KR101859576B1 - Antibodies for specifically detecting KIF5B-RET fusion protein and uses thereof - Google Patents

Abstract

The present invention relates to an antibody that specifically detects a KIF5B-RET fusion protein and a use thereof, and more particularly to an antibody that specifically detects a KIF5B-RET fusion protein or a variant thereof and a composition for diagnosing lung cancer comprising the antibody And a kit, and provides a method of providing information on lung cancer diagnosis using the antibody.

Description

Antibodies specifically detecting KIF5B-RET fusion proteins and uses thereof KIF5B-RET fusion proteins and uses thereof < RTI ID = 0.0 >

The present invention relates to an antibody that specifically detects a KIF5B-RET fusion protein and a use thereof, and more particularly to an antibody that specifically detects a KIF5B-RET fusion protein or a variant thereof and a composition for diagnosing lung cancer comprising the antibody And a kit, and provides a method of providing information on lung cancer diagnosis using the antibody.

Lung cancer is one of the leading causes of cancer death worldwide. Lung cancer is classified into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Non-small cell lung cancer is classified into adenocarcinoma, squamous cell carcinoma, And epithelial cancers. The types of these different types of tissues, which are likely to occur, various types of clinical features such as progression type, speed, and symptoms, and treatment methods are also various.

In particular, non-small cell lung cancer accounts for about 85% of lung cancer. However, surgical treatment of the tumor is the only effective treatment because the chemotherapy alone can not cure the disease. However, lung cancer usually causes symptoms such as invasion of surrounding tissues or airway obstruction due to the growth of cancer cells, lymph node metastasis, and most lung cancer is diagnosed in a state of progression, .

Therefore, it is necessary to diagnose lung cancer early and reduce mortality from lung cancer.

Smoking is a major risk factor for lung cancer, but the incidence of lung cancer in non-smokers is often due to genetic changes. For example, somatic mutations of EGFR, KRAS and ALK genes (so called triple markers) in non-small cell lung cancers have been found in lung cancer patients. Recently, the inventors of the present invention have firstly elucidated the existence of a KIF5B-RET fusion gene generated by fusion of KIF5B gene and RET gene by chromosome inversion as a result of DNA analysis from a sample of lung cancer patients (Genome Res. 2012. 22: 436-455). In addition, this fusion gene is also detected in a cancer that shows a negative response to a known triple marker, and it can be used as an effective marker for lung cancer diagnosis because it is detected in liver or bone metastasis as well as lung tissue as a primary lesion.

Thus, although the importance of the KIF5B-RET fusion gene as a new marker of lung cancer has emerged, there is little research on its function. As an antibody used to detect KIF5B-RET fusion gene using an immune response in lung cancer tissue, an antibody that reacts with the end of RET protein is known (Hidefumi Sasaki et al., Cancer Medicine 1 (1): 68 -75, 2012), this antibody fails to detect various variant types of the KIF5B-RET fusion gene and fails to reproduce the degree of gene expression in clinical practice.

Therefore, it is necessary to develop an antibody capable of specifically detecting the KIF5B-RET fusion gene as well as detecting various mutants thereof, and this antibody not only diagnoses lung cancer, but also identifies the function of the KIF5B-RET fusion gene in lung cancer It is expected to be used.

Young Seok Ju et al., Genome Res. 22: 436-445 (published online on Dec. 22, 2011) Hidefumi Sasaki et al., Cancer Medicine 1 (1): 68-75 (published online on July 12, 2012)

Accordingly, the present inventors completed the present invention by developing an antibody capable of specifically detecting KIF5B-RET fusion gene as well as detecting various mutants thereof.

Accordingly, one object of the present invention is to provide an antibody that specifically binds to a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (SQEDPK), or a fragment comprising the antigen binding site thereof.

It is another object of the present invention to provide a composition for diagnosing lung cancer comprising the antibody or a fragment comprising the antigen binding site thereof.

Another object of the present invention is to provide a kit for diagnosing lung cancer, which comprises the antibody or a fragment containing the antigen binding site thereof.

It is yet another object of the present invention to provide a method for detecting a KIF5B-RET fusion protein or its variant of an isolated biological sample of an individual suspected of lung cancer using the antibody or a fragment containing the antigen binding site thereof through an antigen- And a method for providing information for lung cancer diagnosis.

For example, the present invention relates to an antibody that specifically binds to a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (SQEDPK) or a fragment comprising an antigen binding site thereof.

In another specific example, the antibody may comprise the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6.

In another specific example, the antibody may comprise the heavy chain of SEQ ID NO: 7 and the light chain of SEQ ID NO: 8.

In another specific example, the antibody or fragment comprising its antigen binding site can specifically detect KIF5B (rearranged during transfection) fusion protein or variant thereof.

In another aspect, the present invention relates to a composition for diagnosing lung cancer comprising the antibody or a fragment comprising an antigen binding site thereof.

In another aspect, the present invention relates to a kit for lung cancer diagnosis comprising the antibody or fragment comprising the antigen binding site thereof.

In another aspect, another object of the present invention is to provide a method for screening a KIF5B-RET fusion protein or a variant thereof of an isolated biological sample of a subject suspected of lung cancer using the antibody or a fragment comprising the antigen binding site thereof, The method comprising the steps of: detecting the presence or absence of cancer in the lung cancer;

Hereinafter, the present invention will be described in more detail.

As used herein, the term "antibody" means a substance produced by stimulation of an antigen in the immune system, and the type thereof is not particularly limited. In the present invention, the antibody includes all monoclonal antibodies, polyclonal antibodies, recombinant antibodies, animal antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two antibodies . In the present invention, an antibody also includes an antigen-binding fragment of an antibody having an antigen-binding ability. For purposes of the present invention, an antibody is an antibody that specifically binds to an antigenic epitope of SEQ ID NO: 2, or an antibody that specifically detects a KIF5B-RET fusion protein, comprising an antibody produced by the antigenic peptide of SEQ ID NO: 1, As well as their antigen binding fragments.

A complete antibody is a structure with two full length light chains and two full length heavy chains, each light chain linked by a disulfide bond with a heavy chain.

Means a fragment having an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv or single chain antibody molecule molecules, and the like. Among the antibody-binding fragments, Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. F (ab ') differs from Fab in that it has a hinge region containing at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F (ab ') ₂ is produced by disulfide bonding of cysteine residues in the hinge region of Fab'. Fv refers to the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme. For example, when the whole antibody is cleaved with papain, a Fab can be obtained. When cut with pepsin, an F (ab ') ₂ fragment can be obtained, Can be produced through recombinant DNA technology.

As used herein, the term "variable region" means a portion of an antibody molecule that exhibits a number of mutations in the sequence while specifically binding to an antigen, and the variable region includes complementarity determining regions (CDRs) CDR2 and CDR3 are present. A framework region (FR) portion exists between the CDRs to support the CDR ring.

