KR20190038174A - Anti c-Met antibody and uses thereof - Google Patents

Abstract

The present invention relates to an anti-c-Met antibody and uses thereof and more specifically to: an antibody or a fragment thereof, which specifically binds to a human c-Met protein; a method for producing the same; a c-Met-specific detection method using the same; a circulating tumor cell (CTC) detection method using the same; and a kit for detecting CTC, comprising the same as an active ingredient. The methods according to the present invention can be usefully employed in detecting c-Met antibodies and detecting CTC in blood by means of the antibodies.

Description

Anti-c-Met antibodies and uses thereof < RTI ID = 0.0 >

The present invention relates to an anti-c-Met antibody and a use thereof, and more particularly to an antibody or a fragment thereof that specifically binds to a human-derived c-Met protein, a production method thereof, a c-Met- A method for detecting circulating tumor cells (CTC) using the same, and a kit for detecting circulating cancer cells containing the same as an active ingredient.

c-Met is a typical RTK (Receptor Tyrosine Kinase) present on the cell surface. It binds with its ligand, HGF / SF (Hepatocyte Growth Factor / Scattering Factor), promotes intracellular signal transduction and promotes cell growth It is overexpressed in many kinds of cancer cells and is widely involved in cancer development, cancer metastasis, cancer cell migration, cancer cell infiltration, and neovascularization. Also, as the name of the ligand implies, c-Met signaling through HGF / SF is a typical early stage cancer protein that causes scattering by weakening cell-cell contact of almost all types of epithelial tumors (Nat Rev Cancer. Jan 24; 12 (2): 89-103). In particular, it is well known that the presence of hypoxiaresponse elements upstream of the c-Met gene increases the expression of the gene in the absence of oxygen (Oral Oncol 2006 Jul; 42 (6): 593-8). In addition, c-Met and its ligand HGF have been the leading candidates for targeted cancer therapy because c-Met contributes to the different stages of cancer development from onset to progression through metastasis ([Comoglio et al. Nat Rev Drug Discov 7: 504; Knudsen and Vande Woude 2008. Curr Opin Genet Dev 18:87). In particular, c-Met is known to be involved in drug resistance in the mechanism of action of known anticancer drugs, and thus it is recognized that it is more important for personalized treatment. .

On the other hand, Circulating Tumor Cells (CTCs) are known to be a cancer cell that circulates the body along with blood separated from primary tumor cells and plays a key role in the transformation of cancer to other organs. It can be used as a prognostic factor for predicting the recurrence of cancer. It is a useful means to monitor efficacy and evaluation at the same time as administering a therapeutic agent. It is possible to identify micrometastasis clinically difficult to diagnose It can be used as a useful biomarker. DNA and protein can be extracted from circulating cancer cells, and various downstream analysis techniques can be used for analysis, thus wider analysis data can be obtained. However, since circulating cancer cells are present in a very small amount in the blood (1 to 10 per 100,000 blood cells), it is essential to have advanced separation technology based on accuracy and quick separation technique in order to detect circulating cancer cells in the patient's blood do.

Therefore, it is necessary to develop a c-Met antibody which can bind specifically with higher affinity to c-Met and which has a human-derived sequence and is less likely to induce an immune response upon administration and exhibits a variety of activities There is a need to develop diagnostic methods that exhibit high sensitivity to detect blood tumor cells present in a patient ' s body.

Thus, the present inventors have made intensive efforts to develop an antibody exhibiting various physiological activities while targeting c-Met. As a result, the present inventors have found that a complementary crystal domain derived from a human specifically binding to c-Met (CDR) and framework region (FR) exhibit similar activity to HGF, and that this c-Met antibody binds to circulating cancer cells in the blood, thereby completing the present invention.

Therefore, an object of the present invention is to provide an antibody or a fragment thereof that specifically binds to a human-derived c-Met protein.

Another object of the present invention is to provide a polynucleotide encoding said antibody or fragment thereof, a vector and a cell transformed with said vector.

It is still another object of the present invention to provide a method for producing an antibody or fragment thereof binding to human c-Met and a c-Met specific detection method.

Another object of the present invention is to provide a method for detecting circulating tumor cells (CTC) using the antibody, a composition for detection, and a kit for detection.

In order to accomplish the above object, the present invention provides a complementary crystal region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 1, a complementary crystal region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 2, An antibody light chain variable region (VL) comprising a complementary crystal region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 3 and a complementary crystal region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: (VH) comprising a complementary crystal region (CDR) H2 comprising an amino acid sequence represented by SEQ ID NO: 5 and a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6 an antibody or a fragment thereof that specifically binds to a c-Met protein.

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding said antibody or fragment thereof.

In order to achieve still another object of the present invention, the present invention provides a vector comprising the polynucleotide.

In order to achieve still another object of the present invention, the present invention provides a cell transformed with said vector.

In order to accomplish still another object of the present invention, the present invention provides a method for producing a polypeptide comprising the steps of: culturing the cell under a condition that expresses a polynucleotide, producing a polypeptide comprising a light chain and a heavy chain variable region; And recovering the polypeptide from human c-Met, or a method for producing the antibody.

In order to achieve still another object of the present invention, the present invention provides a c-Met specific detection method comprising contacting the antibody or a fragment thereof with a sample and detecting the antibody or fragment thereof.

In order to accomplish still another object of the present invention, the present invention provides a method for detecting a protein comprising the steps of: a) contacting a sample obtained from an individual with the antibody of claim 1; b) separating the complex formed by binding the antibody of claim 1 to the sample from the non-complexed part; And c) obtaining a complex isolated in step b). The method of detecting a circulating tumor cell (CTC) comprises the steps of:

In order to accomplish still another object of the present invention, the present invention provides a composition for detecting circulating tumor cells (CTC) comprising the antibody as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a kit for detecting circulating tumor cells (CTC) comprising the antibody as an active ingredient.

Hereinafter, the present invention will be described in detail.

(CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 1, a complementary crystal region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: An antibody light chain variable region (VL) comprising a complementary crystal region (CDR) L3 comprising an amino acid sequence represented by SEQ ID NO: 4 and a complementary crystal region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: Specific c-Met protein comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H2 and a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: 6 Lt; / RTI > antibody or fragment thereof.

The 'anti-c-Met antibody', 'anti-c-Met antibody', 'humanized anti-c-Met antibody' (Polyclonal antibodies), multispecific antibodies (e. G., Bispecific antibodies), and antibody fragments (e. G., Monoclonal antibodies, including full length monoclonal antibodies) For example, a variable region and other portions of the antibody exhibiting the desired biological activity (e. G., Binding to c-Met).

The antibody of the present invention is an antibody in which a specific amino acid sequence is contained in a light chain and a heavy chain CDR so as to be capable of selectively binding to c-Met, and includes both monoclonal antibodies and polyclonal antibodies, preferably monoclonal antibodies Lt; / RTI > In addition, the antibody of the present invention includes both a chimeric antibody, a humanized antibody, and a human antibody, and may preferably be a human antibody.

A monoclonal antibody of the invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies bind to single antigen epitopes very specifically.

The term " monoclonal " in the present invention means that the antibody is obtained from a substantially homologous population and is characteristic of the antibody, and does not necessarily mean that the antibody should be produced by a specific method. For example, a monoclonal antibody of the invention can be prepared by the hybridoma method first described in Kohler et al. (1975) Nature 256: 495), or by recombinant DNA methods 4,816, 567). (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597 and Presta (2005) J. Allergy Clin. Immunol. 116: 731).

The antibody of the present invention specifically includes a chimeric antibody, wherein a portion of the heavy chain and / or light chain originates from a particular species or is homologous or homologous to the corresponding sequence of a particular antibody, May be of another species or may be identical or homologous to the corresponding sequence of another antibody as long as it exhibits the desired biological activity (e. G., Selective binding to NRS) (U.S. Patent No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

Humanized antibodies are antibodies that include both human and non-human (e.g., rat, rat) antibodies. Generally, the remainder of the epitope binding site (CDR) is of a human antibody, (CDR) may comprise a non-human derived sequence. A complete human antibody refers to an antibody comprising only a human immunoglobulin protein sequence and can be produced in a hybridoma originating from a mouse, mouse cell, or mouse cell, or produced by a phage display method.

