KR100824505B1 - A METHOD FOR THE MASS PRODUCTION OF IMMUNOGLOBULIN Fc REGION DELETED INITIAL METHIONINE RESIDUES - Google Patents

Abstract

The present invention relates to a method for mass production of immunoglobulin Fc regions in the form of monomers or dimers from which a starting methionine residue has been removed, using a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region. will be.

Immunoglobulins, Fc regions, aggregates, dimers

Description

A METHOD FOR THE MASS PRODUCTION OF IMMUNOGLOBULIN Fc REGION DELETED INITIAL METHIONINE RESIDUES}

Figure 1 is an electrophoresis result of the formation of monomers and dimers of the Fc region according to the human immunoglobulin IgG4 Fc region expression vector.

Figure 2 shows the results confirming the binding capacity of the human immunoglobulin IgG 4Fc region with C1q.

Figure 3 shows the results confirming the binding ability of the human immunoglobulin IgG Fc region with FcγRI.

4 shows the results of confirming the binding ability of the human immunoglobulin IgG Fc region with FcγRIII.

5 shows the results of confirming the binding ability of the human immunoglobulin IgG Fc region with FcRnαβ2.

FIG. 6 shows the blood half-life of the binding protein EPO-PEG-Fc conjugate prepared using the human immunoglobulin IgG Fc region as a carrier.

7 is a result of inoculating the microbial transformants obtained in Example 2 by inoculation into the fermentor, and then mixing a portion of the fermentation broth with 2 × protein drop buffer in the same amount, followed by electrophoresis on 15% SDS-PAGE.

FIG. 8 shows the protein bands by refolding the products expressed as aggregates in each transformant in Example 2 and electrophoresing under the above conditions.

9 is the result of inoculating the microbial transformants obtained in Example 3 by inoculation and expressing the fermentation broth in the same amount with 2 × protein drop buffer, followed by electrophoresis on 15% SDS-PAGE.

FIG. 10 shows the results of electrophoresis on 15% SDS-PAGE after mixing the respective expression products purified in Example 3 with a protein drop buffer in which a reducing agent such as DTT or beta-mecaptoethanol was removed.

The present invention relates to a method for mass production of immunoglobulin Fc regions in the form of monomers or dimers from which a starting methionine residue has been removed, using a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region. will be.

With the development of genetic engineering technology, many kinds of protein medicines have been manufactured and used. However, in the case of protein medicine, it has a fatal disadvantage that it is not easily denatured or easily degraded by in vivo protease and the like, so that the concentration and titer in vivo cannot be sustained for a long time. Therefore, increasing the stability of the protein, and maintaining the blood and in vivo concentration of the protein drug is an important problem not only for effective treatment, but also for the patient's discomfort and economic reasons for receiving the protein by frequent injections.

Therefore, in order to increase the in vivo stability of protein drugs, various methods have been tried for a long time, such as changing the formulation of a protein, fusing another protein, or chemically or biologically attaching a suitable polymer to a protein surface. .

One of the attempts to increase protein stability by fusion with other proteins is the fusion of immunoglobulin Fc with a protein.

The Fc region is responsible for effector functions such as complement-depentent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC) in addition to antigen-binding ability, which is an inherent function of immunoglobulins. In addition, the FcRn sequences present in the Fc region play a role in regulating serum IgG levels by increasing IgG transport and half-life to neonates (Ghetie and Ward, Immunology Today 18: 592-598, 1997), protein A and Regulates interaction with protein G. In order to increase the stability of the therapeutic protein through the fusion of the Fc region and the therapeutic protein has been actively studied.

Korean Patent No. 249572 connects the carboxyl ends of various proteins such as IL4 receptor, IL7 receptor, G-CSF receptor, and EPO receptor to the amino terminus of IgG1 heavy chain constant region (Fc), and then fusions prepared from mammalian cells. Protein is disclosed. U. S. Patent No. 5,605, 690 reports a fusion protein prepared from animal cells by fusion of a human IgGl Fc derivative to the carboxyl terminus of a tumor necrosis factor receptor. Tanox also reported in US Pat. Nos. 5,723,125 and 5,908,626 that the carboxyl ends of human interferon alpha and beta genes and native human IgG4 Fc genes were produced in animal cells using peptide linkers, and Lexigen applied for PCT applications. In WO 00/69913, a native IgG1 Fc carboxyl terminus and an amino terminus of human interferon were produced in animal cells by linkage of the amino terminus without a linker. US Patent Publication No. 20030082679 reports that the fusion protein produced in animal cells has increased blood half-life after linking the carboxyl end of human G-CSF gene with the amino end of IgG1 Fc using a peptide linker. U.S. Patent Publication Nos. 20010053539, 6,030,613, PCT Application Publication Nos. WO 99/02709, WO 01/03737, and European Patent No. EP 0464533B1, and the like disclose an IgG1 Fc gene or a derivative of the Fc gene at its amino terminus. After insertion of the peptide linker or without the peptide linker to the carboxyl terminus of the human EPO gene, TPO gene, human growth hormone gene, or human interferon beta gene, each fusion was produced using animal cells. Reported that the half-life in blood was higher than that of the native protein.

However, the fusion protein with Fc has a problem that the half-life of the target protein is increased, but at the same time, the effector function of the Fc region is exerted (US Pat. No. 5,349,053). By the effector function of the Fc region, it complements or binds to cells expressing FcγRs, destroying specific cells and inducing the production and secretion of several cytokines that cause inflammation, causing unwanted inflammation. In addition, since the protein sequence of the fusion site is a new protein sequence that does not exist in the human body, there are various disadvantages such as the possibility of inducing an immune response upon long-term administration.

To this end, studies have been made to use immunoglobulins or immunoglobulin fragments that maintain long blood half-lives but lack effector function. Cole et al. Demonstrated that the ADCC activity was inhibited by substitution of alanine for residues 234, 235, and 237, which are known to play an important role in binding to the Fc receptor in the CH2 region, to produce Fc derivatives with reduced affinity for the Fc receptor. Reported (Cole et al., J. Immunol . 159: 3613-3621, 1997). However, all of them may have greater immunity or antigenicity due to the presence of native human Fc regions and other inappropriate amino acids and may lose desirable Fc functions.

One way to remove or reduce unwanted effector function while maintaining high blood levels of immunoglobulins has been to remove sugars from immunoglobulins. U.S. Pat.No. 5,585,097 prepared non-glycosylated antibody derivatives by substituting the 297th asparagine residue of the CH2 domain, the glycosylated residue of the antibody, with another amino acid in the preparation of the CD3 antibody. It showed reduced effector function while maintaining without changing the blood half-life. However, this method also has a problem in that it can be recognized and rejected as an external substance in the immune system by generating a new recombinant construct having an abnormal sequence. US Patent Publication No. 20030073164 describes a method for producing antibodies by fusing heavy and light chains to secretory sequences using E. coli cells without glycosylation to produce therapeutic antibodies lacking effector function.

Amgen, USA, manufactures therapeutic proteins or therapeutic protein peptide mimics in US Pat. Nos. 6,660,843, US Patent Publication Nos. 20040044188 and 20040053845, the first 5 of a human IgG1 Fc hinge at its amino or carboxyl terminus. A method for the production of an E. coli host after fusion of a derivative having dog amino acids deleted is described. However, since a fusion protein expressed without a signal signal is expressed and exists in the form of an inclusion body in the cytoplasm, it has a disadvantage of undergoing a separate refolding process after separation. Through the refolding process, the yield of protein is lowered, and in the case of a protein present as a homologous or heterologous dimer, the yield produced by the dimer is significantly reduced. In addition, a protein expressed in E. coli without a signal signal has a methionine residue added to its amino terminus due to the nature of the E. coli protein expression system. The expression products of Amgen's mentioned have methionine residues added at the amino terminus, and these methionine residues may cause an immune response upon repeated or overdose administration to the human body. In addition, since they express genes in the form of fusion proteins in Escherichia coli by connecting genes encoding therapeutic proteins with genes encoding Fc, they are difficult to be expressed in Escherichia coli or have a problem with activity when they are expressed in Escherichia coli. Fusions have the disadvantage of being difficult to produce. In addition, since the fusion site of the two proteins is an abnormal sequence that does not exist in vivo, it may be recognized as an external substance in the immune system and cause an immune response.

To improve this, the inventors have prepared the Fc region and protein drug as individual polypeptides in each of the best expression systems, rather than fusion by conventional recombinant methods, and then covalently bind the Fc as a carrier of the drug. There is a bar. In this case, a combination of glycated polypeptide drug and non-glycosylated Fc can be prepared, thereby eliminating an inappropriate immune response while satisfying all of physiological drug activity, biological persistence and stability.

In this case, since Fc is preferably in a non-glycosylated form, a prokaryotic expression system such as E. coli is used. Production method using the expression system of E. coli has a number of advantages over the conventional method using animal cells, E. coli is easy to produce the expression vector can be quickly verified whether the expression, and because the growth rate is very low cost As it can be mass-produced and relatively simple fermentation method can be applied, it is more useful than other host cells in terms of commercial use.

In the case of overexpressing the Fc region in E. coli, most of the Fc region is expressed in the form of aggregates. In order to industrially use the Fc region, there have been attempts to use the Fc region expressed in a water-soluble state. European patent EP0227110 describes a process for producing an immunoglobulin G1 Fc region by overexpressing an immunoglobulin G1 Fc region and then taking only a cell lysate that is soluble. However, when only the immunoglobulin expressed in the water-soluble state is separated, the yield is too low as 15mg / L has the disadvantage of low industrial use value. In order to improve the above point, Korean Patent Application No. 0092783 expresses an immunoglobulin Fc region in a form fused with an E. coli signal sequence to express it in a water-soluble form rather than an aggregate. In this case, it was confirmed that the signal peptide was expressed in the soluble form from which the protein was removed, and the protein production yield was greatly improved to 50 to 600 mg / L.

