KR102083884B1 - Minicircle vector for treating transplantation disease - Google Patents

Abstract

The present invention relates to a vector for inhibiting a transplant rejection response and an antibody expressed therefrom. When the first and second minicircle vectors of the present invention are expressed in the body, an anti-IL-2Rα fusion antibody to which IL-10 is linked is As a result, it is possible to reduce the development cost than existing biological immunosuppressants, and to compensate for the disadvantage of the nonspecific immunosuppressive effect that also reduces the regulatory T cell population and balances immune tolerance, which is more effective for various transplant rejection diseases. Has

Description

Minicircle vector for the treatment of transplant rejection disease {MINICIRCLE VECTOR FOR TREATING TRANSPLANTATION DISEASE}

The present invention relates to vectors and antibodies expressed therefrom for inhibiting transplant rejection.

The immune system must be able to distinguish between self and nonmagnetic. Without distinguishing between magnetic and nonmagnetic, the immune system destroys cells and tissues in the body, resulting in autoimmune diseases. Regulatory T cells actively inhibit activation of the immune system to prevent pathological self-response and consequently to prevent autoimmune diseases. Regulatory T cells (Tregs) are a class of CD4 ⁺ CD25 ⁺ T cells that inhibit the activity of other immune cells. The immune system is well equipped to quickly identify and quickly destroy alien, germ or inflammatory tissue. This has been a major barrier to gene therapy as well as tissue, organ and cell transplantation. The main issues were generally chronic immunosuppression, encapsulation and immune sequestration. Unwanted side effects of chronic immunosuppression include increased susceptibility to opportunistic infection and tumor formation.

The mechanism by which T cell responses to foreign (homologous or heterologous) proteins or cells or organs act is well known. Antigen presenting cells (APCs) are attracted to the site of inflammation or injury (induced by transplantation), and peripheral T cell repertoire constantly monitors the presence (exogenous or heterologous) or pathogens of external tissue. Once these warning signals are recognized, APCs swallow the protein, digest it, and present it to the host's immune system. Allogeneic or syngeneic tumor cells were engineered to express viral IL-10, which induces a local immune response to the tumor, but this treatment did not affect the rejection of non-transformed tumors at a distance (Suzuki et al., 1995). Topically administered IL-10 is thought to change the T cell repertoire responsive to transplanted cells into a Th2 phenotype that may be cytolytic but not protective.

Organ, tissue, or cell transplantation can be used to save the lives of patients suffering from various kinds of diseases. Allotransplantation of organs such as kidneys, liver, heart, kidneys, lungs, and pancreas, tissues such as skin, bone marrow, etc. has already been commonly performed in hospitals to treat intractable diseases such as terminal organ failure. . In addition, xenotransplantation with donors from mammals other than humans has also been actively studied as a method to replace allograft donors. In particular, recently, transplantation of stem cells capable of permanently regenerating themselves and differentiating into various kinds of cells constituting the body under appropriate conditions has emerged as one of cell replacement treatments for various refractory diseases. According to the National Organ Transplantation Center, more than 20,000 patients in Korea are waiting for organ transplants, but the number of donations is less than 10% of demand. In addition, in the United States, there is an additional waiting time every 16 minutes for the need for organs, but 11 of the waiting days die without surgery. In addition to this situation, the development of life science and technology is the background for the development of xenotransplantation technology.In 2010, a total of 28,664 transplants were completed in the United States, including 16,899 kidneys, 6291 livers, 2333 heart and 1770 lungs. (Engels et al., 2011, JAMA, 306 (17): 1891-1901). Significant improvements have been made in immunosuppressive therapy and in the treatment of pre- and post-transplant patients, but graft rejection still affects approximately 60% of individuals transplanted, so graft rejection is less than 40% of individuals transplanted within the first year after transplantation. Is a risk factor for multiple graft loss, with observation of graft rejection in Jani et al., 2000, Ann Surg. 232 (4): 490-500. Acute rejection is also a risk factor known to progress to chronic rejection and therefore the detection and treatment of acute rejection episodes as soon as possible is a major goal to minimize graft damage and prevent downstream rejection episodes. In most cases, adaptive immune response to transplanted tissue is a major obstacle to successful transplantation. Rejection is caused by an immune response to the allogeneic antigen on the graft, which is a protein that varies from species to species and is therefore recognized as a foreign by the recipient (Janeway, et al., 2001, Immunobiology: The Immune System). in Health and Disease.5th edition.New York: Garland Science).

Advances in surgical techniques, immunosuppressive and infectious monitoring and treatment over the past decade have revolutionized patient and graft survival. However, despite this success, transplant recipients still exhibit much higher morbidity and mortality than the general population. This is due in part to the effects of chronic allograft injury, but the main cause is comorbidity affected by chronic immunosuppressive drug use (Soulillou and Giral 2007, Transplantation 72 (Suppl 12): S89-93, Hourmant et al., 1998, Lancet 351: 623-628; Halloran, 2004, N Engl J Med 351: 2715-2729).

Immunosuppression related toxicities can be significant. For example, many studies in adult liver transplant recipients have demonstrated a time-dependent successive decline in renal function following exposure to immunosuppressive therapy. Other important complications of long-term immunosuppression include new onset of post-transplant diabetes (NODAT), hypertension, hyperlipidemia and the need for statin therapy (Srinivas et al., 2008, CJASN: (Supplement 2) S101-S116]). . To remedy this situation, research priorities in organ transplantation are shifting from the search for new potent immunosuppressive drugs to a strategy to minimize immunosuppression, and to ensure that transplanted tissues are maintained for long periods of time without sustained immunosuppression. It becomes a goal.

On the other hand, the IL-2 receptor has three forms: (1) low affinity receptor IL-2RA without signaling; (2) intermediate affinity receptors (IL-2Rαβγ) consisting of IL-2RB and IL-2RG, which are widely expressed in conventional T cells (Tcon), NK cells, eosinophils and monocytes; And (3) a high affinity receptor (IL-2Rαβγ) consisting of IL-2RA, IL-2RB and IL-2RG, transiently expressed in activated T cells and constantly expressed in Treg cells. In addition, interleukin-10 (IL-10), initially known as cytokine synthesis inhibitor factor or CSIF, is a potent immunomodulator of hematopoietic cells, especially immune cells. Cells such as activated Th2 cells, B cells, keratinocytes, monocytes and macrophages produce IL-10 (Moore et al., Annu. Rev. Immunol. 11: 165 (1993)). IL-10 inhibits the activation and effector function of many cells, including T cells, monocytes and macrophages. In particular, IL-10 inhibits cytokine synthesis, including the synthesis of IL-1, IFN-γ, and TNF, by cells such as Th1 cells, natural killer cells, monocytes, and macrophages (Fiorentino et al., J. Exp. Med., 170: 2081-2095 (1989); Fiorentino et al., J. Immunol. 146: 3444 (1991); Hsu et al., Int. Immunol. 4: 563 (1992); Hsu et al., Int. Immunol. 4: 563 (1992); D'Andrea et al., J. Exp. Med. 178: 1041 (1993); de Waal Malefyt et al., J. Exp. Med. 174: 915 (1991); Fiorentino et al., J. Immunol. 147: 3815 (1991)).

