KR101556011B1 - Human Monoclonal Antibody against Jagged 1 - Google Patents

Abstract

The present invention relates to a human monoclonal antibody against Jagged 1, more particularly to a CDR1 site consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2 site consisting of the amino acid sequence of SEQ ID NO: 4, a CDR3 site consisting of the amino acid sequence of SEQ ID NO: And a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region comprising a CDR2 region comprising the amino acid sequence of SEQ ID NO: 7.
The J1J1 human monoclonal antibody according to the present invention is screened in a human antibody library through a phage display so that it can specifically target Jagged 1, a ligand of Notch 1 that is specifically overexpressed in cancer cells, It has been confirmed that it does not require the humanization process required and can be applied to human beings and reduces the growth, migration and invasive ability of cancer cells. Therefore, it can be used as an antibody for diagnosing and treating cancer related to Jagged 1 signal .

Description

Human Monoclonal Antibody against Jagged 1 < RTI ID = 0.0 > 1 < / RTI &

The present invention relates to a human monoclonal antibody against Jagged 1, more particularly to a CDR1 site consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2 site consisting of the amino acid sequence of SEQ ID NO: 4, a CDR3 site consisting of the amino acid sequence of SEQ ID NO: A CDR1 region consisting of the amino acid sequence of SEQ ID NO: 7, a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 8, and a heavy chain variable region comprising the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: Lt; RTI ID = 0.0 > monoclonal < / RTI >

Since the development of monoclonal antibody-producing technologies, attempts have been made to use them in the diagnosis and treatment of diseases. Accordingly, many monoclonal antibodies for diagnosis have been developed and marketed. However, monoclonal antibodies for treatment are delayed in commercialization because of various constraints. One of them is the immune response of the human body.

The development of antibody therapeutics has been directed towards reducing immunogenicity. Orthoclone-OKT3, the first to succeed in commercialization, is an immunosuppressant that inhibits the rejection of kidney transplantation and is a mouse antibody with mouse sequences. In order to reduce the HAMA (human anti-mouse antibody) reaction that can be induced by the mouse antibody, chimeric antibodies containing human sequences other than the antibody variable domain sequence have been developed and the CDR (complementarity determining region) Humanized antibodies, all of which have human sequences, have been developed in sequence.

Since the development of this technology, about 10 humanized antibody therapeutics have been approved by the FDA and are currently being used as a therapeutic agent for cancer and autoimmune diseases and as a preventive agent for viral infections. Herceptin, a Her2 binding antibody, and Avastin, a VEGF neutralizing antibody, It is a blockbuster antibody product as a therapeutic agent.

As such, efforts have been made to produce a fully human monoclonal antibody having a human antibody sequence in addition to a method of manipulating an antibody derived from a mouse to maximally resemble a human antibody.

Phage display technologies such as Cambridge Antibody Technolgy (CAT) and Dyax, among the existing human antibody technologies, have been applied to phage coat The transgenic mouse technology, which Medarex and Abgenix have, is a method for finding an antibody fragment that binds to a specific antigen by genetically manipulating an antibody fragment containing a CDR sequence to a part of the pIII protein constituting the protein, A transgenic mouse in which an antibody-expressing gene is substituted with a human antibody-expressing gene is used. Taking the antibody of VEGFR-2 as an example, Tanibirumab is a fully human antibody developed using phage display technology. The major difference from other VEGFR-2 neutralizing antibodies currently being developed is that it reacts with mouse / rat VEGFR-2, Both Ramucirumab of Imclone and CDP-791 of UCB pharma are unable to bind to mouse VEGFR-2. Thus, the ability of the two antibodies to bind to mouse VEGFR-2 in Tanibirumab can be assessed for neovascular inhibitory effects in animal models, while it is difficult to assess the efficacy of these two antibodies in animal models. This feature is expected to be of great help in selecting the most appropriate indication for Tanibirumab during clinical trial development. In addition, the human monoclonal antibody produced by the phage display method is an antibody against TNF-α (adalimumab, product name: Humira), a strong antibody product approved by the FDA in 2002 for treating arthritis.

Notch signaling is an evolutionarily well - preserved signaling system that is known to regulate cell crystallization, differentiation, proliferation and death. The Notch protein is a single membrane penetrating protein that acts as a cell surface receptor and nuclear transfer modulator. The Notch gene was first discovered in the Drosophila phenotypic study when it found that there was a notch at the end of the flapping wings and named the related gene Notch. The mammal has four Notch (Notch 1, 2, 3, 4), each Notch is synthesized with a protein of 300-350 kDa in size, and the S1 is cleaved by furin-like convertase in the Golgi, to form a heterodimer.

Activated Notch signaling is known to induce tumorigenesis in several cancer models (Wang Z. et < RTI ID = 0.0 > al ., Anticancer Res . , ≪ / RTI > 28: 3621, 2008; Santagata S. et al., Cancer Res . , 1; 64: 6854, 2004). T-cell leukemia / lymphomas are caused by expression of NICD, an active Notch, in murine hematopoietic cells, and T-cell acute lymphoblastic leukemia (T-ALL) is caused by tumor growth and embryo development. About 50% of activated Notch 1 was found (Weng AP et al ., Science, 306: 269, 2004). In addition, notch carcinogenic effects have been reported in breast cancer rat models.

On the other hand, Notch's ligand, Jagged 1, has a poor prognosis in breast cancer patients (Reedijk M. et al ., Cancer Res, 15; 65:.. . 8530, 2005) and in prostate cancer is known to be associated with cancer metastasis and (Santagata S. et al, Cancer Res , 64: 6854, 2004), beginning with the Notch / ligand binding Notch signal transduction systems do not require enzymatic signal amplification such as kinase, so that signal intensity can be adjusted relatively accurately, and Notch signal transduction is attracting attention in cancer research. Notch signaling can be inhibited at the early stage of Notch signaling using a monoclonal antibody that interferes with Notch / ligand binding, and a monoclonal antibody targeting the Notch ligand, Dll4, has been developed to inhibit Notch signaling in endothelial cells There have been reports of limiting the production of angiogenic blood vessels (Ridgway J. et al ., Nature , 444: 1083, 2006), many monoclonal antibodies are now being developed that can interfere with Notch-related antibodies for cancer diagnosis and Notch signaling (Yan M. et al ., Nature , 464: 1052, 2010).

Therefore, the present inventors have made intensive efforts to provide a monoclonal antibody of Jagged 1, which is a ligand of Notch 1, which can be a main target of new chemotherapy, and as a result, it has been found that antibody J1J1 specifically binding to Jagged 1 in a human antibody library is selected Migration, and invasive ability of cancer cells, thereby completing the present invention.

An object of the present invention is to provide a human monoclonal antibody that specifically binds to Jagged 1, a gene encoding the same, a recombinant vector containing the gene, and a recombinant cell transformed with the gene or the recombinant vector.

It is yet another object of the present invention to provide a method for producing a human monoclonal antibody against Jagged 1 by culturing the recombinant cells.

It is still another object of the present invention to provide a cancer diagnostic composition comprising a human monoclonal antibody against Jagged 1 and a composition for treating cancer.

In order to achieve the above object, the present invention provides a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 4, a light chain variable region comprising the CDR3 region consisting of the amino acid sequence of SEQ ID NO: Comprising a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 8, a human monoclonal antibody to Jagged 1 do.

