KR20120043207A - A fusion protein of a fibronectin fragment and an immunoglobulin fragment and use thereof - Google Patents

Abstract

PURPOSE: A fusion protein of immunoglobulin fragment and fibronectin fragment is provided to highly maintain drug activity in vivo. CONSTITUTION: A fusion protein contains an immunoglobulin(IgG) Fc fragment and fibronectin fragment containing a binding domain, which are fused together. The fibronectin fragment has an amino acid of sequence number 4, 11, 17, 24, or 30. The IgG Fc fragment is an Fc fragment of IgG, IgA, IgD, IgE, IgM, or mixture or hybrid thereof. The IgG Fc fragment is IgG4 Fc fragment. The fusion protein has an amino acid of sequence number 10, 16, 21, 23, or 29. A pharmaceutical composition contains the fusion protein as a drug carrier.

Description

Fusion proteins of fibronectin fragments and immunoglobulin fragments and their use {A FUSION PROTEIN OF A FIBRONECTIN FRAGMENT AND AN IMMUNOGLOBULIN FRAGMENT AND USE THEREOF}

The present invention relates to a fusion protein of a fibronectin fragment and an immunoglobulin fragment, a polynucleotide encoding the fusion protein, a pharmaceutical composition comprising the fusion protein as a carrier of a drug, and the blood of the drug using the fusion protein as a carrier of the drug. A method of increasing the half-life.

In general, protein / polypeptides are poorly deformed and are easily denatured, and are easily decomposed by proteolytic enzymes in the blood and easily removed by the kidneys or liver. Frequently administered to the patient. However, in the case of protein drugs, which are mostly administered in the form of injections, frequent injections to maintain blood levels of active polypeptides can cause tremendous pain in the patient. To address this, efforts have been made to maximize the drug efficacy by increasing the blood stability of protein drugs and by maintaining high blood drug concentrations for a long time. Such prolonged preparations of protein drugs should increase the stability of the protein drug while maintaining the titer of the drug itself high enough and not elicit an immune response in the patient.

As a method for stabilizing proteins and inhibiting contact with proteolytic enzymes and loss of elongation, conventionally, chemically adding highly soluble polymers such as polyethylene glycol (PEG) to the surface of protein drugs are used. Has been. PEG is known to be non-specifically bound to a specific site or various sites of the protein to increase solubility, stabilize the protein, prevent the hydrolysis of the protein and do not cause any special side effects (Sada et al., J. Fermentation Bioengineering 71: 137-139, 1991). However, although PEG may increase the stability of the protein, the titer of the bioactive protein is significantly lowered, and as the molecular weight of PEG increases, the reactivity with the protein decreases, resulting in a decrease in yield.

As another method for enhancing the in vivo stability of the bioactive protein, a fusion protein produced by binding albumin or a fragment thereof to a desired bioactive protein by gene recombination has been reported (International Patent Publications WO93 / 15199 and WO93 /). 15200, European Patent Publication No. EP413622). In addition, the fusion protein of yeast-produced interferon alpha and albumin from Human Genome Science increased the half-life of interferon from 5 to 93 hours in monkeys, but with less than 5% bioactivity compared to unmodified interferon. (Osborn et al., J. Phar. Exp. Ther 303 (2): 540-548, 2002). Novo has developed Lemirmir, which combines fatty acids with insulin to reduce the frequency of insulin that must be administered before meals. Fatty acid bound to levamir is known to increase its persistence through irreversible binding to albumin in the blood, but it does not exceed 24 hours. Aventis Corp. commercialized Lantus, a long-acting insulin that lowered PI by altering the amino acid sequence of insulin. Lantose is known to increase the duration of effect by slowing the migration time from the injection site into the blood when subcutaneously administered by lowered PI, but this also does not exceed 24 hours.

On the other hand, attempts have been actively made to enhance the stability of protein drugs using immunoglobulins and fragments thereof, such as fusion of Fc fusion proteins produced by genetic recombination, such as interferon-beta or derivatives thereof with immunoglobulin Fc fragments. Proteins (WO00 / 23472), fusion proteins of IL-5 receptors and immunoglobulin Fc fragments (US Pat. No. 5,712,121) are disclosed. However, the Fc fusion protein produced by this genetic recombination method is capable of protein fusion only at specific sites of the immunoglobulin Fc fragment, that is, at the amino or carboxyl terminus, and only between the glycated or non-glycosylated proteins. Fusion of glycated and nonglycosylated proteins has the disadvantage of being impossible.

The present inventors have already developed an Fc fragment that can be usefully used as a carrier of drugs by overcoming the shortcomings of the fusion technology while improving the in vivo persistence and minimizing the decrease in activity (Korean Patent Registration No. 755315, 775343). No. 824505).

On the other hand, fibronectin is a glycoprotein composed of two similar polypeptides with a molecular weight of about 250 KDa. It is present in extracellular matrix, such as the basement membrane of cells and blood vessels. The fibronectin consists of three types of homologous repeating modules, type I, II and III. Fibronectin is known to bind to fibrin, collagen, proteoglycan and glycosaminoglycans to enhance adhesion between cells or between cells and matrix layers, and is involved in cell morphology and organization of cell substrates. (Glosen JB. Et al., J. Dermatol. Surg. Oncol., 14: 198, 1998; Stenman S. et al., J. Exp. Med., 147: 1054, 1978).

Therefore, the present inventors have made diligent research efforts to develop a carrier of a drug which can improve the in vivo persistence of the drug and minimize the decrease in the activity of the drug. As a result, heparin binding sites of the fibronectin were added to the immunoglobulin Fc fragments developed by the present inventors. The present invention has been completed by discovering that fusion can be usefully used as a carrier of drugs by the heparin binding site of Fc carrier and fibronectin.

It is an object of the present invention to provide fusion proteins of fibronectin fragments and immunoglobulin fragments useful as carriers of drugs and polynucleotides encoding them.

Another object of the present invention to provide a pharmaceutical composition comprising the fusion protein as a carrier.

It is another object of the present invention to provide a method of increasing the blood half-life of a drug by using the fusion protein as a carrier of the drug.

In order to achieve the above object, in one aspect, the present invention relates to fusion proteins of fibronectin fragments and immunoglobulin fragments that can be usefully used as carriers of drugs. Preferably the present invention provides a fusion protein of the heparin binding domain of fibronectin with an immunoglobulin Fc fragment.

As used herein, the term "carrier" refers to a substance that is bound with a drug, and is generally bound to the drug to increase or decrease the physiological activity of the drug. However, the carrier in the present invention minimizes the decrease in the biological activity of the drug bound for the purposes of the present invention while at the same time increasing the sustainability of the drug by delaying the in vivo stability of the drug and the migration from the site of administration to the blood upon subcutaneous administration. It is for. Various substances such as lipids and polymers have been studied as carriers of such drugs, but a technique using a fusion of immunoglobulin Fc fragments and fibronectin fragments is not known. That is, the present invention is characterized by providing a fusion protein of a fibronectin heparin binding domain and an immunoglobulin Fc fragment as a carrier for enhancing the in vivo persistence of the drug to be bound and minimizing the decrease in the activity in vivo.

In the present invention, "immunoglobulin Fc fragment" refers to heavy chain constant region 2 (CH2) and heavy chain constant region 3 (except for heavy and light chain variable regions, heavy chain constant region 1 (CH1) and light chain constant region 1 (CL1) of immunoglobulin) CH3) portion, and may include a hinge portion in the heavy chain constant region. In addition, the immunoglobulin Fc fragments of the present invention may have partial or full heavy chain constant region 1 (CH1) and / or light chain constant, except for the heavy and light chain variable regions of the immunoglobulin, so long as they have substantially the same or improved effects as the native form. It may be an extended Fc fragment comprising region 1 (CL1). It may also be a region from which some fairly long amino acid sequences corresponding to CH2 and / or CH3 have been removed. In other words, the immunoglobulin Fc fragments of the present invention are 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) A combination of one or two or more domains with an immunoglobulin hinge region (or a portion of the hinge region), 6) heavy chain constant region dimer of each domain and light chain constant region.

Immunoglobulin Fc fragments are safe for use as carriers of drugs because they are biodegradable polypeptides that are metabolized in vivo. In addition, immunoglobulin Fc fragments have a relatively low molecular weight compared to the whole immunoglobulin molecule, which is advantageous in the preparation, purification and yield of the conjugate, as well as the homogeneity of the material since the amino acid sequence varies from antibody to antibody, resulting in the removal of Fab portions showing high heterogeneity. This effect can be expected to be greatly increased and the likelihood of inducing blood antigens is also lowered.

