KR20210067120A - Antibody specific for cobll1 protein or antigen binding fragment thereof, and its use - Google Patents

Abstract

The present invention relates to antibodies specific for Cobll1 protein or antigen-binding fragments thereof, and uses thereof. The antibody or antigen-binding fragment thereof of the present invention effectively detects Cobll1 protein, and thus can be effectively used in a method for diagnosing advanced stages of chronic myeloid leukemia (CML) or cancer.

Description

Cobll1 protein specific antibody or antigen-binding fragment thereof, and uses thereof {ANTIBODY SPECIFIC FOR COBLL1 PROTEIN OR ANTIGEN BINDING FRAGMENT THEREOF, AND ITS USE}

The present invention relates to antibodies or antigen-binding fragments thereof specific for Cobll1 protein, and uses thereof. The antibody or antigen-binding fragment thereof of the present invention can effectively detect Cobll1 protein, and can be effectively used in a method for diagnosing advanced stages of chronic myeloid leukemia (CML) or cancer.

Leukemia refers to a disease in which white blood cells proliferate as a tumor. The types of leukemia can be divided into myeloid leukemia and lymphocytic leukemia according to the white blood cells from which the leukemia originates. The clinical manifestations of leukemia vary depending on the type of disease and the nature of the cells involved. Lymphocytic leukemia is caused by lymphoid blood cells, myeloid leukemia is caused by myeloid blood cells, and chronic myelogenous leukemia is caused by malignant clones of mutated hematopoietic stem cells (HSCs).

In particular, chronic myelogenous leukemia (CML) is a malignant blood cancer that mainly occurs in adults as a malignant blood disease in which hematopoietic stem cells proliferate in large quantities in the bone marrow and abnormally increased various blood cells invade the peripheral blood or other organs. . Chronic myeloid leukemia progresses through three clinical stages: chronic phase (CP), accelerated phase (AP), and blast crisis (BC). The most common method for diagnosing chronic myeloid leukemia is to find the Philadelphia chromosome through a chromosome test. In fact, in about 95% of chronic myeloid leukemia patients, a translocation between chromosome 9 and chromosome 22 is found, and the translocation is a part of the abelson oncogene (abl) gene present at position q34 of chromosome 9, q11.2 on chromosome 22 It is formed by translocation to the bcr (break point cluster region) gene present in the position, and this rearrangement is called a BCR-ABL1 rearrangement.

In general, the translocation leading to the BCR-ABL1 fusion gene is the major cause of CML initiation, and the BCR-ABL1 fusion gene encodes the BCR-ABL1 fusion protein, which is a constitutively active tyrosine kinase. Imatinib was the first tyrosine kinase inhibitor (TKI) to target BCR-ABL1 and was found to be very effective in the treatment of CML. However, imatinib has a problem in that it cannot completely eliminate CML clones due to the resistance and insensitivity of primitive CML cells to imatinib treatment. In addition, nilotinib is being used to treat CML as a TKI. Despite the development of next-generation BCR-ABL1 TKIs, including ATP and allosteric site inhibitors, some patients still develop resistance to TKIs in the acute stage. This resistance was found to be either BCR-ABL1 kinase dependent or independent. While the majority of patients develop resistance through BCR-ABL1 kinase-dependent mechanisms such as point mutations or amplification, the presence of BCR-ABL1-independent resistance suggests that there are other mechanisms of TKI resistance.

In addition, in the acute stage of chronic myelogenous leukemia, a significant number of patients are resistant to targeted chemotherapy. However, there is a problem that delays in establishing alternative treatment strategies such as bone marrow transplantation while proceeding with chemotherapy without stage diagnosis. also do

Therefore, research to identify the protein as a new biomarker for chronic myeloid leukemia and to analyze its function is in progress.

Cordon-bleu protein-like 1 (Cobll1) protein is known as a negative regulator of apoptosis and is associated with low insulin resistance. In addition, it is known that the clinical outcome of CML patients with high Cobll1 expression is poor. In particular, Cobll1 protein is not expressed in the chronic phase (CP), which is an early stage of CML, but its expression is significantly increased in the acute phase (BP), confirming that the Cobll1 protein is associated with the progression of CML to the acute phase. In addition, it has been studied to have resistance to TKI upon expression of Cobll1 (Patent Document 1). Furthermore, Cobll1 is known to induce TKI resistance and blastic transformation through activation of the NF-κB signaling pathway in CML (Non-Patent Document 1).

Korean Patent No. 10-1820572

Han, S., et al. Cobll1 is linked to drug resistance and blastic transformation in chronic myeloid leukemia. Leukemia 31, 1532 (2017)

In the present invention, the onset or progression of CML is diagnosed early and the patient's prognosis more quickly and accurately by detecting the Cobll1 protein, which is highly expressed in the acute phase cells of chronic myelogenous leukemia (CML) or cells resistant to the existing target anticancer drug aimed at predicting.

For this purpose, it is an object of the present invention to provide an antibody specific for Cobll1 protein.

In order to solve the above problems, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Cobll1 protein.

Specifically, the antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein of the present invention may be selected from the group consisting of the following (1) to (5).

(1) CDRH1 having the sequence of SEQ ID NO: 1; CDRH2 having the sequence of SEQ ID NO:2; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO:3; and CDRL1 having the sequence of SEQ ID NO:4; CDRL2 having the sequence of SEQ ID NO: 5; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 6, an antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(2) CDRH1 having the sequence of SEQ ID NO:7; CDRH2 having the sequence of SEQ ID NO:8; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO: 9; and CDRL1 having the sequence of SEQ ID NO: 10; CDRL2 having the sequence of SEQ ID NO: 11; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 12. An antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(3) CDRH1 having the sequence of SEQ ID NO: 13; CDRH2 having the sequence of SEQ ID NO: 14; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO: 15; and CDRL1 having the sequence of SEQ ID NO: 16; CDRL2 having the sequence of SEQ ID NO: 17; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 18. An antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(4) CDRH1 having the sequence of SEQ ID NO:19; CDRH2 having the sequence of SEQ ID NO: 20; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO:21; and CDRL1 having the sequence of SEQ ID NO: 22; CDRL2 having the sequence of SEQ ID NO:23; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 24, an antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(5) CDRH1 having the sequence of SEQ ID NO:25; CDRH2 having the sequence of SEQ ID NO:26; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO: 27; and CDRL1 having the sequence of SEQ ID NO: 28; CDRL2 having the sequence of SEQ ID NO: 29; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 30. An antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof may be an antibody 1 to antibody 5 or an antigen-binding fragment thereof comprising a heavy chain CDR and a light chain CDR consisting of the amino acid sequences of Table 1 below.