The present invention also provides a gene encoding a human monoclonal antibody to Jagged 1.

The present invention also relates to a method for producing a human monoclonal antibody against Jagged 1, wherein the recombinant vector containing the gene, the gene or the recombinant vector is introduced into the host microorganism, and the recombinant cell and the recombinant cell are cultured .

The present invention also provides a composition for diagnosing cancer comprising the human monoclonal antibody against Jagged 1 and a composition for treating cancer.

The J1J1 human monoclonal antibody according to the present invention is screened in a human antibody library through a phage display so that it can specifically target Jagged 1, a ligand of Notch 1 that is specifically overexpressed in cancer cells, It has been confirmed that it does not require the humanization process required and can be applied to human beings and reduces the growth, migration and invasive ability of cancer cells. Therefore, it can be used as an antibody for diagnosing and treating cancer related to Jagged 1 signal .

FIG. 1 shows data of J1J1 antibody selectively binding to Jagged 1-peptide from third and fourth order panning.
Fig. 2 is a schematic diagram showing the light chain variable region and heavy chain variable region amino acid sequence and CDR region of the J1J1 antibody.
Fig. 3 shows data showing the results of purification of the Fab-type J1J1 antibody after conversion to whole Ig form.
Fig. 4 is data showing the degree of inhibition of growth of OVCAR3 cell line by J1J1 antibody.
FIG. 5 is data showing the degree of inhibition of growth of SKOV 3 cell line by J1J1 antibody.
6 is data showing the degree of inhibition of metastasis and invasion of OVCAR3 cell line by J1J1 antibody.
FIG. 7 is data showing the degree of inhibition of metastasis and invasion of SKOV 3 cell line by J1J1 antibody.
8 is a schematic diagram showing a Notch signal.
Fig. 9 is data showing the degree of inhibition of mRNA expression of the Notch signal by the J1J1 antibody in the OVCAR3 cell line.
10 is data showing the degree of inhibition of mRNA expression of Notch signal by J1J1 antibody in SKOV 3 cell line.
FIG. 11 is data showing inhibition of protein expression of Notch signal by J1J1 antibody in OVCAR3 cell line.
Fig. 12 shows data of anti-cancer effect of J1J1 antibody in vivo.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention provides, in one aspect, a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 4, a light chain variable region comprising the CDR3 region consisting of the amino acid sequence of SEQ ID NO: 5, A CDR1 region consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 8.

In the present invention, the human monoclonal antibody to Jagged 1 may include a light chain variable region represented by the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 2.

In the present invention, 'Jagged 1' corresponds to a notch receptor ligand, and 'Jagged 1' is used in combination with the term 'Jag 1' as used herein.

The term " antibody " in the present invention refers to an immune protein that binds to an antigen and interferes with the action of the antigen or eliminates the antigen. There are five types of antibodies, immunoglobulin (Ig) M, IgD, IgG, IgA and IgE, each containing a heavy chain made up of heavy chain constant region genes μ, δ, γ, α, and ε. In antibody technology, IgG is mainly used. There are four types of isotypes, IgG1, IgG2, IgG3, and IgG4, and their structural and functional characteristics are different.

IgG has a very stable structure (molecular weight, 150 kDa) with two heavy chain (50 kDa) proteins and two light chain (25 kDa) proteins. The light and heavy chains of the antibody are divided into constant regions having the same amino acid sequence as the variable region in which the amino acid sequences of the antibodies differ from each other. The heavy chain constant region includes CH1, H (hinge), CH2, CH3 domains Lt; / RTI > Each domain consists of two β-sheets and an intramolecular disulfide bond is connected between them. Two variable regions of the heavy chain and the light chain are combined to form an antigen binding site. Each of these regions has one Y-shaped arm, which can bind to the antigen binding fragment ), And the part that can not bind antigen is called Fc (crystalizable fragment). Fab and Fc are connected by a flexible hinge region.

Within the heavy and light chain variable regions of the antibody, there are different portions of the antibody that differ in amino acid sequence. These are called hypervariable regions and are called complementarity determining regions (CDRs) because they form binding sites for antigens. As for the stereostructure of the antibody, these CDRs have a loop shape on the surface of the antibody, and below the ring there exists a framework region (FR) that structurally supports the CDRs. There are three ring structures in each of the heavy chain and the light chain, and these six ring structures combine with each other to directly contact the antigen.

The human antibody of the present invention can be easily produced by a known monoclonal antibody production technique. For example, a method for producing a monoclonal antibody can be performed by preparing a hybridoma using B lymphocytes obtained from an immunized animal, or by using a phage display technique, It is not.

Phage display was first introduced by G. Smith in 1985 and first applied to the manufacture of antibodies by MRC in the UK in 1990. Bacteriophage is a kind of virus parasitic to Escherichia coli. In this technology, M13, which is a filamentous bacteriophage, is mainly used.

The phage display of the antibody amplifies the heavy and light chain variable regions of the antibody in the human body by PCR. The antibody is cloned into the phage surface protein (pIII) contained in the phagemid vector and expressed in E. coli. The helper phage Refers to a technique capable of producing an antibody library of various combinations of heavy and light chain variable regions displayed on the surface of the phage upon infection. The type of antibody used here is scFv or Fab. From this library, a human monoclonal antibody that binds to a specific antigen can be separately prepared by a panning method. Panning is a process of binding a phage to a target antigen and removing the unbound phage, collecting the bound phage, infecting the E. coli to amplify the phage number, and repeating this process 2-4 times. With this technology, human monoclonal antibodies can be isolated in only a few weeks. The synthetic antibody library is called a naive antibody library, in which antibody diversity is created from an antibody gene already present in the human body, and randomization sequences are added to the CDRs of antibodies to create diversity. The native library does not have a high affinity for the separated antibody, but it does not cost much to produce. The synthetic library has a high affinity for the separated antibody, but is expensive to produce. If the affinity of the separated antibody clones is not high, the affinity maturation is performed in which residues of the CDRs and FRs of the antibody are mutated and then the antibody clones having higher affinity are selected again using the phage display method.

The phages that can be used to construct the antibody library in the present invention include, for example, filamentous phages, such as fd, M13, f1, If1, Ike, Zj / Z, Ff, Xf, Pf1 or Pf3 phage , But the types of phage that can be used in the present invention are not limited by the above examples. Examples of the vector that can be used for expression of the heterologous gene on the surface of the filamentous phage include a phage vector such as fUSE5, fAFF1, fd-CAT1 or fdtetDOG, or a phage vector such as pHEN1, pComb3, pComb8, pKRIBB- , But the present invention is not limited thereto. In the present invention, the pKRIBB-Fab phagemid vector was used.

Helper phage that can also be used to provide the wild-type coat protein required for successful reinfection of the recombinant phage for amplification include, but are not limited to, for example, M13K07 or VSCM13.

