HK1121768B

HK1121768B - A method for the mass production of immunoglobulin fc region deleted initial methionine residues

Info

Publication number: HK1121768B
Application number: HK09101855.3A
Authority: HK
Inventors: 郑圣烨; 金镇善; 申珍焕; 权世昌; 李宽淳; 宋大海
Original assignee: 韩美科学株式会社
Priority date: 2005-08-16
Filing date: 2006-08-16
Publication date: 2014-01-10

Description

Method for mass-producing immunoglobulin Fc region deleted initial methionine residue

Technical Field

The present invention relates to a method for the large-scale production of monomeric or dimeric immunoglobulin Fc regions free of initial methionine residues by using a recombinant expression vector comprising an Fc region encoding a recombinant immunoglobulin comprising an immunoglobulin hinge region.

Background

With the development of genetic engineering, a large number of protein drugs have been developed and used. However, proteinaceous drugs are susceptible to denaturation or proteolytic degradation in the body and it is difficult to maintain in vivo concentrations and titers for extended periods of time. The improvement in vivo protein stability allows the in vivo concentration of the protein drug to be maintained at an appropriate level, which is very important not only to promote the efficacy of the treatment but also to provide convenience and cost to the patient who needs frequent injections of the protein drug.

Various attempts have been made to enhance the in vivo stability of proteinaceous drugs over a long period of time, for example by changing the protein composition, fusing one protein to another, or attaching suitable multimers to the surface of the protein either chemically or biologically.

One such technique is the preparation of fusion proteins with immunoglobulin Fc fragments.

The Fc fragment mediates effector functions such as Complement Dependent Cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC), and antigen binding ability, which are unique functions of immunoglobulins. The Fc fragment also contains sequences involved in binding to neonatal Fc receptor (FcRn) which act in modulating serum IgG levels by increasing the transport of parent IgG to the neonate and the half-life of the IgG (Ghetie and Ward, Immunology Today 18: 592-598, 1997) and which modulate the interaction between protein A and protein G. Various studies have been conducted through the fusion of the Fc fragment with a therapeutic protein to enhance the stability of the therapeutic protein.

For example, KR 249572 discloses a fusion protein prepared by linking the amino terminus of the Fc region (Fc) of IgG1 heavy chain to the carboxy terminus of a protein (e.g., IL4 receptor, IL7 receptor, G-CSF receptor, or EPO receptor) and producing the resulting fusion protein in mammalian cells. US 5,605,690 describes a fusion protein produced in animal cells, said fusion protein comprising a tumor necrosis factor receptor fused at its carboxy terminus to a human IgG1Fc derivative. In US 5,723,125 and 5,908,626, Tanox inc. also reports a hybrid molecule comprising human interferon alpha or beta linked at its carboxy terminus via a peptide linker to native human IgG4Fc and produced in animal cells. In PCT application WO 00/69913, lexigen inc, native IgG1Fc linked at its carboxy terminus to the amino terminus of human interferon was prepared by genetic recombination without the use of a linker, and the fusion protein was produced in animal cells. U.S. patent application publication No. 20030082679 discloses a fusion protein with an extended serum half-life comprising human G-CSF linked at its carboxy terminus to the amino terminus of IgG1Fc and produced in animal cells. U.S. patent application publication No. 20010053539, US 6,030,613, PCT applications WO 99/02709 and WO 01/03737, and EP0464533B1 disclose Fc fusion proteins having a longer serum half-life than the native protein, which comprise IgG1Fc or an Fc derivative linked at its amino terminus to the carboxy terminus of human EPO, TPO, human growth hormone, or human interferon β via a peptide linker or without a peptide linker, and which are produced in animal cells.

These Fc fusion proteins as described above increase the serum half-life of the target protein, but this also raises problems associated with Fc fragment-mediated effector functions (US 5,349,053). The Fc fusion protein fixes complement or binds Fc γ R-expressing cells through effector functions of the Fc fragment, causing lysis of specific cells and inducing the production and secretion of some inflammation-inducing cytokines, causing unwanted inflammation. Fusion also generates new amino acid sequences in the junction region between the Fc fragment and chaperones, which may induce immune responses if administered for long periods.

In this regard, much effort has been devoted to the preparation of immunoglobulins or immunoglobulin fragments that have a long serum half-life but lack effector function. Co1e et al report that ADCC activity is inhibited when amino acid residues 234, 235 and 237 of CH2 region, which are known to play an important role in binding Fc receptor, are substituted with alanine to produce Fc derivatives with reduced Fc receptor binding affinity (Cole et al, J.Immunol.159: 3613-3621, 1997). However, in all of these variants, the Fc may have enhanced immunogenicity or antigenicity due to the presence of unsuitable amino acids, and may lose the desired Fc function, as compared to the native human Fc fragment.

Among the methods of removing or reducing unwanted effector functions while maintaining high serum concentrations of immunoglobulins, one method is based on the removal of the carbohydrate portion of the immunoglobulin. Derivatives of aglycosylated antibodies (e.g. anti-CD 3 antibodies) may be prepared by substituting the glycosylated residue (asparagine residue at position 297 of CH2 domain) of the antibody with another amino acid, as described in US 5,585,097. The non-glycosylated antibody derivative exhibits reduced effector function, but retains its binding affinity to the FcRn receptor and does not alter its serum half-life. However, this derivative also has a problem in that it may be recognized as a foreign substance by the immune system and rejected due to the generation of a new recombinant construct having an abnormal sequence. U.S. patent application publication No. 20030073164 discloses a method of producing Fc derivatives using e.coli (e.coli) without glycosylation ability to produce therapeutic antibodies lacking effector function.