The present invention overcomes economic disadvantages by inducing the production of a biological therapeutic agent expressing a fusion antibody anti-IL-2Rα antibody / IL-10 in the body, and complements the non-specific immunosuppressive effects of existing therapeutic agents, Its purpose is to use in various transplant rejection diseases.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting transplant rejection.

The present invention also provides anti-IL-2Rα antibody / IL-10 antibody or antigen-binding fragment.

The present invention also provides an antibody composition for inhibiting transplant rejection.

The present invention also provides a method of producing a minicircle vector.

In addition, the present invention provides a method for preparing an anti-IL-2Rα antibody to which IL-10 protein is bound.

According to the present invention, the minicircle vectors of the present invention can reduce the development cost than existing biological immunosuppressive agents by expressing the fusion antibody anti-IL-2Rα antibody / IL-10 in the body, and the IL-expressed therefrom is expressed. The 10-linked (linked) anti-IL-2Rα antibody can compensate for the shortcomings of non-specific immunosuppressive effects that also reduce regulatory T cell populations and balance immunity to have a more effective therapeutic effect on various transplant rejection diseases.

1 is a diagram showing a map of a plasmid containing a minicircle vector having a gene expression cassette.
Figure 2 is a schematic diagram showing the process of recombining the minicircle vector from the plasmid in the host strain.
3 is a diagram showing an anti-IL-2Rα heavy chain / IL-10 fusion protein bound to the C-terminus of an IL-10-linked anti-IL-2Rα antibody heavy chain expressed from a first minicircle vector of the present invention.
4 is a diagram showing an anti-IL-2Rα antibody light chain expressed from the second minicircle vector of the present invention.
FIG. 5 shows the anti-IL-2Rα antibody heavy chain / IL-10 and anti-IL-2Rα antibody light chains expressed by combining the first minicircle vector and the second minicircle vector of the present invention, respectively. Figure shows IL-2Rα antibody / IL-10.
FIG. 6 is a diagram illustrating whether a minicircle vector is produced by treating arabinos or restriction enzymes with moplasmid. FIG.
FIG. 7 is a diagram illustrating whether anti-IL-2Rα antibody heavy chain / IL-10 and anti-IL-2Rα antibody light chain were introduced into the prepared minicircle vectors, respectively.
8 is a diagram confirming the expression of the RFP protein in the moplasmid and minicircle vector.
9 is a diagram confirming the concentrations of anti-IL-2Rα and IL-10 present in the serum on day 3 after minicircle vector administration to rats.
10 is a graph showing survival dates of transplanted skin of rats:
saline: control group treated with PBS;
mc-mock: mice transformed with empty minicircle vectors;
mc-IL-2Rα: Mice transformed with minicircle vectors inserted with gene cassettes expressing heavy and light chains of the anti-IL-2Rα antibody;
mc-IL-10: mouse transformed with a minicircle vector into which a gene cassette expressing IL-10 protein was inserted; And
mc-DTA: Inserted a minicircle vector (first minicircle vector) and a gene cassette expressing an anti-IL-2Rα antibody light chain inserted with a gene cassette expressing an anti-IL-2Rα antibody heavy chain / IL-10 fusion protein Mice transformed with the minicircle vector (second minicircle vector).
Figure 11 is a graph showing the results of calculating the mean survival time (Mean Survival Time) of the skin transplanted through the survival curve.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments are presented by way of illustration of the present invention, when it is determined that the detailed description of the well-known technology or construction known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. However, the present invention is not limited thereto. The invention is susceptible to various modifications and applications within the scope of the following claims and the equivalent scope thereof.

In addition, terms used in the present specification (terminology) are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of the user, the operator, or customs in the field to which the present invention belongs. Therefore, the definitions of the terms should be made based on the contents throughout the specification. Throughout the specification, when a part is said to "include" a certain component, it means that it may further include other components, except to exclude other components unless specifically stated otherwise.

In one aspect, the invention provides an anti-interleukin-2 receptor alpha (IL-2Rα) antibody heavy chain gene and a first minicircle vector comprising an IL-10 (interleukin-10) gene; And a second minicircle vector comprising an anti-IL-2Rα antibody light chain gene.

In one embodiment, the first minicircle vector comprises a polypeptide of SEQ ID NO: 1 operably linked with an anti-IL-2Rα antibody heavy chain gene of SEQ ID NO: 3 and an IL-10 gene of SEQ ID NO: 4, and an anti-IL The C-terminus of the -2Rα antibody heavy chain may express a fusion protein (SEQ ID NO: 5) (FIG. 3) bound to the IL-10 protein.

In one embodiment, the second minicircle vector comprises the polypeptide of SEQ ID NO: 2 and may express an anti-IL-2Rα antibody light chain (SEQ ID NO: 6) (FIG. 4).

In one embodiment, the anti-IL-2Rα antibody heavy chain / IL-10 fusion protein and the anti-IL-2Rα light chain expressed in the two minicircle vectors are bound in vivo to bind the anti-IL- with IL-10 protein. 2Rα antibodies can be achieved (FIG. 5).

In one embodiment, the polynucleotide of SEQ ID NO: 1 encoding the anti-IL-2Rα antibody heavy chain / IL-10 protein comprises a polypeptide encoding the anti-IL-2Rα antibody heavy chain (SEQ ID NOs: 3 and IL-10 protein). In-frame gene linkage of the polypeptide (SEQ ID NO: 4) results in a single "fusion protein" in which the IL-10 protein is bound to the C-terminus of the anti-IL-2Rα heavy chain upon transcription / translation. Is intended to mean a functional link between two or more elements. For example, an operative linkage between two polypeptides fuses the two polypeptides together in frame to produce a single polypeptide fusion protein. In, the fusion protein may further comprise a third polypeptide, which may further comprise a linker sequence.

The terms "CD25" or "IL-2 receptor α" or "IL-2Rα" as used herein refer to mammals such as primates (eg, humans) and rodents (eg, mice and rats) and otherwise. If not indicated, any natural or recombinant IL-2Rα from any vertebrate source, including livestock or agricultural mammals. The term also encompasses naturally occurring variants of IL2Rα, eg, splice variants or allelic variants or non-naturally occurring variants. Human IL-2 exerts its biological effect through signaling through its receptor system, IL-2R.

When preparing the expression cassettes included in the minicircle vectors of the present invention, various polynucleotides can be engineered to provide the polynucleotide sequences in the proper orientation and, where appropriate, in the appropriate reading frame. To this end, polynucleotides can be linked using adapters or linkers or manipulations may be involved to provide convenient restriction sites, removal of unnecessary DNA, removal of restriction sites, and the like. For example, linkers such as two glycines can be added between the polypeptides. Methionine residues encoded by atg nucleotide sequences may be added to allow for initiation of gene transcription. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution, such as transitions and transversions can be included.