In one embodiment of the present invention, the antigen of Jagged 1 was reacted with a phage displaying a human antibody library for 1 hour, 10 times for the first panning, 20 times for the second panning, 30 times for the third panning, In the case of 4th panning, it was washed 40 times. After washing, the phage-antibodies bound to the antigen were separated and then infected with TG1 E. coli and cultured overnight. At this time, the number of the phage used at the beginning of each panning was input, and after the washing process, the number of colonies formed by the TG1 Escherichia coli after being separated by the glycine solution was calculated as the number of output phage, And the colonies obtained as a result of the first panning were cultured and then cultured in the same manner as described above to prepare the phage on which the antibody was displayed. The same amount of antigens were subjected to secondary and tertiary , And 4th panning. In order to confirm that the antibodies having high antigen binding ability were amplified as the number of panning was increased, the antigen binding ability of the antibodies purified after the library and the first, second and third panning was measured by an indirect ELISA method. As a result, After panning, it was confirmed that the antibody specific for Jagged 1-peptid was concentrated.

Seven positive clones that selectively bind to the Jagged 1-peptide without binding to the control (negative control) were selected. Among them, a clone having a strong binding ability to the Jagged 1-peptide was selected and named J1J1 (FIG. 1) As a result of analyzing the base sequence and amino acid sequence of the selected J1J1 antibody, the J1J1 antibody was composed of a light chain variable region represented by the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 2, 9, and a heavy chain variable region represented by the nucleotide sequence of SEQ ID NO: 10. The light chain variable region comprises a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 4, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: A CDR1 region consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 8 (FIG. 2).

As a result of comparing the base sequence of the J1J1 antibody of the present invention with the human germline base sequence, it was found that the heavy and light chain variable region sequences of J1J1 corresponded to any heavy and light chain sequences in the IMGT database (IMGT data base) Not identical, it was confirmed that J1J1 was not a pointline antibody but a new antibody that had undergone maturation in the human body.

Antibody fragments are antigen binding domains except for the Fc region that exhibits an effector function in the antibody molecule, and mainly include Fab, single-chain antibody fragment (scFv) and third-generation antibody fragment (eg, single domain, minibody, etc.).

Since antibody fragments are small in size compared to whole IgG, they have good permeability to tissues and tumors, can be produced in bacteria, have a low production cost, and have no effector function because they do not have Fc. Antibody fragments are suitable for biopsy because of their short half-life in the human body. However, when replacing some basic, acidic or neutral amino acid among the amino acids constituting the antibody, the unique isoelectric point of the antibody alone may be changed. The change in isoelectric point of the antibody may induce changes such as toxicity side effects of the antibody in vivo or increase the water solubility. Therefore, in the case of the therapeutic antibody, the whole Ig is used considering the affinity and the structural form.

In another aspect, the invention relates to a gene encoding a human monoclonal antibody to Jagged 1.

In the present invention, the gene may include a light chain variable region encoded by the DNA sequence of SEQ ID NO: 9 and a heavy chain variable region encoded by the DNA sequence of SEQ ID NO: 10.

In another aspect, the present invention relates to a recombinant vector having the ability to produce a human monoclonal antibody against Jagged 1, wherein the recombinant vector containing the gene, the gene or the recombinant vector is introduced into a host cell will be.

In the present invention, a "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Since the plasmid is the most commonly used form of the current vector, 'plasmid' and 'vector' are sometimes used interchangeably in the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

The recombinant cell according to the present invention can be produced by inserting the gene on a chromosome of a host cell according to a conventional method, or introducing the recombinant vector onto a plasmid of a host cell.

A variety of expression host / vector combinations may be used to express human monoclonal antibodies according to the invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, cow papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. Expression vectors that can be used for bacterial hosts include bacterial plasmids obtained from Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9 and derivatives thereof, , Phage DNA which may be exemplified by a wide variety of phage lambda derivatives such as gt10 and gt11, NM989, and other DNA phages such as M13 and filamentous single stranded DNA fragments. An expression vector useful for yeast cells is 2 plasmid and its derivatives. The vector useful for insect cells is pVL941.

The recombinant vector of the present invention may be transformed into an appropriate host cell such as a yeast cell, an animal cell, etc., and then the transformed host cell may be cultured to mass-produce the human antibody or fragment thereof of the present invention.

A suitable host cell of the vector is E. coli, Bacillus subtilis (Bacillus subtilis), genus Streptomyces (Streptomyces sp.), Pseudomonas species (Pseudomonas sp.), Proteus ( Proteus mirabilis ) or Staphylococcus sp.). < / RTI > In addition, the genus Aspergillus sp.), fungi such as Pichia pastoris), Celebi as Saccharomyces My jiae access (Saccharomyces S. cerevisiae , Schizosaccharomyces eukaryotic cells such as yeast such as Neurospora crassa , other lower eukaryotic cells, and cells of higher eukaryotes such as cells from insects. It can also be derived from plants and mammals. Preferably, the cells are selected from the group consisting of monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) MDCK, myeloma cell line, HuT 78 cell, and HEK293 cell, but are not limited thereto, and more preferably HEK293 cells.

As a method of introducing the recombinant vector of the present invention into a host cell and transforming it, a commonly known gene manipulation method can be used. For example, microinjection (direct insertion of DNA into cells), liposome, directed DNA uptake, receptor ~ mediated DNA transfer, or DNA transport using Ca ⁺⁺ . Is widely used. Examples include retrovirus vectors, adenovirus vectors, adeno-associated viral vectors, herpes simplex virus vectors, poxvirus vectors, or lentiviral vectors. Particularly, retroviruses have a high gene transfer efficiency, gross deletion It can be used in a wide range of cells without binding by host DNA and rearrangement (resulting in alteration of host DNA function by altering the region of the host DNA similar to that of the host DNA).

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer may be operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, " operably linked " means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

In another aspect, the present invention provides a method for producing Jagged 1 comprising: (a) culturing a recombinant cell having the ability to produce human monoclonal antibody against Jagged 1 to produce a human monoclonal antibody against Jagged 1; And (b) recovering the human monoclonal antibody against the resulting Jagged 1.

The human monoclonal antibody to Jagged 1 of the present invention is preferably obtained by expressing and purifying by a recombinant method. Specifically, the human monoclonal antibody for Jagged 1 is obtained by expressing the gene sequence encoding the heavy chain variable region or the entire heavy chain region of the antibody and the light chain variable region The gene sequence encoding the region may be expressed separately in one or two vectors, but is not limited thereto.

Specifically, the method for producing a human monoclonal antibody against Jagged 1 comprises the steps of: preparing a recombinant vector by inserting a gene encoding a human monoclonal antibody against Jagged 1 of the present invention into a vector; Transforming the recombinant vector into a host cell and culturing it; And separating and purifying a human monoclonal antibody against Jagged 1 from the cultured transformant.

Specifically, a human monoclonal antibody against Jagged 1 can be produced in large quantities by culturing the transformant expressing the recombinant vector in a nutrient medium. The medium and culture conditions can be suitably selected depending on the host cell. The conditions such as the temperature, the pH of the medium and the incubation time can be appropriately adjusted so as to be suitable for cell growth and mass production of the protein at the time of culturing.

Such recombinantly produced peptides or proteins can be recovered from the medium or cell lysate. If membrane bound, it can be liberated from the membrane by using a suitable surfactant solution (e.g., Triton-X 100) or by enzymatic cleavage.

Cells used for antibody expression can be disrupted by various physical or chemical means such as freeze-thawing, sonication, mechanical disruption or cell disruption, and can be isolated and purified by conventional biochemical separation techniques (Sambrook . et al, Molecular Cloning:. .. A laborarory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol 182. Academic Press Inc., San Diego, CA (1990)). Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immuno adsorption chromatography, size exclusion chromatography, etc.), isoelectric focusing and various variations and combinations thereof But are not so limited.

