KR101334207B1 - Antigen Derived from Extracellular Domain of Multi-transmembrane Protein and Uses Thereof - Google Patents

Abstract

The present invention includes a polypeptide of an extracellular loop of a multi-transmembrane protein, wherein the N-terminus and C-terminus of the polypeptide are attached to a solid sunstrate to fix it. Or a transmembrane protein antigen, wherein the N-terminus and C-terminus are attached to both ends of the same linker, respectively, to form a cyclic structure, an antibody or an antigen-binding fragment thereof that specifically binds to the antigen, and the antigen It relates to a method of screening for an antibody that specifically binds to.
The present invention can be usefully used for the efficient production of antibodies to complex transmembrane proteins that are important for disease-related phenomena such as cell signal transduction.
The looped antigen of the present invention has a much stronger affinity than conventional linear antigens by perfectly replicating the three-dimensional structure of the loop of the complex transmembrane protein in vivo.
Not only can the present invention be used for efficient screening of antibodies, it can be usefully used as a composition for preventing and treating cancer, a composition for preventing and treating inflammation, and a highly reliable diagnostic composition according to the characteristics of the transmembrane protein used.

Description

Anti-membrane Peptide Antigens of Complex Transmembrane Proteins and Their Uses {Antigen Derived from Extracellular Domain of Multi-transmembrane Protein and Uses Thereof}

The present invention relates to an antigen consisting of a peptide of an extracellular loop region of a complex transmembrane protein and a method of using the same for antibody production and screening.

Transmembrane proteins have been selected as major target antigens for antibody therapeutics because they have a large number (more than 7,000) on the human genome and are important for disease-related phenomena such as cell signal transduction. Receptor proteins (about 400 species), enzyme proteins (about 500 species), and other cell membrane proteins (about 500 species) are present as proteins that pass through the membrane once or twice. Proteins vary widely, including receptor proteins (about 1000 species), most transporter proteins (about 800 species), and proteins needed for tight junctions between cells. In order to produce and develop useful antibodies using various transmembrane proteins, it is essential to obtain high quality antigens in their natural state. In particular, in the case of protein antigens, it is well known that high-quality antibodies can be produced only when protein purity, as well as three-dimensional structure and physiological activity are verified. Many existing researchers have made antigens using a variety of transmembrane proteins, but it is difficult to produce excellent antibodies due to the characteristics of the transmembrane proteins. In most cases, the antibody is prepared by using the antigen exposed to the cytoplasm or extracellular membrane at the N-terminal or C-terminal site, and in this case, most cases do not bring sufficient expected effects as the antibody. Transmembrane proteins play an important role in vivo between cells and between cells or between tissues and tissues, and are very good materials for disease research, but have two or more complex transmembrane sites when manufactured with recombinant proteins. It is difficult to have the natural form combined with the membrane in the state, and it is difficult to obtain a high quality antibody because it is very difficult to use as an antigen due to mixing with lipid components. Many researchers have used biological products to express transmembrane proteins as antigens, or to selectively synthesize specific sites as antigens. Other researchers are conducting various researches in which the Fc of the transmembrane protein and the human antibody are fused and expressed individually through animal cell lines to produce antigens of various transmembrane proteins. Not only is the probability of being expressed as a transmembrane protein low, but also has a disadvantage that a lot of time and economic difficulties in development. In view of the fact that a complex transmembrane protein having two or more transmembrane sites naturally has an extracellular membrane loop, the present inventors have simulated the site as it is and use a linker or the like to use a single looped peptide as an antigen. Devised.

Antibodies are proteins produced by the immune system that have a high degree of specificity for specific antigens compared to small molecules, and inactivate or remove antigens by using the body's natural immune system. This can be a very effective treatment with fewer side effects. For example, it can be applied as an antibody therapeutic for removing cancer cells by using immune cells or immunomodulators targeted to transmembrane proteins showing specific expression on cancer cell surfaces. Antibodies can also be used as carriers of radioisotopes and toxic substances to selectively treat only cancer cells (Antibody-directed enzyme prodrug therapy, ADEPT). On the other hand, although human antibodies are the most ideal forms as therapeutic antibodies, their development has been delayed since the production by human hybridoma technology using human cells is impossible. Recently, however, phage display technology, which can produce human monoclonal antibodies without relying on human hybridoma technology, has received great attention.

