KR20080095141A - A fusion polypeptide comprising a human fc including human albumin linker and a biologically active polypeptide and a method of preparing the same - Google Patents

Abstract

A fusion polypeptide is provided to minimize non-specific immuno-reaction in vivo and optimize the activity by using a hybrid Fc, increase in vivo stability of a physiologically active polypeptide and maintain the activity by coupling an antibody Fc fragment through a linker derived from human albumin to the active polypeptide. A fusion polypeptide comprises an antibody Fc fragment, a linker derived from human albumin linked to a C-terminal of the Fc fragment, and an activated polypeptide, of which an N-terminal is linked to the linker. A host cell transformed by a recombinant vector includes a gene consisting of a nucleic acid sequence coding the fusion polypeptide to prepare the fusion polypeptide. A pharmaceutical composition comprises the fusion polypeptide and a pharmaceutically acceptable carrier.

Description

Fusion polypeptide comprising a human Fc including human albumin linker and a biologically active polypeptide and a method of preparing the same}

1 is a schematic diagram of an antibody fusion polypeptide prepared by linking a human albumin linker to the C-terminus of an Fc of a human antibody.

FIG. 2 shows the structure of human interferon-beta antibody fusion polypeptides comprising various types of human albumin linkers. FIG.

FIG. 3 shows the results of in vitro bioactivity of human interferon-beta antibody fusion polypeptides comprising various kinds of human albumin linkers. FIG.

The present invention relates to a fusion polypeptide in which an active polypeptide is linked to the C-terminus of an Fc fragment of an antibody, and more particularly, to an Fc fragment of an antibody and a human albumin linker linked to the C-terminus of the Fc fragment. And an active polypeptide having an N-terminus linked to the linker, and a method for producing the same.

Existing protein drugs that use physiologically active polypeptides have low stability and are easily degraded by proteolytic enzymes in vivo and removed through the kidneys or liver. It is necessary to administer it frequently. In order to solve the problems of the first generation protein drugs, the method of enhancing the half-life in vivo by changing the glycosylation of the active polypeptide, or the high solubility high molecular material such as polyethylene glycol (PEG) The chemical addition method, albumin or its fragments have been used to combine the active polypeptide with the recombinant method to produce a fusion polypeptide. However, both of these methods can increase the stability of the protein, but the disadvantage is that the titer of the bioactive protein is significantly lowered.

Therefore, a more efficient method has been required, and recently, methods for fusion of an Fc fragment of human immunoglobulin G (human IgG) and an active polypeptide, which are known to have a long half-life in serum, have been developed. This method is known to increase the half-life of proteins to be delivered as the neonatal Fc receptor (FcRn) and human IgG bind and stabilize among the Fc receptors (Nat. Biotechnol. 1997 15: 637-640). However, this method has a problem in that IgG increases antigenic nonspecific immune response in vivo through enhancing phagocytosis through activation of FcRn and other Fc receptors and activation of the complement system.

As a representative method for solving this problem, there is a method of causing mutations in amino acids located at these binding sites. In other words, N-terminal, more specifically, 234-237 amino acids in the CH2 region of IgG are known to be important for binding to FcR class I (Nature, 1998 332: 563-564). Amino acid numbers are determined and used according to the EU index (Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, 1991). On the other hand, binding to C1q is known to be important for the C-terminal region of CH2 of human IgG (Nature, 1998 332: 563-564). In particular, mutation of Proline 331 of IgG1 to Serine significantly reduces C1q binding. (Tao et al, J Exp Med 178: 661-667, 1993). In addition, mutations of Glutamate 318, Lysine 320, and Lysine 322 have been reported to significantly reduce complement system activation (J. Immunol., 1995 154: 5590-5600). Thus, based on these findings, Fc of mouse IgG2a mutated with Glutamate 318 and Lysine 320, which are known to be important for C1q binding and Leucine 235, which is recently important for binding to FcR, in order to enhance the function of Fc as a carrier of active proteins. There have been reports of binding erythropoietin (EPO) to fragments (WO 99/02709). In this report, however, no specific validation of the function of the mutated Fc was made. Other studies have shown that the persistence of human-derived Fc with modified effector functions has been increased to increase serum half-life by mutating Serine 228 of human IgG4 Fc to Proline and Leucine 235 to Alanine. 7,030,226).

In addition to these methods, Fc receptor binding sites of CH2 of IgG1 or IgG4 (233, 234, 235, 236) in 1999 (Eur. J. Immunol. 1999. 29; 2613-2624) and patents (WO 2005/040217) Replacing amino acids with the corresponding amino acids of IgG2 has been reported to significantly reduce the ability to bind Fc receptors. However, there is no report on whether the chimeric IgG invented in this report actually has a long half-life in vivo, and when the fusion protein such as EPO is fused, the fusion protein stays active for a long time. There has also been no report on whether it is possible.

 Accordingly, there is a need for a method of overcoming the problem of increasing the nonspecific immune response of the Fc as a carrier of the active protein and at the same time improving the in vivo persistence of the active protein bound to the Fc.

