RU2800558C1 - IGG4 Fc FRAGMENT CONTAINING A MODIFIED HINGE REGION - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a modified IgG4 Fc fragment having a low risk of inducing chain exchange and monomer formation in vivo, as well as to a nucleic acid encoding it. A vector, a host cell containing the above nucleic acid, and a method of obtaining an IgG4 Fc fragment using the above cell are disclosed. The invention also relates to a medicinal product conjugate containing the above IgG4 Fc fragment conjugated to the medicinal product using a linker.

EFFECT: effective minimization of the effector function of IgG4 Fc and the exchange of chains with IgG in vivo, preservation of the activity of the medicinal product conjugate in vivo and improvement of its prolonged release in vivo.

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Description

Field of invention

The present invention relates to an IgG4 Fc fragment useful as a drug carrier, and more particularly to an IgG4 Fc fragment which can minimize effector functions by Fc but does not induce chain exchange with IgG in vivo and can improve the half-life of the conjugated drug. means in vivo .

Prior Art

The development of genetic engineering technologies has led to the manufacture and use of various types of protein drugs. However, protein drugs have critical problems in that they are easily denatured or easily degraded in vivo by proteases and thus cannot maintain their in vivo concentrations or titers over a long period of time . Therefore, it is very important to maintain the concentrations of protein drugs in the blood and in vivo at an appropriate level by increasing the stability of the protein in order to ensure effective treatment of patients while reducing the burden of patients to frequently inject proteins, etc. and cost reduction.

Accordingly, various attempts have been made for a long time to improve the stability of protein drugs in vivo by changing the type of protein drug, fusion with other proteins, or attaching a suitable polymer to the surfaces of proteins by chemical or biological methods.

One attempt to improve the stability of a protein by fusion with other proteins is to fuse an immunoglobulin Fc fragment to a protein.

The Fc region is responsible for effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), in addition to antigen-binding capacity, which is the main function of immunoglobulins. In addition, the FcRn sequence present in the Fc region plays a role in regulating serum IgG levels by increasing the in vivo half-life when conjugated to the FcRn receptor in vivo . In this regard, active research has been carried out to improve therapeutic proteins through Fc-region fusions with therapeutic proteins.

However, Fc fusion proteins produced by genetic recombination have disadvantages in that protein fusion is possible only in a specific region of the Fc region, i.e. with the amino or carboxy terminus, and only between glycosylated proteins or between aglycosylated proteins. proteins, but not between a glycosylated protein and an aglycosylated protein. In addition, Fc fusion proteins produced by genetic recombination have problems in that an immune response may occur due to the new amino acid sequence created by the fusion, as well as a possible increase in the sensitivity of the proteinase to the linker region.

In addition, Fc fusion proteins have an increased serum half-life of the target protein, but at the same time they have the problem of exhibiting the effector functions that the Fc region has (US Pat. No. 5,349,053). Through the effector functions of the Fc region, fusion proteins can fix complements or bind to FcR expressing cells to kill specific cells and induce the production and secretion of various cytokines that cause inflammation, thereby causing inflammation. In addition, the protein sequences at the confluence regions are novel protein sequences that are not present in the human body and thus have various disadvantages, including the possible induction of an immune response in case of long-term administration.

Accordingly, research has focused on the use of immunoglobulins or immunoglobulin fragments in which effector functions have been removed while maintaining serum half-life. Cole et al . previously reported that ADCC activity was inhibited by replacing residues 234, 235, and 237 with alanine in the CH2 domain, which is known to play an important role in binding to Fc receptors, to produce Fc derivatives with reduced affinities for Fc receptors (Cole et al. , J. Immunol . 159: 3613-3621, 1997). However, they all have mismatched amino acids that differ from the amino acids in the natural human Fc region, and may thus have higher immunity or antigenicity, and the preferred Fc functions may be lost.

As a method for removing or reducing unwanted effector functions while maintaining a high concentration of immunoglobulins in the blood, a method for removing saccharides in immunoglobulins has been studied. In US Pat. FcRn without changing their serum half-life. However, this method also has the problem that they can be recognized as foreign substances and, as such, rejected by the immune system due to the production of a new recombinant construct.

When preparing fusion proteins using native IgG Fc sequences, IgG4 Fc can be selected to minimize Fc effector functions. It is known that IgG4 has a similar in vivo half-life to IgG1, but has relatively small effector functions due to the difference in amino acid sequence. However, despite the advantages of IgG4 having reduced effector functions, in vivo chain exchange can occur between IgG4 due to its specific hinge sequence and thus many difficulties have been reported in the use of fusion proteins for therapeutic purposes (van der Neut Kolfschoten , et al ., Science, 317:1554-1557, 2007). That is, there is a problem that when using IgG4 Fc as a carrier for the fusion protein, there is an exchange of chains with IgG4 present in vivo , resulting in a hybrid with native IgG4, or it may be present in the form of monomers, thereby changing the original structure and have a structure with low therapeutic activity. A common problem is whether a fusion product between an IgG4 Fc fragment and a physiologically active material can be obtained through genetic engineering or in vitro .

Description of the invention

Technical problem

Under these conditions, the present inventors, as a result of research into the development of an IgG Fc fragment capable of acting as a drug carrier with a low risk of inducing Fab region exchange reaction with IgG in vivo and effector functions, but able to overcome the disadvantages of fusion technology through genetic recombination, found that a drug conjugate with increased longevity but without the risk of inducing Fab-IgG exchange reaction in vivo and effector functions can be formed when the hinge sequence of an IgG4 Fc fragment that is mutated to have only one cysteine residue, was produced in E. coli and was conjugated with a drug, thereby carrying out the present invention.

Technical solution

The aim of the present invention is to provide an IgG4 Fc fragment that has a low risk of inducing IgG chain exchange reaction in vivo or effector functions and can act as a drug carrier. More specifically, it is an object of the present invention to provide a modified IgG4 Fc fragment comprising a modified hinge region where a portion of the hinge sequence is removed so that only one cysteine residue is contained.

Another object of the present invention is to provide a nucleic acid that encodes a modified IgG4 Fc fragment comprising a modified hinge region, where part of the hinge sequence is deleted so that only one cysteine residue is contained.

