KR20130126534A - The therapy using a stem cell comprising minicircle vector expressing physiologically active protein - Google Patents

Abstract

The present invention relates to a stem cell therapeutic agent containing a minicircle vector which expresses physiologically active proteins and, more particularly, to stem cells containing a minicircle vector which expresses physiologically active proteins and a pharmaceutical composition and a composition for drug delivery containing the same.

Description

Therapies using a stem cell comprising minicircle vector expressing physiologically active protein

The present invention relates to a stem cell therapeutic agent comprising a minicircle vector expressing a bioactive protein, and more particularly, to a stem cell comprising a minicircle vector expressing a bioactive protein, a pharmaceutical composition comprising the same, and a drug delivery method. It relates to a composition.

Gene therapy 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. The injected gene is successfully delivered to a nucleus of a target cell, whereby a large amount of gene is derived from the gene. It is important to allow expression.

Gene transfer technology for this purpose is largely a physical method such as a method using a virus as a carrier, a non-viral method using synthetic phospholipids or synthetic cationic polymers, and an electrical permeation method to introduce a gene by applying a temporary electric stimulation to the cell membrane It can be divided into Here, the method of using a virus carrier has advantages such as excellent delivery efficiency and long-term and stable gene expression, but the administered gene permanently changes the structure of the gene in the human chromosome, or has the ability to reproduce itself or to reproduce itself. Biological safety issues, such as activating the immune defense system of the system, are limited in practical application. Non-viral gene delivery methods can increase the biological safety and freely alter the chemical composition and structure of the transporter. However, the non-viral gene delivery method is very inferior to the case of using the virus transporter in terms of its efficiency and time of expression. It is a difficult problem to use in the clinic. Therefore, there is a need for a gene delivery system that specifically acts on a target cell or target tissue and has high expression efficiency.

Recently, with the development of stem cell science, research and therapeutic applications for human stem cells are emerging as an alternative to overcome the problems of safety and efficiency of existing gene therapy and to enable new cell and gene therapy for the human body.

However, current stem cell research is still in its infancy worldwide, requiring a lot of basic research and many problems to be solved for actual clinical application. In particular, the mechanism of engraftment, migration, differentiation, external gene expression, and integration into the host upon administration of stem cells in vivo should be elucidated and confirmed that the administered stem cells achieve appropriate functional improvement in the host. .

Therefore, the present inventors have developed a gene therapy vector system that efficiently delivers a therapeutic gene to a target cell, expresses a therapeutic gene continuously and for a long time, and has high biological safety. To provide a new concept of stem cell therapeutics that can improve the, to complete the present invention.

It is an object of the present invention to provide a stem cell therapeutic agent comprising a non-viral vector expressing a bioactive protein.

To this end, one object of the present invention is to provide a stem cell into which a non-viral vector expressing a bioactive protein is introduced.

Still another object of the present invention is to provide a pharmaceutical composition comprising the stem cells.

Another object of the present invention to provide a composition for drug delivery comprising the stem cells.

In one embodiment, the invention relates to stem cells comprising non-viral vectors expressing bioactive proteins.

In a preferred embodiment, the present invention comprises a gene expression cassette consisting of a promoter, a gene encoding a bioactive protein and a transcription terminator, and a site specific recombination region located outside of the gene expression cassette, wherein the replication initiation and selection marker genes are It relates to a stem cell into which a non-viral vector is introduced.

As used herein, the term "stem cell" refers to a cell having the ability to differentiate into various cells and having self-proliferative capacity, embryonic stem cells isolated from early embryos, and embryonic primitive reproduction. Embryonic germ cells isolated from cells, pluripotent adult stem cells isolated from adult bone marrow, and the like.

Stem cells usable in the present invention include all pluripotent stem cells derived from mammalian embryos, fetuses, umbilical cord blood, or adult tissues such as adult organs or bone marrow, skin or blood, and have similar characteristics to embryonic stem cells. It includes stem cells having a. In this case, the term "embryonic stem cell-like trait" means that there are surface (antigen) markers specific for embryonic stem cells, express embryonic stem cell-specific genes, or have teratoma-forming functions, Or it can be defined as cell biological properties specific to embryonic stem cells, such as chimeric mouse formation ability. Examples of adult stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, and adipose stem cells.

The present invention introduces various physiologically active proteins having a therapeutic effect to the stem cells in the form of genes and delivers them to the diseased parts of the body and expresses them at the corresponding site, thereby reducing the half-life and biological activity of the existing protein therapeutics. It can solve the problems such as the reduction of the problem, solve the problem of delivery efficiency and stability of the existing gene therapy, and can provide a continuous and stable therapeutic effect.

In addition, in the present invention, in order to solve both the problem of biological stability such as immunogenicity of the viral vector system used in gene therapy and the problem of delivery efficiency of the non-viral vector system, the therapeutic gene is a target cell. The vector system has been applied to stem cells with high efficiency and long-term expression of therapeutic genes and high biological safety.

Specifically, the vector system of the present invention includes a gene expression cassette consisting of only minimum essential elements such as a promoter, a therapeutic gene and a transcription terminator, and a selection marker such as a replication initiation point and an antibiotic resistance gene used in a conventional vector system. Genes are not included.

The origin of replication is primarily bacterial sequences, which can cause unwanted immune responses in the human body, and selectable marker genes, such as antibiotic resistance genes, can be passed on to bacteria in the body. If administered, there is a problem that can cause unnecessary antibiotic resistance. Therefore, in the present invention, by not including the replication start point and the selection marker gene, it is possible to eliminate the problem of unnecessary immune response and antibiotic resistance. In addition, unmethylated CpG motifs derived from prokaryotic cells can be removed to reduce the immune response. In addition, because of this, the vector of the present invention has a smaller physical size than the conventional vector, thereby making it easy to manufacture, improve delivery efficiency, and improve biological stability.

The vector of the present invention is characterized in that the double-stranded DNA in the form of a circular supercoil.

The promoter used in the vector of the present invention may use a promoter used in the art without particular limitation, and may include an inducible or constitutive promoter, and preferably may be a cytomegalovirus (CMV) promoter.

The transcription terminator used in the vector of the present invention may use a terminator used in the art without particular limitation, but may preferably include an SV40 polyadenylation sequence.

The vector of the present invention further includes site specific recombination outside of a gene expression cassette consisting of a promoter, a gene encoding a bispecific protein as a therapeutic gene, and a transcription terminator.

