KR101535219B1 - Human Monoclonal Antibody against Notch 3 - Google Patents

Abstract

The present invention relates to human monoclonal antibodies against Notch 3, and more particularly to human monoclonal antibodies capable of specifically targeting Notch 3 specifically overexpressed in cancer cells.
The Notch 3 human monoclonal antibody according to the present invention can be screened in a human antibody library through phage display so as to specifically target Notch 3 specifically overexpressed in cancer cells, Can be applied to humans immediately, and can be easily used as an antibody for cancer diagnosis and treatment.

Description

Human Monoclonal Antibody Against Notch 3 < RTI ID = 0.0 >

The present invention relates to human monoclonal antibodies against Notch 3, and more particularly to human monoclonal antibodies capable of specifically targeting Notch 3 specifically overexpressed in cancer cells.

Since the development of monoclonal antibody-producing techniques, attempts have been made to use them in the diagnosis and treatment of diseases. Accordingly, many products for diagnostic monoclonal antibodies have already been developed and marketed. However, in the case of monoclonal antibodies for therapeutic use, the commercialization is delayed due to various restriction conditions. 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 change it to 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 has been shown to induce tumorigenesis in several cancer models (Wang Z. et al., Anticancer Res. , 28: 3621, 2008; Santagata S. et al., Cancer Res. , 1; : 6854, 2004). T-cell leukemia / lymphomas are caused by expression of NICD, an active Notch, in rat hematopoietic cells, and T-cell acute lymphoblastic leukemia (T-ALL) 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.

Recently, a number of studies have reported on the cancer-causing action of Notch. Notch receptors, ligands and Notch targets have been reported to be active in cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer (Miele L. et al., Oncogene, Sep 1; 27: 5124, 2008) , Notch 1 and Jagged-1 are known to be associated with a poor prognosis in breast cancer patients (Reedijk M. et al., Cancer Res., 15; 65: 8530, 2005) and cancer metastasis in prostate cancer (Santagata S. et al., Cancer Res ., 64: 6854, 2004).

Notch signaling is a focus of attention in cancer research because it can be a major target for new chemotherapy. First, the Notch signaling system, which is initiated by Notch / ligand binding, does not require enzymatic signal amplification, such as kinase, and can regulate signal intensity relatively accurately. As a result, the Notch target genes can be restricted in a concentration-dependent manner. Second, the active Notch half-life is short. This means that sustained signaling inhibition may not be necessary and that adequate inhibition of the intermediate intensity may limit the Notch signaling pathway. Third, Notch signaling is different depending on tissues and organs. Therefore, the Notch signal can be used for other purposes depending on the cell characteristics.

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 (Ridgway J. et al., Nature, 444: 1083, 2006), many monoclonal antibodies are currently under development that can interfere with Notch signaling (Yan M. et al. Nature , 464: 1052, 2010).

Accordingly, the present inventors have made intensive efforts to provide a Notch monoclonal human antibody that can be a main target of new chemotherapy. As a result, the Notch 3 human monoclonal antibody N37 has been reported to be specific for Notch 3, which is overexpressed in various kinds of cancer cells And the present invention has been completed.

It is an object of the present invention to provide a human monoclonal antibody that can specifically target Notch 3 that is specifically overexpressed in cancer cells.

Another object of the present invention is to provide a cancer diagnostic composition comprising a human monoclonal antibody that specifically binds to Notch3.

It is still another object of the present invention to provide a composition for treating cancer comprising a human monoclonal antibody that specifically binds to Notch3.

In order to achieve the above object, the present invention provides a human monoclonal antibody against Notch 3 containing the light chain variable region represented by SEQ ID NO: 1 and the heavy chain variable region represented by SEQ ID NO: 2.

In addition, the present invention provides a composition for cancer diagnosis comprising the human monoclonal antibody against Notch 3.

In addition, the present invention provides a composition for treating cancer comprising a human monoclonal antibody against Notch3.

The Notch 3 human monoclonal antibody according to the present invention can be screened in a human antibody library through phage display so as to specifically target Notch 3 specifically overexpressed in cancer cells, Can be applied to humans immediately, and can be easily used as an antibody for cancer diagnosis and treatment.

