KR102014383B1 - Human antibodies regulating functions of a G-protein coupled receptor - Google Patents

Abstract

The present invention relates to a monoclonal antibody or fragment thereof that recognizes and specifically binds an extracellular site of entotheliner receptor A as an antigen. The present invention also relates to a screening method for an antibody or fragment thereof that specifically recognizes an extracellular site of GPCR as an antigen. The screening method of the present invention can be used for antibody detection specifically binding to the extracellular site of GPCR, and the antibody discovered through this method can be usefully used as a therapeutic agent for hypertension or cancer associated with entotelin receptor A. .

Description

Human antibodies regulating functions of a G-protein coupled receptor

The present invention relates to an antibody having a signaling inhibitory activity of human GPCR and a method for screening an extracellular-specific antibody of GPCR. More specifically, the antibody of the present invention inhibits the signaling function of ET _A to inhibit cancer disease or It can be used for the treatment of hypertension.

GPCR (G-protein coupled receptor) is a multi-membrane-permeable protein with seven membrane permeable sites. It delivers extracellular signals into cells to activate G-protein heterotrimers, resulting in physiology as well as responses to signal transduction agents. Plays an essential role in awards. In addition, GPCR not only directly regulates major biological phenomena such as cell growth, proliferation, migration, grouping and death, but also directly related to diseases such as cancer and cardiovascular diseases. I am getting it.

Endothelial receptor type _A (ET _A ) is mainly expressed in vascular smooth muscle in normal individuals and is known to cause vasoconstriction through intracellular signal transduction by binding of the ligand ET-1 (endothelin-1). Thus, drugs such as bosentan, an endothelin receptor antagonist, have been used to treat hypertension (Nature reviews drug discovery, 2011, Vol. 10, 47). Recently, many basic biological and clinical studies have shown that ET _A is overexpressed in various cancers such as bladder cancer, lung cancer, ovarian cancer, kidney cancer, colon cancer, prostate cancer, breast cancer, uterine cancer, rhabdomyosarcoma and glioblastoma. It is reported to lower the survival rate of patients by being directly involved in major cancer processes such as migration, invasion, metastasis, and angiogenesis, etc. (Nature review cancer, 2013, Vol. 13, 637).

Antibody drugs target ET _A in spite of advantages such as high specificity, low side effects, high blood half-life, and mechanisms of apoptosis (ADCC, ADCP, CDC) due to Fc region compared to low molecular weight synthetic drugs. Antibody drugs have not been reported as low-molecular compounds such as, Zibotentan, Atrasentan, etc., and the only approved antibody drug for sale even when expanded to GPCR is Kyowa Hakko Kirin's Poteligeo (Mogamulizumab) targeting CCR4. Therefore, there is a need for the discovery of novel antibodies that specifically bind to ET _A and regulate its activity. In addition, in order to develop novel antibody drugs with GPCR targets, it is necessary to specifically bind to the extracellular domain (ECD) of the antigen. The proportion of the extracellular portion of the total surface area of the GPCR is about 10%, which is very limited. Because of this, there are limitations to existing antibody discovery strategies.

The matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and should not be taken as acknowledging that they correspond to the related art already known to those skilled in the art.

The present inventors made a diligent effort to discover novel antibodies that specifically bind to GPCR and inhibit signal transduction. As a result, we devised the MSP lipid bilayer of the nano disc and utilizing GPCR extracellular above specific antibody screening strategies utilizing protein, the ET-1 signaling inhibited mechanism in which the ET _A stabilized by using this expression cell line The present invention was completed by confirming.

Accordingly, an object of the present invention is to provide a method for screening an antibody or fragment thereof that specifically recognizes an extracellular domain of GPCR (G-protein coupled receptor) as an antigen.

Another object of the present invention is to provide a monoclonal antibody or fragment thereof that recognizes and binds specifically to an extracellular domain of an endothelin receptor type A.

Still another object of the present invention is to provide a nucleic acid molecule encoding the monoclonal antibody or fragment thereof.

Still another object of the present invention is to provide a vector containing the nucleic acid molecule.

Another object of the present invention to provide a host cell comprising the vector.

Still another object of the present invention is to provide a composition comprising the monoclonal antibody, nucleic acid molecule or vector.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a method for screening an antibody or fragment thereof that specifically recognizes an extracellular domain of GPCR (G-protein coupled receptor) as an antigen.

The present inventors have tried to find a novel antibody that specifically binds to the GPCR and inhibits the signal transduction process. As a result, the directionality is regulated using the binding partner G-protein of the nanodisk and GPCR, which is a lipid double layer of the disk type using MSP protein. It was confirmed that the screened antibody effectively inhibited the signaling function of GPCR through the biopanning method using the extracellular site of the GPCR as an antigen.