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

Bioactive proteins introduced into the stem cells of the present invention include all proteins used for therapeutic effects. For example, the bioactive protein may be human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, interferon receptors, colony stimulating factor, glucacon-like peptides (GLP-1, etc.) Protein-coupled receptors, interleukins, interleukin receptors, enzymes, interleukin binding proteins, cytokine binding proteins, macrophage activators, macrophage peptides, B cell factors, T cell factors, protein A , Allergic inhibitors, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, alpha-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin , High glycated erythropoietin, angiopoietin, hemoglobin, chrombin, thrombin receptor active peptide, thrombomodulin, blood factor VII, VIIa, VII, VII, and XI II, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet derived growth factor, Epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone formation growth factor, bone formation promoter, calcitonin, insulin, atriopeptin, cartilage inducer, elkatonin, connective tissue activator, tissue factor pathway inhibitor, Follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, corticosteroids, glucagon, cholecystokinin, pancreatic polypeptide, gastrin Releasing peptides, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, May be selected from for retention, receptor antagonists, cell surface antigens, virus derived vaccine antigens, monoclonal antibodies, the group consisting of polyclonal antibodies and antibody fragments flow, but is not the scope of the present invention is not limited to this.

Preferably, in the present invention, the physiologically active protein, human growth hormone, interferon, interleukin, tumor necrosis factor, granulocyte colony stimulating factor, erythropoietin, high frequency when administered to the human body for the purpose of treating or preventing disease And antibody fragments. In addition, any derivative or derivative is included within the scope of the bioactive protein of the present invention so long as it has a function, structure, activity or stability substantially equivalent to or increased with the natural form of the bioactive polypeptide.

In one embodiment, the bioactive protein may be an anti-IL-6R protein.

Interleukin is a type of cytokine that acts as a chemical signal between red blood cells. Of these, interleukin-6 (IL-6) has been shown to be abnormally produced in inflammatory diseases, autoimmune diseases and tumors and has been shown to be involved in the pathogenesis of such diseases. In the present invention, the anti-IL-6R protein has a high binding affinity for the IL-6 receptor and may exhibit a particularly excellent effect in the treatment of autoimmune diseases by inhibiting the interaction between IL-6 and IL-6 receptor.

In another embodiment, the bioactive protein may be a TNFR2 protein or a fragment comprising an extracellular region of the TNFR2.

Tumor necrosis factor (TNF) is a pleiotropic cytokine, in particular TNF-α plays an important role in the inflammatory response and immune system. TNFR2 is one of the receptors that bind to TNF-α. In the present invention, the TNFR2 protein or its extracellular domain fragment has a high binding affinity for TNF-α and inhibits the action of TNF-α. It can exhibit excellent effects.

In another embodiment, the physiologically active protein comprises a dual comprising an anti-IL-6R (Interleukin-6 receptor) protein and a Tumor necrosis factor receptor type 2 (TNFR2) protein or a fragment comprising an extracellular region of the TNFR2. May be a specific protein.

As used herein, the term "bispecific protein" or "bispecific antibody" refers to a protein or antibody having an antigen binding site in the same molecule that can recognize two or more different epitopes.

The bispecific protein herein is a protein that specifically recognizes TNF-α and IL-6R and is capable of double antagonism against them, wherein the constant region of the anti-IL-6R monoclonal antibody has a TNFR2 protein at the C-terminus or The fragment comprising the extracellular region of the TNFR2 is bound to a bispecific antibody structure.

In the present invention, the bispecific protein has a high binding affinity for IL-6R and TNF-α and may exhibit a particularly excellent effect in the treatment of autoimmune diseases by inhibiting the action of IL-6R and TNF-α.

As used herein, the term "variable region" refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and in the variable region CDR1, a complementarity determining region (CDR), CDR2 and CDR3 are present. A framework region (FR) portion exists between the CDRs to support the CDR rings.

Also herein, the term “complementarity determining region” is a ring-shaped region involved in the recognition of an antigen, and as the sequence of this region changes, the specificity of the antibody to the antigen is determined.

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

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

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

As another preferred embodiment, the anti-IL-6R protein in the present invention, heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: A heavy chain variable region comprising a; And a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 10, a light chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 12.

More preferably, the monoclonal antibody specific for IL-6R may include a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 27, and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 28.

The anti-IL-6R monoclonal antibody specified by the heavy chain variable region and the light chain variable region as described above will be referred to herein as "B10".

As another preferred embodiment, the anti-IL-6R protein in the present invention, heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 14, heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 15 A heavy chain variable region comprising a; And a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 16, a light chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 18.

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

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

As another preferred embodiment, the anti-IL-6R protein in the present invention, heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 19, heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 20, heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 21 A heavy chain variable region comprising a; And a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 22, a light chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 23, and a light chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 24.

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

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

In addition, in the present invention, the TNFR2 protein or the fragment including the extracellular region of TNFR2 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33. Alternatively, the amino acid sequence of SEQ ID NO: 33 may be a polypeptide comprising 23 to 179 from the N terminal.

Vectors expressing bioactive proteins in the present invention are known in a number of methods, such as transient transfection, microinjection, transduction, electroporation, DEAE Dextran mediated traits. It can be introduced into cells or tissues by infection, monocationic liposome fusion, polycationic liposome fusion, plasma fusion, lipofectamine, naked DNA delivery, and the like.

Stem cells into which a vector expressing a physiologically active protein is introduced in the present invention can be expected to have the following effects greatly.

First, stem cells can serve as effective drug carriers of genes encoding bioactive proteins. Since stem cells exhibit a homing effect when they are injected into a living body (homing effect), in the present invention, stem cells can act as a delivery medium for targeting to a lesion site, thereby delivering a therapeutic gene to the lesion site. And expression to provide the effect of the gene therapy agent. In addition, stem cells in the present invention is capable of continuously and stably expressing a therapeutic gene, and in a specific embodiment of the present invention, it was confirmed that the stem cells to which the vector system of the present invention was successfully expressed protein.

Second, the physiologically active protein introduced into the stem cells enhances the intrinsic immunological function of the stem cells, can exhibit an effect as a cell therapeutic agent enhanced the effect of disease treatment by stem cells. As used herein, "treatment" means any action that inhibits, alleviates or beneficially alters the clinical situation associated with a disease. Treatment can also mean increased survival compared to the expected survival if untreated. Treatment includes simultaneously prophylactic measures in addition to therapeutic means.

Stem cells according to the present invention are administered in a manner that is directly transplanted or migrated to a desired tissue site, for example, induces the death of disease-related cells, affects the signaling system, modulates immune function, or functionally. The therapeutic effect can be provided by reconstitution or regeneration of the deficient site.

Therefore, as another aspect, the present invention relates to a gene drug delivery composition comprising stem cells into which a minicircle vector expressing a physiologically active protein is introduced.

In another aspect, the present invention relates to a pharmaceutical composition comprising stem cells into which a minicircle vector expressing a bioactive protein is introduced.

The composition 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 irritate an organism and does not inhibit the biological activity and properties of the administered component. 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 composition of the present invention may further include a filler, an excipient, a disintegrant, a binder and a lubricant. The compositions of the present 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.

As used herein, the term "administration" refers to introducing a composition of the present invention to a patient in any suitable manner, wherein the route of administration of the composition of the present invention is via oral or parenteral various routes as long as the target tissue can be reached. May be administered. 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 the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved and whether other agents are used in some cases, the age, weight, general state of health, sex of the patient. And various factors and similar factors well known in the medicinal art, including diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or concurrent with the specific composition.