Against this background, the inventors have tried to find a way to industrially produce large quantities of active immunoglobulin Fc regions in an improved, yieldable, industrialized manner in which sugar chains are eliminated and there is no risk of inducing an immune response. When expressed by linking to the N-terminus of the immunoglobulin Fc region, the immunoglobulin Fc region expressed in aggregate form is dimer or monomer form in which the starting methionine residue is removed through solublization and refolding. Confirmed that the production of the immunoglobulin Fc region, and completed the present invention to produce a large amount of immunoglobulin Fc region from which the methionine start codon was removed.

One object of the present invention is to prepare a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; Transforming the vector into prokaryotic cells; Culturing the transformant; And separating and purifying the immunoglobulin Fc region expressed in aggregate form from the transformant, thereby providing a method for mass-producing an immunoglobulin Fc region from which the starting methionine residue has been removed.

Another object of the present invention is to provide an immunoglobulin Fc region in the form of a dimer or monomer in which the starting methionine residues prepared by the above method are removed.

In one aspect, the present invention provides a method for preparing a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; Transforming the vector into prokaryotic cells; Culturing the transformant; And isolating and purifying the immunoglobulin Fc region expressed in aggregate form from the transformant, the method for mass production of immunoglobulin Fc regions from which the starting methionine residue has been removed.

The present invention relates to a method for producing a large amount of the Fc region of an immunoglobulin that can be usefully used as a carrier of a drug, and when the hinge region is expressed by connecting to the N terminal of the immunoglobulin Fc region, aggregates It was confirmed that the immunoglobulin Fc region expressed in the form was solublized and refolded into the Fc region in the form of an active dimer or monomer in which the starting methionine residues encoded by the initiation codon were removed. It was found that the hinge region acts as a determinant regulatory element that binds the Fc region of the immunoglobulin to effect processing and refolding of the initiating methionine residue encoded by the initiation codon without loss of activity and in the form of a native sequence removed. The present invention has a significant meaning in that.

Hinge regions that can be combined with immunoglobulin Fc regions in recombinant form to be used for mass production of Fc regions include human, goat, pig, mouse and rabbit. It is a hinge region derived from IgG, IgA, IgM, IgE or IgD originating from animals such as hamsters, rats, guinea pigs, more preferably the hinge region derived from IgG1, IgG2, IgG3 or IgG4 (SEQ ID NO: 14). To 17). The hinge region can be a full length hinge region as well as a fragment thereof. Preferably the hinge region fragment is a fragment having two or more contiguous amino acid sequences, more preferably a fragment having two or more contiguous amino acid sequences comprising one or more cysteine residues. In a specific embodiment of the present invention, fragments derived from the hinge region derived from IgG4 described in SEQ ID NO: 17 are used and are described in SEQ ID NO: 18, 19, 20 or 21. When using the SEQ ID NO: 18, 19, 20 hinge region, it is possible to prepare an immunoglobulin Fc region in the form of dimers and monomers, and an immunoglobulin Fc region in the monomer form can be efficiently prepared using the hinge region of SEQ ID NO: 21. In addition, in a specific embodiment of the present invention, a fragment derived from a hinge region derived from IgG1 described in SEQ ID NO: 14 was used, which is described as SEQ ID NO: 48, 49, 50, 51, 52, 53, 54 or 55, SEQ ID NO: Fragments derived from the hinge region derived from IgG2, described as 15, were used, which were set forth in SEQ ID NOs: 56, 57, 58, 59 or 60, and when using them, an immunoglobulin Fc region in dimeric form could be prepared.

Immunoglobulin Fc regions that can be produced by the invention include human, goat, pig, mouse, rabbit. It may be a recombinant or a derivative thereof obtained from a natural type, transformed animal cell or microorganism isolated in vivo of an animal such as hamster, rat, guinea pig. Preferably, it may be an Fc region of human-derived IgG, IgA, IgM, IgE, IgD, a combination thereof, or a hybrid thereof. By "combination" is meant that a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of different origin, and "hybrid" means two or more different origins of the immunoglobulin Fc region of the short chain. The term means that there is a sequence corresponding to an immunoglobulin Fc region. The immunoglobulin may preferably be an Fc region derived from IgG1, IgG2, IgG3, IgG4, or a combination thereof. Nucleic acid sequences encoding human immunoglobulin Fc regions useful herein and amino acid sequences defining them may be those encoded by the nucleotide sequences disclosed in the GenBank and / or EMBL databases.

The immunoglobulin Fc region of this invention contains an amino acid sequence derivative. Amino acid sequence derivatives mean that a natural amino acid sequence and one or more amino acid residues have a different sequence and can occur naturally or artificially. Fc regions of immunoglobulins include derivatives by deletions, insertions, non-conservative or conservative 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. 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. Neurode, 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. Such derivatives may be prepared by chemical peptide synthesis methods known in the art or by recombinant methods based on DNA sequences (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).

In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.

The immunoglobulin derivatives may be functional equivalents that exhibit the same biological activity as natural proteins, or derivatives whose properties are modified as necessary. Preferably, the immunoglobulin Fc region derivatives are structurally stable to the heat, pH, etc. of the protein by induction and modification on the amino acid sequence within a range in which the protein sequence of the resulting derivative is not too different from that of the human to induce an immune response. Or derivatives that have increased solubility, improved sulfide bond formation, affinity with the expression host, binding with complement, binding with Fc receptors, antibody dependent cytotoxicity, and the like. At this time, the site of change in the Fc region derivatives is different from that of humans and should not induce an immune response when administered to humans. Examples of preferred derivatives include reduced binding to Fc receptors that cause antibody dependent cytotoxicity. Specific residues of the IgG1 Fc region can be derived. The derivative may include the deletion of 234 leucine residues present in the IgG1 CH2 sequence (see numbering in the Kobat database for substitution) or substitution with other residues, with the most preferred induction being an amino acid residue corresponding to the same position in IgG4. It is substituted with phosphorus phenylalanine.

delete

The hinge region that binds according to the immunoglobulin Fc region can be selected, preferably using a hinge region of the same origin as the immunoglobulin Fc region. In a specific embodiment of the present invention, a recombinant immunoglobulin Fc having an amino acid sequence of SEQ ID NO: 7, 9, 11 or 13 is linked to a hinge region having an amino acid sequence of SEQ ID NO: 18, 19, 20 or 21 to an Fc region derived from IgG4. Nucleic acid sequences encoding the regions were prepared. The nucleic acid encoding said recombinant immunoglobulin Fc region is preferably set forth in SEQ ID NO: 6, 8, 10 or 12, respectively.

In a specific embodiment of the present invention, SEQ ID NOs: 23, 25, 27, in which a hinge region having an amino acid sequence of SEQ ID NO: 48, 49, 50, 51, 52, 53, 54 or 55 is linked to an Fc region derived from IgG1 Nucleic acid sequences encoding recombinant immunoglobulin Fc regions having amino acid sequences of 29, 31, 33, 35, or 37 were prepared. The nucleic acid encoding the recombinant immunoglobulin Fc region is preferably described by SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34 or 36, respectively.

In a specific embodiment of the present invention, the amino acid sequence of SEQ ID NO: 39, 41, 43, 45 or 47 is linked to the hinge region having the amino acid sequence of SEQ ID NO: 56, 57, 58, 59 or 60 to the Fc region derived from IgG2 A nucleic acid sequence encoding a recombinant immunoglobulin Fc region having was prepared. The nucleic acid encoding said recombinant immunoglobulin Fc region is preferably set forth in SEQ ID NO: 38, 40, 42, 44 or 46, respectively.

The nucleic acid sequence encoding the recombinant immunoglobulin Fc region is provided by operably linked to a vector capable of expressing it.

As used herein, the term "vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express a gene insert.

In the present invention, "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. It can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be facilitated using enzymes generally known in the art, etc. Suitable expression vectors include promoters, Expression control element sequences such as codons, stop codons, polyadenylation signals, and enhancers.The initiation codon and stop codons must be functional in the individual when the gene construct is administered and must be in frame with the coding sequence. The general promoter can be either constitutive or inducible. And a selectable marker for selecting a host cell containing the vector, and a replication origin in the case of a replicable expression vector .. In specific embodiments of the present invention, pmSCPFc, pmPSCFc, pmCPSFc, pmCPFc, pMEPKFC1, pMSCKFc1, pMDKTFc1, pMCPAFc1, pMPKSFc1, pMCPPFc1, pMPPCFc, pMPCPFc, pmPPCG2Fc, pmPCPG2Fc, pmCPG2Fc, pmCCVG2Fc and pmCVE2Fc were prepared.

The recombinant expression vector expressing the 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. These prokaryotic cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Or, preferably E. coli. Escherichia coli is Escherichia coli XL-1 Blue, Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH series, Escherichia coli TOP10 and Escherichia coli HB101, more preferably Escherichia coli BL21 (DE3), but is not limited thereto. When E. coli is used as a host cell, since E. coli has no system for linking sugar chains to proteins, the Fc region of immunoglobulins can be produced in a form in which sugar present in the CH2 domain of the native immunoglobulin 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, the production of Fc regions of unglycosylated immunoglobulins and binding to therapeutic proteins can maintain blood levels of therapeutic proteins for long periods of time without inducing the effector functions of undesirable immunoglobulins.