The culture and recovery of the recombinant cells for producing the human monoclonal antibody against Jagged 1 of the present invention can be applied to any culture method and separation and purification method of recombinant proteins conventionally known in the art without any particular limitation.

In another embodiment of the present invention, the J1J1 heavy chain variable region and the light chain variable region were amplified by polymerase chain reaction to transform Fab-type J1J1 into whole Ig form, and J1J1 antibody was purified by transforming HEK293T cells 3).

In another aspect, the present invention relates to a composition for treating cancer comprising a human monoclonal antibody (J1J1) for Jagged 1 containing a light chain variable region represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2 from another viewpoint.

In the present invention, the term 'anti-cancer' includes 'prevention' and 'treatment'. Here, 'prevention' means all actions in which cancer is suppressed or delayed by administration of a composition containing an antibody of the present invention, Quot; means any action that improves or alleviates the symptoms of cancer by administration of the antibody of the present invention.

In the present invention, the cancer may be selected from the group consisting of cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer. However, the present invention is not limited thereto, and the Notch, particularly Jagged 1, The cancer or carcinoma in which the signal of Notch 1 is abnormally expressed is not particularly limited and includes solid tumors and blood cancers.

In another embodiment of the present invention, in order to confirm the therapeutic effect of J1J1 antibody as a human antibody against Jagged 1, ovarian adenocarcinoma cells OVCAR-3 cells and ovarian carcinoma cells SKOV3 cells are used Proliferation, migration and invasion of cancer cells were observed. J1J1 antibody was treated with cancer cells to observe cell viability after 24 hours, 48 hours, and 72 hours. As a result, it was confirmed that the survival rate of OVCAR cells was reduced by about 70% as compared with the control (Fig. 4) It was confirmed that the survival rate was reduced by about 60% (FIG. 5), and it was confirmed that the growth of cancer cells was inhibited by the J1J1 antibody. Also, in After checking the metastasis and invasion ability of cancer cells by J1J1 antibody on vitro, when processing the J1J1 antibody, confirming that the metastasis and invasion of OVCAR3 (FIG. 6) and SKOV3 (Fig. 7) compared to the control is suppressed, J1J1 It was confirmed that the ability of the antibody to inhibit metastasis and invasion of cancer cells was inhibited.

In another embodiment of the present invention, the degree of mRNA and protein expression of Notch 1, NICD 1, Notch 3, Jagged 1, and Hes 1 associated with Notch signal was confirmed in order to confirm the degree of inhibition of Notch signal by J1J1 antibody. As shown in FIG. 8, activated Notch signaling activates NICD 1, Hes 1, so that when Jagged 1, which is a ligand of Notch 1, is blocked by the J1J1 antibody, the expression of Notch 1, NICD 1 and Hes 1 is decreased do. When the J1J1 antibody of the present invention was treated, mRNA expression of Notch 1, NICD 1, Notch 3, Jagged 1 and Hes 1 related to Notch signal was significantly decreased in OVCAR3 (FIG. 9) and SKOV3 (FIG. 10) In OVCAR3, protein expression of Notch 1, NICD 1, Notch 3, Jagged 1 and Hes 1 was significantly reduced (FIG. 11).

In another embodiment of the present invention, the anticancer effect of the J1J1 antibody in a mouse model xenografted with ovarian adenocarcinoma cells OVCAR-3 was examined. As a result, H & E staining revealed that in the group treated with J1J1 antibody, And TUNEL staining revealed apoptosis in the group treated with J1J1 antibody (FIG. 12).

That is, it was confirmed that the J1J1 antibody of the present invention reduces cancer cell growth, metastasis and infiltration ability and inhibits Notch signal, and thus can be used as a cancer therapeutic agent.

The anti-cancer composition of the present invention may further comprise a pharmaceutically acceptable carrier. In the case of oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment and a flavoring agent may be used. , A stabilizer, and the like. In case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration, and in the case of injections, unit dosage ampoules or a plurality of dosage forms. In addition, the anti-cancer composition may typically comprise a surfactant that facilitates migration through the membrane. Such surfactants are those derived from steroids or cationic lipids such as N- [1- (2,3-dioloyl) propyl-N, N, N-trimethylammonium chloride (DOTMA), or cholesterol hemi- , Phosphatidylglycerol, and the like.

In another aspect, the present invention provides a method for treating cancer and inhibiting cancer growth by administering a composition comprising the human monoclonal antibody or the human monoclonal antibody to the Jagged 1. A composition comprising a human monoclonal antibody to Jagged 1 according to the present invention may be administered in a pharmaceutically effective amount to treat cancer cells or their metastasis or to inhibit cancer growth. The type of cancer, the age and weight of the patient, the nature and severity of symptoms, the type of current treatment, the number of treatments, the route and route of administration, and can be readily determined by those skilled in the art. The composition of the present invention may be administered together with the above-described pharmacological or physiological components or may be administered sequentially, or may be administered in combination with additional conventional therapeutic agents, and administered sequentially or concurrently with conventional therapeutic agents. Such administration may be single or multiple administrations. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

In the present invention, the term 'individual' means a mammal having or at risk for a condition or disease that can be alleviated, suppressed or treated by administering an antibody of the present invention, preferably a human.

In the present invention, 'administering' means introducing a predetermined substance into a subject by any suitable method, so that the administration route of the composition comprising the antibody of the present invention is administered through any conventional route so long as it can reach the target tissue . But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. However, at the time of oral administration, since the protein is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top. In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell.

In another aspect, the present invention relates to a cancer diagnostic composition comprising a human monoclonal antibody (J1J1) for Jagged 1 of the present invention.

In the present invention, the cancer that can be diagnosed by detecting the expression of Jagged 1 may include cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, etc. However, the present invention is not limited thereto, Because Jagged 1 or Jagged 1 peptides can be recognized with hyper sensitivity, it is possible to diagnose a disease associated with the Notch 1 signal, which includes Notch, especially Jagged 1 as a ligand.

The diagnostic composition of the present invention comprises a human monoclonal antibody against Jagged 1 containing a light chain variable region represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2. Inhibitors, < / RTI > accelerators and / or inhibitors.

The cancer diagnosis of the present invention can be performed using a biosensor using surface plasmon resonance, immunoprecipitation, immunodot or glass chip, In particular, biosensors using surface plasmon resonance provide qualitative information (whether two molecules specifically bind) and quantitative information (reaction rate, kinetics, equilibrium constants) It can be detected in real time and is particularly useful for measuring antigen and antibody binding.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Preparation of human antibody libraries and screening of antibodies binding to Jagged 1

1-1: Preparation of phage display human antibody library

To select antibodies binding to Jagged 1, a human antibody library was prepared by cloning. The pKB-Fab100 phagemid vector was digested with restriction enzymes Apa (New England Biolabs) and EcoR-HF (New England Biolabs) The light and heavy chain genes were cloned in situ to prepare and clone the library.