In addition, the immunoglobulin Fc fragments of the present invention include native amino acid sequences as well as sequence derivatives thereof. Amino acid sequence derivatives mean that a natural amino acid sequence and one or more amino acid residues have a different sequence and may occur naturally or artificially. Fc fragments of immunoglobulins include derivatives by deletions, insertions, non-complementary or complementary substitutions, or combinations thereof. Insertions typically consist of a contiguous sequence of about 1 to 20 amino acids, although larger insertions are also possible. Deletion typically consists of about 1 to 30 residues. Techniques for preparing sequence derivatives of such immunoglobulin Fc fragments are disclosed in International Patent Publications WO97 / 34631, WO96 / 32478, and the like, which are incorporated herein by reference. 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). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amylation, etc. may be modified.

The immunoglobulin Fc derivatives described above are derivatives which exhibit the same biological activity as the Fc fragments of the present invention but increase the structural stability against heat, pH, etc. of the Fc fragments.

In addition, such immunoglobulin Fc fragments may be obtained from natural forms isolated in vivo from animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, and the like. It may be a recombinant or derivative thereof obtained from. Here, the method obtained from the natural form can be obtained by separating the whole immunoglobulin from the human or animal living body, and then treating the protease. When treating papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F (ab ') ₂ . This may be separated by Fc or pF'c using size-exclusion chromatography. Preferably the immunoglobulin Fc fragments according to the invention are recombinant immunoglobulin Fc fragments obtained from microorganisms of immunoglobulin Fc fragments derived from humans.

In addition, the immunoglobulin Fc fragments may be in the form of natural sugar chains, increased sugar chains compared to the natural form, reduced sugar chains or the sugar chains removed from the natural form. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or decrease such immunoglobulin Fc sugar chains. Herein, immunoglobulin Fc fragments in which the sugar chain is removed from the Fc are significantly reduced in binding strength of the complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, thereby not inducing unnecessary immune responses in vivo. Do not. In this regard, a form more consistent with the object of the present invention as a carrier of the drug is an immunoglobulin Fc fragment in which sugar chains are de-chained or unglycosylated.

In the present invention, "deglycosylation" refers to an immunoglobulin Fc fragment in which sugar is removed by an enzyme, and "aglycosylation" refers to an immunoglobulin Fc produced in prokaryotic cells, preferably Escherichia coli, which is not glycosylated. Means a fragment.

On the other hand, the immunoglobulin Fc fragment may be of animal origin, such as human, bovine, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., preferably human origin. In addition, the immunoglobulin Fc fragments may be Fc fragments derived from IgG, IgA, IgD, IgE, IgM or combinations thereof or hybrids thereof. It is preferably derived from IgG or IgM, which is most abundant in human blood, and most preferably from IgG known to enhance the half-life of ligand binding proteins.

On the other hand, "combination" in the present invention, when forming a dimer or multimer, means that the polypeptides encoding the same origin single chain immunoglobulin Fc fragment form a bond with the single chain polypeptides of different origin. That is, it is possible to prepare dimers or multimers from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE.

In the present invention, "hybrid" means that there is a sequence corresponding to two or more different immunoglobulin Fc fragments of different origin in an immunoglobulin Fc fragment of short chain. In the case of the present invention, various types of hybrids are possible. That is, hybridization of a domain consisting of 1 to 4 domains from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible, and may include a hinge region. .

On the other hand, IgG can also be divided into subclasses of IgG1, IgG2, IgG3 and IgG4, and the present invention includes combinations thereof and hybrids thereof. Preferred are the IgG2 and IgG4 subclasses, most preferably the Fc fragment of IgG4 with little effector function, such as complement-dependent cytotoxicity (CDC).

On the other hand, the immunoglobulin Fc fragment of the present invention may be a recombinant form having a hinge region connected to its N-terminus, in which case the immunoglobulin Fc fragment expressed in aggregate form is encoded by the initiation codon without causing loss of activity. The advantage is that the starting methionine residues are solublized and refolded into the Fc fragment in the form of an active dimer or monomer in which the starting methionine residue has been removed.

Description of immunoglobulin Fc fragments suitable for the present invention as described above is described in detail in Korean Patent Nos. 755315, 775343 and 824505, which are incorporated herein by reference.

That is, the most preferred immunoglobulin Fc fragments for use in fusion proteins with fibronectin fragments according to the invention as carriers of drugs are non-glycosylated Fc fragments derived from human IgG4. Human-derived Fc fragments are preferred over non-human-derived Fc fragments that can cause undesirable immune responses, such as acting as antigens in human living organisms to produce new antibodies against them.

In one embodiment of the present invention, the immunoglobulin Fc fragment is prepared in the form of a fusion protein with the fibronectin fragment and may be usefully used as a carrier of the drug.

In the present invention, fibronectin is composed of three types of homologue repeating modules (types I, II and III) in rod-like form. The Type I module consists of about 45 amino acids and is known to be involved in the binding action between fibrin and collagen, which is present at the amino and carboxyl ends. Type II modules consist of about 60 amino acids and are known to be primarily involved in the binding action with collagen. The Type III module is the largest form of fibronectin protein and consists of about 90 amino acids, consists of 15 domains, and contains the receptor binding motifs RGD (Arg-Gly-Asp) and heparin binding motifs. . The heparin binding motif is present in the 12th, 13th and 14th domains.

In the present invention, the "fibronectin fragment" may be selected from heparin binding domains 12, 13 and 14 and combinations thereof, such as fragments including 13 and 14 domains, fragments including all 12 to 14 domains, and the like. Preferably heparin binding domain 13 may be selected, more preferably heparin binding motif (HBM) may be selected in heparin binding domain 13. In a preferred embodiment of the invention, the fibronectin fragment is heparin binding domain 13 having an amino acid sequence as set out in SEQ ID NO: 11, heparin binding domain 14 having an amino acid sequence as set out in SEQ ID NO: 17, as set forth in SEQ ID NO: 24 Heparin binding motifs in heparin binding domains 13 and 14 having an amino acid sequence, heparin binding domains 12 to 14 having the amino acid sequence set forth in SEQ ID NO: 4, and heparin binding domains 13 having the amino acid sequence set forth in SEQ ID NO: 30 Used.

Accordingly, the present invention provides a fusion protein of a fibronectin fragment and an immunoglobulin Fc fragment that can be usefully used as a carrier of a drug, a polynucleotide encoding the fusion protein, and a recombinant expression vector including the same.

In one aspect of the invention, the fibronectin fragment that may be linked to an immunoglobulin Fc fragment in the fusion protein of the invention may be heparin binding domains 12, 13 and 14 of fibronectin and combinations thereof, preferably SEQ ID NO: 4, Fibronectin fragments having amino acid sequences described as 11, 17, 24, and 30. The fusion protein to which the fibronectin fragment is linked to the immunoglobulin Fc fragment according to the invention preferably has an amino acid sequence set forth in SEQ ID NOs: 10, 16, 21, 23 and 29, and the polynucleotide encoding the same is SEQ ID NO: 9, Base sequences described in 15, 20, 22 and 28.

The polynucleotide sequence encoding the fusion protein of the fibronectin fragment and the immunoglobulin Fc fragment is provided by operably linked to a vector capable of expressing it.

As used herein, a "vector" refers to a genetic construct that is capable of expressing a protein of interest in a suitable host cell, and includes a genetic construct that contains essential regulatory elements operably linked to express a gene insert. In the present invention, the term "operably linked" refers to a functional link between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. Operational linkage with the vector can be prepared using genetic recombination techniques known in the art, and site-specific DNA cleavage and ligation can be readily performed using enzymes or the like generally known in the art. Suitable expression vectors can include expression control sequences such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. The start codon and the stop codon must be functional in the subject when the gene construct is administered and must be in frame with the coding sequence. Generic promoters can be either constitutive or inducible. The expression vector also includes a selectable marker for selecting a host cell containing the vector, and, in the case of a replicable expression vector, may contain the origin of replication.

Preferably, the fibronectin fragment to be linked to the immunoglobulin Fc fragment can be selectively amplified by a polymerase chain reaction (PCR) or a DNA synthesizer by selectively amplifying the type III heparin binding domains 12-14 in the fibronectin cDNA. Amplified / synthesized fibronectin fragment was digested with an appropriate restriction enzyme to prepare an insert fragment, and then inserted into an expression vector containing an immunoglobulin Fc fragment, thereby preparing an expression vector expressing a fusion protein linked to the fibronectin fragment to immunoglobulin Fc. .

