WO2014047973A1

WO2014047973A1 - Monoclonal antibodies specifically recognizing b-raf mutant proteins and preparation method and use thereof

Info

Publication number: WO2014047973A1
Application number: PCT/CN2012/082763
Authority: WO
Inventors: 黄文俊
Original assignee: 武汉纽斯特生物技术有限公司
Priority date: 2012-09-28
Filing date: 2012-10-11
Publication date: 2014-04-03
Also published as: CN103172744A; CN103172744B

Abstract

Disclosed in the present invention are monoclonal antibodies specifically recognizing B-Raf mutant proteins and the preparation method and use thereof. The three monoclonal antibodies prepared by the method of the present invention can specifically recognize three mutant proteins, respectively, i.e., B-Raf N581S, B-Raf V600E and B-Raf V600K mutant proteins, and can be used to detect whether there are N581S, V600E and V600K mutations in the B-Raf proteins of the tissues of an animal or a human in order to provide the basis for the diagnosis and treatment of skin cancer and other various cancers clinically.

Description

Monoclonal antibody for specifically recognizing B-Raf mutein, preparation method and application thereof

The present invention is in the field of biological sciences and diagnostic reagents, and in particular relates to monoclonal antibodies that specifically recognize B-Raf mutant proteins, methods for their preparation, and their use as diagnostic and therapeutic agents. Background technique

The B-Raf protein (v-raf murine sarcoma viral oncogene homolog Bl) is a member of the Raf/Mi l serine/threonine protein kinase family. This protein plays an important role in the mid-term regulation of Ras/Raf/MEK/ERK/MAP cascade signaling pathways that regulate cell proliferation. Mutations in the B-Raf protein are present in a variety of human cancers, such as melanoma, papillary thyroid, rectal cancer, and ovarian cancer (Garnett and Marais, Cancer Cell, 6: 313-319 (2004)). These mutations result in sustained activation of the B-Raf protein, which in turn promotes cell proliferation and cancer cell growth. The V600E mutein (the mutation of Valine (V) at position 600 to a mutant protein of Glutamic Acid (E)) is very common in melanoma and papillary thyroid carcinoma (Capper et al., Acta Neuropathol, 122 (1): 11-19 (2011)). Other B-Raf mutations include N581S and V600K.

Inhibitors against the V600E mutein are under development (Chapman et al., NEng JMed, 364 (26): 2507-2516 (2011)). In the use of such inhibitors, an antibody that specifically binds to the mutated B-Raf protein but does not bind to the wild-type B-Raf protein is required. 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 B-Raf muteins are also urgently needed to treat cancer. Introduction to invention

The object of the present invention is to provide a specific recognition of B-Raf separately for solving the above problems.

Three monoclonal antibodies, preparation methods and applications of N581S, B-Raf V600E and B-Raf V600K mutant proteins. Monoclonal antibodies prepared by the method of the present invention are capable of specifically recognizing B-Raf N581S, B-Raf V600E and B-Raf V600K mutant proteins, respectively, without identifying wild-type B-Raf protein.

In some embodiments, the monoclonal antibody or antigen binding site that specifically recognizes a B-Raf mutein is characterized by a heavy chain and a light chain; the CDK CDR2 and CDR3 of the heavy and light chains The amino acid sequences are the amino acid sequences of CDR1, CDR2 and CDR3 of monoclonal antibodies produced by hybridoma cells of China National Type Culture Collection (CCTCC) under the accession numbers CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122 and CCTCC C2012112, respectively. Decide. In a further example of the operation, the monoclonal antibody or antigen-binding site that specifically recognizes a B-Raf mutein is characterized by a heavy chain and a light chain of a monoclonal antibody produced by the hybridoma cell, respectively. A heavy chain and a light chain of the amino acid sequence of the variable region of the chain. In still further examples of the operation, 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.

The monoclonal antibody of the present invention may be a humanized or chimeric antibody. In some examples of practice, this monoclonal antibody is IgG.

In one aspect, the invention includes hybridoma cells deposited in the China Type Culture Collection (CCTCC) under the accession numbers CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, and CCTCC C20121 12.

The present invention encompasses a drug containing the monoclonal antibody or antigen binding site or a drug usable carrier. The present invention also encompasses a diagnostic kit comprising the monoclonal antibody or antigen binding site thereof as referred to in the present invention.

In some application examples, the present invention can provide a method for cancer diagnosis (for example, melanoma, thyroid cancer, pancreatic cancer, esophageal cancer, colon cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer, and liver cancer), which is mainly The method comprises: contacting the biological sample from the detection target with the monoclonal antibody of the present invention; detecting whether the antibody or a part thereof binds to the sample; if the antibody is combined with the sample, the detected object is cancer, or the presence is developed The risk of cancer.

In another application example, 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 further provides a method for treating melanoma, thyroid cancer, pancreatic cancer, esophageal cancer, colon cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer, and liver cancer using the monoclonal antibody of the present invention or an antigen binding site thereof as an agent.

