KR101783793B1 - An anti-relaxin-2 antibody and use of the same for anti-cancer - Google Patents

Abstract

The present invention provides a heavy chain variable region having a heavy chain CDR (complementarity determining region) amino acid sequence of CDRH1 of Sequence Listing 1 sequence, CDRH2 of Sequence Listing 2 sequence and CDRH3 of Sequence Listing 3 sequence; And an antibody to Relaxin-2 comprising a light chain variable region having the light chain CDR amino acid sequence of CDRL3 consisting of SEQ ID No. 5 sequence, CDRL2 consisting of SEQ ID No. 6 sequence and CDRL3 consisting of SEQ ID No. 7 sequence Or antigen-binding fragment thereof, the monoclonal antibody of the present invention is applicable to the treatment of cancer in humans.

Description

An anti-relaxin-2 antibody and use thereof for the same anti-cancer.

The present invention relates to antillaaxin-2 antibodies and anticancer uses thereof.

Cancer or tumor is a tumor mass that results from uncontrolled abnormal cell growth, which may be benign or malignant. Benign tumors are usually localized. Malignant tumors are collectively referred to as cancer. The term "malignant" generally means that a tumor can invade neighboring bodily tissues and destroy it, then spread to distant sites and lead to death (Robbins and Angell, 1976, Basic Pathology, 2d Ed ., WB Saunders Co., Philadelphia, pp. 68-122].

Cancer can be induced in many parts of the body and works differently depending on its origin. Cancer cells destroy some of their body parts and then spread to other parts of the body and begin to grow new, causing more destruction.

The treatment of cancer has not yet been clarified. Current treatment options, such as surgery, chemotherapy, and radiation therapy, often result in ineffective or serious side effects.

Current cancer therapies may include surgery, chemotherapy, hormone therapy and / or radiation therapy to eradicate tumor cells in patients (see, for example, Stockdale, 1998, "Principles of Cancer Patient Management," in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV].

Recent cancer therapies may be associated with biological therapy or immunotherapy. All of these approaches can provide significant side effects to the patient. For example, surgery may be contraindicated by the patient's health condition or may not be allowed to the patient. In addition, surgery may not be able to completely remove tumor tissue. Radiation therapy is effective only when the sensitivity to radiation is higher in tumor tissue than in normal tissue, and radiation therapy can often cause serious side effects. Hormone therapy is rarely used as a single agent, and although it may be effective, it is often used to prevent or delay cancer recurrence after removal of the majority of cancer cells using other therapies. Biotherapy / immunotherapy is limited in number, and each therapy is generally only effective for very specific types of cancer.

With regard to chemotherapy, there are various chemotherapeutic agents available for treating cancer. Most cancer chemotherapy works by inhibiting DNA synthesis by directly or indirectly inhibiting the biosynthesis of deoxyribonucleotide triphosphate precursors to prevent DNA replication and subsequent cell division (see, for example, Gilman et < RTI ID = 0.0 > al., Goodman and Gilman ' s: The Pharmacological Basis of Therapeutics, 8th Ed. (Pergamom Press, New York), 1990]. Such as, for example, alkylating agents such as nitroso urea, antimetabolites such as methotrexate and hydroxyurea, and other agonists such as etoposides, campathecins, bleomycin, doxorubicin ), Daunorubicin, etc., are not necessarily cell cycle specific, but they kill cells during the S phase due to their effect on DNA replication. Other agonists, specifically colchicine and vinca alkaloids such as vinblastine and vincristine, interfere with microtubule assembly and result in a fission arrest. Chemotherapy protocols generally involve increasing the therapeutic efficacy by concomitantly administering chemotherapeutic agents.

Despite the availability of various chemotherapeutic agents, chemotherapy has many drawbacks. (See, for example, Stockdale, 1998, "Principles Of Cancer Patient Management" in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds., Ch. 12, sect. 10]. Nearly every chemotherapeutic agent is toxic, and chemotherapy is a significant and often dangerous side effect. These side effects include severe nausea, bone marrow suppression, and immunosuppression. Additionally, even when coadministered with chemotherapeutic agents, many tumor cells are resistant or resistant to these chemotherapeutic agents. In fact, cells resistant to the particular chemotherapeutic agent used in this treatment protocol have been shown to be resistant to other drugs, even drugs that act by mechanisms that differ from the mechanism of action of the drugs used in the particular treatment ; This phenomenon is called pleiotropic drug or multiple drug resistance. Thus, due to drug resistance, it has proved difficult to treat many cancers using standard chemotherapy treatment protocols.

Relaxin-2 is important for cancer cell proliferation, invasion and angiogenesis. However, the possibility of relaxin-2 as a target for immunotherapy has never been directly confirmed.

[Prior Patent Literature]

Korean Patent Publication No. 1020130048242

Disclosure of Invention Technical Problem [8] The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a novel anti-rat lactic-2 antibody.

Another object of the present invention is to provide a novel anticancer agent.

In order to achieve the above object, the present invention provides a CDRH1 comprising the sequence of SEQ ID No. 1, the CDRH2 consisting of the sequence of SEQ ID No. 2 and the heavy chain variable having the CDR3 amino acid sequence of CDRH3 comprising the sequence of SEQ ID No. 3 domain; And

An antibody to Relaxin-2 comprising a light chain variable region having a light chain CDR amino acid sequence of CDRL1 of Sequence Listing Fifth Sequence, CDRL2 of Sequence Listing Sequence No. 6 and CDRL3 of Sequence Listing Seventh Sequence, or Binding fragment thereof.

In one embodiment of the invention, the heavy chain variable region preferably has the amino acid sequence of SEQ ID NO: 4, but is not limited thereto,

In another embodiment of the present invention, the light chain variable region preferably has the amino acid sequence of SEQ ID NO: 8, but is not limited thereto.

The present invention also provides a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the invention.

In one embodiment of the present invention, the nucleic acid molecule preferably has a nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.

The present invention also provides a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention.