G-protein coupled receptors (GPCRs) form the largest family of membrane proteins in the human genome (more than 800), and seven transmembrane helices form extracellular loops (ECL) and It is connected to intracellular loops (ICL). GPCR activates G-protein heterotrimers by transmitting extracellular signals into cells, and plays an essential role in physiology such as sensation as well as response to signaling agents. In addition, GPCR not only directly regulates major biological phenomena such as cell growth, proliferation, migration, grouping and death, but also directly related to diseases such as cancer and cardiovascular diseases. I am getting it. The extracellular domain of the GPCR (ECD) contains three ECLs and N-terms, and the intracellular domain (ICD) contains three ICLs and C-terms (M Wheatley et al., Br J Pharmacol. 2012 Mar; 165 (6): 16881703).

In accordance with a preferred embodiment of the invention, the antibody or fragment thereof of the invention specifically binds to an extracellular site other than the intracellular site of the GPCR as an antigen (e.g., an epitope).

According to a preferred embodiment of the present invention, the screening method of the present invention comprises the following steps:

(a) preparing a nanodisk by mixing a membrane scaffold protein (MSP), a lipid, and a GPCR;

(b) coupling magnetic beads to the nanodisks;

(c) exposing an extracellular site by binding an intracellular domain of GPCR included in the nanodisk to a plate on which G-protein is fixed; And

d) screening an antibody or fragment thereof that specifically binds the extracellular site as an antigen.

Nanodisks are formed by non-covalent assembly of lipids and the membrane scaffold protein (MSP). MSP may be MSP1 or MSP2.

The present invention provides a method for discovering extracellular specific antibodies in the development of GPCR specific antibody drugs. According to an embodiment of the present invention, the screening method of the present invention uses a binding partner G-protein of a nanodisk, which is a disk-type lipid bilayer using the MSP1 protein illustrated in Examples 1 to 3 and 3, and a GPCR used. It may be a biopanning method using an extracellular site of GPCR whose direction has been adjusted. The embodiment to over-expression in E. coli as a scAb form schematized in Figure 4 combines the Cκ a dig antibody according to the example in FIG. Purification by chromatography affinity cell in ET _A expression mammalian cells (ET _A expressed CHO-K1 stable cell line) As a result of measuring the function regulating ability by external gastric binding, it was confirmed that the inhibitory activity K _i exhibited excellent function regulating ability at 56 nM even under a natural ligand of 10 nM.

The screening methods of the present invention can use fluorescence labeled cell separation (FACS) screening, or other automated flow cytometry techniques. Instruments for performing flow cytometry are known to those skilled in the art. Examples of such devices are FACSAria, FACS Star Plus, FACScan and FACSort devices (Becton Dickinson, Foster City, CA), Epics C (Coulter Epics Division, Hialeah, FL), MOFLO (Cytomation, Colorado Springs, Colo.), MOFLO- XDP (Beckman Coulter, Indianapolis, IN). Flow cytometry techniques generally include the separation of cells or other particles in a liquid sample. Typically the purpose of a flow cytometer is to analyze the separated particles for their one or more properties (eg the presence of labeled ligands or other molecules). Particles are passed one by one by the sensor and are classified based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, and the like.

According to another aspect of the present invention, the present invention provides a monoclonal antibody or fragment thereof that recognizes and specifically binds an extracellular domain of an endothelin receptor type A as an antigen. .

The entotellin receptor A is responsible for intracellular signal transduction by the binding of the ligand ET-1 (endothelin-1), and has attracted much attention as a drug for treating hypertension and a major anticancer target, but has previously been used to target ET _A. These are low-molecular compounds such as Bosentan, Zibotentan, Atrasentan and the like, and no monoclonal antibody drug that recognizes the extracellular site of Entotheliner Receptor A as an antigen has been reported. The entotellin receptor A preferably comprises the first sequence of SEQ ID NO.

According to a preferred embodiment of the present invention, the extracellular region consists of an N-term region of SEQ ID NO: 2, ECL 1 of SEQ ID NO: 3, ECL 2 of SEQ ID NO: 4, and ECL 3 of SEQ ID NO: 5 It is one or more sites selected from the group.

As used herein, the term “epitope” refers to a localized site of an antigen to which an antibody or fragment thereof can specifically bind. For example, a contiguous amino acid in an antigen polypeptide may be an epitope, and two or more sites that are discontinuous by tertiary structural folding in the polypeptide may be epitopes together. Epitopes contain at least two, at least three, at least four, at least five, at least six, at least seven, and at least eight consecutive or discontinuous amino acids in the unique three-dimensional structure of an antigen. At least 9, at least 10, at least 11, at least 12, at least 12, at least 13, at least 14 or at least 15 may be included. The antibody or fragment thereof of the invention recognizes and specifically binds an extracellular site of GPCR (e.g., Entotellin Receptor A) as an antigen, wherein the extracellular site of GPCR has one N-term and three ECLs. (ECL1, ECL2 and ECL3), and may specifically bind one or more extracellular sites selected from the group consisting of N-term, ECL1, ECL2 and ECL3 as epitopes, wherein the epitope is 1 or It may contain more amino acids.

Methods of determining the epitope to which a given antibody binds (eg, epitope mapping) include various methods, such as immunoblotting and immunoprecipitation tests through reactivity tests for antibodies. Determination of the three-dimensional spatial structure of the epitope can be performed using various methods such as x-ray crystallography, 2-dimensional nuclear magnetic resonance, and HDX-MS. Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996).

