KR20130031137A - Fusion protein comprising ctla4 and il21r and composition for preventing or treating arthritis comprising the same - Google Patents

Abstract

PURPOSE: A fusion protein is provided to suppress T cell activity and to prevent or treat arthritis. CONSTITUTION: A fusion protein contains CTLA4(cytotoxic T-lymphocyte antigen 4) and IL21R(Interleukin-21 receptor). The fusion protein has an amino acid of sequence number 1. IL21R and CTLA4 have amino acid sequences of sequence numbers 2 and 3, respectively. The fusion protein additionally contains an immunoglobulin Fc region at N-terminal or C-terminal. A transformant is prepared by transforming a host cell with a recombinant vector containing a gene encoding the fusion protein. [Reference numerals] (AA) Stimulation (-); (BB) Stimulation (+); (CC) CTLA4 +IL-21Fc dual target(fusion protein)

Description

Fusion protein comprising CTLA4 and IL21R and composition for preventing or treating arthritis comprising the same}

The present invention relates to a fusion protein comprising CTLA4 and IL21R and a composition for treating arthritis comprising the same. The present invention also relates to a gene encoding the fusion protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, and a method for producing a fusion protein comprising culturing the host cell. .

Arthritis-related diseases are representative degenerative and refractory diseases in which about 12% of the world suffers. There are more than 2 million patients in Korea. Arthritis is a general name that causes inflammatory changes in the musculoskeletal and connective tissues of the body, resulting in systemic symptoms in the musculoskeletal system. The disease is characterized by chronic inflammation that affects joints, bones, cartilage tissue or spinal cord, often causing permanent tissue damage, malformations, degeneration and disorders (Hofbause, LC, et.al., Arthritis and Rheumatism 44: 253-259, 2001).

Osteoarthritis is the most commonly seen arthritis, also called degenerative arthritis, which causes degenerative changes in articular cartilage, such as pain, stiffness, and gradual dyskinesia in weight-bearing joints such as knee and hip joints as the articular cartilage wears away. Disease. In the past, as a cause of aging caused by the aging of the past, but now it is not a single cause, but the symptoms, such as age, genetic factors, obesity, joint shape, hormones, etc. It is thought.

Rheumatoid arthritis is an inflammatory autoimmune disease that occurs multiple times in multiple joints. The disease indicates persistent inflammatory synovitis, which causes cartilage destruction and bone erosion, causing structural malformations of the surrounding joints. Symptoms associated with rheumatoid arthritis include joint swelling, joint tenderness, inflammation, landscape stiffness and especially pain when bending the joint. This includes.

In addition, arthritis includes ankylosing spondylitis, reactive arthritis, psoriatic arthritis, systemic lupus erythematosus, polymyositis or rheumatoid polymyalgia, and the ideal goal for all arthritis treatments is to protect and maintain cartilage and bone tissue in the diseased joints. will be.

Currently used treatment methods for arthritis are as follows. Drugs commonly used in early treatment are nonsteroidal anti-inflammatory drugs (NSAIDs), which may slightly improve symptoms but do not prevent joint cartilage loss or disease progression. This severely manifested case requires about half of patients to stop treatment within a year. Next steps include: drugs modifying anti-rheumatic drugs such as gold drugs (gold sodium thiomalate, gold sodium thiosulfate), penicillamine, anti-malarials; DMARDs) and the like. However, these agents also slightly reduce the progression of arthritis but can have serious side effects, so only 5-15% of patients adhere to the use of these drugs after 5 years of treatment with DMARD. If the above treatments are no longer effective, it may be necessary to replace the joints with rheumatoid joints with external joints (Simon LS, Int. J. Clin. Pract., 54, pp 243-249, 2000).

Therefore, there is an urgent need for the development of new materials effective for the prevention and treatment of arthritis having a new function and skeletal structure that minimize the toxicity and side effects that are the limitations of current therapeutic agents of arthritis.

