WO2012138102A2 - Dlk1-specific human antibodies and pharmaceutical composition containing same - Google Patents

Dlk1-specific human antibodies and pharmaceutical composition containing same Download PDF

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Publication number
WO2012138102A2
WO2012138102A2 PCT/KR2012/002496 KR2012002496W WO2012138102A2 WO 2012138102 A2 WO2012138102 A2 WO 2012138102A2 KR 2012002496 W KR2012002496 W KR 2012002496W WO 2012138102 A2 WO2012138102 A2 WO 2012138102A2
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seq id
dlk1
heavy chain
light chain
variable region
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PCT/KR2012/002496
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French (fr)
Korean (ko)
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WO2012138102A3 (en
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박영우
조기원
이동희
윤선하
손명호
김경진
김동진
이유라
박찬웅
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한국생명공학연구원
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Priority to KR10-2011-0030871 priority Critical
Priority to KR20110030871 priority
Priority to KR1020120001457A priority patent/KR101438265B1/en
Priority to KR10-2012-0001457 priority
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Publication of WO2012138102A2 publication Critical patent/WO2012138102A2/en
Publication of WO2012138102A3 publication Critical patent/WO2012138102A3/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/485Epidermal growth factor [EGF] (urogastrone)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Abstract

The present invention relates to human antibodies that binds specifically to the extracellular water-soluble domain of DLK1 and fragments which include antigen-binding sites thereof; a polynucleotide which encodes for the human antibodies or fragments thereof; a recombinant vector containing the polynucleotide; transformants transformed with the recombinant vector; method for manufacturing the human antibodies or fragments thereof; a pharmaceutical composition for preventing or treating disease which includes the human antibodies or fragment thereof; and diagnostic kits. Human antibodies and fragments which include antigen-binding sites that bind specifically to the extracellular water-soluble domain of DLK1 of the present invention show anti-tumor activity. By significantly raising the binding capacity of the DLK1 extracellular water-soluble domain to the Activin receptor type II B (ACVR2B), utility is demonstrated for prevention and treatment of diseases associated with ACVR2B signal transduction including related metabolic disorders, immune disease, liver diseases, and cancer.

Description

DLK1 specific human antibodies and pharmaceutical compositions containing them

The invention DLK1 (delta-like 1 homolog) human specifically binding to other regions on the water-soluble domain cell antibody or fragment thereof comprising an antigen binding site, the human antibody or a polynucleotide encoding a fragment thereof, wherein said polynucleotide recombinant vector, the recombinant vector transformed with the transformant, the human antibody or a method of making a fragment thereof, the human antibody or a pharmaceutical for the prevention and treatment of diseases, including fragments thereof compositions and diagnostic kits containing such It relates.

Cancer are characterized as "uncontrolled cell growth", is called the tumor (tumor) by these abnormal cell growth, cell mass is formed to also be the case and penetrating into the tissues around, severe spread to other organs of the body. The cancer is incurable chronic disease that gives pain to the patient, even if treated with surgery, radiation and chemotherapy does not cure the underlying in many cases, ultimately leading to death.

Globally, patients suffering from cancer, had million over 2000, there are more than 6 million people die each year from cancer, in 2020, because the 11 million people expected to die of cancer, cancer is important to naeeoya urgently find the cure the disease. Cancer is the case, but the difference is, in every developed country or countries account for over 20% of all deaths. Cancer is classified as blood cancers and solid tumors, it occurs in the lung, stomach, breast and oral cancer, liver cancer, cervical cancer, esophageal cancer, almost all of the body parts such as skin cancer. These tumors are collectively called the first chemotherapy except for surgery or radiation therapy of the methods used to treat cancer, and the most of the material mainly showing the anticancer activity by inhibiting the synthesis of nucleic acids.

On the other hand, Notch / Delta / up-rate system (notch / delta / serrate family) DLK1 (delta-like 1 homolog) belonging to the 383 amino acid glycoprotein is encrypted in dlk1 gene transmembrane (transmembrane) in which is located on chromosome 14q32 It consists of a. The protein has a 280 cell divided into the outer region and the 24 transmembrane regions, in the region of 56 cells, the double membrane outside of the six epidermal growth factor similar Repeats (epidermal growth factor like repeat) domains, three It has an N- glycosylation (glycosylation) and 7 O- glycosylation site. It is well known to; (TACE Tumor necrosis factor alpha converting enzyme) shown outside of the cell membrane is located in the cell membrane by (shedding), proteins of the airway function separately DLK1 is also a membrane protein, however, tumor necrosis factor alpha - converting enzyme.

A study on the relevance of DLK1 cancer is the expression of DLK1 is overexpressed in brain cancer (glioma), and if over-expression of cDNA of DLK1 brain cancer increased growth of brain cancer cells (proliferation) metastases (migration) increases There was a report that (D Yin et al, Oncogene, 25:. 1852-1861, 2006), compared to the up DLK1 expression in liver cancer and normal stem cells, the size of the tumor when sikyeoteul DLK1 reduce expression of the siRNA through experiments It was reported that reduced (Huang J et al, Carcinogenesis, 28 (5):. 1094-1103, 2007).

In addition, the present inventors first identified hanba that the Republic of Korea Patent No. 10-0982170 arc soluble extracellular region domains and Fc domains are joined DLK1-Fc fusion proteins of the domains and the IgG antibody of DLK1 this may indicate anticancer activity . However, there was a bar the anticancer action of the extracellular domain of the soluble domain DLK1 known at all before Whether by any functional group.

Thus, the inventors have combined the embodiment efforts result, in this area, a water-soluble domain extracellular fluid activin in DLK1 and competitive liquid activin receptor type 2B (Activin receptor type II B) to clarify the anticancer mechanism of action of extracellular region soluble domain of DLK1 to find a represents an antitumor effect by blocking the activin signaling solution, and have completed the present invention.

Further, not only a human antibody that specifically binds to a region-accepting domain outside of the DLK1 cells can suppress the metastasis of cancer cells, liquid activin significantly increase the binding affinity to receptor type 2B the areas soluble domain outside of DLK1 cells It confirmed that this can and have completed the present invention.

An object of the present invention to provide a DLK1 (delta-like 1 homolog) fragment containing a human antibody or an antigen-binding site that specifically binds to the soluble extracellular region domain of the cell.

Another object of the present invention to provide a polynucleotide encoding a fragment containing the human antibody or an antigen-binding site.

Another object of the invention to provide a recombinant vector comprising said polynucleotide.

Another object of the invention to provide a transformant transformed with the recombinant vector.

A further object of the invention is to provide a manufacturing method of a fragment containing the human antibody or an antigen-binding site.

A further object of the invention is to provide a pharmaceutical composition for preventing or treating cancer comprising a fragment containing the human antibody or an antigen-binding site.

In some embodiments in order to achieve the above object, the present invention provides a DLK1 (delta-like 1 homolog) fragment containing a human antibody or an antigen-binding site that specifically binds to the soluble extracellular region domain of the cell.

The present invention the term, "DLK1 (delta-like 1 homolog)" means in the encrypted in the glycoprotein gene dlk1 pass film which is composed of 383 amino acids which is located on chromosome 14q32.

As used herein, the term "extracellular domain soluble domain of DLK1" is the extracellular region, a transmembrane region and a cell means the split DLK1 protein of the soluble domain of the extracellular region in the region, and cancer of the extracellular domain soluble domain of DLK1 effect is the identified first bar by the present inventors.

The extracellular domain of the soluble domain DLK1 herein can be expressed by mixing the water-soluble DLK1.

Preferably, the water-soluble DLK1 of the present invention may be formed of a 300 amino acid 200 to which the water-soluble DLK1 activity, more preferably, be made of the amino acid sequence set forth in SEQ ID NO: 1. However, the water-soluble amino acid sequence showing a DLK1 activity is limited without It may be included.

"Human antibody" terminology, in the present invention means that the entire amino acid sequence constituting the antibody, including all of the complementarity determining regions, framework regions as a molecule derived from human immunoglobulin is composed of a human immunoglobulin.

In general, one of the antibody molecule has two heavy chains (heavy chain) and has two light chains (light chain), each of the heavy and light chains contains a variable region in its N- terminus. Each variable region consists of three complementarity determining regions (Complementarity determining region, CDR) and four structural formation region (framework regions, FRs), and complementarity determining regions are determining the antigen binding specificity of the antibody, to form the structure of the variable region present in a relatively short peptide sequence is maintained in the region.

Fragment containing the human antibody or an antigen-binding site of the present invention has a specific activity that bind to soluble DLK1. In addition, the fragment containing the human antibody or an antigen-binding site of the present invention may have, by specifically binding to soluble DLK1, activity to ultimately increase the binding force for the liquid of the water-soluble activin receptor type 2B DLK1. Therefore, it is a by fragment containing the human antibody or an antigen-binding site of the present invention increases the affinity for the liquid activin receptor type 2B of the extracellular domain soluble domain of DLK1, liquid activin receptor type 2B combine to DLK1 and competitive to block the binding of other ligands, it is possible to suppress the transmission signal.

In addition, the fragment containing the human antibody or an antigen-binding site of the present invention can inhibit the signal transduction in hypoxic cells.

In an embodiment of the present invention, by ensuring that it can be combined with human antibody typically B09 antibody is soluble DLK1 of the present invention, it was confirmed that the human antibody of the invention can specifically bind to soluble DLK1 ( 21, 22 and 27), when treated with the human antibody of the invention and, together with the water-soluble DLK1 was confirmed that the binding force of a water-soluble liquid DLK1 for activin receptor type 2B hayeoteum significantly increase (Fig. 25, 27 and 29).

In one embodiment of the present invention, by ensuring that it is possible to inhibit the signal transduction the cells in the human antibody of the representatively the B09 antibody hypoxia of the present invention, the human antibodies of the invention the cellular response in hypoxic It was confirmed that this can be inhibited (Fig. 28).

Preferably, the fragment containing the human antibody or an antigen-binding site of the present invention as part of a heavy chain variable region, SEQ ID NO: 2 (SYAMN), SEQ ID NO: 8 (DYAIH), SEQ ID NO: 14 (EHAMH), SEQ ID NO: 20 ( DYAMH), SEQ ID NO: 26 (any one of the heavy chain CDR1 sequence selected from the group consisting of LYGMS) and SEQ ID NO: 32 (DYYMS); SEQ ID NO: 3 (TITATSGKTYYADSVKG), SEQ ID NO: 9 (WINPGSGNTKYSHNFEG), SEQ ID NO: 15 (GINWNSGKTGYADSVKG), SEQ ID NO: 21 (GISWNSGSIGYADSVKG), SEQ ID NO: 27 (SIPGSGTRTHYADSVKG) and SEQ ID NO: any one of the heavy chain selected from the group consisting of 33 (YISGSGITTYYADSVKG) CDR2 sequence; Or SEQ ID NO: 4 (GESCSGGACSDFDY), SEQ ID NO: 10 (SVSAYGSNYFDP), SEQ ID NO: 16 (SGGYGGNTNWYFDL), SEQ ID NO: 22 (GPGLATGKGYADY), SEQ ID NO: 28 (STAYLFDY) and SEQ ID NO: 34 which is selected from the group consisting of (LQGHCSGGACSNWFDA) one can be a fragment containing a human antibody or an antigen-binding site comprising a heavy chain CDR3 sequence, also as part of a light chain variable region, SEQ ID NO: 5 (TGTSSDIGRYNRVS), SEQ ID NO: 11 (QASQDISNYLN), SEQ ID NO: 17 (IGTSSNIGVGYDVH), SEQ ID NO: 23 (RASQRISSWLA), SEQ ID NO: 29 (RASQSIRHYLA) and SEQ ID NO: any one of the light chain CDR1 sequence selected from the group consisting of 35 (RASQSILTYLN); SEQ ID NO: 6 (DVTTRPS), SEQ ID NO: 12 (STSNLQS), SEQ ID NO: 18 (GNNNRPS), SEQ ID NO: 24 (SASTLHN), any one of the light chain selected from the group consisting of SEQ ID NO: 30 (GASSRAT) and SEQ ID NO: 36 (AASSLQR) CDR2 sequence; Or SEQ ID NO: 7 (GSYAGSYTY), SEQ ID NO: 13 (QQLNSYPL), SEQ ID NO: 19 (QSYDSRLGV), SEQ ID NO: 25 (QQGHSFPY), SEQ ID NO: 31 (QHYGSPLH) and SEQ ID NO: one selected from the group consisting of 37 (QQGYGTPY) one comprising a light chain CDR3 sequence may be a human antibody or fragment thereof comprising an antigen binding site.

