KR101356075B1 - Novel polypeptide binding with interleukin-6 - Google Patents

Abstract

The present invention provides a polypeptide capable of binding to interleukin-6 and inhibiting its activity, a polynucleotide encoding the polypeptide, an expression vector including the polynucleotide, a transformant into which the expression vector is introduced, and the transformation. It relates to a method for producing the polypeptide using a sieve. Since the polypeptide of the present invention can bind to IL-6 and inhibit its activity with a higher affinity than the IL-6 receptor (IL-6Ra) which exists in nature, the polypeptide for preventing or treating IL-6-related diseases It can be widely used for development.

Description

Novel polypeptide binding with interleukin-6}

The present invention relates to a novel polypeptide capable of binding to interleukin-6, and more particularly, the present invention relates to a polypeptide capable of binding to interleukin-6 and inhibiting its activity, a polynucleotide encoding the polypeptide, An expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, and a method of producing the polypeptide using the transformant.

Among various substances present in vivo, proteins play a wide range of biological roles as macromolecules that perform the maintenance and function of life phenomena. To play this role, protein-protein interactions must first occur, which is the basis of all life phenomena. If protein interactions are not properly regulated, homeostasis in the body is broken, resulting in various diseases. Therefore, various methods for treating diseases by artificially regulating protein interactions have been researched and developed, and various medicines that can be used for therapeutic purposes have been successfully developed.

Antibodies are important proteins in immune responses that perform biological functions through specific interactions with antigens. This specific binding force enables high therapeutic effects without side effects, unlike conventional low molecular weight chemicals. Therefore, many research institutes and pharmaceutical companies are actively working to develop antibodies that bind to well-known therapeutic targets using various screening techniques. Therapeutic antibodies developed in recent decades have shown significant success in the overall therapeutic market by showing high efficacy and low side effects in the treatment of various diseases, and continue to maintain high growth in the overall therapeutic market. In addition, it is widely used in various fields, such as separation and purification of biological material and molecular medical diagnostic technology, not just for therapeutic purposes. However, despite these advantages, the development of new therapeutics using antibodies has not been achieved due to problems such as low tissue penetration due to large molecular weight, high product cost due to complex production process, and entry barriers due to existing patents. It is true. To overcome these limitations, several research groups are actively researching new protein backbones to replace antibodies. As a result, a protein called Repebody was developed. The Repebody refers to a polypeptide optimized by consensus design by fusing based on the similarity of the structure of the VLR with the N-terminus of an internalin having a Leucine-rich repeat (LRR) structure, wherein the Repebody is a water-soluble monomer form in E. coli. Because it is expressed in large quantities, it is possible to lower the production cost, and has a high physicochemical stability to facilitate modification of the protein. In addition, it has the advantage of being free from existing patents because it is a new protein skeleton that has not been researched and developed.

The present inventors have successfully prepared a specific binding protein for MD-2 (Myeloid differentiation protein-2), a target protein related to pulmonary disease, by using the Repebody, and biologically inhibited by a cell-based detection method. Although it has been proved to be effective, the applied research is still in its infancy, and further research is being actively conducted, but no results have been reported yet.

Against this background, the inventors have made intensive efforts to develop Repebody as a universal binding protein backbone with binding ability to various proteins, and based on a random mutation library constructed through modular structural analysis and overall structural analysis of Repebody The present invention was completed by selecting a novel protein having binding ability to interleukin-6 and improving binding ability through an affinity increasing method based on a repeat module.

One object of the present invention is to provide a polypeptide that can effectively bind to interleukin-6 and inhibit its activity.

Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

Still another object of the present invention is to provide an expression vector comprising the polynucleotide.

It is still another object of the present invention to provide a transformant into which the above expression vector has been introduced.

Still another object of the present invention is to provide a method for producing the polypeptide using the transformant.

As one embodiment for achieving the above object, the present invention comprises the amino acid sequence of any one of SEQ ID NO: 9 and 11 to 21, and can effectively bind to interleukin-6 (IL-6) to inhibit its activity To provide a polypeptide.