Yet another object of the present invention is to provide a vector comprising a nucleic acid that encodes a modified IgG4 Fc fragment comprising a modified hinge region, where part of the hinge sequence is deleted so that only one cysteine residue is contained.

Another object of the present invention is to provide a microorganism with an introduced vector comprising a nucleic acid that encodes a modified IgG4 Fc fragment comprising a modified hinge region, where part of the hinge sequence is deleted so that only one cysteine residue is contained.

Yet another object of the present invention is to provide a method for producing a modified IgG4 Fc fragment, comprising culturing a microorganism into which a vector comprising a nucleic acid encoding a modified IgG4 Fc fragment is introduced.

Yet another object of the present invention is to provide a drug conjugate wherein the drug and a modified IgG4 Fc fragment are conjugated with a linker.

Another object of the present invention is to provide a pharmaceutical composition comprising a drug conjugate, wherein the drug and a modified IgG4 Fc fragment are conjugated with a linker.

Useful effects of the invention

The present invention can provide a modified IgG4 Fc fragment that has a minimized effector function without substitution or addition of amino acids or addition of glycans, and also does not have a chain exchange reaction with IgG4 in vivo . The modified IgG4 Fc fragment of the present invention, regardless of its drug conjugation method such as genetic engineering method and in vitro covalent method, can inhibit the in vivo chain exchange reaction of the conjugated drug when it is conjugated to the drug, and can way to provide a significant therapeutic superiority compared with the native Fc fragment of IgG4.

BRIEF DESCRIPTION OF GRAPHICS

On FIG. 1 shows the presence/absence of a human IgG4 chain exchange between biotinylated hIgG4 and a human granulocyte colony stimulating factor-PEG (polyethylene glycol)-IgG4 Fc fragment conjugate in rat blood.

On FIG. 2 shows the presence/absence of a human IgG4 chain exchange between biotinylated hIgG4 and a human granulocyte colony stimulating factor-PEG (polyethylene glycol)-IgG4 Fc fragment conjugate in human blood.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the present invention will be described below in more detail with reference to the accompanying drawings. The present invention may, however, be embodied in various forms and should not be construed as being limited to the embodiments set forth in this specification. Rather, these embodiments are presented so that this description is complete and comprehensive and fully indicates the scope of the present invention to those skilled in the art.

In one aspect, in order to achieve the above objects, the present invention provides a modified IgG4 Fc fragment useful as a drug carrier. More specifically, the present invention provides a modified IgG4 Fc fragment comprising a modified hinge region where a portion of the hinge sequence is removed so that only one cysteine residue is contained.

The modified IgG4 Fc fragment of the present invention includes a modified hinge region where the portion of the hinge region represented by the following amino acid sequence has been removed so that only one cysteine residue is contained:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 1).

The inventors of the present invention, seeking to solve the problem of the Fc region of IgG4 having low utility due to the chain exchange reaction with IgG4 in vivo despite its usefulness as a carrier for increasing the half-life of drugs, found that the chain exchange reaction in vivo and the formation no monomer occurs when the IgG4 Fc fragment is modified by removing, by deletion, one of the two cysteine residues present in the IgG4 Fc hinge region as well as part of the hinge region, thereby confirming that the modified IgG4 Fc fragment may be effectively used as a drug carrier.

In one embodiment, an IgG4 Fc fragment of the present invention may include a hinge region that is modified by deletion of 1-8 amino acids, including the Cys residue at position 8 of the amino acid sequence of SEQ ID NO: 1.

In addition, in one embodiment, the IgG4 Fc fragment of the present invention may include a hinge region that is modified by deletion of 1-8 amino acids, including the Cys residue at position 11 of the amino acid sequence of SEQ ID NO: 1.

Alternatively, in one embodiment, an IgG4 Fc fragment of the present invention may include a hinge region that is modified by deletion of 1-5 amino acids, including the Cys residue at position 8, or 1-5 amino acids, including the Cys residue at position 11 of the amino acid sequences SEQ ID NO: 1.

Alternatively, in one embodiment, an IgG4 Fc fragment of the present invention may include a hinge region that is modified by deletion of 1-3 amino acids, including the Cys residue at position 8, or 1-3 amino acids, including the Cys residue at position 11 of the amino acid sequences SEQ ID NO: 1.

Amino acid residues removed as above may be continuous or discontinuous.

The hinge Fc region of the IgG4 fragment of the present invention is characterized in that it is modified to include only one cysteine residue between the two cysteine residues at the 8th and 11th positions of the amino acid sequence of SEQ ID NO: 1, and so that both of these two cysteine residues were not removed.

The hinge region modified to include only one cysteine residue of the two cysteine residues in the hinge region affects without chain exchangein vivo, monomer formation, etc. on a modified IgG4 Fc fragment.

As used herein, the term “carrier” refers to a substance that is conjugated to a drug and, when conjugated to the drug, typically enhances or eliminates the physiological activities of the drug. However, the carrier of the present invention increases the in vivo stability of the drug while minimizing the decrease in the physiological activity of the drug, and the carrier of the present invention is characterized in that it does not have any pharmacological effects to counteract the therapeutic activities of the drug conjugated to the carrier, such as apoptosis or complement activation, and binding to a specific protein.

As used herein, the term “IgG4 Fc fragment” refers to heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), excluding heavy and light chain variable regions, heavy chain constant region 1 (CH1) and constant region 1 light chain (CL1) IgG4, but including a modified hinge region in the heavy chain constant region. In addition, an IgG4 Fc fragment of the present invention may refer to an extended Fc region comprising part or all of the heavy chain constant region 1 (CH1) and/or light chain constant region 1 (CL1), excluding the heavy and light chain variable regions. immunoglobulin, since the Fc fragment of IgG4 provides essentially the same effect or an improved effect compared to the effect of the native type.