More preferably, the fragment containing the human antibody or an antigen-binding site of the present invention is a heavy chain variable region, comprising a heavy chain CDR1, a heavy chain CDR2, and SEQ ID NO: heavy chain CDR3 according to the four set forth in SEQ ID NO: 3 set forth in SEQ ID NO: 2 the heavy chain variable region; A heavy chain CDR1 defined by SEQ ID NO: 8, a heavy chain variable region comprising the heavy chain CDR2 and heavy chain CDR3 defined by SEQ ID NO: 10 set forth in SEQ ID NO: 9; In SEQ ID NO: 14 set forth the heavy chain CDR1, a heavy chain variable region comprising the heavy chain CDR2 and heavy chain CDR3 SEQ ID NO: 16 as set forth described in SEQ ID NO: 15; In SEQ ID NO: 20 set forth the heavy chain CDR1, a heavy chain variable region comprising the heavy chain CDR2 and heavy chain CDR3 SEQ ID NO: 22 as described in described in SEQ ID NO: 21; SEQ ID NO: 26 as described in the heavy chain CDR1, a heavy chain variable region comprising the heavy chain CDR2 and heavy chain CDR3 SEQ ID NO: 28 as described in described in SEQ ID NO: 27; Or it may be a fragment containing a human antibody or an antigen-binding site comprising a heavy chain variable region comprising the heavy chain CDR2 and heavy chain CDR3 SEQ ID NO: 34 according to the described in the heavy chain CDR1, SEQ ID NO: 33 set forth in SEQ ID NO: 32.

Further, as the light chain variable region, the light chain variable region comprising the light chain CDR1, light chain CDR2 SEQ ID NO: 6 and SEQ ID NO: light chain CDR3 according to 7 described as set forth in SEQ ID NO: 5; A light chain CDR1 defined by SEQ ID NO: 11, a light chain variable region comprising a light chain CDR3 defined by SEQ ID NO: 13 and a light chain CDR2 defined by SEQ ID NO: 12; In SEQ ID NO: 17 set forth the light chain CDR1, a light chain variable region comprising the light chain CDR3 according to the light chain CDR2, and SEQ ID NO: 19 set forth in SEQ ID NO: 18; In SEQ ID NO: 23 set forth the light chain CDR1, a light chain variable region comprising the light chain CDR3 according to the light chain CDR2, and SEQ ID NO: 25 set forth in SEQ ID NO: 24; In SEQ ID NO: 29 set forth the light chain CDR1, a light chain variable region comprising the light chain CDR3 according to the light chain CDR2, and SEQ ID NO: 31 set forth in SEQ ID NO: 30; Or it may be a fragment containing a human antibody or an antigen-binding site comprising the light chain variable region comprising the light chain CDR2 and light chain CDR3 SEQ ID NO: 37 as described according to the light chain CDR1, SEQ ID NO: 36 set forth in SEQ ID NO: 35.

Even more preferably, the fragment containing the human antibody or an antigen-binding site of the present invention to a heavy chain variable region sequence, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46 or SEQ ID NO: 48 described may be a fragment containing a human antibody or an antigen-binding site comprising the amino acid sequence, a light chain variable region sequence, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 49 comprising the amino acid sequence set forth in may be a human antibody or fragment thereof comprising an antigen binding site. In addition, the heavy and light chains may be used separately or together depending on the purpose, it is possible to a large number of CDR sequences, and any combination of the light chain and the heavy chain according to conventional genetic engineering methods by those skilled in the art that the bar purpose.

Holding the antigen-binding function as a fragment having immunological activity of a human antibody molecule of the present invention that can achieve the antibody binding - The term "fragment comprising the antigen binding site of human antibodies" means antigens (soluble DLK1) in the present invention It means that the fragment. Examples of such fragments are Fab fragment consisting of (i) a light chain variable yeokyeok (VL) and the heavy chain variable region (VH) and the constant light chain yeokyeok (CL) and the first constant domain (CH1) of the heavy chain; (Ii) Fd fragment consisting of the VH and CH1 domains; (Iii) Fv fragment consisting of the VL and VH domains of a monoclonal antibody; (Iv) dAb fragment consisting of VH domain (Ward ES et al, Nature, 341:. 544-546,1989); (V) isolated CDR regions; (Vi) 2 interconnecting the divalent fragment F comprising a Fab fragment (ab ') 2 fragment; (Vii) single chain Fv molecules (scFv) coupled by a peptide linker to combine the VH domain and VL domain to form an antigen binding site; And the like, whereby; (viii) bispecific is of a single chain Fv dimers (PCT / US92 / 09965) and (ix) a multivalent or multi-specific fragments of dia body (WO94 / 13804 diabody) produced by gene fusion it is not limited.

Human antibodies of the invention can be easily produced by known monoclonal antibody production techniques. For example, a method for producing a monoclonal antibody using B lymphocytes obtained from the immunized animal may be performed by preparing a hybridoma, but may be performed using phage display (phage display) technology, limited to it is not.

Preferably, the human antibodies of the invention may be a human antibody produced from a phage display. Antibody library using the phage display technology is a method to obtain the antibody genes from B-lymphocytes directly without making a hybridoma expressing the antibody on phage (phage) surface. When using the phage display technology can be a conventional difficulties involved in generating monoclonal antibodies overcome by the immortalized B- cells (immortalization). Phage display technique of a common antibody is 1) coat protein (coat protein of the phage) inserting the sequence comprising an antibody variable region of random sequence in the gene region for the N- terminal p (or p); 2) expressing a fusion protein of an antibody variable region encoded by a random sequence with the coat protein portion of the wild-type; 3) treating an antigen capable of binding with said antibody fusion protein library; 4) the antibody binds to the antigen-step of the phage particles eluted using a low pH or competitive binding molecules; 5) the step of amplifying the eluted phage from the host cell; 6) repeating the method to obtain the desired amount; And 7) it may be constructed from the DNA sequence of the phage clone selected by panning with the step of determining the sequence of the antibody variable region which is active.

Phage which can be used to construct an antibody library in the above step is, for example, filamentous phage (filamentous phage) as fd, M13, f1, If1, Ike, Zj / Z, Ff, Xf, but the Pf1 or Pf3 phage and is not the type of available phage used in the present invention limited by the examples. In addition, examples of vectors that can be used for the expression of the heterologous gene on the surface of the filamentous phage, fUSE5, fAFF1, fd-CAT1 or fdtetDOG such as phage vectors or pHEN1, pComb3, pComb8, pKRIBB-Fab or pSEX etc. Although in the phagemid vector, without being limited thereto.

Further, the helper phage can be used to provide a wild-type coat protein that is required for a successful re-infection of recombinant phage for amplification, for example but not intended to be, or the like M13K07 VSCM13, limited.

In another one aspect, the invention provides a recombinant vector comprising a human antibody or a polynucleotide encoding a fragment thereof comprising an antigen binding site (hereinafter a fragment thereof) and the poly New Clare suited according to the invention.

A polynucleotide of the present invention, nucleotide units (monomer) is a covalently linked chain-shaped polymer (polymer) a predetermined length or more of DNA (deoxyribonucleic acid) or (ribonucleic acid) RNA strand in the oligonucleotide that resulted in a hold by a human according to the invention an antibody or a polynucleotide encoding a fragment thereof. Preferably, the polynucleotide of the invention may be a polynucleotide that comprises a light chain variable region described in SEQ ID NO: 51 and a heavy chain variable region described in SEQ ID NO: 50.

A polynucleotide encoding a human antibody or fragment thereof of the invention is a human being in consideration of the codon-preferred organisms intended to express the result or the human antibody or fragment thereof to the degeneracy (degeneracy) of the codon, it expressed from the coding region to the extent that does not change the amino acid sequence of the antibody or fragment thereof, and various modifications may be made to the coding region, and in the portions other than the coding region may be made various changes or equation in a range which does not affect the expression of the gene, such a modified gene will also be appreciated by those skilled in the art well is included in the scope of the invention. That is, polynucleotides of the invention may be mutated by a one or more nucleotide substitution, deletion, insertion or a combination thereof encoding a protein having equivalent activity, these are also included within the scope of the present invention.

The recombinant vector of the present invention as a means to express a human antibody or fragment thereof of the present invention by introducing the DNA into a host cell from a microorganism, at the time of production of the recombinant vector in a host cell to produce the human antibody or fragment thereof depending on the type can select a promoter (promoter), a terminator (terminator), enhancer expression control sequences, such as sequences for membrane targeting or secretion, such as (inhancer) as appropriate, and various combinations in accordance with the object.

The recombinant vector of the present invention is not one limited to, plasmid vectors and the like, Coe mid vector, bacteriophage vector, and virus vector. A suitable recombinant vector comprising in addition a membrane signal sequence or leader sequence for targeting or secretion promoter, an operator, an initiation codon, expression control elements, such as termination codons, polyadenylation signals and enhancers, and can be variously made in accordance with the purpose. Promoter of the recombinant vector may be constitutive or inducible. If the signal sequence is the host is yeast (yeast), the MFα signal sequence, SUC2 signal sequence, etc. When the host is an animal cell include, but to use the insulin signal sequence, α- interferon signal sequence, antibody molecule signal sequence, this is not limited. In addition, the combination vector may comprise a selection marker for selecting host cells containing the vector, in the case of replicable recombinant vector may include a replication origin.

In yet another aspect, the present invention provides a method for preparing a recombinant vector comprising a polynucleotide encoding a transformed and the transformant (a) a human antibody or fragment thereof of the invention with a recombinant vector of the present invention; (B) the transformed as transformants by introducing the recombinant vector into a host the cells; And (c) provides a method for producing a human antibody or fragment thereof according to the present invention comprising culturing said transformant.

After transfection of the recombinant vector of the invention such as suitable host cells, e.g., yeast cells, animal cells by culturing the transformed host cell can be mass-produced human antibody or fragment thereof of the present invention.

As used herein, the term "transgenic" is meant to be introduced into the gene in the host cell, expression in a host cell and transformed gene if it can be expressed in a host cell within the chromosome of the host cell insertion or it is located besides the chromosome it includes but are not limited somehow. In addition, the gene comprises DNA and RNA as a polynucleotide capable of encoding a polypeptide. The gene as long as it can be expressed is introduced into a host cell does not matter, whether it is introduced in any form. For example, the gene can be introduced into a host cell in the form of there is expressed by itself in an expression cassette (expression cassette) A polynucleotide construct containing all the elements required. The expression cassette typically comprises a promoter that is operably linked to the gene (promoter), a transcription termination signal, ribosome binding site and translation termination signal.

To transformed by introducing the recombinant vector of the invention into a host cell are known methods of recombinant vector containing the DNA of the present invention in the art, for example, but are not limited to, transient transfection (transient transfection), mediated-microinjection, transduction (transduction), cell fusion, calcium phosphate precipitation, liposome-mediated transfection (liposem-mediated transfection), DEAE-dextran-mediated transfection (DEAE dextran-mediated transfection), polybrene transformants can be transformed by introducing into a host cell by known methods, such as infection (polybrene-mediated transfection), electric invasion method (electroporation).

The host cell is Saccharomyces as MY processes Sergio non jiae (Saccharomyces cerevisiae) and the like yeast, insect cells may be a eukaryotic cell derived from a plant cell, an animal cell, preferably wherein the animal cell is autologous or allogeneic be animal cells is. Autologous or allogeneic switch is manufactured by introducing a transgenic animal cell body may be used for cell therapy that is administered to a subject to treat cancer.

How to culture the transformant of the present invention can be used by appropriately selecting any of the methods known in the art to produce human antibody or fragment thereof of the present invention.

In another one embodiment, the fragment containing the human antibody or an antigen-binding site of the present invention is to, by specifically binding to the soluble domain outside of DLK1 cells, liquid activin receptor type 2B the areas soluble domain outside of DLK1 cells has an activity to increase the binding force, whereby the liquid by blocking the binding of another ligand that can on activin receptor type 2B combine to DLK1 and competitive, useful for preventing or treating diseases which are associated with liquid activin signaling pathway of the receptor type 2B it can be used.

A liquid activin ligand binding to the receptor type 2B is liquid activin (activin A, activin B and inhibin A) (Derynck, Zhang et al. 1998), Nodal (Nodal) (Oh and Li 2002), BMP6 (Ebisawa, Tada et al. 1999), BMP7 (Yamashita, ten Dijke et al. 1995), GDF5 (Nishitoh, Ichijo et al. 1996), GDF11 (Oh, Yeo et al. 2002) there is known. When the ligands are coupled to the ACVR2B and form a complex in combination as a type I receptor or ACVR1A ACVR1B (complex), ACVR2B is phosphorylation, thereby activating the type I receptor. The activated type I receptor activates the transcription control design Smad performing cellular signaling (Derynck, Zhang et al. 1998). These ligands are performing a wide variety of signal transduction through the ACVR2B, when the regulation of signal transduction in particular of fault signaling and activation by overexpression of the ligand is known to exhibit a variety of disorders.

Thus, yet another one embodiment, the present invention provides a pharmaceutical composition for preventing or treating a disease comprising a fragment containing a human antibody or an antigen-binding site of the present invention.

More specifically, the present invention provides a comprising a fragment containing a human antibody or an antigen-binding site according to the present invention, cancer, metabolic disorders, immune diseases or liver pharmaceutical composition for prevention or treatment.

In yet another one embodiment the present invention provides a method of treating cancer comprising administering to a pharmaceutical composition for cancer, metabolic disorders, immune system disease, an object that has one or onset possibility of onset of liver disease according to the invention It provides.

The term "prevention" means any action that to suppress the ACVR2B related diseases, including cancer, by administration of the pharmaceutical composition of the present invention, or delay the onset, and the term "treatment" in the present invention is the pharmaceutical composition of the present invention It means any of the acts to improve the symptoms caused by ACVR2B related diseases including cancer, by administration or benefit changes.

