereof.
  • Human mutation The EGFR proteins include: G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D, and V769L.
  • the nucleic acid molecule encodes only the heavy or light chain of the monoclonal antibody or antigen binding site.
  • the nucleic acid molecule simultaneously encodes the heavy and light chains of the monoclonal antibody and its antigen binding site.
  • the nucleic acid molecule encodes a monoclonal antibody produced by hybridoma cells with accession numbers CCTCC C20130K CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the accounting can be a purified nucleic acid molecule.
  • a nucleic acid molecule encoding an antibody of the invention can be obtained from any source capable of producing the antibody.
  • nucleic acids can be obtained from hybridoma cells having the accession numbers CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • a method of isolating and purifying a nucleic acid encoding an antibody is a known common technique. Specific methods can be found in the book: [United States] J. Sambrook D. W. Russell, Huang Peitang, etc., "Molecular Cloning Experiment Guide", Science Press, Third Edition, 2002.
  • the nucleic acid sequences of the heavy chain of the monoclonal antibody of the present invention may be composed of nucleic acid sequences encoding V H domains of an antibody of the present invention and encoded by an encoding of any other source of the antibody heavy chain constant region of The same frame coding sequence.
  • a nucleic acid molecule encoding a light chain of an anti-EGFR protein of the invention may also be in the same frame as the composition of the nucleic acid sequence encoding the V domain of an antibody of the invention and a light chain constant region encoding an antibody of any other source. Coding sequence.
  • a nucleic acid molecule encoding a heavy chain variable region ( VH ) and/or a light chain variable region (VL) is transformed into a full length antibody encoding gene.
  • the nucleic acid sequences encoding the V H or VL domains are respectively inserted into the already containing heavy chain constant region (C H) or light chain of the expression vector constant region (CL) in to construct a full Long antibody gene. Fragments of a full-length antibody gene fragment and the C H CL is connected so created in the carrier, and / or Fragment encoding the VL fragment encoding the V H are connected in the carrier.
  • the encoding v H and / or V domain nucleic acid sequence fragment, respectively by the above recombinant nucleic acid technology is coupled to a fragment encoding c H and / or code C.
  • Genes encoding the constant domains of human immunoglobulin heavy and light chains are known. See the book Kabat et al. for details.
  • a nucleic acid molecule encoding a full-length heavy chain and/or a light chain can be expressed in a cell line into which the EGFR antibody is introduced and purified.
  • nucleic acid molecules can also be used to produce chimeric antibodies, bispecific binding antibodies, single chain antibodies, immunoadhesives, double-headed antibodies, mutant antibodies, and antibody derivatives. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule may also be used to effect humanization of the antibody.
  • the present invention also encompasses the insertion of a nucleic acid molecule encoding an antibody heavy chain, a light chain, or both an EGFR antibody of the present invention or an antigen binding site thereof.
  • the vector may be suitable for expressing an antibody of the invention or an antigen binding site thereof in a prokaryotic host cell, such as a yeast cell, an insect cell or a mammalian cell.
  • Common mammalian cells including many immortal cell lines, can be used as expression hosts. These cell lines include, but are not limited to, Chinese hamster ovary cells (CH0), NS0 cells, SP2 cells, HEK293T cells, 293 Free styl e (Inv i trogen), NIH3T3 cells, HeLa cells, baby hamster kidney cells (BHK), African green monkey kidney cells, human liver cancer cells (such as HepG2), and A549 cells. Other cells that can be used for expression are insect cells, including sf9 and sf2 1 cells. Plant host cells include: tobacco, Arabidopsis thaliana, duckweed, corn, wheat, sauerkraut, rice-derived cells. Bacterial host cells include E. coli and actinomycetes. Yeast hosts include fission yeast, Saccharomyces cerevisiae, Pichia pastoris.
  • glycosylation patterns can be obtained using different cell lines under different culture conditions, or using antibodies expressed by transgenic animals. However, regardless of the glycosylation pattern, all of the antibodies encoded by the nucleic acid or the sequence containing the nucleic acid are within the scope of the invention.
  • Chimeric and humanized antibodies The chimeric antibody referred to herein is characterized by having an antibody composed of domains of two or more different antibodies.