Fig. 1 shows the results of measuring the purity of N37 after conversion of N37 in the form of Fab to whole Ig.
FIG. 2 shows the results of analysis of the reaction of the N37 antibody with the Notch 3 domain 20-22.
FIG. 3 shows the result of FACS analysis of the specific binding of human monoclonal antibody N37 to cells expressing L1CAM.

In one aspect, the present invention relates to a human monoclonal antibody against Notch 3 comprising the light chain variable region represented by SEQ ID NO: 1 and the heavy chain variable region represented by SEQ ID NO: 2.

In the present invention, the human monoclonal antibody to Notch 3 comprises (a) a step of preparing a Notch 3 antigen protein; (b) selecting the human antibody that binds to Notch3; (c) analyzing the nucleotide sequence and amino acid sequence of human antibody, N37, which binds to Notch3; (d) conversion and production of N37 to whole IgG; (e) Preparation of human monoclonal antibody N37 of Notch 3 through the step of analyzing the binding ability to Notch 3 expressed on the cancer cell surface of the N37 antibody and confirming the binding ability to Notch 3.

In one embodiment of the present invention, the sequence of the domain 20-22 (amino acid sequence 771-885) of Notch 3 is amplified by a polymerase chain reaction (PCR), and the leader sequence is linked to Fc Notch 3 antigen was prepared.

In the present invention, the term "antibody" means 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.

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 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 just 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. Na ㅿ and library do not have high affinity of separated antibody, but they are not expensive to manufacture, and the synthetic library has a high affinity for the separated antibody, but it 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.

In another embodiment of the present invention, the antigen of Notch 3 was reacted with a phage displaying a human antibody library for 1 hour, 10 times in the case of the first panning, 20 times in the case of the second panning, 30 times in the case of the third panning Respectively. After washing, the phage-antibodies bound to the antigen were separated and then infected with TG1 E. coli and cultured overnight. 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, 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 phages displayed with the antibodies. Car panning was carried out. 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 first, second and third panning of the library was measured by an indirect ELISA method, After that, it was confirmed that the clones having binding ability to the Notch 3 domain 20-22 were amplified. After the plasmid DNAs were isolated, the nucleotide sequences were analyzed.

As a result of comparing the base sequence of the Notch 3 antibody of the present invention with the human germline base sequence, the light chain variable region was derived from the human antibody point line Kappa group IGKV1-5 * 03 (93.55% identical) (SEQ ID NO: 1) and the heavy chain variable region of the N37 antibody was analyzed to be derived from the heavy chain group IGHV1-46 * 01 (94.10% identical) of the human antibody gland line (SEQ ID NO: 2) Were not 100% identical to any of the heavy and light chain sequences in the IMGT data base (IMGT data base), confirming that N37 is a new antibody that has undergone maturation in the human body, rather than a pointline antibody.

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 embodiment of the present invention, the heavy chain variable region and the light chain variable region were amplified by polymerase chain reaction from N37 in order to convert the Fab form of N37 into whole Ig form, and the N37 antibody was purified by transforming into HEK293T cells, 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 light chain of the antibody, was observed (Fig. 1). The binding of Notch 3 domain 20-22 Fc protein to N37 antibody was confirmed (Figure 2).

In another embodiment of the present invention, to test whether the N37 antibody, a human antibody to Notch3, is able to recognize Norch3 present on the surface of cells expressing Notch3, a human cervical cancer cell line OVCAR3, SKOV3 MDH2774 and human breast cancer cell lines MDAMB231 and T47D showed that N37 binds to human cancer cell lines expressing Notch 3 (OVCAR3, T47D), and cells not expressing Notch 3 (2774) or Notch 3 (Fig. 3). Fig. 3 shows the results of immunohistochemical staining for MDAMB231 and SKOV3 cells.

In another aspect, the present invention relates to a composition for cancer diagnosis comprising a human monoclonal antibody against Notch 3, which comprises a light chain variable region represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2.

In the present invention, the cancer that can be diagnosed by detecting the expression of Notch 3 includes cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, prostate cancer and the like.

The diagnostic composition of the present invention comprises a human monoclonal antibody against Notch 3 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.