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 B-Raf protein of the present invention. Further the invention encompasses a vector comprising the nucleic acid sequence; the vector is optimized to contain 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 example, 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 B-Raf 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 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

The B-Raf described in this patent refers to the gene or protein of the murine sarcoma viral oncogene homolog Bl (B-RAF) (Sithanandam et al., Oncogene , 5 (12) : 1775-1780 (1990) ). This gene or protein is also known as B-Raf, BRAF, RAFB1, p94, NS7 or B-Raf proto-oncogene serine/threonine protein kinase (p94). The wild type B-Raf protein sequence includes SEQ ID NO: 4, and the wild type B-Raf protein encoding mRNA sequence includes SEQ ID NO: 5.

B-Raf is a member of the serine/threonine protein kinase family of the ral/mi l family. The B-Raf protein is involved in the regulation of the RAS/RAF/MEK/ERK/MAP cascade signaling pathway. A mutant version of the B-Raf protein includes insertions, deletions, substitutions, and the like of one or more amino acids. The 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). Antibody-antigen binding site

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. Heavy chains can be divided into μ, δ, γ, α, ε, and the combination of different heavy and light chains determines whether the type of antibody is IgM, IgD, IgG, IgA or IgE, respectively (Fundamental Immunology Ch. 7, Paul, W. , ed., 2nd ed. Raven Press, NY (1989)).

Each heavy chain (sometimes referred to as H chain or HC) contains a heavy chain variable region (also known as V _H) and a heavy chain constant region (also called 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 VL) and a light chain constant region (CL). The light chain constant region contains only one structure and CL. Within the light and heavy chains, 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-terminus to the C-terminus as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. 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)).

One of the contents of the present invention is an antigen binding site of a monoclonal antibody that specifically binds to a mutant B-Raf protein. "Antigen-binding site" refers to an antibody that specifically recognizes and binds to an antigen (eg, An antibody fragment, such as a fragment of a B-Raf protein that contains a polypeptide fragment of a particular mutation. It is currently believed that the antigen binding function of an antibody can be achieved by a partial fragment of a full length antibody. Wherein "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). Furthermore, although the two domains _VH and VL of the F _V fragment are encoded by different independent genes, they can be recombined by synthesizing a link region to form a single-chain protein. V _H and VL are paired into monovalent molecules, also known as single chains

Fv (scFv) (Bird et al. Science 242: 423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). Such single chain antibodies are also included within the scope of "antigen binding sites". A portion of antibodies, such as Fab and F(ab') ₂ fragments, 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. anti-B-Raf antibody and its antigen binding site

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 B-Raf 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.

Specific disease-associated mutant B-Raf proteins include B-Raf V600E, B-Raf N58 IS, B-Raf V600K. An antibody that specifically binds to the B-Raf V600E protein in the present invention can be obtained by immunizing a mouse with the polypeptide KIGDFGLATEKSRWSGS (SEQ ID NO: 1). 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 numbers are CCTCC C201283, CCTCC C201284 and CCTCC C201285. An antibody that specifically binds to the B-Raf V600K protein in the present invention can be obtained by immunizing a mouse with the polypeptide KIGDFGLATKKSRWSGS (SEQ ID NO: 2). The hybridoma cells obtained by immunization of the polypeptide in the present invention have It is deposited at the China Center for Type Culture Collection in accordance with the Budapest Agreement, under the accession number CCTCC C2012122. Antibodies that specifically bind to the B-Raf N581S protein in the present invention can be obtained by immunizing mice with the polypeptide SIIHRDLKSNSIFLHED (SEQ ID NO: 3). 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 C20121 12.

The present invention further comprises a monoclonal antibody or antigen binding site thereof, which comprises a heavy chain amino acid sequence of a monoclonal antibody produced by a hybridoma cell having the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, CCTCC C2012112. And light chain amino acid sequences. In some embodiments, the monoclonal antibodies of the present invention may comprise only the heavy chain amino acid sequence of a monoclonal antibody produced by hybridoma cells having accession numbers CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, CCTCC C2012112 or Light chain amino acid sequence.

In certain embodiments, the anti-B-Raf antibody or antigen binding site of the invention comprises one or more single cells produced by hybridoma cells having accession numbers CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, CCTCC C2012112 The amino acid sequence of the heavy or light chain or CDR of the cloned antibody. In certain embodiments, the antibody or antigen binding site includes all CDRs of the heavy and light chains of the monoclonal antibodies produced by hybridoma cells deposited under the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, CCTCC C2012112. , CDR2 and CDR3 amino acid sequences.

In certain embodiments, 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 amino acid sequence of the variable region of the strand or light chain. In a specific example of the operation, 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.