First, the pKB-Fab100 phagemid vector was digested with restriction enzyme Apa (New England Biolabs) at 25 ° C for 1 hour and EcoR-HF (New England Biolabs) at 37 ° C for 2 hours to prepare a library. % Agarose gel (Cambrex). Using the purified vector and purified genes at a molar ratio of 2, 20 l of 10 x ligase buffer, 10 l of T4 DNA ligase and sterile distilled water were added to make 200 l , And reacted overnight at 16 ° C. The next day, the ligation product was added with glycogen, concentrated by ethanol precipitation, and then used for transformation of E. coli.

Transformation of E. coli was performed by electroporation. Electrocompetent E. coli (TG1) was electroporated (using BioRad Gene Pulser Xcell) under the conditions of 2.5 kV, 200 O and 25 D in the above-described ligation product. The transformed Escherichia coli was cultured on an SOB solid culture plate containing 23.5 cm x 23.5 cm x 20.0 cm of ampicillin, glucose and magnesium and incubated overnight at 37 ° C. Ampicillin and glucose , And 2YT liquid medium containing magnesium. Glycerol was added to a final concentration of 20% and stored at -80 ° C. The size of the prepared library was determined by measuring the number of cfu (colony forming units) obtained after electroporation, multiplying the obtained Escherichia coli by a multi-stage dilution, plating on a 2YT plate containing ampicillin, incubating overnight at 37 ° C, I was able to get a library of ⁹ cfu.

The E. coli cells containing the human antibody library (CDR3-24) were cultured to an OD ₆₀₀ value of 0.6-0.7, added with a helper phage (VCSM13), allowed to stand for about 30 minutes, Shaking for one minute, kanamycin (70 μg / ml), and then cultured overnight. After culturing, the library phage was recovered from the culture solution.

1-2: Selection of antibody binding to Jagged 1 peptide from prepared antibody library

100 μg of Jagged 1-peptide was mixed with 1 ml of a coating buffer solution (15 mM Na ₂ CO ₃ , 34.84 mM NaHCO ₃ , pH 9.6), and the mixture was coated overnight at 4 ° C. in an immune tube (Maxisorp Immunotube, Nunc) Tween 20-added PBS solution (PBST) and blocked with 4% skim milk for 1 hour at room temperature. After two washes again, the phage (3.8 x 10 ¹² ) displaying the antigen prepared in Example 1 and the human antibody library were mixed in a final concentration of 2% skim milk and placed in a 1 ml immune tube and incubated at 37 ° C for 1 hour And reacted with the antigen. The following 0.5% Tween 20-added PBS was washed 10 times for the first panning, 20 times for the second panning, 30 times for the third panning, and 40 times for the fourth panning. After adding the washing solution, the solution is pipetted up and down 10 times using a pipette pipetman, then 4 ml of washing solution is added, and the solution is allowed to stand for 5 minutes. After washing, phage that was bound to the antigen-antibody were separated by the use of 0.1M glycine (pH 2.5) and immediately 1㎖ OD ₆₀₀ value was neutralized by adding 2 M Tris HCl pH 9.0 in a 60㎕ 0.8, cultured in 2YT (2YTA) solid medium containing ampicillin (100 μg / ml), and then cultured overnight at 37 ° C. The number of the phages used at the beginning of each panning was input, and after the washing process, the number of colonies made by the TG1 Escherichia coli after being separated by the glycine solution was calculated as the number of output phage, Was used as an indicator of the degree of amplification of the antibody.

Number of input and output phage of panning with Jagged 1-peptide as antigen Primary panning input 3.8 x 10 < ¹² > / ml output 5.28 × 10 ⁴ Secondary panning input 7.9 × 10 ¹² / ml output 6.05 × 10 ⁵ 3rd panning input 1.27 x 10 < ¹³ > / ml output 5.28 × 10 ⁶ 4th panning input 1.08 x 10 < ¹³ > / ml output 9.79 x 10 ⁷

The colonies obtained as a result of the first panning were collected and cultured, and the second phage was added in the same manner and cultured to prepare the phage displayed with the antibody, and the second, third and fourth panning was performed on the same amount of antigen. In order to confirm the amplification of antibodies with high antigen binding ability as the number of panning increases, the library and the selected colonies after the 1st, 2nd, 3rd and 4th panning were collected and OD ₆₀₀ value 0.8, IPTG was added to the final concentration of 1 mM and expressed overnight at 28 ° C. Then, the antigen binding ability of the antibodies present in the supernatant was measured by an indirect ELISA method.

In an indirect ELISA, Jagged 1-peptide was coated on a 96-well plate (MaxiSorp, Nunc), and 100 쨉 l of the culture supernatant was added to each well, followed by reaction at room temperature for 30 minutes. After washing three times with PBST, the primary antigen KR127 was diluted 1: 3000 in 0.05% PBST, and attached at 37 ° C for 1 hour. Then, it was washed 3 times with PBST, and secondary antigen mouse Fc-HRP (2 ^nd antibody mouse Fc-HRP) was reacted at 37 ° C for 1 hour with 1: 3000 dilution in 0.05% PBST. After washing three times with PBST, 100 μl of TMB (BD) and hydrogen peroxide solution were mixed at a ratio of 1: 1, and reacted for 5 minutes. Then, 50 μl of 2 M H2SO4 was added to stop the reaction. Absorbance was read at 450 nm wavelength using a spectrophotometer (VERSA max microplate reader) and antibody expression was measured.

As a result, it was confirmed that the antibody specific to the Jagged 1-peptide was enriched after the third panning.

1-3: Selection of positive antibodies

In order to screen clones bound to Jagged 1-peptide, the phages eluted after the 3rd and 4th panning were diluted, infected with TG1 E. coli, plated on 2 YTA solid medium, placed in a 37 ° C thermostat, and about 1000 single colonies Lt; / RTI > Single colonies were each inoculated into 3 ml of 2 YTA liquid medium and incubated at 37 ° C in a shaking incubator until the absorbance of each clone was between 0.6 and 0.7 at 600 nm. Next, IPTG was added to a final concentration of 1 mM, incubated overnight at 28 ° C in a shaking incubator, and then the culture supernatant was recovered.

In order to measure the antigen binding ability of the antibodies present in the supernatant, an experiment was conducted in the same manner as in the indirect ELISA method of Example 1-2.

Through these ELISA procedures, seven positive clones were selectively selected for binding to the Jagged 1-peptide without binding to the control (negative control). Among them, a clone having a strong binding ability to the Jagged 1-peptide was selected and named as J1J1 1), the plasmid DNA was isolated and the nucleotide sequence was analyzed.

As a result, the selected J1J1 antibody was analyzed to include a light chain variable region encoded by the DNA sequence of SEQ ID NO: 9 and a heavy chain variable region encoded by the DNA sequence of SEQ ID NO: 10. As a result of amino acid sequence analysis, the J1J1 antibody A light chain variable region represented by the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 2. The light chain variable region comprises a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 4, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: A CDR1 region consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 8 (FIG. 2).

Jagged  1 < / RTI > < RTI ID = 0.0 >

The nucleotide sequence of the J1J1 antibody selected in Example 1 was compared with a human germline base sequence using IMGT software (http://imgt.cines.fr/ IMGT_vquest / share / textes /). As a result, The variable region was analyzed to be derived from IGKV1-5 * 03 (93.55% identical) of the human antibody dotted line Kappa group. The heavy and light chain variable region sequences of J1J1 were not 100% identical to any heavy and light chain sequences in the IMGT data base. This demonstrates that J1J1 is not a pointline antibody but a new antibody that has undergone maturation in the human body.