On the other hand, as arthritis is known to be induced by an immune response associated with antigen-nonspecific intercellular interactions between T-lymphocytes and antigen presenting cells, one development of new substances for the treatment of arthritis, such as rheumatoid arthritis As a strategy, attempts have been made to develop therapeutic molecules that block the costimulatory signal between T-lymphocytes and antigen presenting cells by blocking the interaction of CTLA4 (cytotoxic T-lymphocyte antigen 4) with its ligands. To this end, soluble CLTA4 molecules, etc., modified to bind to ligands and block costimulatory signals with greater binding than CTLA4 or antibodies that bind CTLA4, have been developed (Korean Patent Publication No. 2003-0017606, National Publication No. 2011-0014180, etc.).

In addition, IL-21 is a type of cytokine having an alpha-helix structure, and is known to cause an inflammatory response through a signaling process using the receptor and gamma chain of IL-21.

The present inventors have studied to develop a new therapeutic agent effective for arthritis, and as a result, a fusion of CTLA4 and IL12R (Interleukin-21 receptor) that antagonizes CTLA4 and IL-21 at the same time, compared to the case of using CTLA4 or IL12R alone The present invention was completed by confirming that CTLA4 and IL12R were expressed as a fusion, and thus, particularly effective in the treatment of arthritis.

One object of the present invention is to provide a fusion protein comprising cytotoxic T-lymphocyte antigen 4 (CTLA4) and Interleukin-21 receptor (IL12R).

It is another object of the present invention to provide a gene encoding the fusion protein.

It is another object of the present invention to provide a recombinant vector comprising the gene.

Still another object of the present invention is to provide a transformant transformed from a host cell with the recombinant vector.

Another object of the present invention to provide a method for producing a fusion protein comprising the step of culturing the transformant.

Still another object of the present invention is to provide a composition for preventing or treating arthritis, including a fusion protein including CTLA4 and IL21R.

It is another object of the present invention to provide a method for treating arthritis comprising administering a pharmaceutical composition comprising a fusion protein comprising CTLA4 and IL21R.

As one aspect for achieving the above object, the present invention relates to a fusion protein comprising a cytotoxic T-lymphocyte antigen 4 (CTLA4) and Interleukin-21 receptor (IL21R).

As used herein, the term "fusion protein" refers to a protein in a form in which two or more proteins are artificially linked, and in the present invention, a protein linked to CTLA4 and IL21R. Such fusion proteins can be obtained by chemical synthesis after each partner has been determined or by expression and purification by genetic recombination methods. Preferably, a fusion gene combining the nucleic acid encoding CTLA4 and the nucleic acid encoding IL21R can be obtained by expression in a cell expression system. In a preferred embodiment, the amino acids constituting the fusion protein of the present invention are shown by the amino acid sequence of SEQ ID NO: 1.

In the present invention, IL21R preferably uses an extracellular region of IL21R, but is not limited thereto, and more preferably, may have an amino acid sequence of SEQ ID NO: 2.

In the present invention, CTLA4 preferably uses an extracellular region, but is not limited thereto. More preferably, CTLA4 may be one having an amino acid sequence of SEQ ID NO.

The fusion protein of the present invention may further comprise an Fc region of an immunoglobulin at the N-terminus or C-terminus. The Fc region is preferably selected from the group consisting of IgA, IgD, IgE, IgG and IgM, and more preferably includes all or part of the CH2 and CH3 constant domains, but is not limited thereto. In addition, the CTLA4-IL21R fusion protein preferably includes a form having a constant domain region of the heavy chain of two or more equivalents, but is not limited thereto. These two Fc heavy chains are preferably connected by disulfide bonds or other covalent bonds, but are not limited thereto.

Such fusion recombinant proteins may be linked directly to CTLA4 and IL21R or via a linker such as a linker. In the present invention, the linker refers to a peptide inserted between the protein and the protein so that when the CTLA4 and IL21R are linked to make a recombinant fusion protein, the structural flexibility of the protein may be increased to enhance the activity of each protein to which it is bound. The linker does not reduce the overall physiological activity, and the type or number of amino acids is not limited as long as it can minimize the immune response, but preferably 1 to 20 amino acids, more preferably 1 to 5 amino acids are preferred. .