Binding specificity of B-Raf The antibody or antigen-binding site of the present invention is capable of specifically binding to the mutant B-Raf V600E, B-Raf V600K, and B-Raf N581S proteins, respectively, but does not bind to the wild-type B-Raf protein (not bound at all or at some To a lesser extent). In other words, the antibody or antigen binding site is capable of distinguishing between mutant and wild type human B-Raf proteins. The specificity of this binding can be confirmed by known commonly used detection methods, such as Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometric sorting, co-immunoprecipitation, immunofluorescence, immunohistochemistry Dyeing, surface ion resonance (eg BIAC0RETM) or radioimmunoassay.

In some embodiments, the antibody is capable of specifically preferentially binding to a mutant B-Raf protein, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 250 for the mutant B-Raf protein. Multiple, or at least 500 times stronger than its ability to bind to wild-type B-Raf protein. In certain embodiments, the antibody or antigen binding site of the invention specifically binds to the mutant B-Raf 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 Times, at least 500 times, or at least 1000 times stronger than the dissociation equilibrium constant (K _D ) for binding to wild-type B-Raf 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 B-Raf mutein and not to other B-Raf muteins (eg, one of B-Raf V600E, B-Raf V600K, B-Raf N581S) . For example, 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.

In some embodiments, the antibody may only bind to the B-Raf V600K protein but not to other mutant B-Raf proteins (e.g., B-Raf V600E, B-Raf N581S protein). For example, 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 other Binding ability of mutant B-Raf proteins (eg, B-Raf V600E, B-Raf N581S protein).

In other examples of manipulation, the antibody may only bind to the B-Raf N581S protein and not to other mutant B-Raf proteins (eg, B-Raf V600E, B-Raf V600K protein). For example, the ability of the antibody to specifically bind to the B-Raf N581S protein may be at least 10 fold, at least 20 Times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times stronger than its ability to bind to any other mutant B-Raf protein (eg, B-Raf V600E, B_Raf V600K protein).

In some embodiments, the antibody or antigen binding site of the invention is capable of specifically binding to a B-Raf polypeptide comprising SEQ ID NO: 1, 2, or 3. In some embodiments, the antibody or antigen binding site of the invention specifically binds to the epitope of a monoclonal antibody produced by a hybridoma cell having the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, or CCTCC C20121 12 Or an antigenic epitope that coincides with it. Recombinant method for producing antibodies

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. In some embodiments, 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. In some embodiments, the nucleic acid molecule encodes a heavy chain, a light chain, a heavy chain variable of a monoclonal antibody produced by a hybridoma cell having the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, or CCTCC C20121 12 Zone or light chain variable zone. 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. For example, nucleic acids can be obtained from hybridoma cells having the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, or CCTCC C20121 12. 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 et al., Molecular Cloning Experimental Guide, Science Press, Third Edition, 2002.

In some instances the operation, the nucleic acid sequences of the heavy chain of the monoclonal antibody of the present invention may be composed of a nucleic acid sequence encoding the 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. Similarly, a nucleic acid molecule encoding a light chain of an anti-B-Raf protein of the invention may also be a nucleic acid sequence encoding a VL domain of an antibody of the invention. A in-frame coding sequence encoding the composition of the light chain constant region of an antibody of any other source. In a further example of the operation, 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. In some operating instances, 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 (C), and going to build a whole Long antibody gene. Fragment C _H C segment of the full-length antibody genes thus constructed is connected in the carrier, and / or a fragment encoding the VL fragment encoding the V _H connected in the carrier. In a further example of operation, encoding V _H and / or nucleic acid sequence of the VL domain fragment, respectively by the above recombinant nucleic acid technology is coupled to a fragment encoding the C _H and / or code C. Genes encoding the constant domains of human immunoglobulin heavy and light chains are known. See, in particular, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publ. No. 91-3242, 1991. 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 B-Raf antibody is introduced and purified.

Recombinant expression of a large number of B-Raf antibodies can be achieved using nucleic acid molecules. 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 a B-Raf 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 Freestyle (Invitrogen), NIH3T3 cells, HeLa cells, baby hamster kidney cells (BHK), African green monkey kidneys. 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 sf21 cells. Plant host cells include: tobacco, Arabidopsis thaliana, duckweed, corn, wheat, tomato, rice-derived cells. Bacterial host cells include E. coli and actinomycetes. Yeast hosts include fission yeast, Saccharomyces cerevisiae, Pichia mother.

Different 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 antibodies encoded by the nucleic acid or sequences 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. For example, the variable region domain of a chimeric antibody may be a monoclonal antibody produced by a hybridoma cell having the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, or CCTCC C20121 12, but the constant region thereof may be From human antibodies. Such chimeric antibodies have less immunogenicity when used as therapeutic agents for humans.