As used herein, the term "complementarity determining region" refers to a region that imparts binding specificity with an antigen among the variable regions of the heavy chain and light chain of the antibody. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide the major contact residues in binding the antibody to the antigen or epitope. In the present specification, the terms "specifically binding" or "specifically recognizing" are the same as those conventionally known to those skilled in the art, and the antigen and the antibody specifically interact to effect an immunological reaction .

In the present invention, the term "epitope " refers to a site that determines the antigen specificity present in an antigen molecule, and may be used in combination with an" antigenic determinant "

As used herein, the term "heavy chain" refers to a variable region domain V _H comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen and three constant region domains C _H 1, C _H 2 and C _H 3, Lt; RTI ID = 0.0 > and / or < / RTI > fragments thereof.

As used herein, the term "hinge region" refers to a region contained in the heavy chain of an antibody that is between the CH1 and CH2 regions and which functions to provide flexibility of the antigen binding site in the antibody it means.

The term "light chain" as used herein also refers to both the full length light chain and its fragments, including variable region domains V _L and constant region domains C _L comprising amino acid sequences with sufficient variable region sequences to confer specificity to the antigen do.

IgG, IgD, IgG, IgG (IgG1, IgG1, IgG2, IgG1, IgG2, IgG3, IgG3, IgG3, IgG3, IgG3, , IgG2, IgG3, IgG4), IgM, etc.), and may be, for example, from a light chain constant region and / or a heavy chain constant region of all subtypes of immunoglobulins.

When the antibody of the present invention is a monoclonal antibody, it can be easily produced by a known monoclonal antibody production technique. For example, a method for producing a monoclonal antibody can be performed by preparing a hybridoma using B lymphocytes obtained from an immunized animal (Koeher and Milstein, 1976, Nature, 256: 495), phage display ) Technology, but is not limited thereto.

In the present invention, the antigen peptide used for preparing the antibody comprises an 8-mer amino acid sequence present at a site where the 16th exon of KIF5B and the 12th exon of RET are fused, and is shown in SEQ ID NO: 1. In a preferred embodiment, the antibody of the present invention is obtained from the serum of an animal immunized with the peptide of SEQ ID NO: 1.

Since the antibody or antigen-binding fragment thereof of the present invention recognizes the fusion site of the KIF5B-RET fusion protein, it can be used to diagnose lung cancer by specifically detecting KIF5B-RET fusion protein existing in the body or in a biological sample , And even if the structure of the KIF5B-RET fusion protein is somewhat modified in lung cancer patients, the detection possibility for these mutants is very high.

The KIF5B-RET fusion protein to be detected herein is a fusion protein in which KIF5B is located in the 5 'direction and RET is located in the 3' direction. Recently, KIF5B gene and RET gene have been fused by the chromosomal inversion And the resulting KIF5B-RET fusion gene was reported to be present.

The KIF5B protein, also called the kinesin-1 heavy chain, is encoded by the KIF5B gene. The KIF5B protein can be derived from mammals, including humans. The human KIF5B gene encoding the human KIF5B protein is located on chromosome 10 (10q11.22) and contains 26 exons. The human KIF5B protein is encoded in the NCBI by the KIF5B gene, which is represented by gene number NM_004521.

For example, KIF5B as a fusion partner of a KIF5B-RET fusion protein herein may comprise an amino acid sequence encoded by a polynucleotide of a first exon to a 16th exon. For example, the KIF5B protein may comprise at least about 329 consecutive amino acids from the first position of the KIF5B protein encoded by NM_004521. In addition, the KIF5B protein may further comprise two or more double helical domains starting from the amino acid at position 329 of the KIF5B protein encoded by NM_004521. For example, the two double helical domains may further have an amino acid sequence at position 329 to 638 of the KIF5B protein encoded by NM_004521.

Thus, in another example, the KIF5B protein as a fusion partner comprises about 329 to about 900 consecutive amino acids, about 329 to about 700 consecutive amino acids, about 329 to about 650 consecutive amino acids, Amino acids, or consecutive about 329 to 638 amino acids.

The numbers of exons used in the present invention are numbered according to the number of exons located in NCBI.

The RET protein is a transmembrane receptor tyrosine kinase. RET consists of an intracellular domain comprising the extracellular domain (including the Cadherin-like domain), the transmembrane domain, and the tyrosine kinase domain. The RET protein can be derived from mammals, including humans. The human RET gene encoding the human RET protein is located on chromosome 10 (10q11.2) and contains 24 exons, depending on the species. The human RET protein can be encoded from the human RET gene, which is represented by NCBI (Gene Accession No. NM_020630 or NM_020975).

For example, RET as a fusion partner of a KIF5B-RET fusion protein herein may comprise an amino acid sequence encoded by the polynucleotide of the last exon in the 12th exon (exon 12) to the C terminus. For example, towards the C-terminus of the RET protein encoded by NM_020630 or NM_020975 from the start position of the twelfth exon (e. G., Position 713 of the RET protein encoded by NM_020975), at least about 300 consecutive Lt; / RTI > amino acids. For example, the C-terminal domain of the RET protein may contain a RET protein encoded by NM_020630 (19 exons) or NM_020975 (20 exons) from the start of the 12th exon (eg, 713th digit) Towards the C-terminus, about 300-450 consecutive amino acids, about 300-420 consecutive amino acids, or about 300-440 consecutive amino acids.

Fusion in the KIF5B-RET fusion protein may occur between the 16th exon of the KIF5B gene and the 12th exon of the RET gene, but is not limited thereto. For example, the 15th exon of the KIF5B gene and the 12th exon of the RET gene, Fusion at various sites such as the 22nd exon of the KIF5B gene and the 12th exon of the RET gene, the 23rd exon of the KIF5B gene, the 12th exon of the RET gene, the 24th exon of the KIF5B gene and the 11th exon of the RET gene are possible .

As a preferred example, the KIF5B-RET fusion protein to be detected herein may refer to a fusion protein between the 16th exon of the KIF5B gene and the 12th exon of the RET gene, which have been reported to date in lung cancer patients . As well as various variations thereof. In a preferable example, the mutation form is a fusion protein in which a fusion between the 16th exon of the KIF5B gene and the 12th exon of the RET gene occurs, and the remaining part except for the fusion site, for example, the N-terminal part, the C- , Or variants in which some amino acids in the internal region other than the fusion region are deleted, inserted and / or substituted.

Furthermore, it can be useful for various research purposes, for example, in the study of KIF5B-RET fusion protein-related diseases.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of lung cancer, and further to ascertain whether the disease progresses or not.

Herein, lung cancer includes small cell carcinoma and non-small cell carcinoma, and non-small cell carcinoma includes histology such as adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and squamous cell carcinoma. Preferably, it may be non-small cell lung cancer, more preferably, lung cancer of non-small cell lung cancer, but is not limited thereto.