Fusion proteins of the invention include variants of fusion proteins as well as proteins having their wild type amino acid sequences. A variant of a fusion protein means a protein in which the normal amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Such variants have a protein which preferably has at least 75%, more preferably 85%, even more preferably 90% and most preferably at least 95% homology with the amino acid sequence of SEQ ID NO. It may include.

In addition, the fusion protein may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like. The variant or modifier is a functional equivalent that exhibits the same biological activity as the native protein, but may be a variant or modifier that modifies the properties of the protein as needed. Preferably, the structural stability against heat, pH, etc. of the protein is increased or protein activity is increased by the variation and modification on the amino acid sequence.

The fusion protein of the present invention may be chemically synthesized or produced through genetic recombination methods. Preferably, the recombinant protein may be transformed into a host cell to isolate and purify the protein expressed therefrom.

Thus, as another aspect, the present invention relates to a gene encoding the fusion protein and a recombinant vector comprising the gene.

As another aspect, the present invention relates to a transformed host cell transformed with the recombinant vector and a method for producing a fusion protein comprising culturing the host cell.

The process of producing the fusion protein of the present invention by the genetic recombination method includes the following steps.

First, a recombinant vector containing a gene encoding a fusion protein is prepared.

Gene sequences encoding CTLA4 and IL21R can be prepared by various methods known in the art, such as chemical synthesis, RT-PCR, respectively, and preferably a primer capable of amplifying the target gene using DNA of the target protein as a template. It can be prepared via PCR using. The obtained CTLA4 and IL21R nucleic acids can be operably linked to a vector to prepare a recombinant expression vector. In the present invention, any expression vector generally used in the art may be used without limitation, and preferably, a vector including Fc may be used. For the purposes of the present invention, expression vectors may further comprise signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, introns, polyadenylation signals and enhancers. In a specific embodiment of the present invention, the pYK602-Fc vector was used. In addition, a vector into which a nucleic acid encoding IL21R and CTLA4 was inserted to express the fusion protein of the present invention was named pYK602-Fc :: IL21R / CTLA4, and its structure is shown in FIG. 1, and the nucleic acid sequence of the vector is illustrated. 2 is shown.

Next, the host cell is transformed using the recombinant vector.

A host cell that can be transformed with a recombinant vector according to the present invention includes both prokaryotic and eukaryotic cells, and a host having high DNA introduction efficiency and a high expression efficiency of introduced DNA is commonly used. Bacteria, for example, known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera fruitgifer (SF9), CHO, COS 1, COS 7 Animal cells such as, BSC 1, BSC40, BMT 10 and the like can be used, preferably Escherichia coli can be used.

Methods for preparing transformants by introducing recombinant vectors into host cells include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, and agrobacterial mediated transformation. , PEG, dextran sulfate, lipofectamine mediated transformation methods, and the like.

Next, the transformant is cultivated in a suitable medium and conditions to obtain a fusion protein.

The medium and the culture conditions can be used by appropriately selecting the tolerable according to the host cell. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for growth of cells and mass production of proteins during the culture. The expressed fusion protein can be isolated from the cell's lysate. Specifically, cell pellets may be obtained by centrifugation or filtration, and an eluate including cell lysate may be obtained by removing media components including a ruptured cell wall or cell membrane. Specifically, cell pellets are well known in the art, such as alkalis, surfactants (CHAPS and SDS, etc.), the use of organic solvents and enzymes (lysozyme, etc.), sonication and the use of French Presses. And lysis by periodic high temperature, pressure, application of freezing and thawing cycles, and the like. The cell filtrate obtained above can obtain a clean eluate by removing the minimum area of biomass in the cell filtrate through cell fractionation, e.g., the unbroken part of the cell or the supernatant free of insoluble cell debris by centrifugation. Clear eluents can be obtained by taking these and, if necessary, can be subjected to additional purification procedures known in the art.