In some embodiments, 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 acceptor antibody. Thus, the FR and constant regions of chimeric antibodies are derived from humans. Such humanized B-Raf antibodies containing human FR regions are less immunogenic in humans than chimeric antibodies derived from humans that are only constant regions. In some embodiments, the CDR sequences of the humanized antibody may be monoclonal antibodies produced by hybridoma cells derived from CCTCC C201283/CCTCC C201284/CCTCC C201285, CCTCC C2012122, or CCTCC C20121 12. The method of humanizing antibodies is a known mature technique. Modification and tag antibody

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. For example, 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, ¹³¹ 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, --galactosidase, luciferase, alkaline phosphatase, glucose oxidase, acetylcholinesterase). In a further example of the operation, 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. In still further examples of the operation, any of the antibodies or portions thereof may be modified with polyvinyl alcohol (PEG), methyl or ethyl or a sugar group. Diagnostic use of anti-B-Raf antibodies or antigen binding sites thereof

One aspect of the present invention is to use the anti-B-Raf 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, melanoma, thyroid cancer (eg, papillary thyroid carcinoma), pancreatic cancer, esophageal cancer, colon cancer, rectal cancer, prostate cancer, ovarian cancer, breast Cancer, lung cancer (eg non-small cell lung cancer), brain cancer, liver cancer, gastric cancer or hematopoietic tissue cancer (eg hairy cell leukemia).

Diagnosis includes detecting the extent of cancer development, confirming the presence of cancer, or typing or classifying cancer. In some embodiments, the invention may provide a method of diagnosing cancer in a subject, the steps comprising: contacting a biological sample from a detection target with an anti-B-Raf monoclonal antibody of the invention; detecting an antibody or Whether the part binds to the sample; 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-B-Raf 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 B-Raf antibody and capable of being detected can be used. According to common knowledge, a specific secondary antibody is capable of specifically binding to a primary antibody of a particular species and subtype. For example, if the anti-B-Raf antibody is a mouse IgG, the secondary antibody must be a labeled anti-mouse IgG antibody. Other molecules 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. E.g 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, luminescent materials, magnetic materials, and radioactive materials (described above). In some embodiments, the antibodies of the invention, or portions thereof, can be provided in a kit comprising a detection antibody binding reagent.

Anti-B-Raf antibodies or parts thereof 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 the B-Raf mutation. The treatment regimen can be surgery, radiation therapy, medication (including chemotherapy and targeted therapy), or a combination of the above. For example, the invention includes a method of treating a cancer patient, comprising: examining whether a biological sample from a patient carries a V600E, V600K or N581S mutation; and if a mutation is present, administering a B-Raf inhibitor to the patient treatment. The B-Raf inhibitor may be an siRNA, an antisense RNA, a microRNA drug, an anti-B-Raf antibody or a small molecule compound. For example, the B-Raf inhibitor can be a sirolimus tablet (ZELB0RAF®), sorafenib (BAY43_9006, NEXAVAR®), panitumumab, GDC-0879, PLX-4720, GSK2118436/SB590885, AZ628, RAF265 (CHIR-265) or XL281. Testing for the presence of a B-Raf mutation prior to administering a targeted therapy to a patient with anti-B-Raf drugs is of great interest for treatment. Therapeutic application of anti-B-Raf antibody and its antigen binding site

The present invention also provides an application of an antibody and an 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. These human cancers include melanoma, thyroid cancer, pancreatic cancer, esophageal cancer, colon cancer, rectal cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer, brain cancer, liver cancer, gastric cancer or hematopoietic tissue cancer. Detailed cancer types can also be found in the description above.

In some embodiments, 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.

The components of the therapeutic drug of the present invention may include an acceptable pharmaceutical carrier in addition to the monoclonal antibody or a portion 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. In many cases, preferred carriers include osmotic balance agents such as saccharides, polyalcohols such as mannitol, sorbitol, and 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.

In some examples of the operation, the anti-B-Raf antibody of the present invention or a part thereof is mixed with other ones and/or a plurality of therapeutic drugs, diagnostic drugs, or prophylactic agents. Therapeutic agents include, but are not limited to, anti-B-Raf antibodies that bind to different targets with different subtle specific differences, and B-Raf inhibitors, such as inhibitors can be vemurafenib tablets (ZELBORAF®), sorafenib ( BAY43-9006, NEXAVAR®), panitumumab, GDC-0879 (Hoefl ich et al., Cancer Res, 69 (7): 3042-3051 (2009)), PLX-4720 (Tsai et al. PNAS, 105 : 3041 - 3046 (2004) ), GSK2118436/SB590885 (Kefford et al., J Clin Oncol 2010 ; 28 : (suppl ; abstr 8503) ) , AZ628 , RAF265 (CHIR-265 ) ( Mordant et al. , Mol Cancer Ther 2010 ; 9 : 358-368 (2010), Su et al., Clin Cancer Res, 18 (8) : 2184-2198 (2012) ) or XL281 (Schwartz et al., J. Clin. Oncol. , 27 (Suppl ) : 3513. 39 (2009) ).

The pharmaceutical compositions of the invention may contain a "therapeutically effective dose" or a "prophylactically effective amount" of an antibody or antigen binding site of the invention. "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" means an effective drug that has a preventive effect at a certain course of treatment and dosage. the amount. Usually, since the prophylactic agent is used before or during the disease, the prophylactically effective amount may be less than the therapeutically effective amount.

