KR20170037247A - A method for the production of immunoglobulin Fc region comprising initial Methionine residue - Google Patents

Abstract

The present invention provides a method for producing an immunoglobulin Fc region in the form of a monomer or dimer comprising an initiating methionine residue using a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region, To an immunoglobulin Fc region in the form of a monomer or dimer comprising an initiating methionine residue prepared by the method. The recombinant immunoglobulin Fc region containing the immunoglobulin hinge region of the present invention can be used to produce a recombinant immunoglobulin Fc region in the form of an aggregate in E. coli. The immunoglobulin Fc region expressed and produced in a form containing methionine is expressed in physiological May be used to increase the half-life of the physiologically active polypeptide bound to the active peptide.

Description

FIELD OF THE INVENTION The present invention relates to a method for producing an immunoglobulin Fc region comprising an initiation methionine residue,

The present invention provides a method for producing an immunoglobulin Fc region comprising an initiation methionine residue using a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region and a method for producing an initiation methionine ≪ / RTI > residues in the form of monomers or dimers.

With the development of genetic engineering technology, many kinds of protein drugs have been produced and used in medicine field. However, protein drugs have a fatal disadvantage that they can not be easily denatured or easily decomposed by in vivo protease, so that in vivo concentration and activity can not be sustained for a long time. Therefore, increasing the stability of the protein and keeping the blood and in vivo concentration of the protein drug at an appropriate level is an important issue not only for effective treatment but also for the inconvenience reduction and economical reasons for the patient to be supplied with the protein through frequent injection.

Therefore, various methods such as changing the formulation of a protein, fusing other proteins, or attaching a suitable polymer to the surface of a protein by chemical or biological methods have been attempted for a long time in order to increase the in vivo stability of the protein drug . In particular, one of the attempts to increase the stability of proteins by fusion with other proteins is the fusion of immunoglobulin Fc with proteins.

The Fc region is responsible for effector functions such as complement-depenent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC), which are other than the antigen-binding ability inherent to immunoglobulins. In addition, the FcRn sequence present in the Fc region plays a role in regulating the level of IgG in serum by increasing IgG transport and half-life to the newborn (Ghetie and Ward, Immunology Today 18: 592-598, 1997) And regulates the interaction with protein G. Studies have been actively carried out to increase the stability of the therapeutic protein through the fusion of the Fc region with the therapeutic protein.

Korean Patent No. 249572 discloses a method of ligating a carboxyl terminal of various proteins such as an IL4 receptor, an IL7 receptor, a G-CSF receptor, and an EPO receptor to an amino terminal of an IgG1 heavy chain constant region (Fc) Protein. ≪ / RTI > U.S. Patent No. 5,605,690 reports fusion proteins prepared from animal cells by fusing human IgG1 Fc derivatives to the carboxyl terminus of tumor necrosis factor receptor. In addition, Tanox reported in US Pat. Nos. 5,723,125 and 5,908,626 that the carboxyl terminus of the human interferon alpha and beta genes and the native human IgG4 Fc gene are produced in animal cells using a peptide linker, In WO 00/69913, wild-type IgG1 Fc carboxyl terminus and amino terminus of human interferon are ligated in a recombinant manner without linker and produced in animal cells. U.S. Patent Publication No. 20030082679 reports that the fusion protein produced in animal cells after binding the carboxyl terminal of the human G-CSF gene and the amino terminal of IgG1 Fc using a peptide linker shows an increased blood half-life. U.S. Patent Publication Nos. 20010053539, 6,030,613, PCT Application Publication Nos. WO 99/02709, WO 01/03737, EP 0464533B1 disclose the use of an IgG1 Fc gene or a derivative of an Fc gene at its amino terminus A peptide linker or a peptide linker and ligated to the carboxyl terminal of the human EPO gene, the TPO gene, the human growth hormone gene, and the human interferon beta gene, and then the respective fusions were produced using animal cells. These Fc fusion proteins Of the total protein was elevated than that of the native protein.

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

Therefore, studies have been made to use immunoglobulin or immunoglobulin fragments that retain long half-life of blood but lack effector functions. Cole et al. Reported that 234, 235, and 237 residues, which are known to play an important role in binding to Fc receptors in the CH2 domain to produce Fc derivatives with reduced affinity for the Fc receptor, are replaced with alanine to inhibit the activity of ADCC (Cole et al., J. Immunol. 159: 3613-3621, 1997). However, all of these may have greater immune or antigenicity due to the presence of naturally occurring human Fc regions and other inappropriate amino acids, and may also lose desirable Fc functions.

A method for removing sugar from immunoglobulins has been studied as one of the methods for eliminating or reducing undesired effector functions while maintaining a high blood concentration of immunoglobulin. U.S. Patent No. 5,585,097 describes the preparation of a CD3 antibody by substituting another amino acid for the 297th asparagine residues of the CH2 domain, the glycosylation residue of the antibody, to produce a non-glycosylated antibody derivative. In this case, the binding ability of the derivative to the FcRn receptor And exhibited a reduced effector function while keeping the blood half-life unchanged. However, this method also has a problem in that it can be recognized and rejected as an external substance in the immune system by the generation of a new recombinant construct having an abnormal sequence. U.S. Patent Publication No. 20030073164 describes a method for producing an antibody by fusing a heavy chain and a light chain to a secretion sequence using an Escherichia coli cell without saccharifying function to produce a therapeutic antibody in which the effector function is deleted.

Amgen in the United States manufactures therapeutic proteins or therapeutic protein peptide mimics in U.S. Patent No. 6,660,843, U.S. Patent Nos. 20040044188 and 20040053845, and discloses that the first 5 of the human IgG1 Fc hinge at its amino terminus or carboxyl terminus A method is described in which a derivative in which an amino acid is deleted is fused and then produced using an E. coli host. However, since the fusion protein expressed without the signal signal is expressed and is present as an inclusion body in the cytoplasm, it has a disadvantage that it must undergo a separate refolding process after separation. When the refolding process is performed, the yield of the protein is lowered, and in the case of a protein that exists as a homologous or heterodimeric dimer, the yield of the dimeric product is remarkably reduced.

To improve this point, the present inventors have found that Fc region and protein drug are not produced by conventional recombinant methods, but are produced by the individual polypeptides in each of the best expression systems, covalently bound to each other, and Fc is used as a carrier of the drug There is a bar. In this case, a combination of the glycosylated polypeptide drug and the non-glycosylated Fc can be prepared, so that the inappropriate immune response is eliminated, thereby satisfying all of the physiological drug activity, biological continuity and stability.

In the above case, Fc is preferably a non-glycosylated form, so that a prokaryotic cell expression system such as E. coli is used. The production method using the expression system of Escherichia coli has various advantages over the method using the existing animal cells. Escherichia coli can express expression easily because it is easy to produce the expression vector, and since the growth rate is very fast, Can be mass-produced, and a relatively simple fermentation method can be applied, which is more advantageous than using other host cells in terms of commercial use.

There has been an attempt to use an Fc region expressed in a water-soluble state to industrially use the Fc region. European Patent EP0227110 describes a process for producing an immunoglobulin G1 Fc region by overexpression of an immunoglobulin G1 Fc region followed by taking only a cell lysate expressed in a water-soluble state. However, in the case of isolating only immunoglobulin expressed in a water-soluble state, the yield is too low as 15 mg / L, which is disadvantageous in that the value for industrial use is low. When the Fc region is overexpressed in E. coli, most of the Fc region is expressed in aggregate form.