  • the variable region domain of a chimeric antibody may be derived from a hybridization of the CCTCC C20130 K CCTCC C2012127, CCTCC C20121 18, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the humanized antibody of the antibody of the present invention is a CDR domain of an antibody derived from a mouse of the present invention inserted into a human receptor antibody.
  • the FR and constant regions of chimeric antibodies are derived from humans.
  • This humanized EGFR antibody containing the human FR region has less immunogenicity in humans than chimeric antibodies derived from humans only in the constant region.
  • the CDR sequences of the humanized antibody may be derived from the accession numbers CCTCC C20130 K CCTCC C2012127, CCTCC C20121 18, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304, and CCTCC C201316.
  • the present invention also encompasses an antibody against which the anti-EGFR 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).
  • Common detection labels include, but are not limited to: radioisotopes (eg 125 I, m I, 35 S or 3 ⁇ 4), fluorescent complexes (eg fluorescein, dichlorotriazine fluorescein, fluorescein isothiocyanate, rhodamine , 5-dimethylamino-1-naphthalenesulfonyl chloride, fluorescein PE, rare earth luminescent, umbelliferone, dansyl chloride), and enzymes (eg horseradish peroxidase, 0-galactosidase, beta _galactosidase, luciferase, alkaline phosphatase, glucose oxygen Chemical enzyme, acetylcholinesterase).
  • radioisotopes eg 125 I, m I, 35 S or 3 ⁇ 4
  • fluorescent complexes eg fluorescein, dichlorotriazine fluorescein, fluorescein isothiocyanate,
  • the antibody or portion thereof of the invention may be labeled with biotin, or may have been shown to be conjugated to another reporter molecule (eg, a leucine zipper pairing region, a secondary antibody binding site, a metal collection domain, Epitope tag) The recognized epitope of the polypeptide.
  • another reporter molecule eg, a leucine zipper pairing region, a secondary antibody binding site, a metal collection domain, Epitope tag
  • any of the antibodies or portions thereof may be modified with polyvinyl alcohol (PEG), methyl or ethyl or a sugar group.
  • One aspect of the present invention is to use the anti-EGFR antibody or antigen-binding site thereof as a cancer diagnostic reagent.
  • Cancer refers to any type of malignancy, including cancer at any stage and level.
  • Specific examples of cancers that may be diagnosed with the monoclonal antibodies of the present invention include, but are not limited to: lung cancer, non-small cell lung cancer, rectal cancer, colon cancer, papillary thyroid cancer, pancreatic cancer, esophageal cancer, prostate cancer, ovarian cancer, colloid Tumor, brain cancer.
  • Diagnosis includes detecting the extent of cancer development, confirming the presence of cancer, or typing or classifying cancer.
  • the invention may provide a method of diagnosing cancer in a target, the method comprising: contacting a biological sample from a detection target with an anti-EGFR monoclonal antibody of the invention; detecting the antibody or a portion thereof Whether the sample binds; if the antibody is combined with the sample, it indicates that the subject is cancer, or there is a risk of developing cancer.
  • the sample to be tested may be blood, serum, lymph, tissue (e.g., puncture or formaldehyde and paraffin fixed coated sections).
  • the binding of the antibody to the sample can be detected using common known techniques, including but not limited to: ELISA, RIA, flow cytometry, immunocytochemistry, immunohistochemistry, immunofluorescence, immunoblotting, or co-immunoprecipitation.
  • Anti-EGFR antibodies or portions thereof can also be directly labeled with a detectable label. If the antibody is not labeled, a secondary antibody or other molecule capable of binding to the EGFR antibody and capable of being detected can be used.
  • a specific secondary antibody is capable of specifically binding to a primary antibody of a particular species and subtype. For example, if the anti-EGFR antibody is a mouse IgG, the secondary antibody must be a labeled anti-mouse IgG antibody.
  • proteins capable of binding to an antibody include, but are not limited to, Protein A and Protein G. They all come in a variety of commercial forms. For example, Protein A and Protein G from Pierce.
  • Molecules suitable for labeling antibodies or secondary antibodies include, but are not limited to: enzymes, prosthetic groups, fluorescent materials, luminescence Materials, magnetic materials and radioactive materials (see above).
  • the antibodies of the invention, or portions thereof can be provided in a kit comprising a detection antibody binding reagent.