Unless otherwise stated, all technical or scientific terms used herein are consistent with common knowledge of those of ordinary skill in the art. Exemplary methods and materials are described in detail below, although methods and materials similar or equivalent to those described herein can be used to test the invention. All references are detailed. Although a large number of documents are cited, these references do not indicate that any document is common knowledge in the field. The use of "including" or "comprising", as used in the description of this patent, should be understood to include the inclusion of the complete object or group of objects, rather than excluding any other object or group of objects. The materials, methods and examples are merely illustrative of the technical solutions of the present invention and are not limiting.

The following examples are intended to describe the methods and materials of the present invention. Appropriate modifications or equivalent substitutions of the described conditions or parameters are generally apparent to those skilled in the art and are therefore still within the spirit and scope of the invention. DRAWINGS

Figure 1 shows an immunoblotting assay map of an antibody produced by a cell line with the accession number CCTCC C2012112 that specifically binds to the B-Raf N581S protein but does not bind to the wild-type B-Raf protein. Lane 1 contains the wild-type B-Raf protein expressed by E. coli. Lane 2 contains the mutant B-Raf N581S protein expressed in E. coli. Lane 3 contains the wild-type B-Raf protein expressed by HEK293T cells. Lane 4 contains the mutant B-Raf N581S protein expressed by HEK293T cells.

Figure 2 shows an immunoblotting assay map of antibodies produced by a cell line with the accession number CCTCC C201283/CCTCC C201284/CCTCC C201285 that specifically binds to the B-Raf V600E protein but does not bind to the wild-type B-Raf protein. Lane 1 contains the wild-type B-Raf protein expressed by E. coli. Lane 2 contains the mutant B-Raf V600E protein expressed by E. coli. Lane 3 contains the wild-type B-Raf protein expressed by HEK293T cells. Lane 4 contains the mutant B-Raf V600E protein expressed by HEK293T cells.

Figure 3 shows an immunoblotting assay map of an antibody produced by a cell line with the accession number CCTCC C2012122 that specifically binds to the B-Raf V600K protein but does not bind to the wild-type B-Raf protein. Lane 1 contains There is a wild-type B-Raf protein expressed by E. coli. Lane 2 contains the mutant B-Raf V600K protein expressed in E. coli. Lane 3 contains the wild-type B-Raf protein expressed by HEK293T cells. Lane 4 contains the mutant B-Raf V600K protein expressed by HEK293T cells.

Figure 4 shows the immunofluorescence assay map of the antibody produced by the cell line with the accession number CCTCC C2012112 that specifically binds to the B-Raf N581S protein but does not bind to the wild-type B-Raf protein. The green fluorescent protein (GFP) signal indicates the expression of wild-type B-Raf (top) and mutant B-Raf N581S protein (lower), 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 B-Raf N581S protein (below).

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).

Figure 6 shows the antibody-specific binding of B-Raf produced by a cell line with the accession number CCTCC C2012122.

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).

Figure 7 Antibody specific binding to B-Raf produced by a cell line with CCTCC C20121 12

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.

Figure 8 shows the cell line of CCTCC C201283/CCTCC C201284/CCTCC C201285. The antibody produced specifically binds to the B-Raf V600E protein but does not bind to the immunohistochemical map of wild-type B-Raf protein. The brown signal indicates that the antibody binds to the B-Raf V600E mutein in cancer cells. The blue signal indicates the nucleus.

Figure 9 Antibody specific binding to B-Raf produced by a cell line with accession number CCTCC C2012122 The V600K 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 V600K mutein in cancer cells. The blue signal indicates the nucleus. detailed description

Example 1

Production of monoclonal antibodies that specifically bind to B-Raf muteins

A polypeptide containing the amino acid sequence of the B-Raf mutein is obtained by synthesis. Detailed polypeptide sequences are listed in the table below, and the mutated amino acids of the wild-type protein are underlined:

Table 1 Mutant B-Raf peptides

Each synthetic peptide was coupled to succinylated keyhole limpet hemocyanin (KLH) to immunize mice and to enhance the immune effect by mixing Freund's adjuvant. The spleen cells of anti-B-Raf N581S, V600E, V600K mice immunized with the above polypeptide were fused with mouse myeloma SP2/0 cells by polyvinyl alcohol (PEG), respectively. The ffi EUSA experiment screened for positive clones. Positive cloned cells were injected into the peritoneal cavity of mice of the same strain to produce ascites. Purified antibodies in plexus ascites.

Hybridoma cells produced by immunizing mice with a polypeptide containing a mutated B Raf protein are deposited with the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, Hubei Province, China, 430072) 'The name and deposit number of the hybridoma cells are in the table below. Listed.