In a preferred embodiment of the invention, pET22b-CarrierA-Hep13 expressing a fusion protein linked to an immunoglobulin Fc fragment to which heparin binding domain 13 of fibronectin is linked; PET22b-CarrierA-Hep14, which expresses a fusion protein to which an immunoglobulin Fc fragment is linked to heparin binding domain 14 of fibronectin; PET22b-CarrierA-Hep12-14 expressing a fusion protein to which an immunoglobulin Fc fragment is linked with heparin binding domains 12, 13 and 14 of fibronectin; PET22b-CarrierA-Hep12-13 that expresses a fusion protein to which an immunoglobulin Fc fragment is linked with heparin binding domains 12 and 13 of fibronectin; And pET22b-CarrierA-HBM expressing a fusion protein in which an immunoglobulin Fc fragment is linked to a heparin binding motif in heparin binding domain 13 of fibronectin.

The present invention provides a transformant transformed with the recombinant expression vector and a method for mass production of a fusion protein using the same.

In one aspect of the invention, the recombinant expression vector expressing the fusion protein is transformed into a host cell. For the purposes of the present invention, host cells are prokaryotic cells in which glycosylation does not occur. Such prokaryotic cells include Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus. sp.) and the like, preferably Escherichia coli. E. coli suitable for the present invention includes E. coli XL-1 blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH series, E. coli TOP10 and E. coli HB101, preferably, but is not limited to E. coli BL21 (DE3). When E. coli is used as a host cell, since E. coli has no system for linking sugar chains to proteins, it is possible to produce Fc fragments of immunoglobulins in a form in which sugar present in the CH2 domain of native immunoglobulins is naturally deleted. Sugars in the immunoglobulin's CH2 domain do not affect the structural stability of immunoglobulins, but immunoglobulins bind to cells expressing Fc receptors, resulting in antibody-dependent cytotoxicity, and immune cells secrete cytokines to cause inflammatory reactions. It is known to cause the complement fixation reaction by binding to the C1q element of the complement. Thus, production of Fc fragments of unglycosylated immunoglobulins and binding to therapeutic proteins can maintain blood levels of therapeutic proteins for long periods of time without causing undesirable effector functions of immunoglobulins.

The method for transforming the recombinant expression vector into prokaryotic cells may include any method for introducing nucleic acids into cells, and may be performed by selecting appropriate standard techniques according to host cells as known in the art. These methods include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, PEG, dextran sulfate, lipofectamine, etc. , But not limited to these.

In a preferred embodiment of the present invention, the recombinant expression vector pET22b-CarrierA-HBM expresses a fusion protein linked to an heparin binding motif (HBM) in heparin binding domain 13 of fibronectin to an immunoglobulin Fc fragment to E. coli BL21 (DE3). To prepare a fusion protein.

The transformant transformed with the recombinant expression vector is cultured in a conventional manner. This culture process can be used by those skilled in the art can be easily adjusted according to the strain selected. The medium used for culture should generally contain all the nutrients necessary for cell growth and survival. The medium contains various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, linoleic acid Fatty acids such as, alcohols such as glycelol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL), and soybean wheat, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate Inorganic nitrogen sources are included. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium-containing salts. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and appropriate precursors may be included. During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (eg, air) may be injected into the culture. The temperature of the culture is usually 20 to 45 ° C, preferably 25 to 40 ° C. In addition, a fermentor may be used. When producing a protein using the fermenter, various factors such as the growth rate of the host cell and the amount of the expression product should be considered. In appropriate culture conditions, IPTG may be added to induce the expression of proteins.

Fusion proteins of immunoglobulin fragments and fibronectin fragments expressed from the transformants can be purified in a conventional manner known in the art. After the fusion protein produced from the transformant is crushed by a method such as a French press or an ultrasonic mill, only insoluble aggregates containing the fusion protein are separated using a centrifuge, and the obtained fractions are dissolved and denatured. And re-overlap with agents such as urea, guanidine, arginine cysteine, beta-mercaptoethanol and the like, followed by column chromatography and ultrafiltration, such as dialysis, gel filtration, ion exchange, precipitation, adsorption, electrophoresis, reverse phase column chromatography, and the like. The techniques can be applied alone or in combination to obtain purely the fusion protein of the invention.

The fusion protein of the immunoglobulin fragment and fibronectin fragment isolated and purified from the transformant as described above may be usefully used as a carrier of the drug. Accordingly, the present invention provides a pharmaceutical composition comprising the fusion protein as a carrier. The present invention also provides a method of increasing the blood half-life of a drug by using the fusion protein as a carrier of the drug.

In one aspect of the invention, the fusion protein of the immunoglobulin fragment and the fibronectin fragment according to the invention may act as a carrier of the drug to form a conjugate with the drug.

As used herein, "drug conjugate" or "conjugate" means that one or more drugs are interconnected with a fusion protein of a fusion protein of one or more immunoglobulin fragments and fibronectin fragments.

As used herein, the term "drug" refers to a substance that exhibits therapeutic activity when administered to humans or animals, and includes, but is not limited to, polypeptides, compounds, extracts, nucleic acids, and the like. The drug is preferably a polypeptide drug. In the present invention, "physiologically active polypeptide drug", "polypeptide drug" and "protein drug" are used in the same sense, it is characterized in that the physiologically active form exhibiting antagonism to various physiological phenomena in vivo.

Such polypeptide drugs have a disadvantage in that they cannot sustain physiological activity for a long time due to their easily denatured or well degraded by protease present in vivo. However, in the conjugate in which the polypeptide is bound to the fusion protein of the immunoglobulin fragment and the fibronectin fragment according to the present invention, the structural stability of the drug is increased, the degradation half-life is increased, and the drug is sustained by binding to heparin when administered subcutaneously. It has the effect of increasing time.

Examples of protein drugs that can be linked to fusion proteins of immunoglobulin fragments and fibronectin fragments of the present invention include liver growth hormone, growth hormone releasing hormone, growth hormone releasing peptides, interferons and interferon receptors (e.g., interferon-alpha, -beta). And-gamma, water soluble type I interferon receptors, etc.), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), glucacon-like peptides (GLP-1, etc.), G G-protein-coupled receptors, interleukins (eg IL-1 receptor, IL-4 receptor, etc.), enzymes (eg glucocerebrosidase, iduronate-2- Sulfatase, alpha-galactosidase-A, agalsidase alpha, beta, alpha-L-iduronidase, butyrylcholine Butyrylcholinesterase, chitinase, glutamate decarboxylase (gl utamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, myo Eroperoxidase (myeloperoxidase, etc.), interleukins and cytokine binding proteins (eg, IL-18bp, TNF-binding proteins, etc.), macrophage activators, macrophage peptides, B cell factor, T cell factor, protein A , Allergic inhibitors, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein- E, erythropoietin, high glycated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor active peptide, thrombomodulin, blood factor 혈액, blood factor 인 a, blood Ⅷ, blood factor Ⅸ, blood factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, super Oxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone formation growth factor, bone stimulating protein, calcitonin, insulin, atriopeptin, Cartilage inducer, elcatonin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor (e.g. nerve growth) Factors, ciliary neurotrophic factor, axogenesis factor-1, brain-natriuretic peptide, glial Glial derived neurotrophic factor, netrin, neutrophil inhibitor factor, neurotrophic factor, neuturin, etc., parathyroid hormone, relaxin, secretin, somatomedin, insulin Similar growth factors, corticosteroids, 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 (e.g. scFv, Fab, Fab ', F (ab') 2 and Fd), virus-derived vaccine antigens, and the like, but are not limited to the types exemplified above.