Various studies on the claudine family have been focused among the studies on the relationship between transmembrane proteins and cancer. The Clauden family is a family of cell membrane proteins of molecular weight approximately 20-34 kDa, consisting of four transmembrane regions and with tight junctions. Claudein contains 23-24 groups in humans and mice, and each member of Claudein is known to have a unique pattern of expression for each type of epithelial cell (Furuse and Tsukita, TRENDS). in Cell Biology 16: 181 (2006)), Wilcox, et al., Cell 104: 165 (2001), Rahner, et al., GASTROENTEROLOGY 120: 411 (2001). In sheets of epithelial cells, the mechanism works to prevent the release (diffusion) of substances in the intercellular space, and cell-cell attachment systems, called tight junctions, appear to actually play a central role as a "barrier" in the mechanism of preventing leakage. have. The general structure of the cloudin protein is shown in Figure 1 (Lai-Nag M et.al Genomic). Biology 2009, 10: 235 (2009)). The expression of claudine protein is closely related to cancer and reported, in particular, claudine-1 suppresses breast cancer and prostate cancer, and the reduction of claudine-7 expression in esophageal carcinoma cells is a cancer cell caused by loss of E-cadherin. It is known to cause an increase. The increase or decrease in the expression of high specific cladin protein in human cancer tissue can be used as a predictive indicator of cancer production and can be used as a useful biomarker in the detection, diagnosis and treatment of cancer. In particular, CLDN 3 and 4 have been shown to increase expression in a wide variety of cancers, in particular intensive studies as a biomarker of ovarian cancer has not yet developed a useful therapeutic drug (Choi et al., Histol Histopathol 22: 1185 ~ 1195) (2007)). Most of the development of antibodies using CLDN 3 and 4 is made by using a linear virtual peptide as an antigen.

CD151 is a membrane protein belonging to the tetraspanin family, also called PETA-3 or SFA-1 (Boucheix and Rubinstein, Cell). Mol . Life Sci . 58: 1189-1205 (2001); Republic of Korea Patent Application Publication No. 2011-0010708; Hemler ME, J. Cell Biol . 155: 1103-1107 (2001). In humans, CD151 has 253 amino acids, four membrane fragments and two extracellular domains called the extracellular loops EC1 (18 amino acids, [40-57] sequence) and EC2 (109 amino acids, [ 113-221]). CD151 is a mutant having two variants of CD151, nucleotides A and C, at positions 395 and 409, respectively, in the nucleotide sequence (Fitter et al., Blood 86 (4): 1348-1355 (1995)) and the same position. Other variants with nucleotides G and T instead of nucleotides A and C (Hasegawa et al., J. Virol . 70 (5): 3258-3263 (1996)) have been identified to date. It was confirmed that the K (Lys) and P (Pro) residues are mutated to R (Arg) and S (Ser) residues at positions 132 and 137, respectively, on the peptide sequence. CD151 interacts with various membrane proteins on the surface of cells. In particular, very stable complexes have been identified that resist the action of laminin receptor integrins and, more specifically, certain detergents that form a preferred ligand with integrin α3β1 or α6β4, which is also laminin 5 ( Yauch et al., Mol . Biol. Cell 9: 2751-2765 (1998); Lammerding et al., Proc . Natl . Acad . Sci USA 100: 7616-7621 (2003). This complex contains the extracellular domain of CD151 and integrin. The QRD [194-196] sequence of CD151, located in the ECL2 loop, is very important for this linkage because mutations in this site lose interaction with certain integrins (Kazarov et al., J. Cell). Biol . 158: 1299-1309 (2002).

Several previous studies have shown that overexpression of CD151 is associated with the aggressiveness of certain cancers, such as lung, rectal and prostate cancers, which may be considered as a factor for poor prognosis (Tokuhara et al., Clin . Cancer Res . 7: 4109-4114 (2001); Hashida et al, Br J. Cancer 89:.... 158-167 (2003); Ang et al, Cancer Epidemiol Biomarkers Prev . 13: 1717-1721 (2004). In these cases, in fact, the average survival rate decreased in patients with tumors expressing CD151 compared to the survival rates of patients with tumors not expressing CD151. In various human tumor lines (HeLa, RPMI14788, A172, HT1080), overexpression of CD151 caused by the transformation of the gene of interest results in increased movement, migration and invasion of infected cells, which is an anti-CD151 antibody Inhibited in the presence of (Testa et al., Cancer Res . 59: 3812-3820 (1999); Kohno et al., Int . J. Cancer 97: 336-343 (2002)), which may be a very important target in the development of anticancer therapies.