In one embodiment of the present invention, the nucleic acid molecule preferably has a nucleotide sequence of SEQ ID NO: 10, but is not limited thereto.

The present invention also relates to a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the antibody or its antigen-binding fragment thereof to the reliloxin-2 of the present invention; And (b) a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

In one embodiment of the invention, the cancer is selected from the group consisting of breast cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, lung cancer, gastric cancer, rectal cancer, soft-tissue sarcoma, colon cancer, carcinoma of the papilla vateri, nonendocrine lung tumor, and bronchial carcinoid tumor, but is not limited thereto.

The present invention also provides a kit for cancer diagnosis comprising the above-mentioned antibody against rilacsin-2 or antigen-binding fragment thereof.

In one embodiment of the invention, the cancer is selected from the group consisting of breast cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, lung cancer, gastric cancer, rectal cancer, soft-tissue sarcoma, colon cancer, carcinoma of the papilla vateri, nonendocrine lung tumor, and bronchial carcinoid tumor, but is not limited thereto.

I. Section Relaxin -2 antibody and its antigen-binding fragment

The antibody of the present invention has a specific binding ability to Relaxin-2.

The term " antibody " used herein to refer to Relaxin-2 is a specific antibody to Relaxin-2 that specifically binds to a specific epitope of Relaxin-2, Antigen-binding fragment (antibody fragment) of the molecule.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has kappa (kappa) and lambda (lambda) types.

An antigen binding fragment or an antibody fragment of an antibody molecule refers to a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') 2 and Fv. Fabs in the antibody fragment have 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 (C _H1 ) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain C _H1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for producing Fv fragments with minimal antibody fragments having only a heavy chain variable region and a light chain variable region are described in PCT International Publication Nos. WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086, WO 88/09344. The double-chain Fv is a non-covalent linkage between a heavy chain variable region and a light chain variable region. A single-chain Fv generally shares a variable region of a heavy chain and a variable region of a short chain via a peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. 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, and when cut with pepsin, an F (ab ') 2 fragment can be obtained) It can also be produced through recombinant technology.

According to one embodiment of the present invention, the antibody in the present invention is in the form of an antibody or a complete antibody form. In addition, the heavy chain constant region may be selected from any one of gamma (gamma), mu (mu), alpha (alpha), delta (delta) or epsilon (epsilon). In one particular example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4), and in other specific examples the constant region is IgG2a isotype. The light chain constant region may be kappa or lambda form. In one particular example, the light chain constant region is kappa type.

As used herein, the term "heavy chain" includes 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 _H1 , C _H2 and C _H3 Lt; / RTI > and the fragments thereof. The term "light chain" as used herein also includes full-length light chains, 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, It means all fragments.

The antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising CDRH1 comprising SEQ ID NO: 1, CDRH2 comprising SEQ ID NO: 28 and CDRH3 comprising SEQ ID NO: 3.

As used herein, the term "complementarity determining region" (CDR) refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy chain and light chain (Kabat et al . Sequences of proteins of Immunological Interest , 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDRH1, CDRH2 and CDRH3) and light chains (CDRL1, CDRL2 and CDRL3) each contain three CDRs. CDRs provide the major contact residues for antibody binding to an antigen or epitope.

The antibody or antibody fragment of the present invention may include variants of the amino acid sequence described in the Sequence Listing attached thereto within a range that specifically recognizes Relaxin-2. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and / or substitution of the amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of the 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 both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoruicin (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in imparting the interactive biological function of proteins. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When a mutation is introduced with reference to a hydrophobic index, substitution is performed between amino acids showing hydrophobic index differences within ± 2 in one specific example, within ± 1 in another specific example, and within ± 0.5 in another specific example.

On the other hand, it is also well known that the substitution between amino acids having similar hydrophilicity values leads to proteins with homogeneous biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When a mutation is introduced with reference to the hydrophilicity value, substitution is made between amino acids showing a difference in hydrophilic value within ± 2 in one specific example, within ± 1 in another specific example, and within ± 0.5 in another specific example.

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

Considering the mutation having the above-mentioned biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted as including a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the above-described sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, 70% homology according to one particular example, 80% homology according to another specific example, and 90% homology according to another specific example. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv . Appl . Math . (1981) 2: 482, Needleman and Wunsch, J. Mol . Bio . (1970) 48: 443; Pearson and Lipman, Methods in Mol . Biol . (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73: 237-44; Higgins and Sharp, CABIOS (1989) 5: 151-3; Corpet et al . Nuc. Acids Res . (1988) 16: 10881-90; Huang et al . Comp. Appl . BioSci . (1992) 8: 155-65 and Pearson et al . Meth . Mol . Biol . (1994) 24: 307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al ., J. Mol . Biol . (1990) 215: 403-10) is accessible from NBCI and the like and has sequences such as blastp, blasm, blastx, tblastn and tblastx on the Internet It can be used in conjunction with the analysis program. BLSAT is available at www.ncbi.nlm.nih.gov/BLAST/. A comparison of sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to one embodiment of the invention, the antibody of the present invention comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4.

According to one embodiment of the present invention, the antibody of the present invention additionally comprises a light chain variable region having the following light chain CDR amino acid sequence: CDRL1 comprising SEQ ID NO: 5, CDRL2 comprising SEQ ID NO: 6, and CDRL3 comprising SEQ ID NO: 7 sequence.

According to one embodiment of the present invention, the antibody of the present invention comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 8.

The antibodies of the present invention can be used in combination with monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, short chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide- And anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of such antibodies, and the like.

According to the present invention, the antibodies of the present invention can be produced in various forms of antibodies. For example, as described in the Examples below, antibodies of the invention can be made into Fab antibodies, and recombinant with human constant regions using the light and heavy chain variable regions obtained in Fab antibodies, ) ≪ / RTI >

According to one embodiment of the present invention, the antibody of the present invention is a monoclonal antibody. The term " monoclonal antibody " refers to a single molecule composition of antibody molecules obtained in a substantially identical population of antibodies, wherein the monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope.