In the present invention, a method for detecting a KIF5B-RET fusion protein can be performed by treating the antibody of the present invention with a sample of a control group (for example, a healthy person) and an experimental group (for example, The level of the antigen-antibody reaction in the present invention refers to the amount of the KIF5B-RET fusion protein in the sample and the amount of the antibody or its antigen-binding site recognizing the KIF5B-RET fusion protein in the sample, Means the amount of complex. The level of the reaction of such an antigen-antibody can be compared using an unlimited number of assay methods commonly used in the art, for example quantitatively measuring the size of the signal of a detection label.

Immunoassays may include any method capable of measuring the binding of an antigen to an antibody. Such methods are well known in the art and include, for example, Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunofluorescence assay Immunoblotting, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complete fixation assay, FACS (immunohistochemistry) Fluorescence Activated Cell Sorting, or protein chip, but are not limited thereto.

Preferably, the KIF5B-RET fusion protein in the sample can be detected using an ELISA method.

The ELISA can be performed by direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of antibody and antigen attached to the solid support or with another antibody recognizing the antigen in the complex of antibody and antigen attached to the solid support And indirect sandwich ELISA using a labeled secondary antibody recognizing the antibody. For example, an antibody may be attached to a solid support, reacted with a sample, labeled with an antibody that recognizes an antigen of an antigen-antibody complex, and then labeled with an antibody that recognizes the antigen of the antigen-antibody complex The secondary antibody can be detected and detected by a sandwich ELISA method in which the secondary antibody is enzymatically developed. The KIF5B-RET fusion protein in the sample can be detected by confirming the complex formation degree of the antigen and the antibody.

The term "biological sample" in the present invention refers to a tissue, cell, whole blood, plasma, serum, blood, saliva, synovial fluid, urine, sputum, lymph, cerebrospinal fluid, tissue autopsy sample (brain, skin, lymph node, Culture supernatant, or ruptured eukaryotic cells, and includes samples derived from metastatic lesions as well as primary lesions of cancer. These biological samples can be reacted with the antibody of the present invention without manipulation or manipulation to confirm the presence of KIF5B-RET fusion protein and the presence or absence of lung cancer.

The term "individual" in the present invention includes mammals, birds, and the like, including cows, pigs, sheep, chickens, dogs,

The lung cancer diagnostic kit of the present invention may further comprise tools or reagents known in the art used for immunological analysis in addition to the antibody against the KIF5B-RET fusion protein.

Immunological analysis may include any method capable of measuring the binding of an antigen to an antibody. Such methods are well known in the art and include, for example, Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, immunofluorescence, immunoblot, ooclonus immunodiffusion, rocket immunoelectrophoresis, Assay methods, complement fixation assays, FACS, protein chips, and the like, but are not limited thereto.

Tools or reagents used in immunological assays may include suitable carriers or supports, labels capable of generating a detectable signal, solubilizers, detergents, and stabilizers, and the like. Suitable carriers include, but are not limited to, a substrate capable of measuring the enzyme activity, a suitable buffer solution, a secondary antibody labeled with a chromogenic or fluorescent substance, a chromogenic substrate, and a reaction stop when the labeling substance is an enzyme And the like.

Antibodies to KIF5B-RET fusion proteins, included in the kits of the present invention, can be immobilized, preferably using a variety of methods as described in the literature, to suitable carriers or supports ( Antibodies : A Labotory Manual, Harlow & Cold Spring Harbor, 1988). Examples of suitable carriers or supports are PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, But are not limited to, liposomes, liposomes, carboxymethylcellulose, polyacrylamide, polysterine, gabbro, filter paper, ion exchange resins, plastic films, plastic tubes, polyamine- Nylon, metal, glass, glass beads, or magnetic particles. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip).

A label capable of producing a detectable signal enables the formation of an antigen-antibody complex to be qualitatively or quantitatively measurable, and examples thereof include an enzyme, a fluorescent substance, a ligand, a luminescent material, a microparticle, a redox molecule, Isotopes and the like can be used. Examples of the enzymes include? -Glucuronidase,? -D-glucosidase, urease, peroxidase (such as hosadradia peroxidase), alkaline phosphatase, acetylcholinesterase, glycosidoxidase, Dehydrogenase, glucose-6-phosphate dehydrogenase, invertase and the like can be used. Examples of the fluorescent substance include fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, fluorine synthase, and the like. Examples of the ligand include biotin derivatives, and examples of the luminescent material include acridinium ester, luciferin, and luciferase. Examples of the fine particles include colloidal gold and colored latex. Examples of redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like. Radioactive isotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re. However, in addition to those exemplified above, any of them can be used as long as they can be used for immunological assays.

As an enzyme coloring substrate, for example, when hosadradici peroxidase (HRP) is selected as the enzyme label, 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o (3-ethylbenzothiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3 ', 5,5'-tetramethylbenzidine, o- Dianisidine, or 3,3-dimethoxybenzidine-containing solution may be used. Further, when alkaline phosphatase is selected as the enzyme label, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, or p-nitrophenyl phosphate as a substrate can be used. When β-D-galactosidase is selected as the enzyme label, o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indole- Lactopyranoside-containing solutions may be used. In addition, various enzyme and enzyme chromogenic substrates known in the art can be used.

The antibodies provided in the present invention can be used to detect KIF5B-RET fusion proteins and thus can be used for diagnosis of lung cancer and further useful as an experimental material in laboratories to study the function of KIF5B-RET fusion protein in lung cancer Can contribute to the development of therapeutic agents for diseases.