In another aspect, the present invention relates to a composition for treating cancer comprising a human monoclonal antibody against Notch 3 containing a light chain variable region represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2.

The administration of human monoclonal antibodies to Notch 3 according to the invention consists of a variety of standard procedures. For a more detailed description of this technique, see Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988). With antibodies, it is desirable to administer pharmaceutically acceptable adjuvants such as complete or non-complete Freund's adjuvant, RIBI (muramyl dipeptides) and ISCOM (immunostimulating complexes). Preferably, the composition may comprise a water-in-oil emulsion or aluminum hydroxide as an adjuvant.

The antibody composition is administered to the patient or treating animal one or more times in a treatment series. The most effective mode and dosage regimen upon administration will depend on the level of immunology, the particular agent and / or adjuvant used in the treatment, the extent and course of the anticipated infection, the previous treatment, the health status of the patient or animal, And response to immunological treatment. For example, it depends on the immune component patient, the most highly immunogenic polypeptide, the smaller dose and the number of immune measures needed. Similarly, the dosage and treatment time will be lowered when the antibody is administered as an adjuvant.

The human monoclonal antibody against Notch 3 containing the light chain variable region represented by SEQ ID NO: 1 and the heavy chain variable region represented by SEQ ID NO: 2 of the present invention can prevent or prevent various diseases caused by viruses, bacteria and parasites It will be appreciated by those skilled in the art that many animal models will be used. Any animal susceptible to germs will be used. Balb / c mice are suitable animal models for activated immunosurveillance screens, and immunodeficient mice are also desirable animal models for passive screens. Thus, by administering a particular polypeptide or antibody to this animal model, one of ordinary skill in the art can determine whether the polypeptide or antibody is useful for the methods and compositions described herein without undue experimentation.

It will be understood that it can change. The therapeutic composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

Injection of the therapeutic composition according to the present invention can be injected into muscle cells or other cells in muscle tissue and injected into visceral cells in the abdominal cavity.

In a preferred embodiment, the therapeutic composition can be prepared by mixing the active ingredient together with solid excipient and can be prepared in the form of granules for preparation in tablets or dragee forms. Suitable excipients include sugars such as lactose, sucrose, mannitol and sorbitol or starches from corn, wheat flour, rice, potato or other plants, cellulose such as methylcellulose, hydrocyclopropylmethylcellulose or sodium carboxymethylcellulose , Arabic gum, carbohydrates such as gum including Tagakans gum, or protein fillers such as gelatin and collagen. If necessary, disintegrating or solubilizing agents in the form of respective salts such as cross-linked polyvinylpyrrolidone, agar and alginic acid or sodium alginic acid may be added.

In a preferred embodiment, for parenteral administration, the therapeutic composition of the present invention can be prepared as a water-soluble solution. Preferably, physically suitable buffer solutions such as Hank's solution, Ringer's solution or physically buffered saline can be used. Water-soluble injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In addition, suspensions of the active ingredient may be prepared in suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension may employ a suitable stabilizing agent or agent to increase the solubility of the compound and to produce a high concentration of solution.

The monoclonal antibody in the therapeutic composition is adsorbed on a glass container such as a vial and a syringe, is unstable, and easily deactivated by various physicochemical factors such as heat, pH and humidity. Therefore, a stabilizer, a pH adjuster, a buffer, a solubilizing agent, a surfactant, and the like are added for formulation in a stable form. Examples of the stabilizer include amino acids such as glycine and alanine, sugars such as dextran 40 and mannose, sugar alcohols such as sorbitol, mannitol, xylitol, etc., and two or more of these may be used in combination. The amount of these stabilizers to be added is preferably 0.01 to 100 times, more preferably 0.1 to 10 times, the weight of the antibody. By adding these stabilizers, the storage stability of the liquid preparation or freeze-dried preparation can be improved. Examples of the buffer include phosphate buffer, citrate buffer, and the like. The buffer adjusts the pH of the aqueous solution after the redissolution of the liquid preparation or lyophilized preparation, and contributes to the stability and solubility of the antibody. The amount of the buffering agent to be added is, for example, 1 to 10 mM relative to the amount after the redispersion of the liquid formulation or the lyophilized formulation. As the surfactant, polysorbate 20, pullulanic F-68, polyethylene glycol and the like can be used, and polysulfate 80 is particularly preferable, and two or more kinds of these may be used in combination.