According to a preferred embodiment of the present invention, the epitope to which the antibody or fragment thereof of the present invention can bind may be NMR spectroscopy, X-ray diffraction crystallography, ELISA analysis, HDX-MS (hydrogen / deuterium exchange coupled with mass spectrometry), array Can be determined via array-based oligo-peptide scanning assays, and / or mutagenesis mapping (Giege R et al. , (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4). ): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).

As used herein, the term “antibody” may be any type of antibody (eg, IgG, IgE, IgM, IgD, IgA, or IgY) of an immunoglobulin molecule, and any subtype of antibody (eg, human IgG1, IgG2, IgG3, and IgG4), and IgG1, IgG2a, IgG2b, and IgG3) in mice. Immunoglobulins (eg, IgG1) may have various allotypes, and the term "antibody" herein includes generally known isotypes and allotypes. In addition, the term “antibody” herein may be IgG1, IgG2, IgG3, or IgG4, or a hybrid type thereof (eg, a hybrid of IgG2 and IgG4).

As used herein, the term “monoclonal antibody” or “monoclonal antibody” refers to an antibody that exhibits single binding specificity and affinity for a specific epitope.

In the present specification, the monoclonal antibody is used as a meaning including a fragment thereof, and the fragment preferably means an antigen binding fragment. Such fragments can be prepared using various methods known in the art. Fab and F (ab ') 2 fragments, for example, through proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (production of Fab fragments) or pepsin (F (ab') 2). Can be prepared.

As used herein, the term "fragment" may be Fab, Fab ', F (ab') 2, Fv, single chain Fv (scFV), or sdAb comprising the VH or VL domain of a monomer, for which fragment Well known in the industry.

Monoclonal antibody or fragment thereof of the present invention preferably comprises a heavy chain variable region selected from the group consisting of CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7 and CDR3 of SEQ ID NO: 8, and Preferably, CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7 and CDR3 of SEQ ID NO: 8 are included.

Monoclonal antibody or fragment thereof of the present invention preferably comprises a light chain variable region selected from the group consisting of CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10 and CDR3 of SEQ ID NO: 11 Preferably, CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11 are included.

The VH domain, or one or more CDRs, may be linked to a constant domain to form a heavy chain. In addition, the VL domain, or one or more CDRs, may be linked to a constant domain to form a light chain. Full length heavy and full length light chains combine to form a full length antibody.

According to a preferred embodiment of the present invention, the monoclonal antibody or fragment thereof comprises a heavy chain variable region of SEQ ID NO: 12 and a light chain variable region of SEQ ID NO: 13.

According to another aspect of the invention, the present invention provides a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule or a host cell comprising the vector.

Nucleic acid molecules of the invention can be isolated or recombinant and include single and double stranded DNA and RNA as well as corresponding complementarity sequences. An isolated nucleic acid is a nucleic acid isolated from a naturally occurring source, in the case of a nucleic acid isolated from the surrounding genetic sequence present in the genome of the individual from which the nucleic acid is isolated. In the case of nucleic acids, such as PCR products, cDNA molecules, or oligonucleotides synthesized enzymatically or chemically from a template, the nucleic acid resulting from this procedure can be understood as an isolated nucleic acid molecule. Isolated nucleic acid molecules refer to nucleic acid molecules in the form of separate fragments or as components of larger nucleic acid constructs. Nucleic acids are operably linked when placed in a functional relationship with other nucleic acid sequences. For example, the DNA of a presequence or secretion leader is operably linked to the DNA of a polypeptide when expressed as a preprotein, which is the form before the polypeptide is secreted, and the promoter or enhancer is a polypeptide sequence. Operably linked to a coding sequence when affecting the transcription of the ribosome binding site, or when the ribosome binding site is arranged to facilitate translation. In general, operably linked means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, they are contiguous within the same reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional methods.

As used herein, the term vector refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences can be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids and viruses (eg bacteriophages). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994, etc.).

As used herein, the term expression vector refers to a vector comprising a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, RNA molecules are then translated into proteins, polypeptides, or peptides. Expression vectors can include various regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors can also include nucleic acid sequences that provide additional functionality.

As used herein, the term host cell refers to any transgenic organism that includes eukaryotes and prokaryotes and is capable of replicating the vector or expressing a gene encoded by the vector. The host cell may be transfected or transformed by the vector, which means a process in which exogenous nucleic acid molecules are delivered or introduced into the host cell.

The host cell of the present invention preferably includes bacterial cells, CHO cells, HeLa cells, HEK293 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, NIH3T3 cells and the like, but is not limited thereto. no.

According to another aspect of the invention, the invention provides a composition comprising the monoclonal antibody or fragment thereof, the nucleic acid molecule or the vector.

According to a preferred embodiment of the present invention, the composition of the present invention is a pharmaceutical composition for the prevention or treatment of hypertension or cancer.

The pharmaceutical composition of the present invention comprises (a) the antibody or fragment thereof, the nucleic acid molecule or the vector comprising the nucleic acid molecule; And (b) a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a method for preventing or treating hypertension or cancer, comprising administering the pharmaceutical composition.