1 shows the cleavage map of the lentiviral vector pLOC, and B shows the construct of the KIF5B-RET fusion gene.
FIG. 2 shows the results of confirming that the KIF5B-RET fusion gene was correctly expressed through the lentiviral vector cloned with the KIF5B-RET fusion gene.
FIG. 3 shows the result of confirming the expression of the fusion protein in the HEK-293T cell line transfected with the lentiviral vector cloned with the KIF5B-RET fusion gene without fluorescence (DIC: Differential interference contrast, left photograph) (Right picture).
FIG. 4A is a photograph showing a tumor formation in a xenograft animal model prepared using a KIF5B-RET fusion gene transfected cell line, and B shows a result of confirming the expression of a KIF5B-RET fusion protein in a tumor tissue formed.
FIG. 5 shows the amino acid sequence (SEQ ID NO: 4) of the KIF5B-RET fusion protein prepared and the site where the antigen peptide selected for antibody production is located.
FIG. 6 shows the result of confirming hydrophobicity and antigenicity of the whole KIF5B-RET fusion protein, and the red circle shows the result of analyzing the antigenicity of the antigen peptide region.
FIG. 7 shows results of first ELISA in which rabbit (designated rabbit 1) was subcutaneously injected with an antigen peptide to induce an immune response, and serum was collected at 5th week.
Fig. 8 shows the results of primary ELISA in which rabbit (designated rabbit 2) was subcutaneously injected with an antigen peptide to induce an immune response and then serum was collected at 5th week.
FIG. 9 shows results of secondary ELISA in which sera were collected at week 7 after subcutaneous injection of antigen peptide into rabbit 1 to induce an immune response.
FIG. 10 shows results of second ELISA after subcutaneous injection of antigen peptide into rabbit 2 to induce an immune response and serum at 7th week.
FIG. 11 shows the results of the final ELISA after raising the immune response by subcutaneously injecting the antigen peptide into rabbit 1 and then collecting the serum at the ninth time.
FIG. 12 shows results of a final ELISA in which rabbit (rabbit 2) was subcutaneously injected with an antigen peptide to induce an immune response, and serum was collected at the ninth week.
FIG. 13 shows the structure of a KIF5B-RET long form containing exons 12 to 20 of RET, a structure of KIF5B-RET short form in which a part of RET end (exon 20) is truncated, and a plasmid structure used for expressing them in cells .
FIG. 14A shows that a construct of KIF5B-RET long form containing exons 12-20 of RET and a construct of KIF5B-RET short form in which a part of RET end (exon 20) was cleaved was expressed in the cell line, B shows the result of Western blotting after immunoprecipitation using the anti-c-RET antibody that has been previously marketed to the cell lysate.
FIG. 15 shows the results of a single treatment (denoted by ab) of an antibody (designated as antibody No. 2) isolated from a second rabbit (rabbit 2) to a fragment of HEK293T cell line expressing KIF5B / RET short form expression, Peptide, pre-incubated (denoted by ab + peptide), and then subjected to Western blotting.
FIG. 16 shows the result of Western blotting after treatment of a commercially available antibody with a fragment of HEK293T cell line expressing KIF5B / RET short form expression, and comparing this with the results of FIG.
17 shows the results of immunohistochemical staining of HEK293T cells expressing the KIF5B-RET fusion gene using the anti-c-RET antibody and the anti-KIF5B-RET antibody (antibody No. 2) .
FIG. 18 shows a comparison between the position where the GFP fluorescence transferred together with the KIF5B-RET fusion gene is developed and the DAB coloration reaction position by the anti-KIF5B-RET antibody of FIG.
Figure 19 shows the epitope mapping results of the anti-KIF5B-RET antibody.
FIG. 20 shows three peptide regions exhibiting strong affinity and one peptide region exhibiting the weakest affinity for the anti-KIF5B-RET antibody against the fusion protein of KIF5B-RET.
FIG. 21 shows the results of SDS-PAGE of the purified anti-KIF5B-RET antibody for sequencing of the anti-KIF5B-RET antibody and then identifying the bands corresponding to the heavy chain and the light chain, respectively.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1 Establishment of Transformed Cell Lines Using Lentiviral Vectors

The lentiviral system not only enables infection in both cleaved and non-cleaved cells, but also permits sustained and stable gene expression. Herein, the construct of the KIF5B-RET fusion gene in which the exotoxin 16 of KIF5B and the exon 12-19 of RET are fused to the Multi Tag Cloning Site of the lentivirus system pLOC (Fig 1A) (Surin Bioscience, Korea) 1B; SEQ ID NO: 3) was inserted to prepare a lentiviral system in which a KIF5B-RET fusion gene was cloned. The above procedure was performed by asking Surin Bioscience (Korea).

The constructed KIF5B-RET fusion gene construct For confirmation, the two restriction enzymes used in the viral vector production were reacted at 37 ° C for one hour. Thereafter, it was confirmed by electrophoresis on 1% agarose gel that a KIF5B-RET fusion gene of 3000 bp or more was inserted into the viral vector (FIG. 2). As a result of comparison with the inserted construct, the expressed protein expressed KIF5B / RET It is confirmed that this is due to a fusion gene.

A 50% dense HEK (human embryonic kidney) -293T cell line (purchased from ATCC), which had been previously cultured and diluted with 100 μl of the lentivirus containing the constructed KIF5B-RET fusion gene, To the culture dish. After about 24 hours, the culture medium was removed, and the cells were washed with PBS (phosphate buffer saline), and a new culture medium was added. After 3 days, the expression level of GFP was confirmed by fluorescence microscopy and the degree of transformation of cells by lentivirus was confirmed (Fig. 3).

Therefore, a transformed cell line expressing the KIF5B-RET fusion gene was established by transfecting the KIF5B-RET fusion gene with the lentivirus system in which the KIF5B-RET fusion gene was cloned.

Example  2. KIF5B -RET fusion gene transfected cell line Tumor formation ability  Confirm

An xenograft animal model was prepared using the KIF5B-RET fusion gene transfected cell line prepared in Example 1 above. 1 x ¹⁰⁶ transgenic cell lines were transplanted subcutaneously in the upper back of 4-week-old nude mice and periodically observed to confirm tumor formation ability. Tumor formation capacity was determined by volumetric measurement and the short axis and long axis length were measured and calculated as the short axis * long axis / 2. As a result, it was confirmed that the same number of HEK293 cells that were not transformed with the fusion gene (i.e., including an empty vector) had a faster tumor growth rate than the subcutaneously transplanted xenograft model (Fig. 4A).

In order to confirm the expression of the KIF5B-RET fusion protein in the tumor tissue formed, the tumor tissues of the animal model transformed with the fusion gene and the animal model using the empty vector without the fusion gene were separated and digested, and genomic DNA Respectively. The expression of KIF5B-RET fusion gene was confirmed by PCR and electrophoresis using DNA extracted from each tissue as a template. DNA of a known sequence used in the construct was used as a positive control. As a result, in the tumor tissue of the animal model transformed with the fusion gene Expression of the KIF5B-RET fusion protein was confirmed (FIG. 4B).

Example 3. Preparation of antibody against KIF5B-RET fusion gene

3-1. Selection of antigen peptides

First, it was confirmed that there is no transmembrane domain and signal peptide in the KIF5B-RET fusion gene (including the fusion region between exon 16 of KIF5B and exon 12 of RET). In addition, the domain portion was excluded to exclude the possibility of cross reactivity with other proteins. Thus, the kinesin motor domain of KIF5B and the protein kinase domain of RET were excluded. Glycosylation site, myristoylation site, and phosphorylation site were excluded from the epitope for antibody production due to high possibility of structural variation by post-translational modification. Finally, a peptide was prepared by targeting a site where fusion of KIF5B-RET fusion protein was formed, and then an antibody was produced (FIG. 5). Generally, the length of the antigen peptide is selected to be more than 10 mer. However, in the fusion region of the KIF5B-RET fusion protein, when the peptide has a long length, it is considered that there is a possibility of producing an antibody that detects only the anterior or posterior part. (RISQEDPK; SEQ ID NO: 1). Based on the nucleotide sequence of the entire fusion protein, using a peptide analysis system As a result of confirming hydrophobicity and antigenicity, it was confirmed that the peptide portion selected above has a possibility of producing an antigen (Fig. 6).