In addition, the Fc fragment of IgG4 of the present invention includes not only its natural amino acid sequence, but also a derivative of its sequence (mutein). As used herein, the term "IgG4 Fc mutein" refers to an IgG4 Fc fragment having an amino acid sequence that differs from its natural type by a deletion, insertion, conservative substitution, non-conservative substitution of at least one amino acid residue, or a combination thereof in the naturally occurring amino acid sequence in region that does not include its hinge region. In addition, various types of derivatives are possible in which the region capable of forming a disulfide bond has been removed, several amino acids have been removed from the N-terminus of natural Fc, or a methionine residue may have been added to the N-terminus of natural Fc. In addition, a complement binding region, such as a C1q binding region or an ADCC (antibody dependent cellular cytotoxicity) region, can be removed to abolish effector functions. Methods for producing Fc region muteins are disclosed in international patent publications WO 97/34631, WO 96/32478, etc.

The replacement of amino acids in proteins or peptides without a complete change in the activity of the molecules has been previously described (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions 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/Pro , Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly. In some cases, the modification may be by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

Meanwhile, the IgG4 Fc fragment of the present invention can be obtained from humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and preferably from humans.

The IgG4 Fc fragment of the present invention may be a recombinant type of IgG4 Fc fragment in which an Fc region originating from humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc. , was obtained from a microorganism.

In addition, the IgG4 Fc fragment can be in the form of native glycans, glycans with an increased amount compared to the native glycan, or in the form without glycans. The increase, decrease or removal of Fc-glycans in immunoglobulins can be carried out using a conventional method such as a chemical method, an enzymatic method, a genetic engineering method using microorganisms, etc. In particular, since the Fc region of an immunoglobulin where the glycans are removed from the Fc exhibits a significant deterioration in complement binding capacity (c1q) and a reduction or elimination of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, unnecessary in vivo immune responses are not induced. In this regard, a more suitable type of IgG4 Fc fragment, which better suits the original purpose of use as a drug carrier, may be an aglycosylated IgG4 Fc fragment.

As used herein, the term “deglycosylation” refers to an Fc region where saccharides are removed by an enzyme, and “aglycosylation” refers to an aglycosylated Fc fragment produced in a prokaryotic cell, preferably E. coli .

In addition, the IgG4 Fc fragment of the present invention can be modified with a non-peptide polymer. Preferably, the IgG4 Fc fragment of the present invention may be modified with polyethylene glycol. An IgG4 Fc fragment modified with polyethylene glycol can be obtained by reaction with polyethylene glycol at pH 7 or higher, preferably at pH 7.5 - pH 9, and more preferably at pH 8.0.

As used herein, the term “modified hinge region” refers to a hinge region in which any one cysteine residue is removed from the cysteine residues at positions 8 and 11 of the amino acid sequence of SEQ ID NO: 1, which is the hinge sequence of the native Fc-fragment of IgG4 and additionally removed part of the amino acids.

In the present invention, the number of amino acid residues removed in the hinge region may range from 1 to 8, and in particular, the amino acid residues may be contiguous or interrupted. In particular, for example, the modified hinge region of the present invention may include the deletion of only one cysteine residue at the 8th position or at the 11th position; or a deletion of 2 to 8 continuous or discontinuous amino acids, including a cysteine residue in the 8th position; or a deletion of 2-8 continuous or discontinuous amino acids including a cysteine residue at position 11.

The modified hinge region of the present invention may have, for example, at least one amino acid sequence from the amino acid sequences shown below:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro, Glu-Ser-Lys- Tyr-Gly-Pro-Pro-Cys-Pro-Ser, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro, Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys, Glu-Lys-Tyr-Gly-Pro-Pro-Cys, Glu-Ser-Pro-Ser-Cys-Pro, Glu-Pro-Ser-Cys- Pro, Pro-Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro, Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro, Glu- Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys, Lys-Tyr-Gly-Pro-Pro-Cys-Pro, Glu-Ser- Lys-Pro-Ser-Cys-Pro, Glu-Ser-Pro-Ser-Cys-Pro, Glu-Pro-Ser-Cys.

In a typical embodiment of the present invention, in order to investigate the mechanism of chain exchange between IgG4 present in rat blood and human blood and the modified IgG4 Fc fragment and physiologically active protein conjugate of the present invention, rat blood and human blood, respectively, were mixed with an IgG4 Fc conjugate. and physiologically active protein, and samples were collected according to each time zone and subjected to Western blot analysis using antibodies to physiologically active protein. As a result, it was confirmed that molecules that could be generated by chain exchange with rat IgG4 or human IgG4 were not generated.

Accordingly, when the modified IgG4 Fc fragment of the present invention is used as a drug carrier, it can be effectively used to increase the serum half-life of drugs and improve the physiological activity of drugs without in vivo exchange with native immunoglobulins.

According to another aspect, the present invention provides a nucleic acid encoding a modified IgG4 Fc fragment having a hinge region that is mutated to include only one cysteine residue by deleting a portion of the amino acids in the hinge region, as well as a vector comprising the nucleic acid.

The nucleic acid encoding a modified IgG4 Fc fragment of the present invention includes nucleic acids encoding a modified IgG4 Fc fragment comprising the amino acid sequence of SEQ ID NO: 2 (Pro-Ser-Cys-Pro-Ala-Pro-Glu-Phe-Leu -Gly- Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys- Pro-Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr- Pro-Glu-Val-Thr -Cys-Val-Val-Val-Asp-Val- Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-Tyr-Val-Asp-Gly-Val-Glu-Val-His -Asn- Ala-Lys-Thr-Lys-Pro-Arg-Glu-Glu-Gln-Phe- Asn-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu- Thr-Val-Leu-His -Gln-Asp-Trp-Leu-Asn-Gly- Lys-Glu-Tyr-Lys-Cys-Lys-Val-Ser-Asn-Lys- Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr -Ile- Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro- Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys -Asn-Gln-Val-Ser-Leu-Thr- Cys-Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-Ser-Asn-Gly -Gln- Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro- Val-Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu- Tyr-Ser-Arg-Leu -Thr-Val-Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-Met-His-Glu-Ala-Leu-His-Asn-His -Tyr-Thr-Gln-Lys-Ser-Leu-Ser-Leu-Ser-Leu-Gly-Lys). For example, the nucleic acid of the present invention may include the nucleotide sequence of SEQ ID NO: 3

As used herein, the term "vector" refers to a recombinant vector capable of expressing a target protein in a suitable host cell, which is a genetic construct comprising regulatory factors operably linked to allow expression of the gene insert.