On the other hand, fusion of an active polypeptide to an Fc fragment results in a dimer fusion polypeptide, and as a result, the two active polypeptides are adjacent to each other, thereby spatially inhibiting the binding of the active polypeptide to a target receptor and the like. This decreases. Therefore, in the case where the C-terminus of the active protein is bound to the N-terminus of the Fc fragment, the N-terminus of the Fc fragment preferably includes a hinge domain with good flexibility. However, when the active polypeptide is fused to the C-terminus of the Fc, it is preferable to insert a linker because there is no flexible region such as a hinge. Currently well known linkers for maintaining the activity of the active polypeptide fused to the C-terminus of the Fc are those consisting of amino acids such as Gly, Ser (WO 2004/022088; US5,908,626; WO2006 / 000448), requiring the development of more desirable linkers. It is becoming.

Based on this background, the inventors have found that the use of hybrid Fc in combination with Fc fragments present in vivo results in minimizing the in vivo nonspecific immune response and optimizing the activity of the fusion polypeptide. When the linker derived from human albumin is used when the active polypeptide is fused to the terminal, the present invention was completed by confirming that the activity of the fused active polypeptide is maintained to the maximum.

Accordingly, one object of the present invention is to provide a fusion polypeptide of an active polypeptide coupled with an antibody Fc fragment via a linker derived from human albumin, which can increase the in vivo stability and maintain activity of the physiologically active polypeptide.

Another object of the present invention is to provide a nucleic acid sequence encoding the fusion polypeptide.

Another object of the present invention is to provide an efficient recombinant vector for preparing the fusion polypeptide.

Another object of the present invention to provide a transformant transformed by the recombinant vector.

It is another object of the present invention to provide a method of culturing the transformant to produce a fusion polypeptide comprising a biologically active polypeptide coupled to a hybrid Fc fragment.

Another object of the present invention is to provide a pharmaceutical composition comprising the fusion polypeptide.

The present invention relates to a fusion polypeptide in which an active polypeptide is linked to the C-terminus of an Fc fragment of an antibody, and more particularly, to an Fc fragment of an antibody and a human albumin linker linked to the C-terminus of the Fc fragment. And an active polypeptide having an N-terminus linked to the linker, and a method for producing the same.

Accordingly, the present invention provides, in one aspect, an antibody Fc fragment, a linker derived from human albumin linked to the C-terminus of the Fc, and a fusion polypeptide comprising an active polypeptide linked to the linker with an N-terminus.

In the present invention, immunoglobulin (hereinafter referred to as “Ig”) is a generic term for polypeptides that selectively interact with antigens and participate in biological immunity, and may be derived from humans. Looking at the general structure of the immunoglobulin, immunoglobulin is composed of a heavy chain (light chain) and heavy chain (light chain), heavy chain constant region gamma (γ), mu (μ), alpha (α), delta (δ) , Epsilon (ε) type and subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types (Coleman et al., Fundamental Immunology, 2nd Ed., 1989, 55-73). By these immunoglobulins can be divided into isotypes of IgG, IgA, IgD, IgE and IgM. IgG is further divided into subclasses IgG1, IgG2, IgG3 and IgG4. In addition, immunoglobulins are structurally known as fragments. Among the fragments of immunoglobulins, Fab (antibody binding fragment) is a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain. It has one antigen binding site. Fc (crystalizable fragment) is divided into three parts, the hinge region (CH2 domain) and the CH3 domain connected by disulfide bond.

The present invention provides a fusion polypeptide in which an Fc fragment of an immunoglobulin is fused with a biologically active polypeptide via a linker derived from human albumin, thereby minimizing a decrease in the biological activity of the biologically active polypeptide in the fusion polypeptide, while at the same time living in a biologically active polypeptide. It has been found that the stability can be increased.

In the present invention, the Fc fragment may be a human IgG subclass of IgG1, IgG2, IgG3, IgG4, more preferably a hybrid Fc (hybrid Fc) consisting of a combination of these IgG subclasses, or a combination of human IgD and IgG4. hFc) fragments may be used. As used herein, the term "hybrid" is a term used to mean that a sequence corresponding to two or more different immunoglobulin fragments of different origins exists in an immunoglobulin fragment of a single chain. The hybrid Fc used in the present invention was selected from the various Ig subtypes as Fc, which is based on IgG having a long half-life through binding to FcRn. In particular, in order to solve the problem of increasing the antigenic nonspecific immune response in vivo through activation of the complement system, IgG4 without complement activity was used, unlike other IgG subclasses. In addition, the hybrid Fc according to the present invention was used to inhibit phagocytosis and the like by binding to other Fc receptors other than FcRn, IgD having low binding capacity to the IgG receptor or Fc receptor with Fc receptor was used. . More specifically, in the case of a hybrid Fc (thFc-1) consisting of a combination of IgG2 and IgG4, N-terminal six amino acids (231-236 amino acids) in the CH2 region of IgG4 was replaced with the corresponding amino acids of IgG2, In the case of hybrid Fc (thFc-2) of IgD and IgG4, the N-terminal 10 amino acids (amino acids 231-240) in the CH2 region of IgG4 were replaced with 8 amino acids of IgD (see FIG. 1). In addition, a hinge domain having a region capable of forming an interdisulfide bond is required to prepare a dimeric Fc fusion polypeptide. In the case of thFc-1, a hinge of all 15 amino acids of IgG1 was used. In the case of thFc-2, 15 amino acids (amino acids 277-291) were used among the 64 amino acids of the IgD hinge. In addition, the signal sequence of 23 amino acids of the human tissue plasminogen activator (tpa) is used in both types of thFc in order to allow the fusion polypeptide to be secreted out of the cell efficiently. It was.