3-2. Preparation of Antibodies to KIF5B-RET Fusion Proteins

1 mg of the peptide of SEQ ID NO: 1 was mixed with CFA (Complete Freund's adjuvant) in two rabbits (rabbit 1, rabbit 2, respectively, 8 to 10 week old male New Zealand white rabbit supplied by Koatec) Each of them was subcutaneously injected to induce an immune response. After 4 weeks, 500 ug of the peptide was injected subcutaneously with IFA (Incomplete Freund's adjuvant) to activate the immune response once more. One week later (week 5), 1 ml of serum was collected from the ear vain and immunity to the antigen was confirmed by primary ELISA analysis. Specifically, peptides prepared as antigens were diluted to 2 ug / ml at a concentration of 50 쨉 l per well in a 96-well plate, followed by storage at 4 째 C for 12 hours, followed by additional storage at 37 째 C for 3 hours. Serum isolated from the rabbits was diluted in various ratios, and 100 ul of each antigen was added to the plate. The mixture was reacted at 37 ° C for 2 hours. The secondary antibody with HRP was further reacted at 37 ° C for 1 hour, and the color development of TBM was induced and the degree of color development was analyzed with a colorimeter. ELISA of the first serum revealed that the OD value was 1.0 or more at 1: 1000 dilution fold, and that the immunizing effect was obtained (FIGS. 7 and 8).

At 6 weeks, 500 μg of peptide was injected subcutaneously again with IFA to induce activation of the second immune response. One week later (week 7), 1 ml of serum was collected from the ear vain and secondary ELISA analysis was performed in the same manner as above. As a result, the OD value was higher than that of the first bleeding ELISA, and it was confirmed that it had an immunization effect (FIGS. 9 and 10).

At week 8, 500 ug of the peptide was injected subcutaneously again with IFA for the third final immunization reaction, and the final antigenic serum (50 ml) was obtained through final whole blood after 1 week (9 weeks) by sacrificing the heart (puncture). The immune response was confirmed by the final ELISA analysis in the same manner as above. As a result, the OD value was higher than that of the second bleeding ELISA, and it was confirmed that the slope of the graph showing titer was gentle, indicating that it had an immunization effect (FIGS. 11 and 12).

After the final ELISA analysis above, the antibodies were finally separated from each of the two rabbits and named 1 antibody (KR1) and 2 antibody (KR2), respectively.

Example 4. Validation of the efficacy of anti-KIF5B-RET antibody

4-1. Detection of KIF5B-RET fusion gene using anti-c-RET antibody

A construct of a KIF5B-RET long form containing exons 12 to 20 of RET and a KIF5B-RET short form having a portion of RET end (exon 20) cleaved was prepared (Fig. 13). Specifically, genes for KIF5B-RET long form and KIF5B-RET short form were synthesized from Macrogen (Korea). The pcDNA3 vector was digested with KpnI and XbaI restriction enzymes, and the terminal region of the fusion gene was cleaved with the same restriction enzyme. Then, the ligated gene was ligated with ligase enzyme. The confirmation of the connection was confirmed by sequencing analysis and it was confirmed that the construction with proper direction and length was made. This pcDNA-based construct was transformed into NIH3T3 cell line using transfection reagent (Lipofectamin, Life technology), and DNA and protein were extracted. From the extracted DNA, it was confirmed that the gene was expressed in both long and short forms by PCR. The DNA of the identified sequence used in constructing the construct was used as a positive control (Fig. 14A).

Next, Western blotting was performed after immunoprecipitation using an anti-c-RET antibody (cell signaling company, USA) that was previously sold on the lysate of stomach cells, and the antibody was detected with KIF5B. Specifically, the cells were lysed with RIPA buffer containing phosphatase and protease inhibitor, and the intracellular proteins were separated using a centrifuge. A portion of the protein was electrophoresed on 10-12% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Bioscience). The blotted membrane was incubated with blocking buffer (1 × TBS, 0.1% Tween-20, 5% And reacted with the corresponding primary antibody for 12 hours. After reacting for 1 hour with secondary antibody, reaction was carried out with chemiluminescence detection kit (ECL, Amersham Bioscience). Protein expression was confirmed using a multi-image analysis system. As a result, although KIF5B-RET long form and KIF5B-RET short form genes were expressed in cells, only KIF5B-RET long form was detected in Western blot (FIG. 14B). This suggests that the presence of KIF5B-RET mutations in the patient sample can not detect all of these mutations with conventional antibodies.

4-2. Detection of the KIF5B-RET fusion gene using the anti-KIF5B-RET antibody of the present application

Western blot analysis was performed using the lysates of HEK293T cells in which the KIF5B-RET short form was confirmed to express the two antibodies prepared in Example 3, as described above. The membrane was directly reacted with each of the two antibodies, and the membrane was pre-incubated with the antigen peptide (SEQ ID NO: 1). As a result, as shown in FIG. 15, in the case of the membrane reacted with the antibody # 2, the intensity of the band indicated by the arrow was decreased by the competition reaction of the peptide, and the target band, that is, KIF5B-RET short form was expressed (Figure 15).

In addition, western blot analysis was performed on an antibody (cell signaling company, USA) which was previously sold in the same manner. As a result of comparing with the membrane reacted with antibody No. 2 as described above, it was found that the target band was not observed in the membrane reacted with the existing antibody, and KIF5B-RET fusion protein could not be detected (FIG. 16).

4-3. Validation of the efficacy of anti-KIF5B-RET antibody

Immunocytochemistry was performed on HEK293T cells expressing the KIF5B-RET fusion gene. Specifically, 1% BSA was dissolved in 4% paraformaldehypa for 10 minutes to attach the HEK293T, which had been confirmed to express the KIF5B-RET fusion gene, on the slide for 10 minutes to aid intracellular antibody penetration Of PBS in 0.1% triton-X for 10 minutes. After washing with PBS containing 20% Tween 20, the cells were reacted with blocking buffer containing glycine for 30 minutes in PBS containing 1% BSA. The resulting polyclonal antibody was used for cold reaction overnight with the primary antibody. After reacting with DAB detection kit, the nuclei were stained with hematoxylin.

As a result, as shown in FIG. 17, the DAB coloration reaction was mainly observed in the cytoplasm of the KIF5B-RET fusion protein polyclonal antibody, and a color development reaction was also observed in the nucleus. On the other hand, when the immunocytochemistry was carried out for the antibody (abcam), which has been sold in the same way, a coloring reaction was observed in most of the nuclei.

In addition, as shown in FIG. 18, it was confirmed that the position where the GIF fluorescence emitted when the KIF5B-RET fusion gene was introduced and the DAB coloration reaction site were observed in the cytoplasm by the KIF5B-RET fusion gene polyclonal antibody.

Example 5. Epitope mapping for anti-KIF5B-RET antibodies

For the analysis of the epitope mapping of the KR2 antibody prepared in Example 3 above, a peptide microarray platform PEPperCHIP ( R ) technology ( PepperPrint ( www.pepperprint.com/high-content-peptide-microarrays/ ) PEPperMAP®).