The term "interleukin-6 (IL-6)" of the present invention is a glycoprotein having a molecular weight of 210,000 separated by B cell stimulating factor 2 (BSF-2), which induces the final differentiation of B cells into antibody-producing cells. It refers to a kind of cytokine produced by various cells such as lymphocytes, B lymphocytes, macrophages, and fibroblasts. It is involved in immune response, proliferation and differentiation of hematopoietic and nervous system cells, acute reactions, and growth and differentiation of various cells.

In order to develop novel polypeptides that can effectively bind IL-6 and inhibit its activity, the inventors of the N-terminus, Variable Lymphocyte Receptor (VLR) of the Internalin B protein A library was constructed containing randomly repeat modules of polypeptides fused with LRR (Leucine-rich repeat) protein portions. The polypeptide contained in the library may be coded according to the polynucleotide sequence of SEQ ID NO: 1 or 75%, preferably 85%, more preferably 90%, more preferably 95% or more of the polynucleotide sequence of SEQ ID NO: Lt; / RTI > sequence homologous to the nucleotide sequence of SEQ ID NO.

In addition, the library may be in the form of a phagemid containing the polynucleotide. The term "phagemid" of the present invention refers to a circular polynucleotide molecule derived from phage, which is a virus of Escherichia coli as a host, and includes sequences of proteins and surface proteins necessary for propagation and propagation. Recombinant phagemids can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes or the like generally known in the art. The phagemid may include a signal sequence or leader sequence for secretion in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, an enhancer, and mainly fused a desired protein with the surface protein of the phage to label the phage surface. It can be used for the method. The promoter of phagemid is mainly inducible and may include a selectable marker for selecting host cells. For the purpose of the present invention, the above-mentioned phagiimide includes MalEss, DsbAss or PelBss, which is a signal sequence or leader sequence for expressing and secreting a polynucleotide encoding a polypeptide constituting the library, and a recombinant protein A polynucleotide encoding the gp3 domain, which is a kind of surface protein of the M13 phage for expression to the phage surface, and a histidine-tag for confirming the expression, but is not limited to the polynucleotide of SEQ ID NO: Do not.

The present inventors used a phage display method using the library containing phagemid to select a novel polypeptide (SEQ ID NOs. 3 to 6) in the form of Repebody having excellent binding to IL-6. However, since these selected polypeptides showed a lower level of binding to IL-6 than IL-6 receptors in nature (IL-6Ra), the selected polypeptides were mutated to IL-6. It was intended to produce a mutant with improved binding capacity. To this end, a second library was constructed in which four amino acid residues were mutated to the polypeptide of SEQ ID NO: 5 having the highest binding capacity among the selected polypeptides (FIG. 8), and the phage display method using the second library was described. Using a second polypeptide (SEQ ID NOS: 8 to 10) in the form of a Repebody excellent binding capacity to IL-6 (Fig. 9), the highest binding capacity of the SEQ ID NO: 9 of the second selected polypeptide A third library was constructed by performing the same mutations on the polypeptide (FIG. 11), and a novel polypeptide in the form of Repebody having excellent binding ability to IL-6 was obtained using the phage display method using the third library (sequence). Nos. 11-21) were selected secondarily (Table 1).

As used herein, the term "internaline B protein" refers to a type of LRR family protein expressed in Listeria strains, which is different from other LRR family proteins in which other hydrophobic cores are uniformly distributed throughout the molecule. N-terminal structure is known to be stably expressed in microorganisms. The N-terminus of this internalin protein is stable in the microorganism because the N-terminus most important for the folding of the repeat module is derived from the microorganism and its form contains a more stable structure comprising alpha helix. Can be used for expression.

As used herein, the term "N-terminus of an internalin protein" refers to the N-terminus of an internalin protein necessary for water soluble expression and folding of the protein, and refers to an alpha helical capping motif and repeat module of an internalin protein. it means. The N-terminus of the internalin protein includes without limitation the N-terminus of the internalin protein necessary for the water soluble expression and folding of the protein, but may include, for example, the alpha helical capping motif "ETITVSTPIKQIFPDDAFAETIKANLKKKSVTDAVTQNE" and a repeat module. Preferably, the repeating module pattern may include "LxxLxxLxLxxN". L in the repeating module pattern is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine or tryptophan; N means asparagine, glutamine, serine, cysteine or threonine, x means hydrophilic amino acid. The N-terminus of the internalin protein can be selected as the N-terminus having high structural similarity according to the type of LRR family protein that can be fused, and the most stable amino acid can be selected through calculation of binding energy and the like. Variation is possible.