The polynucleotide encoding the anti-IL-2Rα antibody heavy chain / IL-10 fusion protein of the present invention has a coding region due to degeneracy of codons or in consideration of codons preferred in organisms to express the antibody. Various modifications may be made to the coding region within the range of not changing the amino acid sequence of the antibody expressed from, and various modifications or modifications may be made within the range not affecting the expression of the gene even in a portion except the coding region. Those skilled in the art will appreciate that such modified genes are also within the scope of the present invention. That is, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such polynucleotides may be single or double chained and may be DNA molecules or RNA (mRNA) molecules.

A "minicircle vector" or "minicircle DNA" of the present invention refers to a circular DNA having an antibody or genetically engineered antibody gene expression cassette, wherein the expression cassette is expressed by the gene sequence of the signal peptide and / or the gene sequence of the tag. Or a promoter operably linked to a genetically engineered antibody gene. In one embodiment, minicircle vectors of the invention may comprise a CMV7 promoter, an antibody or genetically engineered antibody gene, an EF1 promoter, a reporter gene, polyA tailing and attR.

In one embodiment, the anti-IL-2Rα antibody heavy chain (HC) / IL-10 construct encoding a fusion protein fused IL-10 to the C-terminus of the anti IL-2 Ra heavy chain is represented by SEQ ID NO: 1. The polynucleotide encoding the anti-interleukin-2 receptor alpha (anti-IL-2Rα) antibody heavy chain, which may include the nucleotide sequence, may include the nucleotide sequence represented by SEQ ID NO: 3, and the anti-IL-2Rα antibody. The polynucleotide encoding the light chain of may comprise a nucleotide sequence represented by SEQ ID NO: 2, the polynucleotide encoding the IL-10 protein may comprise a nucleotide sequence represented by SEQ ID NO: 4.

In one embodiment, the first minicircle vector may comprise a nucleotide sequence represented by SEQ ID NO: 1, wherein the anti-IL-2Rα antibody heavy chain gene and the IL-10 gene are operably linked, and the second minicircle The vector may include the nucleotide sequence represented by SEQ ID NO: 2.

In one aspect, the invention provides a kit comprising a first minicircle vector comprising an anti-IL-2Rα antibody heavy chain gene and an IL-10 gene; And a second minicircle vector comprising an anti-IL-2Rα antibody light chain gene as an active ingredient.

In one embodiment, the first minicircle vector may comprise a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 to which the anti-IL-2Rα antibody heavy chain gene and the IL-10 gene are operably linked, and the second minicircle The vector may comprise a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 encoding the light chain of the anti-IL-2Rα antibody.

In one aspect, the invention relates to an antibody specific for IL-2Rα to which an IL-10 protein is linked.

In one embodiment, the antibody comprises an anti-IL-2Rα antibody light chain comprising the amino acid sequence of SEQ ID NO: 6; And an anti-IL-2Rα antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 7, wherein an IL-10 protein comprising the amino acid sequence of SEQ ID NO: 8 is bound to the C-terminus of the antibody heavy chain. It may be a binding fragment.

In one embodiment, the antibody may comprise an anti-IL-2Rα antibody heavy chain / IL-10 fusion protein comprising the amino acid sequence of SEQ ID NO: 5.

Anti-IL-2Rα antibody or CD25 antibody of the present invention is another antibody used as a therapeutic agent for xenograft rejection and is an antibody against CD25, an alpha chain of IL-2 receptor on T cells. Antibodies bind to CD25 on T cells and block IL-2's binding and response to T cells to inhibit proliferation of T cells or to remove activated T cells expressing CD25, similar to the mechanism of OKT3 antibody. It is used to prevent tissue transplant rejection. CD25 is mainly expressed in activated T cells as well as in activated B cells, some thymic cells and promyelocytic cells. Anti-CD25 antibodies have recently been used clinically to prevent the destruction of CD4 + T and CD8 + T cell dependent glioma cells.

The antibodies are in the form of whole antibodies as well as functional fragments of antibody molecules. The whole antibody has a structure having two full length light chains and two full length heavy chains, and each light chain is connected with a heavy chain and a disulfide bond. The functional fragment of an antibody molecule means a fragment having an antigen binding function. Examples of antibody fragments include (i) the variable region (VL) of the light chain, the variable region (VH) of the heavy chain, the constant region (CL) of the light chain, and Fab fragment consisting of the first constant region (CH1) of the heavy chain; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341: 544-546 (1989)); (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments F (ab ') 2 fragments; (vii) single-chain Fv molecules (scFv) bound by peptide linkers that bind the VH and VL domains to form antigen binding sites; (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965) and (ix) diabody WO94 / 13804, a multivalent or multispecific fragment produced by gene fusion.

The first and second minicircle vectors according to the present invention can be transformed into appropriate host eukaryotic cells, followed by culturing the transformed host cells, thereby mass-producing the antibodies according to the present invention. Suitable culture methods, media conditions, etc. according to the type of host cell can be easily selected by those skilled in the art from known techniques known to those skilled in the art. The host cell may be a eukaryotic cell derived from yeast, insect cells, plant cells, animal cells such as Saccharomyces cerevisiae . The expression vector introduction method into the host cell may be any method known to those skilled in the art.

Antibodies or fragments thereof of the present invention can be generated using various phage display methods known in the art and described in Brinkman et al., 1995, J. Immunol. Methods, 182: 41-50; Ams et al., 1995, J. Immunol. Methods, 184, 177-186; Kettleborough et al. 1994, Eur. J. Immunol, 24, 952-958; Persic et al., 1997, Gene, 187, 9-18; And Burton et al., 1994, Advances in Immunol, 57, 191-280; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; And WO 95/20401; And US 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; And 5,969,108.

Other organisms, including transgenic (eg, genetically engineered) mice, or other mammals, can be used to produce antibodies to which the proteins of the invention are bound (see, eg, US 6,300,129). For example, only the variable regions of the mouse immune genes (heavy chain V, D, and J segments, and the light chain V and J segments) are replaced with corresponding human variable sequences so that the engineered mice have a high affinity antibody having human variable sequences. It is known that it can be used for mass production (see, eg, US 6,586,251; US 6,596,541, and US 7,105,348).

The pharmaceutical composition of the present invention may further include a known immunosuppressant or a transplant rejection inhibitor in addition to the first minicircle vector and the second minicircle vector, or the antibody or fragment thereof expressed as the active ingredient. And other treatments known for the treatment associated with these transplant rejection diseases.