CD9 is also a cell surface glycoprotein receptor belonging to members of the tetraspanin family of about 24-27 kD, regulating signal transduction events that play an important role in regulating cell development, activity, growth and motility. Cell attachment (Anton, ES, et al., J. Neurosci. 15: 584-595, 1995) and cell migration (Klein-Soyer, C., et al., Arterioscler Thromb Vasc Biol. 20: 360 -369, 2000) and involved in platelet-induced endothelial cell proliferation (Masellis Smith, A., and Shaw, AR, J. Immunol . 152: 2768-2777 (1994) It is known to induce platelet activation and aggregation, and is known to be involved in various phenomena in cells, such as promoting muscle cell fusion and contributing to myotube maintenance. CD9 also reports that a second extracellular loop2 (ECL2) is important for cell attachment (George, A., et al., Blood 100: 4502-4511, 2002), and DTR for Diphteria Toxin (DT) It is reported that it is important to increase the activity of (Hidetoshi, H., et al. 289: 782-790 (2001)). In addition, many proteins in the tetraspanin family have glycosylated ECL2 domains, but CD9 is differentiated because glycosylated ECL1 domains. CD9 has also been reported to be associated with cell motility and tumor metastasis (Miyake, M. and Hakomori, S., Biochemistry 30: 3328-3334, (1991)). It is assumed to exhibit properties that are tissue-specific. CD9 is known as colorectal cancer (Mori, M., et al., Clin . Cancer Res . 4: 1507-1510 (1998)), Breast Cancer (Miyake, M., et al., Cancer Res . 55: 4127-4131 (1995)), lung cancer (Higachiyama, M., et al., Cancer Res . 55: 6040-6044 (1995); Funakoshi, T., et al., Oncogene 22: 674-687 (2003)) and pancreatic cancer (Sho, M., et al., Int. J. Cancer 79: 509-516 (1998)) in patients with reduced expression, which has been reported to be associated with invasion, metastasis and poor prognosis of patients. However, squamous cell carcinoma of the head and neck (Erovic, BM, et al., Head Neck 25: 848-857 (2003) and gastric cancer (Hori, H., et al., J. Surg. Res . 117: 208-215 (2004)), the higher the cancer progression, the higher the expression of CD9. Reported.

Europlakin 1B (UPK1B) is another cell surface protein characterized by the presence of four hydrophobic regions as another tetraspanin family member. It mediates signal transduction, which plays an important role in cell development, activation, growth and motility. In particular, the interaction between the unbalanced unit membrane (AUM) and the cytoskeleton is associated with cancer, and the methylation of the CpG island of the UPK1B promoter Associations with bladder cancer are well known (Varga AE, Leonardos L, Jackson P, Marreiros A, Cowled PA., Neoplasia . Mar-Apr; 6 (2): 128-35 (2004)).

The various membrane proteins mentioned above have recently been linked to many cancers, and the development of antibodies using specific sites has been continued, but most of them have antigens using linear virtual peptides or antigens using some expressed proteins. Minutes.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors made diligent research efforts to develop an efficient method for producing an antibody against a complex transmembrane protein, which plays an important role in disease-related phenomena such as cell signal transduction. As a result, the present invention has been completed by discovering that a very good and efficient antibody can be obtained by using as a antigen a single loop-type peptide that mimics a loop connecting the transmembrane sites.

It is therefore an object of the present invention to provide a multi-transmembrane protein antigen.

Another object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to a complex transmembrane protein antigen.

It is another object of the present invention to provide a method for screening antibodies that specifically bind to a complex transmembrane protein antigen.

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

According to one aspect of the present invention, the present invention comprises a polypeptide of an extracellular loop of a multi-transmembrane protein, wherein the N-terminus and C-terminus of the polypeptide are solid substrates. It provides a complex transmembrane protein antigen, characterized in that it is attached and fixed to (solid sunstrate).

The present inventors made diligent research efforts to develop an efficient method for producing an antibody against a complex transmembrane protein, which plays an important role in disease-related phenomena such as cell signal transduction. As a result, it was found that a very good and efficient antibody can be obtained by using as a antigen a single looped peptide that mimics a loop connecting the transmembrane sites.

As used herein, the term “polypeptide” refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds. Polypeptides of the present invention can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques, together with molecular biological methods (Merrifield, J. Amer . Chem . Soc . 85) . 2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis , 2nd. ed., Pierce Chem. Co .: Rockford, 111 (1984)).

As used herein, the term “multi-transmembrane protein” refers to a membrane protein that has two or more transmembrane sites and has extracellular loop sites that connect them.

According to the present invention, when an antigen is produced by replicating a polypeptide of an extracellular membrane loop region, a much superior affinity and affinity for an antibody are compared with an existing antigen using a linear imaginary polypeptide expressing a transmembrane protein as it is. Have The present invention immobilizes both ends of the polypeptide of the extracellular loop region to a solid substrate, thereby perfectly replicating the three-dimensional structure of the loop connecting adjacent transmembrane sites in a complex transmembrane protein attached to the membrane in vivo. Significantly increased efficiency and specificity as antigen.

The solid sunstrate used in the present invention is capable of attaching a polypeptide chain through covalent or non-covalent bonds with peptide residues, and any solid substrate commonly used in the art may be used without limitation.

Due to the above-mentioned high efficiency and specificity, the antigen of the present invention is used for the regulation of membrane protein receptor protein, the regulation of membrane transport pathway proteins and the regulation of tight junction related proteins between cells of membrane proteins. As a result, it may be usefully used to study cellular signaling mechanisms.

According to a preferred embodiment of the invention, the polypeptide of the extracellular loop of the complex transmembrane protein of the invention is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 21 sequences.