In another embodiment, the antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein of the present invention may be selected from the group consisting of the following (6) to (10).

(6) a heavy chain variable region having the sequence of SEQ ID NO: 31; and a light chain variable region having the sequence of SEQ ID NO: 32, an antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(7) a heavy chain variable region having the sequence of SEQ ID NO: 33; and a light chain variable region having the sequence of SEQ ID NO: 34. An antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(8) a heavy chain variable region having the sequence of SEQ ID NO: 35; and a light chain variable region having the sequence of SEQ ID NO: 36. An antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(9) a heavy chain variable region having the sequence of SEQ ID NO: 37; and a light chain variable region having the sequence of SEQ ID NO: 38. An antibody or antigen-binding fragment thereof that specifically binds to a Cobll1 protein.

(10) a heavy chain variable region having the sequence of SEQ ID NO: 39; and a light chain variable region having the sequence of SEQ ID NO: 40, an antibody or antigen-binding fragment thereof that specifically binds to Cobll1 protein.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof may be an antibody 1 to antibody 5 or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region consisting of the amino acid sequences of Table 2 below.

In another embodiment, the antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein of the present invention may be selected from the group consisting of the following (11) to (15).

(11) a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 41; and a light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 42, an antibody or antigen-binding fragment thereof that specifically binds to Cobll1 protein.

(12) a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 43; and a light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 44, an antibody or antigen-binding fragment thereof that specifically binds to Cobll1 protein.

(13) a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 45; and a light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 46, an antibody or antigen-binding fragment thereof that specifically binds to Cobll1 protein.

(14) a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 47; and a light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 48, an antibody or antigen-binding fragment thereof that specifically binds to Cobll1 protein.

(15) a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 49; and a light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 50, an antibody or antigen-binding fragment thereof that specifically binds to Cobll1 protein.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof is antibody 1 to antibody 5 or antigen-binding thereof comprising a heavy chain variable region and a light chain variable region consisting of amino acid sequences each encoded by the nucleotide sequences of Table 3 below. It can be short.

In the present invention, “antibody” refers to a polypeptide that specifically binds to and recognizes an antigen, such as Cobll1 protein. The present invention includes not only the complete antibody form that binds to the Cobll1 protein, but also antigen-binding fragments of the antibody molecule.

In the present invention, "antibody" may include monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies). An antibody is composed of two heavy chains and two light chains, and has a variable region in which the amino acid sequence is different depending on the type of target antigen and a constant region in which the sequence does not change. have

Preferably, the antibody in the present invention may be a monoclonal antibody. Most preferably, the antibody may be a monoclonal antibody referred to herein as 3A11, 4H8, 5C12, 6A2 or 18D12. As used herein, the term "monoclonal antibody" means that the antibody molecule is prepared as a single molecule. Monoclonal antibodies have specificity to respond only to antigens having the same epitope, and also show affinity for only a specific epitope. The monoclonal antibody of the present invention may be one that specifically binds to the Cobll1 protein. Alternatively, the monoclonal antibody of the present invention may specifically bind to a small fragment or isoform of the Cobll1 protein having a size of 140 kDa or 180 kDa. In one embodiment, the 18D12 monoclonal antibody of the present invention can specifically bind to a small fragment or isoform of the Cobll1 protein having a size of 180 kDa. The 3A11 or 6A2 monoclonal antibody of the present invention can specifically bind to a small fragment or isoform of the Cobll1 protein having a size of 140 kDa.

In one embodiment of the invention, the monoclonal antibody may be produced in a hybridoma cell line. The hybridoma cells can be prepared using methods known in the art. For example, the hybridoma cells immunize an animal with an antigenic protein, fuse B cells, which are antibody-producing cells derived from the immunized animal, with myeloma cells to prepare a hybridoma, and then It can be prepared by a method of selecting a hybridoma that produces a monoclonal antibody that specifically binds. The animal to be immunized may be not only the mouse used in Examples, but also animals such as goats, sheep, guinea pigs, rats, or rabbits.

Accordingly, the present invention also provides a hybridoma cell line producing the antibody or antigen-binding fragment thereof.

A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond. The heavy chain has the types of IgG, IgM, IgD, and IgA, and the light chain has the kappa (κ) and lambda (λ) types. Preferably, in the antibody of the present invention, the heavy chain may be IgM, and the light chain may be a kappa type.

The variable regions of the light and heavy chains contain three highly variable regions called "complementarity-determining regions (CDRs)". The CDRs bind to an epitope of an antigen, and the CDRs of each chain typically have three regions (CDR1, CDR2 and CDR3). Herein, the CDRs of the heavy chain are also referred to as CDRH1, CDRH2 and CDRH3, and the CDRs of the light chain are also referred to as CDRL1, CDRL2 and CDRL3. These CDRs are very important regions in antigen specificity for each antibody, and thus are the most important parts for specifying the antibody.

As used herein, the term "antigen-binding fragment (of an antibody)" refers to a fragment retaining antibody function, and includes Fab, F(ab'), F(ab') ₂ and Fv, and the like. Among the antibody fragments, Fab (fragment antigen binding) has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1), and has one antigen-binding site. F(ab') differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of F(ab'). Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is known in the art. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and single-chain variable fragment (scFv) is generally a heavy chain variable region and a light chain variable region through a peptide linker. The regions may be linked by covalent bonds or linked directly at the C-terminus to form a dimer-like structure like a double-stranded Fv. Such antibody fragments can be obtained using proteolytic enzymes (e.g., papain-restricted digestion of the whole antibody yields Fab and pepsin digestion yields F(ab') ₂ fragments), or It can be produced through genetic recombination technology.

As used herein, the term “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues, also referred to as CDRs. The FRs of a variable domain generally consist of four FR domains FR1, FR2, FR3 and FR4. Thus, HVR (CDR) and FR sequences generally appear in the heavy chain variable region (VH) in the following order:

FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4.

In addition, the HVR (CDR) and FR sequences generally appear in the light chain variable region (VL; also denoted Vκ or Vλ depending on the type of light chain) in the following order:

FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4.

As used herein, the term “specifically binds” or the like means that an antibody or antigen-binding fragment thereof, or other construct such as an scFv, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may be characterized by an equilibrium dissociation constant of at least about 1 x 10 ^-6 M or less (eg, a KD less than this indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

The antibody of the present invention may be a human antibody, a humanized antibody, or a chimeric antibody.