In the present invention, when "linker" is bound to the C-terminus of the active polypeptide to form a fusion polypeptide, between the C-terminus and the active polypeptide of the Fc to increase the structural flexibility to promote the activity of the bound active polypeptide Refers to the peptide to be inserted. The linker is preferably capable of minimizing the immune response, and for this purpose, it is preferable to use fragments already present in vivo. In the present invention, as a preferred example of the linker, a peptide linker consisting of 33 amino acids located in the 282 to 314th part of the human albumin most present in the blood, more preferably consisting of 13 amino acids located in the 292 to 304th part A peptide linker is provided, which is a part that is mostly exposed to the outside of the three-dimensional structure is a part that minimizes the possibility of inducing an immune response in the body.

By "active polypeptide" in the present invention is meant any kind of biologically active polypeptide which is capable of increasing its half-life in vivo while maintaining its activity by deficient in a hybrid Fc fragment comprising an albumin linker according to the present invention. In a specific embodiment of the invention, human interferon beta was used as the active polypeptide to bind to the C-terminus of the Fc fragment. However, biologically active polypeptides that can be prepared as fusion polypeptides are not limited thereto, and particularly preferred bioactive polypeptides are human growth hormone, interferon (interferon) which is frequently administered when administered to a human body for the purpose of treating or preventing a disease. -α, -β, -γ, etc.), granulocyte colony stimulating factor, erythropoietin, betacellulin (BTC) and antibody fragments.

In addition, growth hormone releasing hormone, growth hormone releasing peptide, interferon receptors (e.g. interferon-alpha, -beta and -gamma, water soluble type I interferon receptors, etc.), granulocyte-macrophage colony stimulating factor (GM-CSF), gluca Cone-like peptides (eg GLP-1), G-protein-coupled receptors, interleukins (eg IL-1 receptor, IL-4 receptor, etc.), enzymes (eg glucocerebro) Sidase (glucocerebrosidase, etc.), interleukin and cytokine binding proteins (eg, IL-18bp, TNF-binding proteins, etc.), macrophage activators, macrophage peptides, B cell factors, T cell factors, protein A, allergic inhibitors , Cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythrocyte production Factor, high glycated red blood cells Adult, angiopoietin, hemoglobin, thrombin, thrombin receptor active peptide, thrombomodulin, blood factor Ⅶ, blood factor 인 a, blood factor Ⅷ, blood factor Ⅸ, blood factor 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, elcatonin , Connective tissue activators, tissue factor pathway inhibitors, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth Magnetic flux (e.g. nerve growth factor, ciliary neurotrophic factor, axogenesis factor-1, brain-natriuretic peptide, glial derived neurotrophic factor ), Netrin, neutrophil inhibitor factor, neurotrophic factor, neuturin, etc.), parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenal cortex hormone, Glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors (e.g. TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, B cell activator receptor, etc.), receptor antagonists (eg IL1-Ra, etc.), cell surface antigens (eg CD 2, 3, 4, 5, 7, 11a , 11b, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc ), Monoclonal antibodies, polyclonal antibodies, antibody fragments (eg scFv, Fab, Fab ', F (ab') 2 and Fd), virus derived vaccine antigens and the like.

In addition, fusion polypeptides, including Fc fragments, linkers, and active polypeptides of the invention include variants of the fusion polypeptide as well as proteins having their wild type amino acid sequences. A variant of a fusion polypeptide refers to a polypeptide in which the normal amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). In addition, the fusion polypeptide may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like. The variant or modifier is a functional equivalent that exhibits the same biological activity as the native protein, but may be a variant or modifier that modifies the properties of the protein as needed. Preferably, the polypeptide has increased structural stability against heat, pH, etc., or increased polypeptide activity of the protein by variation and modification on amino acid sequence.

In another aspect, the present invention provides a nucleic acid sequence encoding a fusion polypeptide comprising an antibody Fc fragment, an albumin linker and an active polypeptide.

Nucleic acid sequences encoding the fusion polypeptides of the invention can be isolated from nature or prepared using chemical synthesis. The nucleic acid having the above-described nucleic acid sequence may be single or double stranded, and may be a DNA molecule (genome, cDNA) or an RNA molecule. When chemically synthesizing nucleic acid sequences encoding fusion polypeptides, synthesis methods well known in the art, for example, as described in Engels and Uhlmann, Angew Chem Int Ed Engl. 37: 73-127, 1988 Methods may be used, such as tryster, phosphate, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on solid supports, and the like.

In addition, the nucleic acid sequence according to the present invention includes codon-optimized to increase the expression of the protein by increasing the stability of the mRNA by increasing the G-C content of the nucleic acid base encoding the hybrid Fc fragment and the biologically active polypeptide. Preferably, the nucleic acid sequence includes a codon-optimized base sequence of at least one of an Fc gene and a gene of an active polypeptide such as human interferon beta. More preferably, the nucleic acid sequence is a base sequence of an active polypeptide gene or an Fc gene. All contain nucleic acid sequences consisting of optimized codons.

In another aspect, the present invention provides an expression vector comprising the nucleic acid sequence.

As used herein, an "expression vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express a gene insert. As used herein, the term “operably linked” refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. Operable linkage of a promoter and a nucleic acid sequence encoding a protein may affect the expression of the encoding nucleic acid sequence.Operative linkages with recombinant vectors may be prepared using genetic recombination techniques well known in the art. Site-specific DNA cleavage and ligation uses enzymes generally known in the art, and the like.

Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and adenovirus vectors, retroviral vectors, viral vectors such as adeno-associated viral vectors, and the like. For the purposes of the present invention, the expression vector may further comprise signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, start codons, stop codons, introns, polyadenylation signals and enhancers.

In the present invention, the promoter may be constitutive or inducible. Prokaryotic cells include, but are not limited to, lac, tac, T3 and T7 promoters. Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example the long terminal repeat (LTR) promoter of HIV, moroni virus, cytomegalovirus (CMV). ), Epstein bar virus (EBV), Loose sacoma virus (RSV) promoters, as well as promoters derived from β-actin promoter, human hemoglobin, human muscle creatine, human metallothionein. The promoter included in the expression vector of the present invention is preferably a promoter of an early gene of cytomegalovirus (CMV), which is known as the strongest promoter to date. Initiation and termination codons are generally considered to be part of the nucleic acid sequence encoding the immunogenic target protein and must be functional in the individual when the gene construct is administered and must be in frame with the coding sequence. In addition, mammalian genes include introns, which have been reported to have significantly increased expression levels than in the absence of introns (Korb et al., Nucleic Acids Res, 25, 5901-5908, 1993). This is known because intron stabilizes mRNA and increases transcription efficiency. Accordingly, the expression vector of the present invention is preferably the second intron sequence of the beta globin gene of rabbits (globin intervening sequence, hereinafter abbreviated as "gIVS"). Polyadenylation motifs (pA) are important for enhancing the stability of mRNA (Drummond et al., Nucleic Acids Res, 25, 7375-7394, 1985). The expression vector of the present invention comprises a polyadenylation motif, the polyadenylation motif is preferably a polyadenylation motif (polyadenylation motif) of bovine growth hormone (BGH) gene.

In addition, a selectable marker gene is required to select for cell lines that transduce the expression vector of the invention to overexpress the fusion polypeptide. Accordingly, the expression vector of the present invention is prepared to include a selection marker gene, and is selected for facilitating selection in CHO / dhfr- cells, which are cell lines derived from Chinese hamster ovary (abbreviated as “CHO”). It is preferable to use a marker gene, dihydrofolate reductase (hereinafter abbreviated as “DHFR”) gene. CHO / dhfr- cells are CHO cells in which the DHFR gene is damaged and thus the nucleic acid biosynthesis process is incomplete, and when the fusion polypeptide expression vector of the present invention including the DHFR gene is transduced into the cells, the nucleic acid synthesis ability is restored. Fusion polypeptide expression vectors can be introduced to specifically select cells expressing the fusion polypeptide.

In another aspect, the present invention provides a host cell transformed with the recombinant expression vector.

In the present invention, "transformation" or "transfection" into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue or organ and selects a suitable standard technique according to the host cell as known in the art. Can be done. These methods include electroporation, plasma fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agrobacterial mediated transformation, PEG, dextran sulfate, lipofectamine and dry / inhibition mediated Transformation methods, and the like.

In the present invention, since the expression amount and formula of the protein are different depending on the host cell, the host cell may be selected and used most suitable for the purpose. Although host cells include prokaryotic or eukaryotic cells, it is preferable to use eukaryotic cells capable of glycosylation for the purposes of the present invention, and in particular, it is preferable to use CHO cells, but the present invention is not limited thereto. More preferably, the host cell may be used having a function as a marker that can determine whether the transformation. In a specific embodiment of the present invention, in order to prepare a fusion polypeptide high expression cell line exhibiting high activity in the human body, while having a relatively well glycated glycosylation system or glycosylation system, it is safe and high in vivo activity without causing antigenicity in the human body CHO cells, which are hamster cell types, are used as host cells among mammalian cells that produce glycostructures capable of expressing. In addition, CHO-dhfr- cells, which are CHO-derived cell lines, are CHO cells in which the nucleic acid biosynthesis process is incomplete because the DHFR gene is damaged, and CHO cells having the expression vector restore nucleic acid synthesis ability due to the DHFR gene included in the expression vector of the present invention. Done. Therefore, by culturing CHO cells in a cell culture medium free of nucleic acid, it is possible to select cells with an expression vector. In addition, the growth of cells in a culture medium containing methotrexate (MTX), which is a DHFR substrate analogue, is possible by amplifying the DHFR gene in the cells. Since the expression vector of the present invention contains the DHFR gene, the fusion polypeptide gene is also amplified at the same time. do. Thus, by adding MTX, which is a substrate analog of DHFR, to cell cultures, cell lines that express high fusion polypeptides can be made.

In another aspect, the present invention provides a method for producing a fusion polypeptide comprising a physiologically active polypeptide coupled to a hybrid Fc by culturing the transformed host cell.

Cultivation of the transformant in the production of the immunoglobulin fragments may be made according to suitable media and culture conditions known in the art. This culture process can be used by those skilled in the art can be easily adjusted according to the strain selected. The immunoglobulin fragments of the present invention obtained by culturing the transformant may be used without purification, and may be further purified by high purity using various conventional methods such as dialysis, salt precipitation, and chromatography. have. Among them, the method using chromatography is the most widely used, and there is no law that can be applied in any case to the selection of column type and order, and ion exchange chromatography, size exclusion chromatography, etc. , Affinity chromatography, protein-A affinity column, and the like. In the present invention, purified using a protein-A affinity column, SP Sepharose FF column and the like.

In another aspect, the present invention provides a pharmaceutical composition comprising the fusion polypeptide and a pharmaceutically acceptable carrier.