Methods of transforming the vector into prokaryotic cells include any method of introducing nucleic acids into cells, and may be carried out by selecting appropriate standard techniques according to host cells as known in the art. Such methods include, but are not limited to, electroporation, plasma fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation with silicon carbide fibers, PEG, dextran sulfate, lipofectamine, and the like. Do not.

In a specific embodiment of the present invention, BL21 / pmSCPFc (HM11200), BL21 / pmPSCFc (HM11201), BL21 / pmCPSFc (HM11204), BL21 / pmCPAFc (HM11205), BL21 / pMEPKFc1 (HM11206), BL21 / pMSCDFc1 (HM11207), BL21 / pMDKTFc1 (HM11208), BL21 / pMCPAFc1 (HM11209), BL21 / pMPKSFc1 (HM11210), BL21 / pMCPPFc1 (HM11211), BL21 / pMPPCFc1 (HM11212), BL21 / pMPCPFc1 (HM11213) BL21 / H21 / pmPCPG2Fc (HM11215), BL21 / pmCPG2Fc (HM11216) and BL21 / pmCCVG2Fc (HM11217), BL21 / pmCVEG2Fc (HM11218) were prepared.

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 potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts as a person. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, appropriate precursors, and the like can 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) is injected into the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. It is also possible to use fermenters. When producing proteins using fermenters, several factors, such as the growth rate of host cells and the amount of expression products, must be taken into account. In appropriate culture conditions, IPTG may be administered to induce the expression of proteins.

Immunoglobulin Fc regions that are overexpressed in aggregate form can be purified in a conventional manner. The immunoglobulin Fc region produced from the transformant was crushed by using a French press, an ultrasonic mill, or the like. After separating only the insoluble aggregate containing the immunoglobulin Fc region using a centrifuge, the fractions were dissolved and By denaturation and refolding with refolding agents such as urea, guanidine, arginine cysteine, beta-mercaptoethanol and the like, or techniques such as column chromatography and ultrafiltration, such as dialysis, gel filtration, ion exchange, reverse phase column chromatography, etc. It can be applied to obtain a protein encoding the immunoglobulin constant region of the present invention. This refolding process is a relatively complex process, and the refolding efficiency of the protein is known to be very low and the activity of the protein after refolding is lower than that of the soluble protein.

However, by using the method of the present invention, it is possible to prepare a large amount of the active type Fc immunoglobulin from which the starting methionine is removed from the aggregate by overcoming the above point. When E. coli expresses and produces a heterologous protein, an initiation codon, methionine, is added, and a methionine-added protein product may cause an immune response upon repeated or overdose in the human body for the purpose of treatment, thereby reducing its efficacy and causing toxicity. However, when the recombinant immunoglobulin Fc region of the present invention is expressed in E. coli, the N-terminal cleavage of the methionine residue modified by the initiation codon by an enzyme called aminopeptidase, one of the enzymes originally present in the cytoplasm of E. coli Sequence analysis showed that (Adams et al., J. Mol. Biol. 33: 571-589, 1968, Takeda, Proc. Natl. Acad. Sci. USA 60: 1487-1494, 1968) activity of these aminopeptides Is dependent on the amino acid sequence and structure of the protein (Moerschell et al., J. Biol. Chem. 265: 19638-19643, 1990, James et al., Protein Expression and Purification 41: 45-52, 2005). When bound to this immunoglobulin Fc region, it can be seen that the starting methionine residue is processed by being affected by aminopeptidase activity, and the degree of processing depends on the amino acid sequence of the hinge region to which it binds.

Since post-translational modification by protease is different depending on the characteristics of the hinge region to be added, the ratio of dimers and monomers can be effectively controlled by appropriately selecting and using the hinge region. In addition, when the aggregates are refolded, the formation of accurate dimers is hindered by mismatch of cysteine when forming disulfide bonds, but the active type dimers can be produced correctly by using the method of the present invention. It is very desirable.

In addition, the immunoglobulin Fc region can be produced at a larger capacity than the existing invention for mass production of immunoglobulin Fc regions. Specifically, in European Patent EP0227110, the immunoglobulin G1 Fc region is overexpressed, and only the product that is expressed in water-soluble state (cell lysate) is used to obtain the immunoglobulin G1 Fc region in 15 mg / L yield. Was expressed in a fused form with the E. coli signal sequence and expressed in a water-soluble form rather than in aggregate to produce 50-600 mg / L yield. However, when the immunoglobulin Fc region is produced from the aggregates using the recombinant immunoglobulin Fc region including the hinge region of the present invention, it can be produced in a yield of 3 to 6 g / L. Therefore, the method for producing the immunoglobulin Fc region of the present invention is a very useful system for producing industrial immunoglobulin Fc region with a much higher yield than the conventional method.

In another aspect, the present invention relates to an immunoglobulin Fc region prepared by the above method.

Industrial application of the immunoglobulin Fc region produced in prokaryotic cells such as Escherichia coli according to the above method is not particularly limited. One exemplary application is to use as a carrier for the formation of a conjugate with any drug. The form of the conjugate in which the immunoglobulin Fc region and the drug are combined is not particularly limited. For example, the immunoglobulin Fc region and the drug can be bound in a variety of blanks, and binding may be mediated by a linker or the like.

Drugs include polypeptides, compounds, extracts, nucleic acids, and the like, but are preferably polypeptides (used in terms equivalent to proteins). The linker includes both a peptidic linker or a nonpeptidic linker, but is preferably a nonpeptidic linker, more preferably a nonpeptidic polymer. A preferred example of an immunoglobulin heavy chain region is Fc.

As the bioactive polypeptide that can be used in combination with the immunoglobulin Fc region prepared by the method of the present invention, any one that needs to increase blood half-life can be used without particular limitation. For example, cytokines, interleukins, interleukin binding proteins, enzymes, antibodies, growth factors, transcriptional regulators, blood factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens used for the purpose of treating or preventing human diseases. And various bioactive polypeptides, such as receptor antagonists, derivatives and analogs thereof.

Specifically, the physiologically active polypeptides include human 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.), colonies Stimulators, interleukins (e.g. interleukin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 , -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, etc.) And interleukin receptors (e.g. IL-1 receptor, IL-4 receptor, etc.), enzymes (e.g. glucocerebrosidase, iduronate-2-sulfatase, alpha Galactosidase-A, agalsidase alpha, beta, alpha-L-iduronidase, butyrylcholinesterase, chitinase , Glutamate decarboxylase, imiglucerase, Lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, etc., interleukin and Cytokine binding proteins (e.g. IL-18bp, TNF-binding protein, etc.), macrophage activators, macrophage peptides, B cell factor, T cell factor, protein A, allergic inhibitor, cell necrosis glycoprotein, immunotoxin, lympho Toxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, high glycated erythropoietin, anji Opoietin, hemoglobin, thrombin, thrombin receptor active peptide, thrombomodulin, blood factor Ⅶ, blood factor 혈액 a, blood factor Ⅷ, blood factor Ⅸ, blood factor XIII, Razminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet derived growth factor , Epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone formation growth factor, bone formation promoting protein, calcitonin, insulin, atriopeptin, cartilage inducer, elcatonin, Connective tissue activators, tissue factor pathway inhibitors, follicle stimulating hormones, luteinizing hormones, luteinizing hormone releasing hormones, nerve growth factors (e.g. nerve growth factor, ciliary neurotrophic factor, Axogenesis factor-1, brain-natriuretic peptide, and glial derived neurotrophic factor trophic factor, netrin, neutrophil inhibitor factor, neurotrophic factor, neuturin, etc., parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenal cortex Hormones, 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 thereto. The physiologically active polypeptides applicable in the present invention may be natural or may be produced by genetic recombination in prokaryotic cells such as Escherichia coli or yeast cells, yeast cells, insect cells or animal cells, and may also have activity equivalent to that of the natural type. And derivatives in which mutations are made at one or more amino acid positions.

In a specific embodiment of the present invention, an EPO-PEG-immunoglobulin Fc region protein conjugate was prepared by binding an immunoglobulin Fc region produced from transformant HM11201 to human erythropoietin (EPO) using polyethylene glycol. It was confirmed that the protein conjugate exhibited better blood half-life than Aranesp (Argensp, Amgen), known as second-generation EPO, which exhibits natural EPO and increased blood half-life. Thus, immunoglobulin Fc regions from which the starting methionine residues generated from aggregates using the hinge region have been removed can be usefully used to increase blood half-life and improve physiological activity of bioactive polypeptides without the risk of eliciting an immune response. It can be seen that.

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> human immunoglobulin IgG4 Fc  Construction of region expression vectors, IgG4 Fc  Expression and Purification of Regions and Identification of N-terminal Sequences

<1-1> IgG4 Fc  Construction of Region Expression Vector

In order to clone the heavy chain Fc region including the hinge region of IgG4, RT-PCR was performed using RNA of blood cells obtained from human blood as a template. First, total RNA is isolated from about 6 ml of blood using the Qiamp RNA Blood Kit (Qiagen), which is then templated and amplified using the One-Step RT-PCR kit (Qiagen). It was. At this time, a primer pair described in SEQ ID NOs: 1 and 2, 3 and 2, 4 and 2, and 5 and 2 was used to amplify genes of different N-terminal sequences. In order to facilitate the subsequent cloning process, the 5'-promoter of SEQ ID NOS: 1, 3, 4 and 5 was inserted with the NdeI restriction enzyme recognition site and ATG, an initiation codon required for protein expression, and the 3'- of SEQ ID NO: 2 In the primer, a BamHI restriction enzyme recognition site including a stop codon was inserted. Fc region products amplified therefrom were digested with NdeI and BamHI, respectively, and then inserted into plasmid pET22b (Novagen) treated with the same restriction enzyme to prepare respective plasmids. Each plasmid was designed to contain the following sequence in Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro, the full amino acid sequence of the IgG4 hinge.