As used herein, the term “human antibody” or “humanized antibody” refers to an antibody produced by a human or human cell, or from a non-human source using human antibody repertoires or other human antibody coding sequences. has an amino acid sequence corresponding to the amino acid sequence of an antibody derived from

As used herein, the term "chimeric antibody" means that a portion of the heavy and/or light chain is derived from a particular source or species and the remainder of the heavy and/or light chain is derived from a different source or species. means antibodies.

The antibody or antigen-binding fragment thereof of the present invention may include a variant of the amino acid sequence described in the accompanying sequence list within a range capable of specifically recognizing Cobll1 protein. For example, changes can be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody.

Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.

Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

The nucleic acid of the present invention encoding the antibody or antigen-binding fragment thereof of the present invention is interpreted to include a nucleotide nucleotide sequence showing substantial identity to the nucleotide nucleotide sequence described above. The substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. homology, more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981)]; [Nedleman and Wunsch, J. Mol. Bio. 48:443 (1970)]; [Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988)]; [Higgins and Sharp, Gene 73:237-44 (1988)]; [Higgins and Sharp, CABIOS 5:151-3 (1989)]; [Corpet et al., Nuc. Acids Res. 16:10881-90 (1988)]; [Huang et al., Comp. Appl. BioSci. 8:155-65 (1992)] and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994), et al. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLAST can be accessed through the BLAST page of the ncbi website. A method for comparing sequence homology using this program can be found on the BLAST help page of the ncbi website.

The present invention also provides an expression vector comprising the nucleic acid.

As used herein, the term "expression vector" refers to a vector comprising recombinant nucleotides comprising an expression control sequence operably linked to a nucleotide base sequence to be expressed in order to express a gene of interest in a host cell. The expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be provided by a host cell or an in vitro expression system. The expression vector may include a plasmid vector containing a recombinant polynucleotide; cozmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, lentiviral vectors, retroviral vectors and adeno-associated viral vectors. In one example, the nucleic acid molecule encoding the switch molecule in the vector of the present invention is operatively linked to the promoter of the vector. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence to regulate the transcription and/or translation of said other nucleic acid sequences.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in [Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001)], etc. disclosed, which is incorporated herein by reference.

As used herein, the term "cloning" is a genetic engineering technique that mass-produces the same gene, and is introduced into a plasmid or virus that acts as a vector to mass-produce a gene or DNA fragment, which is then introduced through cell division. It refers to the technology of cloning entirely identical clones.

As used herein, the term “cDNA” refers to DNA complementary to mRNA.

As used herein, the term “clone” refers to identical copies, and refers to identical copies of the same cell population or specific gene segment.

As used herein, the term “transformation” refers to a change in the genetic properties of a host cell by introducing a foreign gene into a host cell, and when the host is a eukaryotic cell, the term “transfection” is used. Can be used. Transformation used in the present invention may include any method of introducing a foreign gene into an organism, cell, tissue or organ, and preferably means introducing the foreign gene into E. coli. As a transformation method, a method generally used such as electroporation, a method using liposomes, and a DNA transport method using Ca++ can be used.

The present invention also provides a kit for detecting Cobll1 protein, comprising the antibody or antigen-binding fragment thereof.

In the case of Cobll1 protein, Cobll1 was not expressed in the chronic phase (CP), which is the early stage of CML, but the expression level of the Cobll1 protein was significantly increased in the patient sample that reached the acute phase (BC). In addition, it was confirmed that Cobll1 protein was expressed in HeLa cells, which are cervical cancer cells, and U2OS cells, which are osteosarcoma cells.

Accordingly, the kit of the present invention can be used for diagnosing the acute stage of chronic myeloid leukemia, or diagnosing cervical cancer or osteosarcoma.

The kit for detecting the Cobll1 protein of the present invention includes a tool for collecting a test sample or applying a detection reagent, a reagent or device for confirming antigen-antibody binding, etc. using an antibody to check the presence or absence of an antigen protein from a biological sample. Tools, devices, or reagents that may be included in conventional detection or diagnostic kits may further be included.

The kit of the present invention can be used for diagnosing the acute stage of chronic myelogenous leukemia by detecting Cobll1 protein according to an immunoassay method, or for diagnosing cervical cancer or osteosarcoma. This immunoassay may be performed according to various previously developed immunoassays or immunostaining protocols. The immunoassay or immunostaining format can be, for example, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunoassay (ELISA), flow cytometry, immunofluorescence staining, immunoaffinity purification, immunochromatography. (immunochromatography) and the like.

Preferably, the kit of the present invention may be for use in a sandwich enzyme-linked immunoassay (Sandwich ELISA). For example, the kit may comprise the antibody or antigen binding site thereof as a capturing antibody; and a polyclonal antibody against the Cobll1 protein as a detection antibody. It may be a kit for detecting Cobll1 protein. The antibody or antigen-binding site thereof as the capture antibody may be preferably a monoclonal antibody, and more preferably, a monoclonal antibody 3A11, 4H8, 5C12, 6A2 or 18D12. The polyclonal antibody as the detection antibody preferably binds specifically to the C-terminal region of the Cobll1 protein having the amino acid sequence of SEQ ID NO: 53.

The detection antibody may be linked to a detection label. Specific examples of detection labels include chemicals (eg, biotin), enzymes (eg, alkaline phosphatase, β-galactosidase, Horseradish peroxidase (HRP), and cytochrome P450). ), radioactive materials (e.g. C14, I125, P32 and S35), fluorescent materials (e.g. fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET). can Preferably, the detection label is biotin.

The test target sample may be a biological material derived from an individual. The subject may be a vertebrate. The vertebrate may be a mammal, preferably a human. The sample may be collected or isolated from a subject, for example, blood or blood constituent, bodily fluid, saliva, sputum, or a combination thereof.

The present invention provides a method for detecting Cobll1 protein, comprising the step of contacting the capture antibody and the detection antibody with a test target sample using the detection kit, and detecting the antigen-antibody complex formed therefrom. In one embodiment, the detection method of the present invention comprises

(a) (i) the sample to be tested;

(ii) said antibody or antigen binding site thereof as a capture antibody; and

(iii) a polyclonal antibody that specifically binds to the Coll1 protein as a detection antibody

providing a medium comprising a;

(b) incubating the medium under conditions in which the capture antibody and the detection antibody bind to the Coll1 protein; and

(c) measuring the level of the detection label of the detection antibody bound to an epitope other than the portion to which the capture antibody and the Coll1 protein are bound

includes

As used herein, the term “antigen-antibody complex” refers to a combination of the Cobll1 protein antigen in a sample and the antibody according to the present invention that recognizes it. Such antigen-antibody complexes are prepared by a colormetric method, an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment and a scintillation method. counting method) can be detected by any method selected from the group consisting of. By this detection, the Cobll1 protein can be analyzed qualitatively or quantitatively.