Meanwhile, a fusion protein of an immunoglobulin fragment and a fibronectin fragment according to the present invention may form a conjugate linked to a drug through a gene recombination method or a linker. Linkers include both peptidic linkers or nonpeptidic linkers, but are preferably nonpeptidic linkers, more preferably nonpeptidic polymers. In the present invention, "peptidic linker" means an amino acid, preferably 1 to 20 amino acids linked by peptide bonds, and may be in a glycated chain form. Such a peptidic linker is preferably a peptide having Gly, Ser repeat units and immunologically inactive peptides against T cells. In the present invention, "non-peptidyl polymer" refers to a biocompatible polymer having two or more repeating units bonded thereto. Examples of the non-peptidyl polymer include polyethylene glycol (PEG), polypropylene glycol, PPG), ethylene glycol-propylene glycol copolymer (co-poly (ethylene / propylene) glycol), polyoxyethylene (polyoxyethylene, POE), polyurethane (polyurethane), polyphosphazene, polysaccharide (polysaccharide) , Dextran, polyvinyl alcohol, polyvinyl pyrrolidones, polyvinyl ethyl ether polyacryl amide, polyacrylate, polycyano Acrylates (polycyanoacrylates), lipid polymers, chitin, hyaluronic acid, heparin and the like, and preferably polyethylene glycol. The number of linkers capable of binding to the fusion protein of the immunoglobulin fragment and the fibronectin fragment according to the present invention is not particularly limited. Preferably, in the drug conjugate of the present invention, the fusion protein and linker of the immunoglobulin fragment and the fibronectin fragment may be bound in a molar ratio of 1: 1 to 1:10, preferably 1: 1 to 1: 2.

In a preferred embodiment of the present invention, polyethylene glycol is PEGylated to the fusion protein of the immunoglobulin fragment and the fibronectin fragment. As used herein, "pegylation" refers to the process by which polyethylene glycol is bound, and for the purposes of the present invention, means that polyethylene glycol is covalently bound to an immunoglobulin Fc fragment. PEGylation of immunoglobulin fragments using non-peptidyl polymers is described in detail in Korean Patent Registration No. 754667, which is incorporated herein by reference.

The number of drugs that can bind to the fusion protein of the immunoglobulin fragment and the fibronectin fragment according to the present invention is not particularly limited. Preferably, in the drug conjugate of the present invention, the fusion protein of the immunoglobulin fragment and the fibronectin fragment and the drug may be bound in a molar ratio of 1: 1 to 1:10, preferably 1: 1 to 1: 4. The binding of the fusion protein, any linker, and the drug of the immunoglobulin fragment and the fibronectin fragment is a peptide bond when the polypeptide drug is expressed in the form of a fusion protein to the fusion protein of the immunoglobulin fragment and the fibronectin fragment by a genetic recombination method. All covalent bonds, except), and all types of non-covalent bonds, such as hydrogen bonds, ionic bonds, van der Waals affinity, and hydrophobic interactions. However, in view of the biological activity of the drug is preferably covalent.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for increasing the in vivo persistence and stability of a drug comprising a drug conjugate linked to a fusion protein of an immunoglobulin fragment and a fibronectin fragment. In this case, the drug conjugate may be fusion protein linked using a linker as described above.

The pharmaceutical compositions described above can be administered via several routes. In the present invention, "administration" means introducing a predetermined substance into a patient by any suitable method and the route of administration of the drug 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, intramuscular administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, but is not limited thereto. However, upon oral administration, since the peptide is digested, it is desirable that the oral composition is coated or formulated to protect it from degradation in the stomach. It may preferably be administered in the form of an injection. In addition, the active agent may be administered by any device that can migrate to the target cell.

The pharmaceutical composition 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, flavoring agents, etc. In the case of injections, buffers, preservatives, An analgesic agent, a solubilizer, an isotonic agent, a stabilizer, etc. can be mixed and used, and a base agent, an excipient, a lubricant, a preservative etc. can be used for topical administration. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above. For example, oral administration can 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 in multiple dosage forms. . Other solutions, suspensions, tablets, capsules, sustained release preparations and the like. Examples of suitable carriers, excipients and diluents for formulation are 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 dosage of the pharmaceutical composition according to the present invention is determined according to the type of drug which is the active ingredient, together with various related factors such as the disease, the route of administration, the age, sex and weight of the patient, and the severity of the disease. Since the pharmaceutical composition of the present invention has very good sustainability in vivo, the frequency and frequency of administration of the pharmaceutical preparations can be significantly reduced. In addition, since the pharmaceutical composition of the present invention does not act as immunogenic in vivo, there is little concern of side effects, and long-term administration is possible and safe.

When the drug is bound to the fusion protein of the immunoglobulin fragment and the fibronectin fragment according to the present invention, the structural stability of the drug may be increased, the degradation half-life may be increased, and the duration of the drug may be increased by binding to heparin when administered subcutaneously. . Therefore, the pharmaceutical composition comprising a fusion protein of an immunoglobulin fragment and a fibronectin fragment according to the present invention as a carrier not only minimizes a decrease in in vivo activity while maintaining blood persistence of the drug, and also has little risk of inducing an immune response. It can be usefully used for the development of long-acting formulations of drugs.

1 is a column chromatography result confirming that the fusion protein of the immunoglobulin Fc fragment and the heparin binding motif of fibronectin (HBM) according to the present invention has an attachment activity to heparin,
Figure 2 is a result of measuring the in vivo pharmacokinetics of a drug conjugate in which insulin is bound to a fusion protein of an immunoglobulin Fc fragment and a heparin binding motif (HBM) of fibronectin according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to 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: immunoglobulin fragments Fibronectin  Preparation of Fusion Proteins of Fragments

<1-1> Fibronectin  Protein Type III Hep12 Preparation of Expression Vectors Containing 13, 14 and 14 Domains

To prepare an expression vector comprising the type III Hep12 to 14 domain of fibronectin, amino acid 1723 corresponding to Hep12 to 14 domain using fibronectin cDNA (NM_212482.1) as a template and primer pairs of SEQ ID NOs: 1 and 2 Portions from 1 to 2075 were amplified by PCR. To facilitate subcloning, both ends of the primers were designed to be cleaved with restriction enzymes NdeI and BamHI, and fibronectin cDNA was used as a Liver cDNA library (Stratagene, USA).

PCR was performed by mixing 150 ng of template DNA, 1 ml of each 100 pM primer, 5 ml of 2.5 mM dNTP, 10 units of pfx polymerase (Invitrogen, USA), and 10 × buffer to prepare a reaction solution. After initial denaturation, amplification was performed 30 times at 95 ° C. for 30 seconds, at 55 ° C. for 30 seconds, and at 68 ° C. for 80 seconds, and finally, at 68 ° C. for 30 seconds. PCR fragments obtained therefrom were extracted using a gel extraction kit (Quiagen, Germany) and then treated with restriction enzymes NdeI and BamHI to prepare insert fragments. The pET22b vector (Novagen, USA) was then digested with the same restriction enzyme and extracted using the same gel extraction kit. The inserted fragment was linked to the vector thus prepared using T4 ligase and transformed into E. coli TOP10 competent cells (Invitrogen, USA).

The transformants were inoculated in ampicillin containing LB solid medium and cultured to randomly select 10 colonies formed on the medium. Selected colonies were inoculated again in ampicillin-containing LB liquid medium and cultured overnight, and then plasmid DNA was extracted using an extraction kit (Qiagen, Germany). The plasmid DNA was digested with restriction enzymes AlwNI and SacI, and electrophoresis confirmed the presence of 789 bp inserted DNA. DNA sequencing confirmed that the type III Hep12-14 domain of fibronectin was correctly inserted into the vector. As a result of sequencing, the type III Hep12 to 14 domain of fibronectin has an amino acid sequence set forth in SEQ ID NO: 4, and the polypeptide encoding it has a nucleotide sequence set forth in SEQ ID NO: 3. The inventors named the expression vector including the type III Hep12 to 14 domain of the fibronectin prepared as above "pET22b-FNHeparin".

<1-2> immunoglobulin Fc  Fragments and Fibronectin  Type III Hep13  Preparation of Expression Vectors Containing Fusion Proteins of a Domain

To prepare a fusion protein in which the Hep13 domain is fused to the C-terminus of an immunoglobulin Fc fragment, first, an N-terminal primer of an immunoglobulin Fc fragment comprising an immunoglobulin Fc fragment as a template and including an NdeI restriction enzyme cleavage site (SEQ ID NO: : 5) and a first PCR using a C-terminal primer (SEQ ID NO: 6) of an immunoglobulin Fc fragment containing a part of the N-terminal portion of Hep13, amplified the immunoglobulin Fc gene fragment, and template Hep13 gene Secondary PCR using a Hep13 N-terminal primer (SEQ ID NO: 7) containing a part of the C-terminus of an immunoglobulin Fc and a Hep13 C-terminal primer (SEQ ID NO: 8) containing a BamHI restriction enzyme cleavage site Was performed to amplify the Hep13 gene segment. PCR conditions were that the first PCR performed 30 times amplification for 30 seconds at 95 ° C, 30 seconds at 55 ° C, and 80 seconds at 68 ° C, and the secondary PCR was 30 seconds at 95 ° C, 30 seconds at 55 ° C and 68 ° C. Amplification was performed in the same manner as in Example <1-1> except that the amplification was performed 30 times for 80 seconds at.