The plasmid containing the genes amplified by SEQ ID NOS: 1 and 2 is pmSCPFc, which encodes a DNA sequence whose N-terminal amino acid sequence begins with methionine-serine-cysteine-proline, and has a nucleotide sequence of SEQ ID NO: 6 as a result of sequencing analysis. It has an amino acid sequence of SEQ ID NO: 7 in expression. The plasmid containing the genes amplified by SEQ ID NOs: 3 and 2 is pmPSCFc, and the N-terminal amino acid sequence encodes a DNA sequence starting with methionine-proline-serine-cysteine-proline, and the nucleotide sequence of SEQ ID NO: 8 is shown by sequencing analysis. It has an amino acid sequence of SEQ ID NO: 9 in expression. The plasmid containing the genes amplified by SEQ ID NOs: 4 and 2 is pmCPSFc, whose N-terminal amino acid sequence encodes a DNA sequence starting with methionine-cysteine-proline-serine-cysteine-proline, and sequence analysis shows SEQ ID NO: It has a base sequence of 10 and has the amino acid sequence of SEQ ID NO: 11 when expressed. The plasmid containing the genes amplified by SEQ ID NOs: 5 and 2 is pmCPFc, and the N-terminal amino acid sequence encodes a DNA sequence starting with methionine-cysteine-proline and has a nucleotide sequence of SEQ ID NO: 12 by sequencing analysis. When expressed it has the amino acid sequence of SEQ ID NO: 13.

The E. coli transformants BL21 / pmSCPFc (HM11200), BL21 / pmPSCFc (HM11201), BL21 / pmCPSFc (HM11204), and BL21 / pmCPFc (HM11205) were prepared by transforming E. coli BL21 (DE3). BL21 / pmSCPFc (HM11200) and BL21 / pmPSCFc (HM11201) were assigned to Korea Microorganism Conservation Center (KCCM) with access numbers KCCM-10659P and KCCM-10660P as of June 20, 2005, respectively, BL21 / pmCPSFc (HM11204) and BL21 /. pmCPFc (HM11205) was deposited on July 28, 2005 under the accession numbers KCCM-10665P and KCCM-10666P, respectively.

<1-2> IgG4 Fc  Expression and Purification of Regions

After inoculating the microbial transformants obtained in Example <1-1> into a fermentor (Marubishi), the expression of immunoglobulin Fc region fragments was confirmed.

First, the transformants were incubated overnight in 100 ml of LB medium, and then inoculated into a fermenter to carry out the main culture. The temperature of the fermenter was maintained at 35 ° C. or 28 ° C., and stirred at 500 rpm while introducing air at 20 vvm to prevent going into an anaerobic state. As fermentation progressed, insufficient energy sources for the growth of microorganisms were administered glucose and yeast extract according to the fermentation state of microorganisms, and when the OD value reached 80 at absorbance of 600 nm expression was induced by administering IPTG, an inducer). This was incubated for 40 to 45 hours and incubated at a high concentration so as to have an OD value of 100 to 120 at an absorbance of 600 nm.

In order to confirm the expression of the immunoglobulin Fc from the E. coli transformants, aggregate formation and dimer formation, the following tests were performed. In order to confirm the total expression in the cytoplasm, a portion of the fermentation broth was mixed in the same amount with 2x protein drop buffer and subjected to electrophoresis on 15% SDS-PAGE (Criterion Gel, Bio-Rad). It was confirmed that globulin Fc was overexpressed. Thereafter, the fermentation broth was crushed using ultra-sonication (Misonix) to obtain cell lysates, and the cell lysates were centrifuged to separate the water-soluble and insoluble materials, and then the water-soluble and insoluble fractions. After electrophoresis on 15% SDS-PAGE, it was confirmed that most of the expressed products exist as insoluble aggregates. The aggregates were refolded in the following manner to confirm the degree of Fc refolding, the presence of dimers, and the degree of formation from the aggregates. First, 10 g of cells are dissolved in 100 mL of lysis buffer (10 mM Tris, pH9.0, 1 mM EDTA, 0.5% Triton X-100, 0.2 M NaCl) for ultra-sonication. After centrifugation at 10,000 rpm for 20 minutes, the aqueous and insoluble fractions were separated, and 2 g of insoluble aggregate was dissolved in 20 mL of 1M Tris (pH9.0) and 20mL of solublization buffer (6M Guanidine, 50mM Tris). React lightly for 30 minutes. After the reaction was completed, the mixture was diluted with 10 times the refolding buffer (2M urea, 50mM Trsi, 0.25M Arginine, 3mM cysteine, pH9.0) at 4 ° C, and reacted by mixing lightly overnight. When the reaction was completed, the mixture was mixed with a protein drop buffer in which a reducing agent such as DTT or beta-mercaptoethanol was removed, followed by electrophoresis on 15% SDS-PAGE (Criterion Gel, Bio-Rad). After the electrophoresis gel was stained with coomassie brilliant dye, the protein band was confirmed. Figure 1 shows the protein bands by refolding the product expressed as aggregates in each transformant and electrophoresed under the above conditions. Lanes 1 and 2 of FIG. 1 refold the aggregates generated at 32 ° C and 28 ° C of transformant HM11201, respectively, and lanes 3 and 4 refold the aggregates generated at 28 ° C and 32 ° C, respectively, of M11200. Lanes 5 and 6 refold the aggregates produced by HM11204 at 28 ° C and 32 ° C respectively, and lanes 7 and 8 refold the aggregates produced by HM11205 at 32 ° C and 28 ° C. Lane C used as a control was Fc purified from E. coli previous researchers. It can be seen that the Fc protein accounts for a large proportion of the total protein, and also forms a dimer after refolding. However, it can be seen that the ratio of dimers and monomers is different due to the difference in the N-terminal amino acid sequence of the Fc protein encoded in the transformant. That is, the Fc protein starting with methionine-proline-serine-cysteine-proline-CH2-CH3, a product of HM11201, exists in a large number of dimers, and the Fc protein starting with methionine-cysteine-proline-CH2-CH3, an expression product of HM11205. It is understood that silver dimers are rarely present and all exist as monomers. This is thought to be a difference in the processing of the aminopeptide proteins of the host cell in E. coli host cells due to differences in the Fc N-terminal sequence.

<1-3> N-terminal sequence identification

In the above embodiment, the Fc expressed in the dimeric form by refolding the aggregate has a sequence different from that of the natural type because methionine, which is a start codon, is added. In order to confirm whether these methionine residues were processed by Escherichia coli protease, N-terminal sequence analysis of the proteins was commissioned at the Seoul Institute of Basic Science Support, and samples to be analyzed were prepared as follows.

First, PVDF membrane (Bio Rad) was activated by soaking in methanol for about 2-3 seconds and sufficiently wetted with blocking buffer (170 mM glycine, 25 mM Tris-HCl (pH 8), 20% methanol). The non-reducing SDS-PAGE gel developed in Example <1-2> was blotted on the PVDF membrane prepared as above using a blotting kit (Hoefer Semi-Dry Transfer unit, Amersham) for about 1 hour. It was. Proteins transferred to PVDF membranes were stained briefly (3-4 seconds) with Coomassie Blue R-250 (Amnesco), a protein dye, and washed with decolorizing solution (water: acetic acid: methanol 5: 1: 4). . N-terminal sequencing was requested by cutting the site containing the protein (dimer and monomer, respectively) from the washed membrane with scissors.

 The IgG4 Fc protein sequence including the hinge is Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys Pro-Ser-Cys-Pro-CH2-CH3 and the amino acid sequence and N-terminal sequence analysis of each transformant The results are shown in the table below.

As a result of the amino acid sequence analysis as described above, it was confirmed that the products forming the dimers of the Fc fragments which are expressed in the aggregate form from the E. coli transformant of the present invention and have the correct N-terminal sequence by methionine processing. Protein products that remain as monomers even after refolding are considered to be unable to form dimers by removing cysteine residues necessary for dimer formation. In addition, as shown in FIG. 1, the ratio of monomers is different for each transformant, and in the case of HM11205, the dimer is not present. Therefore, the amino acid sequence of the N-terminal sequence affects the processing of N-terminal and regulates it from the aggregates. It can be seen that the desired N-terminal sequence can be obtained in the production of the protein. In addition, it can be seen from the following test that even a protein having the same sequence is processed according to the culture conditions of the E. coli host producing the protein, particularly the temperature. In the case of HM11200, when it is cultured at a low temperature (28 ℃-32 ℃), it can be seen that it is in the form of an accepting form as it is in the aggregate. It can be seen that it is present as a monomer. Therefore, the present inventors confirmed that the immunoglobulin Fc of the monomer or the immunoglobulin Fc of the dimer can be obtained by adjusting the amino acid sequence of the N-terminal and the culture conditions of the host cell.

To quantitatively confirm the expression level of the immunoglobulin Fc region expressed from the E. coli transformant Using a protein-A affinity column known to have a strong affinity for the immunoglobulin Fc region in the refolded solution It was purified by the following method.