J1J1 of whole IgG Conversion and production

In order to analyze the selected J1J1 more precisely, it was first transformed into whole form, transfected at 293T cell, and purified using protein A column.

The heavy chain variable region and the light chain variable region were amplified by polymerase chain reaction from Fab J1J1 in order to convert the Fab form of J1J1 into a whole form form, separated through 1.5% agarose gel (Cambrex), and then transferred to a Zymo gel extraction kit , USA). The primers used herein were SEQ ID NO: 12 and SEQ ID NO: 13 for the light chain variable region and SEQ ID NO: 14 and SEQ ID NO: 15 for the heavy chain variable region.

[SEQ ID NO: 12] 5'-TTGTCTGGTGTTGAAGGAGACATCCAGATGACCCAG-3 '

[SEQ ID NO: 13] 5'-GCAGCCGCCGTACGTTTGATCTCCACTTTGGTCCCTTGGCC-3 '

[SEQ ID NO: 14] 5'-ACTACAGGTGTCCACTCCGAAGTGCAGCTGGTGGAG-3 '

[SEQ ID NO: 15] 5'-CACCGGTTCGGGGAAGT-3 '

The following heavy chain and light chain leader sequences were synthesized and separated from pdCMV-dhfrC (Korean Patent Registration No. 10-0476363) by PCR in order to express the following antibodies in animal cells: 1.5% agarose gel (Cambrex) and Zymo gel And extracted and purified through an extraction kit (Zymoresearch). The primers used herein were SEQ ID NO: 16 and SEQ ID NO: 17 for the light chain leader sequence and SEQ ID NO: 18 and SEQ ID NO: 19 for the heavy chain leader sequence. The following heavy chain leader sequences and heavy chain variable regions were ligated to the light chain leader sequence and the light chain variable region using recombination PCR, respectively, and purified by 1.5% agarose gel (Cambrex) and Zymo gel extraction kit.

[SEQ ID NO: 16] 5'-TGCAAAGCTTCGGCACGAGCA-3 '

[SEQ ID NO: 17] 5'-TCCTTCAACACCAGACAAC-3 '

[SEQ ID NO: 18] 5'-GACGAATTCACTCTAACCATGGAA-3 '

[SEQ ID NO: 19] 5'-GGAGTGGACACCTGTAG-3 '

The light chain was digested with restriction enzymes EcoRI (Roche, Switzerland) and ApaI (Roche, Switzerland) and the light chain was cut with HindIII (Roche, Switzerland) and BsiWI (Roche, Switzerland) and digested with the same restriction enzymes to obtain pdCMV-dhfrC vector Cloned at the top of the heavy and light chain constant regions, respectively, and named pdCMV-dhfrC-J1J1.

The pdCMV-dhfrC-J1J1 DNA was transformed into HEK293T cells using Lipofectamine 2000 (Invitrogen, USA). After incubation for 4 to 6 hours at 37 ° C in a 5% CO2 incubator, the medium was replaced with serum-free medium CD293 (Gibco, USA) and incubated for 3 days in the same incubator to collect the supernatant from J1J1. The J1J1 antibody was purified from the supernatant obtained above through a protein A agarose column (Upstate, USA). As a result, when this antibody was reduced, a protein band having a molecular weight of 55 KDa and 25 KDa, which is known as the molecular weight of the heavy chain and the light chain of the antibody, was observed (FIG. 3).

J1J1  Identification of antibody's ability to inhibit cancer cell growth

The ability of the whole form of J1J1 purified in Example 3 to inhibit cancer cell growth was confirmed.

First, each cell of OVCAR3 (KCLB No. 30161) and SKOV3 (KCLB No. 30077) was plated to be 1 × 10 ³ per well of a 96 well plate. Then, a control antibody IgG (Thermo Cat No.31414) and a J1J1 antibody Was mixed with RPMI 1640 (WEL GENE Cat No. LM011-01) medium containing 1% FBS (HyClone Cat No. SH30919.03) at a concentration of 20 μg / ml and treated with 100 μl / well. Then, the cells were cultured in a 5% CO _{2 and} 37 ° C incubator. Cell viability was checked by MTT assay after 24, 48 and 72 hours of culture.

MTT assay was performed by dissolving Thiazolyl Blue Tetrazolium Bromide (MTT) in DPBS to a concentration of 0.5 mg / ml, and then culturing in RPMI 1640 (WEL GENE Cat No. LM011-01) medium containing 1% FBS (HyClone Cat No. SH30919.03) after a six-fold dilution was treated with 120㎕ / well to each well and then were incubated for 4 hours in an incubator of 5% CO _2, 37 ℃ condition, removing the medium, treated with DMSO of 100㎕ / well 562nm Lt; / RTI >

The cell survival rate after 24 hours, 48 hours, and 72 hours after treatment with J1J1 antibody was confirmed to be about 70% that of OVCAR3 cells compared to the control (Fig. 4). SKOV3 cells showed about 60% It was confirmed that the survival rate was decreased (Fig. 5), and it was confirmed that the growth of cancer cells was inhibited by the J1J1 antibody.

J1J1  Cancer cell metastasis of antibody ( migration ) And infiltration ( invasive ) Confirmation of ability inhibition effect

The ability of J1J1 antibody to metastasize and infiltrate cancer cells in vitro was examined. Transfection and infiltration were performed by diluting a 100-fold dilution of Matrigel (BD Science Cat No. 356234) in DPBS and coating it on the upper surface of Transwell (Corning costar Cat No. 3422) for 1 hour at room temperature to observe the infiltration phenomenon .

Each of the cells was coated with 2 × 10 ⁴ cells / well in a tranwell coated with OVCAR3 (KCLB No. 30161) and SKOV3 (KCLB No. 30077), and the concentration of the control antibody IgG (Thermo Cat No. 31414) and J1J1 antibody was 20 μg / ML and mixed with RPMI 1640 (WEL GENE Cat No. LM011-01) medium containing 1% FBS (HyClone Cat No. SH30919.03), and treated with 150 μl / well. After incubation for 4 hours in a 5% CO _{2 ,} 37 ° C incubator, the degree of metastasis and invasion of cancer cells was examined under a microscope.

J1J1 antibody inhibited the metastasis and infiltration of OVCAR3 (Fig. 6) and SKOV3 (Fig. 7) compared to the control group, indicating that the J1J1 antibody inhibited the metastasis and infiltration of the J1J1 antibody It was confirmed that the ability of metastasis and invasion of cancer cells was inhibited.

J1J1  By antibody notch  Signal change confirmation

6-1: notch  Of signal mRNA  Identification of expression changes

Each of OVCAR3 (KCLB No. 30161) and SKOV3 (KCLB No. 30077) cells were cultured in DMEM supplemented with 10% FBS (HyClone) (WEL GENE Cat No. LM011-01) medium containing 5% CO _{2 and} 37 ° C for 1 week. Then, each cell was plated in a 6-well plate (Coning Costar Cat No. CLS3516) at 2 × 10 ⁵ cells / well. Then, 1 μg / ml of a control antibody IgG (Thermo Cat No. 31414) and a J1J1 antibody concentration of 20 μg / And mixed with RPMI 1640 (WEL GENE Cat No. LM011-01) medium containing% FBS (HyClone Cat No. SH30919.03) and treated with 2 ml / well. The RNA was then extracts and incubated for 24 hours in an incubator of 5% CO _2, 37 ℃ conditions.