The present invention refers to all animals including the term "object" is a human invention that can hayeotgeona outbreak ACVR2B related diseases, including cancer.

Preferably, the pharmaceutical composition of the present invention can be changed advantageously reduce or improve the symptoms of the cancer through the cancer metastasis or invasion-inhibitory activity.

The pharmaceutical composition of the present invention may further comprise a water-soluble DLK1, the water-soluble DLK1 can exhibit such antitumor effect by blocking the liquid activin and activin receptor type 2B competitively liquid solution activin signaling by binding to. Further, the liquid activity of activin aqueous DLK1 to inhibit the binding between the receptor type 2B and activin are liquid can be significantly raised by the human antibodies of the invention, it is possible to treat effectively ACVR2B related diseases, including cancer.

In addition, the pharmaceutical composition of the present invention and the water-soluble DLK1 antibody Fc region, and may further comprise a conjugated DLK1-Fc fusion protein, preferably the antibody Fc region may be a human antibody Fc.

In one embodiment of the present invention, the results of processing the representatively B09 antibody of human antibody according to the present invention, it was confirmed the inhibition (Fig. 32), tumor metastasis inhibition of a liquid activin signaling (Figure 34), of the present invention when treated with the human antibody and a water-soluble DLK1 was confirmed that the water-soluble liquid and hayeoteum DLK1 activin significantly increase bonding force between the receptor type 2B (Fig. 25, 27 and 29). Therefore, the pharmaceutical composition of the present invention exhibits an antitumor effect by blocking the liquid activin signaling by inhibiting the binding between activin and liquid solution activin receptor type 2B can be useful in the prevention and treatment of cancer.

When the cancer is in progress pharmaceutical composition of the present invention bean aekti overexpressed in the onset or progression of cancer or liquid activin / liquid activin growth of cancer by a receptor type 2B signaling, transfer, etc., it may be used without limitation, and preferably It may be used for skin cancers, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prevention and treatment of cancer than any one selected from the group consisting of pancreatic cancer and gastric cancer.

The metabolic diseases in the present invention may include diabetes or obesity, the immune system diseases may include autoimmune disease or rheumatoid tube jyeolyeom, the liver disease from chronic hepatitis, alcoholic liver cirrhosis, acute liver failure, liver cancer or liver It may include, but, if the diseases associated with signal transduction caused by the binding of the ligand ACVR2B, and the like.

The route of administration of the pharmaceutical composition of the present invention can be administered via any common route which can be reached with the object tissue or cells. The pharmaceutical composition of the present invention is a stay in direct or indirect intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, blood administration, oral administration, pulmonary administration, rectal administration, the cells can be administered, as desired. The pharmaceutical composition of the present invention To this end, may be administered by any of the devices in the active material is within a target cell.

The pharmaceutical composition of the present invention may comprise an acceptable carrier. The pharmaceutical composition comprising a pharmaceutically acceptable carrier may be in various formulations sphere oral or parenteral. When formulated, the are prepared by using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surface active agents. Solid formulations for oral administration include tablets pills, powders, granules, capsules, etc. These solid preparations may be, for at least one or more excipients, for example, one or more compounds, starch, calcium carbonate, sucrose (sucrose) or lactose (lactose ), it is prepared a mixture of gelatin. Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. are applicable there is a number of excipients in addition to simple diluents such as water and liquid paraffin which is commonly used, for example, can contain wetting agents, sweeteners, aromatics and preservatives, etc. is. Parenteral formulations are sterilized aqueous solution for administration, include a non-aqueous water-insoluble excipients, suspensions, emulsions, lyophilized preparations, suppositories. Non-aqueous water-insoluble excipients, suspensions and the like can be used as is for injectable ester such as propylene glycol (propylene glycol), polyethylene glycol, olive oil and vegetable oils, such as ethyl oleate. Group zero of suppositories and the like above tepsol (witepsol), Macrogol, Tween (tween) 61, cacao paper with gelatin, laurin, glycero- be used.

The pharmaceutical composition of the present invention is selected from tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solution, nonaqueous solvent, suspension, emulsion, the group consisting of freeze-dried preparations and suppositories which it may have any of the formulations.

The pharmaceutical composition of the present invention will be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to any medical treatment, and effective dose level object type and severity, age, gender and activity of the drug, the drug to can be determined according to the sensitivity, administration time and emission rate, duration of treatment, the well-known elements in the element and other medical fields, including drugs that are used simultaneously.

The pharmaceutical composition of the present invention can be used in combination with either alone, surgery, Glands therapy, drug therapy, and the use of biological response modifiers for the treatment of cancer.

In yet another aspect, the present invention is the DLK1 (delta-like 1 homolog), cancer, metabolic disorders, including fragments containing human antibody or an antigen-binding site specifically binding to other regions on the water-soluble domain cells and it provides the immune system disease or liver disease diagnostic kit.

In the present invention, the kit can include but an enzyme immunoassay (ELISA) kit or a sandwich ELISA kit, and the like.

As well as it indicates the enemy human antibody or the anti-cancer effect for an antigen binding site fragments that specifically bind to the extracellular domain soluble domain of DLK1 of the present invention, the binding force of the liquid activin receptor type 2B the areas soluble domain outside of DLK1 cells thereby significantly increased.

Figure 1 is a picture and graph showing the effect of inhibiting metastasis in pancreatic cancer cell line (MIA-PaCa-2) by a water-soluble DLK1.

Figure 2 is a photograph and graph showing the inhibitory effects of the transition from the cervical cancer cell line (HeLa) by the water-soluble DLK1.

3 is a photograph and graph showing the attached independent growth inhibitory effect on pancreatic cancer cell line (MIA-PaCa-2) by a water-soluble DLK1.

Figure 4 is a photograph and graph showing the attached independent growth inhibitory effect of the uterine cancer cell line (HeLa) by the water-soluble DLK1.

Figure 5 is a photo showing a wound healing effect in inhibiting pancreatic cancer cell line (MIA-PaCa-2) and renal cancer cell line (786-O) of the water-soluble DLK1.

Figure 6 is a photograph showing a Western blot results to find out the effect of water-soluble DLK1 on the signaling of the actual cell.

Figure 7 is a Western blot and RT-PCR photo picture showing that the water-soluble DLK1 is overexpressed in a cell line overexpressing the soluble DLK1.

Figure 8 is a photograph by using a cell line over-expressing the soluble DLK1 showing the metastasis, invasion, adhesion independent growth and inhibition of wound healing effect of the water-soluble DLK1.

Figure 9 shows the results with the co-immunoprecipitation method seems that the solution of the water-soluble DLK1 receptor activin receptor type 2B

10 shows the results of the ELISA shown that a water-soluble DLK1 coupled to liquid activin receptor type 2B and specific.

Figure 11 shows the results with the enzyme immunoassay shown a water-soluble DLK1 does not bind activin A than liquid.

12 shows the results of the competitive ELISA shown that the water-soluble DLK1 is combined with liquid activin and activin receptor type 2B competitively liquid measurement.

Figure 13 shows the results of the surface plasmon resonance method exhibit various liquid aqueous DLK1 activin receptor family liquid activin receptor binding to type 2B and specifically of the box.

Figure 14 is a Western blot photograph showing a liquid activin receptor type 2B Expression of cell used in the flow cytometry analysis of movement.

15 shows a water-soluble liquid and DLK1 activin seen moving flow cytometry analysis result that the coupling between the receptor type 2B occurs over the real cell.

Figure 16 shows the ELISA results of a phage polyclonal antibody to a water-soluble DLK1.

Figure 17 shows the result of confirming the variety of single phage clones for soluble antibody DLK1 through fingerprinting.

18 shows a result of analyzing the polypeptide from the CDR of a single phage clone antibodies to soluble DLK1.

Figure 19 shows the cleavage map of pNATAB H vector.

Figure 20 shows the cleavage map of pNATAB L vector.

21 shows a flow cytometry showing that the B09 antibody binds specifically to the soluble part of DLK1 movement analysis (top) and fluorescence immune staining (bottom).

Figure 22 shows the binding force between the measurement result and the water-soluble DLK1 B09 antibodies by surface plasmon resonance method.

Figure 23 shows the binding affinity measurements between liquid activin A and activin receptor type 2B solution by surface plasmon resonance method.

24 shows a binding force measurements between the aqueous solution and DLK1 activin receptor type 2B by the surface plasmon resonance method.

Figure 25 shows the binding force between the measurement result and the liquid water-soluble DLK1 activin receptor type 2B in the case of treatment with the antibody and soluble B09 DLK1 by surface plasmon resonance method.

26 is a schematic diagram showing the influence of the B09 antibody on the binding force between the water-soluble DLK1 and liquid activin receptor type 2B.

27 shows the results with the aqueous solution and DLK1 activin competition ELISA showing the effect of the antibody on the binding between receptor type 2B.

Figure 28 shows the results with the liquid activin A and activin solution competition ELISA showing the effect of the antibody on the binding between receptor type 2B.

29 shows the results with the liquid activin A and activin receptor type 2B combined aqueous solution DLK1, DLK1 antibody solution and activin receptor type competition ELISA showing the effects of antibody on the 2B between.

30 is a Western blot photograph showing the effect of the aqueous solution DLK1 on activin signaling.

31 shows the SEAP reporter analysis showing the effects of water-soluble DLK1 on the Smad signal transduction.

Figure 32 shows the SEAP reporter analysis showing the influence of the B09 antibody on the Smad signal transduction.

Figure 33 shows a reporter assay results (hypoxia response element) HRE showing the effects of hypoxia signal transduction inhibition DLK1 antibody.

Figure 34 shows a liquid activin receptor type 2B metastasis inhibitory effect of the antibody and antibody DLK1.

Figure 35 shows the influence of the liquid activin receptor type 2B DLK1 antibody and antibodies on metastasis-inhibiting effect of the water-soluble DLK1.

Figure 36 shows the tumor incidence rate of DLK1-Fc treated group in pancreatic cancer orthotopic transplant metastasis model animal experiments.

Figure 37 illustrates a hernia formation rate of DLK1-Fc treated group in pancreatic cancer orthotopic transplant metastasis model animal experiments.

Figure 38 shows a pancreatic cancer orthotopic transplantation hayeoteum transition is reduced proliferation of cancer cells in DLK1-Fc treatment groups in animal model experiments.

Figure 39 shows a pancreas, weight and size of the above DLK1-Fc treated group in pancreatic cancer orthotopic transplant metastasis model animal experiments.

Figure 40 shows a pancreatic cancer orthotopic transplantation hayeoteum transition reduced splenomegaly phenomena DLK1-Fc treatment groups in animal model experiments.

Figure 41 shows the in vivo image of a pancreatic cancer orthotopic transplant metastasis animal models.

The present invention will be described in detail through the following examples. The following examples are to illustrate the invention, but the teachings of the present invention is not limited by the following examples.

Reference Example 1: Preparation of water-soluble and DLK1 DLK1-Fc fusion protein

Example soluble extracellular region domain (hereinafter referred to as water-soluble DLK1) and aqueous DLK1 and human antibody Fc region is bonded DLK1-Fc fusion protein (hereinafter DLK1-Fc) of DLK1 used in the present specification, Republic of Korea Patent No. 10- It was prepared according to the procedure described in No. 0.98217 million.

Example 1: Determine the cancer metastasis inhibitory effect of the water-soluble DLK1

It was carried out: (15-22, 2005. Chen HC, Methods in molecular biology 294) method using a movement analysis of cancer cell lines (migration assay) of the reference order to examine the effects of the cancer cell by the water-soluble DLK1. First, cells (MIA-PaCa-2, HeLa cell line) to give a change to serum-free medium when the 50% level, and measuring the number of cells after removed through the trypsinized cells in 24 hours. The combined cells, each protein to a serum-free medium and to be treated and allowed to incubate for, create a 100 ㎕ 1 hour at 37 ℃. I for 1 hour and then raise the transwell (Corning # 3422) having a pore size of 8.0 μm thereon to put the 5 ~ 10% FBS as a chemoattractant (chemo-attractant) of 1 ㎖ a 24-well plate in the insert the cultured cells, and cells, protein 100 ㎕ mixture was incubated at 37 ℃ carbon dioxide incubator for 24 hours to 48 hours. After incubation, remove the medium and of the trans-well, Diff Quick solution (Sysmex, Japan) was used in a completely remove them by after staining the cells did not pass the transwell using a cotton swab. Then, the observing it dry the transwell a cell passing through the transwell with 400 magnifications, and then taking the picture, it is shown in FIGS.

As a result, even if the handle DLK1-Fc on the cell line MIA-PaCa-2 pancreatic cancer cell line as shown in the first positive-dependently the movement (transfer) of the cell was found to inhibited (FIG. 1). Further, when the handle DLK1-Fc in cervical cancer cell line, HeLa cell line as shown in the second amount-dependent manner was found to shift (transition) of the cell it is inhibited (FIG. 2).