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

Example 1: Preparation of Notch 3 antigen

From the pCMV-tag4-N3FL containing the sequence of the domain 20-22 (771-885) of Notch 3, a polymerase chain reaction (PCR) was performed using the domain amino terminal primer SEQ ID NO: 3 and the domain 22 carboxy terminal primer SEQ ID NO: chain reaction, PCR). PCR was performed by first mixing 50 ng of DNA template, 12.5 pmol of sense primer, antisense primer, 10 mM dNTP and DNA polymerase pfu (Solgent, Korea) in a DNA polymerase buffer solution (Solgent, Korea) Pretreatment reaction was carried out. Next, the DNA is denatured at 95 DEG C for 45 seconds, and the primer is bound at 58 DEG C for 45 seconds. The process of synthesizing the DNA at 72 ° C for 2 minutes was repeated 30 times, and at the final step, the reaction was allowed to proceed at 72 ° C for 10 minutes so that the enzyme was fully activated. The DNA fragment thus obtained was ligated with pJK-dhfr2-L1-monomer (Korean Patent Application No. 10-2006-0079969) containing the leader sequence using the domain amino terminal primers SEQ ID NO: 5 and SEQ ID NO: 6, Polymerase chain reaction (PCR) was performed by ligating the obtained DNA fragment with the polymerase chain reaction (PCR). Amino-terminal primer SEQ ID NO: 5 and carboxy-terminal primer SEQ ID NO: 4 were used. After the treatment with EcoRI and XhoI, it was cloned between the EcoRI and XhoI sites of pJK-dhfr2-Fc. The plasmid thus prepared was named pJK-dhfr2-Notch 3 (20-22) Fc.

The expression plasmid pJK-dhfr2-Notch3 (21-22) F containing the cDNA encoding the sequence of Notch 3 domain 20-22 (771-885) was digested with DMEM (Hyclone, USA) containing 10% fetal bovine serum HEK293T cells were transfected with lipofectamine 2000 (Invitrogene, USA) and cultured in a 5% CO 2 incubator at 37 ° C for 4-6 hours. Then, serum-free CD293 (Gibco, USA) medium. Subsequently, the culture medium was changed into a new medium every 3 days while being cultured in a thermostat at 37 ° C and 5% CO 2, and the supernatant was collected three times. The culture supernatant was subjected to affinity chromatography using Protein G column (Upstate, USA) The protein was purified.

SEQ ID NO: 3: 5'-GACGAATTGCTACTCCTCTCCCCC-3 '

SEQ ID NO: 4: 5'-AGTCTAGACTCGAGGGCGCATCGTGGGCC-3 '

SEQ ID NO: 5: 5'-GAGACCCAAAGCTTATC-3 '

SEQ ID NO: 6: 5'-TAGCAATTCGTCTGG-3 '

Example 2. Screening of Human Antibodies Binding to Notch 3

Human antibody libraries were constructed by two-step cloning. In the first step, the pKRIBB-Fab phagemid vector was digested with restriction enzyme BstXI (Roche, Switzerland), and light chain genes were cloned in situ to prepare a light chain library. As a second step, the light chain library was digested with restriction enzymes SfiI , Switzerland) and cloned heavy chain genes in place.

The pKRIBB-Fab phagemid vector was digested with restriction enzyme BstXI and separated and purified using 0.6% agarose gel (Cambrex) in order to construct a light chain library as the first step, and self- was tested this time was used as a vector only if 1 x 10 ^5/1 ㎍ DNA or less.

Using the purified vector and purified light chain genes at a molar ratio of 2, 20 μl of 10 × ligase buffer, 10 μl of T4 DNA ligase and sterile distilled water were added to make 200 μl Then, the reaction was allowed to proceed 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 Ω, and 25 ㎌D. 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 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, incubation at 37 ° C overnight. A library of 2.3 x 10 ⁸ cfu for Vκ and a library of 2.8 x 10 ⁸ cfu for Vλ were obtained.