According to the present invention, the amino acid sequence of SEQ ID NO: 1 to 21 sequence is a complex transmembrane protein CD-151-1 to CD-151-6; Claudine 3 to 7, 9 and 17; CPE; CD9-1 to 3; And the extracellular loop amino acid sequences of UP1b-1 to 3.

More preferably, the polypeptide of the extracellular loop of the complex transmembrane protein of the present invention is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

The amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4 are amino acid sequences for the polypeptides of the extracellular loop sites of Claude 3 (CLDN 3) and Cladine 4 (CLDN 4), respectively. Thus, antibodies produced from these antigens can be usefully applied as a means of screening for diagnostic compositions or therapeutic agents for various cancers in which the expression of CLDN 3 or CLDN 4 is increased.

According to another aspect of the invention, the invention comprises a polypeptide of an extracellular loop of a multi-transmembrane protein, wherein the N-terminus and C-terminus of the polypeptide are the same linker. It is attached to both ends of each to provide a complex transmembrane protein antigen, characterized in that to form a cyclic structure (cyclic structure).

Since the polypeptide of the extracellular loop of the complex transmembrane protein used in the present invention has been described above, it is omitted to avoid excessive duplication.

As used herein, the term “linker” refers to a molecule that binds both ends by covalently or non-covalently binding to the N-terminus and C-terminus of the polypeptide of the present invention. The linker of the present invention makes the entire molecule into a circular structure so that the three-dimensional structure most similar to the natural structure of the loop connecting adjacent transmembrane sites in vivo can be reproduced. The linker of the present invention may be selected of a suitable length so that the polypeptide has an optimal conformation, preferably selected from the group consisting of aminohexanoic acid, glycine polymer and alanine polymer.

According to a preferred embodiment of the invention, the linker of the invention is attached to a water soluble resin.

According to the present invention, the antigen of the present invention can be attached to a resin, which is an aqueous support, to be used for more convenient and efficient screening of antibodies. It is particularly preferred that the linker of the invention is immobilized via a resin when obtaining antibodies through solid homology. The resin used in the present invention includes all natural resins and synthetic resins used in the art, and is preferably chloro trityl chloride (CTL) resin. Preferably the resin of the present invention is surrounded by polyethylene glycol (PEG) to impart water soluble properties.

According to another aspect of the invention, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to the complex transmembrane protein antigen of the invention. The antibodies of the invention are polyclonal or monoclonal antibodies, preferably monoclonal antibodies.

As used herein, the term “antigen binding fragment of an antibody” (or “antibody fragment”) refers to any one or more fragments of an antibody that retain the ability to specifically bind an antigen of the invention. Exemplary antibody fragments include, but are not limited to, Fab fragments, F (ab ′) 2 fragments, scFv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Fab and F (ab ′) 2 fragments lack Fc fragments of complete antibodies, are removed more quickly from the circulation of animals or plants, and may have less nonspecific tissue binding than complete antibodies.

Antibodies to antigens or antigen-binding fragments thereof of the present invention may be prepared by methods conventionally practiced in the art, such as fusion methods (Kohler and Milstein, European). Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Patent No. 4,816,567) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. Mol . Biol, 222:. 58, can be prepared by 1-597 (1991)). The general process for manufacturing antibodies is Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting an antigen of the invention into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques. Antigen binding fragments of antibodies also screen for utility in the same manner as intact antibodies.

The term “specifically binds” herein means that the antibody or antigen-binding fragment thereof stably produces a complex with the antigen under stable physiological conditions, with the specific binding being at least about 1 × 10 ⁻⁶ M or equilibrium The case of dissociation constant. Methods of determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

Antibodies or antigen-binding fragments thereof of the invention can be conjugated with bioactive agents. As used herein, the term “bioactive agent” refers to any synthetic or naturally occurring compound that binds to an antigen and mediates the desired biological effect to increase the desired activity in the cell. For example, an antibody or fragment thereof of the invention may be conjugated to an immunoconjugate such as a cytotoxin, chemotherapeutic drug, immunosuppressant or radioisotope.

According to another aspect of the invention, the invention provides a method of screening for an antibody that specifically binds to a transmembrane protein antigen, comprising contacting the complex transmembrane protein antigen of the invention with a humanized antibody library.

The antigens used in the present invention and antibodies specifically binding thereto are already described above, and thus description thereof is omitted to avoid excessive duplication.

Processes for screening antibodies that specifically bind to specific antigens are well known in the art and include, for example, phage library display (Clackson et al, Nature , 352: 624-628 (1991) and Marks). et al, J. Mol . Biol . , 222: 58, 1-597 (1991). Illustratively, after reacting the antigen peptide of the present invention bound to the beads with the humanized antibody library solution, the beads are recovered and the humanized antibody library is separated from the membrane filter tube. The isolated humanized antibody library solution is inoculated into cells, incubated with helper phage, followed by centrifugation of the culture and recovery of the supernatant. Highly reactive antibodies can be screened by measuring the titers of library candidates panned in cells.