The antibody of the present invention can be used to treat cancer by binding to a Relaxin-2 protein known as a tumor-specific antigen and reducing / inhibiting / eliminating its activity.

Ⅱ. Nucleic acid molecules and recombinant vectors

According to another aspect of the invention, the present invention provides a nucleic acid molecule encoding a heavy chain variable region of an antibody or an antigen-binding fragment thereof for said Relaxin-2.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding a light chain variable region of an antibody or an antigen-binding fragment thereof for said Relaxin-2.

As used herein, the term "nucleic acid molecule" is intended to encompass both DNA (gDNA and cDNA) and RNA molecules, and nucleotides that are basic constituent units in nucleic acid molecules are not only natural nucleotides, (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90: 543-584). The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the invention can be modified. Such modifications include addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.

According to one embodiment of the present invention, the nucleic acid molecule encoding the heavy chain variable region comprises the nucleotide sequence of SEQ ID NO: 9, and the nucleic acid molecule encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO: 10 .

The nucleic acid molecule of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art. Homology, at least 90% homology in one particular example, and at least 95% homology in another specific example.

According to another aspect of the invention, the present invention provides a nucleic acid molecule comprising (a) a nucleic acid molecule encoding a heavy chain variable region of the invention; And (b) a nucleic acid molecule encoding a light chain variable region of the invention.

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmeptide vector; And viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors.

According to one embodiment of the invention, the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region in the vector of the present invention are operatively linked to the promoter.

As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, Whereby the regulatory sequence regulates transcription and / or translation of the different nucleic acid sequences.

According to a preferred embodiment of the present invention, the recombinant vector of the present invention comprises (a) a nucleic acid molecule encoding a heavy chain variable region of SEQ ID NO: 9; And (b) a nucleic acid molecule encoding a light chain variable region of SEQ ID NO: 10.

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

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL 貫 promoter, , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of decoding, and a transcription / translation termination sequence. The promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C. J. Bacteriol . 158: 1018-1024 (1984)) when E. coli (e.g. HB101, BL21, And the leftward promoter of phage lambda (pL? Promoter, Herskowitz, I. and Hagen, D. Ann. Rev. Genet . 14: 399-445 (1980)). When the Bacillus bacterium is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis ( Appl. Environ. Microbiol . 64: 3932-3938 (1998); Mol . Gen. Genet . 250: 734-741 (1996) ) Or any promoter capable of expressing in Bacillus may be used as a regulatory region.

Meanwhile, the recombinant vector of the present invention can be prepared by using a plasmid (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pGEX series, pET series, pUC19, etc.), phage (e.g., λgt4 · λB, λ-Charon, λΔz1 and M13) or viruses (eg SV40 and the like).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell such as a metallothionein promoter, a beta -actin promoter, a human heterologous promoter, A promoter derived from mammalian viruses such as adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse breast tumor virus (MMTV) promoter , The LTR promoter of HIV, the promoter of Moloney virus promoter Epstein Barr virus (EBV), and the promoter of Roussacomavirus (RSV)), and generally has a polyadenylation sequence as a transcription termination sequence.

The recombinant vectors of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Fused sequences include, for example, glutathione S-transferase (Pharmacia, USA); Maltose binding protein (NEB, USA); FLAG (IBI, USA); Tag sequences such as 6x His (hexahistidine; Quiagen, USA), Pre-S1, and c-Myc; OmpA, and PelB. In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like, without any additional sequence for purification.

On the other hand, the recombinant vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The vector expressing the antibody of the present invention can be a vector system in which a light chain and a heavy chain are expressed simultaneously in one vector or a system in which a light chain and a heavy chain are separately expressed in separate vectors. In the latter case, both vectors are introduced into host cells via co-transfomation and targeted transformation. Co-transfection is a method of introducing each vector DNA encoding a light chain and a heavy chain into a host cell simultaneously, and then selecting cells expressing both the light chain and the heavy chain. Target transduction is carried out by selecting cells transformed with a vector containing the light chain (or heavy chain) and transforming the selected cells expressing the light chain with a vector containing the heavy chain (or light chain) to express both light and heavy chains Cells are finally selected. In the following examples, antibodies were prepared using a vector system in which light chains (V _L and C _L ) and heavy chains (V _H and C _H1 ) were expressed simultaneously in a single vector.

Ⅲ. Transformant

According to another aspect of the present invention, the present invention provides a host cell transformed with the recombinant vector of the present invention.

Host cells capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any host cell known in the art and include, for example, Escherichia coli coli), Bacillus sp, Streptomyces, such as Bacillus subtilis and Bacillus Chuo ringen sheath (Streptomyces), Pseudomonas (Pseudomonas) (e.g., Pseudomonas footage is (Pseudomonas but are not limited to, prokaryotic host cells such as putida ), Proteus mirabilis or Staphylococcus (e.g. Staphylococcus carnosus ) .

Suitable eukaryotic host cells of such vectors include fungi such as Aspergillus species , fungi such as Pichia < RTI ID = 0.0 > pastoris), Mai processes Sergio a saccharide ratio cyano (Saccharomyces such as yeast, such as E. cerevisiae , Schizosaccharomyces and Neurospora crassa , other sub-eukaryotic cells, cells of higher eukaryotes such as insect-derived cells, and plants or mammals One cell can be used.

As used herein, "transformation" and / or "transfection" to a host cell includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, Technology can be selected and performed. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran sulfate, Pectamine, and dry / depressant-mediated transformation methods, and the like.

IV. Antillaxin-2 antibody Mutant  Or a method for producing the antigen binding fragment thereof

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising: (a) culturing a host cell transformed with the recombinant vector of the present invention; And (b) expressing an anti-rilacoxin-2 antibody or an antigen-binding fragment thereof in the host cell, or a method for producing an antigen-binding fragment thereof.