In the present invention, the term "inhibition of transplant rejection" means any prophylactic and transplant rejection that inhibits or delays the occurrence, proliferation and recurrence of transplant rejection or immunity induced by transplantation by administration of the pharmaceutical composition according to the invention. Any therapeutic action that improves or beneficially alters the symptoms of any disease caused by the response. Those skilled in the art to which the present invention pertains can refer to the data presented by the Korean Medical Association and the like to know the exact criteria of the disease for which the composition is effective, and to determine the extent of improvement, improvement and treatment. will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount effective for preventing or treating a disease caused by transplant rejection, and the therapeutically effective amount of the composition of the present invention may be determined by various factors, For example, it may vary depending on the administration method, the target site, the condition of the patient, and the like. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing. Such considerations when determining the effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects, and an effective dose level refers to Factors including health status, type of transplant, severity, drug activity, drug sensitivity, method of administration, time of administration, route of administration and rate of administration, duration of treatment, combination or simultaneous use of drugs, and other well-known factors in the medical field It can be determined according to. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The pharmaceutical compositions of the present invention may comprise carriers, diluents, excipients or combinations of two or more thereof conventionally used in biological preparations. As used herein, the term "pharmaceutically acceptable" means to exhibit properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used as needed. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injectable solutions and sprays, with oral or injectable formulations being more preferred.

As used herein, the term "administration" means providing a substance or substance to an individual or patient in any suitable manner, and according to the method desired, non-oral administration (eg, intravenous, subcutaneous, intraperitoneal). Or topically applied as an injection formulation) or orally, and the dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. Liquid preparations for oral administration of the compositions of the present invention include suspensions, solvents, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. And the like may be included together. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like. The pharmaceutical composition of the invention may be administered by any device in which the active substance may migrate to the target cell. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial growth Pharmaceutical carriers such as preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like).

As used herein, the term "individual" means monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or humans, including humans with or may develop cancer. Means all animals, including guinea pigs, and by effectively administering the pharmaceutical composition of the present invention to the subject can be effectively prevented or treated diseases caused by the transplant rejection or transplant rejection. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate , Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 parts by weight to 90 parts by weight based on the composition, but is not limited thereto.

In one aspect, the present invention relates to an antibody composition for inhibiting transplant rejection, comprising an antibody or antigen-binding fragment expressed and assembled from the first and second minicircle vectors as an active ingredient.

In one aspect, the invention relates to a plasmid comprising an anti IL-2Rα antibody heavy chain / IL-10 fusion gene construct or an anti IL-2Rα antibody light chain gene expression cassette. In one embodiment, the moplasmid completes the recombination process of minicircle DNA in the host strain to exclude the prokaryotic plasmid element to produce the first minicircle vector and the second minicircle vector of the present invention (FIG. 2).

In one aspect, the present invention comprises the steps of inserting a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 to the plasmid comprising the nucleotide sequence of SEQ ID NO: 9; Inserting a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 2 into a moplasmid comprising a nucleotide sequence of SEQ ID NO: 9; Transforming the plasmids prepared in steps 1) and 2) into host cells; Treating the minicircle induction formulation in the host cell; And purifying the minicircle vector.

In one embodiment, the moplasmid can be transformed into the host cell separately or simultaneously.

In one embodiment, the minicircle induction formulation may comprise arabinose.

In one embodiment, the host cell transformed with the moplasmid may be E. coli.

In one aspect, the invention provides a host cell comprising a first minicircle vector comprising an anti-IL-2Rα antibody heavy chain gene and an IL-10 gene, and a second minicircle vector comprising an anti-IL-2Rα antibody light chain gene. Transforming; Culturing the host cell; And it relates to a method for producing an anti-IL-2Rα antibody bound to the IL-10 protein, comprising the step of separating the antibody from the host cell.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Anti-IL-2Rα Antibody / IL-10 Expression Minicircle Vector Preparation

1-1. Construction of anti-IL-2Rα antibody heavy chain (HC) and IL-10 expressing minicircle vectors

1-1-1. Generation of parental plasmid

In order to prepare a miniplasmid for preparing a minicircle vector expressing a protein linked to IL-10 at the FC site of the anti-IL-2Rα heavy chain (HC), an anti-IL-2Rα antibody heavy chain (HC) and an IL-10 gene are included. The plasmid of FIG. 1 was prepared. Specifically, the anti-IL-2Rα antibody heavy chain (HC) gene (SEQ ID NO: 3) and the gene of IL-10 (SEQ ID NO: 4) were synthesized using PCR techniques, respectively, followed by C- of the anti IL-2 Ra heavy chain. An anti-IL-2Rα antibody heavy chain (HC) / IL-10 construct (SEQ ID NO: 1) encoding a fusion protein fused to IL-10 at the end was constructed. Subsequently, they were digested with restriction enzymes BamHI (New England Biolabs inc., Cat # .R0136S) and XbaI (New England Biolabs inc., Cat # .R0145S), respectively, and then cut and loaded onto an agarose gel. The size of the fused construct was confirmed and an insert was inserted into the vector using a gel extraction kit (QIAEX II Gel Extraction Kit, Qiagen, Cat. # 20021). The produced anti-IL-2Rα antibody heavy chain (HC) / IL-10 insert was a minicircle induction moplasmid pMC.CMV-MCS-EF1α-RFP-SV40PolyA vector (System Bioscience, Cat. # MN512A-1) (SEQ ID NO: No. 9) was introduced using a T4 DNA ligase (New England Biolabs Inc., Cat # .M0202S).

1-1-2. Construction of anti-IL-2Rα antibody heavy chain (HC) and IL-10 expressing minicircle vectors

In order to downsize the plasmid, a minicircle vector expressing the anti-IL-2Rα antibody heavy chain to which IL-10 is linked was prepared from the plasmid as shown in FIG. 2. Specifically, 1 μg of the plasmid DNA prepared in Example 1-1-1 was introduced into a competent cell (ZYCY10P3S2T) using a heat shock method. S.O.C. After adding culture medium (invitrogen, Cat #REF 15544-034) for 1 hour at 37 rpm at 220 rpm, 100 μl of this was plated on an LB plate medium containing kanamycin (Lysogeny Broth agar plate). Incubated overnight at 37 ° C. Receptor cells transformed with moplasmid were formed into colonies in LB plate medium, and one of the colonies was inoculated into 5 ml of LB medium containing kanamycin and stirred incubated at 37 ° C. at 200 rpm for 8 hours. Subsequently, 12 g of Tryptone (BD Bioscience, Cat. # 211705), 24 g of Yeast (BD Bioscience, Cat. # 212750) and Glycerol in TB (Terrific Broth) medium containing kanamycin (total volume 1 L, total volume 1 L) in 900 ml of sterile distilled water. (amresco, Cat. # 0854-1L) 4ml dissolved and prepared by adding 100ml PB buffer) was inoculated in 900ml and expanded in the same conditions overnight. To prepare a minicircle induction mix by adding 36 ml of 1N NaOH and 900 µl of 20% L-arabinose to 900 ml of LB broth, mixed with cells inoculated in the TB medium and 200 rpm at 30 ° C. Stirred culture for 5 hours. After incubation, the cells were collected by centrifugation at 4 ° C. at 7000 rpm for 15 minutes, and then extracted with Nucleobond Xtra Midi kit (Macherey-nagel, Cat #REF 740410.50.). Minicircle vectors expressing IL-2Rα antibody heavy chain (HC) were extracted.