In the present invention, "administration" means introducing a predetermined substance into a patient by any suitable method and the route of administration of the conjugate may be administered through any general route as long as the drug can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. However, upon oral administration, since the peptide is digested, it is desirable to formulate the oral composition to coat the active agent or to protect it from degradation in the stomach. It may preferably be administered in the form of an injection. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.

Pharmaceutical compositions comprising a fusion polypeptide of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings, etc., in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be used in combination, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. It may also be formulated as a solution, a suspension, a tablet, a sustained release formulation and the like. Examples of suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

The actual dosage of the fusion polypeptide drug of the invention is determined by the type of drug that is the active ingredient, along with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

In another aspect, the present invention provides a pharmaceutical composition comprising a fusion polypeptide and a pharmaceutically acceptable carrier, wherein the active polypeptide is human interferon-beta.

Human interferon-beta contained in the pharmaceutical composition of the present invention has been mainly used as a therapeutic agent for multiple sclerosis, but also for the treatment of cancers such as breast cancer or cervical cancer, autoimmune disorders, viral infections, diseases related to HIV, hepatitis C, and the like. There are reports that it can be used (Pilling et al., European Journal of Immunology 29: pp1041-1050,1999) and its pharmacological effects continue to be reported. For this reason, it should be understood that the pharmacological effect in the pharmaceutical composition of the present invention includes not only the pharmacological effect as a therapeutic agent for multiple sclerosis, but also all the pharmacological effects of other human interferon-beta. Nevertheless, the pharmacological effects are such that human interferon beta is still mainly used as a therapeutic agent for multiple sclerosis, and that the therapeutic effects of cancer, autoimmune disorders, viral infections, HIV-related diseases, hepatitis C, etc. have already been identified. It is preferable that it is a pharmacological effect.

Hereinafter, the present invention will be described by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example  1. Preparation of human interferon-beta antibody fusion polypeptide expression vector

The hybrid Fc produced in the present Example is largely classified into two types, and tpa hybrid Fc-1 (thFc-1) is a tpa signal sequence, 15 amino acids of IgG1 hinge, and 6 amino acids of N-terminus (231) of CH2 region of IgG4. -236 amino acid) is replaced with the corresponding amino acid of IgG2, tpa hybrid Fc-2 (thFc-2) is the tpa signal sequence, 15 amino acids of the IgD hinge (277-291 amino acids), CH2 of IgD N-terminal eight amino acids (292--299 amino acid) of the region, and CH2 region C-terminal region (241-340 amino acid) derived from IgG4 and the entire CH3 region (341-447 amino acid) ( See FIG. 1). 33 albumin linkers (the 282-314 amino acids) are linked to the C-terminus of thFc-1 and thFc-2, and the N-terminus of the amino acid of human interferon-beta is linked to the entire fusion. The amino acid sequence corresponding to the polypeptide was determined and named as thFc-1-AL (6) -IFN-beta (SEQ ID NO: 1) and thFc-2-AL (6) -IFN-beta (SEQ ID NO: 2). 2). The codon optimized sequence of each gene constituting the fusion polypeptide was obtained by requesting gene synthesis from TOP gene Tech (www.topgenetech.com).

During synthesis, an EcoR I restriction enzyme recognition site was created upstream of ATG, the synthesis initiation codon of the amino terminus of the tpa signal sequence gene, ie at the 5 'end, followed by a Not I restriction enzyme recognition site following the stop codon of human interferon-beta Put it. After cutting with the two restriction enzymes, the resultant clone was cloned into the pAD11 expression vector (SEQ ID NO: 3) prepared by the present inventors, and finally the pAD11 thFc-1-AL (6) -IFN-beta expression vector and pAD11 thFc-2-AL (6 ) -IFN-beta expression vector was prepared. The pAD11 vector is a vector containing a strong promoter of cytomegalovirus, gIVS, and pA of bovine growth hormone. It is composed of the highest expression level when the various elements related to expression were tested.

In addition, in order to prepare fusion polypeptides comprising albumin linkers of various lengths, the Pst I restriction enzyme recognition site in CH3 of the Fc region and the Sca I restriction enzyme recognition site located at the N-terminus of human interferon-beta Was used to prepare fusion polypeptides with each albumin linker. In other words, it synthesizes from the C-terminal Pst I position of Fc to the Sca I position of N-terminus of interferon-beta, but each synthesis includes genes encoding albumin linker amino acids of different lengths. Synthesis was carried out at www.topgenetech.com (see FIG. 2). Finally, AL (0) linked directly to IFN-beta without linker, AL (1) with 3 albumin linker-derived amino acids, AL (2) with 8 albumin linker-derived amino acids, 13 albumin linker-derived amino acids And AL (3) having 18 albumin linker-derived amino acids and AL (5) having 23 albumin linker-derived amino acids, respectively, thFc-1-AL (6). Corresponding portions were replaced in) -IFN-beta and thFc-2-AL (6) -IFN-beta (see FIG. 2). In addition, as a control, a GS fragment including a glycine-serine linker was synthesized, and this portion was placed between Fc and IFN-beta instead of the albumin linker site. The synthesized one was cut into Pst I and Sca I to make an intact fusion polypeptide in the pUC57 vector state, and then cut into EcoR I and Not I and put into pAD11 vector to prepare all final vectors.