As another aspect, the present invention relates to a composition for preventing or treating arthritis, including a fusion protein comprising CTLA4 and IL21R.

In a specific embodiment of the present invention, TLR and APC (antigen-presenting cells) when stimulated with CD3 and cultured together, MLR analysis to determine the effect of CTLA4-IL-21R fusion protein to inhibit the T cell activity As a result, it was confirmed that CTLA4-IL-21R fusion protein has the ability to inhibit the immune response induced by T cells more strongly than when CTLA4 alone was used (FIG. 5). Thus, the fusion proteins of the present invention can be usefully used to prevent and treat arthritis by inhibiting immune responses associated with antigen-nonspecific cell interactions between T-lymphocytes and antigen presenting cells.

In the present invention, the term "treatment or prophylaxis" means any action that inhibits, alleviates or beneficially alters the clinical situation associated with arthritis by using a pharmaceutical composition comprising as an active ingredient a fusion protein comprising CTLA4 and IL21R. . Preferably the composition comprising a fusion protein comprising CTLA4 and IL21R can protect and maintain cartilage and bone tissue of the diseased joint by inhibiting the T-lymphocyte mediated immune response that causes arthritis.

In the present invention, the arthritis is osteoarthritis, rheumatoid arthrits, ankylosing spondylitis, reactive arthritis, psoriatic arthritis, systemic lupus erythematosus, polymyositis Or polymyalgia rheumatica and the like, preferably, rheumatoid arthritis or degenerative arthritis.

Pharmaceutical compositions comprising a fusion protein comprising CTLA4 and IL21R of the present invention may be formulated further comprising a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not significantly stimulate the organism and does not interfere with the biological activity and properties of the administered compound. The pharmaceutically acceptable carrier in the present invention may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, If desired, other conventional additives such as antioxidants, buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, or tablets such as aqueous solutions, suspensions, emulsions and the like.

Also preferably, the pharmaceutical composition of the present invention may further include fillers, excipients, disintegrants, binders and lubricants. The pharmaceutical compositions of the invention may also be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable drugs, sterile powders.

In another aspect, the invention relates to a method for treating arthritis comprising administering a pharmaceutical composition comprising a fusion protein comprising CTLA4 and IL21R.

As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be various oral or parenteral routes as long as it can reach the desired tissue. It can be administered through.

The method of treatment of the present invention comprises administering a pharmaceutically effective amount of a fusion protein comprising CTLA4 and IL21R. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. Preferably the dosage of the pharmaceutical composition may be used in the range of about 10 mg to about 200 mg per kg, and may be administered one time or divided into several times. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

Since the fusion protein of the present invention exhibits significantly superior T cell activity inhibitory effect than CTLA4 or IL21R alone, it can be usefully used for the prevention or treatment of arthritis.

Figure 1 shows the structure of the vector pYK602-Fc :: IL21R / CTLA4 for preparing the fusion protein of the present invention.
Figure 2 shows the nucleic acid sequence of the vector pYK602-Fc :: IL21R / CTLA4 for preparing the fusion protein of the present invention.
Figure 3 shows the structure and amino acid sequence of the fusion protein of the present invention.
Figure 4 shows the Western blot results confirming the expression and purification of the fusion protein of the present invention.
Figure 5 shows the degree of inhibition of T cell activation by concentration of CTLA treatment group (Group A) and CTLA4-IL21R fusion protein treatment group (Group B) when T cells and APC (antigen-presenting cells) were stimulated with CD3 and cultured together. It is shown.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. IL21R  And CTLA4 Preparation of fusion proteins comprising