Table 2 Anti-B-Raf hybridoma cell line and preservation number Antibody and its specificity

Anti-B-Raf N581S CCTCC C2012112 Anti-B-Raf V600E CCTCC C201283

Anti-B-Raf V600E CCTCC C201284

Anti-B-Raf V600E CCTCC C201285

Anti-B-Raf V600K CCTCC C2012122 Example 2

Western blot analysis of monoclonal antibodies specifically binding to E. coli and animal cells expressing mutant proteins

When B-Raf protein is expressed in E. coli, it will encode B-Raf wild type and 581S,

The cD sequence of the V600E, V600K mutant protein was cloned into the bacterial expression vector pET28a with six histidine tags. The constructed plasmid was transformed into Escherichia coli BL21 (DE3) strain, and the transformed bacteria were spread on a LB medium plate containing kanamycin and cultured at 37 Torr. Monoclonal colonies were picked from the plates and placed in an Erlenmeyer flask containing 500 ml of LB medium containing kanamycin. The Erlenmeyer flask was placed in a shaker at 37 ° C until the 0D600 was close to L 0 . Then, IPTG of 0.2 rnM was added to the Erlenmeyer flask for 3 hours at 37 Ό. 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 obtain pure B-Raf protein according to the nickel column manufacturer's instructions. The loading buffer was then mixed 0. 1 μ ₈ each purified B-Raf proteins were mixed with an equal volume of 2 X SDS on, and heated at 95Ό 10 minutes.

When expressed in mammalian cells, the cD A sequence encoding the B Ra.f wild type and the N581S V600E, V600K mutant protein was cloned into the mammalian expression vector pCNDA3. 0, and fused to a sequence encoding green fluorescent protein (GFP) to form an open reading frame. The constructed plasmid was transformed into DH5a large intestine T bacteria to amplify and extract the plasmid. One day before the transfection, HEK293T cells were plated at a density of 30% into 24-well cell culture plates and cultured in a 37-inch 5% CO ₂ incubator. Then 0. 5 μ ₈ containing the expression of each Β- - a plasmid using standard calcium f Raf protein transfection transfected into HEK293T cells. The transfected cells were further cultured for 24 hours. The transfected cells were then collected and 20 μM of cell lysate was added to each well of cells and placed on ice for 10 minutes. Then put the cells at 12000 Centrifuge for 10 minutes at the speed of rotation and collect the supernatant. The supernatant of each cell lysate was taken 10 μl, mixed with an equal volume of 2 X SDS loading buffer, and heated at 95 Torr for 10 minutes.

10 μL of each prepared B-Raf protein sample was added to 10% SDS-PAGE protein gel. Electrophoresis was carried out at 90 volts for 90 minutes. The protein separated on the gel is then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk. The PVDF membrane to which the B-Raf protein was transferred was then incubated in TBS buffer containing the antibody purified in Example 1 at 4 C for 16 hours. The antibody was then removed and the PVDF membrane was rinsed three times with TBS buffer containing 0.1% Tween®-20 for 5 minutes each rinse. The membrane was then incubated for 30 minutes in an HRP-labeled anti-mouse secondary antibody. Remove the secondary antibody and rinse the PVDF membrane three times with TBS buffer containing 0.1% Tween®-20 for 5 minutes each time. The film 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 and Figure 3 show that B-Raf N581S, anti-B-Raf V600E and anti-B-Raf V600K monoclonal antibodies produced in Example 1 are capable of specifically binding to B-Raf, respectively, in immunoblotting experiments. N581S, B-Raf V600E and B-Raf V600K muteins, but do not bind to the wild-type B-Raf protein, and whether the protein is expressed in E. coli or expressed in mammalian cells. Example 3

Immunofluorescence assay to analyze the specific binding of these monoclonal antibodies to B-Raf proteins expressed in mammalian cells

The PCDNA 3.0 plasmid capable of expressing the wild type with the GFP tag and the N581S, V600E, V600K mutant B-Raf protein was constructed in accordance with 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% (3⁄4 incubator). calcium phosphate transfection. a mixture containing _0. 5 μ β expression plasmids using standard each B- Raf protein is transfected into HEK293T cells. transfected cells were cultured for 36 hours. the cells were then 3.7 % paraformaldehyde was fixed for 10 minutes. Cells were treated with PBS buffer containing 1% TritonTM X-100 for 10 minutes to increase their permeability. The cells were then blocked with PBS containing 5% of bovine serum for 10 minutes. Then place these treated cells separately The buffer containing the anti-B-Raf monoclonal antibody produced in Example 1 was 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. At the same time, DAPI dye was added to the secondary antibody solution to stain the nucleus. The secondary antibody was removed and rinsed again three times with PBS buffer for 5 minutes each time. The glass-attached slides were then removed, fixed to glass slides and sealed with nail polish. The cells were observed under a fluorescence microscope and photographed.