1-2. Construction of Anti-IL-2Rα Light Chain (LC) Expression Minicircle Vectors

1-2-1. Generation of parental plasmid

The plasmid of FIG. 1 comprising the anti-IL-2Rα light chain (LC) (SEQ ID NO: 2) gene was prepared using the method of Example 1-1-1.

1-2-2. Construction of anti-IL-2Rα Light Chain (LC) Expression Minicircle Vectors

1 μg of the plasmid DNA prepared in Example 1-2-1 was introduced into a competent cell (ZYCY10P3S2T) using a heat shock method. S.O.C. After addition of the medium, the culture was stirred at 220 rpm for 1 hour at 37 ° C, and 100 µl of this was plated on LB plate medium containing kanamycin (Lysogeny Broth agar plate) and incubated overnight at 37 ° C. Receptor cells transformed with moplasmid were formed as colonies in LB plate medium, and one of the colonies was inoculated into 5 ml of LB medium containing kanamycin and stirred incubated at 37 ° C. at 200 rpm for 8 hours. Thereafter, the cells were inoculated in 900 ml of TB (Terrific Broth) medium containing kanamycin and expanded overnight under the same conditions. To prepare a minicircle induction mix by adding 36 ml of 1N NaOH and 900 µl of 20% L-arabinose to 900 ml of LB broth, mixed with cells inoculated in the TB medium and 200 rpm at 30 ° C. Stirred culture for 5 hours. After incubation, the cells were collected by centrifugation at 7000 rpm for 15 minutes at 4 ° C., and then extracted with Nucleobond Xtra Midi kit (Macherey-nagel, Cat #REF 740410.50.) To extract the anti-IL-2Rα antibody light chain (LC) of FIG. 4. The expressing minicircle vector was extracted.

As a result, the anti-IL-2Rα antibody heavy chain (HC) / IL-10 protein and the anti-IL-2Rα light chain (LC) protein respectively expressed from the two minicircle vectors prepared in the above examples are shown in FIG. Bound together to form a bispecific antibody in which IL-10 is bound to the C-terminus of the anti-IL-2Rα antibody.

Example 2. Confirmation of Anti-IL-2Rα Antibody / IL-10 Expression Minicircle Vector Expression

2-1. Production and gene transfer confirmation of moplasmid and minicircle vectors

In order to confirm that the minicircle vector was produced from the plasmid produced in Example 1, the plasmid without the antibody gene (pp-mock), including the anti-IL-2Rα heavy chain (HC) / IL-10 gene, were included. Treated arabinose, or restriction enzymes BamHI and XbaI (enzynomics), to each of the plasmids (pp-DTA-LC) containing the moplasmid (pp-DTA-HC), and the anti-IL-2Rα light chain (LC) genes After that, electrophoresis on 0.6% agarose gel was performed to confirm the production of minicircles. In addition, to determine whether each gene was introduced into the minicircle DNA vector, a minicircle vector containing no antibody gene (mc-mock), a mini containing an anti-IL-2Rα heavy chain (HC) / IL-10 gene Each of the circle vector (mc-DTA-HC) and the minicircle vector (mc-DTA-LC) containing the anti-IL-2Rα light chain (LC) gene was treated with restriction enzymes BamHI and XbaI (enzynomics) at 37 ° C. The restriction enzyme reaction was performed for 30 minutes. The reacted samples were compared by electrophoresis on 0.6% agarose gel.

Treatment of arabinos or restriction enzymes with moplasmid confirmed that each minicircle vector was produced (FIG. 6), and the minicircle vectors prepared therefrom were anti-IL-2Rα antibody heavy chain (HC) / IL. -10 and anti-IL-2Rα light chain (LC) was confirmed to be introduced (Fig. 7).

2-2. Confirmation of Protein Expression of Moplasmid and Minicircle Vectors ( in vitro )

In order to confirm whether the plasmid and minicircle vectors produced in Example 1 normally express each protein in cells, reporter gene RFP was introduced into the plasmid and minicircle vectors to confirm the expression of RFP protein. First, a HEK293T cell, including, but the 96-well plate (SPL life Sciences, Pocheon, Korea) for 2 X 10 ⁴ / of 100㎕ divided by the number of cells, 7.5% FBS and antibiotics and cultured for one day in DMEM medium without. Then, PBS (pH 7.4) (control), moplasmid (pp-mock), empty minicircle vector (mc-mock), minicircle vector expressing anti-IL-2Rα antibody (mc-IL-2Rα), Minicircle expressing IL-10 protein only (mc-IL10), and two minicircle vectors mc-DTA-HC of the present invention (minicircle vector comprising anti-IL-2Rα heavy chain (HC) / IL-10 gene) And (mc-DTA) DNAs mixed with mc-DTA-LC (minicircle vector comprising anti-IL-2Rα light chain (LC) gene) were added to opti-MEM and lipofectamine 2000 (invitrogen, Carlsbad, CA, USA). ) Transformed into HEK293T cells and incubated at 37 ° C. for 16 hours, exchanged with growth medium, incubated for 24 hours, and RFP protein expression was confirmed by an inverted fluorescence microscpe.

 As a result, it was confirmed that the expression of the RFP protein is normal in all of the plasmid and minicircle vectors except the control group (FIG. 8).

2-3. In Vitro Protein Expression of Minicircle Vectors by ELISA

In order to confirm the expression of the two minicircle vectors of the present invention and the operation of the protein, the two minicircle vectors of the present invention including the mc-DTA-HC (anti-IL-2Rα heavy chain (HC) / IL-10 gene) 40 μg of DNA (mc-DTA) mixed with minicircle vector) and mc-DTA-LC (minicircle vector containing anti-IL-2Rα light chain (LC) gene) was extracted from BALB / c (Orient Bio Inc., Seongnam -si, Korea) Injected by hydrodynamic delivery via the tail vein of male rats. Thereafter, rat serum was collected until 30 days and subjected to ELISA for anti-IL-2Rα and IL-10, respectively. For anti-IL-2Rα, 100 μl of human sIL-2Rα (1 μg / ml; TONBO biosciences, San Diego, Calif., USA) was dispensed into 96 well plates and coated overnight at room temperature. The coated plate was blocked (1% BSA in PBS, pH 7.2-7.4, 0.2um filtered) to block nonspecific reactions after washing. Subsequently, 100 μl / well of the serum collected from Basiliximab (simulated injection; Novartis, Rotkreuz, Switzerland) and rats used as a standard were dispensed at 100 μl / well for reaction for 2 hours at room temperature and washed. HRP (EMD Millipore Corporation, Temecula, Calif., USA) was diluted at a ratio of 1: 5,000 and treated for 2 hours at room temperature. After the final washing, the reaction was performed with TMB substrate solution (iNtRON Biotechnology, Inc., Seongnam-si, Korea) for 20 minutes at room temperature, followed by reaction solution (2NH ₂ SO ₄ ), and absorbance was measured at 450 nm and 570 nm. . In addition, in the case of IL-10, 100 μl of anti-hIL-10 (10 μg / ml; Peprotech, Rocky Hill, NJ, USA) was dispensed and reacted at room temperature overnight, followed by coating and washing. Blocked. Subsequently, 100 μl / well of the serum collected from recombinant hIL-10 (R & D systems Inc., Minneapolis, MN, USA) and rats used as a standard was dispensed at 100 μl / well for 2 hours at room temperature, washed, and detected. Anti-hIL10-biotinylated antibody (0.4 μg / ml; R & D systems Inc.) was treated for 2 hours at room temperature. After further washing, the anti-steptavidin-HRP (R & D systems Inc.) was diluted at a ratio of 1:40 and reacted at room temperature for 20 minutes, and after the last washing, the TMB substrate solution and the reaction stopping solution were treated in the same manner as above. Absorbance was measured at nm and 570 nm.