The type of cancer to be prevented or treated by the present invention is not limited, leukemias and acute lymphocytic leukemia, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic lymphocytic leukemia Lymphomas, brain tumors, glioblastoma, such as myelogenous leukemia, Hodgkin's Disease, non-Hodgkin's lymphomas, and multiple myeloma Childhood solid tumors such as neuroblastoma, rhabdomyosarcoma, retinoblastoma, Wilms Tumor, bone tumors and soft-tissue sarcomas tumors, lung cancer, breast cancer, prostate cancer, urinary cancers, uterine cancers, oral cancers, pancreatic cancer reatic cancer, melanoma and other skin cancers, stomach cancer, ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroid cancer It can be administered to treat multiple cancers, including common solid tumors of adults such as thyroid cancer, esophageal cancer, testicular cancer and the like.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be administered orally or parenterally, preferably parenterally, for example, by intravenous infusion, topical infusion and intraperitoneal infusion.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The pharmaceutical composition of the present invention may be used as a single therapy, but may also be used in combination with other conventional chemotherapy or radiation therapy, and when the combination therapy is performed, cancer treatment may be more effectively performed. Chemotherapeutic agents that can be used with the compositions of the present invention are cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, phospho Ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin, daunorubicin, doxorubicin , Bleomycin, plecomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluoro 5-fluorouracil, vincristin, vinblastin, methotrexate, and the like. Radiation therapy that can be used with the composition of the present invention is X-ray irradiation and γ-ray irradiation and the like.

According to another aspect of the invention, the invention provides a method for quantifying endothelin receptor type A (Endothelin receptor type A) contained in a sample comprising the step of treating a monoclonal antibody or fragment thereof.

In the description of the quantitative method, the same parts as the description of the screening method of the present invention described above will be referred to the part.

Since the monoclonal antibody or fragment thereof of the present invention specifically binds to the entotellin receptor A, the monoclonal antibody or fragment thereof can be used to accurately measure the amount of the entotelin receptor A contained in the sample.

According to another aspect of the present invention, there is provided a method for providing information for diagnosing a disease caused by overexpression of Endothelin receptor A (Endothelin receptor type A) comprising the following steps:

(a) obtaining a sample separated in vitro from the subject;

(b) treating the sample with the monoclonal antibody or fragment thereof of claim 1; And

(c) confirming whether the expression level of the entotellin receptor A contained in the sample of the subject is higher than the expression level of the entotellin receptor A included in the normal group sample.

In the description of the method for providing information for diagnosing a disease due to overexpression of the entotelin receptor A, the same part as the description of the screening method of the present invention described above will be referred to the part.

Entotelin receptor A undergoes vasoconstriction by binding of the ligand ET-1 (Nature reviews drug discovery, 2011, Vol. 10, 47); Major cancer progression such as proliferation, migration, invasion, metastasis and angiogenesis of cancer cells due to overexpression in various cancers including bladder cancer, lung cancer, ovarian cancer, kidney cancer, colon cancer, prostate cancer, breast cancer, uterine cancer, rhabdomyosarcoma and glioblastoma Since it is directly involved in the process (Nature review cancer, 2013, Vol. 13, 637), by comparing the expression level of the entotellin receptor A with a normal person, information for diagnosing a disease caused by overexpression of the entotelin receptor A is obtained. Can provide.

According to a preferred embodiment of the present invention, the disease caused by overexpression of the entotelin receptor A is cancer or hypertension.

According to another aspect of the present invention, the present invention provides an endothelin receptor type A quantitative kit comprising the monoclonal antibody or fragment thereof.

The quantitative kit of the present invention can quantify the amount of entotelin receptor A by analyzing the antigen for the antibody through an antigen-antibody binding reaction, and the antigen-antibody binding reaction is a conventional ELISA (Enzyme-linked immunosorbent assay), RIA ( Radioimmnoassay, Sandwich assay, Western blot on polyacrylamide gel, Immunoblot assay and Immunohstochemical staining are preferably selected from the group consisting of but not limited thereto. It doesn't work.

Fixtures for the antigen-antibody binding reaction include a well plate synthesized with a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin or a polystyrene resin, and a slide glass made of glass. Any one selected from the group can be used, but is not limited now.

The secondary antibody is preferably labeled with a conventional color developing agent that performs a color reaction, and includes HRP (Horseradish peroxidase), Alkaline phosphatase, Colloid gold, Poly L-lysine-fluorescein isothiocyanate ), Any one label selected from the group consisting of Fluorescein and Dye such as Rhodamine-B-isothiocyanate (RITC) may be used. Substrate that induces color development is preferably used according to the labeling reaction, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline -6-sulfonic acid) and OPD (ophenylenediamine) is preferably used, but not limited to any one selected from the group consisting of.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a monoclonal antibody or fragment thereof that recognizes and binds specifically to an extracellular site of entotheliner receptor A as an antigen.

(Ii) The present invention also provides a method for screening an antibody or fragment thereof that recognizes and specifically binds an extracellular site of GPCR as an antigen.