The RNA was extracted using the RIzol® RNA Isolation Reagents (Ambion Life technology Cat No. 15596018) and a protocol similar thereto. After extraction, 50 ng of DNA generated by reverse transcription using SuperScript First-Strand (Invitrogen Cat No. 11904-018) was used as a template strand and 2 μl of 2 pmol primer and aldAmp GT PCR Master Mix (TaKaRa Cat No. RR310A), followed by pretreatment at 94 ° C for 5 minutes, denaturing the DNA at 94 ° C for 30 seconds, and binding the primer at 55 ° C for 30 seconds. The process of synthesizing DNA at 72 ° C for 30 seconds was repeated 25 times. In the final step, the reaction was carried out at 72 ° C for 7 minutes to amplify the gene so that the enzyme exhibited sufficient activity.

Primers used for gene amplification were designed and synthesized to amplify each gene (Bioneer, Korea)

Human GAPDH primer

[SEQ ID NO: 20] 5'-GAGCTGAACGGGAAGCTCACTGG-3 '

[SEQ ID NO: 21] 5'-CAACTGTGAGGAGGGGAGATTCAG-3 '

Human β-actin primer

[SEQ ID NO: 22] 5'-GCGAGAAGATGACCCAGATCATGTT-3 '

[SEQ ID NO: 23] 5'-GCTTCTCCTTAATGTCACGCACGAT-3 '

Human Jagged-1 Primer

[SEQ ID NO: 24] 5'-CAACCGTGCCAGTGACTATTTCTGC-3 '

[SEQ ID NO: 25] 5'-TGTTCCCGTGAAGCCTTTGTTACAG-3 '

Human Hes1 primer

[SEQ ID NO: 26] 5'-TACCTCTCTCCTTGGTCCTGGAAC-3 '

[SEQ ID NO: 27] 5'-CAGATGCTGTCT TTGGTTTATCCG-3 '

Human NOTCH3 primer

[SEQ ID NO: 28] 5'-gttcatgcattgacctcgtg-3 '

[SEQ ID NO: 29] 5'-gtagccaggaagacactcac-3 '

Human NOTCH1 primer

[SEQ ID NO: 30] 5'-tccaccagtttgaatggtca-3 '

[SEQ ID NO: 31] 5'-ggacttgcccaggtcatcta-3 '

Human NICD3 primer

[SEQ ID NO: 32] 5'-AGCACTTTGGGAATTCTGGA-3 '

[SEQ ID NO: 33] 5'-ACGTCCTTGTGCAGTGAGAA-3 '

Human NICD1 primer

[SEQ ID NO: 34] 5'-cctgagggcttcaaagtgtc-3 '

[SEQ ID NO: 35] 5'-cggaacttcttggtctctagag-3 '

The amplified gene was subjected to electrophoresis (1.5% agarose gel, 100 V for 15 minutes) to examine the mRNA expression level of Notch 1, NICD 1, Notch 3, Jagged 1 and Hes 1 associated with Notch signal. As a result, J1J1 antibody was treated , MRNA expression of Notch 1, NICD 1, Notch 3, Jagged 1 and Hes 1 of OVCAR3 (FIG. 9) and SKOV3 (FIG. 10) was significantly decreased compared to the control group. . ≪ / RTI >

6-2: notch  Identification of protein expression changes in signal

To observe protein expression levels of Notch 1, NICD 1, Notch 3, Jagged 1 and Hes 1 associated with Notch signal, OVCAR3 (KCLB No. 30161) cells were cultured in DMEM supplemented with 10% FBS (HyClone Cat No. SH30919.03) And stabilized for 1 week in an incubator of 5% CO _{2 and} 37 ° C using RPMI 1640 (WEL GENE Cat No. LM011-01) medium. Then, each cell was plated in a 6-well plate (Coning Costar Cat No. CLS3516) at 2 × 10 ⁵ cells / well. Then, 1 μg / ml of a control antibody IgG (Thermo Cat No. 31414) and a J1J1 antibody concentration of 20 μg / And mixed with RPMI 1640 (WEL GENE Cat No. LM011-01) medium containing% FBS (HyClone Cat No. SH30919.03) and treated with 2 ml / well. After incubation for 24 hours in a 5% CO _{2 ,} 37 ° C incubator, proteins were extracted.

The proteins were mixed with Laemmli's Sample Buffer Reducing (GenDEPOT Cat No. L1100-011) at a ratio of 3: 1 and reacted at 95 ° C for 5 minutes and electrophoresed (10% acrylamide gel, 90V for 180 minutes). Then, it was transferred to a PVDF membrane (Roche Cat No. 03010040001) and Western blotted. The primary antibodies were notCH1 (Santacruz SC-37389), NOTCH3 (Santacruz SC-5596), Jaggeg1 (Santacruz SC-135955), Hes1 (Santacruz SC-166410), NICD1 (Cellsignaling 2421S) and GAPDH ) And goat anti-rabbit IgG-HRP (Santacruz SC-2004) and goat anti-mouse IgG-HRP (Santacruz SC-2005) were used as secondary antibodies.

Notch 1, NICD 1, Notch 3, Jagged 1, and Hes 1 in Notch 1, NICD 1, Notch 3, and Jagged 1 in OVCAR3 cells when treated with J1J1 antibody , It was confirmed that the expression of Hes 1 protein was greatly reduced (FIG. 11), confirming that the J1J1 antibody of the present invention inhibits the Notch signal.

in vivo  On J1J1  Antitumor effect of antibodies

The anticancer effect of J1J1 antibody in nude mice (5-week-old BALB / c-nu) transplanted with ovarian adenocarcinoma cells OVCAR-3 was confirmed.

The human cancer cells were OVCAR-3 cells, which were ovarian adenocarcinoma cells. Tumor cells were injected subcutaneously (sc 0) at the left side of the mouse at a cell concentration of 4 x 10 6 cells (DPBS) / 100 μl injected.

The J1J1 antibody was injected intravenously (I V. injection) and the injection of antibody was started from day 7 using 'day 1' after 24 hours from 'day 0' injection of tumor cells, and was performed with three schedules per week. Five mice were used as one group and the experiment was divided into two groups. Group 1 was injected with the FC group (control) (= 10 mg / kg) and Group 2 with J1J1 antibody at 100 μg / ea (= 10 mg / kg). The size of the tumor on the mouse was measured every 4 weeks using the formula below.

The tissues of mouse were then separated and fixed in 30% formalin, followed by 1 hour in 70% EtOH, 1 hour in 80% EtOH, 1 hour in 90% EtOH, 1 hour in 100% EtOH, 1 hour in 100% EtOH , EtOH 1: Xylene for 1 hour, Xylene for 1 hour, Paraffin 1: Xylene for 1 hour. After dehydration and transparency, the block was made with paraffin. Thereafter, it was separated into 10 mu m. Hematoxylin 10 min, running water 30 sec, ottix shaper 1 min, eosin 20 sec, ottix shaper 3 min, ottix plus 5 min, ottix plus 15 min, ottix shaper 3 min, runing water 5 min Min, and ottix plus 10 min. TUNEL staining was performed by in situ Cell Death Detection Kit, POD (Roche Cat No. 11684817910), and TUNEL staining was carried out with 10 mM citric acid at 370 W for 5 min.