As a result, unlike the serum of mice injected with the empty minicircle vector, the concentrations of anti-IL-2Rα and IL-10 were significantly increased in the serum of mice injected with the two minicircle vectors of the present invention. (FIG. 9) (* P <0.05 vs. mc-mock.). That is, it was found that the anti-IL-2Rα and IL-10 proteins are co-expressed in the injected minicircle vector.

Example 3 Confirmation of Treatment Effect of Minicircle Vector Transplant Rejection

An allograft dermal animal model was used to investigate the therapeutic effect of minicircle vectors expressing anti-IL-2Rα and IL-10 of the present invention in transplant rejection. In an allogeneic dermal transplant model, the tail skin of a donor male C57BL / c mouse (Orient Bio Inc.) was stripped and implanted into the back skin of a recipient male BALB / c mouse (Orient Bio Inc.). One day before transplantation, the beneficiaries were divided into five groups, minicircle-IL2Rα (mc-IL-2Rα) expressing PBS (pH 7.4), empty minicircle (mc-mock), anti-IL-2Rα antibody single protein. DNA mixed with minicircle-IL10 (mc-IL10) expressing IL-10 single protein, and mc-DTA-HC and mc-DTA-LC minicircle vectors expressing anti-IL-2Rα / IL10 ( mc-DTA) was injected respectively. After transplantation, the change of skin was observed every day, and survival rate was calculated by rejection when scab formation or skin atrophy occurred in the transplanted skin.

As a result, the transplanted skin of the mice injected with the two minicircle vectors (mc-DTA) survived the longest (Fig. 10), and in particular, the mean survival time of the skin transplanted through the survival curve was calculated. As a result, the transplanted skin survival time of the mice was longer than that of the minicircle vector expressing a single protein (mc-IL-2Rα or mc-IL-10) (FIG. 11). (# P <0.05 vs. saline or mc-mock; @ P <0.001 vs. the other groups.).