Figure 4, Figure 5 and Figure 6 show that in the immunofluorescence experiment, the B-Raf N581S, anti-B-Raf V600E and anti-B-Raf V600K monoclonal antibodies produced in Example 1 can specifically bind to mammals, respectively. The B-Raf N581S, B-Raf V600E and B-Raf V600K mutant proteins expressed in the cells, but did not bind to the wild-type B-Raf protein. Example 4

Immunohistochemistry assay for anti-B-Raf anti-antibody specific binding to mutations in tumor tissues B-Raf protein

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 serum was removed, and the sections were separately placed in a buffer containing the anti-B-Raf 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 TBS buffer containing 0.1% Tween®-20 for 5 minutes each rinse. HRP-labeled goat anti-mouse secondary antibody was then added dropwise to the sections and incubated for 30 minutes at room temperature. The antibody was then removed and the cells were rinsed three times with TBS buffer containing 0.1% Tween®-20 for 5 minutes each rinse. DAB chromogenic substrate was added dropwise to the sections. After 60 seconds, the majority of the substrate was washed off with water to terminate the color reaction. 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.

Figure 7, Figure 8 and Figure 9 show that in the immunohistochemical staining experiment, the B-Raf N581S, anti-B_Raf V600E and anti-B_Raf V600K monoclonal antibodies produced in Example 1 can be specifically combined to form formaldehyde fixed. Paraffin sections of human tumor tissue B-Raf N581S, B-Raf V600E and B-Raf V600K muteins, but do not bind wild-type B-Raf protein.

The above embodiments are only used to illustrate the technical solutions of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that the technical solutions of the present invention may be modified or equivalently replaced. The spirit and scope of the present invention should be construed as being included in the scope of the appended claims.

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The following descriptions are mentioned here with the instructions in this application.

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-3-1 Name of the depositary unit CCTCC China Type Culture Collection

—3—2 Deposited Address Wuhan University, Wuhan, Hubei Province, China Post Code: 430072, Hube i

(GN).

-3-3 Deposit Date June 18, 2012 (18. 06. 2012)

—3—4 Deposit No. CCTCC NO : C201283

-4 Supplementary description Classification: Hybridoma cell line M43-1

-5 This note is for the following designated countries

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-3 Preservation

-3-1 Name of the depositary unit CCTCC China Type Culture Collection

—3—2 Deposited Address Wuhan University, Wuhan, Hubei Province, China, 430072, Hube i

(GN).

-3-3 Deposit Date June 18, 2012 (18. 06. 2012)

—3—4 Deposit No. CCTCC NO : C201284

-4 Supplementary description Classification: Hybridoma cell line M43-2

-5 This note is for the following designated countries

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-3-1 Name of the depositary unit CCTCC China Type Culture Collection

(GN).

-3-3 Deposit Date June 18, 2012 (18. 06. 2012)

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-4 Supplementary description Classification: Hybridoma cell line M43-3

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-3-3 Date of Deposit August 24, 2012 (24. 08. 2012)

-3-4 Deposit No. CCTCC NO : C20121 12

-4 Supplementary description Classification: Hybridoma cell line M73-48

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-4 Supplementary explanation Classification: Hybridoma cell line M60-28

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Claims

Claim

A monoclonal antibody that specifically recognizes a B-Raf mutein, which is characterized in that a monoclonal antibody that does not recognize a B-Raf wild-type protein, or an antigen-binding protein, a B-Raf mutein specifically recognized by the monoclonal antibody These include: the 581th place of asparagine (Asparagine, N) is mutated to a serine (Serine, S) mutant protein (B-Raf N581S); the 600th valine (Valine, V) is mutated to glutamic acid ( Glutamic Acid, E) mutant protein (B-Raf V600E); or the 600th valine (Valine, V) is mutated to lysine (K) mutant protein (B-Raf V600K).

The method for producing a monoclonal antibody which specifically recognizes a B-Raf mutein according to claim 1, which specifically binds to a B-Raf protein comprising the polypeptide sequence of SEQ ID NO: 3.

The method for producing a monoclonal antibody which specifically recognizes a B-Raf mutein according to claim 1, which specifically binds to a B-Raf protein comprising the polypeptide sequence of SEQ ID NO: 1.

The method for producing a monoclonal antibody which specifically recognizes a B-Raf mutein according to claim 1, which specifically binds to a B-Raf protein comprising the polypeptide sequence of SEQ ID NO: 2.

The method for producing a monoclonal antibody which specifically recognizes a B-Raf mutein according to claim 1, comprising an antigen binding site consisting of a heavy chain and a light chain; and a heavy chain and a light chain The amino acid sequences of CDR1, CDR2 and CDR3 are CDR1 of monoclonal antibodies produced by hybridoma cells of China National Type Culture Collection (CCTCC) under the accession numbers CCTCC C2012112, CCTCC C201283/CCTCC C201284/CCTCC/C201285 and CCTCC C2012122, respectively. The amino acid sequences of CDR2 and CDR3 are determined.

The method for producing a monoclonal antibody that specifically recognizes a B-Raf mutein according to claim 5, which comprises an antigen binding site, and a monoclonal antibody heavy chain and a light chain produced by the hybridoma cell. The composition consists of variable regions of heavy and light chains, respectively.

The method for producing a monoclonal antibody that specifically recognizes a B-Raf mutein according to claim 5, comprising an antigen-binding site comprising the hybridoma cell The heavy chain and light chain amino acid sequences of the monoclonal antibodies.