  • Anti-EGFR antibodies can also be used to examine the progression of a cancer patient and then decide which treatment regimen to take. For example, in the accompanying test of a treatment regimen. Medical staff may determine the optimal treatment for a particular patient based on the results of EGFR mutations.
  • the treatment regimen can be surgery, radiation therapy, medication (including chemotherapy and targeted therapy), or a combination of multiple treatments.
  • the invention includes a method of treating a cancer patient, comprising: examining whether a biological sample from a patient carries an EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutation If the mutation is present, the patient is treated with an EGFR inhibitor.
  • the EGFR inhibitor may be an anti-sense RNA, a small interfering RNA (s iRNA), a microRNA (miRNA) drug, or an anti-EGFR monoclonal antibody drug.
  • s iRNA small interfering RNA
  • miRNA microRNA
  • the detection of the presence of EGFR mutations prior to targeted therapy to patients with anti-EGFR drugs is of great interest for treatment.
  • the present invention also provides the use of an antibody and antigen-binding site thereof as a therapeutic drug, including the use of the antibody of the present invention or a part thereof as a therapeutic human, and the use of the antibody or a part thereof as an application for the manufacture of a medicament for treating human cancer.
  • human cancers include rectal cancer, papillary thyroid cancer, pancreatic cancer, esophageal cancer, prostate cancer, ovarian cancer, and lung cancer. Detailed cancer types can also be found in the description above.
  • 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, and 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, it is 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.
  • the components of the therapeutic agents of the invention may include an acceptable pharmaceutical carrier in addition to the monoclonal antibodies or portions thereof.
  • An acceptable pharmaceutical carrier is characterized by being a solvent, a dispersing agent, a coating agent, an antibacterial agent and an antifungal agent, an osmotic balance agent and an adsorption retardant, which are characterized by physiological compatibility.
  • preferred carriers include osmotic balance agents such as saccharides, polyols such as mannitol, sorbitol, sodium chloride components.
  • Other acceptable pharmaceutical materials include humectants or minor amounts of adjuvants such as wetting agents, emulsifiers, protectants or buffers which increase the shelf life or effectiveness of the antibody.
  • the anti-EGFR antibodies of the invention, or portions thereof, are mixed with one or more of a therapeutic, diagnostic, or prophylactic agent.
  • Therapeutic agents include, but are not limited to, anti-EGFR antibodies that bind different targets with different fine specific differences, and EGFR inhibitors, for example, the inhibitor may be cetuximab (cetuxmab), panitumumab
  • Therapeutically effective dose refers to the amount of an effective drug to achieve a therapeutic effect at a certain course of treatment and dosage.
  • the therapeutically effective dose of the antibody and its antigen binding portion may vary depending on various factors such as disease progression, age, sex, body weight, and the ability of the antibody or antibody portion to be responsive within the individual.
  • the therapeutically effective dose also encompasses the beneficial effects of the antibody or antigen binding site over any toxic or detrimental effects.
  • Preventive effective amount refers to the amount of effective drug to achieve a preventive effect at a certain course of treatment and dosage. Usually, since the prophylactic agent is used before or during the disease, the prophylactically effective amount may be less than the therapeutically effective amount.