Gene fragments amplified in the first and second PCR were used as templates, and after performing the third PCR using primer pairs of SEQ ID NOs: 5 and 8, the PCR amplification products were digested with restriction enzymes NdeI and BamHI to immunoglobulin Fc. Insertion fragments of -Hep13 fusion protein were prepared. The pET-22b vector was then digested with the same restriction enzyme and the insert fragment of the immunoglobulin Fc-Hep13 fusion protein prepared above was ligated via T4 ligase and transformed into E. coli TOP10 qualified cells (Invitrogen, USA).

The transformants were cultured as in Example <1-2> to extract plasmid DNA, and sequencing confirmed that the immunoglobulin Fc-Hep13 fusion protein was correctly inserted into the vector. As a result of sequencing, the immunoglobulin Fc-Hep13 fusion protein according to the present invention has an amino acid sequence set forth in SEQ ID NO: 10, which is encoded by a polypeptide set forth in SEQ ID NO: 9, wherein the Hep13 protein in the fusion protein is sequenced. Has an amino acid sequence set forth as No. 11. The present inventors named the expression vector containing the immunoglobulin Fc-Hep13 fusion protein prepared as above "pET22b-CarrierA-Hep13".

<1-3> immunoglobulin Fc  Fragments and Fibronectin  Type III Hep14  Preparation of Expression Vectors Containing Fusion Proteins of a Domain

To prepare a fusion protein in which the Hep14 domain is fused to the C-terminus of an immunoglobulin Fc fragment, first, an N-terminal primer of an immunoglobulin Fc fragment comprising an immunoglobulin Fc fragment as a template and including an NdeI restriction enzyme cleavage site (SEQ ID NO: : 7) and the first PCR using the C-terminal primer (SEQ ID NO: 12) of the immunoglobulin Fc fragment to amplify the immunoglobulin Fc gene fragment, Hep14 gene as a template and containing a StuI restriction enzyme cleavage site Hep14 gene fragments were amplified by a secondary PCR using a Hep14 N-terminal primer (SEQ ID NO: 13) and a Hep14 C-terminal primer (SEQ ID NO: 14) including a BamHI restriction enzyme cleavage site. PCR conditions were performed in the same manner as in Example <1-2>.

The amplified gene was cut with NdeI, StuI and BamHI, respectively, to prepare an insertion fragment of an immunoglobulin Fc-Hep14 fusion protein, which was then linked to a pET-22b vector digested with the same restriction enzyme through T4 ligase, and qualified for E. coli TOP10. Cells (Invitrogen, USA) were transformed.

The transformant was cultured as in Example <1-2> to extract plasmid DNA, and sequencing confirmed that the immunoglobulin Fc-Hep14 fusion protein was correctly inserted into the vector. As a result of sequencing, the immunoglobulin Fc-Hep14 fusion protein according to the present invention has an amino acid sequence set forth in SEQ ID NO: 16, which is encoded by a polypeptide set forth in SEQ ID NO: 15, wherein the Hep14 protein in the fusion protein is sequenced. Has an amino acid sequence set forth as No. 17. The present inventors named the expression vector containing the immunoglobulin Fc-Hep14 fusion protein prepared as above "pET22b-CarrierA-Hep14".

<1-4> immunoglobulin Fc  Fragments and Fibronectin  Type III Hep12 Preparation of Expression Vectors Containing Fusion Proteins of ~ 14 Domains

To prepare a fusion protein in which the Hep12-14 domain is fused to the C-terminus of an immunoglobulin Fc fragment, first, an N-terminal primer of an immunoglobulin Fc fragment comprising an immunoglobulin Fc fragment as a template and including an NdeI restriction enzyme cleavage site ( A first PCR was performed using the C-terminal primer (SEQ ID NO: 18) of an immunoglobulin Fc fragment comprising SEQ ID NO: 3) and a Hep12 N-terminal site to amplify the immunoglobulin Fc gene fragment, and the Hep12-14 gene. Using a Hep12 N-terminal primer (SEQ ID NO: 19) containing a C-terminal site of an immunoglobulin Fc fragment (SEQ ID NO: 19) and a Hep14 C-terminal primer (SEQ ID NO: 14) containing a BamHI restriction enzyme cleavage site. Differential PCR was performed to amplify the Hep12-14 gene fragment. PCR conditions were performed in the same manner as in Example <1-2>.

The amplified gene was digested with NdeI and BamHI, respectively, to prepare an insertion fragment of an immunoglobulin Fc-Hep12-14 fusion protein, which was then linked to a pET-22b vector digested with the same restriction enzyme through T4 ligase and qualified for E. coli TOP10. Cells (Invitrogen, USA) were transformed.

The transformants were cultured as in Example <1-2> to extract plasmid DNA, and sequencing confirmed that the immunoglobulin Fc-Hep12-14 fusion protein was correctly inserted into the vector. As a result of sequencing, the immunoglobulin Fc-Hep12-14 fusion protein according to the present invention has an amino acid sequence set forth in SEQ ID NO: 21, and the polypeptide encoding the same has a nucleotide sequence set forth in SEQ ID NO: 20. The present inventors named the expression vector containing the immunoglobulin Fc-Hep12-14 fusion protein prepared as above "pET22b-CarrierA-Hep12-14".

<1-4> immunoglobulin Fc  Fragments and Fibronectin  Type III Hep13 Preparation of Expression Vectors Containing Fusion Proteins of the -14 Domain

To prepare a fusion protein in which the Hep13-14 domain is fused to the C-terminus of an immunoglobulin Fc fragment, first, an N-terminal primer of an immunoglobulin Fc fragment comprising an immunoglobulin Fc fragment as a template and including an NdeI restriction enzyme cleavage site ( The first PCR was performed using a C-terminal primer (SEQ ID NO: 6) of an immunoglobulin Fc fragment comprising SEQ ID NO: 5) and a Hep13 N-terminal site to amplify the immunoglobulin Fc gene fragment, and the Hep13-14 gene. Using a Hep13 N-terminal primer (SEQ ID NO: 7) containing the C-terminal site of an immunoglobulin Fc fragment (SEQ ID NO: 7) and a Hep14 C-terminal primer (SEQ ID NO: 14) containing a BamHI restriction enzyme cleavage site; Differential PCR was performed to amplify the Hep13-14 gene fragment. PCR conditions were performed in the same manner as in Example <1-2>.

The amplified gene was digested with NdeI and BamHI, respectively, to prepare an insertion fragment of an immunoglobulin Fc-Hep13-14 fusion protein, which was then linked to a pET-22b vector digested with the same restriction enzyme via T4 ligase and qualified for E. coli TOP10. Cells (Invitrogen, USA) were transformed.

The transformant was cultured as in Example <1-2> to extract plasmid DNA, and sequencing confirmed that the immunoglobulin Fc-Hep13-14 fusion protein was correctly inserted into the vector. As a result of sequencing, the immunoglobulin Fc-Hep13-14 fusion protein according to the present invention has an amino acid sequence as set out in SEQ ID NO: 23, which is encoded by the polypeptide set forth in SEQ ID NO: 22, in which the Hep13- The 14 protein has the amino acid sequence set forth in SEQ ID NO: 24. The present inventors named the expression vector including the immunoglobulin Fc-Hep13-14 fusion protein prepared as above "pET22b-CarrierA-Hep13-14".

<1-5> Immunoglobulins Fc  Fragment and heparin binding motifs ( HBM Preparation of Expression Vectors Containing Fusion Proteins)

In order to prepare a fusion protein comprising 20 amino acids (SEQ ID NO: 30) of the heparin binding motif (HBM), which is the intermediate region of the Hep13 domain, at the C-terminus of the immunoglobulin Fc fragment, first an immunoglobulin Fc fragment is prepared. N-terminal primer (SEQ ID NO: 5) of an immunoglobulin Fc fragment as a template and containing an NdeI restriction enzyme cleavage site and C-terminal primer of an immunoglobulin Fc fragment comprising an HBM N-terminal site (SEQ ID NO: 25) The first PCR was performed to amplify the immunoglobulin Fc gene fragment, and the HBM N-terminal primer (SEQ ID NO: 26) including the C-terminal region of the immunoglobulin Fc fragment and the HBM including a BamHI restriction enzyme cleavage site. Secondary PCR was performed using the C-terminal primer (SEQ ID NO: 27) to amplify the HBM gene fragment. PCR conditions were performed in the same manner as in Example <1-2>.