Natural antibodies produced in vivo are typically about 150,000 daltons, heterotetrameric glycoproteins, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, but the disulfide chain number varies between the heavy chains of the different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that a particular amino acid residue forms an interface between the light chain variable domain and the heavy chain variable domain. &Quot; Variable domain " or " variable domain " of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable region of the heavy chain is referred to as " VH ", and the variable region of the light chain is referred to as " VL ". These domains are generally the most variable part of the antibody and comprise an antigen binding site.

In the present invention, 'hypervariable' means that several sequences within the variable region are broadly different in the sequence between the antibodies and have residues that are directly related to the binding and specificity of each particular antibody to its specific antigenic determinants Quot; are included. In both the light chain and heavy chain variable regions, the hypervariability is focused on three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparisons in the literature (Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. and Leu, 1987, J. Mol. Biol. 196: 901-917), which is structurally defined according to the three-dimensional structure of the variable region.

The three CDRs in each of the heavy and light chains are separated by a framework region (FR), which includes sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable regions, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The large < RTI ID = 0.0 > ss < / RTI > sheet arrangement of FRs brings the CDRs within each chain closer to each other as well as to the CDRs from the other strands. The form produced contributes to the antigen binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), but not all CDR residues need to be directly involved in antigen binding.

In the present invention, the fragment is a fragment selected from the group consisting of diabodies, Fab, Fab ', F (ab) 2, F (ab') 2, Fv and scFv.

In the present invention, a fragment of an antibody refers to a fragment of an antibody that retains the antigen-specific binding force of the whole antibody. Preferably, the fragment has at least 20%, 50%, 70% %, 80%, 90%, 95% or 100% or more. Specifically, it may be in the form of Fab, F (ab) 2, Fab ', F (ab') 2, Fv, diabody, scFv and the like.

Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one constant domain of each of the heavy and light chains. F (ab ') 2 is a fragment produced by hydrolyzing an antibody to pepsin, and two Fabs are linked from a heavy chain hinge to a disulfide bond. F (ab ') is a monomer antibody fragment in which a heavy chain hinge is added to a Fab obtained by reducing disulfide bonds of F (ab') 2 fragments. Fv (variable fragment) is an antibody fragment consisting of only variable regions of heavy and light chains, respectively. A single chain variable fragment (scFv) is a recombinant antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible peptide linker. A diabody is a fragment of a form in which VH and VL of scFv are linked by very short linkers and can not bind to each other, but form a dimer by binding with VL and VH of another scFv of the same type, respectively.

For the purposes of the present invention, the fragment of the antibody is not limited in structure or form as long as it retains the binding specificity for the human-derived c-Met protein, but may be preferably scFv. The scFv according to the present invention has a CDR structure specific to the aforementioned human-derived c-Met protein or a structure of VH and VL. If the C-terminus of VH and the N-terminus of VL are linked through a linker, It is not limited. The type of the linker is not particularly limited as long as it is known in the art as a linker applicable to scFv.

The antibody or fragment thereof of the present invention may comprise conservative amino acid substitutions (referred to as conservative variants of the antibody) that do not substantially alter its biological activity.

In addition, the antibody or fragment thereof of the present invention may be conjugated with an enzyme, a fluorescent substance, a radioactive substance and a protein, but is not limited thereto. Methods of conjugating such materials to antibodies are also well known in the art.

The antibody of the present invention may be derived from any animal including mammals including humans, birds, and the like. Preferably, the antibody may be an antibody of human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken, most preferably human or mouse.

A human antibody is an antibody having the amino acid sequence of a human immunoglobulin, including an antibody isolated from a human immunoglobulin library or an antibody isolated from an animal that is transgenic for one or more human immunoglobulin and does not express an endogenous immunoglobulin See Patent No. 5,939,598).

The antibody of the present invention may be conjugated with an enzyme, a fluorescent substance, a radioactive substance and a protein, but is not limited thereto. Methods of conjugating such materials to antibodies are also well known in the art.

The present invention provides a polynucleotide encoding said antibody or fragment thereof.

In the present invention, a 'polynucleotide' may be described as an oligonucleotide or a nucleic acid and may be expressed using DNA molecules (eg, cDNA or genomic DNA, RNA molecules (eg, mRNA), nucleotide analogs (E. G., Peptide nucleic acids and non-naturally occurring nucleotide analogs) and hybrids thereof. The polynucleotides may be single-stranded or double- The polynucleotide means a nucleotide sequence encoding a CDR construct specific to the KRS N-terminal region or an antibody comprising a heavy chain and a light chain having a structure of VH and VL.

The polynucleotide of the present invention is not particularly limited as long as it encodes the antibody or fragment thereof of the present invention. The polynucleotide encoding the above-described CDR sequence in the antibody according to the present invention described above has a particularly restricted sequence (Heavy chain CDR2), SEQ ID NO: 3 (heavy chain CDR3), SEQ ID NO: 4 (light chain CDR1), SEQ ID NO: 5 (light chain CDR2), SEQ ID NO: 6 CDR3). ≪ / RTI > The polynucleotide encoding VH and VL described above in the antibody according to the present invention is not particularly limited in its sequence.

Polynucleotides encoding the antibodies or fragments thereof of the present invention can be obtained by methods well known in the art. For example, an oligonucleotide synthesis technique well known in the art, for example, a polymerase chain reaction (PCR), or the like may be used based on a DNA sequence or a corresponding amino acid sequence encoding a part or all of the heavy chain and light chain of the antibody . ≪ / RTI >

The present invention provides a vector comprising the polynucleotide.

The 'vector' of the present invention is used for the purpose of replication or expression of the polynucleotide of the present invention for recombinant production of the antibody or fragment thereof of the present invention, and generally includes a signal sequence, a copy origin, An enhancer element, a promoter, and a transcription termination sequence. The vector of the present invention may preferably be an expression vector, and more preferably, may be a vector comprising a polynucleotide of the present invention operably linked to a regulatory sequence, for example, a promoter.

A plasmid, a type of vector, refers to a linear or circular double stranded DNA molecule to which external polynucleotide fragments can be ligated. Other forms of vectors are viral vectors (e. G., Replication defective retroviruses, adenoviruses and adenoassociated viruses), where additional DNA fragments Can be introduced into the viral genome. Certain vectors may be self-replicating within bacterial vectors (bacterial vectors), including host cells into which they are introduced (e.g., bacterial origin and episomal mammalian vectors) autonomous replication. Other vectors (e. G., Non-episomal mammalian vectors) are integrated into the genome of the host cell by introduction into the host cell, Are duplicated together.

In the present invention, an 'expression vector' is a form of a vector capable of expressing a selected polynucleotide. One polynucleotide sequence is " operably linked " to the regulatory sequence when the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the polynucleotide sequence . The modulatory sequence is a sequence that affects the expression (e.g., level, timing, or location of expression) of the nucleic acid to which it is operatively linked. The modulation sequence can be, for example, a nucleic acid whose effect is directly or indirectly affected by the action of one or more other molecules (e. G., Polypeptides that bind to the regulatory sequence and / . The regulatory sequence includes promoters, enhancers, and other expression control elements. The vector of the present invention may preferably be pOptiVEC -TOPO and pcDNA (TM) 3.3-TOPO.

The present invention provides a cell transformed with said vector.

The cell of the present invention is not particularly limited as long as it is a cell that can be used to express an antibody or a polynucleotide encoding the fragment contained in the expression vector of the present invention. Cells (host cells) transformed with an expression vector according to the invention can be transformed into prokaryotic (e. G., E. coli), eukaryotes (e. G., Yeast or other fungi), plant cells (e. (Eg, a cell), an animal cell (eg, a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, an insect cell or a hybridoma derived therefrom) May be cells derived from mammals, including humans.