PEPperMAP® technology enables the analysis of many types of peptides by printing amino acids in the form of microparticles. Specifically, nonspecific protein binding is minimized and approximately 8,500 peptide analyzes per slide are possible, and the length of one peptide is analyzed to a length of 15 to 17 amino acids. Therefore, it is suitable for polyclonal antibody analysis by making 9,11,14 amino acid repeated with peptide having 10,12,15 amino acid length.

In this example, a total of 3,002 peptides were prepared so as to include the KIF5B-RET peptide, and 6,004 peptides were synthesized in total. HA (YPYDVPDYAG, 164 spots) was used as a control group.

The KIF5B-RET fusion protein was diluted to 1: 1000 with the polyclonal antibody. The peptide having a strong affinity for the secondary antibody is shown in FIG. 19 as a three-pattern peptide sequence.

As shown in Fig. 19, the peptide having the strongest affinity corresponded to the 6 amino acid sequence of the 8 amino acids of the target peptide RISQEDPK prepared with SQEDPK. Other peptides with a strong affinity for the secondary antibody were analyzed for FQSSTSQE and NYDQKSQE sequences, with the weakest affinity observed for QLDDKD. These three peptides are consistent with the sequence of the KIF5B protein.

Based on the analysis results, the positions of the four peptides in the KIF5B-RET fusion protein were shown as shown in FIG. Thus, it can be confirmed that the anti-KIF5B-RET antibody of the present invention specifically binds to the fusion site of KIF5B and RET.

Example 6. Sequence analysis of anti-KIF5B-RET antibody

For the sequencing of the KR2 antibody prepared in Example 3, a peptide amino acid sequence analyzer (LC coupled ESI-MS / MS) connected to a two-dimensional electrophoresis method performed by the company Proteotech (www.proteometech.com) .

In order to analyze the sequence of the KR2 antibody prepared in Example 3, proteins were isolated by 10% SDS-PAGE, and the heavy chain and light chain ). The isolated heavy and light chains were analyzed by LC-MS using trypsin treatment. The analyzed protein sequences were compared with the NCBI Database using Mascot software (www.matrixscience.com). The amino acid sequences for the heavy chain variable region and the light chain variable region and the heavy and light chains of the anti-KIF5B-RET antibody are shown in Table 1 below.

order Heavy chain
Variable area QPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPXCPPPELLGGPSVFIFPPKPKDTLMISR (SEQ ID NO: 5) Light chain
Variable area MRAPTQLLGLLLLWLPGARCDVVMTQTPSSAFEPVGGTVNIKCQASQSIGTRLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQ (SEQ ID NO: 6) Heavy chain QPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPXCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPXVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 7) Light chain MRAPTQLLGLLLLLWLPGARCDVVMTQTPSSAFEPVGGTVNIKCQASQSIGTRLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCLGEYSYSSGDGIFFWNAFGGGTEVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFP (SEQ ID NO: 8)