The amplified gene was digested with NdeI and BamHI, respectively, to prepare an insertion fragment of an immunoglobulin Fc-HBM fusion protein, which was then linked to a pET-22b vector digested with the same restriction enzyme through T4 ligase, followed by Escherichia coli TOP10 qualified cells ( Invitrogen, USA).

The transformants were cultured as in Example <1-2> to extract plasmid DNA, and sequencing confirmed that the immunoglobulin Fc-HBM fusion protein was correctly inserted into the vector. As a result of sequencing, the immunoglobulin Fc-HBM fusion protein according to the present invention has an amino acid sequence set forth in SEQ ID NO: 29, and a polypeptide encoding the same has a nucleotide sequence set forth in SEQ ID NO: 20. The present inventors named the expression vector containing the immunoglobulin Fc-HBM fusion protein prepared as above "pET22b-CarrierA-HBM".

Example  2: immunoglobulin Fc  Fragment and heparin binding motifs ( HBM Heparin Adhesion Activity of the Fusion Protein

An expression vector pET22b-CarrierA-HBM comprising the immunoglobulin Fc-HBM fusion protein prepared in Example <1-5> was transformed into E. coli BL21-DE3 (Novagen, USA), and the E. coli transformant was After culturing in a fed-batch in an L fermentor, 0.5 mM IPTG was added and protein expression was induced. The E. coli transformants were recovered and stored in a -80 ° C deep freezer.

20 g of E. coli transformants were dissolved using a lysis buffer (lysis buffer, 50 mM Tris 9.0, 1 mM EDTA, 0.2 M NaCl, 0.5% Triton X-100), and the cells were pulverized using a high pressure grinder. The protein was recovered from the 8 M urea, and the protein was re-overlaid using a 10-fold volume of reoverlapping buffer (2M Urea, 50 mM Tris 8.0, 1 mM Cystein). The superimposed protein was loaded on a Q HP column (GE healthcare) to remove impurities, and the eluted fraction was loaded on a heparin HP column (GE healthcare), and the eluted fraction was analyzed by SDS-PAGE. Adhesion activity was investigated.

As a result, as shown in Figure 1, it was confirmed that the immunoglobulin Fc-HBM fusion protein according to the present invention has an adhesion activity to heparin.

Example  3: immunoglobulin Fc - HBM Preparation of Insulin Binders

<3-1> PEG  Linker Combined  Immunoglobulins Fc  Preparation of Fragments

In order to PEGylate 5K PropionALD (3) PEG (PEG having 3 propylaldehyde groups, NOF, Japan) at the N-terminus of an immunoglobulin Fc fragment, the concentration of the immunoglobulin Fc fragment was 10 mg / ml and the immunoglobulin Fc fragment and PEG were mixed in a molar ratio of 1: 2 and reacted at 4 ° C. for 4.5 hours. At this time, the reaction was performed by adding 20 mM SCB (NaCNBH ₃ ), a reducing agent, in 100 mM potassium phosphate (pH 6.0) solution. After the reaction was completed, the reaction solution was loaded into a Source 15Q purification column (GE healthcare) to purify the mono-pegylated immunoglobulin Fc fragment.

<3-2> immunoglobulin Fc Preparation of Insulin Binders

The mono-pegylated immunoglobulin Fc fragment prepared in Example <3-1> and insulin (Humulin R, Lilly) were mixed so that the molar ratio was 4: 1 and the total protein concentration was 20 mg / ml at 4 ° C. The reaction was carried out for 20 hours at. At this time, the reaction was performed by adding 20 mM SCB (NaCNBH ₃ ), a reducing agent, in 100 mM potassium phosphate (pH 6.0) solution. After the reaction was completed, the reaction solution was first purified by loading on a source 15Q purification column (GE healthcare), and the fraction eluted therefrom was loaded on a source 15ISO purification column to perform second purification. This gave an immunoglobulin Fc-insulin conjugate in which insulin was bound to a mono-pegylated immunoglobulin Fc fragment.

<3-3> PEG  Linker Combined  Immunoglobulins Fc - HBD  Preparation of Fusion Proteins

In order to PEGylate 5K PropionALD (3) PEG (PEG having 3 propylaldehyde groups, NOF, Japan) to the N-terminus of an immunoglobulin Fc fragment having a heparin binding motif, prepared in Example <1-5> The concentration of the immunoglobulin Fc-HBM fusion protein was 6 mg / ml, and the fusion protein and PEG were mixed at a molar ratio of 1: 2 and reacted at 4 ° C for 4 hours. In this case, the reaction was performed by adding 20 mM SCB (NaCNBH ₃ ), a reducing agent, in a 100 mM potassium phosphate (pH 6.0) solution. After the reaction was completed, the reaction solution was loaded into a source 15Q purification column (GE healthcare) to purify the mono-pegylated immunoglobulin Fc-HBM fusion protein.

<3-4> Immunoglobulins Fc - HBM Preparation of Insulin Binders

The mono-pegylated immunoglobulin Fc-HBM fusion protein prepared in Example <3-3> and insulin (Humulin R, Lilly) were mixed in a molar ratio of 4: 1 and the total protein concentration was 20 mg / ml. Reacted at 4 ° C. for 20 hours. At this time, the reaction was performed by adding 20 mM SCB (NaCNBH ₃ ), a reducing agent, in 100 mM potassium phosphate (pH 6.0) solution. After the reaction was completed, the reaction solution was first purified by loading on a source 15Q purification column (GE healthcare), and the fraction eluted therefrom was loaded on a source 15ISO purification column to perform second purification. This yielded an immunoglobulin Fc-HBM-insulin conjugate with insulin bound to a mono-pegylated immunoglobulin Fc fragment.

Example  4: immunoglobulin Fc - HBM Of insulin conjugates In vivo  Pharmacokinetic Measurement

In order to confirm the in vivo persistence of the immunoglobulin Fc-HBM-insulin conjugate according to the present invention, pharmacokinetics were measured in normal male rats as follows.

Normal type male rats (Humulin R, Lilly) alone, immunoglobulin Fc-insulin conjugate prepared in Example <3-2>, and immunoglobulin Fc-HBM prepared in Example <3-4> Insulin conjugates were administered subcutaneously once every 100 μg / kg (insulin basis), and then the change in blood concentration was measured using the insulin ELISA kit (ALPCO) over time. From the measured values, the pharmacokinetic mediation was performed using WinNonlin 5.2. The variable was calculated.

As a result, as shown in Figure 2, the in vivo half-life of the immunoglobulin Fc-HBM-insulin conjugate according to the present invention is 20 hours longer than that of 0.58 hours of native insulin and 17 hours of immunoglobulin Fc-insulin conjugate. It showed persistence, and the clearance was also confirmed to be excellent.