The present invention relates to a method for producing an immunoglobulin Fc region in the form of a monomer or dimer comprising an initiating methionine residue 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 drugs have been manufactured and used. However, protein drugs have a fatal disadvantage that they can not be easily denatured or easily decomposed by in vivo protease, so that in vivo concentration and activity can not be sustained for a long time. Therefore, increasing the stability of proteins and keeping the blood and in vivo concentrations of protein drugs at an appropriate level is a very important issue not only for effective treatment but also for the inconvenience reduction and economical reasons for patients who need to be supplied with protein by frequent injection .

Under these circumstances, the present inventors tried to find a method of efficiently producing an active immunoglobulin Fc region without the risk of inducing an immune response by removing the sugar chain. The specific hinge region is connected to the N terminus of the immunoglobulin Fc region The immunoglobulin Fc region expressed in the aggregate form is produced as an immunoglobulin Fc region in the form of a dimer or monomer containing an initiating methionine residue through solublization and refolding And could produce an immunoglobulin Fc region containing the methionine initiation codon.

We also linked a particular hinge region from which the initiation methionine residue was removed to the N-terminus of the immunoglobulin Fc region, including the hinge region in which the initiation methionine residue was not removed, And an immunoglobulin Fc region expressed in the form of aggregate was expressed as an immunoglobulin Fc region in the form of a dimer or monomer containing an initiating methionine residue through solublization and refolding.

One object of the present invention is to provide a method for producing a recombinant immunoglobulin Fc region comprising the steps of: preparing a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; Transforming said vector into prokaryotic cells; Culturing the transformant; And isolating and purifying an immunoglobulin Fc region expressed in the form of an aggregate from the transformant. The present invention also provides a method for producing an immunoglobulin Fc region comprising an initiation methionine residue.

It is another object of the present invention to provide an immunoglobulin Fc region in the form of a monomer or dimer comprising an initiating methionine residue prepared by the above method.

According to an aspect of the present invention, there is provided a method for producing a recombinant immunoglobulin Fc region comprising: preparing a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; Transforming said vector into prokaryotic cells; Culturing the transformant; And isolating and purifying the immunoglobulin Fc region expressed in the form of aggregates from the transformant. The present invention also provides a method for producing an immunoglobulin Fc region comprising an initiation methionine residue.

One embodiment of the invention provides a method wherein said immunoglobulin Fc region is an immunoglobulin Fc region in monomeric or dimeric form.

Another embodiment of the invention provides a method characterized in that said immunoglobulin Fc region is unglycosylated.

Another embodiment of the present invention provides a method wherein said 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.

Another embodiment of the present invention is a method according to the method wherein said IgG is IgG1, IgG2, IgG3 or IgG4

Another embodiment of the present invention provides a method wherein said hinge region comprises an amino acid sequence of SEQ ID NO: 17, 18, 19 or 20.

Another embodiment of the invention provides a method wherein said immunoglobulin Fc region consists of one to four selected from the group consisting of CH1, CH2, CH3 and CH4 domains.

Another embodiment of the present invention provides a method wherein said immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE or IgM.

Another embodiment of the present invention is a method wherein each domain of the immunoglobulin Fc region is a hybrid of a domain with different origins derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, IgM to provide.

Another embodiment of the present invention provides a dimeric or multimeric method wherein the immunoglobulin Fc region consists of a short chain immunoglobulin of the same origin domain.

Another embodiment of the present invention provides a method wherein said immunoglobulin Fc region is an IgG4 Fc fragment.

Another embodiment of the present invention provides a method wherein said immunoglobulin Fc region is a human unchallenged IgG4 Fc fragment.

Another embodiment of the present invention provides a method wherein the recombinant immunoglobulin Fc region is comprised of an amino acid sequence of SEQ ID NO: 10, 12, 14 or 16.

Another embodiment of the present invention provides a method wherein the nucleic acid sequence encoding the recombinant immunoglobulin Fc region is comprised of a nucleic acid sequence of SEQ ID NO: 9, 11, 13 or 15.

Another embodiment of the present invention provides a method wherein said vector is pMCPSFc, pMYGPFc, pMKYGFc or pMESKFc.

Another embodiment of the present invention provides a method wherein said prokaryotic cell is E. coli.

Another embodiment of the present invention provides a method wherein said transformant is BL21 / pMCPSFc, BL21 / pMYGPFc, BL21 / pMKYGFc, or BL21 / pMESKFc.

Another aspect of the present invention provides an immunoglobulin Fc region in the form of a monomer or dimer comprising an initiating methionine residue prepared by the method.

The recombinant immunoglobulin Fc region containing the immunoglobulin hinge region of the present invention can be used to produce a recombinant immunoglobulin Fc region in the form of an aggregate in E. coli. The immunoglobulin Fc region expressed and produced in a form containing methionine is expressed in physiological May be used to increase the half-life of the physiologically active polypeptide bound to the active peptide.

Figure 1 is the result of protein electrophoresis after expression of an Fc analog comprising initiation methionine (Met).
Figure 2 shows the results of chromatographic analysis of the purity of the Fc analog containing the initiating methionine.
FIG. 3 shows the results of examining the blood half-life of a binding protein CA-Exendin-PEG-Fc analogue conjugate prepared using an Fc analog containing an initiating methionine as a carrier.

Hereinafter, the present invention will be described in detail.

In one embodiment, the present invention provides a method for producing a recombinant immunoglobulin Fc region comprising: preparing a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; Transforming said vector into prokaryotic cells; Culturing the transformant; And isolating and purifying an immunoglobulin Fc region expressed in the form of aggregates from the transformant. The present invention also relates to a method for producing an immunoglobulin Fc region comprising an initiation methionine residue.

The present invention relates to a method for producing an Fc region of an immunoglobulin which can be usefully used as a carrier for medicines and the like. When the hinge region is linked to the N-terminus of an immunoglobulin Fc region, It was confirmed that the globulin Fc region was solublized and refolded to the active monomer or dimeric Fc region containing the initiation methionine residue encoded by the initiation codon. The Fc region expressed so that the hinge region binds to the Fc region of the immunoglobulin and does not lose its activity but is processed and refolded into a form comprising the initiation methionine residues is the active form of the form in which the initiation methionine residues are removed in terms of pharmacokinetics The significance of the present invention is significant in that it confirms a similar blood half-life as compared with the Fc region in monomeric or dimeric form.

The hinge region that can be used in mass production of the Fc region by recombinantly binding to the immunoglobulin Fc region is IgG, IgA, IgM, IgM, IgA, IgA and IgG derived from animals such as human, goat, pig, mouse, rabbit, hamster, rat, Or a hinge region derived from IgD, and more preferably IgG is a hinge region derived from IgG1, IgG2, IgG3 or IgG4. The hinge region may be a full length hinge region as well as a fragment thereof. Preferably, the hinge region fragment is a fragment having two or more consecutive amino acid sequences, more preferably a fragment having two or more consecutive amino acid sequences comprising at least one cysteine residue. In a specific embodiment of the present invention, fragments derived from the IgG4-derived hinge region were used and are represented by SEQ ID NOS: 17, 18, 19 or 20. When the hinge region of SEQ ID NO: 17, 18, 19 or 20 is used, an immunoglobulin Fc region in the form of an active monomer in the form of methionine and a dimer form can be produced.