(Iii) The screening method of the present invention can be used for the detection of an antibody that specifically binds to the extracellular site of GPCR, and the antibody discovered through this method has a function of inhibiting the signal transduction process of entotelin receptor A. It can be usefully used as a therapeutic agent for hypertension or cancer in which entotellin receptor A is involved.

1 shows the expression of ET _A in E. coli and purification results using Ni-NTA.
Figure 2 shows the results of purification and nanodisk production of MSP-1.
Figure 3 shows an extracellular-specific antibody discovery strategy through the direction control of GPCR.
Figure 4 shows the form fused Cκ to the antibody of the present invention for the expression, purification, and stability of the antibody in E. coli.
Figure 5 shows the SDS-PAGE results of the purified antibody of the Figure 4 form.
Figure 6 is a graph showing the inhibition of the signal transduction induced by ET-1 of 10 nM of the ET _A expression mammalian cells (ET _A expressed CHO-K1 stable cell line) according to the concentration of the purified antibody shown in Fig.
FIG. 7 is a graph showing the inhibitory ability of 10 nM ET-1-induced signaling of colorectal cancer cells (HT-29) known to overexpress ET _A according to the concentration of the purified antibody shown in FIG. 5.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example 1 ET Using Escherichia Coli _A Overexpression and purification of

Expression and purification of the ET _A protein (SEQ ID NO: 1) required for antibody discovery is described in Kwangkyu Lee, et al. , Purification and characterization of recombinant human endothelin receptor type A, Protein expression and purification 2012; 84 (1) : 14-18). More specifically, Escherichia coli BL21 (DE3) transformed with pP9-ET _A expression vector was incubated in LB medium at 37 ° C. until optical density at 600 nm (OD _{600 nm} ) became 0.6, followed by isopropyl-bD-thiogalactopyranoside (IPTG). ) Is added to 0.5 mM to induce the expression of the protein. Then, after incubating for 4 hours at 25 ℃ E. coli harvest using a centrifuge. The crushed liquid obtained by crushing using a high-pressure cell homogenizer removes cell debris by using a high-speed centrifuge, and then, the supernatant is centrifuged at 100,000 times the gravity acceleration for 1 hour to obtain a cell membrane of E. coli. E. coli cell membranes are homogenized with 0.5% sarkosyl surfactant and insoluble parts are removed by centrifugation. The supernatant is purified using a column packed with Ni-NTA (Qiagen, USA). Column washes were with 20 mM imidazole followed by elution with 300 mM imidazole. The eluate is removed by imidazole using PD-10 desalting column (GE, USA) and mixed with 10% glycerol and stored at 80 ° C until use. ET _A expression and purification results are shown in FIG.

Example 2 Fabrication of Nanodisc

The expression, purification and preparation of MSP1 proteins required for nanodisk production are described in Timothy H. Bayburt, et al. , Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins, Nano letters 2002; 2 (8): 853-856). More specifically, E. coli Rosetta (DE3) transformed with the pET28a-MSP1 expression vector was incubated in LB medium at 37 ° C. until OD _{600 nm} became 0.9, and the IPTG was added to 1 mM and further incubated for 4 hours. . E. coli is harvested using a centrifuge, and then crushed using a high pressure cell homogenizer, and an inclusion body is obtained through high-speed centrifugation of lysate. Inclusion bodies are washed with buffer solution and solubilized for 3 hours at room temperature using 8 M Urea. The insoluble portion is removed using a centrifuge and the supernatant is purified on a column filled with Ni-NTA. Washing is done with 20 mM imidazole and elution with 100 mM-500 mM imidazole. The eluate is then dialyzed sequentially in 3 M urea and urea free buffer for refolding. Purification results are shown in FIG. 3.

In order to make the nanodisc, the purified MSP1, the lipid POPC (Avanti Polar Lipid, USA) and the purified ET _A were mixed at a molar ratio of 1: 60: 0.1, and Na-cholate was dissolved until all POPCs were dissolved. Add surfactant. Thereafter, biobeads (Bio-bead SM-2, Bio-rad, USA) were mixed to remove the surfactant and reacted at 4 ° C. for 2 hours, followed by centrifugation to obtain a supernatant. The supernatant is again dialyzed with excess buffer without surfactant. After dialysis, gel-filtration chromatography is used to obtain only the fractions in which the ET _A and MSP-1 protein bands are observed simultaneously. The obtained nanodisks were measured in size using dynamic light scattering (DLS), and the results are shown in FIG. 2.

Example 3 ET _A Of Extracellular Specific Antibodies

Expression and purification of G-proteins required for antibody discovery are described in Krishna Moorthy Vukoti, et al ., Molecular dissection of the interaction between hBLT2 and the G protein alpha subunits, Bulletin of the Korean chemical society 2007; 28 (6). ): 1005-1009). Each protein was transformed into E. coli, and then cultured and purified using Ni-NTA chromatography.