1-1. Preparation of expression vector

To prepare a fusion protein containing IL21R and CTLA4, primers were used to secure the extracellular domain of each gene encoding IL21R and CTLA4 using a DNA Library mix (Kidney, placenta, pancrease, liver) as a template. Was designed to obtain the product amplified by PCR. The primers used are shown in Table 1, and PCR reaction conditions were performed for 2 minutes at 95 ° C., followed by 30 cycles of 20 seconds at 95 ° C., 40 seconds at 58 ° C., and 1 minute at 72 ° C., followed by 5 minutes at 72 ° C. It was.

primer Sequence (5'-3 ') SEQ ID NO: IL21R-SfiI-F agggggccgtgggggcccccgacctcgtctgctacaccg 5 IL21R / CTLA4-overlap-R acgtgcattgctttttcctttaactcctctgactgggtctg 6 IL21R / CTLA4-overlap-F ggagttaaaggaaaaagcaatgcacgtggcccagcct 7 CTLA4-SfiI-R gttttggccgacgcggccaagtcagaatctgggcacggttctgga 8

To insert the amplified gene into pYK602-Fc vector, a mammalian cell expression vector, it was cut with restriction enzyme SfiI (# R033S, Enzynomics, Korea), and then agarose gel (# 9002-18-0, BIO BASIC INC, USA), and then used Gel Purification kit (# 1014876, QIAGEN, USA) to obtain the insert to be inserted into the vector. Next, T4 DNA ligase (# M001S, Enzynomics, Korea) was inserted into the vector. Restriction enzyme reaction cuts 1ug DNA. 1ul SfiI (10unit) and 5ul 10x reaction buffer are mixed and sterile distilled water is added to make 50ul of total volume and reacted for 6 hours in a 37 ℃ warmer. Separation purification was performed. In addition, the Ligation was obtained by mixing 1 ul ligase (10 units) and 1 ul 10 × reaction buffer in a 50 ng vector at a ratio of 1: 3 to the vector and the insert, and incubating at 16 ° C. for at least 16 hours. The vector thus prepared was named pYK602-Fc :: IL21R / CTLA4, the structure thereof is shown in FIG. 1, and the nucleic acid sequence of the vector is shown in FIG. 2.

1-2. Preparation of Transformant

The above-prepared vector was transformed into E. coli to obtain a clone correctly inserted into the pYK602-Fc expression vector, and then subjected to midi prep (# 740 410.100, MACHERY-NAGEL, Germany). Transfected to 293E. Plate 293E cells in a 100 mm plate (# 430599 Corning USA) at 70-80% confluence, mix 13 ug of DNA and 26 ug 1: 2 in PEI (# 23966, Polysciences, USA) for 20 minutes at RT Placed and dropped. After 16 ~ 20 hours, exchange media with Serum Free Media (No FBS DMEM (# SH30243.01 Hyclone Thermo. USA)) and sup. The transfection was performed by kicking off.

1-3. Purification of Fusion Proteins

The sup. (Total 600 ml) was purified on days 2, 3 and 5 in 20 plates of 100 mm plates. First full sup. Was filtered using a 0.22 um top-filter (# PR02890 Millipore USA). This sup. Was bound to 500 ul Protein A beads (# 17-1279-03 GE healthcare Sweden) packed in a 5 ml column. 4 at 0.9 ml / min using a Peri-start pump o / n bound, washed with 100 ml or more PBS (# 70011 Gibco USA), eluted in 6 fractions with 0.1 M Glycine-HCl (# G7126, Sigma, USA) and eluted with 1M Tris (# T-1503-5KG.Sigma USA) (pH 9.0). Next, the protein was quantified and collected with 2-3 fractions containing protein, and then concentrated using amicon ultra (# UFC805024, Millipore, USA) and buffer changes about 3 times with 1X PBS (# 70011 Gibco USA).

As a result of purification, a protein of IL21R / CTLA4-Fc was obtained, and the purification yield was 2.4 mg / L. The structure and amino acid sequence of the purified fusion protein are shown in FIG. 3, and the purification results are shown in FIG. 4.