Suitable prokaryotes for this purpose include gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example E. coli. For example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, Serratia, such as Serratia marcescans and Shigella, and Bacilli, e. G. B. subtilis and rain. B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces. The cell of the present invention is not particularly limited as long as it is capable of expressing the vector of the present invention. Lt; / RTI >

Saccharomyces cerevisiae is the most commonly used eukaryotic cell of the present invention. However, many other genera, species and strains, including, but not limited to, Schizosaccharomyces pombe, Kluyveromyces hosts, e.g., K. K.lactis, K. K. fragilis (ATCC 12, 424), Kay. K. bulgaricus (ATCC 16,045), Kay. K.wickeramii (ATCC 24,178), Kay. K. waltii (ATCC 56,500), Kay. K. drosophilarum (ATCC 36,906), Kay. K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Tricot Neurospora crassa; Schwanniomyces, such as Shibaniomycetes occidentalis; and filamentous fungi, such as Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, For example, Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, such as A. nidulans and A. niger (A. niger) are available.

The term 'transformation' refers to a modification of the genotype of a host cell by the introduction of a foreign polynucleotide, which means that the foreign polynucleotide has been introduced into the host cell irrespective of the method used for its transformation. The exogenous polynucleotide introduced into the host cell may be maintained integrated or maintained in the genome of the host cell, but the present invention encompasses both.

The recombinant expression vector capable of expressing an antibody or a fragment thereof that specifically binds to the human-derived c-Met protein according to the present invention can be produced by a method known in the art, for example, transient transfection transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, Introduced into cells to produce antibodies or fragments thereof by known methods for introducing nucleic acids into cells, such as polybrene-mediated transfection, electroporation, gene gun, Lt; / RTI >

In addition, the cell of the present invention is a cultured cell that can be transformed or transfected with the polynucleotide of the present invention or a vector containing it, which can be subsequently expressed in the host cell. A recombinant cell refers to a cell transformed or transfected with a polynucleotide to be expressed. A cell of the invention may also be a cell that comprises a polynucleotide of the invention but does not express it at a desired level unless a regulatory sequence is introduced into the cell to operably link to the polynucleotide.

The cells of the present invention can be cultured in various media. (Sigma-Aldrich Co., St. Louis, MO), minimal essential medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.) And Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co.) are suitable for culturing cells. The medium may be supplemented with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements and glucose or equivalent energy sources, if necessary.

The present invention relates to a method for producing a polypeptide comprising the steps of culturing the above cells under a condition that a polynucleotide is expressed to produce a polypeptide comprising a light chain and a heavy chain variable region and recovering the polypeptide from the cell or a culture medium in which the polypeptide is cultured A method for producing an antibody that binds to human c-Met or a fragment thereof.

The cells of the production method in the present invention are as described above and include a polynucleotide encoding the antibody of the present invention. The polypeptide of the above production method may be an antibody of the present invention or a fragment thereof itself, and may be an antibody or an amino acid sequence other than the fragment of the present invention.

In this case, the antibodies or fragments thereof of the present invention can be removed using methods well known to those skilled in the art. The culture may vary in the composition of the medium and the culture conditions depending on the type of the cells, and can be appropriately selected and controlled by those skilled in the art.

The antibody molecule may be accumulated in the cytoplasm of a cell, secreted from the cell, targeted by a suitable signal sequence to a periplasm or extracellular medium (supernatant), and labeled with a periplasmic or extracellular medium . It is also desirable to refold the engineered antibody molecules using methods well known to those of ordinary skill in the art and to have functional conformation. The recovery of the polypeptide may vary depending on the characteristics of the produced polypeptide and the characteristics of the cells, and those skilled in the art can appropriately select and control the polypeptide.

 The polypeptide may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If a polypeptide is produced in a cell, it can be destroyed to release the protein as a first step. Particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit proteolysis, a protease inhibitor, such as PMSF, may be included in any preceding step and antibiotics may be included to prevent the growth of contingent contaminants. Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, and the antibodies of the invention can be purified, preferably through affinity chromatography have.

The present invention provides a c-Met specific detection method comprising contacting the antibody or fragment thereof with a sample and detecting the antibody or fragment thereof.

The above detection method of the present invention is a method for detecting the presence or absence of KRS (or an KRS N-terminal peptide exposed to an extracellular membrane) using the antibody or fragment thereof according to the present invention before contacting the antibody or the fragment thereof according to the present invention with a sample. And preparing a sample for measuring the concentration (step (1)).

A person skilled in the art can appropriately select a known method for detecting a protein using an antibody and prepare a sample suitable for a selected method. The sample may also be a cell or tissue, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, etc. obtained by biopsy or the like collected from a subject to be diagnosed as cancer or metastasis. The method for detecting a protein using the antibody is not limited to the above examples, and examples thereof include Western blot, immunoblot, dotslab, immunohistochemistry, enzyme immunoassay (ELISA), radioimmunoassay , Competitive binding assays, and immunoprecipitation. For example, in order to perform western blotting, a buffer suitable for electrophoresis may be added to a sample or a cell lysate, followed by boiling. For immunohistochemical staining, a cell or tissue section is fixed, It is possible to perform pre-processing such as blocking.

Next, (Step (2)) of contacting the antibody or the fragment thereof with the sample prepared in the above-mentioned step.

The antibody according to the present invention has the above-described CDR, or VH and VL, and specifically binds to a human-derived c-Met protein, or a fragment thereof. The specific types and sequences of the antibody are as described above.

The antibody or fragment thereof can generally be labeled with a detectable moiety for its detection. See, for example, Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs], using radioimmunoassay or fluorescent labeling. Or various enzyme-substrate labels are available. Examples of such enzymatic labels are luciferase, luciferin, 2,3-dihydrophene, and the like, such as Fusarium luciferase and Bacillus luciferase (US Patent No. 4,737,456) Alkaline phosphatase,? -Galactosidase, glucoamylase, lysozyme, saccharide such as thalidomide, thalidomide, thalidomide, thalidomide, thalidomide, thalidomide, (Such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e. G., Free radicals and xanthine oxidases), lactoperoxidases, Oxydase, and the like. Techniques for conjugating enzymes to antibodies have been described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym., J. Langone & H. Van Vunakis , eds.), Academic press, NY, 73: 147-166. The label can be conjugated directly or indirectly to the antibody using a variety of known techniques. For example, the antibody can be conjugated to biotin and any label belonging to the three broad categories mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and thus this label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve an indirect conjugation of the label to the antibody, the antibody may be conjugated to a small hapten (e. G., Digoxin) and one of the different types of labels mentioned above may be conjugated to an anti- Hapten antibody (e. G., An anti-diphoshin antibody). Thus, indirect conjugation of the label to the antibody can be achieved.

As used herein, the term " contacting " is used in its ordinary sense, meaning that two or more materials are mixed, combined, or brought into contact with each other. The contact may be carried out in vitro or in another container, and may also be performed in situ, in vivo, intracisternally, intracisternally, or intracellularly.

Next, the step ((3)) of detecting the antibody or the fragment thereof according to the present invention is carried out in the sample after the step (2).

The 'detection' refers to an antibody according to the present invention formed in the sample, or a complex of the fragment and an antigen thereof, and is used to detect the presence or absence of the peptide of human c-Met (or a protein containing the same) (Including both qualitative and quantitative measurements). Therefore, a step of removing the extra antibody or fragments thereof that are not complexed with the human-derived c-Met protein may be further included before the detecting step (3) to be described later after the step (2).

When the antibody or fragment thereof used in the step (2) includes a detectable moiety such as a fluorescent moiety, a radioactive isotope, an enzyme, or the like, the moiety can be detected by a method known in the art The detection can be performed. For example, radioactivity can be measured, for example, by scintillation counting, and fluorescence can be quantified using a fluorimeter.

When the antibody or fragment thereof used in the step (2) does not contain the detection moiety mentioned above, the secondary antibody labeled with fluorescence, radioactivity, or enzyme as known in the art may be used It can be detected indirectly. The secondary antibody binds to an antibody according to the present invention or a fragment thereof (primary antibody).