<110> CATHOLIC UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Antibodies for specifically detecting KIF5B-RET fusion protein          and uses <130> 1060716 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 1 Arg Ile Ser Gln Glu Asp Pro Lys   1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 2 Ser Gln Glu Asp Pro Lys   1 5 <210> 3 <211> 2997 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > KIF5B-RET construct sequence (Fig. 1B) <400> 3 atggcggacc tggccgagtg caacatcaaa gtgatgtgtc gcttcagacc tctcaacgag 60 tctgaagtga accgcggcga caagtacatc gccaagtttc agggagaaga cacggtcgtg 120 atcgcgtcca agccttatgc atttgatcgg gtgttccagt caagcacatc tcaagagcaa 180 gtgtataatg actgtgcaaa gaagattgtt aaagatgtac ttgaaggata taatggaaca 240 atatttgcat atggacaaac atcctctggg aagacacaca caatggaggg taaacttcat 300 gatccagaag gcatgggaat tattccaaga atagtgcaag atatttttaa ttatatttac 360 tccatggatg aaaatttgga atttcatatt aaggtttcat attttgaaat atatttggat 420 aagataaggg acctgttaga tgtttcaaag accaaccttt cagttcatga agacaaaaac 480 cgagttccct atgtaaaggg gtgcacagag cgttttgtat gtagtccaga tgaagttatg 540 gataccatag atgaaggaaa atccaacaga catgtagcag ttacaaatat gaatgaacat 600 agctctagga gtcacagtat atttcttatt aatgtcaaac aagagaacac acaaacggaa 660 caaaagctga gtggaaaact ttatctggtt gatttagctg gtagtgaaaa ggttagtaaa 720 actggagctg aaggtgctgt gctggatgaa gctaaaaaca tcaacaagtc actttctgct 780 cttggaaatg ttatttctgc tttggctgag ggtagtacat atgttccata tcgagatagt 840 aaaatgacaa gaatccttca agattcatta ggtggcaact gtagaaccac tattgtaatt 900 tgctgctctc catcatcata caatgagtct gaaacaaaat ctacactctt atttggccaa 960 agggccaaaa caattaagaa cacagtttgt gtcaatgtgg agttaactgc agaacagtgg 1020 aaaaagaagt atgaaaaaga aaaagaaaaa aataagatcc tgcggaacac tattcagtgg 1080 cttgaaaatg agctcaacag atggcgtaat ggggagacgg tgcctattga tgaacagttt 1140 gacaaagaga aagccaactt ggaagctttc acagtggata aagatattac tcttaccaat 1200 gataaaccag caaccgcaat tggagttata ggaaatttta ctgatgctga aagaagaaag 1260 tgtgaagaag aaattgctaa attatacaaa cagcttgatg acaaggatga agaaattaac 1320 cagcaaagtc aactggtaga gaaactgaag acgcaaatgt tggatcagga ggagcttttg 1380 gcatctacca gaagggatca agacaatatg caagctgagc tgaatcgcct tcaagcagaa 1440 aatgatgcct ctaaagaaga agtgaaagaa gttttacagg ccctagaaga acttgctgtc 1500 aattatgatc agaagtctca ggaagttgaa gacaaaacta aggaatatga attgcttagt 1560 gatgaattga atcagaaatc ggcaacttta gcgagtatag atgctgagct tcagaaactt 1620 aaggaaatga ccaaccacca gaaaaaacga gcagctgaga tgatggcatc tttactaaaa 1680 gaccttgcag aaataggaat tgctgtggga aataatgatg taaagcagcc tgagggaact 1740 ggcatgatag atgaagagtt cactgttgca agactctaca ttagcaaaat gaagtcagaa 1800 gtaaaaacca tggtgaaacg ttgcaagcag ttagaaagca cacaaactga gagcaacaaa 1860 aaaatggaag aaaatgaaaa ggagttagca gcatgtcagc ttcgtatctc tcaagaggat 1920 ccaaagtggg aattccctcg gaagaacttg gttcttggaa aaactctagg agaaggcgaa 1980 tttggaaaag tggtcaaggc aacggccttc catctgaaag gcagagcagg gtacaccacg 2040 gtggccgtga agatgctgaa agagaacgcc tccccgagtg agcttcgaga cctgctgtca 2100 gagttcaacg tcctgaagca ggtcaaccac ccacatgtca tcaaattgta tggggcctgc 2160 agccaggatg gcccgctcct cctcatcgtg gagtacgcca aatacggctc cctgcggggc 2220 ttcctccgcg agagccgcaa agtggggcct ggctacctgg gcagtggagg cagccgcaac 2280 tccagctccc tggaccaccc ggatgagcgg gccctcacca tgggcgacct catctcattt 2340 gcctggcaga tctcacaggg gatgcagtat ctggccgaga tgaagctcgt tcatcgggac 2400 ttggcagcca gaaacatcct ggtagctgag gggcggaaga tgaagatttc ggatttcggc 2460 ttgtcccgag atgtttatga agaggattcg tacgtgaaga ggagccaggg tcggattcca 2520 gttaaatgga tggcaattga atcccttttt gatcatatct acaccacgca aagtgatgta 2580 tggtcttttg gtgtcctgct gtgggagatc gtgaccctag ggggaaaccc ctatcctggg 2640 attcctcctg agcggctctt caaccttctg aagaccggcc accggatgga gaggccagac 2700 aactgcagcg aggagatgta ccgcctgatg ctgcaatgct ggaagcagga gccggacaaa 2760 aggccggtgt ttgcggacat cagcaaagac ctggagaaga tgatggttaa gaggagagac 2820 tacttggacc ttgcggcgtc cactccatct gactccctga tttatgacga cggcctctca 2880 gaggaggaga caccgctggt ggactgtaat aatgcccccc tccctcgagc cctcccttcc 2940 acatggattg aaaacaaact ctatggtaga atttcccatg catttactag attctag 2997 <210> 4 <211> 998 <212> PRT <213> Artificial Sequence <220> <223> KIF5B-RET fusion protein (Fig. 5) <400> 4 Met Ala Asp Leu Ala Glu Cys Asn Ile Lys Val Met Cys Arg Phe Arg   1 5 10 15 Pro Leu Asn Glu Ser Glu Val Asn Arg Gly Asp Lys Tyr Ile Ala Lys              20 25 30 Phe Gln Gly Glu Asp Thr Val Val Ile Ala Ser Lys Pro Tyr Ala Phe          35 40 45 Asp Arg Val Phe Gln Ser Ser Thr Ser Gln Glu Gln Val Tyr Asn Asp      50 55 60 Cys Ala Lys Lys Ile Val Lys Asp Val Leu Glu Gly Tyr Asn Gly Thr  65 70 75 80 Ile Phe Ala Tyr Gly Gln Thr Ser Ser Gly Lys Thr His Thr Met Glu                  85 90 95 Gly Lys Leu His Asp Pro Glu Gly Met Gly Ile Ile Pro Arg Ile Val             100 105 110 Gln Asp Ile Phe Asn Tyr Ile Tyr Ser Met Asp Glu Asn Leu Glu Phe         115 120 125 His Ile Lys Val Ser Tyr Phe Glu Ile Tyr Leu Asp Lys Ile Arg Asp     130 135 140 Leu Leu Asp Val Ser Lys Thr Asn Leu Ser Val His Glu Asp Lys Asn 145 150 155 160 Arg Val Pro Tyr Val Lys Gly Cys Thr Glu Arg Phe Val Cys Ser Pro                 165 170 175 Asp Glu Val Met Asp Thr Ile Asp Glu Gly Lys Ser Asn Arg His Val             180 185 190 Ala Val Thr Asn Met Asn Glu His Ser Ser Arg Ser Ser Ile Phe         195 200 205 Leu Ile Asn Val Lys Gln Glu Asn Thr Gln Thr Glu Gln Lys Leu Ser     210 215 220 Gly Lys Leu Tyr Leu Val Asp Leu Ala Gly Ser Glu Lys Val Ser Lys 225 230 235 240 Thr Gly Ala Glu Gly Ala Val Leu Asp Glu Ala Lys Asn Ile Asn Lys                 245 250 255 Ser Leu Ser Ala Leu Gly Asn Val Ile Ser Ala Leu Ala Glu Gly Ser             260 265 270 Thr Tyr Val Pro Tyr Arg Asp Ser Lys Met Thr Arg Ile Leu Gln Asp         275 280 285 Ser Leu Gly Gly Asn Cys Arg Thr Thr Ile Val Ile Cys Cys Ser Pro     290 295 300 Ser Ser Tyr Asn Glu Ser Glu Thr Lys Ser Thr Leu Leu Phe Gly Gln 305 310 315 320 Arg Ala Lys Thr Ile Lys Asn Thr Val Cys Val Asn Val Glu Leu Thr                 325 330 335 Ala Glu Gln Trp Lys Lys Lys Tyr Glu Lys Glu Lys Glu Lys Asn Lys             340 345 350 Ile Leu Arg Asn Thr Ile Gln Trp Leu Glu Asn Glu Leu Asn Arg Trp         355 360 365 Arg Asn Gly Glu Thr Val Pro Ile Asp Glu Gln Phe Asp Lys Glu Lys     370 375 380 Ala Asn Leu Glu Ala Phe Thr Val Asp Lys Asp Ile Thr Leu Thr Asn 385 390 395 400 Asp Lys Pro Ala Thr Ala Ile Gly Val Ile Gly Asn Phe Thr Asp Ala                 405 410 415 Glu Arg Arg Lys Cys Glu Glu Glu Ile Ala Lys Leu Tyr Lys Gln Leu             420 425 430 Asp Asp Lys Asp Glu Glu Ile Asn Gln Gln Ser Gln Leu Val Glu Lys         435 440 445 Leu Lys Thr Gln Met Leu Asp Gln Glu Glu Leu Leu Ala Ser Thr Arg     450 455 460 Arg Asp Gln Asp Asn Met Gln Ala Glu Leu Asn Arg Leu Gln Ala Glu 465 470 475 480 Asn Asp Ala Ser Lys Glu Glu Val Lys Glu Val Leu Gln Ala Leu Glu                 485 490 495 Glu Leu Ala Val Asn Tyr Asp Gln Lys Ser Gln Glu Val Glu Asp Lys             500 505 510 Thr Lys Glu Tyr Glu Leu Leu Ser Asp Glu Leu Asn Gln Lys Ser Ala         515 520 525 Thr Leu Ala Ser Ile Asp Ala Glu Leu Gln Lys Leu Lys Glu Met Thr     530 535 540 Asn His Gln Lys Lys Arg Ala Ala Glu Met Met Ala Ser Leu Leu Lys 545 550 555 560 Asp Leu Ala Glu Ile Gly Ile Ala Val Gly Asn Asn Asp Val Lys Gln                 565 570 575 Pro Glu Gly Thr Gly Met Ile Asp Glu Glu Phe Thr Val Ala Arg Leu             580 585 590 Tyr Ile Ser Lys Met Lys Ser Glu Val Lys Thr Met Val Lys Arg Cys         595 600 605 Lys Gln Leu Glu Ser Thr Gln Thr Glu Ser Asn Lys Lys Met Glu Glu     610 615 620 Asn Glu Lys Glu Leu Ala Ala Cys Gln Leu Arg Ile Ser Gln Glu Asp 625 630 635 640 Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly Lys Thr Leu                 645 650 655 Gly Glu Gly Glu Phe Gly Lys Val Val Lys Ala Thr Ala Phe His Leu             660 665 670 Lys Gly Arg Ala Gly Tyr Thr Thr Val Ala Val Lys Met Leu Lys Glu         675 680 685 Asn Ala Ser Pro Ser Glu Leu Arg Asp Leu Leu Ser Glu Phe Asn Val     690 695 700 Leu Lys Gln Val Asn His Pro His Val Ile Lys Leu Tyr Gly Ala Cys 705 710 715 720 Ser Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr Gly                 725 730 735 Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg Lys Val Gly Pro Gly Tyr             740 745 750 Leu Gly Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His Pro Asp         755 760 765 Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe Ala Trp Gln Ile     770 775 780 Ser Gln Gly Met Gln Tyr Leu Ala Glu Met Lys Leu Val His Arg Asp 785 790 795 800 Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly Arg Lys Met Lys Ile                 805 810 815 Ser Asp Phe Gly Leu Ser Arg Asp Val Tyr Glu Glu Asp Ser Tyr Val             820 825 830 Lys Arg Ser Gln Gly Arg Ile Pro Val Lys Trp Met Ala Ile Glu Ser         835 840 845 Leu Phe Asp His Ile Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly     850 855 860 Val Leu Leu Trp Glu Ile Val Thr Leu Gly Gly Asn Pro Tyr Pro Gly 865 870 875 880 Ile Pro Pro Glu Arg Leu Phe Asn Leu Leu Lys Thr Gly His Arg Met                 885 890 895 Glu Arg Pro Asp Asn Cys Ser Glu Glu Met Tyr Arg Leu Met Leu Gln             900 905 910 Cys Trp Lys Gln Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile Ser         915 920 925 Lys Asp Leu Glu Lys Met Met Val Lys Arg Arg Asp Tyr Leu Asp Leu     930 935 940 Ala Ala Ser Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly Leu Ser 945 950 955 960 Glu Glu Glu Thr Pro Leu Val Asp Cys Asn Asn Ala Pro Leu Pro Arg                 965 970 975 Ala Leu Pro Ser Thr Trp Ile Glu Asn Lys Leu Tyr Gly Arg Ile Ser             980 985 990 His Ala Phe Thr Arg Phe         995 <210> 5 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of KR2 antibody <400> 5 Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp   1 5 10 15 Thr Pro Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu              20 25 30 Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly          35 40 45 Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu      50 55 60 Ser Ser Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn  65 70 75 80 Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro                  85 90 95 Ser Thr Cys Ser Lys Pro Xaa Cys Pro Pro Pro Glu Leu Leu Gly Gly             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile         115 120 125 Ser Arg     130 <210> 6 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of KR2 antibody <400> 6 Met Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro   1 5 10 15 Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ser Ser Ala Phe              20 25 30 Glu Pro Val Gly Gly Thr Val Asn Ile Lys Cys Gln Ala Ser Gln Ser          35 40 45 Ile Gly Thr Arg Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro      50 55 60 Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Ser Ser  65 70 75 80 Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln                  85 90 <210> 7 <211> 322 <212> PRT <213> Artificial Sequence <220> <223> heavy chain of KR2 antibody <400> 7 Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp   1 5 10 15 Thr Pro Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu              20 25 30 Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly          35 40 45 Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu      50 55 60 Ser Ser Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn  65 70 75 80 Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro                  85 90 95 Ser Thr Cys Ser Lys Pro Xaa Cys Pro Pro Pro Glu Leu Leu Gly Gly             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile         115 120 125 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp     130 135 140 Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg 145 150 155 160 Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg                 165 170 175 Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys             180 185 190 Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu         195 200 205 Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr     210 215 220 Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu 225 230 235 240 Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp                 245 250 255 Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Xaa Val             260 265 270 Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro         275 280 285 Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His     290 295 300 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro 305 310 315 320 Gly Lys         <210> 8 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> light chain of KR2 antibody <400> 8 Met Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro   1 5 10 15 Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ser Ser Ala Phe              20 25 30 Glu Pro Val Gly Gly Thr Val Asn Ile Lys Cys Gln Ala Ser Gln Ser          35 40 45 Ile Gly Thr Arg Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro      50 55 60 Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Ser Ser  65 70 75 80 Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser                  85 90 95 Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Glu Tyr             100 105 110 Ser Tyr Ser Ser Gly Asp Gly Ile Phe Phe Trp Asn Ala Phe Gly Gly         115 120 125 Gly Thr Glu Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu     130 135 140 Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile 145 150 155 160 Val Cys Val Ala Asn Lys Tyr Phe Pro                 165