<110> Hanmi Holdings Co., Ltd <120> A FUSION PROTEIN OF A FIBRONECTIN FRAGMENT AND AN IMMUNOGLOBULIN          FRAGMENT AND USE THEREOF <130> PA100654 / KR <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 1 ggaattccat atgattcctg caccaactga cctg 34 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 2 cgcggatcct cagggctcgc tcttctgatt attct 35 <210> 3 <211> 792 <212> DNA <213> Artificial Sequence <220> <223> Fibronectin Type III Hep12 ~ 14 domain <400> 3 atgattcctg caccaactga cctgaagttc actcaggtca cacccacaag cctgagcgcc 60 cagtggacac cacccaatgt tcagctcact ggatatcgag tgcgggtgac ccccaaggag 120 aagaccggac caatgaaaga aatcaacctt gctcctgaca gctcatccgt ggttgtatca 180 ggacttatgg tggccaccaa atatgaagtg agtgtctatg ctcttaagga cactttgaca 240 agcagaccag ctcagggagt tgtcaccact ctggagaatg tcagcccacc aagaagggct 300 cgtgtgacag atgctactga gaccaccatc accattagct ggagaaccaa gactgagacg 360 atcactggct tccaagttga tgccgttcca gccaatggcc agactccaat ccagagaacc 420 atcaagccag atgtcagaag ctacaccatc acaggtttac aaccaggcac tgactacaag 480 atctacctgt acaccttgaa tgacaatgct cggagctccc ctgtggtcat cgacgcctcc 540 actgccattg atgcaccatc caacctgcgt ttcctggcca ccacacccaa ttccttgctg 600 gtatcatggc agccgccacg tgccaggatt accggctaca tcatcaagta tgagaagcct 660 gggtctcctc ccagagaagt ggtccctcgg ccccgccctg gtgtcacaga ggctactatt 720 actggcctgg aaccgggaac cgaatataca atttatgtca ttgccctgaa gaataatcag 780 aagagcgagc cc 792 <210> 4 <211> 263 <212> PRT <213> Artificial Sequence <220> <223> Fibronectin Type III Hep12 ~ 14 domain <400> 4 Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr Ser   1 5 10 15 Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg              20 25 30 Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn          35 40 45 Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala      50 55 60 Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser  65 70 75 80 Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro                  85 90 95 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser             100 105 110 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val         115 120 125 Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp Val     130 135 140 Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile 145 150 155 160 Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val Ile                 165 170 175 Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala             180 185 190 Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg         195 200 205 Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg     210 215 220 Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr 225 230 235 240 Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys                 245 250 255 Asn Asn Gln Lys Ser Glu Pro             260 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 5 ggaattccat atgccatcat gcccagcacc tgag 34 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 6 ccctgagctg gtcttttacc cagagacagg gagag 35 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 7 ctgtctctgg gtaaaagacc agctcaggga gttg 34 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 8 cgcggatcct caggagctcc gagcattgtc attc 34 <210> 9 <211> 942 <212> DNA <213> Artificial Sequence <220> 223 IG Fc-Hep13 fusion polynucleotide <400> 9 atgccatcat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 60 aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 120 gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 180 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 240 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 300 aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 360 ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 420 acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 480 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 540 ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 600 tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 660 ggtaaaagac cagctcaggg agttgtcacc actctggaga atgtcagccc accaagaagg 720 gctcgtgtga cagatgctac tgagaccacc atcaccatta gctggagaac caagactgag 780 acgatcactg gcttccaagt tgatgccgtt ccagccaatg gccagactcc aatccagaga 840 accatcaagc cagatgtcag aagctacacc atcacaggtt tacaaccagg cactgactac 900 aagatctacc tgtacacctt gaatgacaat gctcggagct cc 942 <210> 10 <211> 313 <212> PRT <213> Artificial Sequence <220> 223 IG Fc-Hep13 fusion protein <400> 10 Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu   1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu              20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln          35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys      50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu  65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                  85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys             100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser         115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys     130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln             180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg Pro Ala     210 215 220 Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg Arg Ala 225 230 235 240 Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser Trp Arg Thr                 245 250 255 Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val Pro Ala Asn             260 265 270 Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp Val Arg Ser Tyr         275 280 285 Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr     290 295 300 Thr Leu Asn Asp Asn Ala Arg Ser Ser 305 310 <210> 11 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> Fibronectin Type III Hep13 domain <400> 11 Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro   1 5 10 15 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser              20 25 30 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val          35 40 45 Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp Val      50 55 60 Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile  65 70 75 80 Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser                  85 90 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 12 tttacccaga gacagggaga ggctcttctg tgt 33 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 13 aaaaggcctg tggtcatcga cgcctccact gcc 33 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 14 cgcggatcct cagggctcgc tcttctgatt attc 34 <210> 15 <211> 939 <212> DNA <213> Artificial Sequence <220> 223 IG Fc-Hep14 fusion polypeptide <400> 15 atgccatcat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 60 aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 120 gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 180 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 240 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 300 aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 360 ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 420 acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 480 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 540 ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 600 tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 660 ggtaaacctg tggtcatcga cgcctccact gccattgatg caccatccaa cctgcgtttc 720 ctggccacca cacccaattc cttgctggta tcatggcagc cgccacgtgc caggattacc 780 ggctacatca tcaagtatga gaagcctggg tctcctccca gagaagtggt ccctcggccc 840 cgccctggtg tcacagaggc tactattact ggcctggaac cgggaaccga atatacaatt 900 tatgtcattg ccctgaagaa taatcagaag agcgagccc 939 <210> 16 <211> 312 <212> PRT <213> Artificial Sequence <220> 223 IG Fc-Hep14 fusion protein <400> 16 Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu   1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu              20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln          35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys      50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu  65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                  85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys             100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser         115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys     130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln             180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Pro Val Val     210 215 220 Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu 225 230 235 240 Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala                 245 250 255 Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro             260 265 270 Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile         275 280 285 Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu     290 295 300 Lys Asn Asn Gln Lys Ser Glu Pro 305 310 <210> 17 <211> 91 <212> PRT <213> Artificial Sequence <220> <223> Fibronectin Type III Hep14 domain <400> 17 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu   1 5 10 15 Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro              20 25 30 Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly          35 40 45 Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu      50 55 60 Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val  65 70 75 80 Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro                  85 90 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 18 ccaatcaggg gctgtttacc cagagacagg gagag 35 <210> 19 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 19 ctgtctctgg gtaaaattcc tgcaccaact gacc 34 <210> 20 <211> 1455 <212> DNA <213> Artificial Sequence <220> 223 IG Fc-Hep12-14 fusion polypeptide <400> 20 atgccatcat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 60 aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 120 gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 180 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 240 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 300 aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 360 ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 420 acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 480 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 540 ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 600 tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 660 ggtaaaattc ctgcaccaac tgacctgaag ttcactcagg tcacacccac aagcctgagc 720 gcccagtgga caccacccaa tgttcagctc actggatatc gagtgcgggt gacccccaag 780 gagaagaccg gaccaatgaa agaaatcaac cttgctcctg acagctcatc cgtggttgta 840 tcaggactta tggtggccac caaatatgaa gtgagtgtct atgctcttaa ggacactttg 900 acaagcagac cagctcaggg agttgtcacc actctggaga atgtcagccc accaagaagg 960 gctcgtgtga cagatgctac tgagaccacc atcaccatta gctggagaac caagactgag 1020 acgatcactg gcttccaagt tgatgccgtt ccagccaatg gccagactcc aatccagaga 1080 accatcaagc cagatgtcag aagctacacc atcacaggtt tacaaccagg cactgactac 1140 aagatctacc tgtacacctt gaatgacaat gctcggagct cccctgtggt catcgacgcc 1200 tccactgcca ttgatgcacc atccaacctg cgtttcctgg ccaccacacc caattccttg 1260 ctggtatcat ggcagccgcc acgtgccagg attaccggct acatcatcaa gtatgagaag 1320 cctgggtctc ctcccagaga agtggtccct cggccccgcc ctggtgtcac agaggctact 1380 attactggcc tggaaccggg aaccgaatat acaatttatg tcattgccct gaagaataat 1440 cagaagagcg agccc 1455 <210> 21 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> IG Fc-Hep12 ~ 14 fusion protein <400> 21 Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu   1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu              20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln          35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys      50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu  65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                  85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys             100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser         115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys     130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln             180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Pro Ala     210 215 220 Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala 225 230 235 240 Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val                 245 250 255 Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro             260 265 270 Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr         275 280 285 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro Ala     290 295 300 Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg Arg Ala 305 310 315 320 Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser Trp Arg Thr                 325 330 335 Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val Pro Ala Asn             340 345 350 Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp Val Arg Ser Tyr         355 360 365 Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr     370 375 380 Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val Ile Asp Ala Ser 385 390 395 400 Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro                 405 410 415 Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly             420 425 430 Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val         435 440 445 Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu     450 455 460 Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln 465 470 475 480 Lys Ser Glu Pro                 <210> 22 <211> 1215 <212> DNA <213> Artificial Sequence <220> 223 IG Fc-Hep13-14 fusion polypeptide <400> 22 atgccatcat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 60 aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 120 gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 180 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 240 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 300 aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 360 ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 420 acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 480 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 540 ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 600 tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 660 ggtaaaagac cagctcaggg agttgtcacc actctggaga atgtcagccc accaagaagg 720 gctcgtgtga cagatgctac tgagaccacc atcaccatta gctggagaac caagactgag 780 acgatcactg gcttccaagt tgatgccgtt ccagccaatg gccagactcc aatccagaga 840 accatcaagc cagatgtcag aagctacacc atcacaggtt tacaaccagg cactgactac 900 aagatctacc tgtacacctt gaatgacaat gctcggagct cccctgtggt catcgacgcc 960 tccactgcca ttgatgcacc atccaacctg cgtttcctgg ccaccacacc caattccttg 1020 ctggtatcat ggcagccgcc acgtgccagg attaccggct acatcatcaa gtatgagaag 1080 cctgggtctc ctcccagaga agtggtccct cggccccgcc ctggtgtcac agaggctact 1140 attactggcc tggaaccggg aaccgaatat acaatttatg tcattgccct gaagaataat 1200 cagaagagcg agccc 1215 <210> 23 <211> 404 <212> PRT <213> Artificial Sequence <220> 223 IG Fc-Hep13-14 fusion protein <400> 23 Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu   1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu              20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln          35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys      50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu  65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                  85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys             100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser         115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys     130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln             180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg Pro Ala     210 215 220 Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg Arg Ala 225 230 235 240 Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser Trp Arg Thr                 245 250 255 Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val Pro Ala Asn             260 265 270 Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp Val Arg Ser Tyr         275 280 285 Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr     290 295 300 Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val Ile Asp Ala Ser 305 310 315 320 Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro                 325 330 335 Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly             340 345 350 Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val         355 360 365 Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu     370 375 380 Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln 385 390 395 400 Lys Ser Glu Pro                 <210> 24 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> Fibronectin type III Hep13-14 domain <400> 24 Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro   1 5 10 15 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser              20 25 30 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val          35 40 45 Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp Val      50 55 60 Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile  65 70 75 80 Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val Ile                  85 90 95 Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala             100 105 110 Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg         115 120 125 Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg     130 135 140 Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr 145 150 155 160 Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys                 165 170 175 Asn Asn Gln Lys Ser Glu Pro             180 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 25 tcttggtggg ctgactttac ccagagacag ggag 34 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 26 ctgtctctgg gtaaagtcag cccaccaaga aggg 34 <210> 27 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 27 cgcggatcct cagctaatgg tgatggtggt tctc 34 <210> 28 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> IG Fc-HBM fusion polyprptide <400> 28 atgccatcat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 60 aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 120 gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 180 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 240 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 300 aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 360 ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 420 acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 480 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 540 ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 600 tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 660 ggtaaagtca gcccaccaag aagggctcgt gtgacagatg ctactgagac caccatcacc 720 attagc 726 <210> 29 <211> 241 <212> PRT <213> Artificial Sequence <220> 223 IG Fc-HBM fusion protein <400> 29 Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu   1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu              20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln          35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys      50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu  65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                  85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys             100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser         115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys     130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln             180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Val Ser Pro     210 215 220 Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile 225 230 235 240 Ser     <210> 30 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Fibronectin type III HBM domain <400> 30 Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr   1 5 10 15 Ile Thr Ile Ser              20

