KR101510302B1 - A stem cell transformed by minicircle vector expressing antibody directed to IL-6 receptor - Google Patents

Abstract

The present invention relates to a stem cell therapeutic agent comprising a vector expressing anti-IL-6R. More particularly, the present invention relates to a stem cell comprising a mini-circle vector expressing anti-IL-6R, and a pharmaceutical composition for treating an autoimmune disease comprising the same.

Description

(A stem cell transformed by minicircle vector expressing antibody directed against IL-6 receptor) transformed through a mini-circle vector so that an antibody targeting the IL-6 receptor is produced.

The present invention relates to a stem cell therapeutic agent comprising a vector expressing anti-IL-6R. More particularly, the present invention relates to a stem cell comprising a mini-circle vector expressing anti-IL-6R, and a pharmaceutical composition for treating an autoimmune disease comprising the same.

Immunity refers to the function of protecting the body from external substances by recognizing and eliminating external antigenic substances present in the body. An autoimmune disease is a disease in which the immune system is susceptible to a part of an individual's own body, thereby destroying the body itself, resulting in a deficiency in the ability to distinguish between self and non-self. Rheumatoid arthritis, insulin- Inflammatory bowel disease, ulcerative colitis, myasthenia gravis, multiple myositis, dermatomyositis, Crohn's disease, autoimmune cytopenias, vasculitis syndrome, and systemic lupus erythematosus.

In particular, rheumatoid arthritis, one of the autoimmune diseases, is an inflammatory autoimmune disease that is still unknown, despite the high prevalence of 21 million people, which is about 1% of the world's population. This disease causes structural malformations of the surrounding joints, and it shows persistent inflammatory synovitis that causes cartilage destruction and bone erosion. It also causes joint inflammation and pain as well as joints and surrounding tissues, and penetrates into various organs, It is accompanied by systemic organ involvement and pain, which is invaded by the eye, resulting in a tremendous loss of quality of life for the patient and disabling life like normal people.

Although autoimmune diseases have been increasing worldwide, the underlying causes of these diseases have not been sufficiently elucidated. Currently, the treatment of autoimmune diseases is aimed at regulating the autoimmune response and restoring the damaged immune function of the body. Typically, an immunosuppressant is used to suppress the immune response. Presently used immunosuppressants include prednisone, a corticosteroid drug, and cyclophosphamide, azathioprine, and tacrolimus, which are non-steroidal drugs. Recently, anti-TNF antagonists have been developed to treat various autoimmune diseases.

Tumor necrosis factor (TNF) is a multispecific cytokine that plays an important role in the inflammatory response and immune system and has been reported to play an important role in the bone destruction process. Therefore, monoclonal antibodies and recombinant proteins against TNF ligands have been developed to inhibit TNF signaling, as an autoimmune disease therapeutic agent for inhibiting the action of TNF. Infliximab, etanercept, and adalimumab have been shown to be highly efficacious in clinical use for nearly a decade as a representative treatment for autoimmune arthritis.

However, despite the high efficacy of these anti-TNF therapeutics, there are many problems that must be overcome. For example, as a side effect of treatment, the suspension of the mechanism of TNF increases the risk of infection of the fungus or germs in the immune system, resulting in an increased risk of relapsing pulmonary tuberculosis in particular. According to the Rheumatology Society, patients with rheumatoid arthritis who received anti-TNF therapies are more likely to develop skin cancer than those who received conventional treatments.

Accordingly, the present invention suggests a new concept of gene therapy agent as a therapeutic agent for autoimmune disease which overcomes the disadvantages of safety and efficacy of existing autoimmune disease therapeutic agents.

A gene therapy method is a method of injecting a gene related to a disease into a patient's body and expressing the gene in a cell to treat a disease, wherein the injected gene is successfully transferred to the nucleus of the target cell, It is important to make them appear.

The gene transfer technology for this purpose is largely classified into a method using a virus as a carrier, a nonviral method using a synthetic phospholipid or a synthetic cationic polymer, and a physical method such as an electrotransmission method in which a gene is introduced by applying a temporary electrical stimulus to the cell membrane . Herein, the method using the viral carrier has advantages such as excellent propagation efficiency and long-term stable gene expression. However, when the administered gene permanently changes the structure of the gene in the human chromosome or has a recombinant ability, And the activation of the immune defense system of the biologic safety issues, such as the practical clinical application is subject to restrictions. The non-viral gene transfer method has the advantage of enhancing the biological stability and freely changing the chemical composition and structure of the transporter. However, the transfection efficiency and the time of expression are very inferior to those of the viral transporter, It is difficult to use for clinical use. Therefore, there is a need for a gene delivery system that specifically acts on a target cell or a target tissue and has high expression efficiency.

Recently, research on stem cells and therapeutic application of stem cells have emerged as an alternative to overcome the problems of safety and efficiency of existing gene therapy and to enable new cells and gene therapy for human body.

However, current stem cell research is still in its infancy, so it requires a lot of basic research and many problems must be solved for clinical application. In particular, it is necessary to identify the donor cell engraftment, migration, differentiation, external gene expression, and the mechanism of integration into the host during the administration of the stem cells into vivo, and confirm that the administered stem cells achieve appropriate functional improvement in the host .

Accordingly, the present inventors have developed a gene therapy vector system that efficiently transfers a therapeutic gene to a target cell, expresses the therapeutic gene continuously and for a long time, and has high biological safety, and introduces the gene therapy vector system into a stem cell, The present invention provides a novel stem cell therapeutic agent capable of overcoming the above problems.

It is an object of the present invention to provide a stem cell therapeutic agent comprising a vector expressing anti-IL-6R.

Specifically, one object of the present invention is to provide a stem cell into which a non-viral vector expressing anti-IL-6R is introduced.

It is another object of the present invention to provide a pharmaceutical composition for preventing and treating autoimmune diseases including stem cells.

The present invention relates to a stem cell into which a non-viral vector expressing anti-IL-6R is introduced.

In a preferred embodiment, the stem cells of the present invention are

(a) a gene expression cassette comprising a promoter, a gene encoding an anti-IL-6R monoclonal antibody, and a transcription terminator,

(b) a site-specific recombination region located outside the gene expression cassette, and

(c) does not include the cloning start point and selectable marker gene

It is a stem cell into which a nonviral vector is introduced.

Hereinafter, the present invention will be described in more detail.

As used herein, the term "stem cell" refers to a cell that has the ability to differentiate into various cells and has autoproliferative capacity, and includes embryonic stem cells isolated from the early embryo, Embryonic germ cells isolated from the cells, and pluripotent adult stem cells isolated from the adult bone marrow and the like.

The stem cells usable in the present invention include all pluripotent stem cells derived from mammalian embryos, fetus, umbilical cord blood, adult tissues such as adult organs or bone marrow, skin or blood, Lt; / RTI > cells. In this case, the term "a trait similar to embryonic stem cells" means that a surface (antigen) marker specific to embryonic stem cells is present, an embryonic stem cell-specific gene is expressed, a teratoma- Or a chimeric mouse capable of forming an embryonic stem cell. Examples of adult stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, adipose stem cells, and the like.

The present invention relates to an anti-IL-6R protein having therapeutic effect, which is introduced into a stem cell in the form of a gene to be delivered to a disease site in the body and expressed at the site, thereby reducing half- It is possible to solve problems such as reduction of activity, solve problems of transfer efficiency and stability of existing gene therapy agents, and provide therapeutic effect continuously and stably.