Recent studies have shown that HGF / SF also acts on the nervous system, and many studies have been reported on the protective function of motor neurons (Novak et al., Journal of Neuroscience. 20: 326-337, 2000). In addition, it has been suggested that it plays an important role in defensive physiological mechanisms after general organs such as cardiac injury recovery (Nakamura et al., J Clin Invest. 106: 1511-1519, 2000) It has been demonstrated that HGF is overexpressed in lesions of these degenerative diseases and has a protective activity before the physiological defense period of tissue injury (Comoglio et al., Nature Review Drug Discovery 7: 504-516, 2008). In addition, hyperactivity of HGF / c-Met signaling is involved in malignant tumorigenesis and angiogenesis of various endothelial cells, and in this respect, antagonistic c-Met antibodies targeting c-Met can be used as anticancer agents (Comoglio et al., Nature Review Drug Discovery. 7: 504-516, 2008). For example, it has been reported that the c-Met antibody having one branch has a negative regulation of activation by c-Met dimerization of HGF, effectively inhibiting tumor growth in a transplanted mouse model (Jin et al, Cancer Research 68 (11): 4360-4368, 2008; Comoglio et al., Nature Review Drug Discovery. 7: 504-516, 2008). In addition, T-cell gene therapy, which selectively recognizes cancer cell surface antigens in T-cell therapy, has been applied to tumor targeting for the connection of T cells to antigens overexpressed in cancer cells (Sadelain, The Cancer Journal 15 ): 451-455, 2009).

The present invention

a) contacting the sample obtained from the subject with the antibody of claim 1;

b) separating the complex formed by binding the antibody of claim 1 to the sample from the non-complexed part; And

and c) obtaining the complex isolated in the step b). The present invention also provides a method for detecting circulating tumor cells (CTC).

In the present invention, step a) is characterized by bringing the above-mentioned antibody into contact with the sample obtained from the subject.

The term " subject " in the present invention means an animal to be diagnosed with cancer, and preferably, it may be an animal including a mammal, particularly a human. More preferably, .

The 'sample' of the present invention may be selected from the group consisting of tissue, blood, serum, plasma, saliva, mucosal solution and urine, Blood, serum, plasma.

In the present invention, the 'antibody of claim 1' is characterized in that the antibody is selected from the group consisting of beads, magnetic beads and magnetic materials. Magnetic substance binding to antibodies, but is not limited to this, and may be a magnetic metal or magnetic metal oxide, preferably Co, Mn, Fe, Ni, Gd, MM _'2 O _4, and MxOy (M or M' = Co , Fe, Ni, Mn, Zn, Gd or Cr, x and y represent integers).

In the step b) of the present invention, the antibody binds to the sample to separate the complex from the non-complexed portion.

In the present invention, the term 'complex' refers to a complex formed by specifically binding a c-Met-containing cell and an antibody to a surface, and the overall density is increased as compared with cells in a sample having the same or similar density as the target cell . More preferably by specifically binding to c-Met on purified tumor cells (CTC).

In the present invention, step c) is characterized in that the complex isolated in step b) is obtained.

The sample including the complex formed in the step b) can separate the complex using the magnetic property, and the complex can be extracted automatically or manually using the separation method, and can be used variously according to the purpose of the experimenter .

The 'Circulating Tumor Cell (CTC)' of the present invention is a tumor cell found in the peripheral blood of a malignant tumor patient. It has been shown that the epithelial cells can be transferred to the epithelium through mesenchymal transitions (EMT), which is a change in the cell structure that can be transferred from the origin of the tumor cells to the blood vessels or lymphatic vessels, (Inflammatory or scarred surface) and digest between endothelial cells. At this time, the mesenchymal to epithelial transitions (MET) process is performed again. The EMT process is known to be involved in the metastasis of malignant tumors, as the cells lose their epithelial cell phenotype and convert to a mesenchymal cell phenotype with high mobility. Circulating cancer cells are also involved in the EMT process and are transferred to new tumors and become cancerous in other tissues. However, it is difficult to detect circulating cancer cells because they exist in trace amounts in blood (1 to 10 cells per billion cells). Therefore, in order to detect circulating cancer cells in the blood of a patient, it is essential that advanced separation technology based on accuracy and quick separation technique be ensured. Such a circulating cancer cell separation technique is not limited to cancer treatment before metastasis, And is useful for diagnosis.

As a method for detecting circulating cancer cells in the blood, a method of separating using a cell-specific antibody (antibody-based), a method using size-based separation, a method using electric charge-based separation, Virus-based separation method and a separation method using microfluidics. The inventors of the present invention confirmed that the c-Met protein is present in the tumor cell membrane, and thus the c-Met antibody of the present invention Were used to detect circulating cancer cells.

The present invention provides a composition for detecting circulating tumor cells (CTC) comprising the antibody as an active ingredient.

The antibody of the present invention may be provided in a labeled state and may be provided in combination with a detectable label to facilitate identification, detection, and quantification of the binding of the antibody of the present invention to circulating cancer cells (CTC). Such detectable labels include, but are not limited to, magnetic materials (e.g., magnetic metal, thick metal oxide), chromogenic enzymes (e.g., peroxidase, alkaline phosphatase), radioisotopes, chromophore, Or fluorescent materials such as FITC, RITC, Green Fluorescent Protein (EGFP), Red Fluorescent Protein (RFP), Discosoma red fluorescent protein (CFP), Cyan Fluorescent Protein ), CGFP (Cyan Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), Cy3, Cy5 and Cy7.5).

The present invention provides a kit for detecting circulating tumor cells (CTC) comprising the antibody as an active ingredient.

The 'kit' of the present invention includes an antibody that specifically binds to c-Met protein, and can detect circulating cancer cells (CTC) in blood through an antigen-antibody binding reaction. More preferably, a complex due to antigen-antibody binding can be formed and detected by a centrifugation method. Further, a filtration process using a filter can be further performed if necessary.

In one embodiment of the present invention, the human scFv library screening is performed using the human recombinant c-Met antibody to obtain a sample with increased output, and the binding force is confirmed by ELISA method to select samples showing the binding signal, Sequence analysis was performed. Then, the hits having double different sequences were selected, and the binding force was confirmed by an ELISA method to select 10 hits that were most strongly binding, and converted to human IgG form (see Example 1, Figs. 1 and 2).

In another embodiment of the present invention, in order to confirm that human IgG binds to natural c-Met, 293F cells were transfected with a plasmid, cells were obtained, and the antibody was purified with protein A beads and subjected to SDS-PAGE As a result, it was confirmed that the above-mentioned 10 hits were converted into human IgG form and expressed in cells, and the sizes of light and heavy chains were confirmed. Next, flow cytometric analysis using c-Met positive cells revealed that four antibodies were selected which corresponded to the highest change in the binding pattern of the antibody in the cells and the expression level of c-Met Example 2, Figures 3 and 4).

In another embodiment of the present invention, the blood of a patient is placed in a test tube, reacted with a c-Met antibody (C8) and a magnetic bead complex, and then separated by a magnetic column. As a result, And it was confirmed that circulating cancer cells in the blood can be detected with the c-Met antibody (see Example 3).

Thus, the present invention provides anti-c-Met antibodies and uses thereof. The method of the present invention can be usefully used to detect c-Met antibodies and to detect circulating cancer cells in blood using antibodies.

Fig. 1 shows phage display (a) using human c-Met recombinant protein as an antigen and screen (b) of screening by ELISA.
FIG. 2 shows the result of checking whether or not the selected heat is coupled according to the result of ELISA.
FIG. 3 shows the result of SDS-PAGE to confirm the heavy and light chain sizes of purified antibodies.
FIG. 4 is a graph showing the results of flow cytometry showing the binding ability of 10 c-Met antibodies using A549 cells and MDA-MB-231 cells (a) and A549, H596 and SKBR- (A8, A11, B10, and C8) by flow cytometry. As shown in FIG.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Experimental Method>

reagent

A549 cell line and MDA-MB231 cell line were purchased from ATCC (American Type Culture Collection, USA), and H596 cells and SKBR-3 cells were purchased from Klean Cell Line Bank (KCLB). The antigen used for screening was a human c-Met recombinant protein containing 1-932 amino acids (aa) of the receptor and was purchased from Sinobiological (China). As a control group, anti-c-Met antibody purchased from Abcam (USA) was used.