Culturing of the transformed host cells in the production of the antibody may be carried out according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted according to the strain selected by those skilled in the art. Such various culturing methods are disclosed in various publications (e.g., James M. Lee, Biochemical Engineering , Prentice-Hall International Editions, 138-176). Cell culture can be classified into batch, infusion, and continuous culture depending on the suspension culture, adhesion culture, and culture method according to the cell growth method. The medium used for the culture should suitably satisfy the requirements of the specific strain.

The animal cell culture comprises a variety of carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, Alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that may be used include organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat and inorganic such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate Nitrogen source. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts as a source. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors and the like may be included.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, foaming can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture. In addition, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain the aerobic state of the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C.

The antibody obtained by culturing the transformed host cell can be used in an unpurified state and can be further purified and used in high purity using various conventional methods such as dialysis, salt precipitation and chromatography. Among them, methods using chromatography are most widely used. The types and order of the columns can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography and the like depending on the characteristics of the antibody and the culture method.

V. Pharmaceutical composition for the prevention or treatment of cancer

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of an antibody to Relaxin-2 or an antigen-binding fragment thereof; And (b) a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

The antibody binds to Relaxin-2 with high affinity and can inhibit the growth, invasion and metastasis of cancer expressing Relaxin-2. Therefore, the antibody can be used alone or in combination with a conventional pharmaceutically acceptable carrier for prevention and treatment of cancer It is possible.

As used herein, the term "prophylactic " means any act that inhibits or slows the progress of cancer by administration of the composition of the present invention, and" treatment "refers to inhibition of cancer development, .

According to one embodiment of the present invention, cancer which is a disease to be applied to the composition of the present invention is a cancer expressing Relaxin-2. Examples of such cancer include breast cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, lung cancer, gastric cancer, Soft-tissue sarcoma, colon cancer, carcinoma of the papilla vateri, nonendocrine lung tumor, and bronchial carcinoid tumor.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, And the like. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. The pharmaceutical composition may also be administered by any device that allows the active agent to migrate to the target cell.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, suppositories, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

Antibodies can be injected into vivo in the form of antibody-therapeutic conjugates and used to treat cancer. Therapeutic agents include chemotherapeutic agents, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, biological agents, and enzyme inhibitors. Preferred functional molecules for coupling to an antibody of the invention or fragments thereof are chemicals, cytokines or chemokines. The chemical substance is an anticancer agent, and examples thereof include at least one selected from the group consisting of asibaiicin, aclarubicin, acordazole, acornisine, adozelesin, alanosin, aldosterukin, allopurinol sodium, altretamine, aminoglutethimide, But are not limited to, amorphous, amorphous, amphiphile, amsacrine, androgens, angiidine, ampicillin glycinate, asalay, asparaginase, 5-azacytidine, azathioprine, bacillus calmette- ), Baker's antipol, beta-2-dioxythioguanosine, bisanthrene HCl, bleomycin sulfate, Bupionine sulphoxide, BWA773U82, BW 502U83 / HCl, BW 7U85 mesylate, , Carbothymine, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulphonamide, chlorochomotin, chromomycin A3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum, CPT-11, chinatol, cyclocytidine, But are not limited to, cyclophosphamide, cyclophosphamide, cytarabine, cytemvena, dabis maliate, decarbazine, dactinomycin, daunorubicin HCl, diarylidines, dexarazoic acid, dianhydrogalactitol, Dihydro-5-azacytin, doxorubicin, etchinomycin, dodestrecite, edcellosine, eplonin, and elixirs solutions such as dicyclohexylcarbodiimide, dodecyl B, diethyldithiocarbamate, , Erythromycin, erythromycin, erythromycin, erythromycin, erythromycin, erythromycin, erythromycin, erythromycin, erythromycin, epothilone, , Flurozolidine, fludarabine phosphate, 5'-fluorouracil, Fluosol ™, flutamide, gallium nitrate, gemcitabine, gosserelin acetate, hepesulpham, hexamethylene bisacetate Hydrolizine hydrochloride, diafuracil HCl, iospasmid, 4-iopentanol, isofluratine, isotretinoin, leucovorin calcium, leuprolide, Acetate, levamisole, liposome daunorubicin, liposome capture doxorubicin, romerstine, ronidamine, mytansine, mechlorethamine hydrochloride, melphalan, menogaril, merbarone, 6-mercaptopurine, Methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin C, mitotan, mitoxanthrone hydrochloride, monocyte / macrophage colony-stimulating factor, navelone, Napoxidine, neocarginostatin, octreotide acetate, ormaflatatin, oxaliplatin, paclitaxel, palla, pentostatin, piperazinethione, Porphyromycin, Porphyromycin, Porphyromycin, Porphyromycin, Predominastine, Procarbazine, Progestin, Pyrazofurin, Razalamus, Pyridoxine, Such as tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, tamoxifen, Tumor Injection, Thiazopurine, Topotecan, Toremifene, Tretinoin, Trifluoperazine Hydrochloride, Tripleuridine, Trimethrecate, TNF (tumor necrosis factor), Uracil Mustard, Vinblastine Sulfate, Vin Cystine sulfate, vinethine, vinorelbine, binzolidine, Yoshi 864, zorubicin, cytosine arabinoside, etoposide, melphalan, taxol, taxol and mixtures thereof, Not limited to.

VI. Composition for cancer diagnosis

According to another aspect of the present invention, there is provided a cancer diagnostic kit comprising the antibody or the antigen-binding fragment thereof for Relaxin-2.

The antibody of the present invention can be applied to a biological sample to diagnose the onset of cancer.

As used herein, the term "biological sample" refers to a tissue, a cell, a whole blood, a serum, a plasma, a tissue autopsy sample (brain, skin, lymph node, spinal cord etc.), a cell culture supernatant, a ruptured eukaryotic cell, But is not limited thereto. These biological samples can be reacted with the antibody of the present invention without manipulation or manipulation to confirm the onset of cancer.