In order to solve the problems of biological stability such as immunogenicity of the viral vector system which has been used in the gene therapy method of the present invention and the problem of the delivery efficiency of the non-viral vector system, , A vector system capable of sustaining and expressing the therapeutic gene for a long time and having a high biological safety, was applied to stem cells.

Specifically, the vector system of the present invention includes a gene expression cassette composed of only minimal essential elements such as a promoter, a gene encoding an anti-IL-6R protein, and a transcription terminator. But does not include a selectable marker gene such as an antibiotic resistance gene.

The origin of replication is mainly a bacterial sequence, which can cause an unnecessary immune response in the human body, and a selectable marker gene such as an antibiotic resistance gene can be transmitted to bacteria present in the body, There is a problem that unnecessary antimicrobial resistance may be caused. Thus, the present invention does not include a cloning start point and a selectable marker gene, thereby eliminating the problem of unnecessary immune response and antibiotic resistance. It is also possible to reduce the immune response by removing unmethylated CpG motifs derived from prokaryotic cells. In addition, since the vector of the present invention has a smaller physical size than the conventional vector, the vector of the present invention can be easily manufactured, improve the delivery efficiency, and improve the biological stability.

The vector of the present invention is characterized by being a double-stranded DNA in a circular supercoiled form.

The promoter used in the vector of the present invention may be any promoter used in the art without any particular limitation, including inducible or constitutive promoters, preferably CMV (cytomegalovirus) promoter.

The transcription terminator used in the vector of the present invention may be a terminator used in the art without any particular limitation, but may preferably contain the SV40 polyadenylation sequence.

The vector of the present invention further comprises a site specific recombination outside the gene expression cassette composed of the promoter, the gene encoding the anti-IL-6R protein as a therapeutic gene, and the transcription terminator.

The site-specific recombination region refers to a region where recombination between specific two base sequences on DNA can occur. The gene cloning method using the site-specific recombination region can be performed using a conventional method using a restriction enzyme and a ligase Which can be used as a genetic engineering method. Preferably, in the present invention, the site-specific recombination region may be an att-attached sequence derived from E. coli or bacteriophage lambda, a substrate sequence of attB or attP, or a hybrid sequence of attR or attL, It is useful for gene cloning by the attB + attP < - > attL + attR reaction in the presence of a recombinant enzyme.

The therapeutic protein introduced into the stem cells of the present invention is an anti-IL-6R (Interleukin-6 receptor) protein.

In the present invention, interleukin (IL) is a kind of cytokine, and plays a role of a chemical signal between red blood cells. Among these, interleukin-6 (IL-6) has been found to be abnormally generated in inflammatory diseases, autoimmune diseases and tumors and has been suggested to be involved in the pathogenesis of such diseases.

In the present invention, the anti-IL-6R monoclonal antibody has a high binding affinity for the IL-6 receptor and can exhibit particularly excellent effects in the treatment of autoimmune diseases by inhibiting the interaction between IL-6 and IL-6 receptors.

As used herein, the term "variable region" means a portion of an antibody molecule that exhibits a number of mutations in the sequence while specifically binding to an antigen, and the variable region includes complementarity determining regions (CDRs) CDR2 and CDR3 are present. A framework region (FR) portion exists between the CDRs to support the CDR ring.

The term "complementarity determining region" as used herein also refers to an annular region involved in recognition of an antigen, and the specificity of the antibody to the antigen is determined as the sequence of the region changes.

In a preferred embodiment, the anti-IL-6R monoclonal antibody of the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: And a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

Preferably, the anti-IL-6R monoclonal antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 25, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26.

The anti-IL-6R monoclonal antibody specified as the heavy chain variable region and the light chain variable region as described above will be referred to as "A7" in the present specification.

In another preferred embodiment, the anti-IL-6R monoclonal antibody of the present invention comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: A heavy chain variable region comprising CDR3; And a light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

More preferably, the IL-6R-specific monoclonal antibody may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

The anti-IL-6R monoclonal antibody that is identified as the heavy chain variable region and the light chain variable region as described above will be referred to as "B10" in the present specification.

In another preferred embodiment, the anti-IL-6R monoclonal antibody of the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: A heavy chain variable region comprising CDR3; And a light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 16, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 17, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

More preferably, the IL-6R-specific monoclonal antibody may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 29 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 30.

The anti-IL-6R monoclonal antibody specified as the heavy chain variable region and the light chain variable region as described above will be referred to as "D2" in the present specification.

In another preferred embodiment, the anti-IL-6R monoclonal antibody of the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 20 and a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: A heavy chain variable region comprising CDR3; And a light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 23, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

More preferably, the IL-6R-specific monoclonal antibody may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 31 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 32.

The anti-IL-6R monoclonal antibody specified as the heavy chain variable region and the light chain variable region as described above will be referred to as "F2" in the present specification.

In a specific embodiment of the present invention, anti-IL-6R monoclonal antibodies with high binding affinity for IL-6R were prepared using phage display technology.

The term "panning ", as used in connection with phage display technology, refers to the ability to bind a target molecule (antibody, enzyme, cell surface receptor, etc.) from a phage library that expresses the peptide in the coat of the phage Refers to the process of selecting only the phage expressing the peptide on the surface.

As used herein, the term "binding affinity" refers to the degree to which an antibody and an antigen form a specific binding, and the affinity of the binding is defined by the term k _a (rate constant for association of the antibody from the antibody / , k _d (dissociation constant), and K _d (k _d / k _a ).

The binding or specific binding means the binding affinity (K _d ) of 10 ^-8 mol / l or less, preferably 10 ^-9 M to 10 ^-13 mol / l. Therefore, the antibody according to the present invention is specifically bound to an antigen, and its binding affinity (K _d ) is 10 ^-8 mol / l or less, preferably 10 ^-9 M to 10 ^-13 mol / to be.

More specifically, in the present invention, an IL-6R antigen is obtained by a recombinant technique, and the IL-6R is reacted with and panned with a library phage prepared from human-derived scFv library cells having diversity, 6 monophage clones were screened. After confirming the selected monophage clones by fingerprinting, the respective sequences were analyzed to confirm the CDR regions of the variable regions (V _H and V _L ). The similarity of the variable region and the variable region of the germ line antibody group was confirmed using the Ig BLAST program of NCBI (www.ncbi.nlm.nih.gov/igblast/), and each of these monophage clones was subjected to IgG To produce a complete monoclonal antibody.

Among these, monoclonal antibodies with excellent inhibitory effect on IL-6 were selected through STAT3 reporter analysis.

In the present invention, the vector expressing the anti-IL-6R protein may be prepared by a number of known methods, for example transient transfection, microinjection, transduction, electroporation, DEAE dextran, Can be introduced into cells or tissues by mediated transfection, monovalent cationic liposome fusion, multivalent cationic liposome fusion, protoplast fusion, lipofectamine, and naked DNA delivery.

In the present invention, stem cells into which a vector expressing a physiologically active protein is introduced can be expected to have the following effects.