Phage Display

The human recombinant c-Met protein was used as an antigen and the human scFv library was used for screening hits binding to the extracellular domain of c-Met. Antigen was coated on an immunity tube (Nunc, USA) with a concentration of 10 / / ㎕ and incubated with O / N for binding. The immune tubes and phages were blocked with blocking buffer (3% milk in PBST). The phages were placed in an antigen-coated immune tube and bound. After 1 hour, they were washed 4 times with PBST and 1 time with PBS. The phage were eluted in 100 mM TEA for 7 to 8 minutes and then neutralized with Tris-HCl (pH 8) solution. The eluted phages were infected with Escherichia coli, and some of them were cultured in a solid LA plate with O / N to confirm the output titer. The remaining phages were rescued using a helper phage and the same experiment was repeated three times.

ELISA screening

After the fourth panning, a single colony was injected into each 150 μl of SB containing ampicillin in a 96 well plate. The cells were then cultured in a 37 ° C shaking incubator until the medium became cloudy. After incubation, the culture was added to the plate, and the plate was incubated with 1 nM IPTG at 30 ° C overnight. The c-Met recombinant protein was used as an antigen, coated on ELISA plate (corning 3690) at a concentration of 1 μg / ml in PBS, and incubated overnight at 4 ° C. The next day, the cloned injected plate was centrifuged at 3000 rpm for 15 minutes. The supernatant was removed and the pellet was resuspended in 1X TES buffer at 37 ° C for 5-7 minutes and then lysed by adding 0.2X TES buffer and reacting at 4 ° C for 30 minutes. Antigen coated plates were washed three times with 150 [mu] l TBST and the reaction was inhibited using 3% skim milk. Periplasmic extracts were obtained in the lysed cells and the reaction was inhibited for 1 hour using a 6% skim milk on a new plate. The solution was then added to the antigen-coated plate, incubated at room temperature for 1 hour, and washed three times with TBST. The anti-HA Hrp secondary antibody was then added, incubated for 1 hour, and washed three times with TBST. The reaction was then initiated by treatment with 30 μl of TMB followed by inhibition of the reaction with 1 N H 2 SO 4 and detection at 450 nm.

Sequence analysis and IgG conversion

The sequence of the selected hits in the ELISA screening was analyzed (Cosmogenetech, Korea). After sequencing and ELISA screening, the selected final hits were converted to human IgG. The scFv sequence was converted to human light and heavy chain sequences and was incorporated into the pOptiVEC ™ -TOPO and pcDNA ™ 3.3-TOPO (Thermofisher, USA) vectors by cloning. The plasmid was then amplified using the midi prep (Macherey Nagel, Germany).

Over-expression and antibody purification

The amplified plasmid was transiently expressed using the Freestyle Expression System (Invitrogen, USA). Freestyle cells were thawed and cultured in a Freestyle Expression Medium in an Erlenmeyer flask (Corning, USA). Cells were cultured to a concentration of 3.0 x 10 ⁶ cells / ml and subcultured every 2 to 3 days. After 4 subcultures, the heavy and light chain plasmids were transfected with FreeStyle ™ MAX Transfection reagent (Invitrogen, USA) Infected. The cells were then cultured in a shaker at 8% CO ₂ , 37 ° C. On day 7 after transfection, cells were obtained and the supernatant was collected and filtered. After filtration, the supernatant was applied to MabSelect SuRe protein A beads (GE healthcare, USA) in a chromatographic column (Bio-rad, USA). The size was then confirmed by SDS-PAGE and coomassie blue staining.

Flow cytometry

Flow cytometric analysis was performed using A549, MDA-MP231, H596 and SKBR-3 cells. Cells were detached with cell dissociation buffer (Hyclone, USA), washed with PBS, and then separated into 2.0 × 10 ⁵ cells and placed in tubes. The antibody was diluted with DBPS (Wellgene) solution containing 2% FBS to a concentration of 1 μg / tube, added to the cells, and reacted for 1 hour. Commercial anti-c-Met antibodies were used as controls. The cells were then washed twice and reacted with a secondary antibody conjugated with FITC for 40 minutes. After washing three times, they were analyzed using FACS BD Calibur (BD, USA).

Cell culture and antibody treatment

H596 cells were cultured in RPMI (Wellgene) containing 10% FBS and 1% penicillin / streptomycin (Hyclone). Cells were cultured in 6-well plates in order to observe whether the antibodies could induce phosphorylation signals. The cells were then cultured in RPMI medium without FBS overnight to remove signal interference by FBS. The next day, the medium was removed and the solution containing antibody or HGF at different concentrations was treated for 1 hour.

Western blot

Cells were cultured as described above and cells were obtained using a dissolution buffer containing RIPA (Biosesang), protease inhibitor (Roche) and phosphatase inhibitor (Roche) To dissolve the cells. After dissolution, the cells were centrifuged at 14000 rpm for 15 minutes. The supernatant was then obtained and the protein quantified by the BCA assay (Thermofisher) method. The supernatant was mixed with 5x sample loading buffer and heat was applied for 10 minutes. SDS-PAGE was then performed and the protein was transferred to an activated PVDF (polyvinylidene difluoride) apa brain (Bio-rad). The membranes were blocked with 5% BSA, reacted with primary antibody, and reacted with secondary antibody conjugated with Hrp. And then confirmed by ECL (Amersham) in the dark room.

&Lt; Example 1 >

Screening and identification of scFv binding to c-Met

To identify antibodies that bind to c-Met, human scFv library screening was performed according to the method described above using human recombinant c-Met antibodies containing only the extracellular domain (aa, 1-932) as an antigen . The antigen was bound to the immunotube and 4 cycles were repeated.

As a result, as shown in FIG. 1A, the output increased in the third and fourth cycles, and the samples obtained in the third and fourth cycles were confirmed by ELISA. 1B. In addition, after selecting the signal to be compared with the control plate, sequencing of the nucleotide sequence was performed, 31 of the candidates having different sequences were selected, ELISA was performed again to confirm the binding force, And 10 candidates (hits). Each candidate was named A8, A9, A11, B8, B10, C8, C9, D7, D12, and E10 based on the ELISA results. The 10 hits were then converted to human IgG form (Figure 2).

&Lt; Example 2 >

Determination of natural c-Met binding in human IgG form

In order to confirm that the human IgG prepared in Example 1 binds to natural c-Met, the following experiment was conducted.

First, 293F cells were transfected with the plasmid according to the above experimental method and cultured for 7 days. The cells were then harvested and the antibodies purified using Protein A beads and subjected to SDS-PAGE.

As a result, as shown in FIG. 3, it was confirmed that the 10 hits most strongly binding in Example 1 were converted into human IgG form and expressed in cells, and the sizes of light and heavy chains were confirmed.

After confirming the conversion of IgG, positive cells for c-Met were selected based on the information of CCLE and flow cytometry was performed according to the above experimental method.

As a result, as shown in FIG. 4A, the binding pattern of the antibody showed the highest change in A549 cells and the expression level of c-Met, and the four most similar antibodies were selected The same experiment was carried out using different cell lines. At this time, the H596 cell line was used as a medium-expressing cell line and the SKBR-3 cell line was used as a negative control group.

As a result, as shown in FIG. 4B, the binding pattern of the four antibodies (A8, A11, B10 and C8) was found to be correlated with the c-Met expression level, and the c-Met negative cell line SKBR-3 The combined movement was not visible. Among these, B10 showed similar patterns of expression of the anti-c-Met antibody in the A549, H596 and SKBR-3 cell lines, and the signals were higher than those of the other antibodies.

This confirms that the four antibodies are specific for c-Met receptors.

&Lt; Example 3 >

Separation of circulating cancer cells using c-Met antibody

Experiments for isolating circulating tumor cells (CTC) only in the blood of antibody C8 specifically binding to c-Met prepared in the above example were performed as follows.