In order to use the magnetic biopanning technique, dimethylformamide (DMF) was added to powdery epoxy magnetic beads (Dynabeads M-270 Epoxy beads, Invitrogen, USA), and the number of magnetic beads was diluted to 2 x 10 ⁹ per milliliter. After storage, the reaction was carried out at room temperature for 16 hours with 2.5 x 10 ⁷ magnetic beads activated with epoxy functional groups with 12 μg of nano-disk protein consisting of MSP-1, P9 protein and lipid bilayer, 0.1 M sodium phosphate and 3 M ammonium sulfate. To combine. After the immobilization using a magnetic material to remove the unbound nanodisk protein, washed with 0.5% BSA (Bovine serum albumin) solubilized in PBS (phosphate buffered saline) to complete the conjugate of the epoxy magnetic beads and nanodisks.

In the M13 phage library on which the human single-chain antibody is surface-expressed, covalently binds P9 fused to the N-terminus of ET _A and nanodisks with P9 inserted into epoxy-activated magnetic beads to remove the clones that bind to the nanodisks. After binding to 10 ¹² phage libraries at room temperature, the magnetic beads were used to fix the magnetic beads, and the supernatant was taken to perform negative sorting (negative sorting) at each time of biopanning. The supernatant after the voice is separated G- protein-immobilized ELISA plates (Corning, USA) the nano disc G- protein and ET, using the manufacturing techniques of the disclosed nano disc consisting of MSP-1, ET _A, the lipid bilayer prior to by using a specific reaction within arm _a cell for selectively cells above the exterior of the immobilized and exposed to ET _a so that the external upper is exposed (SEQ ID NO: 2 to the fifth sequence) it was conducted bio panning with the antigen. After the reaction at room temperature for 1 hour, the supernatant was removed, washed with PBS, eluted with the binding phage with a low pH elution buffer (glycine-HCl pH2.2) and neutralized. The eluted phage was amplified using the VCS M13 helper phage and ER2738 Escherichia coli, and the amplified phages were repeatedly subjected to biopanning by increasing the number of washings each time (FIG. 3). Through the above process, a total of five biopanning processes were performed, and titer results of each cycle are shown in Table 1.

TABLE 1

Phage ELISA was performed on 400 clones of the eluted phages after the 5th time, and the 10 clones showing the highest binding ability to ET _A were sequenced. Among these, 5 clones contained the heavy chain variable region of SEQ ID NO. And the light chain variable region of SEQ ID NO: 13 is identical to each other (including CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 8 in the heavy chain variable region; , CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11 in the light chain variable region).

<Example 4> ET of the excavated antibody _A Signal transduction inhibition by extracellular site binding

Cκ fusion method for E. coli expression, purification and stabilization of unearthed antibodies is described by Andrew Hayhurst, et al ., Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis , Journal of immunological methods 2003; 276 (1-2): 185-196) was used to prepare expression vectors, which were expressed and purified in E. coli. The structure of the antibody formed at this time is shown in FIG. SDS-PAGE results of proteins expressed in E. coli and purified with high purity using affinity chromatography are shown in FIG. 5.

Fura-2-AM fluorescent dye signaling pathway measured by ET-1 in the ET _A with the (ThermoFisher, USA) with within the Ca ^{2 +} concentrations measurement cytoplasm Park, Soo - Jin paper (Soo-Jin Park, et al ., Lysophosphatidylethanolamine utilize LPA1 and CD97 in MDA-MB-231 breast cancer cells, Cellular signaling 2013; 25 (11): 2147-2154). 10 ⁵ ET _A expressing animal cell line (ET _A expressed CHO-K1 stable cell line, GenScript, USA) after treatment with 5 μM Fura-2-AM fluorescent dye and the antibody purified in Figure 5 by concentration, 10 nM ET-1 induces the signal transduction process and uses a fluorescence spectrophotometer (Scinco, USA) to determine the emission fluorescence (Emission by 340 nm excitation / Emission by 380 nm excitation) at 510 nm by excitation wavelength 340 nm and 380 nm. measurements derived by the results shown in Figure 6 to determine whether the process has the signal transmission control of the ET _a dig antibody by measuring the cytoplasmic Ca ^{2 +} concentrations in combination on cells outside of the ET _a.

As shown in Figure 6, under which the antibody of the present invention showed an antagonism effect of combination on cells outside of the ET _A inhibits the signaling pathways of the ET _A by ET-1, ET-1 of 10 nM is present K _i value is 56 nM, showing very effective inhibition. Through the above results, it was confirmed that the antibody discovery strategy of the present invention was effective for the detection of the extracellular specific antibody of GPCR, and the antibody discovered through this result was found to have the effect of effectively inhibiting the signaling process of ET _A.

Example 5 ET in Colon Cancer Cells _A Confirmation of signal transduction inhibition

Overexpression of ET _{A in} the HT-29 colorectal cancer cell line is described by Paraskevi Liakou, et al. , Expression patterns of endothelin-1 and its receptors in colorectal cancer, Journal of surgical oncology 2012; 105 (7): 643-649 HT-29 colorectal cancer cell line was used by the Korea Cell Line Bank (Seoul, Korea). Confirmation of the inhibitory ability of the ET _A signaling process in HT-29 colorectal cancer cells of the excavated antibody was used in the same manner as in Example 4 and the results as shown in FIG.