From the above results, it was found that anti-IL-2Rα / IL10 to which proteins expressed from the two minicircle vectors of the present invention bound significantly inhibited transplant rejection.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> MINICIRCLE VECTOR FOR TREATING TRANSPLANTATION DISEASE <130> PN1711-419 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 1932 <212> DNA <213> Artificial Sequence <220> <223> anti-IL-2R alpha HC / IL-10 <400> 1 gccaccatgg gatggagcta tatcatcctc tttttggtgg ccacagcggc cgatgtccac 60 tcgcaactgc agcagtctgg aacagttctg gctagacctg gcgcctccgt gaagatgtct 120 tgtaaagcca gcggctacag cttcacccgg tactggatgc actggatcaa gcagaggcct 180 ggacagggac tcgagtggat cggagccatc tatcccggca acagcgacac cagctacaac 240 cagaagttcg agggcaaagc caagctgaca gccgtgacaa gcgccagcac agcctatatg 300 gaactgagca gcctgacaca cgaggacagc gccgtgtact actgtagcag agactacggc 360 tactacttcg acttctgggg ccagggcacc acactgacag tgtctagcgc ctctacaaag 420 ggccctagcg ttttcccact ggctcctagc agcaagagca catctggtgg aacagccgct 480 ctgggctgcc tggtcaagga ttactttcct gagcctgtga ccgtgtcctg gaattctggt 540 gctctgacaa gcggcgtgca cacctttcca gccgtgctgc aaagcagcgg cctgtactct 600 ctgtctagcg tggtcacagt gcctagctct agcctgggca cccagaccta catctgcaac 660 gtgaaccaca agcctagcaa caccaaggtg gacaagagag tggaacctcc taagagctgc 720 gacaagaccc acacctgtcc tccatgtcct gctccagaac tgctcggcgg accctccgtt 780 ttcctgtttc cacctaagcc taaggacacc ctgatgatca gcagaacccc tgaagtgacc 840 tgcgtggtgg tggatgtgtc tcacgaggac cccgaagtga agttcaattg gtacgtggac 900 ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagtacaa cagcacctac 960 agagtggtgt ccgtgctgac agtgctgcac caggactggc tgaacggcaa agagtacaag 1020 tgcaaggtgt ccaacaaggc cctgcctgct cctatcgaga aaaccatcag caaggccaag 1080 ggccagccaa gagaacccca ggtttacaca ctgcctccaa gcagggacga gctgaccaag 1140 aatcaggtgt ccctgacctg cctcgtgaag ggcttctacc cttccgatat cgccgtggaa 1200 tgggagagca atggccagcc tgagaacaac tacaagacaa cccctcctgt gctggacagc 1260 gacggctcat tcttcctgta cagcaagctg accgtggaca agtccagatg gcagcagggc 1320 aatgtgttca gctgctctgt gatgcacgag gccctgcaca accactacac ccagaaaagc 1380 ctgtctctga gccctggaaa aggtggcgga ggatctggcg gcggaggaag cggaggcggc 1440 ggatcttctc caggacaggg aacacagagc gagaacagct gtacacactt ccccggcaac 1500 ctgcctaaca tgctgagaga tctgcgggac gccttcagca gagtgaaaac attcttccag 1560 atgaaggacc agctggacaa cctgctgctg aaagagagcc tgctggaaga tttcaagggc 1620 tacctgggct gtcaggccct gagcgagatg atccagttct acctggaaga agtgatgccc 1680 caggccgaga atcaggaccc tgatatcaaa gcccacgtga acagcctggg cgagaacctg 1740 aaaaccctgc ggctgagact gcggcggtgt cacagatttc tgccctgcga gaacaagagc 1800 aaggccgtgg aacaagtgaa gaacgccttc aacaagctgc aagagaaggg catctacaag 1860 gccatgtccg agttcgatat cttcatcaac tacatcgagg cctacatgac catgaagatc 1920 cggaattagt ga 1932 <210> 2 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> anti-IL-2R alpha LC <400> 2 gccaccatgg gatggagcta tatcatcctc tttttggtgg ccacagcggc cgatgtccac 60 tcgcagattg tgtctacaca gagccccgcc atcatgagcg cctctccagg cgagaaagtg 120 accatgacat gtagcgccag cagcagcaga tcctacatgc agtggtatca gcagaagccc 180 ggcacaagcc ccaagagatg gatctacgat accagcaagc tggcctctgg cgtgccagcc 240 agattttctg gctctggcag cggcaccagc tacagcctga caatctctag catggaagcc 300 gaggacgccg ccacctacta ctgtcaccag agaagcagct acaccttcgg cggaggcacc 360 aagctggaaa tcaagagaac agtggccgct ccgagcgtgt tcatctttcc accaagcgac 420 gagcagctga agtctggcac agcctctgtc gtgtgcctgc tgaacaactt ctaccctcgg 480 gaagccaagg tgcagtggaa ggtggacaat gccctgcaga gcggcaatag ccaagagagc 540 gtgaccgagc aggacagcaa ggactctacc tactctctga gcagcaccct gacactgagc 600 aaggccgact acgagaagca caaggtgtac gcctgtgaag tgacacacca gggcctgagc 660 agccctgtga ccaagagctt caatagaggc gagtagtga 699 <210> 3 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> anti-IL-2R alpha HC <400> 3 gccaccatgg gatggagcta tatcatcctc tttttggtgg ccacagcggc cgatgtccac 60 tcgcaactgc agcagtctgg aacagttctg gctagacctg gcgcctccgt gaagatgtct 120 tgtaaagcca gcggctacag cttcacccgg tactggatgc actggatcaa gcagaggcct 180 ggacagggac tcgagtggat cggagccatc tatcccggca acagcgacac cagctacaac 240 cagaagttcg agggcaaagc caagctgaca gccgtgacaa gcgccagcac agcctatatg 300 gaactgagca gcctgacaca cgaggacagc gccgtgtact actgtagcag agactacggc 360 tactacttcg acttctgggg ccagggcacc acactgacag tgtctagcgc ctctacaaag 420 ggccctagcg ttttcccact ggctcctagc agcaagagca catctggtgg aacagccgct 480 ctgggctgcc tggtcaagga ttactttcct gagcctgtga ccgtgtcctg gaattctggt 540 gctctgacaa gcggcgtgca cacctttcca gccgtgctgc aaagcagcgg cctgtactct 600 ctgtctagcg tggtcacagt gcctagctct agcctgggca cccagaccta catctgcaac 660 gtgaaccaca agcctagcaa caccaaggtg gacaagagag tggaacctcc taagagctgc 720 gacaagaccc acacctgtcc tccatgtcct gctccagaac tgctcggcgg accctccgtt 780 ttcctgtttc cacctaagcc taaggacacc ctgatgatca gcagaacccc tgaagtgacc 840 tgcgtggtgg tggatgtgtc tcacgaggac cccgaagtga agttcaattg gtacgtggac 900 ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagtacaa cagcacctac 960 agagtggtgt ccgtgctgac agtgctgcac caggactggc tgaacggcaa agagtacaag 1020 tgcaaggtgt ccaacaaggc cctgcctgct cctatcgaga aaaccatcag caaggccaag 1080 ggccagccaa gagaacccca ggtttacaca ctgcctccaa gcagggacga gctgaccaag 1140 aatcaggtgt ccctgacctg cctcgtgaag ggcttctacc cttccgatat cgccgtggaa 1200 tgggagagca atggccagcc tgagaacaac tacaagacaa cccctcctgt gctggacagc 1260 gacggctcat tcttcctgta cagcaagctg accgtggaca agtccagatg gcagcagggc 1320 aatgtgttca gctgctctgt gatgcacgag gccctgcaca accactacac ccagaaaagc 1380 ctgtctctga gccccggcaa gtagtga 1407 <210> 4 <211> 549 <212> DNA <213> Homo sapiens <400> 4 gccaccatgg gatggagcta tatcatcctc tttttggtgg ccacagcggc cgatgtccac 60 tcgagcccag gccagggcac ccagtctgag aacagctgca cccacttccc aggcaacctg 120 cctaacatgc ttcgagatct ccgagatgcc ttcagcagag tgaagacttt ctttcaaatg 180 aaggatcagc tggacaactt gttgttaaag gagtccttgc tggaggactt taagggttac 240 ctgggttgcc aagccttgtc tgagatgatc cagttttacc tggaggaggt gatgccccaa 300 gctgagaacc aagacccaga catcaaggcg catgtgaact ccctggggga gaacctgaag 360 accctcaggc tgaggctacg gcgctgtcat cgatttcttc cctgtgaaaa caagagcaag 420 gccgtggagc aggtgaagaa tgcctttaat aagctccaag agaaaggcat ctacaaagcc 480 atgagtgagt ttgacatctt catcaactac atagaagcct acatgacaat gaagatacga 540 aactagtga 549 <210> 5 <211> 642 <212> PRT <213> Artificial Sequence <220> <223> Anti-IL-2R alpha HC / IL-10 <400> 5 Ala Thr Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala   1 5 10 15 Ala Asp Val His Ser Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg              20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe          35 40 45 Thr Arg Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu      50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Ser Tyr Asn  65 70 75 80 Gln Lys Phe Glu Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser                  85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr His Glu Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ser Arg Asp Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln         115 120 125 Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly                 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     450 455 460 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 465 470 475 480 Gly Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His                 485 490 495 Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe             500 505 510 Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu         515 520 525 Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys     530 535 540 Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro 545 550 555 560 Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu                 565 570 575 Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg             580 585 590 Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn         595 600 605 Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu     610 615 620 Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 625 630 635 640 Arg asn         <210> 6 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> Anti-IL-2R alpha LC <400> 6 Ala Thr Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala   1 5 10 15 Ala Asp Val His Ser Gln Ile Val Ser Thr Gln Ser Pro Ala Ile Met              20 25 30 Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser          35 40 45 Ser Arg Ser Tyr Met Gln Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro      50 55 60 Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala  65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser                  85 90 95 Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser             100 105 110 Ser Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val         115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys     130 135 140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn                 165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser             180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys         195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr     210 215 220 Lys Ser Phe Asn Arg Gly Glu 225 230 <210> 7 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Anti-IL-2R alpha HC <400> 7 Ala Thr Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala   1 5 10 15 Ala Asp Val His Ser Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg              20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe          35 40 45 Thr Arg Tyr Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu      50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Ser Tyr Asn  65 70 75 80 Gln Lys Phe Glu Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser                  85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr His Glu Asp Ser Ala Val             100 105 110 Tyr Tyr Cys Ser Arg Asp Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln         115 120 125 Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val     130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val             180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro         195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys     210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly                 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     450 455 460 Pro Gly Lys 465 <210> 8 <211> 181 <212> PRT <213> Homo sapiens <400> 8 Ala Thr Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala   1 5 10 15 Ala Asp Val His Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser              20 25 30 Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg          35 40 45 Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu      50 55 60 Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr  65 70 75 80 Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu                  85 90 95 Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val             100 105 110 Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg         115 120 125 Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln     130 135 140 Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala 145 150 155 160 Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr                 165 170 175 Met Lys Ile Arg Asn             180 <210> 9 <211> 7044 <212> DNA <213> Artificial Sequence <220> <223> pMC.CMV-MCS-EF1alpha-RFP-SV40PolyA vector <400> 9 acattaccct gttatcccta gatacattac cctgttatcc cagatgacat accctgttat 60 ccctagatga cattaccctg ttatcccaga tgacattacc ctgttatccc tagatacatt 120 accctgttat cccagatgac ataccctgtt atccctagat gacattaccc tgttatccca 180 gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg acataccctg 240 ttatccctag atgacattac cctgttatcc cagatgacat taccctgtta tccctagata 300 cattaccctg ttatcccaga tgacataccc tgttatccct agatgacatt accctgttat 360 cccagatgac attaccctgt tatccctaga tacattaccc tgttatccca gatgacatac 420 cctgttatcc ctagatgaca ttaccctgtt atcccagatg acattaccct gttatcccta 480 gatacattac cctgttatcc cagatgacat accctgttat ccctagatga cattaccctg 540 ttatcccaga tgacattacc ctgttatccc tagatacatt accctgttat cccagatgac 600 ataccctgtt atccctagat gacattaccc tgttatccca gatgacatta ccctgttatc 660 cctagataca ttaccctgtt atcccagatg acataccctg ttatccctag atgacattac 720 cctgttatcc cagataaact caatgatgat gatgatgatg gtcgagactc agcggccgcg 780 gtgccagggc gtgcccttgg gctccccggg cgcgactagt gaattgatac tagtattatg 840 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 900 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 960 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 1020 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 1080 ggcgtgtacg gtgggaggtt tatataagca gagctcgttt agtgaaccgt cagatcgcct 1140 ggagacgcca tccacgctgt tttgacctcc atagaagatt ctagagctag cgaattcgaa 1200 tttaaatcgg atccgcggcc gcgaaggatc tgcgatcgct ccggtgcccg tcagtgggca 1260 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt 1320 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 1380 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 1440 cgcaacgggt ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca 1500 cgcgcccgcc gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct 1560 cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg 1620 agaccgggcc tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg 1680 ctttgcctga ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt 1740 tacagatcca agctgtgacc ggcgcctacg ctagacgcca ccatggccct tagtaagcac 1800 ggcctgacta aggacatgac catgaagtac catatggagg gctcggtgga cgggcacaag 1860 ttcgtgatta cgggccacgg gaacggcaac ccattcgagg ggaaacagac aatgaacctg 1920 tgtgtggtgg aaggcggacc cctgcccttc tcggaggaca tcctgtccgc aacctttgac 1980 tacggcaatc gcgtgttcac agagtatcca cagggtatgg tggatttctt taagaacagc 2040 tgtcccgcag gatacacctg gcaccgcagc ctgctcttcg aggacggggc ggtgtgtacc 2100 acctccgctg atatcaccgt gtccgtcgag gagaactgct tctaccacaa cagcaaattc 2160 cacggcgtga actttcctgc cgacgggcct gtgatgaaga aaatgactac gaattgggag 2220 cccagctgcg agaaaatcat cccggtccct cgtcagggaa tcttgaaggg cgacatcgca 2280 atgtacctcc tgctcaagga cgggggaaga taccgatgcc agttcgatac catctacaag 2340 gcaaagtccg atcccaagga gatgccagag tggcacttca tccagcacaa actcacccgc 2400 gaggaccgct cagatgccaa gaaccagaag tggcaactgg tcgagcacgc cgtggcatcc 2460 cgtagcgccc tgccagggta agtcgacaat caacctctga ttacaaaatt tgtgaaagat 2520 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc 2580 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct 2640 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca 2700 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt 2760 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg 2820 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga 2880 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt 2940 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc gccctgctgc 3000 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt 3060 gggccgcctc cccgcctggt acctttaaga ccaatgactt acaaggcagc tgtagatctt 3120 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagataa 3180 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc 3240 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 3300 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag 3360 tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa 3420 cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt 3480 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 3540 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc 3600 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 3660 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 3720 agaagtagtg aggaggcttt tttggaggcc tagacttttg cagatcgacc catgggggcc 3780 cgccccaact ggggtaacct ttgagttctc tcagttgggg gtaatcagca tcatgatgtg 3840 gtaccacatc atgatgctga ttataagaat gcggccgcca cactctagtg gatctcgagt 3900 taataattca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag 3960 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa 4020 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt 4080 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat 4140 gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg 4200 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca 4260 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg 4320 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag 4380 caaggtgtag atgacatgga gatcctgccc cggcacttcg cccaatagca gccagtccct 4440 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca 4500 cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac 4560 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat 4620 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc 4680 gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagagct 4740 tgatcccctg cgccatcaga tccttggcgg cgagaaagcc atccagttta ctttgcaggg 4800 cttcccaacc ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa 4860 aaccgcccag tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc 4920 gcttgcgttt tcccttgtcc agatagccca gtagctgaca ttcatccggg gtcagcaccg 4980 tttctgcgga ctggctttct acgtgctcga ggggggccaa acggtctcca gcttggctgt 5040 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt 5100 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg 5160 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta 5220 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 5280 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt 5340 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag 5400 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct 5460 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaccaaa atcccttaac 5520 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 5580 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 5640 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 5700 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 5760 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 5820 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 5880 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 5940 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 6000 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 6060 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 6120 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 6180 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 6240 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 6300 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 6360 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 6420 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt 6480 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 6540 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 6600 ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg 6660 gcatgcataa tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc 6720 cgtcaagccg tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt 6780 cactttttct tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa 6840 tacccgcgag aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg 6900 catccgggtg gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct 6960 taagacgcta atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca 7020 aacatgctgt gcgacgctgg cgat 7044