First, 4 ml of the patient's normal blood obtained in accordance with the criteria of the clinical trial screening committee was placed in a test tube, spiking 100 breast cancer cell line MCF-7 cells, and then incubated with a magnetic bead-specific, c-Met specific C8, which is a binding antibody, was added and left for 1 hour. Separation was then performed on a magnetic column.

As a result, it was confirmed that circulating cancer cells bound with antibodies specifically binding to c-Met were attached to the magnetic column. As a result, it was confirmed that circulating cancer cells in the blood can be detected using c-Met antibody (data not shown).

As described above, the method of the present invention can be used to detect a c-Met antibody and detect circulating cancer cells in blood using an antibody.

<110> Seoul National University R & DB Foundation          ABION Inc.          GenoBio Corp. <120> Anti-c-Met antibody and uses thereof <130> NP17-0079 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR1 of c-Met C8 <400> 1 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Asp Val Asn   1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR2 of c-Met C8 <400> 2 Asp Asp Asn Asn Arg Pro Ser   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR3 of c-Met C8 <400> 3 Ala Ser Trp Asp Asp Ser Leu Ser Gly   1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR1 of c-Met C8 <400> 4 Asp Tyr Ala Met Ser   1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR2 of c-Met C8 <400> 5 Gly Ile Tyr Tyr Asp Asp Ser Ser Gln Tyr Tyr Ala Asp Ser Val Glu   1 5 10 15 Gly     <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR3 of c-Met C8 <400> 6 Arg Ile Val Phe Ser Pro Thr Leu Met Ala Tyr Tyr Ser Asp Gly Met   1 5 10 15 Asp Val         <210> 7 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> c-Met C8 VL <400> 7 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Ser Ser Ser Ser              20 25 30 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Asn          35 40 45 Ile Gly Asn Asn Asp Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala      50 55 60 Pro Lys Leu Leu Ile Tyr Asp Asp Asn Asn Arg Pro Ser Gly Val Pro  65 70 75 80 Asp Arg Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser                  85 90 95 Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp             100 105 110 Asp Asp Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr         115 120 125 Val Leu Arg Thr Val Ala Ala Pro     130 135 <210> 8 <211> 147 <212> PRT <213> Artificial Sequence <220> C-Met C8 VH <400> 8 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val              20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr          35 40 45 Phe Ser Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly      50 55 60 Leu Glu Trp Val Ser Gly Ile Tyr Tyr Asp Asp Ser Ser Gln Tyr Tyr  65 70 75 80 Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys                  85 90 95 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala             100 105 110 Val Tyr Tyr Cys Ala Lys Arg Ile Val Phe Ser Pro Thr Leu Met Ala         115 120 125 Tyr Tyr Ser Asp Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr     130 135 140 Val Ser Ser 145 <210> 9 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> c-Met antibody C8 Light chain <400> 9 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Trp Gln Ser Val Leu Thr Gln Pro Ser Ser Ser Ser              20 25 30 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Asn          35 40 45 Ile Gly Asn Asn Asp Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala      50 55 60 Pro Lys Leu Leu Ile Tyr Asp Asp Asn Asn Arg Pro Ser Gly Val Pro  65 70 75 80 Asp Arg Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser                  85 90 95 Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp             100 105 110 Asp Asp Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr         115 120 125 Val Leu Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser     130 135 140 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 145 150 155 160 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala                 165 170 175 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys             180 185 190 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp         195 200 205 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu     210 215 220 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 10 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> c-Met antibody C8 Heavy chain <400> 10 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Trp Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val              20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr          35 40 45 Phe Ser Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly      50 55 60 Leu Glu Trp Val Ser Gly Ile Tyr Tyr Asp Asp Ser Ser Gln Tyr Tyr  65 70 75 80 Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys                  85 90 95 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala             100 105 110 Val Tyr Tyr Cys Ala Lys Arg Ile Val Phe Ser Pro Thr Leu Met Ala         115 120 125 Tyr Tyr Ser Asp Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr     130 135 140 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 145 150 155 160 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val                 165 170 175 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala             180 185 190 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly         195 200 205 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly     210 215 220 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 225 230 235 240 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys                 245 250 255 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu             260 265 270 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu         275 280 285 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys     290 295 300 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 305 310 315 320 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu                 325 330 335 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys             340 345 350 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys         355 360 365 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser     370 375 380 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 385 390 395 400 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln                 405 410 415 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly             420 425 430 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln         435 440 445 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn     450 455 460 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 11 <211> 1408 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens c-Met Protein <400> 11 Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu Val Leu Leu Phe   1 5 10 15 Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys              20 25 30 Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala          35 40 45 Glu Thr Pro Ile Gln Asn Val Ile Leu His Glu His His Ile Phe Leu      50 55 60 Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys  65 70 75 80 Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe                  85 90 95 Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp             100 105 110 Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Asp Asp         115 120 125 Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His     130 135 140 Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys 145 150 155 160 Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val                 165 170 175 Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe             180 185 190 Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp         195 200 205 His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp     210 215 220 Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu 225 230 235 240 Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser Asn                 245 250 255 Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala Gln             260 265 270 Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu         275 280 285 His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg     290 295 300 Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala 305 310 315 320 Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser                 325 330 335 Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp             340 345 350 Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile Lys         355 360 365 Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg     370 375 380 Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg 385 390 395 400 Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr                 405 410 415 Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly             420 425 430 Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly         435 440 445 Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln     450 455 460 Val Val Val Ser Ser Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu 465 470 475 480 Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu His Thr Leu                 485 490 495 Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys             500 505 510 Ile Pro Leu Asn Gly Leu Gly Cys Arg His His Phe Gln Ser Cys Ser Gln         515 520 525 Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys     530 535 540 Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile 545 550 555 560 Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu                 565 570 575 Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg             580 585 590 Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu         595 600 605 Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys     610 615 620 Thr Val Gly Pro Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile 625 630 635 640 Ser Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp                 645 650 655 Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly             660 665 670 Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg         675 680 685 His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn     690 695 700 Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe 705 710 715 720 Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe                 725 730 735 Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser             740 745 750 Phe Ile Ser Thr Trp Trp Lys Glu Pro Leu Asn Ile Val Ser Phe Leu         755 760 765 Phe Cys Phe Ala Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn     770 775 780 Leu Asn Ser Val Val Val Pro Arg Met Val Ile Asn Val His Glu Ala 785 790 795 800 Gly Arg Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile                 805 810 815 Ile Cys Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro             820 825 830 Leu Lys Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr         835 840 845 Phe Asp Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys     850 855 860 Pro Val Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly 865 870 875 880 Asn Asp Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly                 885 890 895 Asn Lys Ser Cys Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys             900 905 910 Thr Val Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu         915 920 925 Trp Lys Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln     930 935 940 Pro Asp Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser 945 950 955 960 Thr Ala Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg                 965 970 975 Lys Gln Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg             980 985 990 Val His Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser         995 1000 1005 Pro Thr Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr    1010 1015 1020 Phe Pro Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg 1025 1030 1035 1040 Gln Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly                1045 1050 1055 Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile Asp            1060 1065 1070 Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln His Val Val        1075 1080 1085 Ile Gly Pro Ser Ser Leu Ile Val His Phe Asn Glu Val Ile Gly Arg    1090 1095 1100 Gly His Phe Gly Cys Val Tyr His Gly Thr Leu Leu Asp Asn Asp Gly 1105 1110 1115 1120 Lys Lys Ile His Cys Ala Val Lys Ser Leu Asn Arg Ile Thr Asp Ile                1125 1130 1135 Gly Glu Val Ser Gln Phe Leu Thr Glu Gly Ile Ile Met Lys Asp Phe            1140 1145 1150 Ser His Pro Asn Val Leu Ser Leu Leu Gly Ile Cys Leu Arg Ser Glu        1155 1160 1165 Gly Ser Pro Leu Val Val Leu Pro Tyr Met Lys His Gly Asp Leu Arg    1170 1175 1180 Asn Phe Ile Arg Asn Glu Thr His Asn Pro Thr Val Lys Asp Leu Ile 1185 1190 1195 1200 Gly Phe Gly Leu Gln Val Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys                1205 1210 1215 Lys Phe Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu            1220 1225 1230 Lys Phe Thr Val Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr        1235 1240 1245 Asp Lys Glu Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro    1250 1255 1260 Val Lys Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr 1265 1270 1275 1280 Lys Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu Met Thr                1285 1290 1295 Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile Thr Val            1300 1305 1310 Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr Cys Pro Asp        1315 1320 1325 Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro Lys Ala Glu Met    1330 1335 1340 Arg Pro Ser Phe Ser Glu Leu Val Ser Arg Ile Ser Ala Ile Phe Ser 1345 1350 1355 1360 Thr Phe Ile Gly Glu His Tyr Val His Val Asn Ala Thr Tyr Val Asn                1365 1370 1375 Val Lys Cys Val Ala Pro Tyr Pro Ser Leu Leu Ser Ser Glu Asp Asn            1380 1385 1390 Ala Asp Asp Glu Val Asp Thr Arg Pro Ala Ser Phe Trp Glu Thr Ser        1395 1400 1405 <210> 12 <211> 6710 <212> RNA <213> Artificial Sequence <220> <223> Homo sapiens c-Met mRNA <400> 12 gcaggtgacc cggaggccct cgccgcccgc ggcgccccga gcgctttgtg agcagatgcg 60 gagccgagtg gagggcgcga gccagatgcg gggcgacagc tgacttgctg agaggaggcg 120 gggaggcgcg gagcgcgcgt gtggtccttg cgccgctgac ttctccactg gttcctgggc 180 accgaaagat aaacctctca taatgaaggc ccccgctgtg cttgcacctg gcatcctcgt 240 gctcctgttt accttggtgc agaggagcaa tggggagtgt aaagaggcac tagcaaagtc 300 cgagatgaat gtgaatatga agtatcagct tcccaacttc accgcggaaa cacccatcca 360 gaatgtcatt ctacatgagc atcacatttt ccttggtgcc actaactaca tttatgtttt 420 aaatgaggaa gaccttcaga aggttgctga gtacaagact gggcctgtgc tggaacaccc 480 agattgtttc ccatgtcagg actgcagcag caaagccaat ttatcaggag gtgtttggaa 540 agataacatc aacatggctc tagttgtcga cacctactat gatgatcaac tcattagctg 600 tggcagcgtc aacagaggga cctgccagcg acatgtcttt ccccacaatc atactgctga 660 catacagtcg gaggttcact gcatattctc cccacagata gaagagccca gccagtgtcc 720 tgactgtgtg gtgagcgccc tgggagccaa agtcctttca tctgtaaagg accggttcat 780 caacttcttt gtaggcaata ccataaattc ttcttatttc ccagatcatc cattgcattc 840 gatatcagtg agaaggctaa aggaaacgaa agatggtttt atgtttttga cggaccagtc 900 ctacattgat gttttacctg agttcagaga ttcttacccc attaagtatg tccatgcctt 960 tgaaagcaac aattttattt acttcttgac ggtccaaagg gaaactctag atgctcagac 1020 ttttcacaca agaataatca ggttctgttc cataaactct ggattgcatt cctacatgga 1080 aatgcctctg gagtgtattc tcacagaaaa gagaaaaaag agatccacaa agaaggaagt 1140 gttcat aggagccagc ctgaatgatg acattctttt cggggtgttc gcacaaagca agccagattc 1260 tgccgaacca atggatcgat ctgccatgtg tgcattccct atcaaatatg tcaacgactt 1320 cttcaacaag atcgtcaaca aaaacaatgt gagatgtctc cagcattttt acggacccaa 1380 tcatgagcac tgctttaata ggacacttct gagaaattca tcaggctgtg aagcgcgccg 1440 tgatgaatat cgaacagagt ttaccacagc tttgcagcgc gttgacttat tcatgggtca 1500 attcagcgaa gtcctcttaa catctatatc caccttcatt aaaggagacc tcaccatagc 1560 taatcttggg acatcagagg gtcgcttcat gcaggttgtg gtttctcgat caggaccatc 1620 aacccctcat gtgaattttc tcctggactc ccatccagtg tctccagaag tgattgtgga 1680 gcatacatta aaccaaaatg gctacacact ggttatcact gggaagaaga tcacgaagat 1740 cccattgaat ggcttgggct gcagacattt ccagtcctgc agtcaatgcc tctctgcccc 1800 accctttgtt cagtgtggct ggtgccacga caaatgtgtg cgatcggagg aatgcctgag 1860 cgggacatgg actcaacaga tctgtctgcc tgcaatctac aaggttttcc caaatagtgc 1920 accccttgaa ggagggacaa ggctgaccat atgtggctgg gactttggat ttcggaggaa 1980 taataaattt gatttaaaga aaactagagt tctccttgga aatgagagct gcaccttgac 2040 tttaagtgag agcacgatga atacattgaa atgcacagtt ggtcctgcca tgaataagca 2100 tttcaatatg tccataatta tttcaaatgg ccacgggaca acacaataca gtacattctc 2160 ctatgtggat cctgtaataa caagtatttc gccgaaatac ggtcctatgg ctggtggcac 2220 tttacttact ttaactggaa attacctaaa cagtgggaat tctagacaca tttcaattgg 2280 tggaaaaaca tgtactttaa aaagtgtgtc aaacagtatt cttgaatgtt ataccccagc 2340 ccaaaccatt tcaactgagt ttgctgttaa attgaaaatt gacttagcca accgagagac 2400 aagcatcttc agttaccgtg aagatcccat tgtctatgaa attcatccaa ccaaatcttt 2460 tattagtact tggtggaaag aacctctcaa cattgtcagt tttctatttt gctttgccag 2520 tggtgggagc acaataacag gtgttgggaa aaacctgaat tcagttagtg tcccgagaat 2580 ggtcataaat gtgcatgaag caggaaggaa ctttacagtg gcatgtcaac atcgctctaa 2640 ttcagagata atctgttgta ccactccttc cctgcaacag ctgaatctgc aactccccct 2700 gaaaaccaaa gcctttttca tgttagatgg gatcctttcc aaatactttg atctcattta 2760 tgtacataat cctgtgttta agccttttga aaagccagtg atgatctcaa tgggcaatga 2820 aaatgtactg gaaattaagg gaaatgatat tgaccctgaa gcagttaaag gtgaagtgtt 2880 aaaagttgga aataagagct gtgagaatat acacttacat tctgaagccg ttttatgcac 2940 ggtccccaat gacctgctga aattgaacag cgagctaaat atagagtgga agcaagcaat 3000 ttcttcaacc gtccttggaa aagtaatagt tcaaccagat cagaatttca caggattgat 3060 tgctggtgtt gtctcaatat caacagcact gttattacta cttgggtttt tcctgtggct 3120 gaaaaagaga aagcaaatta aagatctggg cagtgaatta gttcgctacg atgcaagagt 3180 acacactcct catttggata ggcttgtaag tgcccgaagt gtaagcccaa ctacagaaat 3240 ggtttcaaat gaatctgtag actaccgagc tacttttcca gaagatcagt ttcctaattc 3300 atctcagaac ggttcatgcc gacaagtgca gtatcctctg acagacatgt cccccatcct 3360 aactagtggg gactctgata tatccagtcc attactgcaa aatactgtcc acattgacct 3420 cagtgctcta aatccagagc tggtccaggc agtgcagcat gtagtgattg ggcccagtag 3480 cctgattgtg catttcaatg aagtcatagg aagagggcat tttggttgtg tatatcatgg 3540 gactttgttg gacaatgatg gcaagaaaat tcactgtgct gtgaaatcct tgaacagaat 3600 cactgacata ggagaagttt cccaatttct gaccgaggga atcatcatga aagattttag 3660 tcatcccaat gtcctctcgc tcctgggaat ctgcctgcga agtgaagggt ctccgctggt 3720 ggtcctacca tacatgaaac atggagatct tcgaaatttc attcgaaatg agactcataa 3780 tccaactgta aaagatctta ttggctttgg tcttcaagta gccaaaggca tgaaatatct 3840 tgcaagcaaa aagtttgtcc acagagactt ggctgcaaga aactgtatgc tggatgaaaa 3900 attcacagtc aaggttgctg attttggtct tgccagagac atgtatgata aagaatacta 3960 tagtgtacac aacaaaacag gtgcaaagct gccagtgaag tggatggctt tggaaagtct 4020 gcaaactcaa aagtttacca ccaagtcaga tgtgtggtcc tttggcgtgc tcctctggga 4080 gctgatgaca agaggagccc caccttatcc tgacgtaaac acctttgata taactgttta 4140 cttgttgcaa gggagaagac tcctacaacc cgaatactgc ccagacccct tatatgaagt 4200 aatgctaaaa tgctggcacc ctaaagccga aatgcgccca tccttttctg aactggtgtc 4260 ccggatatca gcgatcttct ctactttcat tggggagcac tatgtccatg tgaacgctac 4320 ttatgtgaac gtaaaatgtg tcgctccgta tccttctctg ttgtcatcag aagataacgc 4380 tgatgatgag gtggacacac gaccagcctc cttctgggag acatcatagt gctagtacta 4440 tgtcaaagca acagtccaca ctttgtccaa tggttttttc actgcctgac ctttaaaagg 4500 ccatcgatat tctttgctct tgccaaaatt gcactattat aggacttgta ttgttattta 4560 aattactgga ttctaaggaa tttcttatct gacagagcat cagaaccaga ggcttggtcc 4620 cacaggccac ggaccaatgg cctgcagccg tgacaacact cctgtcatat tggagtccaa 4680 aacttgaatt ctgggttgaa ttttttaaaa atcaggtacc acttgatttc atatgggaaa 4740 ttgaagcagg aaatattgag ggcttcttga tcacagaaaa ctcagaagag atagtaatgc 4800 tcaggacagg agcggcagcc ccagaacagg ccactcattt agaattctag tgtttcaaaa 4860 cacttttgtg tgttgtatgg tcaataacat ttttcattac tgatggtgtc attcacccat 4920 taggtaaaca ttccctttta aatgtttgtt tgttttttga gacaggatct cactctgttg 4980 ccagggctgt agtgcagtgg tgtgatcata gctcactgca acctccacct cccaggctca 5040 agcctcccga atagctggga ctacaggcgc acaccaccat ccccggctaa tttttgtatt 5100 ttttgtagag acggggtttt gccatgttgc caaggctggt ttcaaactcc tggactcaag 5160 aaatccaccc acctcagcct cccaaagtgc taggattaca ggcatgagcc actgcgccca 5220 gcccttataa atttttgtat agacattcct ttggttggaa gaatatttat aggcaataca 5280 gtcaaagttt caaaatagca tcacacaaaa catgtttata aatgaacagg atgtaatgta 5340 catagatgac attaagaaaa tttgtatgaa ataatttagt catcatgaaa tatttagttg 5400 tcatataaaa acccactgtt tgagaatgat gctactctga tctaatgaat gtgaacatgt 5460 agatgttttg tgtgtatttt tttaaatgaa aactcaaaat aagacaagta atttgttgat 5520 aaatattttt aaagataact cagcatgttt gtaaagcagg atacatttta ctaaaaggtt 5580 cattggttcc aatcacagct cataggtaga gcaaagaaag ggtggatgga ttgaaaagat 5640 tagcctctgt ctcggtggca ggttcccacc tcgcaagcaa ttggaaacaa aacttttggg 5700 gagttttatt ttgcattagg gtgtgtttta tgttaagcaa aacatacttt agaaacaaat 5760 gaaaaaggca attgaaaatc ccagctattt cacctagatg gaatagccac cctgagcaga 5820 actttgtgat gcttcattct gtggaatttt gtgcttgcta ctgtatagtg catgtggtgt 5880 aggttactct aactggtttt gtcgacgtaa acatttaaag tgttatattt tttataaaaa 5940 tgtttatttt taatgatatg agaaaaattt tgttaggcca caaaaacact gcactgtgaa 6000 cattttagaa aaggtatgtc agactgggat taatgacagc atgattttca atgactgtaa 6060 attgcgataa ggaaatgtac tgattgccaa tacaccccac cctcattaca tcatcaggac 6120 ttgaagccaa gggttaaccc agcaagctac aaagagggtg tgtcacactg aaactcaata 6180 gttgagtttg gctgttgttg caggaaaatg attataacta aaagctctct gatagtgcag 6240 agacttacca gaagacacaa ggaattgtac tgaagagcta ttacaatcca aatattgccg 6300 tttcataaat gtaataagta atactaattc acagagtatt gtaaatggtg gatgacaaaa 6360 gaaaatctgc tctgtggaaa gaaagaactg tctctaccag ggtcaagagc atgaacgcat 6420 caatagaaag aactcgggga aacatcccat caacaggact acacacttgt atatacattc 6480 ttgagaacac tgcaatgtga aaatcacgtt tgctatttat aaacttgtcc ttagattaat 6540 gtgtctggac agattgtggg agtaagtgat tcttctaaga attagatact tgtcactgcc 6600 tatacctgca gctgaactga atggtacttc gtatgttaat agttgttctg ataaatcatg 6660 caattaaagt aaagtgatgc aacatcttgt aaaaaaaaaa aaaaaaaaaa 6710