Example  2. MLR  ( mixed - lymphocyte reaction ) analysis

When T cells and APCs (antigen-presenting cells) were stimulated with CD3 and cultured together, MLR analysis was performed to determine the effect of CTLA4-IL-21R fusion protein prepared in Example 1 on inhibiting T cell activity. It was.

Blood from healthy donors was mixed with PBS and carefully floated on Ficoll-PaqueTM PLUS (17-1440-02, GE Healtrhcare). To separate the PBMC layer was centrifuged at 2000 rpm for 20 minutes at 20 ℃. The separated PBMCs were washed with sterile PBS. The supernatant was removed by centrifugation at 1500 ° C. for 5 minutes at 4 ° C., and CD4 + T cells were separated using the MACS CD4 + T cell separation kit (MACS 130-090-860). Non-CD4 T cells were prepared by radioactive radiation. 1x10 ⁵ CD4 + T cells and radiated non-CD4 T cells were seeded in 96 wells prepared with anti-CD3 coating. In addition, CTLA4 (group A) and CTLA4-IL-21R fusion protein prepared in Example 1 (group B) were mixed at different concentrations (0.1, 1, 10 ug / ml), and then cultured for 58 hours. After addition of 1 μCi / mL [ ³ H] -thymidine between 58-72 hours, 16 hours later [ ³ H] -thymidine using a liquid scintillation counter (Beckman Coulter, Palo Alto, Calif., USA) Influx was measured. [ ³ H] -thymidine influx was expressed as mean of 3 wells