First, stem cells can serve as effective drug delivery vehicles for genes encoding bioactive proteins. Since the stem cells exhibit a homing effect when they are injected into a living body, the stem cells in the present invention can serve as a delivery medium for targeting the lesion site, And the effect of the gene therapeutic agent can be provided. In addition, in the present invention, stem cells can continuously and stably express the therapeutic gene. In a specific embodiment of the present invention, it was confirmed that the stem cells into which the vector system of the present invention was introduced successfully expressed the proteins.

Second, the physiologically active protein introduced into the stem cells enhances the inherent immunological function of the stem cells, and can exhibit the effect as a cell therapy agent having an enhanced therapeutic effect of stem cells. "Treatment" herein refers to any act that inhibits, alleviates, or beneficially alters the clinical condition associated with the disease. Treatment can also mean increased survival compared to the expected survival rate when untreated. Treatment includes preventive measures in addition to therapeutic measures.

The stem cells according to the present invention may be administered in a manner such that they are directly transplanted or moved to a desired tissue site, for example, inducing the death of disease-related cells, affecting signal transduction systems, controlling immune function, A therapeutic effect can be provided in such a manner as reconstructing or regenerating a deficient part.

Accordingly, in another aspect, the present invention relates to a pharmaceutical composition for preventing and treating autoimmune diseases, comprising a stem cell into which a vector expressing the anti-IL-6R protein is introduced.

The present invention also relates to a method for treating an autoimmune disease comprising administering a stem cell into which a vector expressing the anti-IL-6R protein is introduced.

In the present invention, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin dependent diabetes mellitus, multiple sclerosis, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, myasthenia gravis, multiple myositis, dermatomyositis, Crohn's disease, , Systemic lupus erythematosus, and the like. More preferably, it is rheumatoid arthritis.

The composition of the present invention may be formulated to further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not substantially stimulate the organism and does not interfere with the biological activity and properties of the administered ingredients. The pharmaceutically acceptable carrier in the present invention may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more components of these components, If necessary, other conventional additives such as antioxidants, buffers and bacteriostats may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulated into injectable solutions, pills, capsules, or tablets such as aqueous solutions, suspensions, emulsions and the like.

Also preferably, the composition of the present invention may further include a filler, an excipient, a disintegrant, a binder, a lubricant, and the like. The composition of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

The term "administering" in the present invention means introducing the composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention may be carried out through various routes of oral or parenteral routes, &Lt; / RTI &gt; But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal.

In the present invention, the composition may be administered in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. For purposes of the present invention, the specific therapeutically effective amount for a particular patient will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition including whether or not other agents are used, the age, weight, And various factors including diet, time of administration, route of administration and minute of composition, duration of treatment, drugs used or co-used with the specific composition, and similar factors well known in the medical field.

The present invention specifically recognizes IL-6R, and an anti-IL-6R antibody capable of antagonistic action against IL-6R can be applied to a new vector system and introduced into a stem cell, . Accordingly, it is an object of the present invention to solve problems such as decrease in half-life and decrease in physiological activity of existing protein therapeutic agents due to in vivo decomposition, to solve problems of delivery efficiency and stability of existing gene therapy agents, And can provide a stable therapeutic effect.

Figure 1 shows the preparation of an IL-6R antigen for the production of an anti-IL-6R monoclonal antibody. (A) is a result of Western blotting of IL-6R secreted in the supernatant by temporarily expressing IL-6R using a pYK 604 vector in a 150 mm dish. (B) is a result of confirming the purity of IL-6R used as an antigen through chromatography, and (C) is a result of analysis using an Agilent 2100 analysis program 6R &lt; / RTI &gt;
Figure 2 shows the results of a poly-phage ELISA assay for the production of anti-IL-6R monoclonal antibodies.
Figure 3 shows the results of monophase ELISA analysis for the production of anti-IL-6R monoclonal antibodies.
Figure 4 shows fingerprinting results for a monophage clone.
FIG. 5 shows the affinity of the six different monophage clones specifically binding to IL-6R in a stepwise dilution and showing the degree of affinity to IL-6R by ELISA. The degree of affinity was changed according to the degree of dilution Both showed high binding affinity.
FIG. 6 shows phage clone screening results using IL-6R full protein among the results shown in FIG. 5. As a result, B10 has the highest binding force and shows binding strength in the order of D2, A7, A10 and A3 .
Figure 7 shows Western blotting that monophage clones were converted to whole IgG form.
Fig. 8 shows the results of purifying IgG antibodies of monophage clones A3, A7 and A10.
FIG. 9 shows the results of purifying IgG antibodies of monophage clones B10, D2 and F2.
FIG. 10 shows the binding affinity (Kd value) of IL-6R monoclonal antibodies A3, A7, A10, B10, D2 and F2 to IL-6.
FIG. 11 shows the results of analyzing IL-6 inhibition of anti-IL-6R monoclonal antibodies A3, A7, A10, B10, D2 and F2 through STAT3 reporter analysis.
Fig. 12 shows the structure of anti-IL-6R monoclonal antibody prepared in the present invention.
13 is a schematic diagram showing a process of manufacturing a mini-circle vector used in the present invention.
FIG. 14 (A) shows the results of electrophoresis after enzyme cut in the parent plasmid, and FIG. 14 (B) shows the result of introducing a parent plasmid into the E. coli host so that amplification and cloning can be performed. (C) shows the results obtained by electrophoresis that a mini circle vector was prepared using Arabidopsis, and (C) shows the results obtained by electrophoresis.
Fig. 15 shows the result of confirming the expression of GFP by introducing the mini-circle vector into a 293T cell line after constructing to express GFP.
FIG. 16 shows the result of confirming the expression of GFP in vivo by administration to a mouse after constructing the mini-circle vector to express GFP.
17 (A) shows the expression of light chain (LC) and heavy chain (HC) after inserting DNA expressing anti-IL-6R antibody (named A7) into the parent plasmid, and (B) 6R antibody, and then transformed into a mini-circle vector with arabinose, and the expression thereof was confirmed. (C) indicates that the mini-circle vector expresses the anti-IL-6R antibody. The results obtained by cutting are shown.
FIG. 18 shows the results of confirming that the mini-circle vector expresses an anti-IL-6R antibody (named A7) through GFP expression in a 293T cell line. In each figure, LD shows a low dose and HD shows a high dose.
FIG. 19 (A) shows the result of once again confirming that the mini-circle vector expresses the anti-IL-6R antibody (named as A7) through GFP expression in the 293T cell line. (B) shows the result of confirming the concentration of anti-IL-6R present in the culture supernatant of the cell.
FIG. 20 shows that the gene expression efficiency of the mini circle vector is superior when the parent plasmid and the minicircle vector expressing GFP in stem cells are respectively introduced.
Figure 21 shows the effect of the A7 antibody on the proliferation of fibroblast-like synoviocytes (FLS).
Figure 22 shows the effect of the A7 antibody on the inhibition of migration of rheumatoid arthritis synovial cells (FLS).
Figure 23 is a graphical representation of the effect of inhibiting the migration of rheumatoid arthritis synoviocytes (FLS) by the A7 antibody.
24 shows the swelling score of the foot for each animal when the A7 antibody is administered to an animal model of collagen-induced arthritis (CIA). Wild type means normal animal, and CIA means collagen-induced arthritis animal.