As a result, it was confirmed through H & E staining that the nuclei of the cells were reduced in the group treated with the J1J1 antibody, and apoptisis was observed in the group treated with the J1J1 antibody through TUNEL staining (Fig. 12).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> korea research institute of bioscience and biotechnology <120> Human Monoclonal Antibody against Jagged 1 <130> P13-B155 <160> 35 <170> Kopatentin 2.0 <210> 1 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable light chain <400> 1 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ile Val Ser Ser Trp              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Ser Asn Phe Pro Val                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 2 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable heavy chain <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr              20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Arg Ile Ser Pro Ala Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Phe Cys Asp Asn Ser Arg Arg Ala Cys Tyr Ala Met Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser         115 120 125 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable light chain CDR1 <400> 3 Arg Ala Ser Gln Ile Val Ser Ser Trp Leu Ala   1 5 10 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable light chain CDR2 <400> 4 Arg Ala Ser Asn Leu Ala Ser   1 5 <210> 5 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable light chain CDR3 <400> 5 Gln Gln His Ser Asn Phe Pro Val Thr Phe Gly Gln Gly Thr Lys Val   1 5 10 15 Glu Ile Lys             <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable Heavy chain CDR1 <400> 6 Ser Tyr Trp Met Ser   1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable Heavy chain CDR2 <400> 7 Arg Ile Ser Pro Ala Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 8 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> J1J1 variable Heavy chain CDR3 <400> 8 Phe Cys Asp Asn Ser Arg Arg Ala Cys Tyr Ala Met Asp Tyr Trp Gly   1 5 10 15 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser              20 25 <210> 9 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> J1J1 variable light chain <400> 9 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtcggaga ccgcgtcacc 60 atcacttgcc gggcaagtca gattgtttct tcttggctgg cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatagg gcatccaact tggcaagtgg ggtcccatca 180 cgcttcagtg gcagtggatc tgggacggat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag cattccaact ttcctgtcac gttcggccaa 300 gggaccaaag tggagatcaa a 321 <210> 10 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> J1J1 variable heavy chain <400> 10 gaagtgcagc tggtggagtc tgggggaggc ttggttcagc ctggagggtc cctgcgtctc 60 tcctgtgcag cctctggatt caccttctct tcttactgga tgagctgggt ccgccaggcc 120 ccagggaagg ggctggagtg ggtttctagg atttctccgg ctggcggtta tactaactac 180 gcagactctg tgaagggccg tttcaccatc tcccgggaca actctaagaa cactctgtat 240 ctgcaaatga acagcctgcg tgccgaggac acggctgtct attactgtgc gagattctgc 300 gacaagaccc ggcgcgcctg ctatgcgatg gattattggg gccaaggcac cctggtcacc 360 gtctcctcgg ccagc 375 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Jagged 1 peptide <400> 11 Arg Cys Gly Pro Asp Cys Phe Asp Asn Tyr Gly Arg Tyr Lys Tyr Cys   1 5 10 15 Phe     <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> VL primer forward <400> 12 ttgtctggtg ttgaaggaga catccagatg acccag 36 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> VL primer reverse <400> 13 gcagccgccg tacgtttgat ctccactttg gtcccttggc c 41 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> VH primer forward <400> 14 actacaggtg tccactccga agtgcagctg gtggag 36 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> VH primer reverse <400> 15 caccggttcg gggaagt 17 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VL leader sequence primer forward <400> 16 tgcaaagctt cggcacgagc a 21 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> VL leader sequence primer reverse <400> 17 tccttcaaca ccagacaac 19 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> VH leader sequence primer forward <400> 18 gacgaattca ctctaaccat ggaa 24 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> VH leader sequence primer reverse <400> 19 ggagtggaca cctgtag 17 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Human GAPDH primer forward <400> 20 gagctgaacg ggaagctcac tgg 23 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human GAPDH primer reverse <400> 21 caactgtgag gaggggagat tcag 24 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human b-actin primer forward <400> 22 gcgagaagat gacccagatc atgtt 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human b-actin primer reverse <400> 23 gcttctcctt aatgtcacgc acgat 25 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human Jagged-1 primer forward <400> 24 caaccgtgcc agtgactatt tctgc 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human Jagged-1 primer reverse <400> 25 tgttcccgtg aagcctttgt tacag 25 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human Hes1 primer forward <400> 26 tacctctctc cttggtcctg gaac 24 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human Hes1 primer reverse <400> 27 cagatgctgt ctttggttta tccg 24 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NOTCH3 primer forward <400> 28 gttcatgcat tgacctcgtg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NOTCH3 primer reverse <400> 29 gtagccagga agacactcac 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NOTCH1 primer forward <400> 30 tccaccagtt tgaatggtca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NOTCH1 primer reverse <400> 31 ggacttgccc aggtcatcta 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NICD3 primer forward <400> 32 agcactttgg gaattctgga 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NICD3 primer reverse <400> 33 acgtccttgt gcagtgagaa 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NICD1 primer forward <400> 34 cctgagggct tcaaagtgtc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human NICD1 primer reverse <400> 35 cggaacttct tggtctccag 20

Claims (13)

A CDR1 region consisting of the amino acid sequence of SEQ ID NO: 3; a light chain variable region comprising the CDR3 region consisting of the amino acid sequence of SEQ ID NO: 5; and a CDR1 region consisting of the amino acid sequence of SEQ ID NO: A human monoclonal antibody against Jagged 1 comprising a heavy chain variable region comprising a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 7 and a CDR3 region consisting of the amino acid sequence of SEQ ID NO:
2. The human monoclonal antibody for Jagged 1 according to claim 1, which comprises a light chain variable region represented by the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 2.
A gene encoding a human monoclonal antibody against Jagged 1 of claim 1 or 2.
The gene according to claim 3, which comprises a light chain variable region represented by the nucleotide sequence of SEQ ID NO: 9 and a heavy chain variable region represented by the nucleotide sequence of SEQ ID NO: 10.
A recombinant vector containing the gene of claim 3.
A recombinant cell having the ability to produce human monoclonal antibody against Jagged 1, characterized in that the gene of claim 3 or a recombinant vector containing said gene is introduced into the host cell.
7. The recombinant cell of claim 6, wherein said host cell is HEK293.
A method for producing a human monoclonal antibody against Jagged 1 comprising the steps of:
(a) culturing a recombinant cell having the ability to produce human monoclonal antibodies against Jagged 1 of claim 7 to produce a human monoclonal antibody to Jagged 1; And
(b) recovering the human monoclonal antibody against the resulting Jagged 1.
The method according to claim 8, wherein the human monoclonal antibody to Jagged 1 is further purified using FPLC with a Protein A affinity column, SP-sepharose column and size exclusion chromatography, Lt; / RTI &gt; antibody.
A composition for treating cancer comprising a human monoclonal antibody against Jagged 1 of claim 1 or 2.
11. The composition according to claim 10, wherein the cancer is selected from the group consisting of cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer.
A cancer diagnostic composition comprising a human monoclonal antibody directed against Jagged 1 of claim 1 or 2.
13. The cancer diagnostic composition according to claim 12, wherein the cancer is selected from the group consisting of cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer.

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