As used herein, the term "operably linked" means that a regulatory nucleic acid sequence is operably linked to a nucleic acid sequence encoding a target protein such that essential functions are performed. Functional binding to the vector can be done using genetic recombination techniques well known in the art and site-specific DNA cleavage, and binding can be easily done using enzymes etc. generally well known in the art. . Suitable expression vectors may include expression control sequences such as a promoter, start codon, stop codon, polyadenylation signal, and enhancer. The start codon and the stop codon must essentially have their effect in the subject when the genetic construct is introduced into it, and must be in reading frame with the coding sequence. A typical promoter may be constitutive or inducible. The expression vector may also include a selectable marker for selection of a host cell containing the vector, and for a replicable expression vector, it may include an origin of replication.

In another aspect of the present invention, a microorganism is provided into which the above vector is introduced, capable of producing modified IgG4 Fc fragments.

For the purposes of the present invention, the microorganism is preferably a eukaryotic cell. The eukaryotic cell may be Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus, etc., and preferably Escherichia coli . Escherichia coli can be E. coli XL-1 blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH species, E. coli TOP10 and E. coli HB101 , and more preferably E. coli BL21 (DE3 ), but is not limited to them. When E. coli is used as a host cell, the Fc region of immunoglobulins can be obtained in a form where the saccharides present in the CH2 domain of native immunoglobulins are initially removed, since E. coli does not have a glycan-to-protein conjugation system. Although the saccharides present in the CH2 domain of immunoglobulins do not affect the structural stability of immunoglobulins, it is known that immunoglobulins can bind to cells expressing the Fc receptor and induce antibody-dependent cellular cytotoxicity, induce cytokine secretion by immune cells, thus inducing inflammatory responses, and bind to the C1q element of complements and induce complement-fixing reactions. Accordingly, if the Fc regions of aglycosylated immunoglobulins are formed and conjugated to therapeutic proteins, the serum concentration of therapeutic proteins can be maintained for a long period of time without inducing the effector functions of the immunoglobulins.

The method for transforming prokaryotic cells with the above vector may include any method that can introduce nucleic acids into the cell, and transformation can be performed by selecting a standard technique suitable for a given host cell known in the art. Examples of the method may include electroporation, protoplast fusion, calcium phosphate precipitation (CaPO ₄ ), calcium chloride precipitation (CaCl ₂ ), agitation using silicon carbide fibers, PEG (polyethylene glycol), dextran sulfate, lipofectamine, etc., but not limited to them.

The microorganism introduced with the recombinant expression vector can be cultured in a conventional manner.

The culture process can be used after slight adjustment according to the selected microorganism. As a rule, the medium used for culturing should contain all the nutrients necessary for cell growth and survival. The environment may contain various carbon sources, nitrogen sources and trace elements. Examples of carbon sources may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose; fats such as soybean oil, sunflower oil, castor oil, coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. Such carbon sources may be used singly or in combination. Examples of nitrogen sources may include organic nitrogen sources such as peptone, yeast extract, meat sauce, malt extract, corn extract (CSL) and soybean meal; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources can be used singly or in combination. In the above medium, dihydrogen phosphate, hydrogen phosphate and the corresponding sodium-containing salts can be contained as sources of phosphorus. In addition, metal salts such as magnesium sulfate or iron sulfate may be present. In addition, amino acids, vitamins and corresponding precursors may also be contained. During the culture period, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture, as appropriate. Furthermore, during the culture period, a defoamer such as a polyglycol fatty acid ester may be added to prevent foaming. Additionally, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (eg air) can be introduced into the culture. The temperature of the culture may generally be from 20°C to 45°C and preferably from 25°C to 40°C. In addition, a fermenter may be used. When proteins are produced using a fermenter, various factors must be considered, including the rate of growth and the amount of products expressed by the host cell. Protein expression can be induced by adding IPTG (isopropylthiogalactoside) or the like under suitable culture conditions. The modified IgG4 Fc fragment of the present invention may be overexpressed in the host cell in the form of an aggregate, or may be expressed in an aqueous form. Regardless of the type of expression, proteins can be purified using conventional protein purification methods.

Accordingly, in another aspect of the present invention, there is provided a method for producing a modified IgG4 Fc fragment, and the method comprises culturing a microorganism introduced into it with a nucleic acid encoding a modified IgG4 Fc fragment.

According to the above method, the industrial use of the above IgG4 Fc fragment produced in a prokaryotic cell such as E. coli is not particularly limited. An example of application would be its use as a carrier for the formation of a conjugate with an arbitrary drug.

Accordingly, in another aspect of the present invention, there is provided a drug and a drug conjugate wherein a modified IgG4 Fc fragment is conjugated thereto via a linker.

As used herein, "drug conjugate" or "conjugate" means that at least one drug is combined with at least one of the modified IgG4 Fc fragments.

As used herein, a drug refers to a substance that can exhibit therapeutic activities when administered to humans or animals, and may include, but is not limited to, a polypeptide, compound, extract, nucleic acid, etc. Preferably, the drug is a polypeptide drug.

As used herein, a physiologically active polypeptide drug, a polypeptide drug, and a protein drug should be understood to have the same meaning, and are characterized by being physiologically active types antagonizing various physiological phenomena in vivo .

The type of conjugate in which the IgG4 Fc fragment is conjugated to a drug is not particularly limited, and the IgG4 Fc fragment and the drug can be conjugated in various ratios.

In the present invention, a linker may refer to both a peptide linker and a non-peptide linker, preferably a non-peptide linker, and more preferably a non-peptide polymer.

A non-peptide polymer refers to a biocompatible polymer to which at least two repeat units are conjugated and the repeat units are linked by arbitrary covalent bonds other than peptide bonds. The non-peptidic polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer such as polylactic acid (PLA), and polylactic-glycolic acid copolymer. (PLGA), lipid polymer, chitins, hyaluronic acid and combinations thereof, and preferably polyethylene glycol. Derivatives known in the art, and derivatives that can be easily obtained using techniques known in the art, are also included in the scope of the present invention.