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

(a) The present invention includes a polypeptide of an extracellular loop of a multi-transmembrane protein, wherein the N-terminus and C-terminus of the polypeptide are in a solid sunstrate. A complex transmembrane protein antigen, an antibody or antigen-binding fragment thereof, that is attached or immobilized, or wherein the N-terminus and C-terminus are attached to both ends of the same linker, respectively, to form a cyclic structure. And it provides a method for screening antibodies that specifically bind to the antigen.

(b) The present invention can be usefully used for the efficient production of antibodies to complex transmembrane proteins that are important for disease-related phenomena such as cell signal transduction.

(c) The looped antigen of the present invention has a much stronger affinity than conventional linear antigens by perfectly replicating the three-dimensional structure of the loop of the complex transmembrane protein in vivo.

(d) The present invention not only can be used for screening efficient antibodies, but also usefully used as a composition for preventing and treating cancer, a composition for preventing and treating inflammation, and a highly reliable diagnostic composition according to the characteristics of the transmembrane protein used. Can be.

1 is a diagram showing a schematic diagram of a complex transmembrane protein.
2 is a diagram showing a schematic diagram for the synthesis of loop-type peptide.
Figure 3 is a HPLC and Mass picture of the first sequence of the sequence of the synthesized peptide.
4 is a diagram showing a schematic diagram for producing a looped protein.
Figure 5 is a diagram showing the SDS-PAGE results for the loop-type peptide of the sequence listing the second sequence.
6 is a diagram showing a schematic diagram of library screening using the produced loop-type antigen.
7 is a diagram showing a panning result of screening the antigen of the complex transmembrane protein.
8 is a diagram showing the results of ELISA analysis of the efficacy of the selected loop type and linear antibody.
9 is a diagram showing the results of analyzing the efficacy of the selected loop type and linear antibodies by FACS.
10 is a diagram showing the results of SPR analysis of the efficacy of the selected loop type and linear antibody. The binding capacity of C3E6 (top left panel), C3B12 (top right panel), and antibody fragment 3CH12 (bottom panel), antibody fragment derived from loop-type antigen, was examined for KD values (dissociation constants) at various concentrations. The binding force was measured.

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

Example

Example 1 Synthesis of Looped Peptides

To prepare a loop-type peptide antigen of SEQ ID NO: 1 sequence was synthesized in the following two steps, the synthesis was attempted as shown in the schematic diagram shown in FIG.

1) Synthesis of Protective Group Attached Peptides

700 mg of chloro trityl chloride resin (CTL resin, Nova biochem Cat No. 01-64-0021) was added to a reaction vessel, and 10 ml of methylene chloride (MC) was added thereto and stirred for 3 minutes. The solution was removed, 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of dichloromethane solution was added to the reactor, 200 mmole of Fmoc-Ile-OH and 400 mmole of diisopropyl ethylamine (DIEA) were added thereto, stirred to dissolve well, and reacted with stirring for 1 hour. After the reaction, the mixture was washed with methanol and DIEA (2: 1) in DCM for 10 minutes, and then washed with excess DCM / DMF (1: 1). The solution was removed, 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred at room temperature for 10 minutes to remove the solution. After adding the same amount of deprotection solution to maintain the reaction for another 10 minutes, the solution was removed and washed twice with DMF, once with MC, once again with DMF for 3 minutes to prepare Ile-CTL resin. 10 ml of DMF solution was added to a new reactor, 200 mmole of Fmoc-Tyr (tBu) -OH, 200 mmole of HoBt, and 200 mmole of Bop were added, followed by stirring to dissolve. 400 mmole DIEA was added to the reactor twice in fractions and stirred for at least 5 minutes until all solids dissolved. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin and reacted with stirring at room temperature for 1 hour. The reaction solution was removed and stirred with a DMF solution three times for 5 minutes and then removed. A small amount of the reaction resin was taken and the degree of reaction was checked using a Nihydrine Test. Tyr (tBu) -Ile-CTL resin was prepared by the same deprotection reaction as described above with the deprotection solution. After sufficient washing with DMF and MC, once again Kaiser test was performed, and the following amino acid attachment experiment was performed as above. Chain reaction was performed based on the amino acid sequence selected as shown in FIG. 1 to prepare Fmoc-KWTLALKSDYISLLASGTYLATAYI-CTL resin. The peptide is protected with all residues. Peptidyl resin is washed three times with DMF, MC, and methanol, dried with a slow flow of nitrogen air, dried under reduced pressure under vacuum under P2O5, and then dried with a fugitive solution (0.1% triple). After adding 30 ml of trifluroacetic acid), the mixture was occasionally shaken at room temperature to maintain a reaction for 2 hours. The resin was filtered off and washed with a small amount of 0.1% TFA solution and then combined with the mother liquor. Neutralized with pyridine or the like, distilled to a half of the total volume using reduced pressure, and 50 ml of cold ether was added to induce precipitation, followed by centrifugation to collect the precipitate and washing with two more cold ethers. The mother liquor was removed and dried sufficiently under nitrogen to synthesize 1.18 g (yield: 47.6%).