In the second step, the light chain library was digested with restriction enzyme BstXI to isolate and purify using 0.6% agarose gel, and subjected to a self-binding test. At this time, if 1 x 10 ^5/1 ㎍ DNA only or less was used as a vector.

Ligation and E. coli transformation were carried out in the same manner as in the production of light chain library. The above experiment was repeated 60 times or more to construct a large human antibody library containing 5 x 10 ¹⁰ cfu of different antibodies.

In a human Fab antibody library, a kappa library and a lambda library of the light chain portion were mixed in a ratio of 6: 4, and 1.7 x 10 < ¹¹ > clones were mixed with 2YT medium containing ampicillin (100 쨉 g / 16 g of yeast extract, 10 g of yeast extract, 5 g of NaCl, and 1 liter of H2O, pH 7.0) and incubated at 37 ° C with shaking at OD 600 = 0.7-0.8 (about 2 hours) Was added 20 times and kanamycin (70 ㎍ / ml) was added and grown at 30 ° C overnight. The supernatant was centrifuged at 5,000 rpm for 15 minutes using a supra22K centrifuge (No. 11 rotor) on the next day. NaCl and PEG8000 were added to final concentrations of 500 mM and 5 mM, respectively, and left in ice for 2 hours. Next, centrifugation was performed using the same centrifuge (No. 9 rotor) at 10,000 rpm for 1 hour to precipitate the phage and then dissolved in 5 ml of PBS. The supernatant was centrifuged at 12,000 rpm for 10 min using a Legend Micro 17 (Sorvall, USA) ultracentrifuge and filtered with a 0.2 μm disposable filter (Sartorius stedim biotech, Switzerland). The number of phages displayed on this occasion was 7.2 x 10 < ^{13 &} gt ;.

100 μg of Notch 3 (20-22) -Fc was mixed with 1 ml of a coating buffer (15 mM Na ₂ CO ₃ , 34.84 mM NaHCO ₃ , pH 9.6) and placed in an immune tube (Maxisorp Immunotube, Nunc) Coated, washed twice with PBS solution (PBST) supplemented with 0.05% Tween 20, and blocked with 4% skim milk for 1 hour at room temperature. After washing again twice, phage (1.6 x 10 < ¹³ >) displaying the antigen and human antibody library prepared in Example 1 were mixed in a final concentration of 2% skim milk and placed in a 1 ml immune tube and incubated at 37 DEG C for 1 hour And reacted with the antigen. Next, the cells were washed 10 times for the first panning, 20 times for the second panning, and 30 times for the third panning with PBS supplemented with 0.5% Tween 20. For 1 wash, 1 ml of wash solution was added, Using a pipetman, the solution is pipetted up and down 10 times, then 4 ml of washing solution is added, and the solution is allowed to stand for 5 minutes. After washing, the phage-antibodies bound to the antigen were separated using 1 ml of 0.1 M glycine (pH 2.5), neutralized by adding 60 μl of 2 M Tris HCl pH 9.0, and immediately diluted with TG1 Escherichia coli (2 YTA) solid medium containing ampicillin (100 μg / ml) and cultured at 37 ° C. overnight. 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. 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 phages displayed with antibodies. Second and third panning were performed on the same amount of antigens. The colonies selected after the library and primary, secondary, and tertiary panning were collected and incubated at 37 ° C with an OD 600 of 0.8, and then IPTG was added to a final concentration of 1 mM, and expressed at 30 ° C overnight. Then, the antigen binding ability of antibodies present in the supernatant was measured by an indirect ELISA method.

Indirect ELISA was performed in the same manner as in Example 1 except that the Notch 3 domain 20-22 (100 ng / well) prepared in Example 1 was coated on a 96-well plate (MaxiSorp, Nunc) and 100 μl of the culture supernatant was added to each well. Lt; / RTI > After washing three times with PBST, 100 μl of AP2 (1/2000), which is a mouse antibody specifically binding to the light chain-linked preS1 tag, was added thereto, followed by washing with PBST four times. Anti-mouse IgG (Fc- -HRP conjugate (Pierce, 1/2000) was added and reacted at room temperature for 1 hour. After washing with PBST 5 times, 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 2M H 2 SO 4 was added to stop the reaction. Absorbance was read at 450 nm using an absorbance analyzer (VERSA max microplate reader) to measure antibody expression.