The present invention can be applied to a variety of physiologically active proteins having a therapeutic effect to the stem cells by applying to a new vector system to be delivered to the disease site in the body and expressed in the site. Accordingly, the present invention can solve problems such as decrease in half-life and decrease in physiological activity due to degradation in the existing protein therapeutics, and solve the problems of delivery efficiency and stability of the conventional gene therapeutics, and It can provide a therapeutic effect stably.

Figure 1 shows the preparation of IL-6R antigen for the production of anti IL-6R monoclonal antibody. (A) is a result of Western expression of IL-6R secreted into the supernatant by temporarily expressing IL-6R using a pYK 604 vector in a 150 mm dish. It can be confirmed that the expression is normal until 9 days after the transient transfection. (B) shows the purity of IL-6R used as an antigen through chromatography, and (C) shows the Agilent 2100 analysis program. It shows the result of confirming the purity of IL-6R used as an antigen through.
Figure 2 shows the results of poly phage ELISA assay for the production of anti IL-6R monoclonal antibody.
Figure 3 shows the results of mono phage ELISA assay for the production of anti IL-6R monoclonal antibody.
4 shows the fingerprinting results for mono phage clones.
FIG. 5 shows the degree of affinity for IL-6R by diluting six different monophage clones that specifically bind to IL-6R. As shown in FIG. I, both showed high binding affinity.
Figure 6 shows the results of screening the phage clones using IL-6R full protein of the results in Figure 5, as a result it can be seen that the binding force of B10 is the highest, in the order of D2, A7, A10, A3. .
Figure 7 confirms via Western blot that mono phage clones were converted to full IgG form.
8 shows the results of purification of IgG antibodies of monophage clones A3, A7 and A10.
Fig. 9 shows the results of the purification of IgG antibodies of monophage clones B10, D2 and F2.
10 shows the binding affinity (Kd value) of the anti-IL-6R monoclonal antibodies A3, A7, A10, B10, D2, F2 to IL-6R.
Figure 11 shows the results of analyzing the IL-6 inhibitory activity of the anti IL-6R monoclonal antibodies A3, A7, A10, B10, D2, F2 through the STAT3 reporter assay.
12 shows the structure (left) of the anti-IL-6R monoclonal antibody prepared in the present invention and the structure (right) of the bispecific antibody using the same.
13 shows the preparation of a fusion construct of anti-IL-6R antibody and TNFR2.
14 shows the results confirming successful expression of the fusion construct of anti-IL-6R antibody and TNFR2.
15 shows the results confirming successful purification of the fusion construct of anti-IL-6R antibody and TNFR2.
Figure 16 shows the binding affinity (Kd value) for TNF-α of bispecific antibodies A7 / TNFR2, B10 / TNFR2, D2 / TNFR2 and F2 / TNFR2.
Figure 17 is a schematic diagram showing the manufacturing process of the minicircle vector used in the present invention.
Figure 18 (A) is a result confirmed by electrophoresis after the cut (enzyme cut) with respect to the plasmid, (B) is a new cell line by introducing the plasmid into the E. coli host to enable amplification and cloning After the preparation, the result was confirmed by electrophoresis by cutting with restriction enzyme, and (C) shows the result of electrophoresis confirming that a minicircle vector was prepared using arabinose.
Figure 19 shows the result of confirming whether the minicircle vector is configured to express GFP and then introduced into 293T cell line expressing GFP.
20 shows the results of confirming whether the minicircle vector is configured to express GFP and then administered to mice to express GFP in vivo.
Figure 21 (A) shows the anti-IL-6R antibody (named A7) to the plasmid, TNFR2 protein (form bound to Fc, named TNFR2 + Fc), and bispecific protein containing them (A7 + TNFR2) DNAs expressing the naming) were inserted and confirmed. LC means light chain and HC means heavy chain. (B) shows the expression of each DNA after arabinos treatment to each of the plasmids and converts it into a minicircle vector, and (C) confirms that the minicircle vector expresses each DNA by restriction digestion. Results are shown.
FIG. 22 shows the results confirmed by GFP expression in 293T cell lines that minicircle vectors express anti-IL-6R antibodies (named A7), TNFR2 proteins, and bispecific proteins containing them (named Dual). In each figure, LD represents a low dose and HD represents a high dose.
FIG. 23A again shows that minicircle vectors express anti IL-6R antibodies (named A7), TNFR2 proteins, and bispecific proteins containing them (named Dual) via GFP expression in 293T cell lines. The result confirmed once. (B) shows the result of confirming the concentration of anti-IL-6R present in the culture supernatant of each cell, (C) shows the result of confirming the concentration of water-soluble TNFR2 present in the culture supernatant of each cell. .
24 shows that the gene expression efficiency of the minicircle vector is better when the plasmid and the minicircle vector expressing GFP are introduced into the stem cells, respectively.
Figure 25 shows the results of confirming the expression in the liver, stomach, and colon when the minicircle vector expressing GFP in mice.
Figure 26 shows the results of confirming the expression in the liver, stomach, and colon when the rat administration of GFP-expressing minicircle vector transfected adipose derived stem cells (ASC).
Figure 27 shows the results of transfection of a minicircle vector expressing water-soluble TNFR2 in adipose derived stem cells (ASC).
FIG. 28 shows two days (2dpi, (A)) and four days (4dpi, (B)) after intravenously injecting adi circle derived stem cells (ASC) transfected with minicircle vectors expressing water-soluble TNFR2 into the tail of mice. Each blood sample was taken to confirm the concentration of water-soluble TNFR2 present in the serum.

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. IL -6R Monoclonal  Preparation of antibodies

1-1. IL Preparation of -6R Antigen Protein

Using the human cDNA library to make IL-6R-Fc antigen, using a specific primer for IL-6R was carried out by PCR and confirmed by sequecing. Then, the cells were cloned into the animal cell expression vector pYK 604 and transfected into HEK293E cells to induce transient expression and cultured the cells. After transfection, the culture supernatant was secured at intervals of 2, 4, 6, and 9 days and purified using protein beads. The protein agilent 230 kit was used as QC data to confirm the purity of IL-6R and gel migration (FIG. 1).

1-2. Phage  Manufacture of library

300 ml 2XYT (+ Glucose, MgCL2, CM) was added to the stock OD 0.1 to raise the final OD 0.5 ~ 0.7. After this growth, helper phage was infected with MOI 20. It was left in place for 30 min at 37 ° C. and then shaken for 10 min. The grown solution was centrifuged at 4,500 rpm for 15 min at 4 ° C. to remove the supernatant, and the medium was replaced with 300 ml 2XYT (+ MgCL 2, CM, IPTG, Kan) and grown at 30 ° C. overnight. After centrifugation at 4 ° C. for 15 minutes at 4,500 rpm, the supernatant was added to PEG600 4% and NaCl 3% and incubated for 1 hr on ice. Centrifuged at 8,500 rpm at 4 ° C. for 20 min. After removing the supernatant, the phage was dried for 5 min and then dissolved in 5 ml of cold PBS and used for panning.