The formation of the above-mentioned antigen-antibody complex can be performed by a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, Can be detected by the scintillation counting method. As used herein, the term " detection " is intended to detect an antigen-antibody complex and may be carried out using various markers. Specific examples of the label include an enzyme, a minerals, a ligand, a luminescent material, a microparticle or a radioactive isotope.

Examples of the enzyme used as the detection label include acetylcholinesterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase,? -Lactamase, and the like, Eu ^{3 +} , Eu ^{3 +} chelate, or cryptate. Ligands include biotin derivatives and the like. Emitters include acridinium esters and isoluminol derivatives. Fine particles include colloid Gold and colored latex, and the radioactive isotopes include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bonton Hunter reagents and the like.

According to one embodiment of the present invention, the antigen-antibody complex can be detected using enzyme immunoassay (ELISA). Enzyme immunosorbent methods include direct ELISA using labeled antibodies that recognize the antigen attached to a solid support, indirect ELISA using labeled secondary antibodies that recognize the capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of an antibody and an antigen attached to the solid support, an antibody that binds to the solid support, And an indirect sandwich ELISA using a labeled secondary antibody. The antibody of the present invention can have a detection label, and when it does not have a detection label, it can be identified by treating another antibody capable of capturing the antibody of the present invention and having a detection label.

Hereinafter, the present invention will be described in detail.

Liposome  Encapsulated in a complex relaxin -2 Peptides Epitope  Production of anti-relaxin-2 antibody by immunization

The present inventors have reported the development of epitope-based peptide vaccines and protocols for the production of monoclonal antibodies (Kim D, Lee Y, Kwon HJ (2015) Methods Mol Biol 1348: 127-135). In the present invention, the inventors have applied the method to the production of monoclonal antibodies against human relaxin-2. First, we synthesized a candidate B cell epitope of human relaxin-2 and formulated a peptide epitope (RLN2-5 peptide) -CpG-DNA-liposome (DOPE: CHEMS) complex. Next, the inventors immunized BALB / c mice with the complex three times at 10-day intervals. After the third immunization, mouse sera were collected and analyzed for antibody titer. ELISA results clearly demonstrated a significant increase in human relaxin-2 peptide epitope-specific IgG (Figure 1A). Mice with the highest IgG titer were selected and injected with the same antigen complex. After 10 days, mouse splenocytes were collected and used to prepare hybridoma cells. splenocytes were fused with SP2 / 0 myeloma cells by conventional hybridoma technology. The cell culture supernatant of the hybridoma cells was analyzed by indirect ELISA to isolate clones producing high peptide epitope-specific antibodies. Through the screening process, we isolated one hybridoma cell (1B11) that responded to the RLN2-5 peptide (FIG. 1B). The hybridoma cells were further analyzed for the production of monoclonal antibodies after subcloning by limiting dilution method. Finally, one hybridoma clone was selected for the production of the monoclonal antibody (1B11F12) (Fig. 1C).

term- relaxin -2 Monoclonal  The variable domain of the antibody Cloning

CDNA sequences encoding the variable domains of the heavy and light chains (V _H and V _L ) were cloned from hybridoma cells (1B11F12) producing anti-relaxin-2 monoclonal antibodies using heavy and light chain primers. The sequence identified by DNA sequencing is shown in Fig.

term- relaxin -2 Monoclonal  Characterization of antibodies

Hybridoma cells (1B11F12) were injected into the peritoneal cavity to generate ascites in BALB / c mice. Using purified protein-A agarose column chromatography, the anti -relaxin-2 mAb was purified from a plurality and the purified antibody was analyzed by SDS-PAGE and Coomassie staining (Fig. 3A). Based on the ELISA data, the monoclonal antibody isotype was identified as IgG2a (Figure 3B). We have analyzed the reactivity of the anti -relaxin-2 monoclonal antibody to the human relaxin-2 peptide epitope, RNL2-5. The present inventors quantitatively measured the binding affinity of the anti -relaxin-2 monoclonal antibody using an ELISA. The anti -relaxin-2 mAb reacted with human relaxin-2 peptide with an equilibrium dissociation constant ( K d) ~250 pM (Fig. 3C).

To identify whether the monoclonal antibody 1B11F12 clone specifically recognizes the relaxin-2 precursor protein and mature relaxin-2, we performed Western blot analysis. The insoluble fraction of bacterial cell lysate containing recombinant relaxin-2 precursor protein and synthetic mature relaxin-2 was subjected to SDS-PAGE and Western blot analysis. Conventional anti -relaxin-2 monoclonal antibodies can detect recombinant relaxin-2 precursor protein and synthetic mature relaxin-2 (Fig. 4A). The anti -relaxin-2 monoclonal antibody 1B11F12 clone of the present invention is capable of recognizing recombinant relaxin-2 precursor protein; however, it is not responsive to synthetic mature relaxin-2 (FIG. 4B). In order to confirm whether the monoclonal antibody of the present invention can detect native synthetic mature relaxin-2, the present inventors performed immunoprecipitation analysis. As shown in FIG. 4C, the anti -relaxin-2 monoclonal antibody 1B11F12 clone of the present invention was able to detect native synthetic mature relaxin-2. These results indicate that the anti -relaxin-2 mAb recognizes recombinant relaxin-2 precursor protein by Western blot analysis and that native synthetic mature relaxin-2 can be recognized by immunoprecipitation .

term- relaxin -2 Monoclonal  Inhibition of cell growth by antibodies

Relaxin-2 has been reported to be involved in the progression of breast and prostate cancer. Therefore, in order to investigate the effect of anti -relaxin-2 antibody in various breast and prostate cancer cells, we treated recombinant relaxin-2 and its cells in the presence of normal IgG or anti-relaxin-2 monoclonal antibody. As shown in FIG. 5, relaxin-2 alone treatment increased the growth of MCF-7 and LNCaP cells and their growth was reduced in the presence of anti -relaxin-2 antibody.