As shown in Figure 8, the antibody of the present invention shows an antagonism effect that inhibits the signaling process of ET _A by ET-1 in HT-29 colorectal cancer cell line, even in the presence of 10 nM ET-1 _{The i} value is 51 nM, indicating a very effective inhibitory effect. Through the above results, it was confirmed that the antibody of the present invention has an activity of effectively inhibiting the signaling process of ET _A in cancer cells and thus is effective as a therapeutic agent.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Kookmin University Industry Academy Cooperation Foundation <120> Human antibodies regulating functions of a G-protein coupled          receptor <130> HP7278 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 427 <212> PRT <213> Homo sapiens <400> 1 Met Glu Thr Leu Cys Leu Arg Ala Ser Phe Trp Leu Ala Leu Val Gly   1 5 10 15 Cys Val Ile Ser Asp Asn Pro Glu Arg Tyr Ser Thr Asn Leu Ser Asn              20 25 30 His Val Asp Asp Phe Thr Thr Phe Arg Gly Thr Glu Leu Ser Phe Leu          35 40 45 Val Thr Thr His Gln Pro Thr Asn Leu Val Leu Pro Ser Asn Gly Ser      50 55 60 Met His Asn Tyr Cys Pro Gln Gln Thr Lys Ile Thr Ser Ala Phe Lys  65 70 75 80 Tyr Ile Asn Thr Val Ile Ser Cys Thr Ile Phe Ile Val Gly Met Val                  85 90 95 Gly Asn Ala Thr Leu Leu Arg Ile Ile Tyr Gln Asn Lys Cys Met Arg             100 105 110 Asn Gly His Asn Ala Leu Ile Ala Ser Leu Ala Leu Gly Asp Leu Ile         115 120 125 Tyr Val Val Ile Asp Leu Pro Ile Asn Val Phe Lys Leu Leu Ala Gly     130 135 140 Arg Trp Pro Phe Asp His Asn Asp Phe Gly Val Phe Leu Cys Lys Leu 145 150 155 160 Phe Pro Phe Leu Gln Lys Ser Ser Val Gly Ile Thr Val Leu Asn Leu                 165 170 175 Cys Ala Leu Ser Val Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg             180 185 190 Val Gln Gly Ile Gly Ile Pro Leu Val Thr Ala Ile Glu Ile Val Ser         195 200 205 Ile Trp Ile Leu Ser Phe Ile Leu Ala Ile Pro Glu Ala Ile Gly Phe     210 215 220 Val Met Val Pro Phe Glu Tyr Arg Gly Glu Gln His Lys Thr Cys Met 225 230 235 240 Leu Asn Ala Thr Ser Lys Phe Met Glu Phe Tyr Gln Asp Val Lys Asp                 245 250 255 Trp Trp Leu Phe Gly Phe Tyr Phe Cys Met Pro Leu Val Cys Thr Ala             260 265 270 Ile Phe Tyr Thr Leu Met Thr Cys Glu Met Leu Asn Arg Arg Asn Gly         275 280 285 Ser Leu Arg Ile Ala Leu Ser Glu His Leu Lys Gln Arg Arg Glu Val     290 295 300 Ala Lys Thr Val Phe Cys Leu Val Val Ile Phe Ala Leu Cys Trp Phe 305 310 315 320 Pro Val His Leu Ser Arg Ile Leu Lys Lys Thr Val Tyr Asn Glu Met                 325 330 335 Asp Lys Asn Arg Cys Glu Leu Leu Ser Phe Leu Leu Leu Met Asp Tyr             340 345 350 Ile Gly Ile Asn Leu Ala Thr Met Asn Ser Cys Ile Asn Pro Ile Ala         355 360 365 Leu Tyr Phe Val Ser Lys Lys Phe Lys Asn Cys Phe Gln Ser Cys Leu     370 375 380 Cys Cys Cys Cys Tyr Gln Ser Lys Ser Leu Met Thr Ser Val Pro Met 385 390 395 400 Asn Gly Thr Ser Ile Gln Trp Lys Asp His Asp Gln Asn Asn His Asn                 405 410 415 Thr Asp Arg Ser Ser His Lys Asp Ser Met Asn             420 425 <210> 2 <211> 69 <212> PRT <213> Homo sapiens <400> 2 Met Glu Thr Leu Cys Leu Arg Ala Ser Phe Trp Leu Ala Leu Val Gly   1 5 10 15 Cys Val Ile Ser Asp Asn Pro Glu Arg Tyr Ser Thr Asn Leu Ser Asn              20 25 30 His Val Asp Asp Phe Thr Thr Phe Arg Gly Thr Glu Leu Ser Phe Leu          35 40 45 Val Thr Thr His Gln Pro Thr Asn Leu Val Leu Pro Ser Asn Gly Ser      50 55 60 Met His Asn Tyr Cys  65 <210> 3 <211> 4 <212> PRT <213> Homo sapiens <400> 3 Gly Arg Trp Pro   One <210> 4 <211> 21 <212> PRT <213> Homo sapiens <400> 4 Val Met Val Pro Phe Glu Tyr Arg Gly Glu Gln His Lys Thr Cys Met   1 5 10 15 Leu Asn Ala Thr Ser              20 <210> 5 <211> 5 <212> PRT <213> Homo sapiens <400> 5 Asn Glu Met Asp Lys   1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 <400> 6 Gly Tyr Thr Phe Thr Ser Ser   1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 <400> 7 Met Asn Pro Lys Asn Gly Asn   1 5 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 <400> 8 Ala Lys Asp Arg Tyr Gly Ser Gly Thr Tyr Gly Phe Gly Met Asp Val   1 5 10 15 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 <400> 9 Gln Gly Ile Asn Asn Tyr   1 5 <210> 10 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 <400> 10 Leu Gln Ser   One <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 <400> 11 Leu Gln His Asn Thr Tyr Pro Leu Thr   1 5 <210> 12 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 12 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Arg Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser              20 25 30 Asp Ile Asn Trp Ile Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Tyr Met Asn Pro Lys Asn Gly Asn Thr Asp Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Ile Ser Met Thr Arg Asp Ser Ser Ile Ser Thr Ala 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 Lys Asp Arg Tyr Gly Ser Gly Thr Tyr Gly Phe Gly Met Asp Val             100 105 110 Trp Gly Pro Gly Thr Leu Val Thr Val Ser         115 120 <210> 13 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 13 Asp Ile Val Met Thr Gln Ser Pro Ser Ala Met Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Asn Tyr              20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Arg Leu Met          35 40 45 Asp Val Gly Ser Ile Leu Gln Ser Gly Ile Pro Ser Arg Phe Arg Gly      50 55 60 Arg Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala  65 70 75 80 Glu Asp Ser Gly Thr Tyr Phe Cys Leu Gln His Asn Thr Tyr Pro Leu                  85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 14 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Anti-ETA-scFV (AG8) <400> 14 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Arg Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser              20 25 30 Asp Ile Asn Trp Ile Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Tyr Met Asn Pro Lys Asn Gly Asn Thr Asp Tyr Ala Gln Lys Phe      50 55 60 Gln Gly Arg Ile Ser Met Thr Arg Asp Ser Ser Ile Ser Thr Ala 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 Lys Asp Arg Tyr Gly Ser Gly Thr Tyr Gly Phe Gly Met Asp Val             100 105 110 Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser Ser         115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp     130 135 140 Ile Val Met Thr Gln Ser Pro Ser Ala Met Ser Ala Ser Val Gly Asp 145 150 155 160 Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Asn Tyr Leu                 165 170 175 Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Arg Leu Met Asp             180 185 190 Val Gly Ser Ile Leu Gln Ser Gly Ile Pro Ser Arg Phe Arg Gly Arg         195 200 205 Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu     210 215 220 Asp Ser Gly Thr Tyr Phe Cys Leu Gln His Asn Thr Tyr Pro Leu Thr 225 230 235 240 Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg                 245 250