The results are shown in Fig. In FIG. 5, group A shows the result of treatment with CTLA4 only by the specified concentrations (0.1, 1, 10 ug / ml), and group B shows CTLA4-IL- by concentrations (0.1, 1, 10 ug / ml). The result of processing the 21R fusion protein is shown. In the group A, the tendency of T cell activity to be suppressed is very low, whereas in the group B treated with the fusion protein of the present invention, the concentration-dependently potent T cell activity was suppressed. This shows that the CTLA4-IL-21R fusion protein of the present invention has the ability to inhibit the immune response induced by T cells more strongly than when CTLA4 alone is used.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION          A & R therapeutics co., Ltd. <120> Fusion protein comprising CTLA4 and IL21R and composition for          preventing and treating arthritis comprising the same <130> DPP20114433KR <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 338 <212> PRT <213> Artificial Sequence <220> <223> fusion protein comprising CTLA4 and IL21R <400> 1 Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile   1 5 10 15 Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp Gln              20 25 30 Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His          35 40 45 Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp      50 55 60 Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp  65 70 75 80 Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala Glu                  85 90 95 Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser Gly             100 105 110 Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe Tyr         115 120 125 Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg Gly     130 135 140 Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp Ser 145 150 155 160 Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser Tyr                 165 170 175 Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln Gly             180 185 190 Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser Glu         195 200 205 Glu Leu Lys Glu Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu     210 215 220 Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro 225 230 235 240 Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser                 245 250 255 Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu             260 265 270 Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln         275 280 285 Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr     290 295 300 Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile 305 310 315 320 Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp                 325 330 335 Ser Asp         <210> 2 <211> 212 <212> PRT <213> Artificial Sequence <220> <223> IL21R <400> 2 Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile   1 5 10 15 Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp Gln              20 25 30 Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His          35 40 45 Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp      50 55 60 Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp  65 70 75 80 Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala Glu                  85 90 95 Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser Gly             100 105 110 Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe Tyr         115 120 125 Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg Gly     130 135 140 Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp Ser 145 150 155 160 Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser Tyr                 165 170 175 Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln Gly             180 185 190 Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser Glu         195 200 205 Glu Leu Lys Glu     210 <210> 3 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> CTLA4 <400> 3 Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg   1 5 10 15 Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr              20 25 30 Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu          35 40 45 Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp      50 55 60 Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr  65 70 75 80 Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val                  85 90 95 Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr             100 105 110 Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp         115 120 125 <210> 4 <211> 1100 <212> DNA <213> Artificial Sequence <220> PYK602-Fc :: IL21R / CTLA4 vector (Fig. 2) <400> 4 tcgacgccac catgggatgg agctatatca tcctcttttt ggtagcaaca gctacagatg 60 tccactcgca gggggccgtg ggggcccccg acctcgtctg ctacaccgat tacctccaga 120 cggtcatctg catcctggaa atgtggaacc tccaccccag cacgctcacc cttacctggc 180 aagaccagta tgaagagctg aaggacgagg ccacctcctg cagcctccac aggtcggccc 240 acaatgccac gcatgccacc tacacctgcc acatggatgt attccacttc atggccgacg 300 acattttcag tgtcaacatc acagaccagt ctggcaacta ctcccaggag tgtggcagct 360 ttctcctggc tgagagcatc aagccggctc cccctttcaa cgtgactgtg accttctcag 420 gacagtataa tatctcctgg cgctcagatt acgaagaccc tgccttctac atgctgaagg 480 gcaagcttca gtatgagctg cagtacagga accggggaga cccctgggct gtgagtccga 540 ggagaaagct gatctcagtg gactcaagaa gtgtctccct cctccccctg gagttccgca 600 aagactcgag ctatgagctg caggtgcggg cagggcccat gcctggctcc tcctaccagg 660 ggacctggag tgaatggagt gacccggtca tctttcagac ccagtcagag gagttaaagg 720 aaaaagcaat gcacgtggcc cagcctgctg tggtactggc cagcagccga ggcatcgcca 780 gctttgtgtg tgagtatgca tctccaggca aagccactga ggtccgggtg acagtgcttc 840 ggcaggctga cagccaggtg actgaagtct gtgcggcaac ctacatgatg gggaatgagt 900 tgaccttcct agatgattcc atctgcacgg gcacctccag tggaaatcaa gtgaacctca 960 ctatccaagg actgagggcc atggacacgg gactctacat ctgcaaggtg gagctcatgt 1020 acccaccgcc atactacctg ggcataggca acggaaccca gatttatgta attgatccag 1080 aaccgtgccc agattctgac 1100 <210> 5 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> IL21R-SfiI-F primer <400> 5 agggggccgt gggggccccc gacctcgtct gctacaccg 39 <210> 6 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> IL21R / CTLA4-overlap-R primer <400> 6 acgtgcattg ctttttcctt taactcctct gactgggtct g 41 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> IL21R / CTLA4-overlap-F <400> 7 ggagttaaag gaaaaagcaa tgcacgtggc ccagcct 37 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> CTLA4-SfiI-R <400> 8 gttttggccg acgcggccaa gtcagaatct gggcacggtt ctgga 45

Claims (13)

A fusion protein comprising CTLA4 (cytotoxic T-lymphocyte antigen 4) and IL21R (Interleukin-21 receptor).
The fusion protein of claim 1, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 1.
The fusion protein of claim 1, wherein said IL21R is an extracellular region of IL21R.
The fusion protein according to claim 1, wherein the IL21R has an amino acid sequence of SEQ ID NO: 2.
The fusion protein of claim 1, wherein said CTLA4 is an extracellular region of CTLA4.
According to claim 1, wherein the CTLA4 is a fusion protein having the amino acid sequence of SEQ ID NO: 3.
The fusion protein of claim 1, further comprising an Fc region of an immunoglobulin at the N-terminus or C-terminus of the fusion protein.
A gene encoding the fusion protein of any one of claims 1 to 7.
Recombinant vector comprising the gene of claim 8.
The transformant transformed the host cell with the recombinant vector of claim 9.
A method for preparing the fusion protein of claim 1, comprising culturing the transformant of claim 10.
A composition for preventing or treating arthritis, comprising the fusion protein of any one of claims 1 to 7.
The arthritis of claim 12, wherein the arthritis is osteoarthritis, rheumatoid arthrits, ankylosing spondylitis, reactive arthritis, psoriatic arthritis, systemic lupus erythematosus ), Polymyositis or polymyalgia rheumatica.

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