  • Figure 1 shows an immunoblotting assay map of antibodies produced by a cell line with the accession number CCTCC C201301 that specifically binds to the EGFR G719A protein but does not bind to the wild-type EGFR protein.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains E. coli expression
  • Figure 2 shows an immunoblotting assay map of antibodies produced by a cell line with the accession number CCTCC C2012127 that specifically binds to the EGFR G719C protein but does not bind to the wild-type EGFR protein.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR G719C protein expressed by E. coli;
  • Figure 3 shows that the antibody produced by the cell line with the accession number CCTCC C20121 18 specifically binds to the EGFR G719S protein, but does not bind to the wild-type EGFR protein immunoblotting assay.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR G719S protein expressed by E. coli;
  • Figure 4 shows that the antibody produced by the cell line with the accession number CCTCC C201303 specifically binds to the EGFR D761Y protein, but does not bind to the wild-type EGFR protein immunoblotting assay.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR D761Y protein expressed by E. coli;
  • Figure 5 shows that the antibody produced by the cell line with the accession number CCTCC C2012125 specifically binds to the EGFR L858R protein, but does not bind to the wild-type EGFR protein immunoblotting assay.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR L858R protein expressed by E. coli;
  • Figure 6 shows that the antibody produced by the cell line with the accession number CCTCC C201315 specifically binds to the EGFR L861Q protein, but does not bind to the wild-type EGFR protein immunoblotting assay.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR L861Q protein expressed by E. coli;
  • Figure 7 shows that the antibody produced by the cell line with the accession number CCTCC C201302 specifically binds to the EGFR A839T protein, but does not bind to the wild-type EGFR protein immunoblotting assay.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR A839T protein expressed by E. coli;
  • Figure 8 is a graph showing the immunoblotting of an antibody produced by a cell line with the accession number CCTCC C201320 specifically binding to the EGFR N826S protein but not binding to the wild type EGFR protein.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains E. coli expression Mutant EGFR N826S protein;
  • Figure 9 shows that the antibody produced by the cell line with the accession number CCTCC C201304 specifically binds to the EGFR G863D protein, but does not bind to the wild-type EGFR protein immunoblotting assay.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR G863D protein expressed by E. coli;
  • Figure 10 shows that the antibody produced by the cell line with the accession number CCTCC C201316 specifically binds to the EGFR V769L protein, but does not bind to the wild-type EGFR protein immunoblotting assay.
  • Lane 1 contains the wild type EGFR protein expressed by E. coli.
  • Lane 2 contains the mutant EGFR V769L protein expressed by E. coli;
  • Figure 1 1 shows the immunofluorescence assay of the antibody produced by the cell line with CCTCC C201301 specifically binding to the EGFR G719A protein but not the wild-type EGFR protein.
  • the green fluorescent protein (GFP) signal indicates the expression of wild-type EGFR (top) and mutant EGFR G719A proteins (below), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR G719A protein (below);
  • Figure 12 shows an immunofluorescence assay map of an antibody produced by a cell line with the accession number CCTCC C2012127 that specifically binds to the EGFR G719C protein but does not bind to the wild-type EGFR protein.
  • the green fluorescent protein (GFP) signal indicates the expression of wild-type EGFR (top) and mutant EGFR G719C protein (lower), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR G719C protein (below);
  • Figure 13 shows an immunofluorescence assay map of antibodies produced by a cell line with the accession number CCTCC C20121 18 that specifically binds to the EGFR G719S protein but does not bind to the wild-type EGFR protein.
  • the green fluorescent protein (GFP) signal indicates the expression of wild-type EGFR (top) and mutant EGFR G719S proteins (below), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR G719S protein (below);
  • Figure 14 shows an immunofluorescence assay map of an antibody produced by a cell line with the accession number CCTCC C201303 specifically binding to the EGFR D761Y protein but not binding to the wild type EGFR protein.
  • Green fluorescent protein (GFP) signals indicate wild-type EGFR (top) and mutant EGFR D761Y, respectively Expression of protein (below).
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR D761Y protein (below);
  • Figure 15 shows the immunofluorescence assay of antibodies produced by a cell line with the accession number CCTCC C2012125 that specifically binds to the EGFR L858R protein but does not bind to the wild-type EGFR protein.
  • Green fluorescent protein (GFP) signals indicate expression of wild-type EGFR (top) and mutant EGFR L858R proteins (below), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR L858R protein (below);
  • Figure 16 shows the immunofluorescence assay of antibodies produced by a cell line with the accession number CCTCC C201315 that specifically binds to the EGFR L861Q protein but does not bind to the wild-type EGFR protein.
  • Green fluorescent protein (GFP) signals indicate expression of wild-type EGFR (top) and mutant EGFR L861Q proteins (below), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR L861Q protein (below);
  • Figure 17 shows the immunofluorescence assay of antibodies produced by a cell line with the accession number CCTCC C201302 that specifically binds to the EGFR A839T protein but does not bind to the wild-type EGFR protein.
  • Green fluorescent protein (GFP) signals indicate expression of wild-type EGFR (top) and mutant EGFR A839T proteins (below), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR A839T protein (below);
  • Figure 18 shows an immunofluorescence assay map of antibodies produced by a cell line with the accession number CCTCC C201320 that specifically binds to the EGFR N826S protein but does not bind to the wild-type EGFR protein.