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  1. The anti- IL -6R Monoclonal  Preparation of antibodies

1-1. IL -6R antigen protein

The human cDNA library for IL-6R-Fc antigen was used to confirm the specificity of IL-6R by sequencing. Then, the cells were cloned into an animal cell expression vector pYK604, transfected into HEK293E cells, induced transient transfection, and cultured. After transfection, the culture supernatant was secured at intervals of 2 days, 4 days, 6 days and 9 days and purified using protein beads. The purity of IL-6R was confirmed using Protein agilent 230 kit as QC data, and gel migration was confirmed (FIG. 1).

1-2. Phage  Manufacture of library

The final OD was adjusted to 0.5-0.7 by adding 300 ml 2XYT (+ Glucose, MgCL2, CM) to the stock OD of 0.1. Helper phage was infected with MOI 20. It was held in place for 30 min at 37 ° C, followed by shaking for 10 min. The cultured solution was centrifuged at 4,500 rpm for 15 min at 4 ° C to remove the supernatant, and the media was replaced with 300 ml of 2XYT (+ MgCl2, CM, IPTG, Kan) and incubated overnight at 30 ° C. After centrifugation at 4,500 rpm for 15 min at 4 ° C, the supernatant was added to 4% PEG600 and 3% NaCl and incubated for 1 hr on ice. And centrifuged at 4 ° C at 8,500 rpm for 20 min. After removal of the supernatant, the phage was dried for 5 min and dissolved in 5 ml of cold PBS to be used for panning.

1-3. 1st ~ 3rd Panning  ( panning )

30 ug of the IL-6R full domain protein was coated on the immunotube at 4 ° C for overnight. PBS was blocked with 3 ml of 4% skim milk for 2 hours at room temperature. After that, 2 ml of phage prepared in 16% skim milk was added and then blocked for 2 hours at room temperature. 1x PBST for 10 min at 2 min. 100 mM TEA, 500 μl each for 2 minutes for 5 minutes, and neutralized with 250 μl each of 1 M Tris (pH 7.5). The elution fraction thus obtained was collected and used for titering. After leaving about 100 μl of the elution fraction, 8 ml of XL1-blue was added to the final O.D 0.5-0.7, and the cells were infected with 2 ml of the immunotube. At this time, the cells were allowed to infect at 37 ° C for 30 minutes. The infected cells were collected and centrifuged at 4,500 rpm at 4 ° C for 15 minutes. Then, the supernatant was discarded and 2 ml 2XYT (+ GLUCOSE, MgCl 2) was added to the plate. The plate was plated on a square plate and incubated at 37 ° C overnight.

As shown in the following Table 1, a human library possessed by A & RT was made into a phage form using M1 helper phage to bind to IL-6R to make an antibody against IL-6R, and the resulting product was bound to IL-6R as a primary panning output 3.1 x 10 ⁶ was obtained. The result of the first panning was converted into the phage form, bound to the IL-6R, and the second panning output was enriched to 1.69 x 10 ⁷ , and the second panning output was again The result of binding to IL-6R in phage form was enriched to 8.2 x 10 ⁸ by the third panning output.

IL-6R full 1st panning Secondary panning Third panning Input 8.5 x 10 ¹³ 2.78 x 10 ¹³ 2.3 x 10 ¹³ output 3.1 x 10 ⁶ 1.69 x 10 ⁷ 8.2 x 10 ⁸ Washing # (PBST + PBS) 7 (5 + 2) 13 (10 + 3) 23 (20 + 3)

1-4. Poly Phage ELISA  analysis

To elucidate the effect of the a-Myc, FC signal, we performed dilution of 5 each by using the supernatant obtained by phage infection. First, 100 ng / well of IL-6R, 100 ng / well of a-Myc and 100 ng / well of FC were coated on 96 well plates overnight at 4 ℃. 2% skim milk in 1X PBS was blocked with 200 μl / well at 37 ° C for 2 hours. The prepared dilution phage was added and incubated at 37 ° C for 2 hours. After washing with 1x PBST using an ELISA washer, anti-M13-HRP was bound at 1: 2000 concentration for 1 hour at 37 ° C, washed again, and read at 490nm using substrate OPD.

FIG. 2 shows the binding affinity with IL-6R by making a phage form of primary, secondary, and tertiary panned cells of the library and the presence of specificity for IL-6R as a result of the polyphage ELISA analysis. In most cases, it is hard to find any specificity in the library and 1 ^st , but after that, we can confirm that the signal is stronger from the second iterative process. Therefore, it can be confirmed that antibodies specific to IL-6R are present in the second and third orders. A-myc and Fc were used as negative controls.

1-5. Mono Phage ELISA  analysis

96-well 3- ^rd panning colonies were picked in 96-well plates (1 ml of 2XYT-glucose-MgCl2-CM) and coated overnight at 37 ° C. 100 μl of 2XYT-glucose-MgCl2-CM was incubated at 37 ° C for 3 to 4 hours at OD of 0.12. And the final OD was 0.5. helper phage was placed at 37 ° C for 10 to 30 minutes. The medium was centrifuged at 3,500 rpm for 20 minutes at 4 ° C, and the media was changed to 2XYT-IPTG-MgCl2-CM-Kan and incubated overnight at 30 ° C. The supernatant was centrifuged at 4 ° C at 3,500 rpm for 20 min, and the supernatant was used for mono-ELAISA. IL-6R full, a-Myc, and FC were coated at 4 ° C for one day in 96-well plates at 100 ng / well. After blocking at 37 ° C for 2 hours with 1 x PBS 200ul / well in 2% skim milk, the phage supernatant obtained above was once again blocked with 100ul at 37 ° C for 2 hours. 1XPBS was washed with an ELISA washer and then anti-M13-HRP was bound at 1: 2000 concentration for 50 min at 37 ° C. After further washing, substrate OPD was read at 490 nm and used.

FIG. 3 shows the results of the monophage ELISA analysis. As a result of the second panning out put and the third panning out put, the colony was selected to make 96 phage forms, and the binding affinity for IL-6R was once again confirmed. Red shades were also excluded because they also bind to Fc. Since IL-6R has Fc, it is excluded because phage binds to Fc nonspecifically.

1-6. Mono Phage  Screening and fingerprinting

The colony was picked using 1ul of the cell stock. To the colony thus obtained, 10 μl of colony PCR and 10 μl of colony PCR product were added and incubated at 37 ° C. for 2 hours in a Bst NI 0.2 μl / 20 μl reation. After that, 8% PAGE gel was prepared and electrophoresed.

6 kinds of phage forms were obtained as a result of fingerprinting of the phage form having good binding affinity to IL-6R (FIG. 4).

1-7. Selected Mono Phage  Sequence analysis of clones

DNA was extracted from the six kinds of monophage clones identified in Examples 1-6 and sequenced. Sequence analysis was performed by SolGent and ABI 3730, an automatic sequencer, was used for the analysis. As a result, CDR regions of the heavy chain (VH) and light chain (VL) of the selected antibody were identified as shown in Tables 2 and 3. Similarity between the antibody and the germ line antibody group was confirmed using NC BLI's Ig BLAST program (www.ncbi.nlm.nih.gov/igblast/). As a result, in the case of the heavy chain, the human germline sequence Homology between 87% and 96%, and between 90% and 95% homology with light chain. Also, the polypeptides used in CDR3 of the heavy chain and light chain of each human antibody were analyzed, and the sequences were found to be different from each other.