The novel antibody or antigen-binding fragment thereof according to the present invention specifically binds to the Cobll1 protein, thereby effectively detecting the Cobll1 protein.

Therefore, by using the antibody or antigen-binding fragment thereof of the present invention, the acute stage of chronic myelogenous leukemia, cervical cancer or osteosarcoma can be quickly, accurately and conveniently diagnosed, and the diagnosis, prevention, treatment, etc. of chronic myelogenous leukemia or cancer It can be used in various fields of

In addition, by using the ELISA kit using the antibody of the present invention, it is possible to test a large amount of samples, and the possibility of application as molecular diagnosis can be expected.

1 shows the results of purifying the antigen of the N-terminal region of the Cobll1 protein using a Ni-NTA column.
FIG. 2 shows the results of Western blot on the antigen of the N-terminal region of the Cobll1 protein expressed and purified in Example 1. FIG.
3 and 4 show that the monoclonal antibody prepared in Example 1 normally detects Cobll1 protein expressed in 293T cells (Myc-Cobll1) and acute CML patient cells (KRCMC 1885 PBMC, BC) overexpressing Myc-Cobll1 protein. Western blot results showing
5 is a Western blot result showing that the polyclonal antibody prepared in Example 2 normally detects Cobll1 protein. K562 denotes human-derived chronic myeloid leukemia cells, 293T denotes human-derived kidney cells, HeLa denotes human-derived cervical cancer cells, and H1299 denotes human-derived lung cancer cells.
6 shows the amino acid sequences of VH and VL of monoclonal antibodies 3A11, 4H8, 5C12, 6A2 and 18D12.
7 to 11 show the amino acid sequences and base sequences of VH and VL of monoclonal antibodies 3A11, 4H8, 5C12, 6A2 and 18D12, respectively.

The present invention will be described in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Antigen production and purification

To prepare a monoclonal antibody against the Cobll1 protein, the N-terminus of the Cobll1 protein was selected through secondary structure analysis and hydrophobicity & antigenicity analysis of the Cobll1 protein (SEQ ID NO: 51) to prepare an antigen.

After PCR using the following two primers to construct a vector expressing the N-terminal 1-330 mutant of Cobll1 protein, the amplified DNA fragment was isolated and cloned into pDONR223, a gateway plasmid system. After preparing the entry vector, LR reaction was performed on the His-tag pDEST17 vector, and this vector was used for antigen purification for antibody development. The vector was transformed into E. coli, BL21 (DE3) strain, and protein expression was induced. The expressed Cobll1 N-terminal antigen was isolated and purified using a Ni-NTA column ( FIG. 1 ). Western blot was performed on the isolated and purified protein using a His-tag antibody to confirm that the desired antigen was purified (FIG. 2).

Preparation of monoclonal antibodies

The antigen purified in Example 1 was injected into BALB/C mice for primary immunization. Two weeks later, the antigen was re-injected for a second immunization. Immune response tests were performed by collecting blood from mice and measuring antibody titers by ELISA. Antigen was injected every 2 weeks to enhance the immune response until the measured titer reached a reference titer (O.D. > 1.5 at serum dilution 1:1000) that could indicate that a suitable antibody was produced.

Thereafter, the spleen of the mouse was excised, and B cells were isolated, fused with mouse myeloma cells, and then cultured. ELISA was performed using the culture supernatant. Cells in wells showing a positive result in ELISA were aliquoted into 96-well plates. After the first cloning, when colonies were formed, ELISA was performed to select a positive well having a high O.D. value and one colony in the well among the wells showing a positive result, and second cloning was performed. Cloning was performed three times in this way, and the clones with the highest ELISA score were selected through ELISA screening to obtain hybridoma cell lines 3A11, 4H8, 5C12, 6A2 and 18D12. This was cultured to obtain monoclonal antibodies 3A11, 4H8, 5C12, 6A2 and 18D12.

In order to confirm whether monoclonal antibodies were produced normally, 293T cells overexpressing Myc-Cobll1 protein (Myc-Cobll1), blood from acute CML patients (KRCMC 1885 PBMC, BC), and blood from chronic CML patients (KRCMC 1922, PBMC) , CP) was used to perform Western blot. Actin was used as a loading control for Western blot. As a result, as shown in FIGS. 3 and 4, all five monoclonal antibodies produced detected Cobll1 protein expressed in Myc-Cobll1 protein overexpressing cells (Myc-Cobll1) and CML acute stage patient cells (KRCMC 1885 PBMC, BC). was confirmed. Cobll1 protein was not detected in CML chronic phase patient cells (KRCMC 1922, PBMC, CP).

Preparation of polyclonal antibodies

For antibody production, the gene sequence of the C-terminal region (SEQ ID NO: 53) of the Cobll1 protein was inserted into a GST-vector, and this vector was first injected into rabbits through subcutaneous injection as an antigen. Two weeks later, after the second injection of the vector, blood was collected on the 55th day, and Rabbit-anti-Cobll1 antibody was isolated from the collected rabbit blood according to the established protocol using the Aminolink Coupling Gel Column Kit.

As a result, as shown in FIG. 5, the prepared polyclonal antibody is a cell expressing the Cobll1 protein, that is, chronic myeloid leukemia cells (K562), kidney cells (293T), cervical cancer cells (HeLa), lung cancer cells (H1299). ) was confirmed to bind to. From this, it was confirmed that the polyclonal antibody binding to the Cobll1 protein was normally prepared.

Sequence analysis of monoclonal antibodies

The amino acid sequences and base sequences of the heavy chain (VH) and light chain (VL) variable regions of the five monoclonal antibodies 3A11, 4H8, 5C12, 6A2 and 18D12 obtained in Example 2 were analyzed.

To this end, total RNA was isolated from the five hybridoma cell lines of Example 2, and cDNA was prepared therefrom. Total RNA was extracted using the RNeasy mini kit according to the manufacturer's method. In addition, PCR, TA cloning and sequencing were performed using the prepared cDNA to analyze the variable region. See Larrick, J. W., et al. 1989. Biochem. Biophys. Res. Comm. 160, 1250] and [Jones, S. T. and Bendig, M. M. 1991], a mouse Ig-Primer set (Millipore, Cat#69831-3) for amplifying the VH and VL genes of the mouse was purchased, and an experiment was performed to amplify each gene part through a PCR experiment. After band isolation, TA cloning was performed using the pMD20-TA vector. By sequencing the TA cloning product, the amino acid sequence and base sequence of the variable region were analyzed.