  • Green fluorescent protein (GFP) signals indicate expression of wild-type EGFR (top) and mutant EGFR N826S proteins (below), respectively.
  • the red fluorescent signal is indicative of binding of the antibody to the EGFR protein and occurs only in cells expressing the EGFR N826S protein (below);
  • Figure 19 shows an immunofluorescence assay map of an antibody produced by a cell line with the accession number CCTCC C201304 that specifically binds to the EGFR G863D protein but does not bind to the wild-type EGFR protein.
  • Green fluorescent protein (GFP) signals indicate expression of wild-type EGFR (top) and mutant EGFR G863D proteins (below), respectively.
  • the red fluorescent signal is capable of indicating the binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR G863D protein (below);
  • Figure 20 shows an immunofluorescence assay map of an antibody produced by a cell line with the accession number CCTCC C201316 that specifically binds to the EGFR V769L protein but does not bind to the wild-type EGFR protein.
  • Green fluorescent protein (GFP) signals indicate expression of wild-type EGFR (top) and mutant EGFR V769L proteins (below), respectively.
  • the red fluorescent signal is capable of indicating binding of the antibody to the EGFR protein and appears only in cells expressing the EGFR V769L protein (below).
  • a polypeptide containing an amino acid sequence of an EGFR mutein is obtained by synthesis. Detailed polypeptide sequences are listed in the table below, and the mutated amino acids relative to the wild-type protein are underlined:
  • Each synthetic peptide was coupled to a succinylated keyhole limpet hemocyanin (KLH) to immunize mice and to enhance the immune effect by mixing Freund's adjuvant.
  • KLH succinylated keyhole limpet hemocyanin
  • Anti-EGFR G719A, G719C, G719S, D761Y, L858R immunized with the above polypeptides Spleen cells from L861Q mice were fused to mouse myeloma SP2/0 cells via polyvinyl alcohol (PEG), respectively. Positive clones were screened by ELISA. Positive cloned cells were injected into the peritoneal cavity of mice of the same strain to produce ascites. The antibody was purified from ascites.
  • Hybridoma cells produced by immunizing mice with a polypeptide containing a mutated EGFR protein are deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, Hubei province, China, 430072).
  • CTCC China Center for Type Culture Collection
  • the cDNA sequence encoding the wild type of EGFR and the EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutant protein was cloned into 6 sets of ammonia.
  • the constructed plasmid was transformed into E. coli BL21 (DE3) strain and transformed The bacteria were spread on a LB medium plate containing kanamycin and cultured at 37 °C.
  • Monoclonal colonies were picked from the plates and placed in an Erlenmeyer flask containing 500 ml of LB medium containing kanamycin. 0 ⁇ The flask was placed in a shaker at 37 ° C to 0D600 close to 1.0. Then, 0.2 mM IPTG was added to the Erlenmeyer flask to induce incubation for 3 hours at 37 °C. The cultured bacteria were collected by centrifugation, and then lysate (20 mM Tris, 100 mM NaCl, 1% TritonTM X-100, and protease inhibitor) was added. The lysed bacterial solution was centrifuged at a speed of 12,000 g for 30 minutes, and the supernatant was collected.
  • the supernatant was added to a nickel column and purified to yield pure EGFR protein following the nickel column manufacturer's instructions. Then loaded onto 2 X SDS 0. 1 ⁇ ⁇ EGFR proteins were each purified with an equal volume of buffer were mixed and heated at 95 ° C 10 min.
  • the cDNA sequence encoding the EGR wild type and the EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutant protein was cloned into the mammalian expression vector pCNDA3.0. And fused with a sequence encoding green fluorescent protein (GFP) to form an open reading frame. The constructed plasmid was transformed into DH5a E. coli to amplify and extract the plasmid.
  • GFP green fluorescent protein
  • HEK293T cells were plated at a density of 30% into a 24-well cell culture plate, and placed in a 5% (0 2 incubator) at 37 ° C. Then it contained 0.5 ⁇ g ⁇ expression of each
  • the EGFR protein plasmid was transfected into HEK293T cells by standard calcium phosphate transfection. The transfected cells were further cultured for 24 hours. The transfected cells were then collected, and 20 ⁇ M of cell lysate was added to each well of the cells. Place on for 10 minutes. Then centrifuge the cells for 10 minutes at 12,000 rpm and collect the supernatant. Take 10 ⁇ M of each cell lysate supernatant, mix with an equal volume of 2 X SDS loading buffer, and Heat at 95 °C for 10 minutes.