Example 2: Effect of the water-soluble DLK1 on the attached independent growth of cancer cell lines confirmed

Attach independence was performed soft agar growth assay (Soft agar assay) to determine the effects of soluble DLK1 on (anchorage independence growth). First, the 10% FBS, 1% with RPMI medium containing agar (agar) 1: Mix 1 presented a 0.5% agarose composition after quasi spread on 60 mm culture dishes, 6 x 10 3 cells (MIA-PaCa-2 , HeLa cell line), the 5 ㎍ / ㎖ of mix with DLK1-Fc or Fc, 10% 0.5% after FBS is such that 0.3% of the concentration of final agarose after which gave mixture of RPMI medium with 1% agarose containing agar It was laid on top. Were cultured in 3 weeks 37 ℃ CO 2 incubator, the baby was to dry every three days was added to the medium containing 5 ㎍ / ㎖ of DLK1-Fc or Fc to prevent. After the colony is produced taking pictures, and then given to dye the colonies generated with 0.05% crystal violet dye and quantified by counting the colony, it is shown in Figs.

As a result, a pancreatic cancer cell line MIA-PaCa-2 when treated with DLK1-Fc to cell growth independent adhesion compared to Fc-treated control group of cells is only to find out inhibited (Fig. 3). In addition, the cervical cancer cell line, were attached independent growth as compared to the Fc-treated control group, only cells when treated with DLK1-Fc on HeLa cell line is found to inhibited (Figure 4).

Example 3: Effect of soluble DLK1 on the wound healing of cancer cell lines confirmed

When the pancreatic cancer cell line MIA-PaCa-2 or renal cancer cell line, and then the O-786 grown in 6-well plates to 90% cell growth hayeoteul was replaced with serum free medium for 16 hours. Since, 5 ~ 25 ㎍ / ㎖ while the concentration of DLK1-Fc or Fc 1 hour after embellish then gave handles wound scrape cells (scratch) the addition of 5% FBS and 786-O cells to cells for 16 hours, MIA -PaCa-2 cell lines were cultured for 48 hours, by observing the patterns being wound spicy, it is shown in Fig.

As a result, the pancreatic cancer cell line (MIA-PaCa-2) and renal cancer cell lines, wound healing as compared to the process control of Fc only cells when treated with DLK1-Fc in the (786-O) is was found that inhibited (Fig. 5) .

Example 4: Verification signaling by soluble DLK1

In order to examine the effects on the water-soluble DLK1 signaling of the actual cell was performed Western blot. To this end, when the in-PaCa-2 pancreatic cancer cell line MIA in culture plate grew up to 70%, to give replaced with serum-free medium and incubated for 16 hours. DLK1-Fc treated group, after the process gave 10 ㎍ / ㎖ of DLK1-Fc was added to 10% FBS, and harvested after 10 minutes. Harvested cells RIPA buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) a protease inhibitor in (protease inhibitor; Roche), phosphine Porta inhibitor cocktail I, II (phosphotase inhibitor cocktail I , II; Sigma) by the addition of a buffer was dissolved in the cell, using the BCA quantitation kit (Thermo) was determined the concentration of the soluble cell lysates. The solution of 30 ㎍ on SDS-PAGE and modified using the 5X sample buffer and then subjected to electrophoresis were transfer to NC membrane (Watman). Blot with using the p-FAK, FAK, p-AKT, AKT, p-eNOS, eNOS, p-ERK1 / 2 and ERK1 / 2 antibody (Cell signaling) made was subjected to Western blot analysis. In this case, used it was a beta-actin antibody (Sigma) as a loading control. After the reaction overnight at 4 ℃ TBST (Tween 20 0.05%) and then subjected to washing three times with using a rabbit-HRP antibody and the mouse-HRP antibody (Santa Cruz) was reacted at room temperature for one hour again TBST ( the washing was carried out three times with Tween 20 0.05%). Then, the blot using the ECL solution (Intron) developing the film by fluorescent, shown in FIG.

As a result, p-FAK, p-AKT, was unknown that they have p-eNOS is significantly reduced, the level of p-ERK1 / 2 was also found that the decrease slightly (Fig. 6). The results of the, p-FAK, p-AKT and a decrease in p-eNOS is to support the effectiveness of Figures 1 to 5, p-ERK1 reduction of / 2 is to support the attached independent growth inhibitory effect of the Figure 3 and 4 will be.

Example 5: cell line stably expressing the soluble building DLK1

To reconfirm the effect of the semi-aqueous DLK1 process from the outside, it has established a cell line overexpressing the soluble DLK1 using a pancreatic cancer cell line MIA-PaCa-2 cell line. The cell line was constructed by giving into a DNA fragment encoding the domain (extracellular domain) of the outer cell area DLK1 the pcDNA 3.1 vector. Cell lines 500 ㎍ / ㎖ G418 screening at (Geneticin, GIBCO / BRL), and cell lines was carried out by RT-PCR in order to ensure that the well-established, the culture grown cell lines and concentrated 10-fold using a 15 (Millpore) Centricon after it was confirmed that the water-soluble DLK1 is well expressed by a Western blot. Western blot was used for DLK1 monoclonal antibody (R & D) products. Comparison group was used as the transfection vector without the insert only.

As a result, as shown in FIG. 7, it was confirmed that the water-soluble DLK1 in the cell line is stably expressed (Fig. 7).

Example 6: Stable cell lines, cancer metastasis, invasion, adhesion independent growth and wound healing effect due to inhibition verification aqueous DLK1 in expressing the soluble DLK1

Transition by using the cell line constructed in Example 5 was performed the analysis of the infiltration mechanism. First, the number of cells was measured after the cells to give change to serum-free medium when the 50% level, has been removed by the trypsin treatment the cells after 24 hours. After raising to a 24-well plate as a chemoattractant (chemo-attractant) of 1 ㎖ to put the 5 ~ 10% FBS transwell (Corning # 3422) having a pore size of 8.0 μm thereon in the 5 × 10 4 then it gave the cells were incubated at 37 ℃ into carbon dioxide incubator for 24 hours to 48 hours. After incubation, remove the medium and of the trans-well, Diff Quick solution (Sysmex, Japan) was used in a completely remove them by after staining the cells did not pass the transwell using a cotton swab. Then, the observation of a complete pass through the trans-well transwell cell and then dried with 400 magnifications, and was taking a picture.

In order to observe the phenomenon infiltration experiments it was performed after coating a 1 mg / ㎖ concentrations of Matrigel (BD Science) 100 ㎕ the bottom surface transwell.

In order to observe the attached independent growth 10% FBS is loaded RPMI medium with 1% agar 1 in: After gave be a mix of 0.5% to 1 agar composition spread on a 60 mm culture dish, 6 × 10 3 cells with 10% FBS after the one to be 0.3%, the concentration of the final agar after which gave mixed with RPMI medium containing 1% agar was laid on a 0.5% agar. Were cultured in 3 weeks 37 ℃ CO2 incubator, it was added to the culture medium every three days in order to prevent the agar dries. Take a picture after a colony was generated was observed results.

Wound healing assay was replaced with serum-free medium when the cells grown in six-well plates hayeoteul after approximately 90% of cell growth. After incubation in serum-free medium for 16 hours out of the wound scrape the cells after addition of 5% FBS and incubated for 48 hours to observe the pattern being wound spicy.

As a result, as in the case where, as shown in Figure 8, the handle DLK1-Fc of external origin to the cell, the transition by an aqueous DLK1 expressed in the cell line, invasion, adhesion independent growth and wound healing it can be confirmed inhibited It was (Fig. 8).

Example 7: Identification of soluble receptor DLK1

Co immunoprecipitation method was carried out to determine the receptor in soluble DLK1. To this end, first, using PEI (Polyscience) method and a water-soluble DLK1 ACVR2B in 293E cells was overexpressed by transfection. Cells were dissolved in cold DPBS, washed with 3 times a modified RIPA buffer (50 mM TrisHCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40). The lysate was pre-clearing with the people IgG (Jackson Lab) and protein G beads (GE). Using a human antibody and protein G beads have been directly produced in the laboratory were performed for each immunoprecipitation, Western blot analysis showed a 9 to perform the sample was separated on SDS-PAGE. The Western blot was used for DLK1 monoclonal antibody (R & D) and ACVR2B affinity purified polyclonal antibody was used (R & D), compared to the group in Lamin B antibody (Santa Cruz) and α-tubulin antibody (Santa Cruz).

As a result, it was confirmed that the binding to the liquid activin receptor type 2B (ACVR2B) participating in, the aqueous solution DLK1 activin signaling as shown in Fig. 9 (Fig. 9).

Example 8: determine binding between soluble and DLK1 ACVR2B

Via the enzyme immunoassay (ELISA) it was confirmed with the water-soluble coupling between ACVR2B DLK1. To this end, it was coated for 16 hours with 1 ㎍ / ㎖ of the FLAG-tagged soluble DLK1 at 4 ℃ the immune plate (Nunc). At this time, the BSA was used as a control to measure the non-specific binding. The coated plate was then given by King block using PBST (0.05% Tween 20) wash 3 times using a 5% skim milk-PBST. After the ACVR2B Fc-tagged Fc the extracellular portion of the ACVR2B perform serial dilutions (serial dilution) for 1 hour was allowed to react at room temperature. After the reaction was PBST (0.05% Tween 20) human IgG-HRP to measure the degree of coupling and wash 3 times using a (1: 5000) was cultured for one hour. The reaction wash using PBST (0.05% Tween 20) 3 times, and then, using a TMB (Sigma) was measured at later given to perform the color development reaction, and to complete the reaction by the addition of 2.5 MH 2 SO 4 450 nm . As a result, as shown in Figure 10, the one associated with the water-soluble DLK1 ACVR2B, it was confirmed the BSA has no binding (Fig. 10).

Further, after coating the liquid activin A to investigate the relationship between the liquid and the water-soluble DLK1 activin A ligand of the ACVR2B it was measured coupling between DLK1-Fc. As a result, the water-soluble DLK1 was found to be not different from the ligand binding of activin A solution of ACVR2B (Fig. 11).

Example 9: Confirmation competitive binding with activin A solution of the water-soluble DLK1 for ACVR2B

A competition ELISA was performed to see if the water-soluble DLK1 the liquid activin A and competitive binding to the receptor ACVR2B. Of 50 ng / ㎖ solution was coated to activin A were measured as given by reacting the ACVR2B-Fc and FLAG-DLK1. At this time, ACVR2B is then treated in a semi-fixed and the 1-FLAG DLK1 to 2 nM concentration to a concentration of 16 nM was measured for competition binding degree. The control group was used as a liquid and does not bind activin A and Fc ACVR1A-Fc.

As a result, as shown in Figure 12, the higher the concentration of the water-soluble DLK1 was found that the combination of the gradually decreasing liquid activin A and ACVR2B (Fig. 12). These results indicate that the water-soluble DLK1 to block fluid activin A and competitively liquid activin signaling by binding to ACVR2B.

Example 10 confirmed specific binding of the water-soluble DLK1 for ACVR2B

It was by using a Bio-Rad ProteOn's XPR36 performing surface plasmon resonance to determine the specific binding of soluble DLK1 for ACVR2B, a summary of those methods follows. 0.1 M given given EDC and 0.025 M sulfo-NHS shed 60 seconds enable a sensor chip and, flowing the protein to be coated with a put of 30 ㎕ 240 chogan / minute in 10 mM sodium acetate (pH 5.0) the adhesion reaction on the chip It was performed. After the channel blocks 1 M ethanolamine -HCl (pH 8.5) in 200 seconds to wash with DPBST (PBS with 0.005% tween 20). Given 2-fold series shed 120 seconds at a dilution rate of 30 ㎕ / minute and then to evaluate the bonding strength was measured for bond strength. To determine the separation force took the separation step 240 seconds (dissociation phase).

As a result, it was confirmed that the water-soluble DLK1 the specific binding to an activin receptor family ACVR2B from a variety of liquid, as shown in Figure 13 (Figure 13).

Example 11: Check the binding between soluble DLK1 ACVR2B on the real cell

Experiments using flow cytometry to verify the binding between soluble and DLK1 ACVR2B happens on a real cell. It was performed using the BD's products CantoII A summary of the experimental method as follows. By using a pcDNA vector contains the entire gene ACVR2B in 293E cells using PEI (Polyscience) method were prepared the cells were trans faction and overexpressing ACVR2B. Two days after the trans faction were harvested with transfected 293E cells that are not set. Further, after the ACVR2B processes the siRNA (Invitrogen) to inhibit the ACVR2B expression in order to reconfirm that the means for coupling the water-soluble DLK1 inhibit expression of ACVR2B were harvest the cells, as a control for siRNA control siRNA (Invitrogen) 293E treated were also prepared. Harvested cells by using the cold DPBS to give a 2 times wash by adding 1 ㎍ the DLK1-Fc-FITC was reacted for 2 hours at 4 ℃. DLK1-Fc-FITC was used for the reaction is produced by using the protein FITC labeling kit, Pierce's. Reaction to 1% by using a PBA (1% BSA in DPBS) performing a wash buffer, and then determines the degree of binding by flow cytometry, is shown in Fig. In addition, the cells (Fig. 14) confirmed the expression pattern of the ACVR2B by Western blot using on.

That the results, the cell line was also as shown in 15, but significantly combined movement by a water-soluble DLK1 in cell lines that overexpress ACVR2B increased, by treatment with siRNA inhibiting expression of ACVR2B it was found that movement was reduced (Fig. 15).