1-3. 1st ~ 3rd Panning  ( panning )

30 ug of IL-6R full domain protein was coated on the immunotube by overlapping overnight at 4 ° C. Blocking was performed at room temperature for 2 hours using 3 ml of 4% skim milk made of PBS. Thereafter, 2 ml of a prepared phage was added to 16% skim milk and blocked again at room temperature for 2 hours. Washing was performed 10 times with 2 min using 1x PBST. 100 mM TEA, 500 ul, and elution in two portions for 5 minutes and neutralized with 250 ul 1 M Tris (pH 7.5), respectively. The elution fraction thus obtained was collected and used for titering. After leaving about 100 ul, 8 ml of XL1-blue with 0.5 ~ 0.7 final O.D was added and 2ml was also infected with immunotube. At this time, it was allowed to infect for 30 minutes at 37 ℃. All the infected cells were collected, centrifuged for 15 minutes at 4,500 rpm at 4 ° C, and the supernatant was discarded. After 2 ml 2XYT (+ GLUCOSE, MgCl2) was added, the cell suspension was plated on a square plate and incubated at 37 ° C overnight.

As shown in Table 1 below, the human library retained by A & RT to make the antibody against IL-6R was made into phage form using M1 helper phage, and the result of binding to IL-6R as the primary panning output. 3.1 x 10 ⁶ was obtained, and the first panning result was re-formed into phage form, and the result of combining with IL-6R was enriched to 1.69 x 10 ⁷ with the second panning output. The result of binding to IL-6R by making phage form was confirmed to enrich 8.2 x 10 ⁸ with 3rd panning output.

IL-6R full 1st Panning 2nd panning 3rd 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

Using the supernatant obtained by phage infection, dilution of each of the five was compared with a-Myc and FC signals. First, 96 well plates were coated with IL-6R full 100 ng / well, a-Myc 100 ng / well and FC 100ng / well at 4 ° C. overnight. Blocking with 2% skim milk in 1X PBS 200 ul / well for 2 hours at 37 ℃. The prepared dilution phage was added and left at 37 ° C. for 2 hours. After washing with 1xPBST using an ELISA washer, anti-M13-HRP was combined at a concentration of 1: 2000 for 1 hour at 37 ° C, and then washed again and read at 490 nm using a substrate OPD.

Figure 2 shows the degree of binding affinity with IL-6R by making a phage form of the library, primary, secondary and tertiary panning cells to determine whether there is specificity for IL-6R as a result of polyphage ELISA analysis. In most cases, it is hard to find specificity in the library and 1 ^st , but after that, we can see that the signal comes out strongly from the second iteration. Therefore, it can be confirmed that the antibody specific for IL-6R exists in the 2nd and 3rd order. A-myc and Fc were used as negative control.

1-5. Mono Phage ELISA  analysis

96 by the 3 ^rd panning colony on a 96 well plate with gae picking them (2XYT-glucose-MgCl2-CM 1ml) were coated for overnight at 37 ℃. 100ul grown by picking on 1ml of 2XYT-glucose-MgCl2-CM were incubated at 37 ° C. for 3-4 hours when the OD was 0.12. The final OD was incubated to 0.5. The helper phage was placed at 37 ° C. for 10-30 minutes with 55ul each. After centrifugation at 4,500 rpm for 20 minutes, the media was changed to 2XYT-IPTG-MgCl 2 -CM-Kan and grown at 30 ° C. overnight. The supernatant was used for mono-ELAISA by centrifugation for 20 min at 3,500 rpm at 4 ° C. IL-6R full, a-Myc and FC were coated at 4 ° C. for 1 day at 100 ng / well in a 96 well plate. After blocking for 2 hours at 1 ℃ PBS 200ul / well in 2% skim milk for 2 hours at 37 ℃ with the phage supernatant obtained above was further blocked at 100ul 37 ℃ for 2 hours. After washing with ELISA washer with 1ⅹPBS, anti-M13-HRP was combined at 1: 2000 for 50min at 37 ° C for 50 min, and then washed once more, and the substrate OPD was read at 490 nm.

Figure 3 shows the results of the mono phage ELISA assay, colony was selected from the secondary panning out put and tertiary panning out put, 96 pieces were made into phage form, and the binding affinity for IL-6R was once again confirmed. Red shades are excluded because they also bind to Fc. IL-6R was excluded because phage binds to Fc nonspecifically because it has Fc.

1-6. Mono Phage  Screening and Fingerprinting

The colony was picked using 1 ul of cell stock. Colony PCR 10 ul reation and colony PCR product 10 ul were added to the colony thus obtained, and incubated at 37 ° C. for 2 hours by adding 0.2 ul / 20 ul reation of Bst NI. Thereafter, 8% PAGE Gel was made and loaded and electrophoresed.

As a result of screening and selecting a phage form having good binding affinity to IL-6R, all six different phage forms were obtained (FIG. 4).

1-7. Select mono Phage  Sequencing of Clones

DNA was extracted from six types of monophage clones identified in Examples 1-6 and subjected to sequencing. The sequencing was commissioned by SolGent, and the analysis method was ABI 3730, an automatic sequencr. As a result, the CDR regions of the heavy chain (VH) and light chain (VL) of the selected antibodies as shown in Table 2 and Table 3 were confirmed. The similarity between the antibody and germ line antibody group was confirmed using NCBI's Ig BLAST program (www.ncbi.nlm.nih.gov/igblast/). 87% to 96% homology, and light chains showed approximately 90% to 95% homology. In addition, the polypeptides used in CDR3 of the heavy and light chains of the respective human antibodies were analyzed, and it was confirmed that the sequences were different from each other.

Clone name 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 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 the ELISA showing the degree of affinity for IL-6R by diluting six different monophage clones that specifically bind to IL-6R. I, both showed high binding affinity.

Figure 6 shows the results of screening the phage clones using IL-6R full protein of the results in Figure 5, as a result it can be seen that the binding force of B10 is the highest, in the order of D2, A7, A10, A3. . F2 showed the lowest binding force.

1-9. all IgG  Transformation analysis

Six monophage clones were converted to full IgG form and confirmed by Western blot for assembly in mammalian cells. A3 was weakly assembled but the other five were all well assembled and high in expression level (FIG. 7).

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

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

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

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

Coating over 6 days with IL-6R 100ng / well. Each well was blocked for 2h at 37 ° C using 200ul of 2% skim milk. Two times dilution from 50nm was placed for 2 hours at 37 ℃ to bind 100ul each. After washing three times with 1xPBST, anti-Fab-HRP was bound at a concentration of 1: 4000 at 100ul for 1 hour at 37 ° C. After washing 3 times with 1xPBST, OPD 1tablet and 6ul H ₂ O ₂ were added to 10ml of PC buffer, and 100ul were processed at room temperature for 7min each, and the reaction was stopped with 50ul of NH ₂ SO ₄ and read at 490nM.