As a result, clones having binding ability to Notch 3 domain 20-22 were amplified after the third panning. After the third round of panning, the eluted phages were diluted and infected with TG1 E. coli, and then plated on 2 YTA solid medium and incubated at 37 ° C in a thermostat. 100 colonies were randomly selected and inoculated into 3 ml of 2 YTA liquid medium, respectively, and incubated at 37 ° C in a shaking incubator until the absorbance of each clone reached between 0.7 and 0.8 at 600 nm. Next, IPTG was added to a final concentration of 1 mM and incubated overnight at 30 ° C with shaking. The culture supernatant was recovered and indirect ELISA was performed to detect antibodies recognizing Notch 3 domain 20-22.

The clone binding to the Notch 3 domain 20-22 was selected and named N37. As a result, the light chain variable region sequence was represented by SEQ ID NO: 1, the heavy chain variable region sequence was represented by SEQ ID NO: 2, the plasmid DNA was isolated, Respectively.

Example 3. Nucleotide Sequence and Amino Acid Sequence Analysis of N37 Antibody

As a result of comparing the nucleotide sequence of Example 3 with a human germline sequence using IMGT software (http://imgt.cines.fr/ IMGT_vquest / share / textes /), the light chain variable region was found to contain a human antibody The heavy chain variable region of the N37 antibody was analyzed to be derived from the heavy chain group IGHV1-46 * 01 (94.10%) of the human antibody point line (germline) The same). The heavy and light chain variable region sequences of N37 were not 100% identical to any heavy and light chain sequences in the IMGT data base. This demonstrates that N37 is not a pointline antibody but a new antibody that has undergone maturation in the human body.

Example 4. Conversion and production of N37 into whole IgG

The heavy chain variable region and the light chain variable region were amplified by polymerase chain reaction from N37 and separated through 1.5% agarose gel (Cambrex) to convert Fab form N37 to whole Ig form. Then, Zymo gel extraction kit (Zymoresearch, USA). The primers used herein were SEQ ID NO: 7 and SEQ ID NO: 8 for the light chain variable region and SEQ ID NO: 7 and SEQ ID NO: 9 for the heavy chain variable region.

SEQ ID NO: 7:

5'-ACTACAGGTGTCCACTCCCAGGTGCAGCTGGTGGAGTCT-3 '

SEQ ID NO: 8:

5'-GCAGCCGCCGTACGTTTGATCTCCACTTTGGTCCCTTGGCC-3 '

SEQ ID NO: 9: 5'-CCGATGGGCCCTTGGTGGAGGCGCTGGCCGAGGA-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: 10 and SEQ ID NO: 11 for the heavy chain leader sequence and SEQ ID NO: 12 and SEQ ID NO: 13 for the light 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. The primers used for linking the heavy chain variable region to the leader peptide of the heavy chain were SEQ ID NO: 10 and SEQ ID NO: 9, and the primers used for linking the light chain variable region to the light chain leader sequence were SEQ ID NO: 13 and SEQ ID NO: 14.

SEQ ID NO: 10: 5'-GACGAATTCACTCTAACCATGGAA-3 '

SEQ ID NO: 11: 5'-GGAGTGGACACCTGTAG-3 '

SEQ ID NO: 12: 5'-TCCTTCAACACCAGACAAC-3 '

SEQ ID NO: 13: 5'-TGCAAAGCTTCGGCACGAGCA-3 '

SEQ ID NO: 14: 5'-TGCAGCCACCGTACGTTTGATCTCCAGCTTGGT-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-N37.

The pdCMV-dhfrC-N37 DNA was transformed into HEK293T cells using Lipofectamine 2000 (Invitrogen, USA). After incubation for 4-6 hours in a 37 ° C 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 produced by N37. The N37 antibody was purified from the supernatant obtained above via protein G agarose column (Upstate, USA). As a result, when the 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. 1).