In the present invention, the non-peptide polymer may have two or three reactive end groups, and the reactive end group of the non-peptide polymer is preferably selected from the group consisting of a reactive aldehyde group, a propionic aldehyde group, a butylaldehyde group, a maleimide group, and a succinimide derivative. Examples of the succinimide derivative may include succinimidyl propionate, hydroxyl succinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate. In particular, when the non-peptide polymer contains a reactive aldehyde group at its end as a reactive group, it can minimize non-specific interactions and is effective in its appropriate binding to a physiological polypeptide and an immunoglobulin Fc fragment. The end product produced by reductive alkylation through aldehyde bonding is more stable than that attached through an amide bond. The aldehyde reactive group reacts selectively at the N-terminus at low pH, and at high pH, eg pH 9.0, it can form a covalent bond with the lysine residue.

The terminal reactive groups of the non-peptide polymer may be the same or different from each other. For example, a non-peptide polymer may have a maleimide group at one end, yet have an aldehyde group, a propionaldehyde group, or a butylaldehyde group at the other end. When using polyethylene glycol or a non-peptide polymer having a hydroxyl reactive group at both ends, the conjugate of the present invention can be obtained by activating the hydroxyl group with various reactive groups according to a known chemical reaction, or using polyethylene glycol having a modified reactive group, which is available in sale.

With regard to physiologically active polypeptides for use by binding to a modified IgG4 Fc fragment of the present invention, any of them requiring an increase in serum half-life can be used without limitation. For example, various physiologically active polypeptides can be used, such as cytokines, interleukins, interleukin-binding proteins, enzymes, antibodies, growth factors, transcription regulation factors, blood coagulation factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists, their derivatives and their analogues.

More specifically, physiologically active polypeptides may include human growth hormone; growth hormone releasing hormone; growth hormone releasing peptide; interferons and interferon receptors (e.g. interferon-α, -β and -γ, soluble type I interferon receptor, etc.), colony stimulating factors, 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, α-galactosidase-A, agalsidase α, β, α-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 activating factor, macrophage peptide, B-cell factors, T-cell factors, protein A, allergy inhibitory factors, necrosis glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, transforming growth factors, α-1-antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobins, thrombin , thrombin receptor activating peptide, thrombomodulin, blood coagulation factor VII, blood coagulation factor VIIα, blood coagulation factor VIII, coagulation factor IX, blood coagulation factor XIII, plasminogen activators, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone morphogenetic growth factor, bone morphogenetic protein, calcitonin, insulin and insulin derivative, atriopeptin, cartilage inducing factor, elkatonin, connective tissue activating factor, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors (eg, nerve growth factor, ciliary neurotrophic factor, axogenesis factor-1, brain natriuretic peptide, neurotrophic factor glial cells, netrin, neutrophil inhibitory factor, neurotrophic factors, neurin, etc.), parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, insulinotropic peptides, including glucagon-like peptide-1 and exendin -4, incretins secreted in the gut, adipocytes including leptons and neurocytokines effective for metabolic syndrome, cholecystokinin, pancreatic polypeptides, gastrin-releasing peptides, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, receptors (e.g. TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, B cell activation receptor, etc.), receptor antagonists (e.g. IL1-Ra, etc.), cell surface antigens (e.g. CD 2, 3, 4, 5, 7, 11a, 11b, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal antibodies, polyclonal antibodies, antibody fragments (eg scFv, Fab, Fab', F(ab') ₂ and Fd), viral vaccine antigen, etc., but are not limited to. The physiologically active polypeptide useful in the present invention may be of the naturally occurring type; obtained by genetic recombination in a prokaryotic cell, such as E. coli, or in a eukaryotic cell, such as a yeast cell, an insect cell, or an animal cell; or a derivative which has an activity equivalent to the natural type and a mutation in at least one amino acid position.

The modified IgG4 Fc fragment of the present invention can be produced in a cell after it is coupled to a physiologically active polypeptide as a single gene sequence using a direct genetic recombination method, or an IgG4 Fc fragment can be produced independently and conjugated to a drug such as a physiologically active polypeptide, in vitro .

In yet another aspect of the present invention, there is provided a pharmaceutical composition comprising the above drug conjugate of the present invention as an active ingredient.

The pharmaceutical composition containing the conjugate of the present invention may contain a pharmaceutically acceptable carrier. Examples of a pharmaceutically acceptable carrier may include a binder, lubricant, disintegrant, excipient, solubilizing agent, dispersing agent, stabilizer, suspending agent, coloring agent, flavoring agent, etc.; for injectable compositions, buffering agent, preservative, analgesic, isotonic agent, stabilizer, etc. may be mixed for use; and for topical formulations, a base, excipient, lubricant, preservative, etc. may be used.

A different type of preparation of the pharmaceutical composition of the present invention can be obtained by combination with a pharmaceutically acceptable carrier. For example, for oral administration, the pharmaceutical composition may be formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, cachets, and the like. For injection, the pharmaceutical composition may be formulated in single dose ampoules or in multi-dose containers. The pharmaceutical composition may also be formulated as solutions, suspensions, tablets, capsules and sustained release preparations.

While examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the composition of the present invention may further contain a filler, an anticoagulant, a lubricant, a humectant, a fragrance, an emulsifier, a preservative, and the like.

In the present invention, the actual dose of drugs where the IgG4 Fc fragment is used as a carrier will be determined based on the types of drugs used as active ingredients, along with various factors such as the disease being treated, route of administration, age, sex and patient weight, disease severity, etc. Since the pharmaceutical composition of the present invention has an excellent duration of action in vivo , the number and frequency of administration of the pharmaceutical composition of the present invention can be significantly reduced.

The pharmaceutical composition of the present invention can be administered in various ways.

As used herein, the term “administering” refers to administering a particular substance to a patient in a suitable manner, and the conjugate drug of the present invention may be administered by any of the usual routes, provided that the drug can reach the target tissue. For example, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration may be performed, but the route of administration is not limited thereto. However, since the peptides are digested upon oral administration, the active ingredients of the composition for oral administration must be coated or formulated to protect against degradation in the stomach. Preferably, the present composition may be administered in injectable form. In addition, the pharmaceutical composition can be administered using a specific device capable of transporting the active ingredients into the target cell.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, these Examples are provided for illustrative purposes only and the invention should not be limited to these Examples.