Table 5 below shows the results of measuring the Kd value of each antibody according to the IL-6R dilution concentration, and FIG. 10 shows the result of changing the graph. The results show that A3, A7 and F2 show very high binding capacity for very small amounts of (0.02441 nM) 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 2.3 x 10 ^-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 2.93 x 10 ^-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 4.3 x 10 ^-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 3.5 x ^{10 -10}
(R ² : 0.98)

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

The STAT3-reporter assay was performed to confirm the function of the anti-IL-6R monoclonal antibody. A cell line continuously expressing the reporter gene with lucifrerase attached to the expression gene of STAT3 was set up, and IL-6 was treated at all doses except the control group at a dose of 50 ng / well. Subsequently, anti-IL-6R prepared for each condition was treated with 0.001ug, 0.01ug, 0.1ug, and 1ug / well, respectively, to determine how the signal induced by IL-6 was induced by anti-IL-6R. It was measured whether it was inhibited. Each cytokine and antibody were treated with the prepared cells and incubated for 24 hours. Afterwards, each cell was treated with a lucifease substrate to induce color development.

As a result, A7, B10, D2 and F2 IL-6R antibodies showed the effect of inhibiting the signal of IL6. Among them, A7 and F2 were superior to other inhibitory effects (FIG. 11), and therefore, it was decided to fuse these antibodies with TNFR2.

Example  3. Bispecific  Antibody ( bi - specific antibody Manufacturing

A bispecific antibody capable of specifically recognizing IL-6R and TNFR2 was prepared using the anti-IL-6R monoclonal antibody prepared in Example 2. 12 shows the structure (left) of the anti-IL-6R monoclonal antibody prepared in the present invention and the structure (right) of the bispecific antibody using the same. Specific production process is as follows.

Specifically, the antispecific IL-IL-6R / TNFR2 (named A7 / TNFR2, B10 / TNFR2, F2 / TNFR2, D2 / TNFR2, respectively) is an Anti-IL-6R heavy chain (HC) (A7, B10). , F2, D2) was made by fusion of the extracellular region of Opti-TNFR2 to the C-terminal end, so that it could be inserted into pNATABH, a heavy chain (HC) mammalian expression vector, to obtain a bispecific antibody protein. PCR techniques were used to fuse A7 heavy chain + TNFR2 (SEQ ID NO: 34), B10 heavy chain + TNFR2 (SEQ ID NO: 35), F2 heavy chain + TNFR2 (SEQ ID NO: 36), and D2 heavy chain + TNFR2 (SEQ ID NO: 37), respectively. Cut with enzyme SfiI (# R033S, Enzynomics, korea) and NheI (# R016M, Enzynomics, korea), load onto Agarose (# 9002-18-0, BIO BASIC INC, USA) gel, and then use Gel Purification kit ( # 1014876, QIAGEN, USA) was used to secure the inserts to be inserted into the vector. Ligation was performed using T4 DNA ligase (# M001S, Enzynomics, Korea). Restriction enzyme reaction and Ligation reaction were performed identically under the following conditions.

Restriction enzyme reaction cuts 1ug DNA. 1ul SfiI (10unit), 1ul NheI, and 5ul 10x reaction buffer are mixed and sterile distilled water is added to make 50ul of total volume and reacted for 6 hours at 37 ℃. Separation purification was performed from the gel. In addition, Ligation was obtained by mixing 1 ul Ligase (10 units) and 1 ul 10x reaction buffer in a 50 ng vector with a ratio of 1: 3 to a vector and an insert ratio, and incubating at 16 ° C. for 16 hr or more.

The obtained ligates were transformed into E. coli cells, and the colony was correctly inserted into the pNATABH heavy chain expression vector by sequencing, and the midi prep (# 740 410.100, MACHERY-NAGEL, Germany) was performed. It was.

In addition, amplification of the A7 light chain, B10 light chain, F2 light chain, and D2 light chain by PCR was performed to allow insertion into pNATABL, a light chain (HC) mammalian expression vector, and then SfiI (# R033S, Enzynomics, korea), BglII (# R010M, Enzynomics, korea), cut with restriction enzymes, and then loaded onto Agarose (# 9002-18-0, BIO BASIC INC, USA) gel, and then Gel Purification kit (# 1014876, QIAGEN, USA) was used to secure the insert to be inserted into the vector. Ligation was performed using T4 DNA ligase (# M001S, Enzynomics, Korea). Restriction and Ligation reactions were performed identically under the following conditions.

Restriction enzyme reaction cuts 1ug DNA. 1ul SfiI (10unit), 1ul BglII, and 5ul 10x reaction buffer are mixed, and the total volume is 50ul by adding sterile distilled water and reacted for 6 hours at 37 ℃. Separation purification was performed from the gel. In addition, Ligation was obtained by mixing 1 ul Ligase (10 units) and 1 ul 10x reaction buffer in a 50 ng vector with a ratio of 1: 3 to a vector and an insert ratio, and incubating at 16 ° C. for 16 hr or more.

The obtained ligates were transformed into E. coli cells, and the colonies obtained were correctly inserted into the pNATABL light chain expression vector through sequencing to perform midi prep (# 740 410.100, MACHERY-NAGEL, Germany). It was.

In order to secure bispecific antibody proteins, the expression rate of Anti-IL-6R / TNFR2 was co-transfected into mammalian cell 293E using a heavy chain vector and a light chain vector at a 3: 7 ratio, and then cultured in large quantities. Anti-IL-6R (A7, B10, F2, D2) / TNFR2 proteins as fusion antibodies were obtained.

Example  4. Bispecific  Binding Affinity Analysis of Antibodies

It was coated with 100 ng of TNF-α (# C001-1MG, enzynomics, Korea) per well using an ELISA plate (# 439454, Nunc, Denmark). Coating was carried out overnight at 4 ° C. A solution containing 4% Skim milk (# 232100, Difco, France) in 200 μl of PBS was added to the coated ELISA plate, and blocking for approximately 1 hour at room temperature to suppress nonspecific reaction (blocking: 4% skim milk / 1xPBS). ) After blocking, the blocking buffer was removed from the ELISA plate, and each purified protein was prepared at 100 nM in 100 µl of a solution containing 1% skim milk in PBS, followed by sequential dilution at 1/4 dilution. After serial dilution, the reaction was performed at room temperature for about 2 hours. The plate was then washed five times with 200 μl PBST and 2 μl of the secondary antibody, anti-Human Fc-HRP (# 31413, Thermo, USA), was used in 4 ml containing 1% skim milk. After mixing in PBS, put 200μl per well in an ELISA plate and reacted for 1 hour at room temperature. After the reaction, the secondary antibody on the ELISA plate was removed, washed five times with 200 μl of PBS, and 10 μl of hydrogen peroxide solution (H 2 O 2, # H1009-100ML, Sigma, USA) and 10 mL of PC buffer (5.1 g) per well. Add Citric acid monohydrate, 7.3 g Sodium phosphate / L (pH5.0), and one OPD tablet (# P8787-100TAB Sigma USA) to add a total volume of 100 μl. After the reaction solution was added, the reaction was allowed to proceed for 10 minutes in a dark state at room temperature. The color was confirmed, and the reaction was terminated by adding 50 μl of a stop buffer to stop the reaction, and the wavelength of 490 nm in the ELISA reader. Kd value was measured at. TNF-α ligands showed a low Kd value with each protein, thereby confirming high binding strength. TNFR2 alone was identified as 7.8x10 ^-11 M, 7.2x10 ^-10 M, 4.3x10 ^-10 M, and 6x10 ^-10 M, respectively, when the anti-IL-6R / TNFR2 antibodies were tested for binding to TNF-a. It was shown to be almost the same as the treatment.