Thus, the inventors have concluded that inhibition of relaxin-2 signaling of the monoclonal antibody may inhibit tumor growth of breast and prostate cancer cells

The anti -relaxin-2 mAb of the present invention has been shown to be capable of specifically recognizing native human relaxin-2 using immunoprecipitation and has inhibited the growth of breast and prostate cancer cells, Relaxin-2 is a good target of therapy, and the monoclonal antibody of the present invention can be applied to the treatment of cancer in humans.

Figure 1: Screening of hybridoma clones producing anti-relaxin-2 monoclonal anti-monoclonal antibodies. (A) Three BALB / c mice were immunized intraperitoneally with RLN2-5 peptide encapsulated in DOPE: CHEMS complex and MB-ODN 4531 (O) four times at 10 day intervals. The serum was collected and the whole IgG was assayed using an ELISA kit. (B) Initial screening-derived ELISA results of cell fusion experiments using splenocytes from RLN2-5 peptide immunized mice. (C) Hybridomas derived from Figure IB (arrows) were selected for the production of monoclonal antibodies followed by subcloning by limiting dilution method.
Figure 2 is a cDNA sequence for the variable domains of heavy and light chains separated from the hybridoma cell clone 1B11F12. (A) heavy chain variable domain sequence. (B) a light chain variable domain sequence. Describe predicted amino acid sequences under the cDNA sequence.
Figure 3: Production of anti-human relaxin-2 monoclonal antibodies. (A) 1B11F12 Hybridoma clones were injected into the multiple membrane steels to prepare a plurality. The anti -relaxin-2 mAb was purified from the ascites of mice injected with hybridoma cell line 1B11F12 clone using protein-A agarose column chromatography. The purified antibody was identified by SDS-PAGE and Coomassie blue staining. (B) titration curves were obtained using purified 1B11F12 monoclonal antibody to determine isotype. (C) 1B11F12 Monoclonal antibody binding affinity determination. The titer for the RLN2-5 peptide of the 1B11F12 mAb was analyzed by ELISA and binding affinities were calculated using the SigmaPlot program.
Figure 4 shows the specificity of the anti -relaxin-2 monoclonal antibody. The insoluble fractions of E. coli lysate containing recombinant relaxin-2 precursor protein (A, B) and synthetic rhRLN2 peptide were separated by SDS-PAGE. Western blotting was performed using conventional rat anti-human relaxin-2 monoclonal antibody (A) and anti-relaxin-2 monoclonal antibody (B) obtained from 1B11F12 hybridoma clone, respectively. (C) rhRLN2 peptide was cultured with normal mouse IgG, rat anti-human relaxin-2 monoclonal antibody or 1B11F12 monoclonal antibody. Immunoconjugates were grafted with protein A beads and Western blot analysis was performed with rat anti-human relaxin-2 mAb. ◀: Relaxin-2 precursor, ◁: mature Relaxin-2. HC: heavy chain, LC: light chain.
Figure 5 shows inhibition of cancer cell growth by anti -relaxin-2 monoclonal antibody. The cultured samples were treated for breast cancer cells (MCF-7) and prostate cancer cells (LNCaP) after rhRLN2 peptide was preincubated with anti-relaxin-2 monoclonal antibody for 1 hour at 4 ° C. The growth of cancer cells was measured by a cell proliferation assay. ***, p < 0.001.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Recombination relaxin -2 expression

To express human pro-relaxin-2 with histidine (His) -tagged protein, whole mRNA was obtained from MCF-7 cells and the human pro-relaxin-2 cDNA was PCR amplified using the following primer set: sense 5'- AAG CTT ATG CCT CGC CTG TTT TTT TTC-3 'and anti-sense 5'- CTC GAG GCA AAA TCT AGC AAG AGA TCT T-3'. The amplified cDNA fragments were cloned into pET-22b (Novagen, Darmstadt, Germany) expression vector containing C-terminal His-tag. Expression of the recombinant pro-relaxin-2 protein was measured in E. coli D-1-thioglactopyranoside (IPTG, Sigma Aldrich, St Louis, USA) in a 1% The bacterial cells were sonicated in lysis buffer (10 mM PBS containing 1 ug / ml lysozyme, 5 mM EDTA, 10 ug / ml leupeptin, 1 mM PMSF, and 1 mM DTT). After centrifugation, soluble and insoluble fractions were used for Western blot analysis.

Example  2: Oligodeoxynucleotides

The present inventors have previously developed a phosphodiester bond CpG-DNA (MB-ODN 4531 (O)) having immunomodulatory activity from Mycobacterium bovis genomic DNA (Lee KW, Jung J, Lee Y, Kim TY, Kim DS, Kwon HJ (2006) Mol Immunol 43 (13): 2107-2118). The synthesized oligonucleotide MB-ODN 4531 (O) was obtained from Samchully Pharmaceutical Co. (Seoul, Korea). MB-ODN 4531 was composed of 20 bases containing three CpG motifs (underlined): AGCAG CG TT CG TGT CG GCCT.

Example  3: B cell epitope Of peptide  Synthesis and selection:

The candidate B cell epitope peptides of the relaxin-2 protein can be used as a substrate for their hydrophobic, hydrophilic, secondary structure, antigenicity index and amphoteric basis as described in Kyte J, Doolittle RF (1982) J Mol Biol 157 (1): 105-132 ( Http://tools.immuneepitpoe.org/main/index.html ).

The sequence of the RLN2-5 peptide was ¹⁶¹ RQLYSALANKCCHVG ¹⁷⁵ (hRLN2-5). The peptide was synthesized using an automated peptide synthesizer (Peptron III-R24, Peptron, Daejeon, Korea) and isolated with a purity of 90% or more.