The hinge region that can be used in mass production of the Fc region by recombinantly binding to the immunoglobulin Fc region is IgG, IgA, IgM, IgM, IgA, IgA and IgG derived from animals such as human, goat, pig, mouse, rabbit, hamster, rat, Or a hinge region derived from IgD, and more preferably IgG is a hinge region derived from IgG1, IgG2, IgG3 or IgG4. The hinge region may be a full length hinge region as well as a fragment thereof. Preferably, the hinge region fragment is a fragment having two or more consecutive amino acid sequences, more preferably a fragment having two or more consecutive amino acid sequences comprising at least one cysteine residue. In the present invention, fragments derived from an IgG4-derived hinge region can be used, and an active type monomer having a methionine-containing form and an immunoglobulin Fc region in the form of a dimer can be produced.

The immunoglobulin Fc region may be linked to the immunoglobulin hinge region to produce a nucleic acid sequence encoding the recombinant immunoglobulin Fc region.

The immunoglobulin Fc region that can be produced by the present invention includes the heavy and light chain variable regions, the heavy chain constant region 2 (CH2) and the heavy chain constant region (CH2), except for the heavy chain constant region 1 (CH1) and the light chain constant region 3 (CH3) (or heavy chain constant region 4 (CH4)), and may also include a hinge portion in the heavy chain constant region. Also, as long as the immunoglobulin Fc region of the present invention has a substantially equivalent or improved effect as compared with the wild type, the immunoglobulin Fc region may contain a heavy chain constant region 1 (CH1) and / or a heavy chain constant region RTI ID = 0.0 > (CL). ≪ / RTI > It may also be a region in which some long amino acid sequences corresponding to CH2 and / or CH3 have been removed. That is, the immunoglobulin Fc region of the present invention includes (1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, (2) a CH1 domain and a CH2 domain, (3) a CH1 domain and a CH3 domain, Domain, (5) a combination of one or more constant domain domains with immunoglobulin hinge regions (or portions of hinge regions), and (6) a heavy chain constant region angular domain and light chain constant region dimer. Constant domains, including immunoglobulin Fc fragments, are biodegradable polypeptides that are metabolized in vivo and are therefore safe for use as carriers for drugs. Since the immunoglobulin Fc fragment has a relatively small molecular weight as compared with the whole immunoglobulin molecule, it is not only advantageous in terms of preparation, purification and yield of the conjugate, but also because the amino acid sequence differs from antibody to antibody, the Fab portion exhibiting high heterogeneity is removed. It is expected that the homogeneity will be greatly increased and the possibility of inducing blood antigenicity will also be lowered.

On the other hand, the immunoglobulin Fc region may be an animal origin such as human or bovine, chlorine, porcine, mouse, rabbit, hamster, rat, guinea pig and the like, preferably human origin. Also, the immunoglobulin Fc region may be selected from the group consisting of IgG, IgA, IgD, IgE, IgM, or a combination thereof, or a constant region by hybridization thereof. Preferably derived from the most abundant IgG or IgM in human blood, and most preferably from IgG known to enhance the half-life of the ligand binding protein. In the present invention, the immunoglobulin Fc region may be a dimer or a multimer composed of a short chain immunoglobulin composed of domains of the same origin. Nucleic acid sequences encoding the human immunoglobulin Fc region useful in the present invention and the amino acid sequences defining the same may be those encoded by the nucleotide sequences disclosed in the GenBank and / or EMBL databases.

In the present invention, the term " combination "means that, when a dimer or multimer is formed, a polypeptide encoding a homologous short chain immunoglobulin Fc region (preferably an Fc region) binds to a short chain polypeptide of a different origin . That is, it is possible to prepare a dimer or a multimer from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE.

The term "hybrid" in the present invention means a sequence corresponding to two or more immunoglobulin Fc regions of two or more different origins in the short chain immunoglobulin Fc region (preferably, Fc region). In the case of the present invention, various types of hybrids are possible. That is, hybrids of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc are possible.

IgG can also be divided into subclasses of IgG1, IgG2, IgG3 and IgG4, and a combination thereof or a hybridized form thereof is also possible in the present invention. Specifically IgG2 and IgG4 subclasses, and more specifically, an Fc region of IgG4 with little effector function such as complement dependent cytotoxicity (CDC).

In addition, the immunoglobulin Fc region may be a natural type sugar chain, an increased sugar chain as compared to the native type, and a reduced sugar chain or sugar chain as compared with the native type. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or decrease the immunoglobulin Fc region sugar chains. Here, the immunoglobulin Fc region in which the sugar chain is removed in the immunoglobulin Fc region is significantly reduced in binding force of the complement (c1q), and the antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, Lt; / RTI > In this regard, forms that are more consistent with the original purpose of the drug as a carrier will be referred to as immunoglobulin Fc regions in which the sugar chain is removed or unglycosylated. Thus, more specifically, non-glycosylated Fc regions derived from human IgG4, i.e., human unchallenged IgG4 Fc regions, can be used. The human-derived Fc region may be preferable to the non-human-derived Fc region which can act as an antigen in a human organism and cause an undesirable immune response such as the generation of new antibodies thereto.

In addition, the immunoglobulin Fc region of the present invention includes a recombinant immunoglobulin Fc region, an immunoglobulin Fc analog or a sequence derivative (mutant) as well as a native amino acid sequence. An amino acid sequence derivative means that at least one amino acid residue in the natural amino acid sequence has a different sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, and the recombinant immunoglobulin Fc region and immunoglobulin Fc analogue Can be used in the same sense as. In particular, in the present invention, a recombinant immunoglobulin Fc region, an immunoglobulin Fc analogue or a sequence derivative may mean an immunoglobulin Fc region including an immunoglobulin hinge region. For example, the amino acid residues 214 to 238, 297 to 299, 318 to 322 or 327 to 331, which are known to be important for binding in the case of IgG Fc, can be an IgG Fc analog prepared to include a hinge region at the N terminus It can be used as a suitable site for deformation. Further, various kinds of derivatives are possible, such as a site capable of forming a disulfide bond is removed, some amino acid at the N-terminus is removed from the native Fc, or a methionine residue is added to the N-terminus of the native Fc. Also, to eliminate the effector function, the complement binding site, for example the C1q binding site, may be removed and the ADCC site may be removed. Techniques for producing sequence derivatives of such immunoglobulin Fc regions are disclosed in WO 97/34631, WO 96/32478, and the like.

The recombinant immunoglobulin Fc region of the invention, or immunoglobulin Fc analog, may be a recombinant immunoglobulin Fc analog having an amino acid sequence of SEQ ID NO: 10, 12, 14 or 16. In a specific embodiment of the present invention, a recombinant immunoglobulin Fc analog having the amino acid sequence of SEQ ID NO: 10, 12, 14 or 16 comprising the hinge region at the N terminus based on the IgG4-derived Fc region and containing the initiation methionine is prepared Respectively.

Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly exchange (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, , 1989). In some cases, the phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, or the like, modification.