The monoclonal antibody specifically recognizing a B-Raf mutein according to claim 1, which comprises an antigen-binding site and is a humanized or chimeric antibody.

The method for producing a monoclonal antibody which specifically recognizes a B-Raf mutein according to any one of claims 2 to 6 and 8, wherein the monoclonal antibody is an IgG.

The monoclonal antibody for specifically recognizing a B-Raf mutein according to claim 1, which is deposited in the China National Type Culture Collection (CCTCC) under the accession number CCTCC C2012112.CCTCC C201283/CCTCC C201284/ Three hybridoma cells of CCTCC C201285 and CCTCC C2012122.

A use of a monoclonal antibody which specifically recognizes a B-Raf mutein, which is characterized by the monoclonal antibody or the drug at the antigen-binding site, and a pharmaceutically acceptable carrier.

12. Use of a monoclonal antibody that specifically recognizes a B-Raf mutein, characterized by a diagnostic kit for diagnosing a B-Raf mutation, the composition comprising the monoclonal antibody of any one of claims 1 to Or antigen binding site.

13. Use of a monoclonal antibody that specifically recognizes a B-Raf mutein, characterized by a method of diagnosing a tumor, comprising the steps of:

(1) contacting a biological sample to be detected with any one of the monoclonal antibodies or antigen-binding sites according to claims 1 to 7;

(2) Detecting whether a monoclonal antibody or antigen-binding site binds to a sample, and the combination indicates that the sample is at risk of developing cancer or developing cancer.

The use of a monoclonal antibody for specifically recognizing a B-Raf mutein according to claim 13, wherein in the diagnostic method, common types of cancer include: rectal cancer, papillary thyroid cancer, pancreatic cancer , esophageal cancer, prostate cancer, ovarian cancer, lung cancer.

15. Use of a monoclonal antibody that specifically recognizes a B-Raf mutein, characterized by a method of treating a cancer patient, characterized in that:

(1) detecting whether the biological sample from the patient contains a B-Raf N581S, V600E or V600K mutation, and

(2) If the above mutation is present, the patient is treated with a B-Raf protein inhibitor.

The use of a monoclonal antibody for specifically recognizing a B-Raf mutein according to claim 15, wherein in the treatment method, the B-Raf protein inhibitor comprises: antisense RNA

(ant i sense RNA), small interfering RNA (siRNA), microRNA (mi RNA) drugs, anti-B-Raf monoclonal antibody drugs. The B-Raf inhibitor can be a sirolimus tablet (ZELBORAF®), sorafenib (BAY43-9006, NEXAVAR®), panitumumab, GDC-0879, PLX-4720, GSK2118436/SB590885, AZ628, RAF265 (CHIR-265) or XL281.

The use of a monoclonal antibody for specifically recognizing a B-Raf mutein according to claim 15, wherein the method for treating a cancer patient is administered to a patient comprising the drug of claim 11.

The use of a monoclonal antibody for specifically recognizing a B-Raf mutein according to claim 15 or 17, wherein in the method for treating a cancer patient, common types of cancer include: rectal cancer, papillary Thyroid cancer, pancreatic cancer, esophageal cancer, prostate cancer, ovarian cancer, lung cancer.

19. Use of a monoclonal antibody that specifically recognizes a B-Raf mutein, characterized in that the monoclonal antibody or antigen-binding site of claims 1 to 8 is used in the production of a cancer therapeutic drug, and common cancer types include: Melanoma, rectal cancer, thyroid cancer, pancreatic cancer, esophageal cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer, or hematopoietic cancer.

20. Use of a monoclonal antibody that specifically recognizes a B-Raf mutein, characterized by a purified nucleic acid molecule comprising a heavy chain capable of encoding the monoclonal antibody of claims 1 to 8 or an antigen binding site thereof Alternatively, it is capable of encoding a light chain or an antigen binding site thereof, or a nucleic acid sequence capable of encoding both.

The use of a monoclonal antibody which specifically recognizes a B-Raf mutein according to claim 20, characterized in that a carrier molecule contains an expression regulatory sequence which can be linked to the nucleic acid molecule.

The use of a monoclonal antibody which specifically recognizes a B-Raf mutein according to claim 20, characterized in that a host cell contains the nucleic acid molecule.

A use of a monoclonal antibody which specifically recognizes a B-Raf mutein, comprising the cell line according to any one of claims 1 to 7, which comprises the monoclonal antibody or an antigen-binding site thereof.

24. Use of a monoclonal antibody that specifically recognizes a B-Raf mutein, characterized by a method for producing a monoclonal antibody or an antigen binding site thereof, which antibody or antigen binding site specifically binds to a human mutant B -Raf protein, but not human wild-type B-Raf protein. The steps include:

(1) cultivating the host cell under appropriate conditions according to claim 22, or culturing the cell strain under the conditions of release according to claim 23;

(2) Purifying the antibody portion.