Antibody antigen
IL-6R antigen
TNF-α A7 2.3 nM - B10 4.3 nM - D2 7.5nM - F2 0.35nM - TNFR2 - 0.068 nM A7 / TNFR2 2.3 nM 0.078 nM B10 / TNFR2 4.3 nM 0.72 nM D2 / TNFR2 7.5nM 0.60 nM F2 / TNFR2 0.35nM 0.43 nM

Example  5. Preparation of Minicircle Vectors

5-1. DNA  Amplification

After dissolving 100 μl of competent cell (DH5a) at 4 ° C. for 5 minutes, mix 1 μg of DNA sample (GFP, anti-IL-6R antibody (A7), TNFR2, A7 + TNFR2) to be amplified in 100 μl competent cell, After incubation at 4 ° C. for 20 minutes, DNA was introduced by thermal shock at 42 ° C. for 45 seconds. It was then incubated at 4 ° C. for 2 minutes. Thereafter, 500 μl of LB (lysogeny broth) liquid medium was added thereto, and the mixture was incubated with stirring at 180 rpm for 1 hour at 37 ° C. After centrifugation at 5000 rpm for 30 seconds, the supernatant was removed from 500 μl. The remaining cells were plated on LB plates containing kanamycin and then incubated for one day in a 37 ° C. incubator. Picking one colony from the LB plate smeared last night in 5ml LB medium containing kanamycin, and incubated with stirring at 180rpm for 7-8 hours. 1 ml of 5 ml LB medium incubated prior to 200 ml LB medium containing kanamycin, and then incubated with stirring at 180 rpm for about 15 hours. After incubation, centrifugation was performed for 15 minutes at 9000 rpm at 4 ° C., and all the supernatant was discarded. DNA was extracted using a Nucleobond Xtra Midi kit (Cat #REF 740410.50) from Macherey-nagel (MN).

5-2. Parental plasmid ZYCY10P3S2T  E. coli Transformed in

100 μl of competent cells (ZYCY10P3S2T) were dissolved at 4 ° C., 1 μg of DNA was mixed with competent cells, and then incubated at 4 ° C. for 30 minutes. After applying a thermal shock at 42 ℃ 30 seconds, the ice was given a low temperature shock for 2 minutes. After the low temperature shock, 0.5 ml of SOC medium was mixed, followed by incubation for 1 hour while stirring at 225 rpm at 37 ° C. Thereafter, 100 µl was collected and plated on LB medium plate containing kanamycin. Plates were incubated overnight in a 37 ° C. incubator.

5-3. Minicircle Vector ( minicircle DNA ) Production

Colonies were picked from 5 ml of kanamycin-containing LB medium and plated with DNA in ZYCY10P3S2T Competent cells. LB medium containing the cells was incubated at 37 ° C. at 200 rpm for 8 hours. 100 μl of the incubated LB medium was added to 200 mL TB (terrific broth) medium containing kanamycin, and the TB buffer was incubated at 37 ° C. 200 rpm overnight (15 hours). The next day, the absorbance was measured at OD600, and when the value was between 4 and 5, the minicircle induction mix was prepared by adding 8 ml 1N NaOH and 200 µl 20% L-arabinose in 200 ml LB medium. After making it, it was mixed in the incubated TB medium. Incubate with stirring at 30 ° C. at 200 rpm for 5 hours. After incubation, centrifugation was performed at 9000 rpm for 15 minutes at 4 ° C, and all the supernatant except pellet was discarded. DNA was extracted using Nucleobond Xtra Midi kit (Macherey-nagel, Cat #REF 740410.50.).

5-4. Minicircle using restriction enzyme DNA Creation of

To confirm that the minicircle DNA was properly produced, restriction enzyme reaction was performed with restriction enzymes BamHI and XbaI (enzynomics). The prepared minicircle DNA, restriction enzyme buffer II, BSA, and tertiary distilled water were mixed in an appropriate amount and incubated at 37 ° C. for 30 minutes. Incubated samples were electrophoresed on 0.8% agarose gel with DNA samples that were not treated with restriction enzymes.

Figure 18 (A) is a result confirmed by electrophoresis after the cut (enzyme cut) with respect to the plasmid, (B) is a new cell line by introducing the plasmid into the E. coli host to enable amplification and cloning After the preparation, the result was confirmed by electrophoresis by cutting with restriction enzyme, and (C) shows the result of electrophoresis confirming that a minicircle vector was prepared using arabinose.

Example  6. Through Minicircle Vectors GFP  Confirmation of Protein Expression

6-1. Check in cell line ( in vitro )

In order to confirm that the minicircle DNA generated in Example 5 properly induces protein synthesis, the GC-inserted minicircle DNA was transfected into 293T cells. One day before transfection, 96 × plates of 293T cells of 3 × 10 ⁴ were plated with 100 μl of DMEM medium containing 7.5% FBS but no antibiotics. 0.2 µg of minicircle DNA was mixed with 25 µl of opti-MEM1, 0.5 µl of lipofectamine 2000 (invitrogen) and 25 µl of opti-MEM were mixed and incubated at room temperature for 5 minutes. Minicircle DNA and lipofectamine 2000 were mixed together and incubated for 20 minutes at room temperature. Thereafter, the mixture was sprayed on 293T cells, incubated at 37 ° C. for 4 hours, and changed to growth medium. Expression was confirmed after 24 hours thereafter.

19 shows the results of confirming that the minicircle vector expresses GFP after constructing it to express GFP.

6-2. In rats  Confirm ( in vivo )

In order to confirm whether the minicircle DNA generated in Example 5 expresses the protein in the body, the minicircle DNA was introduced into the mouse. 6-7 week old female rats were used as DBA1 / J (Oriental Bio), and 7-14 μg of GFP-inserted minicircle DNA was mixed in 1.5-1.8 ml saline corresponding to 10% of the rat's weight. The tail was intravenously injected.

FIG. 20 shows the results of confirming that the minicircle vector expresses GFP and then administered to mice to express GFP in vivo.