The immunoglobulin derivative may be a functional equivalent exhibiting the same biological activity as the natural protein, but may be a derivative in which the properties of the protein are modified, if necessary. Preferably, the immunoglobulin Fc domain derivative is a protein having a structural stability against heat, pH, etc. of the protein by induction and modification on the amino acid sequence within the range that the protein sequence of the derivative to be produced is not so different from human and does not cause an immune response Or increased solubility, improved derivatives of sulfide bond formation, affinity with expression host, binding to complement, binding to Fc receptor, antibody-dependent cytotoxicity, and the like. At this time, the region changed in the Fc region derivative is different from that of human, and should not induce an immune response upon administration to humans. Examples of preferable derivatives include a reduced binding force for Fc receptors inducing antibody-dependent cytotoxicity Specific residues of the IgGl Fc region can be induced. Such derivatives may include deletion or substitution with other residues of the 234 leucine residues present in the IgG1 CH2 sequence (numbering refers to the sequence of the Kobat database), with the most preferred derivative being the amino acid residue corresponding to the same position in IgG4 Phenylalanine. ≪ / RTI >

These immunoglobulin Fc regions may also be obtained from natural forms isolated in vivo in humans and animals such as cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, and the like, Or a derivative thereof. Here, the method of obtaining from the natural form can be obtained by separating the whole immunoglobulin from the living body of human or animal, and then treating the proteolytic enzyme. When papain is treated, it is cleaved into Fab and Fc, and when pepsin is treated, it is cleaved into pF'c and F (ab) 2. Fc or pF'c can be isolated using size-exclusion chromatography or the like.

In the present invention, a nucleic acid sequence coding for a recombinant immunoglobulin Fc region can be prepared by linking an immunoglobulin Fc region with the above immunoglobulin hinge region. In the present invention, the recombinant immunoglobulin Fc region may mean that the hinge region is linked to the N-terminus of the immunoglobulin Fc region by a peptide bond.

A hinge region that binds according to the immunoglobulin Fc region may be selected, and specifically a hinge region of the same origin as the immunoglobulin Fc region may be used. In a specific embodiment of the present invention, a nucleic acid sequence encoding a recombinant immunoglobulin Fc region was constructed in which a hinge region having an amino acid sequence of SEQ ID NO: 17, 18, 19, or 20 was linked to the Fc region derived from IgG4.

Also, in a specific embodiment of the present invention, a nucleic acid sequence encoding a recombinant immunoglobulin Fc region, in which a hinge region having an amino acid sequence of SEQ ID NO: 12 is linked to an Fc region derived from IgG4, is prepared.

The nucleic acid sequence encoding the recombinant immunoglobulin Fc region of the present invention refers to a nucleic acid sequence encoding the recombinant immunoglobulin Fc region. For example, the nucleic acid sequence encoding the recombinant immunoglobulin Fc region may include a sequence comprising an hinge region and an initiation methionine based on an IgG4- A nucleic acid sequence encoding a recombinant immunoglobulin Fc analog having an amino acid sequence of SEQ ID NO: 9, 11, 13, or 15,

In a specific embodiment of the present invention, a recombinant immunoglobulin Fc analog having an amino acid sequence of SEQ ID NO: 10, 12, 14 or 16 comprising a hinge region and an initiation methionine based on the Fc region derived from IgG4 was prepared, Nucleic acid sequences of SEQ ID NOS: 9, 11, 13 and 15 encoding globulin Fc regions were prepared.

The nucleic acid sequence encoding the recombinant immunoglobulin Fc region may be provided by operatively linking to a vector capable of expressing the recombinant immunoglobulin Fc region.

As used herein, the term "vector" refers to a recombinant vector capable of expressing a desired protein in a suitable host cell, including a necessary regulatory element operably linked to the expression of the gene insert.

In the present invention, "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein to perform a general function. The operative linkage with the vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be facilitated using enzymes generally known in the art . Suitable expression vectors may include expression control element sequences such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. The initiation codon and the termination codon must be operative in the individual when the gene construct is administered and in the coding sequence and in frame. Generic promoters may be constitutive or inducible. The expression vector may also include a selectable marker for selecting a host cell containing the vector, and may include a replication origin if it is a replicable expression vector. In a specific embodiment of the present invention, pMCPSFc, pMYGPFc, pMKYGFc and pMESKFc were prepared.

A recombinant expression vector expressing the recombinant immunoglobulin Fc region of the present invention is transformed into a host cell.

For the purposes of the present invention, host cells are prokaryotic cells that do not undergo glycosylation. Such prokaryotic cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus. Or preferably Escherichia coli. E. coli is preferably Escherichia coli XL-1 Blue, Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH series, Escherichia coli TOP10 and Escherichia coli HB101, and more preferably Escherichia coli BL21 (DE3). When Escherichia coli is used as a host cell, E. coli does not have a system for linking sugar chains to proteins, so that the Fc region of immunoglobulin can be produced in a form in which the sugar existing in the CH2 domain of the native immunoglobulin is originally deleted. Although the sugar of the CH2 domain of immunoglobulin does not affect the structural stability of immunoglobulins, immunoglobulin binds to cells expressing Fc receptors, resulting in antibody-dependent cytotoxicity, and immune cells secrete cytokines, , And is known to cause a complement fixation reaction by combining with the complement C1q element. Thus, by producing the Fc region of the unglycosylated immunoglobulin and combining it with the therapeutic protein, the blood concentration of the therapeutic protein can be maintained for a long time without inducing the undesirable effector function of the immunoglobulin.

Transformation of the vector into a prokaryotic cell includes any method of introducing the nucleic acid into a cell, and may be carried out by selecting a suitable standard technique depending on the host cell, as is known in the art. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, stirring with silicon carbide fibers, PEG, dextran sulfate, lipofectamine, It is not limited.

In a specific embodiment of the present invention, BL21 / pMCPSFc, BL21 / pMYGPFc, BL21 / pMKYGFc and BL21 / pMESKFc were prepared.

The transformant transformed with the recombinant expression vector is cultured by a conventional method.

Such a culturing process can be easily adjusted according to the strain selected by those skilled in the art. The medium used for the culture should generally contain all the nutrients essential for cell growth and survival. The medium comprises various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, linoleic acid , Glycerol and alcohols such as ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that may be used include organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL), and soybean wheat and the like, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate And an inorganic nitrogen source. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts as a source. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included. During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, foaming can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture. In addition, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain the aerobic state of the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. A fermenter may also be used. When producing a protein using a fermenter, various factors such as the growth rate of the host cell and the amount of the expressed product must be considered. The expression of the protein may be induced by administering IPTG or the like under appropriate culture conditions.

Immunoglobulin Fc regions overexpressed in aggregate form can be purified in a conventional manner. The immunoglobulin Fc region produced from the transformant was obtained by disrupting cells using a method such as a French press or an ultrasonic disintegrator and then separating only the insoluble aggregate containing the immunoglobulin Fc region using a centrifuge, Denatured and refolded into refolding agents such as urea, guanidine, arginine cysteine, and beta-mercaptoethanol, followed by column chromatography such as dialysis, gel filtration, ion exchange, reverse phase column chromatography and ultrafiltration, To obtain a protein encoding the immunoglobulin Fc region of the present invention. Such refolding process is a relatively complicated process, and the refolding efficiency of the protein is very low and the activity of the protein after refolding is known to be lower than that of the soluble protein.