The term "Variable Lymphocyte Receptor" (VLR) of the present invention refers to a type of LRR family protein expressed in black eel and seven eel, and is a protein that performs immune function in black eel and seven eel. It can be usefully used as a skeleton that can bind. Polypeptides fused with the N-terminus and VLR protein of the internalin B protein have higher water solubility and expression than those of the VLR protein to which the internalin B protein is not fused, and thus can be used for the preparation of novel protein therapeutics based thereon. have.

The term "Leucine rich repeat (LRR) protein" of the present invention means a protein composed of a combination of modules in which leucine is repeated at a predetermined position, and (i) has one or more LRR repeating modules, and (ii) the LRR The repeat module consists of 20 to 30 amino acids, and (iii) the LRR repeat module has "LxxLxxLxLxxN" in a conserved pattern, where L is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine and tryptophan Hydrophobic amino acids, such as N, asparagine, glutamine, serine, cysteine or threonine, x means any, (iv) LRR family protein means a protein having a structure having a three-dimensional structure, such as horseshoe do. The LRR family proteins of the present invention are not only known in the sequence, or newly discovered using a newly derived mRNA or cDNA in vivo, but also have a sequence not known in nature through the design of a consensus design. It may include all variants with skeletal structure of the module.

The term "Repebody" of the present invention refers to a polypeptide that is optimized by consensus design by fusing based on the similarity of the structure of the VLR with the N-terminus of the internalin having an LRR structure. Repebody protein can be divided into concave region (Concave) and convex region (Convex) structurally (Fig. 4). The concave regions are known for their high degree of sequence diversity and important sites for protein interaction. Convex regions, on the other hand, play a role in keeping the overall structure of the protein stable based on highly conserved sequences. Repebody protein may include all of the fusion LRR family proteins that improve the water-soluble expression and protein biophysical properties of all proteins belonging to the LRR family having a repeat module in the above manner.

As another embodiment, the present invention provides a polynucleotide encoding the polypeptide, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.

The polynucleotide provided in the present invention is not particularly limited thereto, but may be a polynucleotide encoding any one of the amino acid sequences of SEQ ID NOs: 9 and 11 to 21, and more preferably 70% or more with the polynucleotide. May be a polynucleotide having a base sequence having at least 80%, more preferably at least 90% homology, but is not particularly limited thereto.

The term "vector" of the present invention means a DNA construct containing a nucleotide sequence of a polynucleotide encoding said target protein operably linked to a suitable regulatory sequence so as to be able to express the protein of interest in the appropriate host. The regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. After the vector has been transformed into a suitable host, it can replicate or function independently of the host genome and integrate into the genome itself.

The vector used in the present invention is not particularly limited as long as it can be replicated in the host, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, phagemids, cosmids, viruses and bacteriophages. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector, and pBR, pUC, and pBluescriptII systems are used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. The vector usable in the present invention is not particularly limited, and known expression vectors can be used. Preferably, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used. Most preferably, pACYC177, pCL, pCC1BAC vectors can be used.

As used herein, the term “transformer” refers to a cell in which a vector having a gene encoding at least one target protein is transfected into a host cell to express the target protein, and includes all eukaryotic cells, prokaryotic cells, and the like. Cells, but are not particularly limited to bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; Yeast cells; Fungal cells such as Pchia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, bow melanoma cells; Or plant cells.

As used herein, the term “transformation” means introducing a vector comprising a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides include all of them, as long as they can be expressed in the host cell, regardless of whether they are inserted into or located outside the chromosome of the host cell. The polynucleotide also includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self expression. The expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked with a sequence required for expression in the host cell.

In another embodiment, the present invention provides a method for producing a culture comprising (i) culturing the transformant to obtain a culture; And (ii) provides a method for producing a polypeptide comprising the step of recovering the polypeptide from the cultured transformant or culture.