The amino acid sequences and base sequences of VH and VL of monoclonal antibodies 3A11, 4H8, 5C12, 6A2 and 18D12 are shown in FIGS. 6 to 11 .

Monoclonal antibody isotype analysis

Using the 5 hybridoma cell lines of Example 2, the isotype of each monoclonal antibody was analyzed using the Isotype ELISA kit. As a result, as shown in Table 4, it was confirmed that all isotypes of monoclonal antibodies were IgG (kappa).

<110> DAEWOONG PHARMACEUTICAL CO., LTD. CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY RESEARCH AND BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY <120> ANTIBODY SPECIFIC FOR COBLL1 PROTEIN OR ANTIGEN BINDING FRAGMENT THEREOF, AND ITS USE <130> DWP19P-0002-KR <160> 53 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 3A11 <400> 1 Gly Phe Thr Phe Ser Ser Tyr Thr 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 3A11 <400> 2 Ile Ser Ser Gly Gly Gly His Thr 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 3A11 <400> 3 Ala Arg Tyr Thr Gly Thr Ser Leu Thr Trp Phe Ala Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 3A11 <400> 4 Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 3A11 <400> 5 Lys Val Ser One <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 3A11 <400> 6 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 4H8 <400> 7 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 4H8 <400> 8 Ile Tyr Pro Gly Asp Gly Asp Thr 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 4H8 <400> 9 Ala Ser Pro Ala Tyr 1 5 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 4H8 <400> 10 Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe 1 5 10 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 4H8 <400> 11 Ala Ala Ser One <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 4H8 <400> 12 Gln Gln Ser Lys Glu Val Pro Tyr Thr 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 5C12 <400> 13 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 5C12 <400> 14 Ile Ser Asn Gly Gly Ser Tyr Thr 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 5C12 <400> 15 Ala Arg Gly Asp Tyr Tyr Phe Asp Tyr 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 5C12 <400> 16 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 17 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 5C12 <400> 17 Lys Val Ser One <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 5C12 <400> 18 Ser Gln Ser Thr His Val Pro Pro Trp Thr 1 5 10 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 6A2 <400> 19 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 6A2 <400> 20 Ile Ser Asn Gly Gly Ser Tyr Thr 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 6A2 <400> 21 Ala Arg Gly Asp Tyr Tyr Phe Asp Tyr 1 5 <210> 22 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 6A2 <400> 22 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 23 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 6A2 <400> 23 Lys Val Ser One <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 6A2 <400> 24 Ser Gln Ser Thr His Val Pro Pro Trp Thr 1 5 10 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 18D12 <400> 25 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 18D12 <400> 26 Ile Ser Asn Gly Gly Ser Tyr Thr 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 18D12 <400> 27 Ala Arg Gly Pro Phe Tyr Phe Asp Tyr 1 5 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 18D12 <400> 28 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 29 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 18D12 <400> 29 Lys Val Ser One <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 18D12 <400> 30 Ser Gln Ser Thr His Val Pro Pro Trp Thr 1 5 10 <210> 31 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 3A11 <400> 31 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Gly His Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Ala Arg Tyr Thr Gly Thr Ser Leu Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 32 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region of 3A11 <400> 32 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 33 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 4H8 <400> 33 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Met Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Ala Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 100 105 110 <210> 34 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region of 4H8 <400> 34 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 5C12 <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asn Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 36 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region of 5C12 <400> 36 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Cys Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 37 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 6A2 <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ala Ile Ser Asn Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 38 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region of 6A2 <400> 38 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 39 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 18D12 <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asn Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Pro Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 40 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region of 18D12 <400> 40 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 41 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 3A11 <400> 41 gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgccg cctctggatt cactttcagt agctatacca tgtcttgggt tcgccagact 120 ccggagaaga ggctggagtg ggtcgcaacc attagtagtg gtggtggtca cacctactat 180 ccagacagtg tgaagggtcg attcaccatc tccagagaca atgccaagaa caacctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acggcctttt attactgtgc aagatacact 300 gggacctccc tgacctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360 360 <210> 42 <211> 337 <212> DNA <213> Artificial