Example 2. Expression of Human Interferon-beta Antibody Fusion Polypeptides

COS-7 cells were cultured using DMEM medium (Gibco BRL) containing 10% FBS (bovine serum bovine). 5 ug 10 ⁶ cells were transformed by electroporation using 10 ug DNA of each human interferon-beta expression vectors, and then cells and cultures were harvested after 48 hours. The procedure was repeated three times, and the expression level of each protein was measured by the enzyme immunoassay using a commercially available IFN-beta expression kit (Endogen, catalog number, 41400-1A). It was.

Example 3. Purification of Human Interferon-beta Antibody Fusion Polypeptides

CHO / dhfr-cells in culture (DG44, ATCC, catalog # CRL-1651) were inoculated at 5 × 10 ⁵ per 60 mm dish plate and incubated for 18 hours. Subsequently, after replacing with 4.5 ml of culture medium (alpha-MEM + 10% dialyzed FBS + HT supplement), about 4 hours later, co-precipitation of CaPO4 was performed using human interferon beta antibody fusion polypeptide expression vector and control vector pAD11 vector, respectively. ) Was transduced by the method. That is, 10 μg of each DNA was first mixed with pure distilled water to have a final volume of 225 μl, and then 25 μl of 2.5 M CaCl 2 solution was added and mixed by vortexer. 250 μl of 2 × HBS solution (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO42H2O, 12 mM dextrose, 50 nM HEPES) was slowly dropped dropwise into the tube containing the DNA. After the reaction for 10 minutes at room temperature, put in a cell culture dish, and incubated for 6 hours. After washing three times with PBS, and added fresh culture medium again and incubated for 48 hours. Defective CHO / dhfr- cells cannot survive on alpha-MEM medium, which is a minimal essential nutrient medium without nucleic acids because of lack of nucleic acid biosynthetic capacity, but recombinant CHO cells with the DHFR gene have nucleic acid biosynthetic capacity. Select cultures may be performed using media that do not provide supplements. Specifically, the cells in culture by transforming with an interferon-beta antibody fusion polypeptide expression plasmid (1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500) and primary screening was performed while incubating in a 100 mm dish in medium without HT supplement. During the screening, the stomach was changed only once a week and the passage was not passed. Afterwards, colony-formed plates were selected for separation after about 2 to 3 weeks. Colonies were obtained from the selected plates and transferred to 24 well plates for subculture. After about one week to two weeks, cells were cloned into 24 well plates to trypsin to obtain cells, and then plated back to 24 well plates at a concentration of 2 × 10 ⁴ cells / well. After 48 hours, supernatants were obtained and the expression level of interferon-beta was measured by enzyme immunoassay. Unlike cells transformed with only the negative control pAD11 vector, expression was confirmed only in clones obtained by transformation with an interferon-beta antibody fusion polypeptide expression plasmid, and clones with high expression were obtained. Serum-free culture from clones showing the highest expression level among them, 5 L of culture supernatant was collected and used for purification. A filtration membrane (Pellicon 2 Cassette Filter, Millipore, MWCO 10K) was mounted with a pump (MasterFlex, Millipore) and the inside of the filtration system was washed with 20 L of tertiary sterile water. After the washing of the filter was completed, the supernatant of the culture supernatant of CHO cells was injected into the filter through a pump, and the filtrate was recovered and reinjected. The HiTrap rProtein A FF (Amersham-pharmacia biotech) column was equilibrated with 20 column sodium phosphate buffer (pH 7.0) by 2 column volumes. Concentrate was injected into the equilibrated column and the column was washed with 20 mM sodium phosphate buffer (pH 7.0). Protein was eluted by injecting 0.1 M sodium citrate buffer (pH 3.0) in 4 column doses. The eluate was added to an activated dialysis membrane (Spectra / Por Spectrum, MWCO 12-14K) and sealed, and then dialyzed three times at 6 hour intervals for 10-fold tertiary sterile water so that the concentration of the salt in the eluate was 5 mM or less. . The in vitro content of the protein eluate was measured by the enzyme immunoassay described in Example 3, through which the yield of each purification step was calculated.

Example 4. Determination of Activity of Human Interferon-beta Antibody Fusion Polypeptides

For activity measurement, specific activity was converted using a CPE assay using a parallel line model against the WHO standard. Experiments were performed when the WISH cells (ATCC, CCL-25) were grown to 90% or more in 100 mm dishes using DMEM / F12 (1: 1) medium containing 10% FBS. WHO international standard (NIBSC, 00/572) for measuring biological activity in cell medium was diluted to 40 IU / ml to create a 1: 2 fold dilution column. Samples were diluted to 40 IU / ml based on protein quantitation to create 1: 2 dilution lines. 100 μl of standard solution and sample solution were added to 4 wells of each concentration in a 96 well plate. WISH cells were added to 3 x 10 ⁴ cells / 50ul per well and incubated for 2 hours at 37 ℃. VSV per well (ATCC, VR-158) was added 1500 PFU / 30ul and incubated for 40 hours. 45 μl of MTT 5 mg / ml solution was added per well, and after 3 hours of incubation, the supernatant was removed, and 100 μl of DMSO was added, and the absorbance was measured at 570 nm. The measured absorbance was substituted into the formula based on the European Pharmacopoeia statistical processing method to calculate the biological activity of the interferon beta stock solution. The biological activity of the stock solution was calculated by multiplying the estimated potency by the relative potency of the sample solution based on the standard solution. That is, the biological activity (IU / mL) of the stock solution is the relative activity of the stock solution x the estimated titer of the stock solution.