In the above method, the step of culturing the transformant is not particularly limited thereto, but is preferably performed by a known batch culture method, continuous culture method, fed-batch culture method, and the like, and the culture conditions are not particularly limited thereto. , Using a basic compound (e.g. sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (e.g. phosphoric acid or sulfuric acid) at an appropriate pH (pH 5-9, preferably pH 6-8, most preferably pH 6.8) Can be controlled to introduce an oxygen or oxygen-containing gas mixture into the culture to maintain aerobic conditions, the incubation temperature can be maintained at 20 to 45 ℃, preferably 25 to 40 ℃, about 10 to 160 hours Incubating is preferred. The polypeptide produced by the culture may be secreted into the medium or remain in the cell.

In addition, the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources. Oils, peanut oils and coconut oils), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid), etc. may be used individually or in combination ; Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination; As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination; Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.

The method for recovering the polypeptide produced in the culturing step of the present invention is a method for recovering the desired polypeptide from the culture medium using a suitable method known in the art according to the culture method, for example, batch, continuous or fed-batch culture method, etc. Can be collected.

Since the polypeptide of the present invention can bind to IL-6 and inhibit its activity with a higher affinity than the IL-6 receptor (IL-6Ra) which exists in nature, the polypeptide for preventing or treating IL-6-related diseases It can be widely used for development.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an overall schematic diagram of an overlapping polymerase chain reaction performed on a module basis. Each yellow part is a variable repeat module with a total of four on the polypeptide. The red bar marked with red is the primer used in the experiment and the green part in the primer refers to the concave region sequence containing the NNK synonymous codon.
FIG. 2 is a graph showing the results of SDS-PAGE analysis of the expression of the repebody in the surrounding cytoplasm through two different signal sequences. Here, E is the number of the eluent, and the middle 27 KDa means the position in the standard marker used.
FIG. 3 is a graph showing Western blot analysis results for confirming the expression of a repebody on the surface of a phage using the phagemid pBEL118M of the present invention, wherein the mM unit means the concentration of IPTG used for inducing a promoter.
Figure 4 is a schematic diagram showing the overall protein structure of the Repebody, it is divided into Concave to recognize the biomolecule and Convex region important for structure maintenance.
FIG. 5 is a schematic diagram showing the entire structure showing amino acid residues in which a random library is to be constructed. FIG.
6 is a view showing the results of experiments to determine whether or not having specificity for the target protein through the enzyme immunoassay.
7 is a view and a diagram showing the results of experiments confirming the binding force of the polypeptide of the present invention to interleukin-6 by an isothermal calorimetry.
8 is a schematic diagram illustrating a method of building a second library. In this case, yellow residues represent residues in which existing libraries are constructed, and green amino acids represent corresponding positions in newly constructed libraries.
9 is a graph showing the measured binding force of binding candidates selected based on the second library.
10 is a schematic diagram illustrating a method of building a third library. In this case, yellow residues represent residues in which existing libraries are constructed, and green amino acids represent corresponding positions in newly constructed libraries.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are merely for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples, and various applications and modifications are possible.

Example  1: random Repebody  For library screening phagemid  design

As a component of the invention, a protein backbone named Repebody was used. The backbone is a water-soluble polypeptide in which the LRR portion including the N-terminus of the internalin B protein and the C-terminus of the VLR protein is fused to have an amino acid sequence identical to SEQ ID NO: 7.

Example  1-1: Signaling in peripheral cytoplasm using signal sequence Repebody  Expression

To determine whether the receptor is applicable to phage display, it is necessary to confirm the periplasmic expression of E. coli to be used as a host and to confirm that the protein is properly expressed on the surface particles of the phage. To this end, two recombinant vectors were constructed using the pMAL-c2x (NEB, USA) vector, in which the distinct signal polypeptides, MalE and DsbA, were inserted immediately after the start codon. Then, the final vector was completed by inserting a DNA fused with Repebody and histitin-tagged between the signal sequence and the stop codon. Two transformed vectors were introduced into the E. coli XL1-blue strain to prepare transformants, and the transformants were cultured until the absorbance (OD600) reached 0.5, followed by 0.1 mM IPTG (Isopropyl β-D- 1-thiogalactopyranoside) was treated to induce the expression of proteins, and then incubated at 30 ° C. for 16 hours. After the incubation, the cells were centrifuged to obtain cells, and the cells were sonicated to obtain a water-soluble protein fraction.