However, when the method of the present invention is used, it is possible to overcome this point to produce an active Fc immunoglobulin containing an initiating methionine from an aggregate. Escherichia coli expresses a heterologous protein and produces an initiation codon, methionine.  Since post-translational modification by the protocol differs according to the characteristics of the hinge region to be added, the production ratio of the monomer and the dimer can be effectively controlled by appropriately selecting and using the hinge region. When the aggregate is refolded, precise dimer formation is disturbed by mismatch of cysteine during disulfide bond formation. However, when the method of the present invention is used, active dimer forming disulfide bond is efficiently produced It is very desirable.

Further, the immunoglobulin Fc region can be produced more efficiently than the existing invention for mass production of the immunoglobulin Fc region. Specifically, in European Patent EP0227110, immunoglobulin G1 Fc region was obtained at a yield of 15 mg / L by taking only the product (cell lysate) expressing a water-soluble state after overexpressing the immunoglobulin G1 Fc region, Korean Patent Application No. 0092783 discloses an immunoglobulin Fc region Was expressed in a form fused with the E. coli signal sequence and expressed in a water-soluble form rather than as an aggregate, and was produced at a yield of 50 to 600 mg / L. However, when the immunoglobulin Fc region is produced from the aggregate using the recombinant immunoglobulin Fc region including the hinge region of the present invention, it can be produced at a yield of 5 g / L or more. Therefore, the method of producing the immunoglobulin Fc region of the present invention is a very useful system for producing the immunoglobulin Fc region at a much higher yield than the conventional method.

In another aspect, the present invention relates to an immunoglobulin Fc region produced by the method of the present invention. Specifically an immunoglobulin Fc region in the form of a monomer or dimer comprising an initiating methionine residue prepared by the above method. The immunoglobulin Fc region prepared by the method of the present invention includes a hinge region and may include methionine as a starting amino acid at the N-terminus.

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 any drug and conjugate formation. The form of the conjugate in which the immunoglobulin Fc region and the drug are bound is not particularly limited. For example, the immunoglobulin Fc region and the drug may be bound in various ratios, and the binding may be mediated by a linker or the like.

Drugs include polypeptides, compounds, extracts, nucleic acids, etc., but preferably polypeptides (used in equivalent terms to proteins). A linker includes both a peptide linker or a non-peptide linker, but is preferably a non-peptide linker, more preferably a non-peptide polymer. A preferred example of an immunoglobulin heavy chain region is Fc.

The physiologically active polypeptide which can be used in combination with the immunoglobulin Fc region prepared by the method of the present invention is not particularly limited as long as it is necessary to increase the half-life of blood. For example, cytokines, interleukins, interleukin binding proteins, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens , Receptor antagonists, derivatives and analogues thereof.

Specifically, the physiologically active polypeptides include human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons and interferon receptor residues such as interferon-alpha, -beta and -gamma, water soluble type I interferon receptor, Stimulating factors, interleukins such as 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, And interleukin receptors (e.g., IL-1 receptor, IL-4 receptor, etc.), enzymes (such as glucocerebrosidase, iduronate-2-sulfatase, -Galactosidase-A, agalsidase alpha, beta-alpha-L-iduronidase, butyrylcholinesterase, chitinase, , Glutamate decarboxylase, imiglucerase, < RTI ID = 0.0 > Such as lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, etc.), interleukin and Cytokine binding proteins (e.g., IL-18bp, TNF-binding protein, etc.), macrophage activator, macrophage peptide, B cell factor, T cell factor, protein A, allergic inhibitor, cytotoxic glycoprotein, immunotoxin, Toxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietic factor, hyperglycemic erythropoietin, angiogenesis factor Angiopoietin, hemoglobin, thrombin, thrombin receptor activator peptide, thrombomodulin, blood factor VII, blood factor VIIa, blood factor VIII, blood factor IX, blood factor XIII, Derived growth factor, leptin, platelet-derived growth factor (FGFR), fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, , Epithelial cell growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic promoting protein, calcitonin, insulin, atriepeptin, cartilage inducer, elcatonin, A tissue factor pathway inhibitor, a follicle stimulating hormone, a luteinizing hormone, a luteinizing hormone releasing hormone, a nerve growth factor such as a nerve growth factor, a cilliary neurotrophic factor, AXogenesis factor-1, brain-natriuretic peptide, glial derived neurotrope, phyric factor, netrin, neurophil inhibitor factor, neurotrophin, neuturin, etc.), parathyroid hormone, rilacsin, cicletin, somatomedin, insulin-like growth factor, (Eg, TNFR (P75)), thyroid stimulating hormone, thyrotropin, autotaxin, lactoferrin, myostatin, and receptors such as hormone, glucagon, cholestystinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, , Receptor antagonists (eg, IL1-Ra), cell surface antigens (eg, CD 2, 3, 4, 5, and 7) , Monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., scFv, Fab, Fab ', F (SEQ ID NOS: 11, 11, 11, 18, 19, 20, 23, 25, 33, ab ') 2 and Fd), virus-derived vaccine antigens, and the like, but are not limited thereto. The physiologically active polypeptide applicable in the present invention may be a native form, or may be produced by genetic recombination in eukaryotic cells such as prokaryotic cells such as Escherichia coli, yeast cells, insect cells or animal cells, Or a mutant at one or more amino acid positions.

In a specific embodiment of the present invention, an Exendin-PEG-immunoglobulin Fc domain protein conjugate was prepared by binding an immunoglobulin Fc region produced from a transformant to Exendin using polyethylene glycol, and the protein complex was Met- Was similar to the Exendin-PEG-Fc protein conjugate using the immunoglobulin Fc region as a carrier. Thus, it can be seen that the immunoglobulin Fc region comprising the initiating methionine residue generated from the aggregate using the hinge region according to the present invention increases the blood half-life of the physiologically active polypeptide.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: methionine (Met) Fc  Construction of Region Analogue Expression Vector

To clone the heavy chain Fc region containing the hinge region of IgG4, RT-PCR was carried out using the RNA of the blood cell obtained from human blood as a template as follows. First, total RNA was isolated from about 6 ml of blood using a Qiamp RNA blood kit (Qiagen), and the gene was amplified using this RNA as a template and using a One-Step RT-PCR kit (Qiagen) . At this time, forward and reverse oligonucleotides were synthesized to amplify genes of different N-terminal sequences, and PCR was performed to amplify each gene.

Primers for gene amplification analog primer Sequences (5 '-> 3') SEQ ID NO: Fc analog 1
(MCPS) Forward GACATATGTGCCCATCATGCCC One Reverse Gactcgagtttacccagagacagggag 2 Fc Analog 2
(MYGP) Forward GACATATGTATGGCCCACCTTGC 3 Reverse Gactcgagtttacccagagacagggag 4 Fc Analog 3
(MKYG) Forward GACATATGAAGTATGGCCCACCTTGC 5 Reverse Gactcgagtttacccagagacagggag 6 Fc Analog 4
(MESK) Forward GACATATGGAATCAAAGTATGGCCCAC 7 Reverse Gactcgagtttacccagagacagggag 8

PCR conditions for methionine-containing Fc analog amplification were repeated for 18 cycles: 95 ° C for 10 seconds, 55 ° C for 5 seconds, and 68 ° C for 6 minutes. The Fc analog fragment containing the methionine obtained under such conditions was inserted into the pET22b vector in order to express it in the form of an inclusion body in a cell, and the expression vector thus obtained was named pET22b-MetFc analog 1 to 4. The expression vector contains a nucleic acid encoding the amino acid sequence of Fc analogs 1 to 4 containing methionine under the control of the T7 promoter and expresses an Fc analogue protein containing methionine in the host in the form of an inclusion body.