As a result, thFc-1-AL (3) -IFN-beta containing 13 albumin-derived peptides (292-304th) as linkers was found to thFc-1-AL (0) -IFN-beta without albumin linker. It showed about 20-fold increased activity compared to that (see FIG. 3).

The hybrid Fc used in the fusion polypeptide of the present invention is a combination of fragments of isotype present in vivo without mutation, and optimizes the activity of the fused protein and minimizes the nonspecific immune response in vivo, thereby inducing the in vivo of the active protein. Half-life can be improved. In addition, the fusion polypeptide of the present invention can maintain the activity of the fused active polypeptide to the maximum by including a linker derived from human albumin. Thus, fusion polypeptides comprising the hybrid Fc and albumin linker according to the present invention will be widely used in the pharmaceutical and medical fields.

<110> Postech Foundation          Progen, Inc. <120> A fusion polypeptide comprising a human Fc including human          albumin linker and a biologically active polypeptide and a method          of preparing the same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> thFc-1-AL (6) -IFN-beta <400> 1 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly   1 5 10 15 Ala Val Phe Val Ser Pro Ser Glu Pro Lys Ser Cys Asp Lys Thr His              20 25 30 Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe          35 40 45 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro      50 55 60 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val  65 70 75 80 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr                  85 90 95 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val             100 105 110 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys         115 120 125 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser     130 135 140 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 145 150 155 160 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val                 165 170 175 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly             180 185 190 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp         195 200 205 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp     210 215 220 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 225 230 235 240 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ser Pro Leu                 245 250 255 Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro             260 265 270 Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Gly         275 280 285 Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe     290 295 300 Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 305 310 315 320 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu                 325 330 335 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu             340 345 350 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp         355 360 365 Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile     370 375 380 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 385 390 395 400 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly                 405 410 415 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp             420 425 430 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg         435 440 445 Leu Thr Gly Tyr Leu Arg Asn     450 455 <210> 2 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> thFc-2-AL (6) -IFN-beta <400> 2 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly   1 5 10 15 Ala Val Phe Val Ser Pro Ser Lys Glu Glu Gln Glu Glu Arg Glu Thr              20 25 30 Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu          35 40 45 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu      50 55 60 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln  65 70 75 80 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys                  85 90 95 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu             100 105 110 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys         115 120 125 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys     130 135 140 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 145 150 155 160 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                 165 170 175 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln             180 185 190 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly         195 200 205 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln     210 215 220 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 225 230 235 240 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Pro Leu Leu                 245 250 255 Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala             260 265 270 Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Met Ser         275 280 285 Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Cys Gln     290 295 300 Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys Asp 305 310 315 320 Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln Phe                 325 330 335 Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn Ile             340 345 350 Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu Thr         355 360 365 Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His Leu     370 375 380 Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg Gly 385 390 395 400 Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu                 405 410 415 His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile Val             420 425 430 Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr Gly         435 440 445 Tyr leu arg asn     450 <210> 3 <211> 5698 <212> DNA <213> Artificial Sequence <220> <223> pAD11 vector <400> 3 gacggatcgg gactagagca ttgggggggg ggacagctca gggctgcgat ttcgcgccaa 60 acttgacggc aatcctagcg tgaaggctgg taggatttta tccccgctgc catcatggtt 120 cgaccattga actgcatcgt cgccgtgtcc caaaatatgg ggattggcaa gaacggagac 180 ctaccctggc ctccgctcag gaacgagttc aagtacttcc aaagaatgac cacaacctct 240 tcagtggaag gtaaacagaa tctggtgatt atgggtagga aaacctggtt ctccattcct 300 gagaagaatc gacctttaaa ggacagaatt aatatagttc tcagtagaga actcaaagaa 360 ccaccacgag gagctcattt tcttgccaaa agtttggatg atgccttaag acttattgaa 420 caaccggaat tggcaagtaa agtagacatg gtttggatag tcggaggcag ttctgtttac 480 caggaagcca tgaatcaacc aggccacctc agactctttg tgacaaggat catgcaggaa 540 tttgaaagtg acacgttttt cccagaaatt gatttgggga aatataaact tctcccagaa 600 tacccaggcg tcctctctga ggtccaggag gaaaaaggca tcaagtataa gtttgaagtc 660 tacgagaaga aagactaaca ggaagatgct ttcaagttct ctgctcccct cctaaagcta 720 tgcattttta taagaccatg ggacttttgc tggctttaga tctttgtgaa ggaaccttac 780 ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat 840 ataaaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag 900 attccaacct atggaactga tgaatgggag cagtggtgga atgcctttaa tgaggaaaac 960 ctgttttgct cagaagaaat gccatctagt gatgatgagg ctactgctga ctctcaacat 1020 tctactcctc caaaaaagaa gagaaaggta gaagacccca aggactttcc ttcagaattg 1080 ctaagttttt tgagtcatgc tgtgtttagt aatagaactc ttgcttgctt tgctatttac 1140 accacaaagg aaaaagctgc actgctatac aagaaaatta tggaaaaata ttctgtaacc 1200 tttataagta ggcataacag ttataatcat aacatactgt tttttcttac tccacacagg 1260 catagagtgt ctgctattaa taactatgct caaaaattgt gtacctttag ctttttaatt 1320 tgtaaagggg ttaataagga atatttgatg tatagtgcct tgactagaga tcataatcag 1380 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa 1440 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg 1500 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 1560 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct cccgatcccc 1620 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc 1680 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc 1740 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc 1800 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat 1860 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 1920 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 1980 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 2040 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 2100 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 2160 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 2220 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 2280 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 2340 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 2400 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata 2460 cgactcacta tagggagacc caagctggct agcgtgagtt tggggaccct tgattgttct 2520 ttctttttcg ctattgtaaa attcatgtta tatggagggg gcaaagtttt cagggtgttg 2580 tttagaacgg gaagatgtcc cttgtatcac catggaccct catgataatt ttgtttcttt 2640 cactttctac tctgttgaca accattgtct cctcttattt tcttttcatt ttctgtaact 2700 ttttcgttaa actttagctt gcatttgtaa cgaattttta aattcacttt tgtttatttg 2760 tcagattgta agtactttct ctaatcactt ttttttcaag gcaatcaggg tatattatat 2820 tgtacttcag cacagtttta gagaacaatt gttataatta aatgataagg tagaatattt 2880 ctgcatataa attctggctg gcgtggaaat attcttattg gtagaaacaa ctacatcctg 2940 gtcatcatcc tgcctttctc tttatggtta caatgatata cactgtttga gatgaggata 3000 aaatactctg agtccaaacc gggcccctct gctaaccatg ttcatgcctt cttctttttc 3060 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg 3120 taatacgact cactataggg cgaattgaag cttggtaccg agctcggatc cactagtcca 3180 gtgtggtgga attcaccgcg gccgctctag agggccctat tctatagtgt cacctaaatg 3240 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 3300 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 3360 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 3420 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 3480 gctctatggc ttctgaggcg gaaagaacca gctggggctc gagagcttgg cgtaatcatg 3540 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 3600 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 3660 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 3720 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 3780 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 3840 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 3900 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 3960 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4020 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4080 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 4140 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4200 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4260 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4320 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4380 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 4440 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 4500 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 4560 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 4620 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 4680 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 4740 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 4800 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 4860 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 4920 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 4980 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 5040 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 5100 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 5160 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 5220 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 5280 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 5340 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 5400 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 5460 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 5520 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 5580 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 5640 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc 5698  