Claims (9)

A first minicircle vector comprising a polynucleotide of SEQ ID NO: 1 to which an anti-interleukin-2 receptor alpha (IL-R-2) antibody heavy chain gene and an IL-10 (interleukin-10) gene are operably linked; And
A pharmaceutical composition for inhibiting transplant rejection comprising a second minicircle vector comprising a polynucleotide of SEQ ID NO: 2, which is an anti-IL-2Rα antibody light chain gene, as an active ingredient. delete delete An anti-IL-2Rα antibody light chain consisting of the amino acid sequence of SEQ ID NO: 6; And
An IL-10 protein comprising an IL-10 protein consisting of the amino acid sequence of SEQ ID NO: 8 linked to the C-terminus of an anti-IL-2Rα antibody heavy chain consisting of the amino acid sequence of SEQ ID NO: 7 and an anti-IL-2Rα antibody heavy chain Bound anti-IL-2Rα antibody. The method of claim 4, wherein
The anti-IL-2Rα antibody heavy chain and the IL-10 protein are anti-IL-2Rα antibody heavy chain / IL-10 fusion protein consisting of the amino acid sequence of SEQ ID NO: 5 IL-2Rα antibody. Antibody composition for inhibiting transplant rejection comprising the antibody of claim 4 as an active ingredient.
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