Claims (27)

A fusion protein of fibronectin fragment and immunoglobulin Fc fragment comprising a heparin binding domain. The fusion protein of claim 1, wherein the fibronectin fragment is selected from the group consisting of heparin binding domains 12, 13, 14 and combinations thereof. The fusion protein of claim 2, wherein the fibronectin fragment has an amino acid sequence of any of the amino acid sequences set forth in SEQ ID NOs: 4, 11, 17, 24, and 30. 4. The fusion protein of claim 1, wherein said immunoglobulin Fc fragment is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and hybrids thereof. The fusion protein of claim 4, wherein said immunoglobulin Fc fragment is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, combinations thereof, and hybrids thereof. 6. The fusion protein of claim 5, wherein said immunoglobulin Fc fragment is an IgG4 Fc fragment. The fusion protein of claim 1, wherein said immunoglobulin Fc fragment is sugar chained or unglycosylated. The fusion protein of claim 1, wherein the fusion protein has an amino acid sequence of any of the amino acid sequences set forth in SEQ ID NOs: 10, 16, 21, 23, and 29. 7. A polynucleotide encoding a fusion protein of a fibronectin fragment and an immunoglobulin Fc fragment according to claim 1. The polynucleotide of claim 9, wherein the polynucleotide has a nucleotide sequence of any one of the nucleotide sequences set forth in SEQ ID NOs: 9, 15, 20, 22, and 28. 11. A pharmaceutical composition comprising a fusion protein of a fibronectin fragment and an immunoglobulin Fc fragment according to claim 1 as a carrier of a drug. The pharmaceutical composition of claim 11, wherein the carrier is in the form of a conjugate in which the drug is linked to the fusion protein. The pharmaceutical composition of claim 12, wherein the fusion protein and the drug are linked in a molar ratio of 1: 1 to 1:10. 13. The conjugate of claim 12, wherein said drug is linked to an immunoglobulin Fc fragment of said fusion protein using a genetically recombinant or nonpeptidic polymer as a linker. The pharmaceutical composition of claim 14, wherein the non-peptidyl polymer is linked to the fusion protein in a molar ratio of 1: 1 to 1:10. The method of claim 14, wherein the non-peptidyl polymer is polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer (poly-ethylene (propylene / propylene) glycol), polyoxyethylene, polyurethane, polyphosphazene, poly Group consisting of saccharide, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylate, lipid polymer, chitin, hyaluronic acid, heparin and combinations thereof The pharmaceutical composition is selected from. The pharmaceutical composition of claim 16, wherein the non-peptidic polymer is polyethylene glycol having two or more reactors. The pharmaceutical composition of claim 11, wherein the drug is a bioactive polypeptide. 19. The method according to claim 18, wherein the physiologically active polypeptide is human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, colony stimulating factors, glucacon-like peptides, exendin-4 peptides. , ANP, BNP, CNP, DNP, G-protein coupled receptors, interleukins, interleukin receptors, enzymes, interleukin binding proteins, cytokine binding proteins, macrophage activators, macrophages Phagocytic peptide, B cell factor, T cell factor, protein A, allergic inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, alpha-1 antitrypsin, albumin, α Lactalbumin, Apolipoprotein-E, Erythropoietin, High glycated erythropoietin, Angiopoietin, Hemoglobin, Thrombin, Thrombin receptor activating peptide, Thrombomodulin, Blood factor Ⅶ, VIIa, VII, VII, and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, Leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone formation growth factor, bone formation promoting protein, calcitonin, insulin, somatostatin, octreotide (somatostatin agonist), atriopeptin, Cartilage inducer, elkatonin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, Insulin-like growth factor, corticosteroids, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticoat Pin release factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, virus-derived vaccine antigens, monoclonal antibodies, polyclonal antibodies and antibody fragments Phosphorus pharmaceutical composition. A method of increasing the blood half-life of a drug using a fusion protein of a fibronectin fragment and an immunoglobulin Fc fragment as a carrier of the drug, comprising linking the drug to a fusion protein of the fibronectin fragment and the immunoglobulin Fc fragment according to claim 1 . The method of claim 20, wherein said drug is linked to said fusion protein in a molar ratio of 1: 1 to 1:10. The method of claim 20, wherein said drug is linked to an immunoglobulin Fc fragment of said fusion protein using a recombinant or nonpeptidic polymer as a linker. The method of claim 22, wherein said nonpeptidyl polymer is linked to said fusion protein in a molar ratio of 1: 1 to 1:10. The method of claim 22, wherein the non-peptidyl polymer is polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer (poly-ethylene / propylene) glycol, polyoxyethylene, polyurethane, polyphosphazene, poly Group consisting of saccharide, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylate, lipid polymer, chitin, hyaluronic acid, heparin and combinations thereof Which is selected from. The method of claim 24, wherein the non-peptidic polymer is polyethylene glycol having two or more reactors. The method of claim 20, wherein the drug is a bioactive polypeptide. 27. The method according to claim 26, wherein the physiologically active polypeptide is human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, interferon receptors, colony stimulating factor, glucacon-like peptide, exendin-4 peptide , ANP, BNP, CNP, DNP, G-protein coupled receptors, interleukins, interleukin receptors, enzymes, interleukin binding proteins, cytokine binding proteins, macrophage activators, macrophages Phagocytic peptide, B cell factor, T cell factor, protein A, allergic inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, alpha-1 antitrypsin, albumin, α Lactalbumin, Apolipoprotein-E, Erythropoietin, High glycated erythropoietin, Angiopoietin, Hemoglobin, Thrombin, Thrombin receptor activating peptide, Thrombomodulin, Blood factor Ⅶ, VIIa, VII, VII, and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, Leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone formation growth factor, bone formation promoting protein, calcitonin, insulin, somatostatin, octreotide (somatostatin agonist), atriopeptin, Cartilage inducer, elkatonin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, Insulin-like growth factor, corticosteroids, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticoat Pin release factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, virus-derived vaccine antigens, monoclonal antibodies, polyclonal antibodies and antibody fragments How to be.

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