The expressed Notch 3 domain 20-22 Fc protein was subjected to 8% SDS-PAGE (polyacrylamide gel electrophoresis), transferred to a PVDF membrane (Millipore, Korea) and Western blotted. At this time, N37 antibody was used as a primary antibody and anti hIgG F (ab ') ₂ -HRP (Pierce, 1/5000) was used as a secondary antibody and detected with ECL reagent (Amersham Biosciences). As a result, the N37 antibody bound to the Notch 3 domain 20-22 Fc (FIG. 2).

Example 5. Analysis of binding ability of N37 antibody to Notch 3 expressed on the surface of cancer cells

In order to test whether the N37 antibody recognizes Norch3 present on the surface of cells expressing Notch 3, the ability to bind to human cervical cancer cell lines OVCAR3, SKOV3 MDH2774, human breast cancer cell lines MDAMB231 and T47D using a flow cytometer (FACS caliber) Respectively. Specifically, the cells were treated with 4 ml of cell isolation buffer (Gibco), treated at 37 ° C for 5 minutes, and then the separation buffer solution was removed using a centrifugal separator. PBS (1% BSA-PBS) The cells were suspended in 1 ml. 100 μl of the solution is placed in a 96-well plate, and 2 μg / well of purified and isolated N37 is added thereto, followed by reaction at 4 ° C. for 1 hour. Centrifuge at 1000 rpm for 3 minutes to remove the reaction solution and wash 3 times with 1% BSA-PBS. 100 μl of 1% BSA-PBS was added to suspend the cells, and the cells were suspended in 1% BSA-PBS so as to be 0.5 μg / well of anti-human Fc-FITC (Sigma) as a detection antibody and 100 μl of each was added thereto at 4 ° C. for 1 hour . After washing 3 times with 1% BSA-PBS, the cells were suspended in 500 μl 1% BSA-PBS and transferred to a tube for FACS. PI (propidium iodide, Sigma, USA) was diluted to 1/10000, reacted at room temperature for 5 minutes, and FACS was performed.

As shown in FIG. 3, N37 binds to human cancer cell lines (OVCAR3, T47D) expressing Notch 3, and MDMAB231 and SKOV3 cells that do not express Notch 3 and 27 not expressing Notch 3 Did not join.

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 and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Human Monoclonal Antibody against Notch 3 <160> 14 <170> Kopatentin 1.71 <210> 1 <211> 121 <212> PRT <213> Homo sapiens <400> 1 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser His              20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Leu Ile Asn Pro Asn Gly Gly Asp Thr Arg Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Val Ala Met Thr Arg Asp Thr Ser Thr Thr Thr Val Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Asp Leu Leu Val Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly             100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser         115 120 <210> 2 <211> 107 <212> PRT <213> Homo sapiens <400> 2 Asp Ile Gln Leu Thr Gln Ser Ser Ser Thr Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Lys Ser Trp              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn Ser Phe Pro Leu                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gacgaattgc tactcctctc cccc 24 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agtctagact cgagggcgca tcgtgggcc 29 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gagacccaaa gcttatc 17 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tagcaattcg tctgg 15 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Antiboy of Notch3 <400> 7 actacaggtg tccactccca ggtgcagctg gtggagtct 39 <210> 8 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gcagccgccg tacgtttgat ctccactttg gtcccttggc c 41 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ccgatgggcc cttggtggag gcgctggccg agga 34 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gacgaattca ctctaaccat ggaa 24 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggagtggaca cctgtag 17 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tccttcaaca ccagacaac 19 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tgcaaagctt cggcacgagc a 21 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tgcagccacc gtacgtttga tctccagctt ggt 33

Claims (5)

A human monoclonal antibody against Notch 3 containing a light chain variable region represented by SEQ ID NO: 1 and a heavy chain variable region represented by SEQ ID NO: 2.
A cancer diagnostic composition comprising a human monoclonal antibody against Notch 3 of claim 1.
The cancer diagnostic composition according to claim 2, wherein the cancer is selected from the group consisting of cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer.
A composition for treating cancer comprising a human monoclonal antibody against Notch 3 of claim 1.
5. The composition according to claim 4, 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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