Example 12: Construction and validation of a human antibody specifically binding to a water-soluble portion of the DLK1

12-1. Production of human antibodies that bind specifically to the soluble part of DLK1

<12-1-1> Preparation of phage libraries

The human-derived scFv library cells having diversity 2.7 × 10 10 2 × YTCM [Tryptone (CONDA, 1612.00) 17 g, Yeast extract (CONDA, 1702.00) 10 g, NaCl (sigma, S7653-5 ㎏) 5 g, chloramphenicol ( sigma, C0857) 34 ㎍ / ㎖ )], 2% glucose (sigma, G5400), and 5 mM MgCl 2 (sigma, M2393 ) were cultured for 2 to 3 hours in a culture medium (3 ℓ) at a temperature of 37 ℃ containing after (OD600 = 0.5 ~ 0.7), a helper phage (helper phage) for by 2 × YTCMK [2 × YT CM, Kanamycin (sigma, K1876) 70 ㎍ / ㎖, 1 mM IPTG (ELPISBIO, IPTG025)] in the medium 30 infection to a temperature of ℃ and incubated for 16 hours. Separating the culture cells centrifuged (4500 rpm, 15 bun, 4 ℃) after, and 4% PEG (Fluka, 81253) in the supernatant was added to 6000 and 3% NaCl (sigma, S7653) is dissolved well in the next one hour at ice It reacted. Centrifuged again (8000 rpm, 20 bun, 4 ℃) one then dissolved by addition of PBS to the pellet followed by centrifugation into the supernatant containing library phage to (12000 rpm, 10 bun, 4 ℃) to a new tube 4 ℃ They were kept in.

<12-1-2> Preparation of monoclonal antibodies

(1) panning (Panning) process

Example 1 A tablet of a coating buffer solution 4 ㎖ the DLK1-Fc in 30 ㎍ Immunosorb tube (Nunc 470319) obtained in [coating buffer; Na 2 CO 3 (sigma, S7795 ) 1.59 g, NaHCO 3 (sigma, S8875) 2.93 g, NaN 3 (sigma, S2002), 0.2 g] in was coated at 4 ℃ in 16 hours rotator (rotator), room temperature in dissolved in PBS for 2 hours using skim milk [(BD, 232100) -4% in 1XPBS] were blocked in immune tube (immunotube). It was added to the phage library prepared in 2 ㎖ immune tube and reacted for 2 hours at room temperature, five times with PBST (0.05%), washed twice with PBS. After washing was amplified by eluting with a specific in 100 mM TEA (Sigma T-0886), only scFv- phage bound, by infecting the eluted phage in E. coli (XL1-Blue, stratagene, 200249). A first panning the phage amplified by increasing only the number of times PBST washing was carried out second, third panning by the same method (2nd: 13 times, third times, 23).

As a result, rise of the antibody titer through panning were as shown in Table 1.

Table 1

Target antigen Fanning times Initial phage Can be combined phage
DLK1-Fc 1st 4.6 × 10 13 6 × 10 7
2nd 2 × 10 12 1.4 × 10 6
3rd 1.49 × 10 14 6.93 × 10 9

(2) Discover the phage antibody phage enzyme-linked immunosorbent assay (ELISA)

① panning result confirmed

A first storage you wrote panning and frozen cells from primary to tertiary water (stock) from 5 ㎖ 2 × YTCM, 2% glucose, 5 mM MgCl, and then semi-put to be a OD600 = 0.1 in 2 medium, 37 ℃ of 2 to 3 hours for (OD600 = 0.5 ~ 0.7) were incubated. Since, and infection of helper phage M1 2 × YTCMK, 5 mM MgCl 2 , 1 mM IPTG at the temperature of 30 ℃ medium and allowed to incubate for 16 hours. Centrifuging the cultured cells after (4500 rpm, 15 bun, 4 ℃) supernatant (the panning poly scFv- phage) was transferred to a new tube. After a 96-well immunological plates (NUNC 439454) coated by treating the antigen with 16 hours of coating buffer at 4 ℃ by 100 ng per well to use the skim milk (4%) was dissolved in PBS to block the wells. Each per well PBS-tween20 (0.05%) into each 100 ㎕ the following gave one a poly scFV- panning phage diluted stock solution and 5, 25, 125, 625, 3125 times the solution washed with 0.2 ㎖ to each well for 2 hours at room temperature and it reacted for. Again each well PBS-tween20 (0.05%) and then washed 4 times using a secondary antibody, wherein 0.2 ㎖ -M13-HRP (Amersham 27-9421-01) 1: 2000 dilution for 1 hour at room temperature It was. PBS-tween20 (0.05%) purified OPD After washing with 0.2 ㎖ (Sigmap 8787-TAB) a PC buffer [C 6 H 8 O 7 · H 2 O (sigma, C0706) 5.1 g, Na 2 HPO 4 (sigma , S7907) the absorbance at 490 ㎚ was made substrate solution was dissolved in 7.3 g] per well into each 100 ㎕ color development for 10 minutes was measured with a spectrophotometer (MolecularDevice, USA).

As a result, as shown, by binding to the third binding capacity began to buildup from secondary polyclonal scFv- phage pools (pools), each of the antigen was confirmed that the reached the saturated state shown in Figure 16 (Figure 16) .

② monoclonal antibody screening

In the binding capacity is large is 2 × YTCM, 2% glucose, 5 mM MgCl 2 with 96-deep well plate (Bioneer 90 030) containing the medium 1 ㎖ Colonies obtained from the clone phage antibody group at a temperature of 37 ℃ incubated for 16 hours It was. In one the OD600 value of the cell culture taken to 100 ~ 200 ㎕ set at 0.1 was diluted to 2 × YTCM, 2% glucose, 5 mM MgCl 2 medium of 1 ㎖ Next, 96-deep temperature of 37 ℃ in well plates OD600 value the cells were cultured for 2 to 3 hours to 0.5 to 0.7. The M1 helper phage at a temperature after the infection of the MOI value so that it is 1:20, 2 × YTCMK, 5 mM MgCl 2, 1 mM 30 ℃ IPTG in the medium and cultured for 16 hours. Separating the culture cells centrifuged (4500 rpm, 15 bun, 4 ℃) which then was dissolved well by taking the supernatant liquid was added to 4% PEG 6000 and 3% NaCl was reacted for 1 hour on ice. Again centrifuged (8000 rpm, 20 bun, 4 ℃) was added to PBS to the pellet and then melted, and then centrifuged (12000 rpm, 10 bun, 4 ℃) to take the supernatant was stored at 4 ℃ transferred to a new tube. Then, after putting ssikeul 100 ng per well of antigen in 96-well immunological plates coated at 4 ℃ for 16 hours using a skim milk (4%) was dissolved in PBS to block the wells. Each each well PBS-tween20 (0.05%) into monoclonal scFv- phage (each 100 scFv-phage) obtained by the following method were washed with a 0.2 ㎖ by 100 ㎕ to each well and reacted at room temperature for 2 hours . Again each well to dilute the PBS-tween20 (0.05%) 2 secondary antibody is anti-M13-HRP after standard using 0.2 ㎖ washed 4 times with 1/2000 was incubated for 1 hour at room temperature. The color development was washed with PBS-tween20 (0.05%) 0.2 ㎖ absorbance was measured at 490 ㎚. As a result, a single phage clones were selected 27 binding capacity is 2 or more for the antigen as shown in Table 2.

Table 2

DLK1 One 2 3 4 5 6 7 8 9 10 11 12
A .1129 .0716 .0482 3.1152 2.9859 .4549 .3612 2.92 .0469 2.8295 .2175 1.0026
B .8858 .8553 2.0914 .788 2.762 2.6351 2.8837 .1342 2.3259 .1396 2.5018 .501
C .4976 .2852 2.466 .2239 .1128 1.2413 2.9255 2.1548 .2169 .0608 .2132 .1591
D .2025 .1882 .1109 .0586 .8865 .0749 .0849 .1145 .8514 .0572 .1653 2.4751
E .0907 .1001 .0418 .047 2.2329 2.3476 2.3778 .7165 .0919 .7527 .1737 .2233
F .1659 .2324 .4055 2.9152 .2405 .933 .3682 .1608 .2258 .1668 2.8944 2.9681
G .0433 .3815 .2245 2.8355 2.5814 3.0216 .752 .3455 .0609 .4363 .1964 .0504
H .1044 2.8427 2.7085 .296 .2403 2.1306 2.8803 1.6389 3.033 .5009 2.7793 3.1994
MYC One 2 3 4 5 6 7 8 9 10 11 12
A .2003 .0474 .0487 .5068 .7838 1.4922 .4492 .5538 .0557 1.2823 .1523 .2432
B 1.357 .7667 1.4038 1.1973 .5932 .8478 .5129 .1191 .0918 .7688 .316 .0526
C .2966 .0741 .2871 .1538 .0683 .6353 .5938 .3595 .6692 .1009 .1206 .2206
D .1631 .4308 .078 .045 .5783 .0632 .0538 .052 2.1443 .0511 .106 .0889
E .0856 .6073 .0465 .0435 .1722 .1883 .4694 .0867 .0639 .272 .3112 .1566
F .1285 .4057 .1421 1.0637 .1115 1.1193 .0898 1.0797 .1751 .1401 .4419 .524
G .0662 .3396 .0844 .9974 2.9974 .6732 .6083 .2278 .0496 .5198 .0561 .0551
H .0479 .5654 1.1204 .4634 .066 .8632 1.0213 .6574 .8562 .1146 .9677 .6741
FC One 2 3 4 5 6 7 8 9 10 11 12
A .0535 .0726 .0731 .0791 .0704 .1111 .0748 .0709 .0535 .0828 .0591 .2558
B .1375 .4065 .0851 .0702 .0575 .0472 2.8291 .0717 .0786 .0743 .0548 .0451
C .0524 .0555 .0521 .0745 .0455 .0825 2.8824 .0559 .0772 .0485 .0663 .061
D .0519 .0686 .0447 .0722 .0431 .0455 .0482 .0528 .0498 .1141 .0651 .0831
E .0496 .0543 .0419 .0587 .0472 .0481 .0558 .2673 .0492 .1508 .0601 .0577
F .0614 .0584 .0528 .0879 .0553 .1223 .0792 .0756 .0661 .0922 .0658 .0757
G .0456 .0687 .0521 .1171 2.079 .0689 .547 .0991 .0874 .2255 .1894 .0457
H .0594 .0642 .049 .0705 .0608 .0766 .1107 .0819 .0701 .1204 .0592 .0539

3 monoclonal phage sorting and inspection

① Verification by fingerprinting

27 monoclonal cell 1 and ㎕ Taq.DNA polymerase (Zhen Dax 5 U / ㎕) 0.2 ㎕, of 50 p / ㎕ forward primer for the first DLK1-Fc screening car (SEQ ID NO: 52: 5'-CTAGATAACGAGGGCAAATCATG- 3 ') and reverse primer (SEQ ID NO: 53: 5'-CGTCACCAATGAAACCATC-3') 0.2 ㎕, 3 ㎕ 10X buffer, 10 mM dNTP mix 0.6 ㎕ distilled water and a mixture of 24.8 ㎕ colony PCR (iCycler iQ, BIO-RAD ) it was performed. Conditions of the PCR program is given in Table 3.

TABLE 3

Temperature time Cycle (cycle)
95 ℃ 5 min
95 ℃ 30 sec 30 cycles
56 ℃ 30 sec
72 ℃ 1 min
72 ℃ 10 min
4 ℃

The colony PCR product was incubated for 1% agarose gel was found in Ross (Seakem LE, CAMERES 50004), BstNI (Roche11288075001, 10 U / ㎕) 2 to 3 hours at 37 ℃ added 0.2 ㎕. The reaction conditions are given in Table 4. The cut product was found in 8% DNA polyacrylamide gel.

TABLE 4

10X Buffer 3 ㎕
Colony PCR products 10 ㎕
BstNI (10 U / ㎕) 0.2 ㎕
Distilled water 16.8 ㎕

As a result, as shown in FIG. 17, by the BstNI diversity have been identified for the fragment of truncated monoclonal phage antibodies was inferred that the antibodies of six kinds of the different screening.

② verified by sequencing

The six types of monoclonal phage for aqueous DLK1 to 2 × YTCM, 2% glucose, at a temperature of 37 ℃ 5 mM MgCl2 medium (5 ㎖) were incubated for 16 hours. Obtained the DNA using a DNA purification kit (Nuclogen 5112) from a monoclonal culture was then asked to sequence analysis using primers of SEQ ID NO: 52 (Sol Gentry, South Korea). As a result, Table 5 and was found to CDR regions of VH and VL of the selected antibody, as shown in Fig.