Example  7. Confirmation of therapeutic protein expression through minicircle vector

7-1. Minicircle using restriction enzyme DNA Creation of

Anti-IL-6R antibody (named A7), TNFR2 protein (form bound to Fc, named TNFR2 + Fc), and bispecifics comprising them according to the methods of Examples <5-1> to <5-4> After preparing a minicircle vector in which DNA expressing an enemy protein (named A7 + TNFR2) was prepared, it was confirmed that minicircle DNA was generated using restriction enzymes.

Figure 21 (A) shows the anti-IL-6R antibody (named A7) to the plasmid, TNFR2 protein (form bound to Fc, named TNFR2 + Fc), and bispecific protein containing them (A7 + TNFR2) DNAs expressing the naming) were inserted and confirmed. LC means light chain and HC means heavy chain. (B) shows the expression of each DNA after arabinos treatment to each of the plasmids and converts it into a minicircle vector, and (C) confirms that the minicircle vector expresses each DNA by restriction digestion. Results are shown.

7-2. Check in cell line ( in vitro )

To confirm that the minicircle DNA generated in Example 5 expresses anti IL-6R antibody, TNFR2 protein, and bispecific protein comprising them, respectively, a minicircle into which DNA expressing GFP and the therapeutic proteins is inserted DNA was transfected into 293T cells and its expression was confirmed according to the method of Example <6-1>.

FIG. 22 shows the results confirmed by GFP expression in 293T cell lines that minicircle vectors express anti-IL-6R antibodies (named A7), TNFR2 proteins, and bispecific proteins containing them (named Dual). In each figure, LD represents a low dose and HD represents a high dose.

7-3. By ELISA  Confirmation of expression

To confirm the expression of the therapeutic protein, 293T cells were transfected with each minicircle DNA, and culture supernatants therefrom were subjected to ELISA for anti-IL-6R. 100 μl of anti-hIL-6R antibody at 1 μg / ml was placed on a 96well corning coaster 9018 plate and placed at 4 ° C. overnight. After washing five times with washing buffer, 100 μl of human sIL-6R was added at 1 μg / ml and incubated at room temperature for 2 hours. After washing again, 1 × assay diluent was treated with 200 μl per well and incubated at room temperature for 1 hour. After rinsing with wash buffer five times again, tociluzumab was standardized at 100 pg / ml in 1x assay diluent. Incubated. After five washes, anti-hIgG-HRP, a detection antibody, was added at a dilution of 1: 10,000 and incubated at room temperature. After seven washes, TMB substrate solution was added and incubated for 15 minutes at room temperature. 50 µl of the reaction stop solution was treated, and the absorbance was measured at 450 nm.

In addition, 293T cells were transfected with each minicircle DNA and culture supernatants therefrom were subjected to ELISA for soluble TNFR2 (soluble TNFR2, sTNFR2), which was kit (Ready-to-use sandwich ELISA, eBioscience). Followed the protocol. In brief, serum samples and samples for standard curves were added to each well on a plate already coated with antibodies to sTNFR2 and incubated at room temperature for 2 hours. Thereafter, the washing process was performed four times, and then treated with 100 µl of TMB substrate solution and incubated at room temperature for 10 minutes. Thereafter, 100 µl of the reaction stop solution was added and the absorbance was measured at 450 nm.

FIG. 23A again shows that minicircle vectors express anti IL-6R antibodies (named A7), TNFR2 proteins, and bispecific proteins containing them (named Dual) via GFP expression in 293T cell lines. The result confirmed once. (B) shows the result of confirming the concentration of anti-IL-6R present in the culture supernatant of each cell, (C) shows the result of confirming the concentration of water-soluble TNFR2 present in the culture supernatant of each cell. .

Example  8. In stem cells through minicircle vectors GFP  Confirm gene expression

8-1. Check in cell line ( in vitro )

In order to confirm that the minicircle vector generated in Example 5 induces gene expression properly in stem cells, the transplasmic moplasmid and minicircle vector expressing GFP were transfected using lipofectamine 2000 (invitrogen), respectively. The expression was confirmed and its expression was confirmed.

Using stem cells derived from adult fat, 1 × 10 ⁶ stem cells were laid in a 60 mm dish. After 24h, minicircle vectors expressing lipofectamine GFP were mixed and treated with cells. Cells and DNA were incubated together for 4 hours and then transferred to new media. After 24 hours, cells were observed using a fluorescence microscope to compare the amount of transfection expression.

24 shows that the gene expression efficiency of the minicircle vector is better when the plasmid and the minicircle vector expressing GFP are introduced into the stem cells, respectively.

8-2. In rats  Confirm ( in vivo )

In order to confirm that stem cells transfected with a minicircle vector containing the GFP gene express GFP in the body, a minicircle vector into which the GFP-expressing DNA is inserted is added to the lipofectamine 2000 (invitrogen). After transfection using, experiments were performed using a method of directly tail vein injection of the thus prepared fat stem cells into mice.

Figure 25 shows the results of confirming the expression in the liver, stomach, and colon when the minicircle vector expressing GFP in the mouse, Figure 26 is a stem cell derived from adipose-derived transfected minicircle vector expressing GFP in the mouse When (ASC) is administered, it shows the result of confirming the expression in liver, stomach and large intestine. 25 and 26 similarly GFP is expressed, but it can be seen that the actual expression is different, which is different from when only the minicircle vectors, stem cells are shifted and settled in accordance with the unique characteristics of fat stem cells it means.

Example  9. Expression of Therapeutic Proteins in Stem Cells Using Minicircle Vectors

9-1. Check in cell line ( in vitro )

In order to confirm that the minicircle vector generated in Example 5 induces gene expression in stem cells properly, lipofectamine 2000 (invitrogen) using a plasmid and a minicircle with DNA expressing water-soluble TNFR2 were used, respectively. Transfection and its expression were confirmed.

Using stem cells derived from adult fat, 1 × 10 ⁶ stem cells were laid in a 60 mm dish. After 24 h, lipofectamine was treated with cells mixed with a minicircle vector expressing water-soluble TNFR2. Cells and DNA were incubated together for 4 hours and then transferred to new media. After 24 hours, cells were observed using a fluorescence microscope to compare the amount of transfection expression.

FIG. 27 shows that the gene expression efficiency of the minicircle vector is better when the plasmid expressing the water-soluble TNFR2 and the minicircle vector are respectively introduced into the stem cells.