The aggregate recovered by centrifugation by the above method was purified by column chromatography after refolding. After 5 ml of protein-A affinity column (Pharmacia) was equilibrated with PBS, the cell lysate was loaded at a flow rate of 5 ml / min. Unbound fractions were washed with PBS and eluted with 100 mM citrate solution (pH 3.0). The eluted fractions were exchanged with 10 mM Tris buffer (pH 8.0) using a desalting column HiPrep 26/10, Pharmacia. Secondary anion exchange column chromatography was performed using 50 ml of Q HP 26/10 column (Pharmacia), which was bound to primary purified recombinant immunoglobulin Fc region fractions and then in 10 mM Tris buffer (pH 8.0). Fractions of high purity were obtained by flowing in a linear concentration gradient (sodium chloride concentration from 0 M to 0.2 M). The amount of expression after partial purification of each product through a Protein-A column is shown in Table 2 below.

< Example  2> human immunoglobulin IgG1 Fc  Construction of region expression vectors, IgG1 Fc  Expression and Purification of Regions and Identification of N-terminal Sequences

<2-1> IgG1 Fc  Construction of Region Expression Vector

In order to clone the heavy chain Fc region including the hinge region of IgG1, the same RT-PCR was performed as the method performed for cloning the IgG4 Fc region in Example <1-1>. At this time, primers of the following sequences were used to amplify genes having different N-terminal sequences.

Sequence of 5 'primer used 5 'primer sequence MEPK 5 'GGA ATT CCA TAT GGA GCC CAA ATC TTG TGA CAA AAC TCA C 3' MSCD 5'GGA ATT CCA TAT GTC TTG TGA CAA AAC TCA CAC ATG CCC 3 ' MDKT 5 'GGA ATT CCA TAT GGA CAA AAC TCA CAC ATG CCC ACC GTG C 3' MCPA 5'GGG AAT TCC ATA TGT GCC CAG CAC CTG AAC TCC TGG GG MPKS 5'GGG AAT TCC ATA TGC CCA AAT CTT GTG ACA AAA CTC AC MCPP 5'GGG AAT TCC ATA TGT GCC CAC CGT GCC CAG CAC CTG AAC TCC MPPC 5'GGA ATT CCA TAT GCC ACC GTG CCC AGC ACC TGA ACT CCT G 3 ' MPCP 5'GGA ATT CCA TAT GCC GTG CCC AGC ACC TGA ACT CCT GGG G 3 '

The 3 'primer used 5' CGC GGA TCC TCA TTT ACC CGG AGA CAG GGA GAG GCT CTT C 3 'and used the same for amplification of genes with different N-terminus. In order to facilitate cloning into the vector afterwards, a NdeI recognition site was inserted into the 5 'primer and a BamHI recognition site was inserted into the 3' primer. When amplified with the above primer pairs, the IgG4 Fc region was inserted into the vector used for cloning and expressed. Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys, which is the entire hinge amino acid sequence of IgG1 Fc, was expressed. The following sequence of -Pro-Pro-Cys-Pro is included. The plasmid containing the gene amplified by the MEPK primer was pMEPKFc1, and the N-terminal amino acid sequence encoded DNA sequences of CH2 and CH3 of IgG1, starting with methionine-glutamate-proline-lysine. Has the amino acid sequence of SEQ ID NO: 23 when expressed. The plasmid containing the gene amplified by the MSCD primer is pMSCKFc1, and the N-terminal amino acid sequence starts with methionine-serine-cysteine-aspartate and encodes the DNA sequences of CH2 and CH3 of IgG1. Has the nucleotide sequence of and has the amino acid sequence of SEQ ID NO: 25 at the time of expression. The plasmid containing the gene amplified by the MDKT primer is pMDKTFc1, and the N-terminal amino acid sequence starts with methionine-aspartate-lysine-threonine, and encodes DNA sequences of CH2 and CH3 of IgG1. Has the nucleotide sequence and has the amino acid sequence of SEQ ID NO: 27 at the time of expression. The plasmid containing the gene amplified by the MCPA primer is pMCPAFc1, and the N-terminal amino acid sequence starts with methionine-cysteine-proline and encodes the DNA sequence of CH2 and CH3 of IgG1. Has the amino acid sequence of SEQ ID NO: 29 at the time of expression. The plasmid containing the gene amplified by the MPKS primer was pMPKSFc1, and the N-terminal amino acid sequence started with methionine-proline-lysine-serine, encoding the DNA sequences of CH2 and CH3 of IgG1. Has the nucleotide sequence and, when expressed, has the amino acid sequence of SEQ ID NO: 31. The plasmid containing the gene amplified by the MCPP primer is pMCPPFc1, and the N-terminal amino acid sequence starts with methionine-cysteine-proline-proline and encodes the DNA sequences of CH2 and CH3 of IgG1. Has the amino acid sequence of SEQ ID NO: 33 when expressed. The plasmid containing the gene amplified by the MPPC primer is pMPPCFc and the N-terminal amino acid sequence starts with methionine-proline-proline-cysteine and encodes the DNA sequences of CH2 and CH3 of IgG1, Has the nucleotide sequence and, when expressed, has the amino acid sequence of SEQ ID NO: 35. The plasmid containing the gene amplified by the MPCP primer is pMPCPFc, whose N-terminal amino acid sequence starts with methionine-proline-cysteine-proline and encodes the DNA sequences of CH2 and CH3 of IgG1, and the nucleotide sequence of SEQ ID NO: 36 is shown by sequencing. Has the amino acid sequence of SEQ ID NO: 37 when expressed. The E. coli transformants BL21 / pMEPKFc1 (HM11206), BL21 / pMSCDFc1 (HM11207), BL21 / pMDKTFc1 (HM11208), and BL21 / pMCPAFc1 (HM11209) BL21 / pMPKSF HM11210), BL21 / pMCPPFc1 (HM11211), BL21 / pMPPCFc1 (HM11212) BL21 / pMPCPFc1 (HM11213).

<2-2> IgG1 Fc  Expression and Purification of Regions

Like the IgG4 Fc, the microbial transformants obtained in Example <2-1> were inoculated and fermented in a fermentor (Marubishi Co., Ltd.) to confirm the expression of immunoglobulin Fc region fragments.

First, the transformants were incubated overnight in 100 ml of LB medium, and then inoculated into a fermenter to carry out the main culture. The temperature of the fermenter was maintained at 35 ° C. or 28 ° C., and stirred at 500 rpm while introducing air at 20 vvm to prevent going into an anaerobic state. As fermentation progressed, insufficient energy sources for the growth of microorganisms were administered glucose and yeast extract according to the fermentation state of microorganisms, and when the OD value reached 80 at absorbance of 600 nm expression was induced by administering IPTG, an inducer). This was incubated for 40 to 45 hours and incubated at a high concentration so as to have an OD value of 100 to 120 at an absorbance of 600 nm.

In order to confirm the expression of the immunoglobulin Fc from the E. coli transformants, aggregate formation and dimer formation, the following tests were performed. In order to confirm the total expression in the cytoplasm, the fermentation broth before and after the induction was aliquoted, and SDS-PAGE was performed under the following reducing conditions.

A portion of the fermentation broth was mixed with 2x protein drop buffer in the same amount and electrophoresed in 15% SDS-PAGE (Criterion Gel, Bio-Rad) is shown in FIG. 7. Lane 1 is IgG4 Fc used as a control, lanes 2 to 4 are the hourly expression of HM11208 transformant, lanes 5 to 7 are the hourly expression of HM11206 transformant, lanes 8 to 13 are HM11207, HM11212 and HM11209, respectively. , HM11210, HM11213 and HM11211 transgenic expression is shown. As shown in FIG. 7, a band (Fc region) corresponding to about 30 KDa, which was not seen before introduction, was clearly seen after introduction in all of the samples used in the expression test, and it was confirmed that the recombinant IgG1 Fc was expressed in comparison with the G4Fc control group. It was confirmed that the amount of the expressed Fc region is overexpressed by about 30% or more of the total protein amount.

Method for purifying IgG4 Fc using a protein-A affinity column known to have a strong affinity for immunoglobulin Fc regions in a refolded solution to quantitatively confirm the expression level of the IgG1 Fc regions. Purification in the same manner as

The expression rates of pMDKTFc, pMEPKFc, pMSCDFc, pMPPCFc, and pMPCPFc transformants with confirmed expression were measured at 133.3 mg, 159 mg, 340 mg, 110 mg, and 120 mg per 10 g of aggregate, and the pMSCDFc plasmid transformant showed the highest expression rate.

The content of the dimer of the expression product of the IgG1 Fc was investigated in the same manner as the content of the dimer in the IgG4 Fc of the above example. The fermentation broth was crushed using ultra-sonication (Misonix) to obtain cell lysates, and the cell lysates were centrifuged to separate the water-soluble and insoluble materials, and then the water-soluble and insoluble fractions, respectively. Electrophoresis on 15% SDS-PAGE confirmed that most of the expressed products exist as insoluble aggregates. The aggregates were refolded to confirm the degree of refolding, dimer formation, and the degree of formation of Fc from the aggregates. When the refolding reaction was completed, purified through a Protein A column, mixed with a protein drop buffer, such as DTT or beta-mercaptoethanol, and then subjected to electrophoresis on 15% SDS-PAGE (Criterion Gel, Bio-Rad). After the electrophoresis gel was stained with coomassie brilliant dye, the protein band was confirmed. FIG. 8 shows the protein bands by refolding the products expressed as aggregates in each transformant and electrophoresing under the above conditions.