  • the PVDF membrane was rinsed three times with TBS buffer containing 0.1% Tween®-20, and rinsed for 5 minutes each time. The membrane was then placed in a solution containing the ECL substrate and blotted with film in a dark room. Wash, develop, fix.
  • Figure 1 Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10 show the anti-EGFR G719A, G719C, G719S produced in Example 1 in an immunoblot experiment.
  • D761Y, L858R, L861Q, A839T, N826S, G863D or V769L monoclonal antibodies can specifically bind to the EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutant proteins, respectively, but do not bind Wild-type EGFR protein, and whether the protein is expressed in E. coli or expressed in mammalian cells.
  • Example 3 Example 3
  • the pCDNA3.0 plasmid capable of expressing the wild type with GFP tag and EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutant EGFR protein was constructed according to the method of Example 2. Prior to transfection, sterile-treated slides were placed in 24-well cell culture plates, and HEK293T cells were plated at 30% density in 5% C0 2 incubator at 37 °C. to cultivate. Calcium phosphate transfection mixture containing 0. 5 ⁇ ⁇ EGFR expression plasmids using standard protein each transfected into HEK293T cells.
  • the transfected cells were further cultured for 36 hours. The cells were then fixed with 3.7% paraformaldehyde for 10 minutes. Cells were treated with 1% TritonTM X-100 in PBS buffer for 10 minutes to increase their permeability. The treatment was then blocked with PBS containing 5% of bovine serum for 10 minutes. These treated cells were then each placed in a buffer containing the anti-EGFR monoclonal antibody produced in Example 1 and incubated at 4 ° C for 16 hours. The antibody was then removed and the cells were rinsed three times with PBS buffer for 5 minutes each time. The anti-mouse secondary antibody labeled with a fluorescent illuminant was then incubated for 30 minutes.
  • DAPI dye was added to the secondary antibody solution to stain the nuclei.
  • the secondary antibody was removed and rinsed again three times with PBS buffer for 5 minutes each time. Then remove the glass slide with the cells attached and fix it to the load. Slide on the slide and seal with nail polish. The cells were observed under a fluorescence microscope and photographed.
  • Figure 1 1, Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17, Figure 18, Figure 20 and Figure 20 show the anti-EGFR G719A, G719C produced in Example 1 in an immunofluorescence experiment.
  • the G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L monoclonal antibodies are capable of specifically binding to the anti-EGFR G719A expressed in mammalian cells,

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Abstract

La présente invention porte sur un anticorps monoclonal reconnaissant spécifiquement les protéines mutantes EGFR, et sur un procédé de préparation et sur l'utilisation de cet anticorps, et la présente invention appartient au domaine de la science biologique et des supports pharmaceutiques. Trois anticorps monoclonaux, préparés respectivement par utilisation du procédé de la présente invention, peuvent spécifiquement reconnaître dix protéines mutantes EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D et EGFR V769L. Ces dix anticorps monoclonaux peuvent être utilisés pour détecter si des protéines EGFR dans des tissus animaux ou humains contiennent ou non des mutations G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D ou V769L, ce qui peut fournir une base pour le diagnostic et le traitement de toute une gamme de cancers cliniques.
PCT/CN2013/074626 2013-04-19 2013-04-24 Anticorps monoclonal reconnaissant spécifiquement des protéines mutantes egfr, et son procédé de préparation et son utilisation WO2014169494A1 (fr)

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CN201310137856.9 2013-04-19
CN201310137856.9A CN103626871A (zh) 2013-04-19 2013-04-19 特异性识别egfr突变蛋白的单克隆抗体、制备方法及其应用

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CN109415441B (zh) * 2016-05-24 2023-04-07 英斯梅德股份有限公司 抗体及其制备方法
CN107236042A (zh) * 2017-07-05 2017-10-10 无锡傲锐东源生物科技有限公司 抗egfr‑l858r蛋白单克隆抗体及其用途

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