Example 1 Preparation of Human Granulocyte Colony Stimulating Factor-PEG-Immunoglobulin Conjugate

<1-1> Construction of a vector expressing the Fc domain of IgG4

To clone the heavy chain Fc region including the IgG4 hinge region, RT-PCR was performed using blood cells harvested from human blood as a template, as described below. First, total RNA was isolated from blood at about pH 6, and the gene was amplified from the RNA template using the Qiamp RNA blood kit (Qiagen). In particular, a pair of SEQ ID NO: 4 (gggcatatgc catcatgccc agcacctgag ttcctgggg) and SEQ ID NO: 5 (gggggatccc tatttaccca gagacaggga ga) were used as primers. To facilitate the subsequent process, a domain capable of recognizing NdeI and ATG restriction sites, a start codon required for protein expression were inserted into the primers, and a domain capable of recognizing BamHI restriction sites was inserted into the 3-primer of SEQ ID NO: 5. Product with Fc The α-site amplified with them was digested with NdeI and BamHI, respectively, and subcloned into pET22b (Novagen Co., Ltd.) to obtain a plasmid. The plasmid was designed in such a way that the Fc fragment of IgG4 could include a hinge sequence where amino acid residues in the 1-8th positions of the complete amino acid sequence of Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser- Cys-Pro in the IgG4 Fc hinge.

The plasmid obtained in this Example was named “pmHMC001”, and the result of analysis of its sequence showed that the nucleic acid encoding the Fc fragment of IgG4 has the nucleotide sequence of SEQ ID NO: 3 and the Fc fragment of IgG4 has the amino acid sequence of SEQ ID NO: 2 during expression.

The expression vector thus obtained was used to transform E. coli BL21 (DE3), and thereby the E. coli transformant E. coli BL21/pmHMC001 (HMC001) was obtained.

<1-2> Expression and purification of the Fc region of IgG4

The microorganism transformant obtained in Example <1-1> was inoculated into a fermenter (Marubishi Co., Ltd.) for fermentation, and expression of the IgG4 Fc fragment was examined.

First, the above transformants, placed in 100 ml of LB medium, were cultured in a shaking water bath overnight and then inoculated into a fermenter to continue cultivation. The fermenter was maintained at 35° C. or 28° C., and culture was started by shaking at 500 rpm with air blowing there at 20 rpm to prevent anaerobic condition. During the fermentation, energy sources insufficient for the growth of the microorganism were replenished using glucose and yeast extract, according to the fermentation state of the microorganism, and expression was induced by adding the inducer IPTG (isopropylthiogalactoside) when the OD value at 600 nm reached 80. Cultivation was continued for 40- 45 hours until the OD value at 600 nm reaches the range from 100 to 120, obtaining a highly concentrated culture.

Expression of Fc IgG4 in the E. coli transformant was confirmed using the experiment described below.

In order to confirm complete expression of Fc IgG4 in the cytoplasm, a portion of the fermentation liquid was mixed with an equal amount of 2x protein drop buffer and subjected to electrophoresis on 15% SDS-PAGE (Criterion Gel, Bio-Rad). As a result, it was confirmed that Fc IgG was overexpressed in the obtained transformant. The overexpressed protein was shown to form coagulants and the protein was purified by refolding and the coagulants were run through the column in the same manner. First, 10 g of cells were dissolved in 100 ml of lysis buffer (10 mM Tris, pH 9.0, 1 mM EDTA, 0.5% Triton X-100 and 0.2 M NaCl) and then sonicated. The resulting product was subjected to centrifugation at 10,000 rpm for 20 minutes to separate into soluble and insoluble fractions, and 2 g of insoluble coagulant was dissolved in 20 ml of solubilization buffer (6 M guanidine and 50 mM Tris) and then allowed to react for 30 minutes at 4 with gentle shaking. At the end of the reaction, the resulting product was diluted by adding 10 volumes of refolding buffer (2 M urea, 50 mM Tris, 0.25 M arginine and 3 mM cysteine, pH 9.0) and then allowed to react overnight with gentle shaking. Upon completion of the reaction, the sample was provided with fresh buffer 10 mm Tris-HCl (pH 8.0) using Sephadex G25. The buffer exchanged sample was eluted with a gradient of Tris-HCl (pH 8.0) and NaCl using DEAE-FF (GE healthcare) and phenyl-FF (GE healthcare) was eluted with a gradient of ammonium sulfate and 10 mM Tris-HCl (pH 7.5) in order to remove a large amount of multimers and monomers. For subsequent column run, the resulting product was desalted with 10 mM Tris (pH 7.5) using Sephadex G25 (GE healthcare) and then eluted with a concentration gradient of 10 mM Tris to obtain high purity IgG4 Fc, 15Q (GE healthcare). -HCl (pH 7.5) and NaCl, and finally received Fc IgG4 .

<1-3> Preparation of drug conjugate I

1) Obtaining a conjugate of granulocyte colony stimulating factors and PEG

ALD-PEG-ALD (Shearwater Inc., USA), poly(ethylene glycol) having a molecular weight of 3.4 kDa with aldehyde reactive groups at both ends was added to 100 mM phosphate buffer in which granulocyte colony stimulating factors were dissolved at a concentration of 5 mg/ml, so that the molar ratio of granulocyte colony stimulating factors:PEG was 1:5. Sodium cyanoborohydride (NaCNBH ₃ ), a reducing agent, was added to a final concentration of 20 mM and reacted for 3 hours at 4 with slow stirring. To prepare a conjugate in which PEG is selectively conjugated to the amino terminus of granulocyte colony stimulating factors and PEG and granulocyte colony stimulating factors are conjugated in a 1:1 ratio, the reaction mixture was subjected to Superdex gel permeation chromatography (Superdex R, Pharmacia, USA). Colony stimulating factors were purified using 10 mM potassium phosphate buffer (pH 6.0) as elution solution, with granulocyte colony stimulating factors that were not conjugated to PEG, unreacted PEG, and dimer by-products where two granulocyte colony stimulating factors were conjugated with PEG were removed. The purified granulocyte colony stimulating factor-PEG conjugate was concentrated to 5 mg/ml.