Lanes 3, 6, and 8 of FIG. 8 are electrophoresed using non-reducing IgG4 Fc as a control group, and lanes 1, 2, 4, 5, and 7 are transformed into HM11208, HM11206, HM11207, HM11212 and HM11213, respectively. The fermented product of the sieve was purified through a Protein A column and electrophoresed under non-reducing conditions. As can be seen in FIG. 8, the IgG1 Fc fragments used in the experiments can be seen to form dimers although there are some differences in the amount.

<2-3> N-terminal sequence identification

In addition, as can be seen in the case of IgG4 Fc, the difference in the N-terminal sequence indicates that the start codon methionine is added, or is cleaved to residues other than the correctly processed or added methionine to have a sequence different from the desired protein sequence. . In the case of IgG1 Fc, IgG1 Fc regions having different N-terminus prepared in the above examples are also N-terminus of IgG4 Fc to confirm that these methionine residues are processed by E. coli protease to maintain the native sequence. In the same way as the sequence was confirmed, the N-terminal sequence analysis of the proteins was commissioned at the Seoul Institute of Basic Science. Each N-terminal sequence analysis results are shown in the table below.

Transformant N-terminal sequence analysis result (dimer) HM11208 Met HM11206 Met HM11207 Ser HM11212 Pro HM11213 Pro

The transformants of HM11208 and HM11206 overexpressed and present as normal dimers exist without the N-terminal methionine residues being processed, but the transformant fermentation products of HM11207, HM11212 and HM11213 are correctly processed with the initiation codon methionine. It was confirmed that there was no N-terminal methionine residue.

Through the experiments described above, the expression of IgG1 Fc region in E. coli differs depending on the presence or absence of expression, amount of expression, formation rate of dimers, N-terminal processing, and the like. It can be seen that the removed Fc region can be obtained in large quantities. The IgG1 Fc regions thus obtained can be usefully used to increase the blood half-life and improve the physiological activity of the physiologically active peptide without the immune response by the addition of foreign amino acids.

< Example  3> human immunoglobulin IgG2 Fc  Construction of Expression Vectors of Regions

<3-1> IgG2 Fc  Construction of Expression Vectors of Regions

In order to clone the heavy chain Fc region including the hinge region of IgG2, the same RT-PCR was performed as in the above example to clone the IgG4 Fc region. In this case, primers of the following sequences were used to amplify genes having different N-terminal sequences.

5 'primer sequence G2MPPCSS 5 'GGG AAT TCC ATA TGC CAC CGT GCC CAG CAC CAC CTG TGG CAG G 3' G2MPCPSS 5 'GGG AAT TCC ATA TGC CGT GCC CAG CAC CAC CTG TGG CAG GAC 3' G2MCPSS 5 'GGG AAT TCC ATA TGT GCC CAG CAC CAC CTG TGG CAG GAC 3' G2MCCVSS 5 'GGG AAT TCC ATA TGT GTT GTG TCG AGT GCC CAC CGT GCC CAG C 3' G2MCVESS 5 'GGG AAT TCC ATA TGT GTG TCG AGT GCC CAC CGT GCC CAG CAC C 3'

The 3 'primer was used as 5' CGC GGA TCC TCA TTT ACC CGG AGA CAG GGA GAG GCT CTT C 3 'and the same gene was used for amplification with different N-terminus. In order to facilitate cloning into the vector afterwards, a NdeI recognition site was inserted into the 5 'primer and a BamHI recognition site was inserted into the 3' primer. When amplified with the above primer pairs, the IgG4 Fc region was inserted into the vector used for cloning and expressed. Glu-Arg-Lys-Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys, which is the entire hinge amino acid sequence of IgG2 Fc, was expressed. -Pro includes the following sequence. The plasmid containing the gene amplified with the G2MPPCSS primer is pmPPCG2Fc, and the N-terminal amino acid sequence starts with methionine-proline-proline-cysteine and encodes the DNA sequences of CH2 and CH3 of IgG2. Has the nucleotide sequence and has the amino acid sequence of SEQ ID NO: 39 at the time of expression. The plasmid containing the gene amplified by the G2MPCPSS primer was pmPCPG2Fc, and the N-terminal amino acid sequence of the DNA sequence of CH2 and CH3 of IgG2, starting with methionine-proline-cysteine-proline, was analyzed by SEQ ID NO: 40. Has the nucleotide sequence and, when expressed, has the amino acid sequence of SEQ ID NO: 41. The plasmid containing the gene amplified with the G2MCPSS primer is pmCPG2Fc, and the N-terminal amino acid sequence starts with methionine-cysteine-proline, and encodes the DNA sequence of CH2 and CH3 of IgG2. Has an amino acid sequence of SEQ ID NO: 43 at the time of expression. The plasmid containing the gene amplified with the G2MCCVSS primer is pmCCVG2Fc and the N-terminal amino acid sequence starts with methionine-cysteine-cysteine-valine-glutamate-cysteine-proline-proline-cysteine-proline, starting with the DNA sequences of CH2 and CH3 of IgG2. It encodes and has the nucleotide sequence of SEQ ID NO: 44 as a result of nucleotide sequence analysis, and has the amino acid sequence of SEQ ID NO: 45 at the time of expression. The plasmid containing the gene amplified with the G2MCVESS primer is pmCVEG2Fc and the N-terminal amino acid sequence encodes the DNA sequences of CH2 and CH3 of IgG2 starting with methionine-cysteine-valine-glutamate-cysteine-proline-proline-cysteine-proline And, as a result of nucleotide sequence analysis, it has the nucleotide sequence of SEQ ID NO: 46, and has the amino acid sequence of SEQ ID NO: 47 at the time of expression. E. coli BL21 (DE3) was transformed into the E. coli transformants BL21 / pmPPCPG2Fc (HM11214), BL21 / pmPCPG2Fc (HM11215), BL21 / pmCPG2Fc (HM11216) and BL21 / pmCCVG2Fc (HM11217), BL2FpmC. (HM11218) was prepared.

<3-2> Expression and Purification, N-terminal Sequence Identification

After inoculating the microbial transformants obtained in the above example with IgG4 Fc in a fermentor (Marubishi), the expression of immunoglobulin Fc region fragments was confirmed. The expression conditions were not significantly different from the methods performed in the above examples, and it was confirmed by SDS-PAGE electrophoresis of reducing conditions that the expression was induced by a variety of conditions such as temperature, expression medium composition, the concentration of the expression inducer. FIG. 9 shows the same amount of fermentation broth mixed with 2 x protein drop buffer and electrophoresed on 15% SDS-PAGE. Lane 1 is an IgG4 Fc fragment used as a control and lanes 2-6 are HM11214, HM11215, HM11216, HM11217 and HM11218. The expression of the transformants is seen. As can be seen in Figure 9 it can be seen that all five kinds of transformants used in the experiment is overexpressed. In order to examine the dimer content of each expression product, the cells were crushed in the same manner as in the above example, and after refolding the insoluble fraction, only the Fc fragment region was purified through the Protein A column. Purified individual expression products were electrophoresed on 15% SDS-PAGE (Criterion Gel, Bio-Rad) after mixing with protein drop buffer which had been reduced with reducing agents such as DTT or beta-mercaptoethanol. After the electrophoresis gel was stained with coomassie brilliant dye, the protein band was confirmed. 10 shows the results of electrophoresis, lanes 1 and 7 are IgG4 Fc fragments used as controls, and lanes 2-6 represent dimers of HM11214, HM11215, HM11216, HM11217 and HM11218 transformants, respectively. As can be seen in Figure 10, the products expressed from each transformant showed a slight difference depending on the difference in the N-terminal sequence or the expression conditions, but it can be seen that forms a dimer. The second amino acid residue was obtained by requesting the N-terminal sequence of the dimer to the Seoul Institute of Basic Science in the same manner as previously performed to confirm whether the starting methionine residue was processed in the N-terminal sequence of the dimers thus formed. It was confirmed that the products of the HM11214 and HM11215 transformants having sequences starting with proline are removed from the starting methionine residue. Through the above experiments, IgG2 Fc regions can also be expressed in E. coli in a large amount, and there are differences in the presence or absence of expression, the amount of expression, the formation rate of dimers, and the N-terminal processing by the difference of N-terminal sequence. It can be seen that a large amount of the Fc region from which the starting methionine residue has been removed can be obtained. The IgG2 Fc regions thus obtained can be usefully used to increase the blood half-life and improve the physiological activity of the physiologically active peptide without the immune response by the addition of foreign amino acids.

< Example  4> By activating enzyme immunoassay C1q Binding capacity  Confirm

In order to confirm whether the immunoglobulin Fc region proteins expressed and purified from the E. coli transformants in Example <1-2> bind human C1q, an activator immunoassay assay (ELISA) was performed as follows. The immunoglobulin Fc region samples produced from the transformants HM11200 and HM11201 were compared with the immunoglobulin (IVIGG-globulin S, green cross PBM) to which the sugar was bound as a control group in a concentration of 1 μg / ml in 10 mM carbonate buffer (pH 9.6). It was prepared. Prepared samples were dispensed in 96-well plates (Nunc) at an amount of 200 ng per well and coated overnight at 4 ° C., and the well plates were then coated with PBS-T solution (137 mM NaCl, 2 mM KCl, 10 mM Na ₂ HPO _4). , 2 mM KH ₂ PO ₄ , 0.05% Tween 20). 250 μl of blocking buffer prepared by dissolving bovine serum albumin in PBS-T solution at a concentration of 1% was added to each well, then left at room temperature for 1 hour and washed three times with the same PBS-T solution. After diluting the standard solution and the sample with an appropriate concentration of PBS-T solution, it was added dropwise to the antibody-coated wells and allowed to react at room temperature for 1 hour and then washed three times with PBS-T solution. After addition of 2 μg / ml C1q (R & D systems) to the plate, the reaction was performed at room temperature for 2 hours, and the plate was washed 6 times with the PBS-T solution. Human anti-human C1q antibody-peroxidase conjugate (Biogenesis, USA) was diluted 1000: 1 in blocking buffer, and 200 μl was added to each well and allowed to react at room temperature for 1 hour. After completion of the reaction, each well was washed three times with PBS-T solution, followed by color solution A and B (Color A-Stabilized peroxide solution and color B-stabilized chromogen [Color B- stabilized chromogen] solution, DY 999, R & D Systems, Inc. was mixed in the same amount, and 200 μl was added to each well and left for 30 minutes. Thereafter, 50 µl of 2 M sulfuric acid, the reaction terminating solution, was added to stop the reaction. The reaction well plate was measured by using a microplate reader (Molecular Device), the absorbance of the standard solution and the sample at 450 nm wavelength, the results are shown in FIG.