Table 5

Clone Name VH Identities CDR3-aa seq VL Identities CDR3-aa seq Group DLK1 a-myc Fc Ratio
DLK1A04 VH1-3 263/294 (89.5%) SVSAYG ---- SNYFDP L8 268/286 (93.7%) QQLNS-YPL One 3.1152 .5068 .0791 6.146803473
DLK1A05 VH3-9 277/290 (95.5%) SGGYGGN - TNWYFDL V1-13 276/295 (93.6%) QSYDSRLGV 2 2.9859 .7838 .0704 3.809517734
DLK1A10 VH3-9 287/292 (98.3%) GPGLATG --- KGYADY L5 268/284 (94.4%) QQGHS-FPY 3 2.8295 1.2823 .0828 2.206581923
DLK1B09 VH3-23 266/294 (90.5%) GESCSGG - ACSDFDY V1-4 263/285 (92.3%) GSYAGSYTY 4 2.3259 .0918 .0786 25.33660131
DLK1H06 VH3-23 267/293 (91.1%) S -------- TAYLFDY A27 257/272 (94.5%) QHYGS-PLH 5 2.1306 .8632 .0766 2.468257646
DLK1H12 VH3-11 279/294 (94.9%) LQGHCSGGACSNWFDA O12 272/284 (95.8%) QQGYG-TPY 6 3.1994 .6741 .0539 4.746180092

These antibodies and germ cell lineages (germ line) of the affinity of the antibody group NCBI Ig BLAST program (www.ncbi.nlm.nih.gov/igblast/) using the results to give the 6 species-specific phage antibodies to the irradiated soluble DLK1 . At this time, in the case of the heavy chain VH3-9 and VH3-23 are two kinds, VH3-11, VH1-3 this was contained in each one jongssik. We analyzed the amino acid sequences used in the area of ​​the antibody heavy and light chain CDR3, it was confirmed that each has a different sequence (Figure 18).

(4) Characterization of the human antibody to the soluble DLK1

① total IgG conversion analysis

To switch to the full vector IgG in the gripping of monoclonal phage antibodies to the water-soluble DLK1 heavy chain monoclonal DNA 1 ㎕, heavy chain forward and reverse primers 10 pmole / ㎕ in Table 6, 10X buffer 5 ㎕, 10 mM dNTP mix 1 ㎕ , pfu DNA polymerase (Sol Gentry, 2.5 U / ㎕) a mixture of distilled water and 0.5 ㎕ was performed colony PCR (iCycler iQ, BIO-RAD). Light chain was also performed colony PCR in the same manner by using the light chain forward and reverse primers shown in Table 6.

Table 6

Clone people HC
Forward primer Reverse primer
A04 HC NATVH3-2 SEQ ID NO: 5 NATJH-ALL SEQ ID NO: 8
A05 HC NATVH7-1 SEQ ID NO: 6
A10 HC NATVH3-2 SEQ ID NO: 5
B09 HC NATVH7-1 SEQ ID NO: 6
H06 HC NATVH1-1 SEQ ID NO: 7
H12 HC
Clone people LC
Forward primer Reverse primer
A04 HC NATVK6 SEQ ID NO: 9 NATJK-R7 SEQ ID NO: 12
A05 HC NATVL13 SEQ ID NO: 10 NATJL2-R SEQ ID NO: 13
A10 HC NATVK6 SEQ ID NO: 9 NATJK-R5 SEQ ID NO: 14
B09 HC NATVL10 SEQ ID NO: 11 NATJL1-R SEQ ID NO: 15
H06 HC NATVK6 SEQ ID NO: 9 NATJK-R4 SEQ ID NO: 16
H12 HC NATJK-R7 SEQ ID NO: 12

After purification to a heavy chain gene obtained by PCR as a DNA- gel extraction kit (Qiagen), pNATAB H vector (Figure 19) 1 ㎕ (10 ng), heavy chain (100 ~ 200 ng) 15 ㎕, 10X buffer 2 ㎕, Lee left for dehydratase (1 U / ㎕) 1 to 2 hours at room temperature, a mixture of 1 ㎕ and distilled water was connected to the vector. After allowed to stand for 30 minutes on ice with transformed cells (XL1-blue) for the transfected vector was transduced was given 90 seconds to heat shock at 42 ℃. Again allowed to stand for 5 minutes on ice, the injection ㎖ LB medium 1 and incubated at 37 ℃ for 1 hour. Then, after plated on Amp LB solid medium and cultured at 37 ℃ for 16 hours. Inoculated with a single colony in LB Amp 5 ㎖ broth and incubated at 37 ℃ for 16 hours. DNA-prep from the culture medium. The DNA was extracted using a kit (Nuclogen). In addition, the light chain DNA was extracted in the same manner as described above using the pNATAB L vector (FIG. 20).

With respect to the CMV-DNA obtained proF primer (SEQ ID NO: 54: AAA TGG GCG GTA GGC GTG) with the request was sequenced (Sol Gentry). As a result, the six sequences of the heavy and light chains in phage clones for the DLK1-Fc converted to whole IgG was confirmed sequence that matched the phage antibodies.

12-2. Verification of a human antibody specifically binding to a water-soluble portion of the DLK1

In order to confirm the binding affinity of a human antibody specifically binding to a water-soluble portion of the produced DLK1 in Example 12-1, to a representative of the six kinds of human antibodies using the B09 antibody using 293E cells expressing DLK1 to perform the flow cytometry analysis of movement, it indicated in Fig. At this time, to measure the binding affinity of the monoclonal antibody and DLK1 ACVR2B antibody purchased from R & D.

As a result, as shown in Figure 21, antibody B09 was confirmed that the specific binding to the soluble part of DLK1. Further, it was confirmed that the binding to the monoclonal antibody and DLK1 ACVR2B antibody also each DLK1 and ACVR2B purchased from R & D (Fig. 21).

Example 13: bond strength measured by surface plasmon resonance method

It was using Bio-Rad's ProteOn XPR36 perform a surface plasmon resonance method to measure the binding affinity, a summary of the method as follows. 0.1 M given given EDC and 0.025 M sulfo-NHS shed 60 seconds enable a sensor chip and, flowing the protein to be coated with a put of 30 ㎕ 240 chogan / minute in 10 mM sodium acetate (pH 5.0) the adhesion reaction on the chip It was performed. After the channel blocks 1 M ethanolamine -HCl (pH 8.5) in 200 seconds to wash with DPBST (PBS with 0.005% tween 20). Given 2-fold series shed 120 seconds at a dilution rate of 30 ㎕ / minute and then to evaluate the bonding strength was measured for bond strength. To determine the separation force took the separation step 240 seconds (dissociation phase).

As shown in Figure 22, the binding force of the B09 antibody to soluble DLK1 Kd value it was determined to be 1.90E-11 (Fig. 22). Further, the binding force of the liquid activin A for ACVR2B was measured with a Kd value of 1.96E-09 (Fig. 23), bonding strength of the water-soluble DLK1 for ACVR2B was determined as Kd value 1.31E-09 (Fig. 24).

In addition, the bonding strength for each was measured for the water-soluble activin DLK1 this solution to quantitatively analyze the impact on the bond between A and ACVR2B. As a result, when the handle during B09 antibody binding between soluble and DLK1 ACVR2B bond increases and was determined to be 4.07E-10 (Fig. 25).

Showed in Figure 26 to synthesize the results, these results are agonist antibodies (agonistic antibody) to the B09 antibody synergistically specifically binding to a water-soluble portion of the DLK1, water-soluble when Compared to remove constants and binding constants to form a more stable structure and, when combined with the DLK1 ACVR2B suggest that giving away the release rate.

Example 14 Competitive enzyme immunoassay (ELISA)

In order to examine the effect of antibodies on the binding between soluble coupling between DLK1 and ACVR2B or liquid activin A and ACVR2B was performed competitive ELISA. 50 ng / ㎖ of after coating the liquid activin A of ACVR2B-Fc or 200 ng / ㎖ was allowed to react for FLAG-DLK1, ACVR2B-Fc or DLK1-Fc This was given observe the competitive binding reaction was added to each antibody .

First, coating the ACVR2B and while combining the aqueous DLK1 In investigating the effects of the antibody on the binding between soluble DLK1 and ACVR2B by processing each of the antibody result, B09 antibodies let increase the bonding strength between the water-soluble DLK1 for ACVR2B exponentially was, liquid activin a and the bond between the water-soluble DLK1 and ACVR2B according to neutralize the effect processing in the R & D AF339 antibody reported to have a (neutralizing effect) for the coupling between the ACVR2B this was confirmed to decrease slightly (Fig. 27). These results indicate that the bond between the water-soluble DLK1 and ACVR2B B09 antibody significantly can be increased, and bonding between the portion and the water-soluble DLK1 and ACVR2B involved in the coupling between the bore to reduce the binding by AF339 antibody solution activin A and ACVR2B this section makes it able to guess that is some overlap involved in.

Next, the coating solution and activin A, and this while combining ACVR2B-Fc between the liquid activin A and ACVR2B in accordance with this result, the process of the R & D AF339 antibody examine the effect of coupling between by processing each antibody solution activin A and ACVR2B bonds are decreased, DLK1 antibodies was confirmed that there is no directly related to the bond between the liquid and the ACVR2B activin a (Figure 28).

Further, the coating liquid for activin A, and this while combining ACVR2B-Fc seen by treating the aqueous DLK1 and each antibody at the effect of water-soluble DLK1, ACVR2B DLK1 antibodies and antibody solution in the coupling between activin A and ACVR2B on. As a result, the water-soluble DLK1 are more stylized concentration is increased reducing the bonding force between the fluid A and activin ACVR2B, when treated with the B09 antibody, confirmed the significantly reducing the bonding force between the fluid A and activin ACVR2B. Further, according to the process of the R & D AF339 antibody it was found to inhibit fluid coupling effect between activin A and ACVR2B by aqueous DLK1 hayeoteum no increase (Fig. 29). These results indicate that to obtain the best effect when given away by treatment with antibodies that interfere with the coupling between the B09 antibody or ACVR2B with activin A solution to the water-soluble DLK1 and synergy to inhibit liquid activin signaling.

Example 15: Western blotting for verifying the effect of the water-soluble liquid DLK1 for activin signaling

When the culture vessel is a pancreatic cancer cell line MIA-PaCa-2 grew up to 70%, to give replaced with serum-free medium and incubated for 16 hours. Of 10 ㎍ / ㎖ in DLK1-Fc treated group to give a process DLK1-Fc, it was added a solution 0.5 nM activin A and harvested after 30 minutes. A cell is used to buffer the addition of RIPA buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) protease inhibitors (Roche) phosphine Porta inhibitor cocktail in I, II (Sigma) Harvest by dissolving the cells, and determining the concentration of the cell lysates was dissolved using a BCA quantitation kit (Thermo). The solution of 30 ㎍ on SDS-PAGE and modified using the 5X sample buffer and then subjected to electrophoretic transfer to PVDF membrane was (GE). Using psamd2 / 3, Smad2 / 3, Smad4 (Cell signaling), Smad7 (Milipore) made by blotting was carried out by Western blot. In this case, used it was a beta-actin antibody (Sigma) as a loading control. On the 4 ℃ subjected to washing three times with TBST (Tween 20 0.05%) after the reaction for one night using a rabbit-HRP antibody and mouse-HRP antibody (Santa Cruz) followed by reaction at room temperature for 1 hour, TBST again ( the washing was carried out three times with Tween 20 0.05%). Then, the blot was developed with the fluorescent film by using ECL solution (Intron).

The result was,, pSmad2 / 3 to decrease the amount activin signaling did not occur by the steps shown in Figure 30, it was also seen that Smad7 of reducing the liquid activin signaling hayeoteum increase (Fig. 30).

Example 16: Effect of the water-soluble Smad signaling on DLK1

The SEAP reporter assay was performed to investigate the effect of water-soluble DLK1 on the Smad neurotransmission. Vector used for the SEAP reporter assay with increasing Smad binding site (Smad binding element, SBE) a system in which the reporter gene expression when the transcription control by Smad in cells made according with that by the liquid activin signaling before SEAP gene is. The MIA-PaCa-2 cell line was introduced into the vector by electroporation. It transferred the next day to give a cell line transfected with a 96-well plate and then cultured for 24 hours in serum-free medium was further cultured for 16 hours. To give a pre-treatment or DLK1 Fc-Fc to the cells for 1 hour, it was performed reporter analysis collected by the cell culture medium after treatment with activin A solution of 0.5 nM, and 48 hours. Analysis was performed using the ABI's Phospha-Light system. Further, by using the B09 antibody it was carried out the same experiment.

As a result, the signal of the reporter systems was reduced according to the process of the DLK1-Fc (FIG. 31), according to the administration amount of the B09 antibody was found that liquid hayeoteum activin signaling is decreased (Fig. 32).

Example 17: Effect of antibody B09 on hypoxia signaling

In order to examine the influence B09 antibody specifically binding to a water-soluble portion of the DLK1 on the signal transfer in the hypoxic conditions that are DLK1 the overexpression was carried out reporter analysis. Vector used in the reporter assay system that has a reporter gene expression, when luciferase gene is made in front of transcription by hypoxia in a cell it has a reactive site hypoxia (hypoxia response element, HRE). The MIA-PaCa-2 cell line was introduced into the vector by electroporation (Invitrogen). Next transferred to a cell line transfected with a 96-well plate it was further incubated for 16 h with serum free medium and then cultured for 24 hours. Given the process 2 (Sigma) of 100 nM CoCl during incubation in serum-free medium it was induced artificially hypoxia. DLK1 the antibody to the cells to give pre-treatment for one hour to perform the Reporter Analysis. Analysis was performed using the ABI's Phospha-Light system.