2) Preparation of water-soluble resin (based on 2 residues)

After precisely measuring the gap of the base of the membrane protein mentioned in PDB Data, etc., a linker containing 1-10 residues of aminohexanoic acid or glycine residues was prepared for the synthesis of looped peptides. L-Lys (Mtt) was positioned at the intermediate residue for smooth attachment to the resin (e.g., Fmoc-Gly-Gly-Glu (OAll) -Gly-Gly-COOH, Fmoc-AHX-AHX-Lys (Mtt) ) -AHX-AHX-COOH). After attaching to Novagel-HMBA resin (Novagen), the N-terminal amino group was selectively left open by deprotecting the N-terminal amino group. The water-soluble resin of the present invention is modified with polyethylene glycol (PEG), mass production is possible, and the linker for each size can be commercialized.

3) Production of Loop Peptidyl Resins

The peptide of SEQ ID NO: 1 prepared in 1) where the entire residue was protected was coupled to the water-soluble resin prepared in 2). Under the HoBt and HBTU conditions, DIEA was appropriately controlled and the reaction was completed by ninhydrin test. Remove all unreacted material, remove allyl group under Pd (Ph3P) 4 / CHCl3 / AcOH / NMN, deprotect N-terminal Fmoc and once again adjust DIEA under HoBt and HBTU conditions. And the end of the reaction was confirmed by the ninhydrin test to prepare a loop-type peptide. The same reactions were able to synthesize peptides in the sequence listing described below.

KWTLALKSDYISLLASGTYLATAYIE-Linker-Resin (1-26 Cyclized)

The synthesized peptide was confirmed to be synthesized correctly by HPLC and Mass analysis as shown in FIG. Sequence information for the synthesized loop-type peptides are shown in Table 1 below.

Synthesized sequence information order Protein name order Remarks One CD-151-1 KWTLALKSDYISLLASGTYLATAYIELinker-Resin Peptides 2  CD151-2 C-GSNNSQDWR DSEWIRSQEA GGRVVPDSCC KTVVALCGQR DHASNIYKVE GG-C protein 3 Claudin 3 SANTIIRDFYNPVVPEAQKREMGAGELinker-Resin
C-SANTIIRDFYNPVVPEAQKREMGAGEC Peptides /
protein 4 Claudin 4 TAHNIIQDFYNPLASGQKREMGASELinker-Resin
C-TAHNIIQDFYNPLASGQKREMGASE-C Peptides /
protein 5 Claudin 5 FANIVVREFYDPSVPVSQKYELGAAELinker-Resin Peptides 6 Claudin 6 TAHAVIRDFYNPLVAEAQKRELGASELinker-Resin Peptides 7 Claudin 7 VANAIIRDFYNSIVNVAQKRELGEAELinker-Resin Peptides 8 Claudin 9 TAHAIIQDFYNPLVAEALKRELGASELinker-Resin Peptides 9 Claudin 17 TANIIIRDFYNPAIHIGQKRELGAAELinker-Resin Peptides 10 CPE Protein KLVMKANSSYSGNYPYSILFQKELinker-Resin Peptides 11 CPE Protein KANSSYSGNYPYSILFQKELinker-Resin Peptides 12 CD151-3 KGSNNSQDWRDSEWIRSQEAGGRVVPDSELinker-Resin Peptides 13 CD151-4 KKTVVALCGQRDHASNIYKVEGGELinker-Resin Peptides 14 CD151-5 WTLALJSDYUSLLASGTYLATAYIELinker-Resin Peptides 15 CD151-6 C-AYYQQLNTELKENLKDTMTKRYHQPGHEAVTSAVDQLQQE FH-C Peptides 16 CD9-1 KRFDSQTKSIFEQETNNNNSSFYTGVE-Linker-Resin Peptides 17 CD9-2 KDEVIKEVQEFYKDTYNKLKTKDEPQRETLE-Linker-Resin Peptides 18 CD9-3 C-GLAGGVEQFISDIC (S) PKKDVLETFTVKS-C protein 19 UP1b-1 KFVSDQHSLYPLLEATDNDDIYGAAWIGIFVE-Linker-Resin 20 UP1b-2 KQMLERYQNNSPPNNDDQWKNNGVTKE-Linker-Resin 21 UP1b-3 C-GVNGPSDWQKYTSFRTENNDADYPWPRQC (S) C (S) VMNNLKEPLNLEAC (S) KLGVPGFYHNQGC-C protein

Example  2: synthesis of looped protein

In order to produce the looped protein of FIG. 4, cloning was performed on the cladin proteins of SEQ ID NO: 3 and 4 in Table 1.