2) Formation of a conjugate between a granulocyte colony stimulating factor-PEG conjugate and an IgG4 Fc fragment

The IgG4 Fc fragment of the present invention was dissolved in 100 mM phosphate buffer. For conjugation of the IgG4 Fc fragment with aldehyde reactive groups of the granulocyte colony stimulating factor-PEG conjugate purified above, the granulocyte colony stimulating factor-PEG conjugate was added to the buffer containing the IgG4 Fc fragment, so that the molar ratio of the granulocyte colony stimulating factor conjugate-PEG : Fc fragment I gG4 was 1:5. To this was added a reducing agent, sodium cyanoborohydride (NaCNBH ₃ ), to a final concentration of 20 mM, and the reaction mixture was reacted for 20 hours at 4 with slow stirring. After completion of the conjugation reaction, the unreacted substance and by-products were removed, and the granulocyte colony stimulating factor-PEG-immunoglobulin protein conjugate was purified by anion exchange chromatography. The granulocyte colony stimulating factor-PEG-Fc fragment IgG4 conjugate was purified by adding the above reaction mixture to a DEAE column (Pharmacia, USA) which had been equilibrated with 20 mM Tris buffer (pH 7.5), followed by washing with the same buffer containing 1 M NaCl, using the linear concentration gradient method (NaCl concentration: 0 M → 0.5 M). Cation exchange chromatography was additionally performed to remove a small amount of unreacted immunoglobulins and human growth hormone admixed with a fraction of the granulocyte colony stimulating factor-PEG-Fc fragment IgG4 conjugate thus obtained. The granulocyte colony stimulating factor-PEG-Fc-fragment IgG4 conjugate fraction was applied to a polyCAT column (PolyLC, USA), which was equilibrated with 10 mM sodium acetate (pH 4.5) and further purified by passing 10 mM sodium acetate buffer (pH 4, 5) containing 1 M NaCl by the linear concentration gradient method (NaCl concentration: 0 M → 0.5 M), and thereby the granulocyte colony stimulating factor-PEG-Fc fragment IgG4 conjugate (HM10460A) was obtained pure.

Example 2 Confirmation of chain exchange between human granulocyte colony stimulating factor-PEG-immunoglobulin conjugate and human IgG4 in rat blood

Human IgG4 at 2 mg was labeled with biotin by mixing with a 20 mg/ml solution of biotin-7-NHL in a 1:10 molecular ratio and purified using a Biotin Protein Labeling Kit (Roche). Blood collected from normal rats was treated with heparin to prevent coagulation and penicillin-streptomycin (1% v/v) was added. 1.5 mg of biotin-labeled IgG4 and 1.32 mg of HM10460A were added to 3 ml of blood, mixed, and the mixture was aliquoted into 6 tubes (0.5 ml/tube) and incubated in an incubator at 37°C. One tube was removed at the time points of 0 hours, 4 hours, 10 hours, 24 hours and 48 hours, respectively, and plasma was separated from them and stored at -20°C. Each of the plasma samples and standards were mixed with a non-reducing protein sample buffer, and the resulting product was subjected to SDS-PAGE using a polyacrylamide gel with a concentration gradient of 4%-15%. Biotin labeled IgG4 and HM10460A were used as reference substances. The gel after completion of electrophoresis was blotted on a PVDF (polyvinylidene fluoride) membrane (Immobilon-P, MILLIPORE) and analyzed using antibodies against human GCSF (granulocyte colony stimulating factor) and streptavidin-HRP (horseradish peroxidase). With regard to antibody binding conditions, anti-human IgG Fc antibodies (Sigma) were used under blocking conditions - 5% skim milk after dilution at a ratio of 1:150000, anti-human GCSF antibodies (Human G-CSF Assay Kit. IBL) under blocking conditions - 1% skim milk after a 1:2000 dilution and streptavidin-HRP under blocking conditions - 5% skim milk after a 1:5000 dilution, respectively. HM10460A was confirmed to form dimers (94 kDa) that have two G-CSFs per IgG4 Fc fragment and IgG4 Fc fragments (50 kDa) by a chain exchange mechanism between HM10460A itself.

In contrast, when HM10460A induces a cross-chain exchange reaction between HM10460A and human IgG4, 100 kD and 122 kD molecules were expected to be generated. However, these molecules were not observed by Western blotting. In contrast, when analyzed with streptavidin-HRP, a 75 kDa band appeared in the human IgG4 lane and this confirms the formation of monomers from human IgG4, which itself is a dimer of human IgG4 (FIG. 1).

Example 3 Confirmation of chain exchange between human granulocyte colony stimulating factor-PEG-immunoglobulin conjugate and human IgG4 in human blood

Penicillin-streptomycin (1% v/v) was added to human blood collected from a donor. 1.32 mg of HM10460A prepared in Example 1 was mixed with 3 ml of blood, and the mixtures were aliquoted into 6 tubes (0.5 ml/tube) and incubated in an incubator at 37°C. One tube was taken out at the following time points of 0 hours, 4 hours, 10 hours, 24 hours and 48 hours, respectively, and plasma was separated from them and stored at -20°C before analysis. Each of the plasma samples, as well as HM10460A and IgG4 Fc fragment at various concentrations as controls, were mixed with a non-reducing protein sample buffer, and the resulting product was subjected to SDS-PAGE using a polyacrylamide gel with a concentration gradient of 4%-15%. The gel after completion of electrophoresis was blotted on a PVDF membrane (Immobilon-P, MILLIPORE) and analyzed using antibodies against human GCSF. Anti-human GCSF antibodies (Human G-CSF Assay Kit, IBL) were used under blocking conditions - 1% skim milk after a 1:2000 dilution. As in rat blood, the 100 kD and 122 kD molecules that could have been produced by chain exchange with human IgG4 were not produced (FIG. 2).

Those skilled in the art will appreciate that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered as illustrative only and not restrictive in all respects. The scope of the present invention is thus defined by the appended claims and not by the preceding description of the invention. All changes that fall within the meaning and equivalence range of the claims are to be included within the scope of the present invention.