DNA and protein sequences of Fc analogs containing methionine analog Sequence (5 '-> 3' or N-terminal-> C-terminal) SEQ ID NO: Fc analog 1
(MCPS) DNA ATG TGC CCA TCA TGC CCA GCA CCT GAG TTC CTG GGG GGA CCA TCA GTC TTC CTG TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAG GAA GAC CCT GAG GTC CAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GGC CTC CCA TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAC AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AGG CTA ACC GTG GAC AAG AGC AGG TGG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CTG GGT AAA 9 protein Met Cys 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 10 Fc Analog 2
(MYGP) DNA ATG TAT GGC CCA CCT TGC CCA TCA TGC CCA GCA CCT GAG TTC CTG GGG GGA CCA TCA GTC TTC CTG TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAG GAA GAC CCT GAG GTC CAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GGC CTC CCA TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AGC CTA ACC GTG GAC AAG AGG AGG TGG CAG GAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CTG GGT AAA 11 protein Met Tyr Gly Pro Pro Cys 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 12 Fc Analog 3
(MKYG) DNA ATG AAG TAT GGC CCA CCT TGC CCA TCA TGC CCA GCA CCT GAG TTC CTG GGG GGA CCA TCA GTC TTC CTG TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAG GAA GAC CCT GAG GTC CAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GGC CTC CCA TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AGC CTA ACC GTG GAC AAG AGG AGG TGG CAG GAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CTG GGT AAA 13 protein Met Lys Tyr Gly Pro Pro Cys 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 14 Fc Analog 4
(MESK) DNA ATG GAA TCA AAG TAT GGC CCA CCT TGC CCA TCA TGC CCA GCA CCT GAG TTC CTG GGG GGA CCA TCA GTC TTC CTG TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAG GAA GAC CCT GAG GTC CAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAT TTC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GGC CTC CCA TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AGG CTA ACC GTG GAC AAG AGC AGG TGG CAG GAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CTG GGT AAA 15 protein Met Glu Ser Lys Tyr Gly Pro Pro Cys 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 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 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 16

Example  2: methionine-containing Fc  Expression of analog

Met containing Fc analog expression under T7 promoter control was performed. Specifically, E. coli BL21-DE3 ( E. coli BF-dcm ompT hsdS (rB-mB-) gal lambda DE3) was used as an Fc analog expression vector containing each methionine prepared in Example 1 above; Novagen). Transfection methods were followed as recommended by Novagen. Each single colony transformed with each of the recombinant expression vectors was inoculated into 2 × Luria Broth (LB) medium containing ampicillin (50 μg / ml) and cultured at 37 ° C. for 15 hours. The recombinant strain culture medium and 2X LB medium containing 30% glycerol were mixed at a ratio of 1: 1 (v / v), and each 1 ml was dispensed into cryo-tubes and stored at -140 ° C. This was used as a cell stock for the production of the recombinant fusion protein.

For expression of recombinant insulin analogs, each cell stock 1 vial was dissolved and inoculated into 500 ml of 2X Luria broth (LB) and incubated at 37 ° C for 14-16 hours with shaking. When the value of OD ₆₀₀ was 4.0 or more, the culture was terminated and used as a seed culture solution. The seed culture was inoculated into a fermentation medium of 1.7 L using a 5 L fermenter (MDL-8C, BEMARUBISHI, Japan), and initial fermentation of the bath was started. The culture conditions were maintained at a temperature of 37 ° C, an air volume of 2 L / min (1 vvm), a stirring speed of 500 rpm and a pH of 6.70 using 20% ammonia water. When the nutrients in the culture medium were limited, the fermentation process was carried out by adding a feeding solution. The growth of the strain was monitored by OD value and was introduced into IPTG at a final concentration of 0.5 mM at an OD value of 120 or higher. The culture was further continued for about 23 to 25 hours after the introduction. After completion of the culture, the recombinant strains were harvested using a centrifuge and stored at -80 ° C until use. Expression after culture was confirmed by SDS-PAGE electrophoresis (Fig. 1).

Example  3: methionine-containing Fc  The recovery of analog and Refolding (refolding)

The cells were disrupted and refolded to convert the Fc analogs containing methionine expressed in Example 2 into a soluble form. Specifically, the cell pellet corresponding to 1 L of each culture was resuspended in 1 L dissolution buffer (50 mM Tris (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M sodium chloride and 0.5% Triton X-100). Cells were disrupted using a microfluidizer processor M-110EH (AC Technology Corp. Model M1475C) at a pressure of 15,000 psi. The shredded cell lysate was centrifuged at 7,000 rpm for 30 minutes at 4 ° C to discard the supernatant and resuspended in 1 L wash buffer (0.5% Triton X-100 and 20 mM Tris (pH 7.0)). The pellet was resuspended in 1 L washing buffer (20 mM Tris (pH 7.0)) by centrifugation at 7,000 rpm for 30 minutes at 4 DEG C, and then centrifuged in the same manner. The pellet was resuspended in 1 L of buffer (8 M Urea, 1.5 mM EDTA, 20 mM Tris (pH 7.0)) and stirred at room temperature for 4 hours. After centrifugation for 4 hours in the same manner, the supernatant was taken, and 0.67 mM cysteine was added thereto, followed by stirring at 4 ° C for 1 hour.

To the refolding of the Fc analog containing the solubilized methionine, 3 L of buffer (1.333 M urea, 0.203 mM cysteine, 0.267 M arginine, 60 mM Tris (pH 9.5)) was added via peristaltic pump At a flow rate of 1000 ml / hr, and the mixture was stirred at 4 ° C for 36 hours.

Example  4: Refolded  Methionine-containing Fc  Replace analogue buffer

In Example 3, the Fc analog containing 4 L methionine refolded was replaced at a rate of 1000 ml / hr using a Sartorius Sartocon cassette (3021405907E-SG) and a peristaltic pump. The buffers used were primary (2M Urea, 10mM Tris (pH 8.5), 0.125M Alginine), secondary (1M Urea, 10mM Tris (pH 8.5)) and tertiary (10mM Tris pH 8.5).

Example  5: Purification by anion-coupled chromatography

After the rejoined sample was bound to a DEAE (GE healthcare) column equilibrated with 10 mM Tris (pH 8.0) buffer, the concentration was 0% using 10 mM Tris (pH 8.0) and 0.5 M sodium chloride buffer, Lt; / RTI > of the Fc analogue protein containing methionine with a linear concentration gradient of 4 column volumes to 45%.