Claims (26)

A fusion polypeptide comprising an antibody Fc fragment, a linker derived from human albumin linked to the C-terminus of the Fc fragment, and an active polypeptide linked to the linker with an N-terminus. The fusion polypeptide of claim 1, wherein the Fc fragment is a hybrid Fc consisting of a combination of human IgG1, IgG2, IgG 3 or IgG4 derived Fc, a combination of IgG subclasses, or a hybrid Fc consisting of a combination of IgD and IgG4. The fusion polypeptide according to claim 2, wherein the Fc fragment has replaced the N-terminal four amino acids (233-236 amino acids) in the CH2 region of IgG4 with the corresponding amino acids of IgG2. The fusion polypeptide according to claim 2, wherein the Fc fragment replaces the N-terminal ten amino acids (231-240 amino acids) in the CH2 region of IgG4 with eight amino acids (292-299 amino acids) of IgD. . The fusion polypeptide of claim 1, further comprising a hinge region at the N-terminus of the Fc. 4. The fusion polypeptide of claim 3, further comprising a hinge region of IgGl at the N-terminus of the Fc. 5. The fusion polypeptide of claim 4, further comprising 15 amino acids (amino acids 277-291) at the N-terminus of the Fc or in the IgD hinge region. The fusion polypeptide of claim 1, further comprising a signal sequence at the N-terminus of the Fc. The fusion polypeptide of claim 8, wherein the signal sequence is a signal sequence of a human tissue plasminogen activator. The fusion polypeptide of claim 1, wherein the linker derived from human albumin consists of 33 or less amino acids located at portions 282 to 314. 12. The linker of claim 10, wherein the linker consists of thirteen amino acids located at portions 292 to 304. The fusion polypeptide of claim 1, wherein the active polypeptide has biological activity. The method of claim 1, wherein the active polypeptide is selected from the group consisting of hormones, cytokines, interferon-alpha, interferon-beta, interferon-gamma, interleukin, costimulatory molecules and betacellulin which are soluble polypeptides. Fusion polypeptide, characterized in that. The fusion polypeptide of claim 1, wherein the active polypeptide is human interferon-beta. A nucleic acid sequence encoding the fusion polypeptide of claim 1. The nucleic acid sequence of claim 15, further comprising a signal sequence. The nucleic acid sequence of claim 16, wherein the signal sequence is a signal sequence of a human tissue plasminogen activator. The nucleic acid sequence of claim 15 which is codon optimized for expression in a mammal. Recombinant vector comprising a gene of claim 15 nucleic acid sequence. 20. The vector of claim 19, wherein the vector further comprises a promoter of cytomegalovirus (CMV) early gene, a globin intervening sequence (gIVS) and a polyadenylation motif (pA) of bovine growth hormone. A host cell transformed with the vector of claim 19. The host cell according to claim 21, which is an animal cell. The host cell according to claim 21, which is a CHO cell. A method of preparing a fusion polypeptide comprising physiologically active polypeptide coupled to a hybrid Fc by culturing the host cell of claim 21. A pharmaceutical composition comprising the fusion polypeptide of claim 1 and a pharmaceutically acceptable carrier. A pharmaceutical composition for treating recurrent and progressive multiple sclerosis, breast cancer and cervical cancer, comprising the fusion polypeptide of claim 14 and a pharmaceutically acceptable carrier.

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