As shown in Figure 2, the immunoglobulin Fc region proteins produced in Escherichia coli according to the present invention shows a markedly lower binding ability to C1q compared to the presence of sugar, so as a carrier when forming a binding protein with a physiologically active polypeptide. Even when used and administered to the human body can be seen that the risk of causing an immune response, such as cytotoxicity, inflammation is very low.

< Example  5> Human by enzyme activity immunoassay Fc γ RI , Rc γ RIII , FcRn αβ ₂ Of Binding capacity  Confirm

Immunoglobulin Fc binds to FcγRI and RcγRIII receptors for blood cells in vivo It is known to perform effector functions such as antibody dependent cytotoxicity. In order to determine whether the immunoglobulin Fc produced in E. coli also causes such effector function, the binding capacity was confirmed by activating enzyme immunoassay after obtaining each receptor. In addition, tests were performed in the same manner to confirm the binding ability with FcRn receptors known to affect the in vivo metabolism of immunoglobulins.

<5-1> human Fc γ RI , Rc γ RIII , FcRn αβ ₂  Establishment of Expression Cell Lines

Whole ribonucleotides were isolated from human peripheral blood monocytes using a kit (Quiagen, Cat. No.), Followed by reverse transcription and polymerization of the extracellular ligand binding portion of human FcγRI, RcγRIII, and FcRnαβ ₂ . Gene sequences were obtained and linked to the GST (glutathione S-transferase) gene and cloned into an animal cell expression vector containing a dihydrofolate reductase expression gene. In order to transform Chinese hamster ovary cells with the prepared pHM000 plasmid, 1 × 10 ⁶ cells were inoculated in a ⁶ cm culture dish and incubated in a 37 ° C., 5% CO ₂ incubator for 24 hours, followed by Opti-MEM (Gibco., Cat no. 31985-070). 1ml of LipofectamineTM Reagent (Invitrogen, Cat. No. 18324-020) was mixed with 1ml Opti-MEM containing 10ug of pHM000 plasmid and reacted at room temperature for 20 minutes, and then evenly put into prepared Chinese hamster ovary cells, and 18 hours After incubation in a 37 ° C., 5% CO ₂ incubator, it was further replaced with DMEM / F12 culture medium containing 10% fetal calf serum and 1% penicillin-streptomycin and cultured for 48 hours. Α- containing 10% dialyzed fetal bovine serum, 1% penicillin-streptomycin, and 800 ug / ml geneticin (Mediatech, Cat. No. 61-234RG) for selection of transformed cell lines. Cell lines obtained through 0.5% trypsin (trypsin, Gibco., Cat. No. 15400-054) treatment and centrifugation in MEM (Welgene, Cat. No.LM008-02) selection medium were transferred to T25 culture vessel (Nunc). After inoculation, the cells were cultured at 37 ° C. in a 5% CO ₂ incubator, and cultured so that the transformed and selected cell lines were 90% or more in the culture vessel. To increase the amount of FcγRI, RcγRIII, and FcRnαβ ₂ expression in selected cell lines, methotrexate (MTX, Sigma, Cat. No.M-8407) was increased every 20 weeks from 20nM to 20nM in 37 ° C, 5% CO ₂ thermostat. Incubated at.

<5-2> human Fc γ RI , Rc γ RIII , FcRn αβ ₂ Production and refining

Inoculate 3.5 × 10 ⁸ cells per factory at Cell Factory (Nunc, Cat. No. 170009) to obtain FcγRI, RcγRIII, and FcRnαβ ₂ , and incubate for 48 hours at 37 ° C., 5% CO ₂ incubator, and factory with PBS. After washing twice with 1 liter of water, add 1 liter of CHO-A-SFM, a production medium containing 0.3mM sodium butyrate (Sigma, Cat. No.B-5887), 33 ℃, 5% CO ₂ The culture supernatant was recovered for 7 times every 2 days while incubating in a thermostat. The recovered supernatant was centrifuged, filtered through a 0.22um filtration device (Corning), concentrated with a concentrator (PALL, Cat. No. PN OS010C70), and then chelating sepharose FF resin (Amersharm pharmacia, Cat. No. 17-0575). G02 of FcγRI, RcγRIII, FcRnαβ ₂ was allowed to bind to nickel by flowing a column bound to 0.1 M nickel sulfide (Sigma, Cat. No. N4887). Bound FcγRI, RcγRIII, FcRnαβ ₂ were purified from the column using 50 mM NaPi (pH 8.0), 300 mM NaCl, 250 mM imidazole.

<5-3> Fc γ RI  Of Binding capacity  Confirm

The FcγRI purified in Example <6-2> was diluted with PBS (pH 7.4) at a concentration of 0.75 ug / ml, and then dispensed by 100ul per well into a 96-well plate (Nunc, Maxisorp) at 4 ° C. in a cold state. The reaction was allowed to bind for 18 hours to the bottom of the well. Dispense a 96-well plate in PBS (pH 7.4) and wash three times with 300 ul per well of flushing buffer with 0.05% Tween-20 (Amresco, Cat. No. 0777)). In order to prevent the binding of substances, analytical buffer solution containing 0.1% Tween-20 and 3% BSA (bovine serum albumin, Amresco, Cat. No. 0332) was added to PBS (pH 7.4) at 300 ul per well. The reaction was continued for 1 hour in a thermostat and the reaction solution was removed from the wells. HC11200 and HM11201 products purified in Example 2, and Fc separated by papain treatment of human-derived serum IgG and serum IgG with sugar chains as a control were diluted to 9 ug / ml in assay buffer, and then analyzed again. Diluted to 7 steps of 1/3 each with a buffer solution, and dispensed 100ul per well to a 96-well plate and reacted for 2 hours with constant shaking at 25 ℃, and washed with a wash buffer six times. HM11200 and HM11201 products bound to FcγRI at the bottom of the well, and antibodies to the goat-derived human antibody heavy chain (Chemicon, AP309P) with HRP attached to identify these controls in assay buffer 1 Dilute with 10000 / well and dispense 100ul per well, shake at 25 ℃ for 2 hours, wash with water buffer 6 times, and react with HRP attached to the antibody (BD bioscience, Cat no.555214) was added to 100ul per well and reacted at 25 ° C for 20 minutes. After the reaction, the reaction stop solution of 2N sulfuric acid solution was added to stop the reaction. The color development was measured at 450 nm using an ELISA reader (Molecular Devices, microplate reader). As can be seen in Figure 3 Fc produced in Escherichia coli hardly binds to FcγRI, it can be seen that in the case of human-derived IgG and Fc with a sugar chain strongly bind.

<5-4> Fc γ RIII Binding capacity  Confirm

Products of HM11200 and HM11201 purified in Example <1-2>, and Fc separated by papain treatment of serum IgG and serum IgG derived from humans with sugar chain as a control group at a concentration of 9 ug / ml After diluting with (pH 9.0), diluting with 1/3 steps of carbonate buffer solution 7 times and dispensing 100ul per well to 96-well plate (Nunc, Maxisorp) and reacting for 4 hours at 4 ℃ refrigerated state It was bound to. The 96-well plate was washed three times by dispensing 300 ul of washing buffer with PBS (pH 7.4) and 0.05% Tween-20 (Amresco, Cat. No. 0777)) and washing three times. In order to prevent the binding of PBS (pH 7.4), 0.05% Tween-20 and 5% skim milk powder (Difco, Cat. No. 232100) were added to the assay buffer solution at 300ul per well for 1 hour in a 37 ℃ thermostat. The reaction was removed from the wells. The FcγRIII purified in Example 4-2 was diluted in the assay solution at a concentration of 1 ug / ml, and 100ul per well was dispensed into a 96-well plate and reacted for 2 hours with constant shaking at 25 ° C. After washing, the rabbit-derived anti-GST antibody (Chemicon, AB3282) capable of binding to HM11200 and HM11201 products and FcγRIII GST (glutathion s-transferase) bound to the control was assayed at 1/10000 in the assay buffer. After diluting, aliquot 100ul per well and constantly reacting at 25 ° C for 2 hours. After completion of the reaction, the cells were washed six times with a washing buffer and the antibody against the rabbit antibody capable of binding to the antibody was diluted to 1/7500 in the assay buffer and dispensed by 100ul per well.

After the reaction for 2 hours with a constant shaking at 25 ℃, washed six times with a wash buffer solution to put the substrate as in Example <6-4> and the color development was measured by ELISA reader. As can be seen in Figure 4 Fc produced in Escherichia coli hardly binds to FcγRIII, and human-derived IgG with Fc chains can be seen that the strong binding.