The obtained water-soluble protein fraction was purified and applied to Ni-NTA (Nickel-nitrilotriacetic acid) resin, and the amount of expression of the produced repebody in the cytoplasm was confirmed by SDS-PAGE analysis (FIG. 2). FIG. 2 is a graph showing the results of SDS-PAGE analysis of the expression of the repebody in the peripheral cytoplasm through two different signal sequences. In FIG. 2, E is the number of the eluent, and 27KDa in the center is the position . As shown in FIG. 2, it was confirmed that the MalE signal sequence exhibits a higher peripheral cytoplasmic expression ratio than the DsbA signal sequence.

Example  1-2: On the gripping surface Repebody  For expression Phagemid  making

Phagemid was designed based on the MalE signal sequence finally determined in Example 1-1. Phagemid was based on pTV118N (Takara, Japan), and the MalE signal sequence was inserted immediately after the start codon, followed by addition of DNA fused with Repebody and histidine-tag. In addition, gp3 was used to label relatively large proteins among several phage surface proteins, and the C-terminus was followed by Amber codon. Phagemid was completed. The phagemid was introduced into XL1-Blue to prepare a transformant, and the produced transformant was cultured in the same manner as in Example 1-1, except that 0.5 mM IPTG was treated, followed by centrifugation. To obtain a culture solution. The culture solution was subjected to polyethylene glycol sedimentation to purify phage.

As a result of western blot analysis using the above-mentioned phage, it was confirmed that Repebody was expressed on the surface of the phage (FIG. 3). FIG. 3 is a graph showing Western blot analysis results for confirming the expression of a repebody on the surface of a phage using the phagemid pBEL118M of the present invention, wherein the mM unit means the concentration of IPTG used for inducing a promoter.

Example  2: protein structure based Repebody  Build a library

Repebody, like the natural LRR proteins, has a modularity in which the repeat units with conserved leucine sequences are continuously connected to maintain the entire protein structure and the concave region due to the curvature of the shape of the whole structure. And a convex region (Fig. 4). FIG. 4 is a schematic diagram showing the entire protein structure of the repebody, which is divided into a Concave that recognizes a biomolecule and a Convex region that is important for maintaining the structure. The hypervariable region, like the complementarity determining region (CDR) of the antibody, is located in the recessed region and mediates protein-protein interactions. In addition, convex regions play an important role in maintaining the overall structure of LRR based on well-conserved sequences. By analyzing the protein structure of such a repebody, a random library was designed in the following manner.

Specifically, the concave region of two consecutive variant modules (LRRV module 1 and 2) located in the amine terminal end direction to escape the steric hinderance caused by the undesigned C-terminal loop structure. Six amino acid residues 91, 93, 94, 115, 117, and 118 located at were selected (FIG. 5). FIG. 5 is a schematic diagram showing the entire structure showing amino acid residues in which a random library is to be constructed. FIG.

Then, a mutagenic primer for constructing a library is constructed by replacing the selected amino acid with a degenerate codon of NNK, constructing a nucleotide sequence of the remaining convex region to include a silent mutation, Were synthesized.

Overlap PCR was then performed on the two modules using the primers, and library DNA was obtained (FIG. 1) and inserted into the phagemid pBEL118M to obtain the final library phagemid. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an overall schematic diagram of an overlapping polymerase chain reaction performed on a module basis. Each yellow part is a variable repeat module with a total of four on the polypeptide. The red bar indicates the primer used in the experiment and the green part in the primer means the sequence of the recessed region containing the codon NNK.

The obtained library was introduced into E. coli XL1-Blue by electroporation to obtain a transformant, thereby constructing a library having a synthetic diversity of 1.8 × ^{10 8} level.