As a result, it was found that it is possible to inhibit, B09 antibodies hypoxia signaling as shown in Fig. 33 (Fig. 33).

Example 18: Antibody DLK1 cancer metastasis inhibitory effects, and the influence of antibodies and ACVR2B DLK1 antibody on cancer metastasis inhibitory effect of the water-soluble DLK1 confirmation of

A cancer metastasis inhibitory effects, and ACVR2B antibodies and antibody DLK1 DLK1 of antibodies In order to examine the influence of the water-soluble DLK1 cancer metastasis inhibitory effect on the metastasis experiment was performed using the MIA-PaCa-2 cell line. First, the number of cells was measured after the cells to give change to serum-free medium when the 70% level has been removed by the trypsin treatment the cells after 24 hours. The combined cells, each protein to a serum-free medium and treated to create a 100 ㎕ were incubated for 1 hour at 37 ℃. For up to a 24-well plate as a chemoattractant (chemo-attractant) of 1 ㎖ to put the 5 ~ 10% FBS transwell (Corning # 3422) having a pore size of 8.0 μm thereon and 1 hours therein DLK1 after the antibody loaded with the pre cultured cells and on the cell, the protein mixture was incubated at 37 ℃ 100 ㎕ carbon dioxide incubator for 24 hours to 48 hours. After the culture, it was completely removed by using them do not pass the after using the removal of the medium of the well and trans, Diff Quick solution (Sysmex, Japan) staining the cells transwell cell swab. Then, the observation of a complete pass through the trans-well transwell cell and then dried with 400 magnifications, and was taking a picture.

As a result, it was confirmed the inhibition of metastasis ability is more excellent than the transition of DLK1 antibody ACVR2B inhibitory ability of the antibody (Fig. 34). In addition, the results mab1144 antibody investigate the effect of the transition time to process with aqueous DLK1 is although large effect on the inhibition transition when the treatment with the water-soluble DLK1, B09 antibody When only treatment as well as with the water-soluble DLK1 treatment showed very good ability to inhibit metastasis even if the. AF339 when antibody treatment rather it was confirmed that it has a metastasis inhibitory effect of the water-soluble DLK1 reduced (FIG. 35).

Example 19: MIA-Paca-2 orthotopic transplantation in animal models of pancreatic cancer (orthotopic animal model)

In order to confirm the DLK1-Fc metastasis and proliferation of cancer cells, inhibitory ability of the cancer in the animal model experiment was performed orthotopic transplantation of MIA-Paca-2 pancreatic cancer. The nude mice used in the experiment was to buy a six-week-old female Can Cg-Foxnl-nu / CrljBgi in Co. Orient. After adapting the one week mice were transplanted in situ the dog pancreatic cancer cell line MIA-Paca-2 cells in the pancreas 5x106. One group is injected with Fc-DLK1 to 5 mpk divided into two groups, and the other one group was injected with 15 mpk, PBS was used as a control. Protein was injected in two injections a week via intraperitoneal injection. After five weeks after orthotopic transplantation was observed through a dissecting the results.

Before dissection in the results DLK1-Fc treated group by checking the tumor incidence was to find out a tumor incidence hayeoteum significantly reduced (Fig. 36), the hernia formation rate in the results DLK1-Fc treated groups investigated hernia formation rate of the anatomical findings significantly It was confirmed that was reduced (Fig. 37).

In addition, liver DLK1-Fc treated group, a significant decrease metastasis to the lungs and the diaphragm, such as to was to find out the proliferation of cancer cells was reduced (Figure 38), dissected and then analyzed to determine the size and weight of the pancreas and stomach result was confirmed that significantly inhibit the formation of cancer in DLK1-Fc treated group (Fig. 39). Further, it was confirmed similarly to the result of measuring the splenomegaly caused by tumor hayeoteum significantly reduced spleen enlargement phenomenon in DLK1-Fc treated group than in the control group (Fig. 40).

Example 20: in vivo imaging (Small Animal In Vivo Imaging; SAIVI )

In order to examine the distribution of the injection of a physical mouse DLK1-Fc is the fluorescence quantum dot band in DLK1-Fc and then attached to (Quantum Dots) in vivo imaging; was performed (Small Animal In Vivo Imaging SAIVI) experiment. The nude mice used in the experiment was to buy a six-week-old female Can Cg-Foxnl-nu / CrljBgi in Co. Orient. Cover quantum dots are used only in the QDs 800 QDs 800 (Invitrogen), the control group. First, the orthotopic transplantation was the mouse pancreatic cancer cell line MIA-Paca-2 cells in 5x106 dog pancreas. By then the cancer tissues formed quantum dots observed fluorescence after injection via the intravenous injection was carried out in vivo imaging. Imaging, the Maestro in vivo imaging system; was used (Maestro in vivo imaging system CRi, Inc.).

As a result, a DLK1-Fc treatment was confirmed that the assembling to where the tumor is located (Fig. 41). This result is thought to be because the ACVR2B is overexpressed in cancer cells than in normal tissue cells.

Including cancer, it can be useful in the prevention or treatment of metabolic disorders, immune disorders, or liver disease associated with signal transduction of ACVR2B.

SEQ. ID. NO. 1 (extracellular water-soluble domain of DLK1): ECFPACNPQN GFCEDDNVCR CQPGWQGPLC DQCVTSPGCL HGLCGEPGQC ICTDGWDGEL CDRDVRACSS APCANNGTCV SLDDGLYECS CAPGYSGKDC QKKDGPCVIN GSPCQHGGTC VDDEGRASHA SCLCPPGFSG NFCEIVANSC TPNPCENDGV CTDIGGDFRC RCPAGFIDKT CSRPVTNCAS SPCQNGGTCL QHTQVSYECL CKPEFTGLTC VKKRALSPQQ VTRLPSGYGL AYRLTPGVHE LPVQQPEHRI LKVSMKELNK KTPLLTEG

SEQ. ID. NO. 2 (CDR1 of heavy chain): SYAMN

SEQ. ID. NO. 3 (CDR2 of heavy chain): TITATSGKTY YADSVKG

SEQ. ID. NO. 4 (CDR3 of heavy chain): GESCSGGACS DFDY

SEQ. ID. NO. 5 (CDR1 of light chain): TGTSSDIGRY NRVS

SEQ. ID. NO. 6 (CDR2 of light chain): DVTTRPS

SEQ. ID. NO. 7 (CDR3 of light chain): GSYAGSYTY

SEQ. ID. NO. 8 (CDR1 of heavy chain): DYAIH

SEQ. ID. NO. 9 (CDR2 of heavy chain): WINPGSGNTK YSHNFEG

SEQ. ID. NO. 10 (CDR3 of heavy chain): SVSAYGSNYF DP

SEQ. ID. NO. 11 (CDR1 of light chain): QASQDISNYL N

SEQ. ID. NO. 12 (CDR2 of light chain): STSNLQS

SEQ. ID. NO. 13 (CDR3 of light chain): QQLNSYPL

SEQ. ID. NO. 14 (CDR1 of heavy chain): EHAMH

SEQ. ID. NO. 15 (CDR2 of heavy chain): GINWNSGKTG YADSVKG

SEQ. ID. NO. 16 (CDR3 of heavy chain): SGGYGGNTNW YFDL

SEQ. ID. NO. 17 (CDR1 of light chain): IGTSSNIGVG YDVH

SEQ. ID. NO. 18 (CDR2 of light chain): GNNNRPS

SEQ. ID. NO. 19 (CDR3 of light chain): QSYDSRLGV

SEQ. ID. NO. 20 (CDR1 of heavy chain): DYAMH

SEQ. ID. NO. 21 (CDR2 of heavy chian): GISWNSGSIG YADSVKG

SEQ. ID. NO. 22 (CDR3 of heavy chain): GPGLATGKGY ADY

SEQ. ID. NO. 23 (CDR1 of light chain): RASQRISSWL A

SEQ. ID. NO. 24 (CDR2 of light chain): SASTLHN

SEQ. ID. NO. 25 (CDR3 of light chain): QQGHSFPY

SEQ. ID. NO. 26 (CDR1 of heavy chain): LYGMS

SEQ. ID. NO. 27 (CDR2 of heavy chain): SIPGSGTRTH YADSVKG

SEQ. ID. NO. 28 (CDR3 of heavy chain): STAYLFDY

SEQ. ID. NO. 29 (CDR1 of light chain): RASQSIRHYL A

SEQ. ID. NO. 30 (CDR2 of light chain): GASSRAT

SEQ. ID. NO. 31 (CDR3 of light chain): QHYGSPLH

SEQ. ID. NO. 32 (CDR1 of heavy chain): DYYMS

SEQ. ID. NO. 33 (CDR2 of heavy chain): YISGSGITTY YADSVKG

SEQ. ID. NO. 34 (CDR3 of heavy chain): LQGHCSGGAC SNWFDA

SEQ. ID. NO. 35 (CDR1 of light chain): RASQSILTYL N

SEQ. ID. NO. 36 (CDR2 of light chain): AASSLQR

SEQ. ID. NO. 37 (CDR3 of light chain): QQGYGTPY

SEQ. ID. NO. 38 (heavy chain VR of B09): QMQLVESGGR LVRPGGSLRL SCAASGFPFT SYAMNWVRQT PGKGLEWVST ITATSGKTYY ADSVKGRFTI SRDNSRNTLF LQMNSLRAED TAVYYCVRGE SCSGGACSDF DYWGQGALVT VSS

SEQ. ID. NO. 39 (light chain VR of B09): QLVLTQPASV SGSPGQSVTI SCTGTSSDIG RYNRVSWYQH HPGKAPKLIL NDVTTRPSGF SNRFSGSKSG NTASLTISGL QAEDEADYSC GSYAGSYTYV FGTGTKVTVL GGG

SEQ. ID. NO. 40 (heavy chain VR of A04): QVQLVESGAE VKKPGASVKV SCKASGYTFK DYAIHWVRQA PGQRLEWMGW INPGSGNTKY SHNFEGRVIL TRDASANTAY LELPSLRSED TAVYYCARSV SAYGSNYFDP WGQGTLITVS S

SEQ. ID. NO. 41 (light chain VR of A04): DIQMTQSPSS LSASVGDRVT ITCQASQDIS NYLNWYQQKP GKAPKLLIYS TSNLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ LNSYPLTFGG GTKVDIKRGG

SEQ. ID. NO. 42 (heavy chain VR of A05): QMQLVESGGG LVQPGRSLRL SCAASGFTFD EHAMHWVRQA PGKGLEWVSG INWNSGKTGY ADSVKGRFTI SRDNGKNSLY LQMNSLRAED TAVYYCAKSG GYGGNTNWYF DLWGRGTLVT VSS

SEQ. ID. NO. 43 (light chain VR of A05): QFVLTQPPSV SGAPGQNVTI SCIGTSSNIG VGYDVHWYQQ VPGTAPKLLV YGNNNRPSGV PDRFSGSKSG TSASLAITGL QAEDEADYYC QSYDSRLGVV FGGGTKLTVL GGG

SEQ. ID. NO. 44 (heavy chain VR of A10): QVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSG ISWNSGSIGY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARGP GLATGKGYAD YWGQGTLVTV SS

SEQ. ID. NO. 45 (light chain VR of A10): DIQMTQSPSS VSASVGDRVT ITCRASQRIS SWLAWYQQKP GRAPKLLIHS ASTLHNGVPS RFSGSASGTD FTLTISSLQP EDYAIYYCQQ GHSFPYTFGQ GTKLEIKRGG

SEQ. ID. NO. 46 (heavy chain VR of H06): QVQLVQSGGG LIQPGGSLRL SCAASGFTFS LYGMSWVRQA PGKGLEWVSS IPGSGTRTHY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCAKST AYLFDYWGPG TLVTVSS

SEQ. ID. NO. 47 (light chain VR of H06): DIQMTQSPAT LSLSPGERAT LSCRASQSIR HYLAWYQQKP GQAPRLLIHG ASSRATGIPD RFSGSGSGTD FTLTISRLEP EDFAVYYCQH YGSPLHSFGP GTKVEIKRGG

SEQ. ID. NO. 48 (heavy chain VR of H12): QVQLVQSGGG LVKPGGSLRL SCAASEFTFS DYYMSWVRQA PGKGLEWLSY ISGSGITTYY ADSVKGRFTI SRDNGKKSLY LEMNSLRAED TAVYYCARLQ GHCSGGACSN WFDAWGQGTL ITVSS

SEQ. ID. NO. 49 (light chain VR of H12): DIQMTQSPSS LSASVGDRLT ITCRASQSIL TYLNWYQQKP GKAPKLLIYA ASSLQRGVPS RFSGSGSGTD FTLTISGLQP EDFATYYCQQ GYGTPYTFGQ GTKVDIKRGG

SEQ. ID. NO. 50 (polynucleotide sequence coding heavy chain of B09 antibody): atgggatgga gctatatcat cctctttttg gtggccacag cggccgatgt ccactcgcag atgcagctgg tggagtctgg gggacgctta gtgcggcctg gggggtccct gagactctcc tgtgcagcct ctggattccc ctttaccagt tatgccatga actgggtccg ccagactcca gggaaggggc tggaatgggt ctctactatc actgctacta gtggtaagac atactacgca gactccgtga agggccggtt caccatctcc agagacaact ccaggaacac gctgtttctg caaatgaaca gtctgagagc cgaggacacg gccgtgtatt actgtgtgag aggggagtct tgtagtggtg gtgcctgctc agattttgac tactggggcc agggagccct ggtcaccgtc tcctcagcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct ctttccccca aaacccaagg acaccctcat gatctcccgg acccctgagg t cacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg ggtaaa