Example  4: Mouse and Immunization

Mice were maintained under specific aseptic conditions. The present inventors purchased 4-week-old male BALB / c (H-2 ^b ) mice as Nara Biotech (Seoul, Korea). This animal study was approved by Hallym University Animal Testing Ethics Committee (Hallym 2015-56). (DOPE) as described in BMB Rep 44 (11): 758-763 as described in Kim D, Kwon S, Ahn CS, Lee Y, Choi SY, Park J, Kwon HY, Kwon HJ Mice were injected intraperitoneally with 50 [mu] g of RLN2-5 peptide supplemented with 50 [mu] g of CpG-DNA (MB-ODN-4531 (O)

Example  5: antigen-specific Ig ELISA Assay

Mouse serum was obtained by orbital bleeding. To measure total IgG, a 96-well immunoplate (Nalgen Nunc International, Penfield, USA) was coated with 5 ug / ml of RLN2-5 peptide, and then 0.05% of PBST containing Tween-20 in After blocking with PBS, whole IgG was measured as described in Park BK, Choi SH, Kim YE, Park S, Lee Y, Lee KW, Kwon HJ (2015) Monoclon Antib Immunodiagn 34 (2): 101-109 Respectively.

Example  6: Mouse anti- relaxin -2 Monoclonal  Production of antibodies

BALB / c mice were intraperitoneally injected with hRLN2-5 peptide (50 ug) and MB-ODN 4531 (O) (50 ug) encapsulated in the DOPE: CHEMS complex three times at intervals of 10 days. Giraffe from immunized mice was used for fusion according to the standard hybridoma method. To obtain claw cell clusters, hybridoma clones in HAT medium and HT medium were selected by standard limiting dilution protocol. To obtain ascites, selected hive dog clones were injected into the peritoneal cavity of BALB / c mice. The anti -relaxin-2 monoclonal antibody was purified from multiple liquids by protein-A column chromatography. To determine the isotype of the anti -relaxin-2 mAb, an isotyping kit (Southern Biotechnology Associates Inc, Birmingham, USA) was incubated with the following reagents: Shang SB, Jin YL, Jiang XT, Zhou JY, Zhang X, Xing G, (2009) Mol Immunol 46 (3): 327-334.

Example  7: anti- relaxin -2 Monoclonal  Variable of antibody Heavy chain  And Light chain  Of the domain Cloning

Hybridoma cells (1B11F12) producing anti -relaxin-2 mAb were cultured and isotyped using a monoclonal antibody isotyping kit (Dipstick format, Bibco BRL or Roche, Mannheim, Germany). Total RNAs were extracted from hybridoma cells (1B11F12) with RNeasy Mini Kit (Qiagen) and cDNAs were prepared with Yokoyama WM, Christensen M, Santos GD, and Miller D (2006) Curr Protoc Immunol Chapter 2, Unit 2.5.

To clone the sequences for the variable heavy and light chain domains (V _H and V _L ) of the anti -relaxin-2 mAb, the resulting cDNAs were amplified using Vent polymerase (NEB) as the following primer set. As the heavy chain primer, IGG2A (5'-GGA AGA TCT CTT GAC CAG GCA TCC TAG AGT CA-3 ') and 5'MH2 (5'-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TCW GG- 3 ') and 5'-GG (5'-GG GAG CTC GAY ATT GTG MTS ACM CAR WCT MCA-3') as kappa chain primers ) Were used (Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R, Ratner D (2000) J Immunol Methods 233, 167-177). Standard PCR reactions were performed for 25 cycles. The PCR products were ligated directly into the pGEM-T easy vector (Promega). Cloned mouse Ig inserts were analyzed by DNA sequencing.

Example  8: Mouse anti- relaxin -2 Monoclonal  Reactivity of antibodies

To determine the binding affinity of the anti -relaxin-2 mAb, 96-well immunoplates were coated with 5 ug / ml hRLN2-5 peptide and blocked with PBST containing 1% BSA. An anti -relaxin-2 mAb was added at 1 uM from the top row of the plate and a 1: 5 serial dilution in PBST was then placed in the column. The plate was incubated at room temperature for 4 hours, washed with PBST and incubated with anti-mouse IgG antibody with horseradish peroxidase for 2 hours. The color development assay was developed with TMB (tetramethylbenzidine) peroxidase substrate (KPL, Gaithersburg, USA), quenched with TMB-stop solution (KPL) and analyzed with Spectra Max 250 microplate reader (Molecular Devices, Sunnyvale, Respectively. The results were analyzed using the SigmaPlot program for the calculation of binding affinity.

Example  9: Western With blut  Immunoprecipitation analysis

Western blot and immunoprecipitation assays were performed with recombinant human relaxin-2 peptide (rhRLN2, Pepprotech, Seoul, Korea) to analyze the specificity of anti -relaxin-2 mAb. In summary, insoluble fractions of E. coli cell lysate containing recombinant pro-relaxin-2 protein and rhRLN2 peptide were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was incubated with rat anti-human relaxin-2 monoclonal antibody (R & D systems, Minneapolis, USA) or anti -relaxin-2 monoclonal antibody (1B11F12) for 2 hours at room temperature. HRP-conjugated anti-mouse IgG or anti-rat IgG (The Jackson Laboratory, Bar Harbor, USA) was used for detection of the primary antibody. For immunoprecipitation, 10 μg of rhRLN2 peptide was incubated with normal mouse IgG, rat anti-human relaxin-2 monoclonal antibody or 1B11F12 monoclonal antibody at 4 ° C for 4 hours, then the sample was mixed with protein A beads and washed with PBST And subjected to Western blot analysis.