Example  3: with phage display IL -6 specific binding protein screening

Example  3-1: Repebody  Purification of library phage and Panning  Combined with interleukin-6 through the process Polypeptide  Selection

The library constructed in Example 2 was cultured by the method of Example 1-1, and the phage in which Repebody was expressed on the surface was selected and purified by the method of Example 1-2. In order to select candidates capable of binding to IL-6, IL-6 was added to immunotubes at a concentration of 100 μg / ml and coated at 4 ° C. for 12 hours. The coated immunotubes were washed three times with PBS and blocked with PBS solution (TPBSA) containing 1% BSA and 0.1% Tween 20 for 2 hours at 4 ° C. The purified phages were then added to the coated immunotubes at a concentration of 10 ¹² cfu / ml and allowed to react at room temperature for 2 hours. After the reaction was completed, the mixture was washed with PBS solution (TPBS) containing 0.1% Tween 20 (TPBS) five times for two minutes and twice with PBS. Finally, 1 ml 0.2 M Gly-HCl (pH 2.2) was added to the immunotubes, and reacted at room temperature for 10 minutes to elute phages expressing candidates capable of binding to IL-6 on the surface. 60 μl of 1.0M Tris-HCl (pH9.1) was added to the eluate, neutralized, and added to a host cell, 10 ml E. coli XL1-Blue solution (OD600 = 0.5), and then bio-panning to a 2 × YT plate. ) Was repeated four times. As a result, it was confirmed that the phages that specifically bind to IL-6 are concentrated through each panning process. The results were analyzed to mean that the library phage binding to IL-6 specifically increased.

Example  3-2: Selected Repebody of IL Specific for -6 Combination  Identification and sequencing

The phage selected by the method of Example 3-1 was subjected to ELISA using a 96-well plate coated with IL-6 and BSA, so that the absorbance (OD450) of IL-6 was 10 times higher than that of BSA. Repebody candidates were selected, their respective amino acid sequences were checked, and WebLogo was performed to confirm consensus sequences. As a result, it was confirmed that a residue having a high frequency of mutation exists in the amino acid sequence of the protein specifically binding to IL-6 expressed in the selected phage. That is, amino acid 91 isoleucine is substituted with tryptophan, valine or threonine, amino acid threonine is substituted with arginine or glutamic acid, amino acid 94 with glycine is substituted with alanine, serine or proline, and amino acid 115 is valine It was confirmed that valine (silent mutation), serine, alanine or asparagine was substituted, 117 amino acid valine was substituted with lysine or tryptophan, and 118 amino acid glutamic acid was substituted with arginine, lysine or leucine.

The results were analyzed to mean that there are residues that play an important role in binding to IL-6.

Example  4: Repebody A module-based affinity enhancement method

Example  4-1: Specific Binding to Interleukin-6 Repebody Characteristic Analysis of

Based on the ELISA results performed in Example 3-2, four candidates (Repebody-B3 (SEQ ID NO: 3) and Repebody-C8 (SEQ ID NO: 4) having the highest absorbance for IL-6 among the Repebody candidates obtained above) were obtained. ), Repebody-D3 (SEQ ID NO: 5) and Repebody-F11 (SEQ ID NO: 6) were subjected to phage ELISA using a plate coated with IL-6, Lysozyme, BSA and Lysozyme (Fig. 6). FIG. 6 is a diagram showing the results of experiments confirming whether or not specificity of the target protein is obtained through enzyme immunoassay. As shown in FIG. 6, the four candidates had target specificity for IL-6.

Meanwhile, dissociation constants of the four candidates for IL-6 were measured. Specifically, using Repebody dissolved in 0.2 mM (6 mg / ml) in PBS and IL-6 dissolved in 0.02 mM (0.5 mg / ml) in PBS, using an isothermal titration calorimetry (ITC) , The dissociation constants of the four candidates for IL-6 at room temperature were measured (FIG. 7). 7 is a view and a diagram showing the results of experiments confirming the binding force of the polypeptide of the present invention to interleukin-6 by an isothermal calorimetry. As shown in FIG. 7, the dissociation constant (KD) of Repebody-D3 for IL-6 is 17 nM, the dissociation constant (KD) of Repebody-B3 is 48 nM, and the dissociation constant of Repebody-C8 among the four candidates. (KD) was 89nM, and the dissociation constant (KD) of Repebody-F11 was 117nM. Therefore, it was found that Repebody-D3 was able to bind IL-6 most effectively among the four candidates.

Example  4-2: Building additional libraries using modules and increasing binding

The dissociation constant for IL-6 of Repebody-D3, which was found to be able to bind to IL-6 most effectively in the results of Example 4-1, was 17 nM, but the receptor for IL-6 (IL-6Ra in nature) exists in nature. Since the dissociation constant for IL-6 of 9) is 9 nM, in order to function as an inhibitor, a dissociation constant lower than 9 nM must be secured. Therefore, the candidate for Repebody of the present invention cannot sufficiently inhibit the activity of IL-6. It was expected.