Example  6: Hydrophobic Bond Chromatography Purification

After 0.6 M ammonium sulfate was inserted into the eluted sample through Example 5, 0.6 M ammonium sulfate was inserted into a Phenyl FF (GE healthcare) column equilibrated with a buffer of 10 mM Tris (pH 8.0) and 0.6 M ammonium sulfate After binding of the samples, Fc analogue proteins containing methionine were eluted with a linear concentration gradient of 4 column volumes to a concentration of 0% to 100% using 10 mM Tris (pH 7.5) buffer.

Example  7: Anion-coupled chromatography purification

The salt was removed from the sample eluted in Example 6 at a rate of 1000 ml / hr using a Sartorius Sartocon cassette (3021405907E-SG) and a peristaltic pump and replaced with buffer solution (10 mM Tris, pH 7.5) . The sample obtained in Example 6 was bound to an anion exchange column (Source Q: GE healthcare) equilibrated with 10 mM Tris (pH 7.5) buffer to separate the Fc analog containing pure methionine, Using a 10 mM Tris (pH 7.5) buffer containing rides, a linear concentration gradient of 2.5 column volumes was made with a linear concentration gradient of 0.6 column volume at 17.1 min and 25% to 32% with a concentration of 0% to 25% The Fc analogue protein containing the Met for 62.5 minutes was eluted.

The purity of the Fc analog containing purified methionine was analyzed using high pressure chromatography (HPLC) (Figure 2).

Example  8: N-terminal sequence analysis

In the above example, methionine, which is an initiation codon, is added to the Fc expressed in a dimeric form by refolding the aggregate. In order to confirm whether the methionine residue is prcessed by E. coli protease, N-terminal sequencing of proteins was commissioned, and samples to be analyzed were prepared as follows.

First, the PVDF membrane (Bio Rad) was immersed in methanol for about 2-3 seconds and immersed in blocking buffer (170 mM glycine, 25 mM Tris-HCl (pH 8), 20% methanol). SDS-PAGE gel of the non-reducing condition developed in Example <1-2> was blotted for 1 hour using a blotting kit (Hoefer Semi-Dry Transfer unit, Amersham) to the prepared PVDF membrane Respectively. The protein transferred to the PVDF membrane was stained briefly (3-4 sec) with a protein stain agent, Coomassie Blue R-250 (Amnesco), and then washed with a decolorizing solution (water: acetic acid: methanol = 5: 1: 4) . In the washed membrane, the site containing the protein (dimer and monomer) was cleaved with scissors to request N-terminal sequence analysis and Met was confirmed.

N-terminal sequencing results Fc region Class Sequence analysis result N-terminal Natural type IgG4 IgG4 ... NTKVDKRVES KYGPPCPSCP ... - - IgG4                    PSCP ... Methionine removal Fc analog 1
(MCPS) IgG4                  MCPSCP ... Check methionine Fc Analog 2
(MYGP) IgG4              MYGPPCPSCP ... Check methionine Fc Analog 3
(MKYG) IgG4             MKYGPPCPSCP ... Check methionine Fc Analog 4
(MESK) IgG4          MES KYGPPCPSCP ... Check methionine

Example  9: CA- Exendin -PEG Connective  Produce

ALD-PEG-ALD (Shearwater), a polyethylene glycol having a molecular weight of 3.4 kDa having an aldehyde reactor at both ends, was added to 100 mM HEPES (pH 7.5) buffer dissolved in CA-Exendin at a concentration of 12 mg / Was 1: 7.5. Sodium cyanoborohydride (NaCNBH ₃ , Sigma), which is a reducing agent, was added thereto to a final concentration of 30 mM and reacted for 4 hours with slow stirring at 4 ° C. PEG was selectively linked to the amino terminal portion of CA-Exendin, and cation-binding chromatography purification was carried out with the above reaction mixture to obtain a 1: 1 binding product of PEG and CA-Exendin.

The rejoined sample was combined with a source S (GE healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) and 45% ethanol buffer, and the column was washed with 20 mM sodium citrate (pH 2.0), 45% ethanol and 0.25 M CA-Exendin-PEG conjugate was purified with a linear concentration gradient of 7 column volumes to a linear concentration gradient of 0.0625 column volume for 0.5 min, 90% to a concentration of 0% to 10% using potassium chloride buffer .

Example  10: CA- Exendin -PEG And  Met included Fc  Analog Coupling Manufacturing

The CA-Exendin-PEG conjugate prepared in Example 9 was combined with the Fc analog containing methionine produced in Example 7. Specifically, the immunoglobulin Fc region fragment prepared in Example 7 was mixed with the CA-Exendin-PEG conjugate so that the molar ratio of the Fc analog fragment containing CA-Exendin-PEG ligand: Met was 1: 2. The reaction solution was made into 100 mM HEPES (pH 8.2) buffer and 3% (v / v) Triton X-100 was added. NaCNBH3 as a reducing agent was added to a final concentration of 50 mM and reacted at RT for 15 hours with slow stirring. Anion-coupled chromatography and hydrophobic chromatography were performed to remove unreacted materials and byproducts after the coupling reaction and purify the Fc analog domain protein conjugate containing the produced CA-Exendin-PEG-Met. The coupling reaction solution was bound to a source Q (GE healthcare) column equilibrated with 20 mM Bistris (pH 6.0) buffer, and the concentration was adjusted to 0% using 20 mM bistris (pH 6.0) and 0.25 M sodium chloride buffer The Fc analog conjugate containing CA-Exendin-PEG-Met was purified at 143.2 minutes with a linear concentration gradient of 16 column volumes to be 100%. The purified fractions were diluted 3.3 times to a final 50 mM potassium phosphate (pH 6.5) and 5M sodium chloride was added to the diluted sample to give a final 1.2 M sodium chloride. The prepared sample was bound to 19 ml of Source Phenyl (GE healthcare) column equilibrated with 20 mM potassium phosphate (pH 6.5) and 1.2 M sodium chloride buffer, and then eluted with 20 mM potassium phosphate (pH 6.5) buffer at a flow rate of 2 ml / To 100% stepwise to purify the Fc analogue conjugate containing CA-Exendin-PEG-Met.

Example  11: Pharmacokinetics

Exendin-PEG-Met-removed Fc-conjugate (control group) and CA-Exendin-PEG-Met-containing Fc analog (test group) obtained in Example 10 were added to 3 SD rats per group at 5 nmol / Kg. &Lt; / RTI &gt; The control and test groups were bled at 1, 4, 8, 10, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, Blood samples were collected in a 1.5 - ml tube, and coagulated. Then, blood cells were removed by centrifugation on an Eppendorf high - speed microcentrifuge for 10 minutes. The amount of protein in serum was measured by ELISA using antibody against Exendin.