Clone name The heavy chain variable region (V _H ) CDR1 CDR2 CDR3 A7 DYAMH
(SEQ ID NO: 1) GVSWNSGTIAYVDSVKG
(SEQ ID NO: 2) DFTYFYESSGYYAFDL
(SEQ ID NO: 3) B10 DYAMF
(SEQ ID NO: 7) GINWNGNGIGYGDSVRG
(SEQ ID NO: 8) PSLYGGNSEFDL
(SEQ ID NO: 9) D2 NYAIN
(SEQ ID NO: 13) RIIPMLGTSDYAEKFQG
(SEQ ID NO: 14) GPRYYGTDSYYLEK
(SEQ ID NO: 15) F2 NYYMH
(SEQ ID NO: 19) IINPSGGNTGYAQKFQG
(SEQ ID NO: 20) GLPWGENGLDV
(SEQ ID NO: 21) A3 EYAFH
(SEQ ID NO: 38) DFTPAFGEGDTAQKFQG
(SEQ ID NO: 39) ETASTY
(SEQ ID NO: 40) A10 DYAMH
(SEQ ID NO: 44) VISGDDGTTHYADSVKG
(SEQ ID NO: 45) DMYPYGDYGYLQH
(SEQ ID NO: 46)

Clone name The light chain variable region (V _L ) CDR1 CDR2 CDR3 A7 TGTNSNIGAGYDVH
(SEQ ID NO: 4) GNTNRPS
(SEQ ID NO: 5) QSFDSSLT
(SEQ ID NO: 6) B10 TGPTIGAGYDVH
(SEQ ID NO: 10) GNLNRPS
(SEQ ID NO: 11) HTYDSSLS
(SEQ ID NO: 12) D2 TGPTIGAGYDVH
(SEQ ID NO: 16) GNLNRPS
(SEQ ID NO: 17) HTYDSSLS
(SEQ ID NO: 18) F2 TGSSSNIGAGYDVH
(SEQ ID NO: 22) GDSDRPS
(SEQ ID NO: 23) QSYDSSLS
(SEQ ID NO: 24) A3 TGPTIGAGYDVH
(SEQ ID NO: 41) GNLNRPS
(SEQ ID NO: 42) HTYDSSLS
(SEQ ID NO: 43) A10 RASQGISSYLA
(SEQ ID NO: 47) AASTLQS
(SEQ ID NO: 48) QQLNTYPL
(SEQ ID NO: 49)

Clone name V _H Homology V _L Homology group IL-6R full a-myc Fc Ratio A03 VH1-69 252/288
(87.5%) V1-13 265/293
(90.4%) One 2.1076 0.2999 0.0418 7.027675892 A07 VH3-9 279/293
(95.2%) V1-13 279/293
(95.2%) 2 2.4774 0.0797 0.0421 31.08406524 A10 VH3-43 270/295
(91.5%) L8 271/286
(94.8%) 3 1.435 0.8478 0.095 1.692616183 6B10 VH3-9 260/287
(90.6%) V1-13 265/293
(90.4%) 4 2.0979 0.1125 0.0406 18.648 D02 VH1-69 267/289
(92.4%) V1-13 265/293
(90.4%) 5 2.3382 0.3031 0.0415 7.714285714 F02 VH1-46 286/295
(96.9%) V1-13 277/292
(94.9%) 6 2.8567 0.2869 0.0405 9.957127919

1-8. IL -6R full , IL -6R FN3 - FN3 Ranking ELISA

FIG. 5 shows ELISA showing the degree of affinity to IL-6R by stepwise dilution of six different monophage clones specifically binding to IL-6R. The ELISA showed a change in binding force according to the degree of dilution Both showed high binding affinity.

FIG. 6 shows phage clone screening results using IL-6R full protein among the results shown in FIG. 5. As a result, B10 has the highest binding force and shows binding strength in the order of D2, A7, A10 and A3 . F2 showed the lowest bond strength.

1-9. all IgG  Conversion analysis

After conversion of the six monophage clones into whole IgG form, Western blot analysis confirmed whether the assemble was successful in mammalian cells. A3 was weakly assemble, but all five were well assimilated and expression levels were high (Fig. 7).

1-10. term IL -6R Monoclonal  Purification of antibodies

The purified purity and gel migration were confirmed using Agilent 230Kit as QC data for each of the 6 antibodies (FIGS. 8 and 9).

Example  2. The anti- IL -6R Monoclonal  Binding Affinity Analysis of Antibodies

2-1. term IL -6R Monoclonal  Antibody Kd  Measure value

IL-6R at 100 ng / well for one day. Each well was blocked with 2% skim milk (200 ul) at 37 ° C for 2 h. Dilution was performed twice at 50 nm, and the mixture was incubated at 37 ° C for 2 hours to bind 100ul each. After washing 3 times with 1xPBST, anti-Fab-HRP was bound at a concentration of 1: 4000 in 100ul at 37 ° C for 1 hour. After washing 3 times with 1xPBST, OPD 1tablet and 6ul H ₂ O ₂ were added to 10ml of PC buffer. After treating with 100ul at room temperature for 7min, the reaction was stopped with 50ul of NH ₂ SO ₄ and read at 490nM.

Table 5 below shows the results of measurement of the Kd value of each antibody according to the dilution concentration of IL-6R, and FIG. These results indicate that A3, A7 and F2 exhibit very high binding affinity for very small amounts (0.02441 nM) of IL-6R protein.

Clone name 50 25 12.5 6.25 3.125 1.5625 0.78125 0.39063 0.19531 0.09766 0.04883 0.02441 kd value A3 2.781 2.2787 1.8467 1.4486 1.1317 0.857 0.6503 0.5904 0.5249 0.4996 0.4894 0.5156 3.5x10 ^-9
(R ² : 0.83) A7 3.0994 2.8431 2.5795 2.1338 1.6383 1.1682 0.7819 0.6175 0.5464 0.4973 0.4983 0.5145 2.3x10 ^-9
(R ² : 0.93) A10 2.5724 2.2591 1.8109 1.4725 1.1259 0.8664 0.6497 0.5731 0.5022 0.4599 0.4693 0.4952 2.93x10 ^-9
(R ² : 0.85) B10 2.0937 1.7787 1.3939 1.0362 0.761 0.6069 0.5 0.4762 0.4289 0.4619 0.4508 0.4679 4.3x10 ^-9
(R ² : 0.75) D2 2.3833 1.9404 1.4694 0.9809 0.7194 0.5843 0.4786 0.4732 0.4688 0.4498 0.4514 0.4486 7.5x10 ^-9
(R ² : 0.8) F2 2.9947 2.8868 2.9209 2.7102 2.7158 2.433 1.9001 1.4102 1.0056 0.7428 0.5836 0.535 3.5x10 ^-10
(R ² : 0.98)

2-2. STAT3 - by reporter analysis IL -6 Inhibition  Test

A STAT3-reporter assay was performed to confirm the function of the anti-IL-6R monoclonal antibody produced. STAT3 was expressed in a cell line expressing the reporter gene with lucifrerase attached thereto. IL-6 was added to the cell line at a dose of 50 ng / well in all conditions except for the control group. After that, anti-IL-6R prepared according to each condition was treated with 0.001 ug, 0.01 ug, 0.1 ug and 1 ug / well, respectively, and the signal induced by IL-6 was detected by anti-IL-6R Was measured. The prepared cells were treated with each cytokine and antibody and then incubated for 24 hours. After that, lucifease substrate was treated with each cell to induce color development, and the degree of expression was confirmed by measuring the color.