DNA sequencing of tcggccaacaccattatccgggacttctacaaccccgtggtgcccgaggcgcagaagcgcgagatgggc gcgggc and keulrawoodin makoe loop DNA sequencing of reverse transcription polymerase chain reaction (RT-PCR) with respect to acggcccacaacatcatccaagacttctacaatccgctggtggcctccgggcagaagcgggagatggg tgcctcg for the sequence of TAHNIIQDFYNPLASGQKREMGAS of 4 or primer overlap extension polymerase for the makoe loop sequence of keulrawoodin 3 SANTIIRDFYNPVVPEAQKREMGA chain Genes were obtained through reaction (overlap extension PCR). To prepare a cyclized recombinant peptide, a gene was obtained by inserting a TGT or TGC, which is a gene encoding a cysteine residue, at the end. In order to connect with the vector, CATATG and CTCGAG, which are cleavage sites of restriction enzymes NdeI and XhoI, were inserted in the 5 'and 3' directions, respectively, and a stop codon (TAA) was inserted in front of the XhoI cleavage site. TGT (C) -tcggccaacaccattatccgggacttctacaaccccgtggtgcccgaggcgcagaagcgcgagatgggcg cgggc-TGT (C) -TAA-CTCGAG The vector is pREP-HTX, which uses a T7 promoter and ampicillin resistance selective marker and contains a preparative Hexa histidine tag and a thrombin cleavage site gene.

After processing the restriction vector NdeI and XhoI for each of the obtained vector and the Claudein peptide genes, the genes were cut on the agarose gel, and the ligation was performed after securing the vector and peptide genes through purification. . T4 ligase was used to proceed with the vector: gene = 1: 3 molecular ratio.

The linking reaction was performed at 15 ° C. for 4 hours. After completion of the ligation, transformation was induced in a chemically prepared E. coli cell line (DH5α). After completion of the transformation induction, the colony was confirmed by plating on agar LB (Luria-Bertani) medium containing ampicillin at a concentration of 50 μg / ml and incubating at 37 ° C. for 18 hours. The colonies containing the target genes were accurately selected through the polymerase chain reaction to confirm whether the genes were included in the obtained colonies.

After incubating in liquid LB containing ampicillin at 37 ° C and 180rpm condition, when the absorbance value of 600nm wavelength reached about 0.7, 1mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added for 4 hours at 30 ° C. Abnormal expression. After the completion of expression, the entire culture medium was recovered only by expressing the cells by centrifugation (4 ℃, 5,000rpm, 15min). Expressing cells were subjected to cell disruption using high pressure. At this time, the expressing cells were suspended and suspended in nickel affinity chromatography buffer containing 5 mM imidazole and 0.5 M sodium chloride in pH 8.0 Tris buffer. The crushed liquid thus obtained was taken only by supernatant using a high-speed centrifuge of 10,000 rpm or more. Nickel affinity chromatography was performed to bind to the pre-prepared column, and then 10 mM Dithiothreitol (DTT) was used to reduce the cysteine residues at the protein ends. Then, the nickel affinity chromatography buffer was changed to perform on-column refolding. The thrombin cleavage solution was then cut using the prepared solution. At this time, the cleavage conditions were left at room temperature for more than 12 hours in the thrombin cleavage solution, but cut to 1 unit per 1mg of protein and flowed through a nickel affinity chromatography buffer solution to ensure only the cleaved protein antigen.

SDS-PAGE gel electrophoresis was performed on the obtained purified protein antigen under non-denatured conditions to confirm that it was a monomer, followed by pure separation of the monomer by gel filtration to obtain a purified cyclic purified protein antigen. SDS-PAGE results for CD151-2 of SEQ ID NO: 2 are shown in FIG. 5.