Claims (12)

A fragment comprising the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6, or an antigen-binding portion thereof, which specifically binds to a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (SQEDPK) delete The fragment of claim 1, wherein the antibody comprises the heavy chain of SEQ ID NO: 7 and the light chain of SEQ ID NO: 8. The fragment according to claim 1, wherein said antibody is capable of specifically detecting a KIF5B (rearranged during transfection) fusion protein or variant thereof. The fragment according to claim 4, wherein the KIF5B-RET fusion protein is a fusion protein in which a fusion between the 16th exon of the KIF5B gene and the 12th exon of the RET gene is formed, or an antigen binding site thereof. The fragment of claim 1, wherein the antibody is capable of specifically detecting lung cancer. The fragment of claim 1, wherein said antibody is a monoclonal antibody, comprising an antibody or antigen-binding portion thereof. The fragment of claim 1, wherein the antibody is obtained from the serum of an animal immunized with a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RISQEDPK). The fragment of claim 1, wherein the fragment comprising the antigen binding site comprises an antibody or antigen binding portion thereof, wherein the fragment comprises Fab, F (ab ') 2, F (ab') ₂ , Fv or a single chain antibody molecule. 9. A composition for diagnosing lung cancer comprising an antibody according to any one of claims 1 to 9 or a fragment comprising an antigen binding site thereof. 9. A kit for diagnosing lung cancer, comprising an antibody according to any one of claims 1 to 9 or a fragment comprising an antigen binding site thereof. Use of an antibody of any one of claims 1 to 9 or a fragment comprising an antigen-binding site thereof to detect a KIF5B-RET fusion protein or variant thereof in an isolated biological sample of a subject suspected of having lung cancer, And detecting the antibody through an antibody reaction.

Images