As a result, the A7, B10, D2 and F2 IL-6R antibodies inhibited IL6 signaling. Among them, A7 and F2 were superior to each other in inhibitory effect (Fig. 11).

Example  3. Manufacture of Mini Circle Vector

3-1. Of DNA  Amplification

100 μl of competent cell (DH5a) was dissolved at 4 ° C for 5 minutes. 1 μg of the DNA sample (GFP or anti-IL-6R) to be amplified was mixed with 100 μl of competent cells and incubated at 4 ° C. for 20 minutes. DNA was introduced by thermal shock at 42 DEG C for 45 seconds. Lt; RTI ID = 0.0 &gt; 4 C &lt; / RTI &gt; for 2 minutes. Thereafter, 500 μl of LB (lysogeny broth) liquid medium was added thereto, followed by incubation at 37 ° C. for 1 hour with stirring at 180 rpm. After centrifugation at 5000 rpm for 30 seconds, 500 쨉 l of the supernatant was removed. The remaining cells were plated on an LB plate containing kanamycin and then incubated in a 37 ° C incubator for one day. One colony was picked from a LB plate streaked overnight in 5 ml LB medium containing kanamycin and incubated for 7 to 8 hours with stirring at 180 rpm. 1 ml of 5 ml LB medium previously incubated with 200 ml LB medium containing kanamycin was added, followed by incubation with stirring at 180 rpm for about 15 hours. After the incubation was completed, the mixture was centrifuged at 4,000 rpm for 15 minutes at 9000 rpm, and the supernatant was discarded. DNA was extracted using the Macherey-nagel Nucleobond Xtra Midi kit (Cat # REF 740410.50).

3-2. A parental plasmid ZYCY10P3S2T  E. coli Transformation into

100 ㎕ of competent cell (ZYCY10P3S2T) was dissolved at 4 째 C, 1 DNA of DNA was mixed with competent cells, and incubated at 4 째 C for 30 minutes. After applying a thermal shock for 30 seconds at 42 캜, the ice was subjected to cold shock for 2 minutes. At the end of the low-temperature impact, 0.5 ml of SOC medium was added, and incubation was carried out for 1 hour while stirring at 225 rpm at 37 占 폚. Then, 100 占 퐇 was taken and plated on an LB medium plate containing kanamycin. Plates were incubated overnight in a 37 &lt; 0 &gt; C incubator.

3-3. Mini Circle Vector ( minicircle DNA ) Production

Colonies were picked from 5 ml of LB medium containing kanamycin in a plate into which DNA was introduced into ZYCY10P3S2T competent cells. The LB medium containing the cells was incubated at 37 DEG C and 200 rpm for 8 hours. 100 μl of the incubated LB medium was added to 200 ml of TB (terrific broth) medium containing kanamycin, and TB buffer was incubated at 37 ° C and 200 rpm overnight (15 hours). On the next day, the absorbance at OD600 was measured. When the value was between 4 and 5, a minicircle induction mix was prepared by adding 8 ml 1N NaOH and 200 l 20% L-arabinose to 200 ml LB medium And then mixed with the incubated TB medium. And incubated at 30 DEG C with stirring at 200 rpm for 5 hours. After the incubation, centrifugation was carried out at 4 DEG C at 9000 rpm for 15 minutes, and the supernatant was discarded except for the pellet. DNA was extracted using a Nucleobond Xtra Midi kit (Macherey-nagel, Cat #REF 740410.50.).

3-4. Mini circle with restriction enzyme DNA Confirm the creation of

To confirm that the mini-circle DNA was properly generated, restriction enzyme reaction was performed with restriction enzymes BamHI and XbaI (enzynomics). The prepared mini-circle DNA was mixed with restriction enzyme buffer II, BSA, and third distilled water in an appropriate amount, followed by incubation at 37 ° C for 30 minutes. The incubated samples were electrophoresed on 0.8% agarose gel along with DNA samples without restriction enzyme treatment.

FIG. 14 (A) shows the results of electrophoresis after enzyme cut in the parent plasmid, and FIG. 14 (B) shows the result of introducing a parent plasmid into the E. coli host so that amplification and cloning can be performed. (C) shows the results obtained by electrophoresis that a mini circle vector was prepared using Arabidopsis, and (C) shows the results obtained by electrophoresis.

Example  4. Through mini circle vector GFP  Identification of protein expression

4-1. Identification from cell lines ( in vitro )

In order to confirm that the mini circle DNA generated in Example 3 correctly induced protein synthesis, mini circle DNA with GFP inserted was transfected into 293T cells. On the day before transfection, 3 x 10 &lt; ⁴ &gt; of 293T cells were plated in 96 well plates with 100 [mu] l of DMEM medium containing 7.5% FBS but no antibiotic. 0.2 μg of Mini Circle DNA was mixed with 25 μl of opti-MEM1, and 0.5 μl of lipofectamine 2000 (Invitrogen) and 25 μl of opti-MEM were mixed, followed by incubation at room temperature for 5 minutes. Mini-Circle DNA and Lipofectamine 2000 were mixed together and incubated at room temperature for 20 minutes. Then, the mixture was sprayed on 293T cells, incubated at 37 ° C for 4 hours, and changed into growth medium. Expression was confirmed 24 hours later.

FIG. 15 shows the result of confirming expression of GFP by introducing the mini-circle vector into a 293T cell line after constructing to express GFP.

4-2. In rats  Confirm ( in vivo )

Mini circle DNA was introduced into rats to confirm that the mini circle DNA generated in Example 3 expressed proteins in the body. Female mice 6-7 weeks old were used with DBA1 / J (Orient Bio). 7-14 mu g of GFP-infected Mini Circle DNA was mixed with 1.5-1.8 ml of saline corresponding to 10% of the body weight of the mice, Intravenously into the tail.

FIG. 16 shows the result of confirming expression of GFP in vivo by administration to a mouse after constructing the mini circle vector to express GFP.

Example  5. Mini-circle vector IL -6R expression

5-1. Mini circle with restriction enzyme DNA Confirm the creation of

A mini-circle vector in which DNA encoding anti-IL-6R was inserted was prepared according to the methods of Examples <3-1> to <3-4>, and mini-circle DNA was generated using restriction enzymes.

FIG. 17 (A) shows a result of inserting a DNA expressing anti-IL-6R (designated as A7) into a parent plasmid and confirming its expression, and (B) (C) shows that the mini-circle vector expresses anti-IL-6R by restriction enzyme cleavage. The results are shown in FIG.

5-2. Identification from cell lines ( in vitro )

In order to confirm that the mini-circle DNA generated in Example 3 expresses anti-IL-6R, mini circle DNA having inserted DNA expressing GFP and anti-IL-6R was transfected into 293T cells, -1 &gt;. &lt; / RTI &gt;

FIG. 18 shows the results of confirming that the mini-circle vector expresses anti-IL-6R (named A7) through GFP expression in a 293T cell line. In each figure, LD shows a low dose and HD shows a high dose.