Claims (16)

A screening method for an antibody or fragment thereof that specifically recognizes an extracellular domain of a G-protein coupled receptor (GPCR) and binds to an antigen, the method comprising the following steps:
(a) preparing a nanodisk by mixing a membrane scaffold protein (MSP), a lipid, and a GPCR;
(b) coupling magnetic beads to the nanodisks;
(c) exposing an extracellular site by binding an intracellular domain of GPCR included in the nanodisk to a plate on which G-protein is fixed; And
(d) screening an antibody or fragment thereof that specifically binds the extracellular site as an antigen.
The method of claim 1, wherein the GPCR is Endothelin receptor type A (Endothelin receptor type A).
delete delete A monoclonal antibody or a fragment thereof screened by the screening method of claim 1, wherein the monoclonal antibody or a fragment thereof has a heavy chain variable region of CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and SEQ ID NO: 8 Monoclonal antibody or fragment thereof comprising CDR3, CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10 and CDR3 of SEQ ID NO: 11 in the light chain variable region.
The monoclonal antibody or fragment thereof according to claim 5, wherein the monoclonal antibody or fragment thereof comprises a heavy chain variable region of SEQ ID NO: 12 and a light chain variable region of SEQ ID NO: 13.
A nucleic acid molecule encoding the monoclonal antibody or fragment thereof of claim 5.
A vector comprising the nucleic acid molecule of claim 7.
A host cell comprising the vector of claim 8.
A pharmaceutical composition for preventing or treating cancer, comprising the monoclonal antibody or fragment thereof of claim 5, the nucleic acid molecule of claim 7, or the vector of claim 8.
A pharmaceutical composition for preventing or treating hypertension, comprising the monoclonal antibody or fragment thereof of claim 5, the nucleic acid molecule of claim 7, or the vector of claim 8.
A method for quantifying endothelin receptor type A, which is included in a sample, comprising the step of treating the monoclonal antibody or fragment thereof of claim 5.
delete delete An endothelin receptor type A quantitative kit comprising the monoclonal antibody of claim 5 or a fragment thereof. delete

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