Example  10: Cell culture

Human breast cancer cell line (MCF-7) was purchased from ATCC (American Type Culture Collection, Manassas, USA). Human prostate cancer cell line, LNCaP, was purchased from Korean Cell Line Bank (Seoul, Korea). MCF-7 cells were maintained in EMEM at 0.01 mg / ml of human recombinant insulin. LNCaP cells were maintained in RPMI1640 (Life Technologies) medium. The medium contains 10% fetal bovine serum (FBS, Life Technologies), 25 mM HEPES, 100 U / ml penicillin and 100 ug / ml streptomycin. The cell lines were cultured in 150 mm dishes and cultured at 37 ° C in 95% air and 5% CO ₂

Example  11: Cell proliferation Assay

96-well cell culture plates (Nalgen Nunc International) were plated with 2 x 10 ³ cells / well of breast (MCF-7) and prostate (LNCaP) cancer cell lines to determine the effect of relaxin- And cultured at 37 ° C in 95% air and 5% CO ₂ or 100% air without CO ₂ . The cells were treated with 500 ng / ml rhRLN2 in top heat of each cell and a 1: 5 dilution of passage was placed in the next column in growth medium. The cells were cultured for 3 days and treated with 100 uL / ml cell counting kit-8 (CCK-8, Dojindo laboratories, Kumamoto, Japan) for 4 hours. The plate was analyzed at 450 nm with a Spectra Max 250 microplate reader (Molecular Devices, Sunnyvale, USA). To determine the effect of anti -relaxin-2 mAb on rhRLN2 peptide-dependent cell growth, rhRLN2 peptide was preincubated with anti-relaxin-2 mAb for 1 hour at 4 ° C. The cultured samples were treated with breast cancer cells and prostate cancer cells, and cell proliferation assays were performed.

<110> Industry Academic Cooperation Foundation, Hallym University <120> An anti-relaxin-2 antibody and the same for anti-cancer <130> OP16-0248HS <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 <400> 1 Gly Phe Ser Leu Thr Thr Tyr Gly Val His   1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 <400> 2 Ile Ile Trp Pro Gly Gly Asn Thr Asn Tyr Asn Ser Ala Leu Met Ser   1 5 10 15 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 <400> 3 Asp Gly Gly Thr Thr Val Asp Tyr Phe Asp Tyr   1 5 10 <210> 4 <211> 156 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 4 Leu Pro Glu Phe Gln Val Gln Leu Glu Gln Ser Gly Pro Gly Leu Val   1 5 10 15 Ala Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser              20 25 30 Leu Thr Thr Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly          35 40 45 Leu Glu Trp Leu Gly Ile Ile Trp Pro Gly Gly Asn Thr Asn Tyr Asn      50 55 60 Ser Ala Leu Met Ser Ser Leu Ser Ile Ser Arg Asp Asn Ser Ser Ser Ser  65 70 75 80 Gln Val Phe Leu Lys Met Asn Ser Leu Gln Ile Asp Asp Thr Ala Met                  85 90 95 Tyr Tyr Cys Ala Arg Asp Gly Gly Thr Thr Val Asp Tyr Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala         115 120 125 Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser     130 135 140 Ser Val Thr Leu Gly Cys Leu Val Lys Arg Ser Ser 145 150 155 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 <400> 5 Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn   1 5 10 15 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 <400> 6 Ala Ala Ser Lys Gln Gly Ser   1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 <400> 7 Gln Gln Ser Lys Glu Val Pro Phe Thr   1 5 <210> 8 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 8 Glu Leu Asp Ile Val Ile Thr Gln Thr Thr Ala Ser Leu Ala Val Ser   1 5 10 15 Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp              20 25 30 Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln          35 40 45 Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Lys Gln Gly Ser Gly Val      50 55 60 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn  65 70 75 80 Ile His Pro Leu Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln                  85 90 95 Ser Lys Glu Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile             100 105 110 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ala Cys         115 120 <210> 9 <211> 456 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain <400> 9 caggttcagc tggagcagtc tggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acttgcactg tctctgggtt ttcattaacc acctatggtg tacactgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggaata atatggcctg gtggaaacac aaattataat 180 tcggctctca tgtccagact gagcatcagc agagacaact ccaagagcca agttttctta 240 aaaatgaaca gtctgcagat tgatgacaca gccatgtact actgtgccag agatggaggt 300 actacggtag actactttga ctactggggc caaggcacca ctctcacagt ctcctcagcc 360 aaaacaacag ccccatcggt ctatccactg gcccctgtgt gtggagatac aactggctcc 420 tcggtgactc taggatgcct ggtcaagaga tcttcc 456 <210> 10 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Light chain <400> 10 gatattgtga tcacacagac tacagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gaactggttc 120 caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa gcaaggatcc 180 ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240 cctttggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccattc 300 acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgcacc aactgtatcc 360 gcatgcacc 369

Claims (11)

A heavy chain variable region having a heavy chain CDR (complementarity determining region) amino acid sequence of CDRH1 consisting of SEQ ID NO: 1, CDRH2 consisting of SEQ ID NO: 2 and CDRH3 consisting of SEQ ID NO: 3; And
An antibody to Relaxin-2 comprising a light chain variable region having a light chain CDR amino acid sequence of CDRL1 of Sequence Listing Fifth Sequence, CDRL2 of Sequence Listing Sequence No. 6 and CDRL3 of Sequence Listing Seventh Sequence, or His antigen binding fragment. The antibody or its antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4. The antibody or its antigen-binding fragment thereof according to claim 1, wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 8. A nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of any one of claims 1 to 3. 5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 9. A nucleic acid molecule encoding a light chain variable region of the antibody or antigen-binding fragment thereof of any one of claims 1 to 3. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 10. (a) a pharmaceutically effective amount of an antibody or an antigen-binding fragment thereof to the subject reliloxin-2 of any one of claims 1 to 3; And (b) a pharmaceutically acceptable carrier.
9. The method of claim 8, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, lung cancer, gastric cancer, rectal cancer, soft-tissue sarcoma, colon cancer, carcinoma of the papilla vateri, , Nonendocrine lung tumor, and bronchial carcinoid tumor. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; A cancer diagnostic kit comprising an antibody against relaxin-2 or an antigen-binding fragment thereof according to any one of claims 1 to 3. 11. The method of claim 10, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, lung cancer, gastric cancer, rectal cancer, soft-tissue sarcoma, colon cancer, carcinoma of the papilla vateri, , Nonendocrine lung tumor, and bronchial carcinoid tumor. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;

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