5-3. By ELISA  Confirmation of expression

In order to confirm the expression of anti-IL-6R, 293T cells were transfected with mini-circle DNA, and culture supernatant was obtained therefrom, and ELISA for anti-IL-6R was performed. 100 μl of anti-hIL-6R antibody (1 μg / ml) was added to a 96-well corning coaster 9018 plate and kept at 4 ° C. overnight. After washing five times with wash buffer, 100 μl of human sIL-6R was added at 1 μg / ml and incubated at room temperature for 2 hours. After washing again, the 1x assay diluent was treated with 200 쨉 l per well and incubated at room temperature for 1 hour. After washing five times with wash buffer, tociluzumab was diluted to a standard of 100 pg / ml in 1x assay diluent, and the remaining samples were diluted with a certain amount of serum samples, and then treated with 100 μl each well. Lt; / RTI &gt; After washing five times, anti-hIgG-HRP as a detection antibody was diluted at a ratio of 1: 10,000, and the mixture was incubated at room temperature. After washing 7 times, TMB substrate solution was added and incubated at room temperature for 15 minutes. 50 [mu] l of the termination solution was treated, and the absorbance at 450 nm was measured.

FIG. 19 (A) shows a result of confirming again that the mini-circle vector expresses anti-IL-6R (named A7) through GFP expression in a 293T cell line, and (B) The results of confirming the concentration of the present anti-IL-6R are shown.

Example  6. Identification of gene expression in stem cells through mini-circle vectors

In order to confirm whether the minicircle vector generated in Example 3 correctly induced gene expression in stem cells, the parent plasmid in which the DNA expressing GFP was inserted and the minicircle vector were transfected with transfectant 2000 (invitrogen) And their expression was confirmed.

Using stem cells derived from adulthood, 1 × 10 ⁶ stem cells were placed on a 60 mm dish. After 24 h, lipofectamine was treated with a mini circle vector expressing GFP. The cells and DNA were incubated together for 4 hours and then replaced with new media. Twenty-four hours later, cells were observed using a fluorescence microscope to compare the amount of transfection expression.

FIG. 20 shows that gene expression efficiency of mini circle vector is better when a parent plasmid and a minicircle vector that express GFP in stem cells, respectively, are introduced.

Experimental Example  One. Rheumatism  arthritis Synovial cell  Proliferation inhibitory effect

Fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis were cultured by primary cell culture and cultured in equal numbers in a 60 mm culture dish. IL-6 (200 ng / ml), IL-6R (200 ng / ml) and TNF-alpha (20 ng / ml) were treated with FBS-free DMEM to induce arthritis and similar environment in the prepared cells. To grow in a situation similar to arthritis.

Next, the A7 antibody prepared in Example 1 was treated with the above-described FLS, and after 2 days of culturing, each treated cell was allowed to stain only living cells using a CCK8 assay kit (CK04, DOJINDO Laboratories) And 1.25 ug of the A7 antibody was used.

As a result, when the A7 antibody was treated, the cell proliferation inhibitory effect was excellent, and it was confirmed that the growth was inhibited very effectively in a concentration-dependent manner (FIG. 21). Therefore, it can be seen that the anti-IL-6R antibody of the present invention is effective in the treatment of arthritis by inhibiting the proliferation of arthritic cells.

Experimental Example  2. Rheumatism  arthritis Synovial cell  Movement inhibitory effect

Fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis were cultured by primary cell culture and cultured in equal numbers in a 60 mm culture dish. Microscopically, the same sized and same tip was used to scrape the surface of the well-grown cells and cause scarring. IL-6 (200 ng / ml), IL-6R (200 ng / ml) and TNF-alpha (20 ng / ml) were treated with FBS-free DMEM to induce arthritis and similar environment in the prepared cells. To grow in a situation similar to arthritis.

Next, 100 ug / ml of the A7 antibody was treated with the FLS cultured on the above, and after 16 hours, how much the cells migrated toward the injured area was observed under a microscope and photographed, The values were compared in terms of area. Experiments were carried out at 37 ℃ incubation conditions.

As a result, when the A7 antibody was treated, the cell migration inhibitory effect was excellent, and it was confirmed that this growth was inhibited very effectively in a concentration-dependent manner (FIGS. 22 and 23). Therefore, it can be seen that the anti-IL-6R antibody of the present invention is effective in the treatment of arthritis by inhibiting the movement of arthritic cells.

Experimental Example  3. Rheumatism  Therapeutic effect in arthritis animal model

An animal model of rheumatoid arthritis was constructed by using mouse monoclonal anti-type II collagen 5 clone antibody cocktail kit (chondrex 53010) in 7-week-old DBA1 / J rats as manual. 6 mg of 5-clone cocktail was intraperitoneally administered, and 3 days later, 25-50 μg of LPS was intraperitoneally administered to construct a disease model. The collagen-induced arthritis (CIA) animal model was subjected to intraperitoneal administration of 5mpk of A7 antibody three times a week.