9-2. In rats  Confirm ( in vivo )

In order to confirm that stem cells transfected with minicircle DNA containing the water-soluble TNFR2 gene express the water-soluble TNFR2 in the body, transgenic plasmids and minicircle vectors containing the water-soluble TNFR2 DNA were transfected into the adipose-derived stem cells. After the specification, the experiment was performed by using tail vein injection of the thus prepared adipose stem cells directly to the mice. Two days after and four days after the stem cell injection, blood was collected twice, and the concentration of sTNFR2 present in the blood was measured by ELISA. Four days later, sTNFR2 was not detected, but these results support the fact that the values from two days later are not non-specific.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> The therapy using a stem cell comprising minicircle vector          expressing physiologically active protein <130> DPP20131625KR <150> KR10-2012-0050445 <151> 2012-05-11 <160> 49 <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> <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 Ala 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 Ala 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 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 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 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> 230 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE (222) (1) .. (230) <223> TNFR2 <400> 33 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser   1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys              20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr          35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu      50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser  65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                  85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Ser Ala Pro Gly Ala Val             180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr         195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly     210 215 220 Pro Ser Pro Pro Ala Glu 225 230 <210> 34 <211> 637 <212> PRT <213> Artificial Sequence <220> <223> A7 heavy chain + TNFR2 <400> 34 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 Ala 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 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser     130 135 140 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 145 150 155 160 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr                 165 170 175 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr             180 185 190 Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln         195 200 205 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp     210 215 220 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro                 245 250 255 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr             260 265 270 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn         275 280 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg     290 295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser                 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             340 345 350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp         355 360 365 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe     370 375 380 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe                 405 410 415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly             420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr         435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala     450 455 460 Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu 465 470 475 480 Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly                 485 490 495 Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp             500 505 510 Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu         515 520 525 Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln     530 535 540 Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp 545 550 555 560 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu                 565 570 575 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr             580 585 590 Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr         595 600 605 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val     610 615 620 Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 635 <210> 35 <211> 633 <212> PRT <213> Artificial Sequence <220> <223> B10 heavy chain + TNFR2 <400> 35 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 Ala 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 Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser     130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys         195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys                 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val             260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp         275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr     290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu                 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg             340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys         355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp     370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser                 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser             420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser         435 440 445 Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr Pro Tyr     450 455 460 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln 465 470 475 480 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys                 485 490 495 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp             500 505 510 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys         515 520 525 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg     530 535 540 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 545 550 555 560 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg                 565 570 575 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val             580 585 590 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr         595 600 605 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly     610 615 620 Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 <210> 36 <211> 635 <212> PRT <213> Artificial Sequence <220> <223> F2 heavy chain + TNFR2 <400> 36 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 Ala         115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser     130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly                 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu             180 185 190 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr         195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg     210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr         275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln             340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu         355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro     370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu                 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val             420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln         435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr     450 455 460 Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr 465 470 475 480 Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His                 485 490 495 Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys             500 505 510 Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu         515 520 525 Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys     530 535 540 Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys 545 550 555 560 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys                 565 570 575 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp             580 585 590 Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser         595 600 605 Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile     610 615 620 Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 635 <210> 37 <211> 632 <212> PRT <213> Artificial Sequence <220> <223> D2 heavy chain + TNFR2 <400> 37 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 Ala Ser Thr Lys         115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly     130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr                 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val             180 185 190 Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn         195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro     210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp                 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp             260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly         275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn     290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro                 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu             340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn         355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile     370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys                 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys             420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu         435 440 445 Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala     450 455 460 Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr 465 470 475 480 Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val                 485 490 495 Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser             500 505 510 Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly         515 520 525 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu     530 535 540 Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser 545 550 555 560 Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro                 565 570 575 Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys             580 585 590 Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp         595 600 605 Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn     610 615 620 Ala Ser Met Asp Ala Val Cys Thr 625 630 <210> 38 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A3 heavy chain variable region <400> 38 Glu Tyr Ala Phe His   1 5 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A3 heavy chain variable region <400> 39 Asp Phe Thr Pro Ala Phe Gly Glu Gly Asp Thr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 40 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A3 heavy chain variable region <400> 40 Glu Thr Ala Ser Thr Tyr   1 5 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A3 light chain variable region <400> 41 Thr Gly Pro Thr Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A3 light chain variable region <400> 42 Gly Asn Leu Asn Arg Pro Ser   1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A3 light chain variable region <400> 43 His Thr Tyr Asp Ser Ser Leu Ser   1 5 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A10 heavy chain variable region <400> 44 Asp Tyr Ala Met His   1 5 <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A10 heavy chain variable region <400> 45 Val Ile Ser Gly Asp Asp Gly Thr Thr His Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 46 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A10 heavy chain variable region <400> 46 Asp Met Tyr Pro Tyr Gly Asp Tyr Gly Tyr Leu Gln His   1 5 10 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A10 light chain variable region <400> 47 Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala   1 5 10 <210> 48 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A10 light chain variable region <400> 48 Ala Ala Ser Thr Leu Gln Ser   1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A10 light chain variable region <400> 49 Gln Gln Leu Asn Thr Tyr Pro Leu   1 5

Claims (9)

(a) a gene expression cassette consisting of a promoter, a gene encoding a bioactive protein and a transcription terminator,
(b) comprises a site specific recombination region located external to the gene expression cassette,
(c) does not include replication initiation and selection marker genes
Stem cells incorporating nonviral vectors.
The stem cell of claim 1, wherein the stem cell is an embryonic stem cell or an adult stem cell.
The stem cell of claim 1, wherein the adult stem cells are mesenchymal stem cells, hematopoietic stem cells, neural stem cells, or adipose stem cells.
According to claim 1, wherein the physiologically active protein is human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, interferon receptors, colony stimulating factor, glucacon-like peptides (GLP-1, etc.) , G-protein-coupled receptor, interleukin, interleukin receptor, enzyme, interleukin binding protein, cytokine binding protein, macrophage activator, macrophage peptide, B cell factor, T cell factor, Protein A, Allergic Inhibitor, Cell Necrosis Glycoprotein, Immunotoxin, Limpotoxin, Tumor Necrosis Factor, Tumor Suppressor, Metastasis Growth Factor, Alpha-1 Antitrypsin, Albumin, α-Lactalbumin, Apolipoprotein-E, Red Blood Cell Production factor, high glycated erythropoietin, angiopoietin, hemoglobin, chrombin, thrombin receptor active peptide, thrombomodulin, blood factor 인, Ⅶa, Ⅷ, Ⅸ, And XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet derived growth factor , Epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone formation growth factor, bone formation promoting protein, calcitonin, insulin, atriopeptin, cartilage inducer, elkatonin, connective tissue activator, tissue factor pathway inhibitor Follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, corticosteroids, glucagon, cholecystokinin, pancreatic polypeptide, Gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostar Stem cells selected from the group consisting of chitin, receptors, receptor antagonists, cell surface antigens, virus-derived vaccine antigens, monoclonal antibodies, polyclonal antibodies and antibody fragments.
The stem cell of claim 1, wherein the promoter is a CMV (cytomegalovirus) promoter.
The stem cell of claim 1, wherein the transcription terminator comprises an SV40 polyadenylation sequence.
The stem cell of claim 1, wherein the site-specific recombination region is an att attachment sequence derived from E. coli or bacteriophage lambda.
The stem cell of claim 1, wherein the non-viral vector is a double-stranded DNA in the form of a circular supercoil.
Gene drug delivery composition comprising the stem cell of any one of claims 1 to 8.

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