In order to solve this problem, we tried to develop a mutant with improved binding capacity to IL-6 using the modularity of Repebody.

Specifically, a module adjacent to the library region constructed in Example 2 (LRRV module 3) was selected to mutate four residues in the concave region in the same manner as in Example 2 (FIG. 8). 8 is a schematic diagram illustrating a method of building a second library. In this case, yellow residues represent residues in which existing libraries are constructed, and green amino acids represent corresponding positions in newly constructed libraries. Yellow residues represent residues in which existing libraries are constructed and green amino acids represent corresponding positions in newly constructed libraries. Four more panning cycles resulted in three candidates (Repebody-D3E5 (SEQ ID NO: 8), Repebody-D3E8 (SEQ ID NO: 9), and Repebody-D3E10 (SEQ ID NO: 10)) having increased binding strength. As a result of measuring the dissociation constant, it was confirmed that the binding force was increased to the level of 2 to 13 nM (FIG. 9). 9 is a graph showing the measured binding force of binding candidates selected based on the second library.

On the other hand, in order to develop a mutant showing more improved binding capacity, a third library was constructed by performing the same method as described above for the adjacent module (LRR2) in the internalin direction (FIG. 10). 10 is a schematic diagram illustrating a method of building a third library. In this case, yellow residues represent residues in which existing libraries are constructed, and green amino acids represent corresponding positions in newly constructed libraries. Candidates with improved binding capacity were selected for the third library using phage display, and candidates with dissociation constants of 400 pM were finally obtained (Table 1).

Sequence and Avidity of Repebody Candidates Repebody order
number Mutation location K _app (10 ⁶ M) K _D (10 ^-9 M) Fold 67 69 71 72 D3E8
D3E8B2
D3E8B3
D3E8B4
D3E8B8
D3E8C4
D3E8C7
D3E8C9
D3E8C11
D3E8H2
D3E8H3
D3E8H5 9
11
12
13
14
15
16
17
18
19
20
21 Y
Y
Y
Y
Y
K
Y
Y
Y
Y
M
V A
T
T
T
T
T
T
A
S
L
I
M G
V
Q
T
R
V
I
S
I
R
R
R G
Q
S
N
N
S
T
S
N
S
S
S 2.35
4.90
6.11
3.24
3.06
14.6
4.47
10.7
1.88
5.58
4.05
5.12 2.47
1.18
0.949
1.04
1.90
0.397
1.30
0.542
3.09
1.43
1.79
1.13 1.0
2.1
2.6
1.4
1.3
6.2
1.9
4.6
0.8
2.4
1.7
2.2

These results suggest that Repebody can rationally design libraries using modularity, which is unique to repetitive proteins, unlike conventional antibodies, and builds libraries based on adjacent modules in order to effectively bind to target proteins. It was confirmed that it can increase.

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Claims (8)

A polypeptide that specifically binds to the interleukin-6 protein, in which the N-terminus of the internalin B protein, the modified repeat module of the Variable Lymphocyte Receptor (VRL) protein, and the C-terminus of the VRL protein are fused, wherein the polypeptide is Polypeptide consisting of the amino acid sequence of any one of SEQ ID NO: 9 or 11 to 21.
delete The method of claim 1,
Said polypeptide binds to interleukin-6 protein and inhibits its activity.
The method of claim 1,
Wherein said modified repeat module of VRL protein comprises the following repeat module pattern:
LxxLxxLxLxxN
In the pattern,
L is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine or tryptophan;
N is asparagine, glutamine, serine, cysteine or threonine, and
x is a hydrophilic amino acid.
A polynucleotide encoding the polypeptide of claim 1.
An expression vector comprising the polynucleotide of claim 5.
A transformant into which the expression vector of claim 6 is introduced.
(Iii) culturing the transformant of claim 7 to obtain a culture; And
(Ii) recovering the polypeptide of claim 1 from the cultured transformant or culture, the method of producing a polypeptide that specifically binds to interleukin-6.

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