<5-5> FcRn αβ ₂ Binding capacity  Confirm

HM11200 and HM11201 products purified in Example <1-2>, and Fc isolated from papain treatment with serum-derived and IgG-derived human serum IgG as a control group at a concentration of 20 ug / ml (pH 9.0) ), Then dilute 7 times with 1/3 steps of carbonate buffer solution and dispense 100ul per well into 96-well plate (Nunc, Maxisorp) and react for 18 hours at 4 ℃ refrigerated to bind to the bottom of well. It was. Dispose of 96-well plates in PBS (pH 7.4) and washing them three times by dispensing 300 ul of washing buffer with 0.05% Tween-20 (Amresco, Cat. No. 0777)) and washing them three times. In order to prevent the binding of PBS (pH 7.4), 0.05% Tween-20 and 0.5% BSA (bovine serum albumin, Amresco, Cat. No. 0332) were added to the assay buffer at 300ul per well. The reaction was carried out for 1 hour, and the reaction solution was removed from the wells. Dilute FcRnαβ ₂ purified in Example 6-2 to a concentration of 3 ug / ml in the assay solution and dispense 100 ul per well into a 96-well plate and react for 2 hours with constant shaking at 25 ° C. After washing six times, rabbit-derived anti-GST antibody (Chemicon, AB3282) capable of binding to HM11200 and HM11201 products and FcRnαβ ₂ GST (glutathion s-transferase) bound to the control group was added to the assay buffer. Diluted to 10000 and dispensed 100ul per well and stirred for 2 hours at constant shaking at 25 ℃. After completion of the reaction, the cells were washed six times with a washing buffer and the antibody against the rabbit antibody capable of binding to the antibody was diluted to 1/7500 in the assay buffer and dispensed by 100ul per well. After the reaction for 2 hours with constant shaking at 25 ℃, washed six times with water-washing buffer solution to put the substrate as in Example 2 and the color development was measured by ELISA reader. As can be seen in Figure 5 Fc produced in E. coli also strongly binds to human-derived native IgG and Fc as in the presence of sugar chains.

< Example  6> Preparation of human erythrocyte production promoter Pharmacokinetics

<6-1> Preparation of Human Red Blood Cell Promoting Factor

To prepare a conjugate of human erythropoietin (EPO), first, an EPO gene was obtained from the blood by RT-PCR, and then cloned into a pBluescript II (Stratagen) vector to prepare a pBlueEP vector. To transfer the EPO gene to the animal cell expression vector pCMV / dhfr- (a vector in which the dhfr gene was inserted into pCDNA3.1 (Invitrogen)), the vector pBlueEP was cut with HindIII and BamHI restriction enzymes, and the fragment containing the EPO gene was cut. After obtaining, it was inserted into the animal cell expression vector treated with the same restriction enzyme to prepare pcmvEP. The pCMV / dhfr- expression vector into which the EPO gene was inserted was transfected into the CHO cell line, which is a protein-expressing cell line, using Lipofectamine (Gibco), and the concentration of MTX was increased to 120 nM in order to increase the expression level. The EPO expression was confirmed at 100 mg or more per liter.

<6-2> human red blood cells Constructor - PEG Connected  Produce

ALD-PEG-ALD (Shearwater), a polyethylene glycol having a molecular weight of 3.4 kDa having an aldehyde reactor at both ends, was used in a 100 mM phosphate buffer in which EPO produced above was dissolved at a concentration of 5 mg / ml. It was added to be 1: 1, 1: 2.5, 1: 5, 1:10 and 1:20. To this was added sodium cyano borohydride (NaCNBH ₃ , Sigma) as a reducing agent to a final concentration of 20 mM, and reacted for 2 hours with gentle stirring at 4 ° C. A size exclusion chromatography with Superdex® (Pharmacia) was used with the reaction mixture to obtain a linker optionally PEG-linked to the amino terminus of EPO and a PEG-to-interferon alpha 1: 1 linkage. Was carried out. EPO-PEG conjugates were purified using 10 mM potassium-phosphate buffer (pH 6.0) as eluent and the dimer by-products with PEG unlinked EPO, unreacted PEG and two EPO were removed. Purified EPO-PEG conjugate was concentrated to 5 mg / ml concentration. From this, it was confirmed that the optimal reaction molar ratio of EPO: PEG having the highest reactivity and low by-products such as dimers was 1: 2.5 to 1: 5.

<6-3> human red blood cells Constructor - PEG With connectors  Recombinant immunoglobulin Fc  Manufacture of conjugates in the region

The EPO-PEG conjugate prepared in Example <6-2> and the immunoglobulin Fc region produced by the HM11201 transformant in Example <1-3> were combined. Specifically, after dissolving the immunoglobulin Fc region fragment (about 53 kDa) prepared in Example <1-3> in 10 mM phosphate buffer, the molar ratio of EPO-PEG conjugate: Fc region fragment was 1: 1, 1, respectively. The reaction was carried out by mixing with EPO-PEG conjugates such that: 2, 1: 4 and 1: 8. The reaction solution was brought to 100 mM phosphate buffer, and NaCNBH ₃ was added as a reducing agent to a final concentration of 20 mM, followed by slow stirring at 4 ° C. for 20 hours. From this, it was confirmed that the optimal reaction molar ratio of the EPO-PEG conjugate: Fc region fragment having the highest reactivity and low by-products such as dimers was 1: 2.

High pressure liquid chromatography was performed to remove unreacted substances and by-products after the binding reaction and to purify the resulting EPO-PEG-immunoglobulin Fc region protein conjugate. The coupling reaction was exchanged with 10 mM Tris buffer (pH 8.0) using a desalted column HiPrep 26/10 (Pharmacia) and then at 50 ml Q HP 26/10 column (Pharmacia) at a flow rate of 8 ml / min. After binding under load, the desired fraction was obtained by a linear concentration gradient (sodium chloride concentration 0 M → 0.2 M). The eluted fractions were rebound to a polyCAT 21.5 × 250 column (polyLC) equilibrated with 10 mM acetic acid buffer (pH 5.2) at a flow rate of 15 ml / min, followed by a linear concentration gradient (sodium chloride concentration 0.1 M → 0.3 M). It was eluted to obtain a high purity fraction.

<6-4> Pharmacokinetics

Three SD rats in each group (Aratsp, Amgen) having increased half-life by increasing the amount of natural EPO and sialic acid obtained in Example <4-3>, and Example <5-3 The EPO-PEG-Fc conjugate (test group) prepared in > The control group was bled 0.5, 1, 2, 4, 6, 12, 24 and 48 hours after injection, and the test group was 1, 12, 24, 30, 48, 72, 96, 120, 144, 168, 192 after injection. Blood was collected after 240, 288, 336 and 384 hours. Blood samples were collected and coagulated in a 1.5 ml tube and centrifuged for 10 minutes in an Eppendorf high speed microcentrifuge to remove blood cells. The amount of protein in the serum was measured by ELISA method using an antibody against EPO.

Table 6 and FIG. 6 show blood half-lives of each native protein and binding protein. The binding protein EPO-PEG-Fc conjugate prepared using the immunoglobulin Fc region produced according to the present invention as a carrier was expressed in the native EPO. It showed a significantly increased blood half-life compared to Aranesp known as second-generation EPO, which shows an increased blood half-life.

As described above, the recombinant immunoglobulin Fc region comprising the hinge region of the present invention can be used to produce a large amount of immunoglobulin Fc in aggregate form in E. coli, and the produced immunoglobulin Fc region binds to a bioactive peptide. Can be usefully used to increase blood half-life and improve physiological activity of bioactive polypeptides without the risk of eliciting an immune response.

Attach sequence list electronic file  

Claims (15)

Preparing a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region whose N-terminus comprises a fragment of an immunoglobulin hinge starting with cysteine, serine or proline; Transforming the vector into prokaryotic cells; Culturing the transformant; And separating and purifying the immunoglobulin Fc region expressed in aggregate form from the transformant, wherein the mass production of the immunoglobulin Fc region from which the starting methionine residue has been removed. The method of claim 1, which is an immunoglobulin Fc region in monomeric or dimeric form. The method of claim 1 wherein the hinge region is a fragment having two or more consecutive amino acid sequences derived from the hinge region of IgG, IgA, IgM, IgE or IgD. delete The method of claim 3, wherein the IgG is IgG1, IgG2, IgG3 or IgG4. The method of claim 5, wherein the hinge region has a sequence of SEQ ID NOs: 18, 19, 20, 21, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. The method of claim 1, wherein the immunoglobulin Fc region is selected from the group consisting of Fc regions that are IgG, IgA, IgM, IgE, IgD, combinations thereof, and hybrids thereof. 8. The method of claim 7, wherein the IgG is selected from the group consisting of Fc regions that are IgGl, IgG2, IgG3, IgG4, and combinations thereof. The method of claim 1, wherein the immunoglobulin Fc region consists of one to three domains selected from the group consisting of CH2, CH3 and CH4 domains. The method of claim 1, wherein the recombinant immunoglobulin Fc region has the amino acid sequence of SEQ ID NO: 7, 9, 11, 13, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47. The vector of claim 1, wherein the vector encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47 Method that is a vector comprising a nucleic acid sequence. The method of claim 1, wherein the prokaryotic cell is Escherichia coli. The amino acid sequence of claim 1, wherein the transformant is selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47. Transformed with a vector comprising a nucleic acid sequence encoding. delete The method of claim 13, wherein the transformant is selected from the group consisting of accession numbers KCCM-10659P, KCCM-10660P, KCCM-10665P, and KCCM-10666P.

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