Example  3: loop type Peptides  And protein library screening

By comparing the loop peptide resin of cladin 3 and the linear peptide resin of SEQ ID NO: 3 sequence prepared as in Example 1, screening antibodies binding specific to claude protein and cladin protein and comparing the significance of steric antigen Phage library display method was used. As shown in the schematic diagram of Figure 6 E. coli TG1 cell line inoculated in LB medium and cultured until reaching the mid-log phase. Only the beads were recovered after reacting the clouded peptide and the humanized antibody library solution bound to the beads at room temperature for 1 hour. After washing three times with TBS-T, the beads were transferred to a membrane filter tube (membrane filter tube) and reacted at room temperature with 100 mM TEA (Triethylamine) for 10 minutes to separate the humanized antibody library bound to the bead-claudein peptide. The isolated humanized antibody library solution was neutralized with Tris-HCl pH7.4, and then inoculated into previously cultured E. coli TG1 cells and incubated at 37 ° C for 1 hour. The culture solution was plated in agar medium containing ampicillin and 2% glucose and incubated at 37 ° C. for 16 hours. 5 ml of LB medium was added to the agar medium, and colonies were scraped with a spreader, and 50 μl was inoculated into LB medium containing ampicillin and incubated at 37 ° C. After reaching the mid-log phase, 500 μl of 1 × 10 ¹¹ / ml VCSM13 helper phage was inoculated and incubated for 1 hour. After 1 hour, 70ng / ml kanamycin was inoculated and incubated at 37 ° C for 16 hours. After centrifugation of the culture solution, the supernatant was collected, and 20% PEG8000 containing 15% NaCl was added thereto, followed by reaction for 30 minutes in the refrigerator. The supernatant was removed by centrifugation, and the pellet was well dissolved in 500 μl PBS, followed by centrifugation at 12,000 rpm for 10 minutes. The supernatant was recovered and the procedure described above was repeated three more times. FIG. 6 is a schematic diagram of library screening using the produced loop-type antigen, and FIG. 7 is a panning result of screening an antigen of a cloudin protein. To determine the titer of panned library candidates, ELISA was performed as follows: biotin-bound Claudin peptide was attached to the microtiter plate at a volume of 100 μl at a concentration of 10 μg / ml per well, followed by sufficient washing. 5% skim milk was used to prevent nonspecific binding. After washing with TBS-Tween 20, antibody fragments expressed in E. coli were attached for 1 hour. After washing the plate sufficiently and washing the plate in the same manner after the reaction, anti-HA-horseradish peroxidase was added to bind the antibody fragment fixed to the plate for 1 hour at room temperature. After sufficient washing, the substrate solution (TMB) of peroxidase was added and developed to measure absorbance at a wavelength of 450 nm. This resulted in the primary screening of candidate antibody fragments. As shown in FIG. 8, the screening results showed that antibody fragments of higher reactivity were screened in the conformation antigen than in the linear antigen. Meanwhile, the inventors further screened antibody fragments or antibodies in the same manner through traditional hybridoma screening using Fab libraries and one synthetic scFv from other sources and mice.

Example  4: selected scFv of FACS  analysis

The primary screened antibody fragments from linear and looped antigens were subcloned into E- coli , expressed, purified and analyzed by FACS to determine the usefulness of the stereotyped looped antigens. For FACS analysis, SNU-8 cell line (Korean Cell Line Bank, Korea), a gastric cancer cell line that is overexpressed by Cladin, was used as the target cell line in this test. Cells were adjusted to density 2 × 10 ⁶ / ml at 1 ml per sample. Cultivation was performed with RPMI 1640 medium (Hyclone) + 10% FBS. The cells were scraped and washed with PBS, and the prepared antibody was attached for 1 hour. After washing again with PBS and attaching HA-FITC Ig for 1 hour, binding results were evaluated using a FacsCalibur ^™ flow cytometer (Becton Dickinson). In addition, OV-90 cell line as a control was confirmed through the same method described above. As confirmed in FIG. 9, it was found that the expressed loop-type antigen showed the best efficacy.

Example  5: selected scFv of SPR  analysis

In order to measure the specificity of the antibody fragments prepared in the present invention for anti-claudin, the clathine peptides were attached to the surface of the ProteOn GLC sensor chip, and the purified antibody fragments screened from the prepared linear and loop peptides were used. Adhesion was analyzed by plasmon resonance. A series of experiments were performed according to the rules provided by Bio-Rad. As shown in Figure 10, the scFv of the loop-type antigen than the scFv of the linear antigen can be seen that the binding force about 1,000 times higher, it was confirmed that the usefulness of the loop-type antigen of the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (11)

A polypeptide of an extracellular loop of a multi-transmembrane protein consisting of the amino acid sequences of SEQ ID NO: 3 or SEQ ID NO: 4, wherein the N-terminus and C of the polypeptide Complex transmembrane protein antigen, characterized in that the end is attached and fixed to a solid sunstrate.
delete delete A polypeptide of an extracellular loop of a multi-transmembrane protein consisting of the amino acid sequences of SEQ ID NO: 3 or SEQ ID NO: 4, wherein the N-terminus and C of the polypeptide Complex transmembrane protein antigens, wherein the ends are attached to both ends of the same linker to form a cyclic structure.
delete delete The complex transmembrane protein antigen according to claim 4, wherein the linker is selected from the group consisting of aminohexanoic acid, glycine polymer and alanine polymer.
5. The complex transmembrane protein antigen according to claim 4, wherein said linker is attached to a water soluble resin.
The complex transmembrane protein antigen according to claim 8, wherein the water soluble resin is a resin surrounded by polyethylene glycol (PEG).
An antibody or antigen-binding fragment thereof that specifically binds to the complex transmembrane protein antigen of any one of claims 1, 4 and 7-9.
10. Claims 1, 4 and 7-9 comprising contacting the complex transmembrane protein antigens of any one of claims 1, 4 and 7-9 with a humanized antibody library. A method of screening for an antibody that specifically binds to any one of the transmembrane protein antigens.

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