Sequence <220> <223> Light Chain Variable Region of 3A11 <400> 42 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120 ttcctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300 tggacgttcg gtggaggcac caagctggaa atcaaac 337 <210> 43 <211> 337 <212> DNA <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 4H8 <400> 43 caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgatgttg 60 tcctgcaagg cttctggcta cacctttact agttactgga tgcagtgggt aaaacagagg 120 cctggacagg gtctggaatg gattggggct atttatcctg gagatggtga tactaggtac 180 actgagaagt tcaagggcaa ggccacattg actgcagata aatcctccag cacagcctac 240 atgcaactca gcagcttggc atctgaggac tctgcggtct attactgtgc ctccccggcc 300 tactggggcc aaggcaccac tctcacagtc tcctcag 337 <210> 44 <211> 334 <212> DNA <213> Artificial Sequence <220> <223> Light Chain Variable Region of 4H8 <400> 44 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gaactggttc 120 caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccaaggatcc 180 ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240 cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtac 300 acgttcggag gggggaccaa gctggaaata aaac 334 <210> 45 <211> 349 <212> DNA <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 5C12 <400> 45 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactattaca tgtattgggt tcgccagact 120 ccggaaaaga ggctggagtg ggtcgcaacc attagtaatg gtggtagtta cacctactat 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caacctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagaggcgac 300 tactactttg actactgggg ccaaggcacc actctcacag tctcctcag 349 <210> 46 <211> 340 <212> DNA <213> Artificial Sequence <220> <223> Light Chain Variable Region of 5C12 <400> 46 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tgcctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttcct 300 ccgtggacgt tcggtggagg caccaagctg gaaatcaaac 340 <210> 47 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 6A2 <400> 47 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactattaca tgtattgggt tcgccagact 120 ccggaaaaga ggctggagtg ggtcgcagcc attagtaatg gtggtagtta cacctactat 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caacctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagaggcgac 300 tactactttg actactgggg ccaaggcacc actctcacag tctcctca 348 <210> 48 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Light Chain Variable Region of 6A2 <400> 48 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttcct 300 ccgtggacgt tcggtggagg caccaagctg gaaatcaaa 339 <210> 49 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Heavy Chain Variable Region of 18D12 <400> 49 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactattaca tgtattgggt tcgccagact 120 ccggaaaaga ggctggagtg ggtcgcaacc attagtaatg gtggtagtta cacctactat 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caacctgttc 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagaggtcct 300 ttctactttg actactgggg ccaaggcacc actctcacag tctcctca 348 <210> 50 <211> 340 <212> DNA <213> Artificial Sequence <220> <223> Light Chain Variable Region of 18D12 <400> 50 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta gatgttgtga tgacccaaac tccactctcc 120 ctgcctgtca gtcttggaga tcaagcctcc atctcttgca gatctagtca gagccttgta 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac gcatgttcct 300 ccgtggacgt tcggtggagg caccaagctg gaaatcaaac 340 <210> 51 <211> 1128 <212> PRT <213> Artificial Sequence <220> <223> CobII1 protein <400> 51 Met Asp Gly Arg Thr Pro Arg Pro Gln Asp Ala Pro Ala Arg Arg Lys 1 5 10 15 Pro Lys Ala Lys Ala Pro Leu Pro Pro Ala Glu Thr Lys Tyr Thr Asp 20 25 30 Val Ser Ser Ala Ala Asp Ser Val Glu Ser Thr Ala Phe Ile Met Glu 35 40 45 Gln Lys Glu Asn Met Ile Asp Lys Asp Val Glu Leu Ser Val Val Leu 50 55 60 Pro Gly Asp Ile Ile Lys Ser Thr Thr Val His Gly Ser Lys Pro Met 65 70 75 80 Met Asp Leu Leu Ile Phe Leu Cys Ala Gln Tyr His Leu Asn Pro Ser 85 90 95 Ser Tyr Thr Ile Asp Leu Leu Ser Ala Glu Gln Asn His Ile Lys Phe 100 105 110 Lys Pro Asn Thr Pro Ile Gly Met Leu Glu Val Glu Lys Val Ile Leu 115 120 125 Lys Pro Lys Met Leu Asp Lys Lys Lys Pro Thr Pro Ile Ile Pro Glu 130 135 140 Lys Thr Val Arg Val Val Ile Asn Phe Lys Lys Thr Gln Lys Thr Ile 145 150 155 160 Val Arg Val Ser Pro His Ala Ser Leu Gln Glu Leu Ala Pro Ile Ile 165 170 175 Cys Ser Lys Cys Glu Phe Asp Pro Leu His Thr Leu Leu Leu Lys Asp 180 185 190 Tyr Gln Ser Gln Glu Pro Leu Asp Leu Thr Lys Ser Leu Asn Asp Leu 195 200 205 Gly Leu Arg Glu Leu Tyr Ala Met Asp Val Asn Arg Glu Ser Cys Gln 210 215 220 Ile Ser Gln Asn Leu Asp Ile Met Lys Glu Lys Glu Asn Lys Gly Phe 225 230 235 240 Phe Ser Phe Phe Gln Arg Ser Lys Lys Lys Arg Asp Gln Thr Ala Ser 245 250 255 Ala Pro Ala Thr Pro Leu Val Asn Lys His Arg Pro Thr Phe Thr Arg 260 265 270 Ser Asn Thr Ile Ser Lys Pro Tyr Ile Ser Asn Thr Leu Pro Ser Asp 275 280 285 Ala Pro Lys Lys Arg Arg Ala Pro Leu Pro Pro Met Pro Ala Ser Gln 290 295 300 Ser Val Pro Gln Asp Leu Ala His Ile Gln Glu Arg Pro Ala Ser Cys 305 310 315 320 Ile Val Lys Ser Met Ser Val Asp Glu Ala Asp Lys Ser Pro Cys Glu 325 330 335 Ala Gly Arg Val Arg Ala Gly Ser Leu Gln Leu Ser Ser Met Ser Ala 340 345 350 Gly Asn Ser Ser Leu Arg Arg Thr Lys Arg Lys Ala Pro Ser Pro Pro 355 360 365 Ser Lys Ile Pro His Gln Ser Asp Glu Asn Ser Arg Val Thr Ala 370 375 380 Leu Gln Pro Val Asp Gly Val Pro Pro Asp Ser Ala Ser Glu Ala Asn 385 390 395 400 Ser Pro Glu Glu Leu Ser Ser Pro Ala Gly Ile Ser Ser Asp Tyr Ser 405 410 415 Leu Glu Glu Ile Asp Glu Lys Glu Glu Leu Ser Glu Val Pro Lys Val 420 425 430 Glu Ala Glu Asn Ile Ser Pro Lys Ser Gln Asp Ile Pro Phe Val Ser 435 440 445 Thr Asp Ile Ile Asn Thr Leu Lys Asn Asp Pro Asp Ser Ala Leu Gly 450 455 460 Asn Gly Ser Gly Glu Phe Ser Gln Asn Ser Met Glu Glu Lys Gln Glu 465 470 475 480 Thr Lys Ser Thr Asp Gly Gln Glu Pro His Ser Val Val Tyr Asp Thr 485 490 495 Ser Asn Gly Lys Lys Val Val Asp Ser Ile Arg Asn Leu Lys Ser Leu 500 505 510 Gly Pro Asn Gln Glu Asn Val Val Gln Asn Glu Ile Ile Val Tyr Pro 515 520 525 Glu Asn Thr