Claims (14)

A complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) L2 including the amino acid sequence represented by SEQ ID NO: 2, and a complementarity determining region An antibody light chain variable region (VL) comprising a crystal region (CDR) L3 and a complementary crystal region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO: 4, a complementary crystal region containing an amino acid sequence represented by SEQ ID NO: 5 An antibody or antibody that specifically binds to a human-derived c-Met protein comprising an antibody heavy chain variable region (VH) comprising a complementarity determining region (CDR) H2 and a complementary crystal region (CDR) H3 comprising an amino acid sequence represented by SEQ ID NO: That piece.
The antibody or fragment thereof according to claim 1, wherein the fragment is a fragment selected from the group consisting of diabodies, Fab, Fab ', F (ab) 2, F (ab') 2, Fv and scFv.
A polynucleotide encoding the antibody of claim 1 or a fragment thereof.
A vector comprising the polynucleotide of claim 3.
A cell transformed with the vector of claim 4.
Culturing the cells of claim 5 under a condition that the polynucleotide is expressed to produce a polypeptide comprising a light chain and a heavy chain variable region and recovering the polypeptide from the cell or a culture medium in which the polypeptide is cultured A method for producing an antibody or a fragment thereof that binds to c-Met.
A c-Met specific detection method comprising contacting the antibody of claim 1 or a fragment thereof with a sample and detecting the antibody or fragment thereof.
a) contacting the sample obtained from the subject with the antibody of claim 1;
b) separating the complex formed by binding the antibody of claim 1 to the sample from the non-complexed part; And
and c) obtaining a complex isolated in step b).
[9] The method according to claim 8, wherein the sample in step a) is selected from the group consisting of tissue, blood, serum, plasma, saliva, mucous membrane fluid and urine.
[Claim 9] The method according to claim 8, wherein the antibody of step a) is further attached to a surface of the antibody selected from the group consisting of beads, magnetic beads and a magnetic material.
11. The method of claim 10, wherein the magnetic material is Co, Mn, Fe, Ni, Gd, MM _'2 O _4, and MxOy (M or M' = Co, Fe, Ni, Mn, Zn, Gd, or Cr, x and y Represents an integer.) &Lt; / RTI &gt;
[9] The detection method according to claim 8, wherein the complex of step b) is specifically bound to a circulating cancer cell having c-Met on its surface and an antibody in the sample.
A composition for detecting circulating tumor cells (CTC) comprising the antibody of claim 1 as an active ingredient.
A kit for detecting circulating tumor cells (CTC) comprising the antibody of claim 1 as an active ingredient.

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