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SEQUENCE LIST

<110> HANMI PHARM. CO., LTD.

<120> IGG4 FC fragment containing a modified hinge region

<130>OPA14085

<140> 10-2013-0063029

<141> 2013-05-31

<160> 21

<170> KopatentIn 2.0

<210> 1

<211> 12

<212> PRT

<213> HOMO SAPIENS

<400> 1

Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro

1 5 10

<210> 2

<211> 221

<212> PRT

<213> HOMO SAPIENS

<400> 2

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

210 215 220

<210> 3

<211> 663

<212> DNA

<213> HOMO SAPIENS

<400> 3

ccatcatgcc cagcacctga gttcctgggg ggaccatcag tcttcctgtt ccccccaaaa 60

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 120

agccaggaag accctgaggt ccagttcaac tggtacgtgg acggcgtgga ggtgcataat 180

gccaagacaa agccgcggga ggagcagttc aacagcacgt accgtgtggt cagcgtcctc 240

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 300

ggcctcccat cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 360

caggtgtaca ccctgccccc atcccaggag gagatgacca agaaccaggt cagcctgacc 420

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 480

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 540

tacagcaggc taaccgtgga caagagcagg tggcaggagg ggaacgtctt ctcatgctcc 600

gtgatgcatg aggctctgca caaccactac acacagaaga gcctctccct gtctctgggt 660

aaa 663

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 4

Glu Ser Lys Tyr Gly Pro Pro Pro Ser Cys Pro

1 5 10

<210> 5

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 5

Glu Ser Lys Tyr Gly Pro Cys Pro Ser Pro

1 5 10

<210> 6

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 6

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser

1 5 10

<210> 7

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 7

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

1 5 10

<210> 8

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 8

Lys Tyr Gly Pro Pro Cys Pro Ser

15

<210> 9

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 9

Glu Ser Lys Tyr Gly Pro Pro Cys

15

<210> 10

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 10

Glu Lys Tyr Gly Pro Pro Cys

15

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 11

Glu Ser Pro Ser Cys Pro

15

<210> 12

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 12

Glu Pro Ser Cys Pro

15

<210> 13

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 13

Pro Ser Cys Pro

1

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 14

Glu Ser Lys Tyr Gly Pro Ser Cys Pro

1 5 10

<210> 15

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 15

Lys Tyr Gly Pro Pro Ser Cys Pro

15

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 16

Glu Ser Lys Tyr Gly Pro Ser Cys Pro

15

<210> 17

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 17

Glu Ser Lys Tyr Gly Pro Pro Cys

15

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 18

Lys Tyr Gly Pro Pro Cys Pro

15

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 19

Glu Ser Lys Pro Ser Cys Pro

15

<210> 20

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 20

Glu Ser Pro Ser Cys Pro

15

<210> 21

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Hinge region option

<400> 21

Glu Pro Ser Cys

1

<---

Claims (12)

1. A modified IgG4 Fc fragment having a low risk of inducing chain exchange and monomer formation in vivo and containing a hinge region represented by any of the amino acid sequences of SEQ ID NOs: 4, 10, 11, 12, 14, 15, 19, 20 and 21 where said hinge region contains only one cysteine residue. 2. A modified IgG4 Fc fragment according to claim 1, which is aglycosylated. 3. Nucleic acid encoding the IgG4 Fc fragment according to claim 1. 4. Expression vector containing the nucleic acid according to claim 3. 5. An E. coli host cell for producing a modified IgG4 Fc fragment according to claim 1, into which the vector according to claim 4 is introduced. 6. A method for obtaining an IgG4 Fc fragment according to claim 1 or 2, including culturing the microorganism according to claim 5. 7. A drug conjugate having a low risk of inducing chain exchange and monomer formation in vivo, wherein the IgG4 Fc fragment of claim 1 or 2 is drug-conjugated with a linker selected from the group consisting of polyethylene glycol, polypropylene glycol, an ethylene glycol copolymer, and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), lipid polymer, chitins, hyaluronic acid, and combinations thereof. 8. The drug conjugate of claim 7, wherein the drug is selected from the group consisting of human growth hormone; growth hormone releasing hormone; a growth hormone releasing peptide; interferons and interferon receptors; colony stimulating factor; interleukins and interleukin receptors; enzymes; interleukin and cytokine binding proteins; macrophage activation factor; macrophage peptide; B-cell factor; T-cell factor; protein A, allergy inhibitory factor, necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, transforming growth factor, α-1-antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin; a peptide that activates the thrombin receptor; thrombomodulin, blood coagulation factor VII, blood coagulation factor VIIα, blood coagulation factor VIII, coagulation factor IX, blood coagulation factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor , collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone morphogenetic growth factor, bone morphogenetic protein, calcitonin, insulin and insulin derivative, atriopeptin, cartilage inducing factor, elkatonin, activating factor connective tissue, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors (eg, nerve growth factor, ciliary neurotrophic factor, axogenesis factor-1, brain natriuretic peptide, glial cell neurotrophic factor, netrin, inhibition of neutrophils, neurotrophic factor and neurin), parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, insulinotropic peptides, including glucagon-like peptide-1 and exendin-4, incretins secreted in the intestine, adipocytes, including leptons, and neurocytokins, effective for metabolic syndrome, cholecystokinin, pancreatic polypeptides, gastrointestinal peptides, corticotropin factor, thyroidimulating hormone, autotaxin, lactoperrin, myostatin, receptor, antagonist, antigenist, antigen of Cleaver accurate surface, monoclonal antibodies, polyclonal antibodies , fragments of antibodies and viral vaccine antigen. 9. The drug conjugate of claim 7, wherein the drug is a granulocyte colony stimulating factor. 10. The drug conjugate of claim 7, wherein the linker is a non-peptidyl polymer. 11. The drug conjugate of claim 10 wherein the non-peptidyl polymer has two or three reactive ends. 12. A pharmaceutical composition having a low risk of inducing chain exchange and monomer formation in vivo and containing the conjugate of claim 7 and a pharmaceutically acceptable carrier.