The degree of paw swelling of each animal was measured and shown in Fig. The swelling of each joint in the rat was scored as 0 ~ 4 points, and the scores were added to the values. The higher the score, the more severe the disease. It was confirmed that rheumatoid arthritis was induced in the disease group (CIA) compared with the wild type, and swelling of the foot was increased. In addition, administration of A7 antibody showed arthritic improvement.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> A stem cell transformed by minicircle vector expressing antibody          directed to IL-6 receptor <130> DPP20131624KR <150> KR10-2012-0050446 <151> 2012-05-11 <160> 44 <170> Kopatentin 1.71 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A7 heavy chain variable region <400> 1 Asp Tyr Ala Met His   1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A7 heavy chain variable region <400> 2 Gly Val Ser Trp Asn Ser Gly Thr Ile Ala Tyr Val Asp Ser Val Lys   1 5 10 15 Gly     <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A7 heavy chain variable region <400> 3 Asp Phe Thr Tyr Phe Tyr Glu Ser Ser Gly Tyr Tyr Ala Phe Asp Leu   1 5 10 15 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A7 light chain variable region <400> 4 Thr Gly Thr Asn Ser Asn Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A7 light chain variable region <400> 5 Gly Asn Thr Asn Arg Pro Ser   1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A7 light chain variable region <400> 6 Gln Ser Phe Asp Ser Ser Leu Thr   1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of B10 heavy chain variable region <400> 7 Asp Tyr Ala Met Phe   1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of B10 heavy chain variable region <400> 8 Gly Ile Asn Trp Asn Gly Asn Gly Ile Gly Tyr Gly Asp Ser Val Arg   1 5 10 15 Gly     <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of B10 heavy chain variable region <400> 9 Pro Ser Leu Tyr Gly Gly Asn Ser Glu Phe Asp Leu   1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of B10 light chain variable region <400> 10 Thr Gly Pro Thr Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of B10 light chain variable region <400> 11 Gly Asn Leu Asn Arg Pro Ser   1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of B10 light chain variable region <400> 12 His Thr Tyr Asp Ser Ser Leu Ser   1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of D2 heavy chain variable region <400> 13 Asn Tyr Ala Ile Asn   1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of D2 heavy chain variable region <400> 14 Arg Ile Ile Pro Met Leu Gly Thr Ser Asp Tyr Ala Glu Lys Phe Gln   1 5 10 15 Gly     <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of D2 heavy chain variable region <400> 15 Gly Pro Arg Tyr Tyr Gly Thr Asp Ser Tyr Tyr Leu Glu Lys   1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of D2 light chain variable region <400> 16 Thr Gly Pro Thr Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of D2 light chain variable region <400> 17 Gly Asn Leu Asn Arg Pro Ser   1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of D2 light chain variable region <400> 18 His Thr Tyr Asp Ser Ser Leu Ser   1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of F2 heavy chain variable region <400> 19 Asn Tyr Tyr Met His   1 5 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of F2 heavy chain variable region <400> 20 Ile Ile Asn Pro Ser Gly Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of F2 heavy chain variable region <400> 21 Gly Leu Pro Trp Gly Glu Asn Gly Leu Asp Val   1 5 10 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of F2 light chain variable region <400> 22 Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of F2 light chain variable region <400> 23 Gly Asp Ser Asp Arg Pro Ser   1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of F2 light chain variable region <400> 24 Gln Ser Tyr Asp Ser Ser Leu Ser   1 5 <210> 25 <211> 129 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > A7 heavy chain variable region <400> 25 Asp Val His Ser Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Thr Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Lys              20 25 30 Phe Glu Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ser Gly Val Ser Trp Asn Ser Gly Thr Ile Ala Tyr      50 55 60 Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys  65 70 75 80 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Asp Phe Thr Tyr Phe Tyr Glu Ser Ser Gly             100 105 110 Tyr Tyr Ala Phe Asp Leu Trp Gly Gln Gly Thr Met Val Thr Val Ser         115 120 125 Ser     <210> 26 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> A7 light chain variable region <400> 26 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Thr Asn Ser Asn              20 25 30 Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Val Pro Gly Thr          35 40 45 Thr Pro Lys Leu Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala  65 70 75 80 Ile Thr Gly Leu Gln Ala Glu Asp Gly Gly Asp Tyr Tyr Cys Gln Ser                  85 90 95 Phe Asp Ser Ser Leu Thr Gly Ser Val Phe Gly Thr Gly Thr Lys Val             100 105 110 Thr Val Leu         115 <210> 27 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> B10 heavy chain variable region <400> 27 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Ile              20 25 30 Phe Asp Asp Tyr Ala Met Phe Trp Val Arg Gln Val Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ala Gly Ile Asn Trp Asn Gly Asn Gly Ile Gly Tyr      50 55 60 Gly Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Arg  65 70 75 80 Asn Ser Leu Asp Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Pro Ser Leu Tyr Gly Gly Asn Ser Glu Phe             100 105 110 Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 <210> 28 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> B10 light chain variable region <400> 28 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Pro Thr Ile Gly              20 25 30 Ala Gly Tyr Asp Val His His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro          35 40 45 Lys Leu Leu Ile Tyr Gly Asn Leu Asn Arg Pro Ser Gly Val Pro Asp      50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr  65 70 75 80 Asp Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Thr Tyr Asp                  85 90 95 Ser Ser Leu Ser Gly Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr             100 105 110 Val Leu         <210> 29 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> D2 heavy chain variable region <400> 29 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Gly Ser Gly Asp Thr              20 25 30 Phe Pro Asn Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Pro Glu Trp Met Gly Arg Ile Ile Pro Met Leu Gly Thr Ser Asp Tyr      50 55 60 Ala Glu Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr  65 70 75 80 Asn Thr Ala Tyr Met Gly Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Val Lys Gly Pro Arg Tyr Tyr Gly Thr Asp Ser Tyr             100 105 110 Tyr Leu Glu Lys Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 30 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> D2 light chain variable region <400> 30 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Pro Thr Ile Gly              20 25 30 Ala Gly Tyr Asp Val His His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro          35 40 45 Lys Leu Leu Ile Tyr Gly Asn Leu Asn Arg Pro Ser Gly Val Pro Asp      50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr  65 70 75 80 Asp Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Thr Tyr Asp                  85 90 95 Ser Ser Leu Ser Gly Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr             100 105 110 Val Leu         <210> 31 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> F2 heavy chain variable region <400> 31 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr              20 25 30 Phe Thr Asn Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Asn Thr Gly Tyr      50 55 60 Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Met Ser Thr  65 70 75 80 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Gly Leu Pro Trp Gly Glu Asn Gly Leu Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 32 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> F2 light chain variable region <400> 32 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Ser Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn              20 25 30 Ile Gly Ala Gly Tyr Asp Val His His Trp Tyr Gln Gln Leu Pro Gly Thr          35 40 45 Ala Pro Lys Val Leu Ile Tyr Gly Asp Ser Asp Arg Pro Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala  65 70 75 80 Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser                  85 90 95 Tyr Asp Ser Ser Leu Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val             100 105 110 Thr Val Leu         115 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A3 heavy chain variable region <400> 33 Glu Tyr Ala Phe His   1 5 <210> 34 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A3 heavy chain variable region <400> 34 Asp Phe Thr Pro Ala Phe Gly Glu Gly Asp Thr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 35 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A3 heavy chain variable region <400> 35 Glu Thr Ala Ser Thr Tyr   1 5 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A3 light chain variable region <400> 36 Thr Gly Pro Thr Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A3 light chain variable region <400> 37 Gly Asn Leu Asn Arg Pro Ser   1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A3 light chain variable region <400> 38 His Thr Tyr Asp Ser Ser Leu Ser   1 5 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A10 heavy chain variable region <400> 39 Asp Tyr Ala Met His   1 5 <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A10 heavy chain variable region <400> 40 Val Ile Ser Gly Asp Asp Gly Thr Thr His Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 41 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A10 heavy chain variable region <400> 41 Asp Met Tyr Pro Tyr Gly Asp Tyr Gly Tyr Leu Gln His   1 5 10 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A10 light chain variable region <400> 42 Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala   1 5 10 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A10 light chain variable region <400> 43 Ala Ala Ser Thr Leu Gln Ser   1 5 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A10 light chain variable region <400> 44 Gln Gln Leu Asn Thr Tyr Pro Leu   1 5

Claims (15)

(a) a promoter;
A gene encoding a monoclonal antibody specific for IL-6R (Interleukin-6 receptor); And
A gene expression cassette comprising a transcription terminator,
(b) a site-specific recombination region located outside the gene expression cassette, and
(c) does not include the cloning start point and selectable marker gene
As a stem cell into which a non-viral vector has been introduced,
The IL-6R-specific monoclonal antibody
A heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; And
A light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.
delete The stem cell according to claim 1, wherein the IL-6R-specific monoclonal antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26.
delete delete delete delete delete delete The stem cell according to claim 1, wherein the promoter is a CMV (cytomegalovirus) promoter.
2. The stem cell of claim 1, wherein the transcription terminator comprises the SV40 polyadenylation sequence.
The stem cell according to claim 1, wherein the site-specific recombination region is an att-attached sequence derived from Escherichia coli or bacteriophage lambda.
2. The stem cell according to claim 1, wherein said vector is double-stranded DNA in the form of a circular supercoil.
A pharmaceutical composition for preventing and treating an autoimmune disease, comprising the stem cell of any one of claims 1, 3, and 10 to 13.
15. The method of claim 14, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin dependent diabetes mellitus, multiple sclerosis, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, myasthenia gravis, multiple myositis, dermatomyositis, Crohn &apos; , Vasculitis syndrome or systemic lupus erythematosus.

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