Glu Asp Asn Met Lys Asn Gly Val Lys Lys Thr Glu Ile 530 535 540 Asn Val Glu Gly Val Ala Lys Asn Asn Asn Ile Asp Met Glu Val Glu 545 550 555 560 Arg Pro Ser Asn Ser Glu Ala His Glu Thr Asp Thr Ala Ile Ser Tyr 565 570 575 Lys Glu Asn His Leu Ala Ala Ser Ser Val Pro Asp Gln Lys Leu Asn 580 585 590 Gln Pro Ser Ala Glu Lys Thr Lys Asp Ala Ala Ile Gln Thr Thr Pro 595 600 605 Ser Cys Asn Ser Phe Asp Gly Lys His Gln Asp His Asn Leu Ser Asp 610 615 620 Ser Lys Val Glu Glu Cys Val Gln Thr Ser Asn Asn Asn Ile Ser Thr 625 630 635 640 Gln His Ser Cys Leu Ser Ser Gln Asp Ser Val Asn Thr Ser Arg Glu 645 650 655 Phe Arg Ser Gln Gly Thr Leu Ile Ile His Ser Glu Asp Pro Leu Thr 660 665 670 Val Lys Asp Pro Ile Cys Ala His Gly Asn Asp Asp Leu Leu Pro Pro 675 680 685 Val Asp Arg Ile Asp Lys Asn Ser Thr Ala Ser Tyr Leu Lys Asn Tyr 690 695 700 Pro Leu Tyr Arg Gln Asp Tyr Asn Pro Lys Pro Lys Pro Ser Asn Glu 705 710 715 720 Ile Thr Arg Glu Tyr Ile Pro Lys Ile Gly Met Thr Thr Tyr Lys Ile 725 730 735 Val Pro Pro Lys Ser Leu Glu Ile Ser Lys Asp Trp Gln Ser Glu Thr 740 745 750 Ile Glu Tyr Lys Asp Asp Gln Asp Met His Ala Leu Gly Lys Lys His 755 760 765 Thr His Glu Asn Val Lys Glu Thr Ala Ile Gin Thr Glu Asp Ser Ala 770 775 780 Ile Ser Glu Ser Pro Glu Glu Pro Leu Pro Asn Leu Lys Pro Lys Pro 785 790 795 800 Asn Leu Arg Thr Glu His Gln Val Pro Ser Ser Val Ser Ser Pro Asp 805 810 815 Asp Ala Met Val Ser Pro Leu Lys Pro Ala Pro Lys Met Thr Arg Asp 820 825 830 Thr Gly Thr Ala Pro Phe Ala Pro Asn Leu Glu Glu Ile Asn Asn Ile 835 840 845 Leu Glu Ser Lys Phe Lys Ser Arg Ala Ser Asn Ala Gln Ala Lys Pro 850 855 860 Ser Ser Phe Phe Leu Gln Met Gln Lys Arg Val Ser Gly His Tyr Val 865 870 875 880 Thr Ser Ala Ala Ala Lys Ser Val His Ala Ala Pro Asn Pro Ala Pro 885 890 895 Lys Glu Leu Thr Asn Lys Glu Ala Glu Arg Asp Met Leu Pro Ser Pro 900 905 910 Glu Gln Thr Leu Ser Pro Leu Ser Lys Met Pro His Ser Val Pro Gln 915 920 925 Pro Leu Val Glu Lys Thr Asp Asp Asp Val Ile Gly Gln Ala Pro Ala 930 935 940 Glu Ala Ser Pro Pro Pro Ile Ala Pro Lys Pro Val Thr Ile Pro Ala 945 950 955 960 Ser Gln Val Ser Thr Gln Asn Leu Lys Thr Leu Glu Thr Phe Gly Ala 965 970 975 Pro Arg Pro Tyr Ser Ser Ser Gly Pro Ser Pro Phe Ala Leu Ala Val 980 985 990 Val Lys Arg Ser Gln Ser Phe Ser Lys Glu Arg Thr Glu Ser Pro Ser 995 1000 1005 Ala Ser Ala Leu Val Gln Pro Pro Ala Asn Thr Glu Glu Gly Lys Thr 1010 1015 1020 His Ser Val Asn Lys Phe Val Asp Ile Pro Gln Leu Gly Val Ser Asp 1025 1030 1035 1040 Lys Glu Asn Asn Ser Ala His Asn Glu Gln Asn Ser Gln Ile Pro Thr 1045 1050 1055 Pro Thr Asp Gly Pro Ser Phe Thr Val Met Arg Gln Ser Ser Leu Thr 1060 1065 1070 Phe Gln Ser Ser Asp Pro Glu Gln Met Arg Gln Ser Leu Leu Thr Ala 1075 1080 1085 Ile Arg Ser Gly Glu Ala Ala Ala Lys Leu Lys Arg Val Thr Ile Pro 1090 1095 1100 Ser Asn Thr Ile Ser Val Asn Gly Arg Ser Arg Leu Ser His Ser Met 1105 1110 1115 1120 Ser Pro Asp Ala Gln Asp Gly His 1125 <210> 52 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> Amino Acids 1-300 of CobII1 protein <400> 52 Met Asp Gly Arg Thr Pro Arg Pro Gln Asp Ala Pro Ala Arg Arg Lys 1 5 10 15 Pro Lys Ala Lys Ala Pro Leu Pro Pro Ala Glu Thr Lys Tyr Thr Asp 20 25 30 Val Ser Ser Ala Ala Asp Ser Val Glu Ser Thr Ala Phe Ile Met Glu 35 40 45 Gln Lys Glu Asn Met Ile Asp Lys Asp Val Glu Leu Ser Val Val Leu 50 55 60 Pro Gly Asp Ile Ile Lys Ser Thr Thr Val His Gly Ser Lys Pro Met 65 70 75 80 Met Asp Leu Leu Ile Phe Leu Cys Ala Gln Tyr His Leu Asn Pro Ser 85 90 95 Ser Tyr Thr Ile Asp Leu Leu Ser Ala Glu Gln Asn His Ile Lys Phe 100 105 110 Lys Pro Asn Thr Pro Ile Gly Met Leu Glu Val Glu Lys Val Ile Leu 115 120 125 Lys Pro Lys Met Leu Asp Lys Lys Lys Pro Thr Pro Ile Ile Pro Glu 130 135 140 Lys Thr Val Arg Val Val Ile Asn Phe Lys Lys Thr Gln Lys Thr Ile 145 150 155 160 Val Arg Val Ser Pro His Ala Ser Leu Gln Glu Leu Ala Pro Ile Ile 165 170 175 Cys Ser Lys Cys Glu Phe Asp Pro Leu His Thr Leu Leu Leu Lys Asp 180 185 190 Tyr Gln Ser Gln Glu Pro Leu Asp Leu Thr Lys Ser Leu Asn Asp Leu 195 200 205 Gly Leu Arg Glu Leu Tyr Ala Met Asp Val Asn Arg Glu Ser Cys Gln 210 215 220 Ile Ser Gln Asn Leu Asp Ile Met Lys Glu Lys Glu Asn Lys Gly Phe 225 230 235 240 Phe Ser Phe Phe Gln Arg Ser Lys Lys Lys Arg Asp Gln Thr Ala Ser 245 250 255 Ala Pro Ala Thr Pro Leu Val Asn Lys His Arg Pro Thr Phe Thr Arg 260 265 270 Ser Asn Thr Ile Ser Lys Pro Tyr Ile Ser Asn Thr Leu Pro Ser Asp 275 280 285 Ala Pro Lys Lys Arg Arg Ala Pro Leu Pro Pro Met 290 295 300 <210> 53 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> CobII1 Antigen <400> 53 Leu Lys Asn Tyr Pro Leu Tyr Arg Gln Asp Tyr Asn Pro Lys Pro Lys 1 5 10 15 Pro Ser Asn Glu Ile Thr Arg Glu Tyr Ile Pro Lys Ile Gly Met Thr 20 25 30 Thr Tyr Lys Ile Val Pro Lys Ser Leu Glu Ile Ser Lys Asp Trp 35 40 45 Gln Ser Glu Thr Ile Glu Tyr Lys Asp Asp Gln Asp Met His Ala Leu 50 55 60 Gly Lys Lys His Thr His Glu Asn Val Lys Glu Thr Ala Ile Gln Thr 65 70 75 80 Glu Asp Ser Ala Ile Ser Glu Ser Pro Glu Glu Pro Leu Pro Asn Leu 85 90 95 Lys Pro Lys Pro Asn Leu Arg Thr Glu His Gln Val Pro Ser Ser Val 100 105 110 Ser Ser Pro Asp Asp Ala Met Val Ser Pro Leu Lys Pro Ala Pro Lys 115 120 125 Met Thr Arg Asp Thr Gly Thr Ala Pro Phe Ala Pro Asn Leu Glu Glu 130 135 140 Ile Asn Asn Ile Leu Glu Ser Lys Phe Lys Ser Arg Ala Ser Asn Ala 145 150 155 160 Gln Ala Lys Pro Ser Ser Phe Phe Leu Gln Met Gln Lys Arg Val Ser 165 170 175 Gly His Tyr Val Thr Ser Ala Ala Ala Lys Ser Val His Ala Ala Pro 180 185 190 Asn Pro Ala Pro Lys Glu Leu Thr Asn Lys Glu Ala Glu Arg Asp Met 195 200 205 Leu Pro Ser Pro Glu Gln Thr Leu Ser Pro Leu Ser Lys Met Pro His 210 215 220 Ser Val Pro Gln Pro Leu Val Glu Lys Thr Asp Asp Asp Val Ile Gly 225 230 235 240 Gln Ala Pro Ala Glu Ala Ser Pro Pro Pro Ile Ala Pro Lys Pro Val 245 250 255 Thr Ile Pro Ala Ser Gln Val Ser Thr Gln Asn Leu Lys Thr Leu Glu 260 265 270 Thr Phe Gly Ala Pro Arg Pro Tyr Ser Ser Ser Gly Pro Ser Pro Phe 275 280 285 Ala Leu Ala Val Val Lys Arg Ser Gln Ser Phe Ser 290 295 300