SEQ. ID. NO. 51 (polynucleotide sequence coding light chain of B09 antibody): atgggatgga gctatatcat cctctttttg gtggccacag cggccgatgt ccactcgcag ctcgtgctga ctcagcctgc ctccgtgtct gggtctcctg gacagtcggt caccatctcc tgcactggaa ccagcagtga cattggtcgt tataaccgtg tctcctggta ccaacaccac cccggcaagg cccccaaact cattcttaat gatgtcacta ctcggccctc agggttttct aatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccatctc tgggctccag gctgaggatg aggctgatta ttcctgcggc tcatatgcag gcagctacac ttatgtcttc ggaactggga ccaaggtcac cgtcctaggt ggaggaagat ctgtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt

SEQ. ID. NO. 52 (forward primer): ctagataacg agggcaaatc atg

SEQ. ID. NO. 53 (reverse primer): cgtcaccaat gaaaccatc

SEQ. ID. NO. 54 (CMV-proF primer): aaatgggcgg taggcgtg

SEQ. ID. NO. 55 (NATVH3-2 primer): ttggtggcca cagcggccga tgtccactcg caggtgcagc tggtggagtc

SEQ. ID. NO. 56 (NATVH7-1 primer): ttggtggcca cagcggccga tgtccactcg cagatgcagc tggtggagtc

SEQ. ID. NO. 57 (NATVH7-1 primer): ttggtggcca cagcggccga tgtccactcg caggtgcagc tggtgcagtc

SEQ. ID. NO. 58 (NATJH-ALL primer): gaggaggcta gctgaggaga cggtga

SEQ. ID. NO. 59 (NATVK6 primer): ttggtggcca cagcggccga tgtccactcg gacatccaga tgacccagtc tcc

SEQ. ID. NO. 60 (NATVL13 primer): ttggtggcca cagcggccga tgtccactcg cagttcgtgc tgactcagcc

SEQ. ID. NO. 61 (NATVL10 primer): ttggtggcca cagcggccga tgtccactcg cagctcgtgc tgactcagcc

SEQ. ID. NO. 62 (NATJK-R7 primer): gaggagagat cttttgatat ccaccttggt

SEQ. ID. NO. 63 (NATJL2-R primer): gaggagagat cttaggacgg tcagcttggt ccc

SEQ. ID. NO. 64 (NATJK-R5 primer): gaggagagat cttttgattt ccagcttggt

SEQ. ID. NO. 65 (NATJL1-R primer): gaggagagat cttaggacgg tgaccttggt ccc

SEQ. ID. NO. 66 (NATJK-R4 primer): gaggagagat cttttgattt ccaccttggt

Claims (27)

  1. DLK1 (delta-like 1 homolog) extracellular region fragment containing a human antibody or an antigen-binding site that specifically binds to the soluble domain.
  2. According to claim 1,
    The extracellular domain of the soluble domain DLK1 is that the fragment containing the human antibody or an antigen-binding site consisting of the amino acid sequence set forth in SEQ ID NO: 1.
  3. According to claim 1,
    Fragment containing the antigen binding sites of the human antibody is a Fab, Fd, Fv, dAb, isolated CDR regions, F (ab '), F (ab') 2, selected from the group consisting of scFv and dia body (diabody) to characterized in that, the human antibody or fragment thereof comprising an antigen binding site.
  4. According to claim 1,
    The human antibodies described in (a) SEQ ID NO: 2 heavy chain according to CDR1, SEQ ID NO: 3 as described in the heavy chain CDR2 and SEQ ID NO: light chain CDR1, SEQ ID NO: 6 described in the heavy chain variable region and SEQ ID NO: 5 comprising a heavy chain CDR3 according to the 4 a light chain CDR2, and SEQ ID NO: light chain variable region comprising the light chain CDR3 as set forth 7;
    (B) SEQ ID NO: heavy chain CDR1, a light chain CDR1, light chain CDR2 and the sequence shown in SEQ ID NO: 12 described in the heavy chain variable region and SEQ ID NO: 11 comprising a heavy chain CDR3 defined by the heavy chain CDR2 and SEQ ID NO: 10 set forth in SEQ ID NO: 9 described in 8 light chain variable region comprising the light chain CDR3 according to the number 13;
    (C) SEQ ID NO: light chain CDR2, and SEQ ID NO: described in a defined heavy chain CDR1, a light chain CDR1, SEQ ID NO: 18 described in the heavy chain variable region and SEQ ID NO: 17 comprising a heavy chain CDR2, and SEQ ID NO: heavy chain CDR3 according to 16 described in SEQ ID NO: 15 14 light chain variable region comprising the light chain CDR3 according to the number 19;
    (D) SEQ ID NO: heavy chain CDR1, a light chain CDR1, light chain CDR2 and the sequence shown in SEQ ID NO: 24 set forth the heavy chain variable region and SEQ ID NO: 23 comprising a heavy chain CDR2, and SEQ ID NO: heavy chain CDR3 according to 22 described in SEQ ID NO: 21 according to the 20 light chain variable region comprising the light chain CDR3 according to the number 25;
    (E) SEQ ID NO: 26 the heavy chain CDR1, a light chain CDR1, light chain CDR2 and the sequence shown in SEQ ID NO: 30 described in the heavy chain variable region and SEQ ID NO: 29 comprising a heavy chain CDR2, and SEQ ID NO: 28 heavy chain CDR3 according to the described in SEQ ID NO: 27 described in light chain variable region comprising the light chain CDR3 according to the number 31; or
    (F) a light chain CDR2 and the sequence shown in SEQ ID NO: heavy chain CDR1 according to 32, SEQ ID NO: 33 as described in the heavy chain CDR2 and SEQ ID NO: 34 the light chain as described for the heavy chain CDR3 in the heavy chain variable region and SEQ ID NO: 35 comprises according to the CDR1, SEQ ID NO: 36 a human antibody or fragment thereof comprising an antigen binding site comprises a light chain variable region comprising the light chain CDR3 according to the number 37.
  5. According to claim 1,
    Human antibodies to the human antibody comprises a light chain variable region amino acid sequence shown in (a) SEQ ID NO: 38 the heavy chain variable region amino acid sequence and SEQ ID NO: 39 as described;
    (B) a human antibody comprising a light chain variable region amino acid sequence shown in SEQ ID NO: 40 the heavy chain variable region amino acid sequence and SEQ ID NO: 41 as described;
    (C) a heavy chain variable region amino acid sequence and SEQ ID NO: human antibody comprising a light chain variable region amino acid sequence shown as 43 described in SEQ ID NO: 42;
    (D) a heavy chain variable region amino acid sequence and SEQ ID NO: human antibody comprising a light chain variable region amino acid sequence shown in SEQ ID NO 44 to 45 as described;
    (E) the heavy chain variable region amino acid sequence and SEQ ID NO: human antibody comprising a light chain variable region amino acid sequence shown in SEQ ID NO 46 to 47 as described; And
    (F) SEQ ID NO: 48 the heavy chain variable region amino acid sequence and SEQ ID NO: 49 the light chain variable region, a human antibody or fragment thereof comprising an antigen binding site is selected from the group consisting of a human antibody comprising the amino acid sequence shown as described by.
  6. According to claim 1,
    The human antibody to a human antibody or fragment thereof comprising an antigen binding site that increases the binding affinity to liquid activin receptor type 2B of the water-soluble region of the outer domain DLK1 cells.
  7. According to claim 1,
    The human antibody fragment containing it in, the human antibody or an antigen-binding site to inhibit the signal transduction in hypoxic cells.
  8. Any one of claims 1 to 7, a polynucleotide encoding any one of the human antibody or fragment thereof comprising an antigen binding site of the anti.
  9. The method of claim 8,
    The polynucleotide which polynucleotide comprises a light chain variable region described in SEQ ID NO: 51 and a heavy chain variable region described in SEQ ID NO: 50.
  10. Claim 8 recombinant vector comprising the polynucleotide.
  11. Claim 8 a transformant transformed with the recombinant vector.
  12. (A) comprising the steps of: 1 to claim 7, wherein producing a recombinant vector comprising a polynucleotide encoding a fragment containing any one of the human antibody or an antigen-binding site of;
    (B) the transformed as transformants by introducing the recombinant vector into a host the cells; And
    (C) method for producing any one of claims 1 to 7 fragment containing any one of the human antibody or an antigen-binding site of the anti comprising the step of culturing the transformant.
  13. Claim 1 to claim 7, wherein in any one of the human antibody or a pharmaceutical composition thereof, including fragments containing the antigen binding site, cancer prevention or treatment.
  14. 14. The method of claim 13,
    Wherein the composition is cancer metastasis or invasion, having inhibitory activity, the pharmaceutical compositions.
  15. 14. The method of claim 13,
    Wherein the composition further comprises a water-soluble region of the outer domain DLK1 cells, the pharmaceutical compositions.
  16. 16. The method of claim 15,
    The extracellular domain of the soluble domain DLK1 is to combine the liquid activin (activin) and competitively liquid activin receptor type 2B (activin receptor type II B), the pharmaceutical compositions.
  17. 14. The method of claim 13,
    The composition of further comprises a DLK1-Fc fusion protein conjugated soluble extracellular region domain and an antibody Fc region DLK1, the pharmaceutical compositions.
  18. 14. The method of claim 13,
    The cancer is skin cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, and would at least any one selected from the group consisting of gastric cancer, the pharmaceutical compositions.
  19. Claim 1 to claim 7, wherein in any one of the human antibodies or derivatives, including fragments containing the antigen binding site, metabolic disorders, immune system disorders, a pharmaceutical composition for the prevention and treatment of liver diseases.
  20. 20. The method of claim 19,
    Wherein the composition further comprises a water-soluble region of the outer domain DLK1 cells, the pharmaceutical compositions.
  21. 20. The method of claim 19,
    The extracellular domain of the soluble domain DLK1 is to combine the liquid activin (activin) and competitively liquid activin receptor type 2B (activin receptor type II B), the pharmaceutical compositions.
  22. 20. The method of claim 19,
    The composition of further comprises a DLK1-Fc fusion protein conjugated soluble extracellular region domain and an antibody Fc region DLK1, the pharmaceutical compositions.
  23. 20. The method of claim 19,
    The metabolic diseases, a pharmaceutical composition, characterized in that diabetes or obesity.
  24. 20. The method of claim 19,
    The immune system disease is self-pharmaceutical composition characterized in that the autoimmune disease or rheumatoid arthritis.
  25. 20. The method of claim 19,
    The liver disease is hepatitis, alcoholic cirrhosis, acute liver failure, hepatocellular carcinoma and liver which is characterized in that the disease is selected from the group consisting of pharmaceutical composition.
  26. Claim 1 to claim 7 comprising the antibody of any one of wherein the cancer, metabolic disorders, immune diseases or liver disease diagnostic kit.
  27. 27. The method of claim 26,
    The kit, a diagnostic kit, characterized in that the enzyme immunoassay (ELISA) kit or a sandwich ELISA kit.
PCT/KR2012/002496 2011-04-04 2012-04-03 Dlk1-specific human antibodies and pharmaceutical composition containing same WO2012138102A2 (en)

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KR10-2011-0030871 2011-04-04
KR20110030871 2011-04-04
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KR10-2012-0001457 2012-01-05

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1702982A1 (en) * 2003-11-28 2006-09-20 Kanagawa Academy Of Science And Technology Method of detecting liver cancer, diagnostic for liver cancer and remedy for cancer
EP1394263B1 (en) * 2001-05-16 2008-10-08 Kanagawa Academy Of Science And Technology Methods for detection and separation of undifferentiated hepatic cells using dlk
KR20090088893A (en) * 2006-11-10 2009-08-20 가부시키가이샤 리부텍쿠 Anti-human dlk-1 antibody showing anti-tumor activity in vivo
KR100982170B1 (en) * 2010-03-16 2010-09-15 한국생명공학연구원 COMPOSITION FOR THE ANTI-CANCER METASTASIS CONTAINING DLK1-Fc FUSION PROTEIN AS AN EFFECTIVE INGREDIENT

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1394263B1 (en) * 2001-05-16 2008-10-08 Kanagawa Academy Of Science And Technology Methods for detection and separation of undifferentiated hepatic cells using dlk
EP1702982A1 (en) * 2003-11-28 2006-09-20 Kanagawa Academy Of Science And Technology Method of detecting liver cancer, diagnostic for liver cancer and remedy for cancer
KR20090088893A (en) * 2006-11-10 2009-08-20 가부시키가이샤 리부텍쿠 Anti-human dlk-1 antibody showing anti-tumor activity in vivo
KR100982170B1 (en) * 2010-03-16 2010-09-15 한국생명공학연구원 COMPOSITION FOR THE ANTI-CANCER METASTASIS CONTAINING DLK1-Fc FUSION PROTEIN AS AN EFFECTIVE INGREDIENT

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