FIG. 3 shows the blood half-life of each sample. The binding protein CA-Exendin-PEG-Fc conjugate prepared using the Met-containing Fc analog domain produced according to the present invention as a carrier was used as a control, CA-Exendin- PEG-Met showed a blood half-life similar to that of the removed Fc.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> HANMI PHARM. CO., LTD. <120> A method for the production of immunoglobulin Fc region          comprising initial Methionine residue <130> KPA151057-KR <160> 20 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 1 forward primer <400> 1 gacatatgtg cccatcatgc cc 22 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 1 reverse primer <400> 2 gactcgagtt tacccagaga cagggag 27 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 2 forward primer <400> 3 gacatatgta tggcccacct tgc 23 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 2 reverse primer <400> 4 gactcgagtt tacccagaga cagggag 27 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 3 forward primer <400> 5 gacatatgaa gtatggccca ccttgc 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 3 reverse primer <400> 6 gactcgagtt tacccagaga cagggag 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 4 forward primer <400> 7 gacatatgga atcaaagtat ggcccac 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Fc analog 4 reverse primer <400> 8 gactcgagtt tacccagaga cagggag 27 <210> 9 <211> 669 <212> DNA <213> Artificial Sequence <220> Human Fc analog 1 (MCPS) <400> 9 atgtgcccat catgcccagc acctgagttc ctggggggac catcagtctt cctgttcccc 60 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 120 gacgtgagcc aggaagaccc tgaggtccag ttcaactggt acgtggacgg cgtggaggtg 180 cataatgcca agacaaagcc gcgggaggag cagttcaaca gcacgtaccg tgtggtcagc 240 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 300 aacaaaggcc tcccatcctc catcgagaaa accatctcca aagccaaagg gcagccccga 360 gaaccacagg tgtacaccct gcccccatcc caggaggaga tgaccaagaa ccaggtcagc 420 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 480 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 540 ttcctctaca gcaggctaac cgtggacaag agcaggtggc aggaggggaa cgtcttctca 600 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 660 ctgggtaaa 669 <210> 10 <211> 223 <212> PRT <213> Artificial Sequence <220> Human Fc analog 1 (MCPS) <400> 10 Met Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val   1 5 10 15 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr              20 25 30 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu          35 40 45 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys      50 55 60 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser  65 70 75 80 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys                  85 90 95 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile             100 105 110 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro         115 120 125 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu     130 135 140 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 145 150 155 160 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser                 165 170 175 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg             180 185 190 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu         195 200 205 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys     210 215 220 <210> 11 <211> 681 <212> DNA <213> Artificial Sequence <220> Human Fc analog 2 (MYGP) <400> 11 atgtatggcc caccttgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 60 ttcctgttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccaggaagac cctgaggtcc agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagg cctcccatcc tccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctctgggtaa a 681 <210> 12 <211> 227 <212> PRT <213> Artificial Sequence <220> Human Fc analog 2 (MYGP) aa <400> 12 Met Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly   1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met              20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln          35 40 45 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val      50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr  65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                  85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile             100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         115 120 125 Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val Ser     130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val             180 185 190 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met         195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     210 215 220 Leu Gly Lys 225 <210> 13 <211> 684 <212> DNA <213> Artificial Sequence <220> Human Fc analog 3 (MKYG) <400> 13 gggaccatc gtcttcctgt tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120 acatgcgtgg tggtggacgt gagccaggaa gaccctgagg tccagttcaa ctggtacgtg 180 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 240 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 300 aagtgcaagg tctccaacaa aggcctccca tcctccatcg agaaaaccat ctccaaagcc 360 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccagga ggagatgacc 420 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 480 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540 tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 600 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660 agcctctccc tgtctctggg taaa 684 <210> 14 <211> 228 <212> PRT <213> Artificial Sequence <220> Human Fc analog 3 (MKYG) aa <400> 14 Met Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu   1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu              20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser          35 40 45 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu      50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr  65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn                  85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser             100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln         115 120 125 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val     130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro                 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr             180 185 190 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val         195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu     210 215 220 Ser Leu Gly Lys 225 <210> 15 <211> 690 <212> DNA <213> Artificial Sequence <220> Human Fc analog 4 (MESK) <400> 15 atggaatcaa agtatggccc accttgccca tcatgcccag cacctgagtt cctgggggga 60 ccatcagtct tcctgttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 120 gaggtcacat gcgtggtggt ggacgtgagc caggaagacc ctgaggtcca gttcaactgg 180 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagttcaac 240 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 300 gagtacaagt gcaaggtctc caacaaaggc ctcccatcct ccatcgagaa aaccatctcc 360 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccaggaggag 420 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 480 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 540 ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg 600 caggagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 660 cagaagagcc tctccctgtc tctgggtaaa 690 <210> 16 <211> 230 <212> PRT <213> Artificial Sequence <220> Human Fc analog 4 (MESK) aa <400> 16 Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu   1 5 10 15 Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp              20 25 30 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp          35 40 45 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly      50 55 60 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn  65 70 75 80 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp                  85 90 95 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro             100 105 110 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu         115 120 125 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn     130 135 140 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 145 150 155 160 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr                 165 170 175 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg             180 185 190 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys         195 200 205 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu     210 215 220 Ser Leu Ser Leu Gly Lys 225 230 <210> 17 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> hinge region 1 <400> 17 Met Cys Pro   One <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> hinge region 2 <400> 18 Met Tyr Gly Pro Pro Cys Pro   1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> hinge region 3 <400> 19 Met Lys Tyr Gly Pro Pro Cys Pro   1 5 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> hinge region 4 <400> 20 Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro   1 5 10

Claims (18)

Preparing a vector comprising a nucleic acid sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; Transforming said vector into prokaryotic cells; Culturing the transformant; And isolating and purifying an immunoglobulin Fc region expressed in aggregate form from the transformant. &Lt; Desc / Clms Page number 12 &gt;
2. The method of claim 1, wherein the immunoglobulin Fc region is an immunoglobulin Fc region in monomeric or dimeric form.
2. The method of claim 1, wherein the immunoglobulin Fc region is unglycosylated.
The method according to 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.
5. The method of claim 4, wherein the IgG is IgGl, IgG2, IgG3 or IgG4.
The method of claim 1, wherein the hinge region comprises an amino acid sequence of SEQ ID NO: 17, 18, 19 or 20.
3. The method of claim 1, wherein the immunoglobulin Fc region comprises one to four selected from the group consisting of CH1, CH2, CH3 and CH4 domains.
2. The method of claim 1, wherein said immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE or IgM.
2. The method of claim 1, wherein each of the domains of the immunoglobulin Fc region is a hybrid of a domain having different origins derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, IgM.
10. The method of claim 9, wherein the immunoglobulin Fc region is a dimeric or multimeric complex consisting of a single chain immunoglobulin of the same origin.
2. The method of claim 1, wherein the immunoglobulin Fc region is an IgG4 Fc fragment.
12. The method of claim 11, wherein said immunoglobulin Fc region is a human unchallenged IgG4 Fc fragment.
10. The method of claim 1, wherein the recombinant immunoglobulin Fc region comprises the amino acid sequence of SEQ ID NO: 10, 12, 14 or 16.
10. The method of claim 1, wherein the nucleic acid sequence encoding the recombinant immunoglobulin Fc region is comprised of a nucleic acid sequence of SEQ ID NO: 9, 11, 13 or 15.
2. The method of claim 1, wherein the vector is pMCPSFc, pMYGPFc, pMKYGFc or pMESKFc.
The method of claim 1, wherein the prokaryotic cell is E. coli.
The method of claim 1, wherein the transformant is BL21 / pMCPSFc, BL21 / pMYGPFc, BL21 / pMKYGFc, or BL21 / pMESKFc.
17. An immunoglobulin Fc region in the form of a monomer or dimer comprising an initiating methionine residue produced by the method of any one of claims 1-17.

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