Claims (30)

CDRH1 having the sequence of SEQ ID NO: 1; CDRH2 having the sequence of SEQ ID NO:2; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO:3; and
CDRL1 having the sequence of SEQ ID NO:4; CDRL2 having the sequence of SEQ ID NO: 5; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO:6.
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
CDRH1 having the sequence of SEQ ID NO:7; CDRH2 having the sequence of SEQ ID NO:8; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO: 9; and
CDRL1 having the sequence of SEQ ID NO: 10; CDRL2 having the sequence of SEQ ID NO: 11; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 12.
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
CDRH1 having the sequence of SEQ ID NO: 13; CDRH2 having the sequence of SEQ ID NO: 14; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO: 15; and
CDRL1 having the sequence of SEQ ID NO: 16; CDRL2 having the sequence of SEQ ID NO: 17; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 18.
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
CDRH1 having the sequence of SEQ ID NO: 19; CDRH2 having the sequence of SEQ ID NO: 20; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO:21; and
CDRL1 having the sequence of SEQ ID NO: 22; CDRL2 having the sequence of SEQ ID NO:23; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 24.
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
CDRH1 having the sequence of SEQ ID NO:25; CDRH2 having the sequence of SEQ ID NO:26; and a heavy chain variable region comprising a CDRH3 having the sequence of SEQ ID NO: 27; and
CDRL1 having the sequence of SEQ ID NO:28; CDRL2 having the sequence of SEQ ID NO: 29; and a light chain variable region comprising a CDRL3 having the sequence of SEQ ID NO: 30.
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having the sequence of SEQ ID NO: 31; and
A light chain variable region having the sequence of SEQ ID NO: 32
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having the sequence of SEQ ID NO:33; and
light chain variable region having the sequence of SEQ ID NO:34
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having the sequence of SEQ ID NO: 35; and
light chain variable region having the sequence of SEQ ID NO:36
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having the sequence of SEQ ID NO: 37; and
light chain variable region having the sequence of SEQ ID NO:38
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having the sequence of SEQ ID NO:39; and
A light chain variable region having the sequence of SEQ ID NO:40
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 41; and
Light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 42
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 43; and
Light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 44
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 45; and
Light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 46
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 47; and
A light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 48
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
a heavy chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 49; and
A light chain variable region having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 50
Including, an antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein.
The antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein according to any one of claims 1 to 15, wherein the antibody is a monoclonal antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the heavy chain of the antibody is immunoglobulin M (IgM).
The antibody or antigen-binding fragment thereof that specifically binds to the Cobll1 protein according to any one of claims 1 to 15, wherein the light chain of the antibody is of the kappa type.
A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-15.
An expression vector comprising the nucleic acid of claim 19 .
A hybridoma cell line producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.
A kit for detecting Cobll1 protein, comprising the antibody of any one of claims 1 to 15.
The kit for detecting Cobll1 protein according to claim 22, characterized in that it is used for the diagnosis of the acute stage of chronic myelogenous leukemia.
The kit for detecting Cobll1 protein according to claim 22, characterized in that it is used for the diagnosis of cervical cancer or osteosarcoma.
16. The antibody of any one of claims 1 to 15 as a capturing antibody; and
Polyclonal antibody that specifically binds to the C-terminal region of the Cobll1 protein having the amino acid sequence of SEQ ID NO: 53 as a detection antibody
A kit for the detection of Cobll1 protein comprising a.
The kit of claim 25, wherein the detection antibody is linked to a detection label.
The kit of claim 26, wherein the detection label is biotin.
The kit of claim 25, wherein the kit is for use in a sandwich enzyme-linked immunoassay (Sandwich ELISA).
The kit for detecting Cobll1 protein according to claim 25, characterized in that it is used for the diagnosis of the acute stage of chronic myelogenous leukemia.
The kit for detecting Cobll1 protein according to claim 25, characterized in that it is used for diagnosis of cervical cancer or osteosarcoma.

Images