In US 6,660,843 and U.S. patent application publication nos. 20040044188 and 20040053845, american Amgen inc. describes a human IgG1Fc derivative with an amino acid deletion at the first 5 amino acid residues of the hinge region, fused to the amino or carboxy terminus of a therapeutic protein mimicked by the therapeutic protein or peptide; and producing the fusion protein using an E.coli host. However, this fusion protein without a signal sequence is expressed in the form of inclusion bodies, and thus an additional refolding process is necessary. This protein refolding process can reduce yield, particularly in proteins that exhibit homodimers or heterodimers, significantly reducing dimer formation. When a protein without a signal sequence is expressed in E.coli, a methionine residue is also added to the N-terminus of the expression product due to the characteristics of the protein expression system of E.coli. The aforementioned expression products of Amgen inc. have an N-terminal methionine residue, which may induce an immune response after repeated or excessive administration to the body. Also, since fusion molecules in the form of these fusion proteins are expressed in E.coli by linking a gene encoding a therapeutic protein to an Fc gene, they are difficult to express in E.coli; or if the therapeutic protein is expressed in the form of a fusion protein in E.coli causing a significant reduction or loss of activity, it is difficult to obtain expression in E.coli. Furthermore, because the fusion of two molecules forms a non-naturally occurring abnormal amino acid sequence at the junction region between the two proteins, the fusion protein may be recognized as "non-self" by the immune system and thus induce an immune response.

To solve these problems, the present inventors previously prepared an Fc fragment and a protein-based drug in the form of isolated polypeptides, did not use a fusion method based on genetic recombination, but used the best expression system, and covalently linked two polypeptides to use the Fc fragment as a drug carrier. In this case, it is possible to prepare conjugates of glycosylated polypeptide drugs and non-glycosylated Fc which do not induce unwanted immune responses but have satisfactory properties of physiological drug activity, in vivo duration and stability.

In the above case, since Fc is preferably in a non-glycosylated form, a prokaryotic expression system such as e.coli is used. The protein preparation method using the E.coli expression system has several advantages as follows compared with the conventional method using animal cells. Coli expression vectors can be easily constructed, thus allowing rapid assessment of protein expression. Coli allows for the production of proteins of interest in large quantities at low cost due to its rapid growth rate. Relatively simple expression methods may also be used. Thus, E.coli is more useful than other host cells for commercial production.

After overexpression in E.coli, most of the Fc region exists in the form of inclusion bodies. For this reason, the industry needs to express the Fc region in a water-soluble form in E.coli. EP0227110 discloses the production of an immunoglobulin G1Fc region using only the product expressed in a water-soluble form (cell lysate) after overexpression of the immunoglobulin G1Fc region. However, the yield of immunoglobulin expressed only in a water-soluble form is as low as 15mg/L, which is not valuable for industrial application. Korean patent application No. 0092783, which overcomes the problems encountered in the prior art, describes a new technique for expressing an immunoglobulin Fc region in escherichia coli in a water-soluble form, not in the form of inclusion bodies, by fusing a nucleotide sequence corresponding to the Fc region with a signal sequence of escherichia coli. Based on the expression in E.coli, the protein of interest exists in a soluble form without signal peptide, and its yield increases to 600 mg/L.

The present invention was made by the long and intensive studies of a method for producing an active, non-glycosylated immunoglobulin Fc region that does not generate an immune response by the present inventors, aiming to increase the yield to a level suitable for industrialization, and as a result, it was found that when a nucleotide sequence encoding the immunoglobulin Fc region is expressed in a form in which its N-terminus is fused with a specific hinge region, the immunoglobulin Fc region is expressed in the form of inclusion bodies that finally form dimers or monomers of the immunoglobulin Fc region without an initial methionine residue through solubilization and refolding processes.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a method for mass-producing an immunoglobulin Fc region free of an initial methionine residue, comprising constructing a vector comprising a nucleotide sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; transforming a prokaryotic cell with the vector; culturing the obtained transformant; and isolating and purifying the immunoglobulin Fc region expressed in the form of inclusion bodies from the transformant.

It is another object of the present invention to provide a dimer or monomer of an immunoglobulin Fc region prepared by the above-described method.

Drawings

The above and other objects, features and other advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

FIG. 1 is a photograph of gel electrophoresis showing formation of monomeric and dimeric Fc region fragments from inclusion bodies expressed using an expression vector having nucleotides encoding the Fc region of human immunoglobulin IgG 4.

FIG. 2 shows the ELISA results for the C1q binding capacity of the Fc region of human immunoglobulin IgG 4.

Figure 3 shows the ELISA results of Fc γ RI binding ability of human immunoglobulin IgG Fc region.

Figure 4 shows the ELISA results of Fc γ RIII binding ability of human immunoglobulin IgG Fc region.

Figure 5 shows the ELISA results of FcRn α β 2 binding ability of the Fc region of human immunoglobulin IgG.

FIG. 6 shows the results of serum half-life of EPO-PEG-Fc conjugates prepared using human immunoglobulin IgG Fc region as a carrier.

FIG. 7 is a photograph of a 15% SDS-PAGE gel on which a portion of fermentation broth obtained by growing the microbial transformant of example 2 in a fermentor under conditions allowing expression was run after mixing with an equal volume of 2X protein sample buffer.

FIG. 8 is a photograph of an SDS-PAGE gel in which proteins refolded from inclusion bodies expressed by the transformants of example 2 were separated and bands were shown.

FIG. 9 is a photograph of a 15% SDS-PAGE gel on which a portion of fermentation broth obtained by growing the microbial transformant of example 3 in a fermentor under conditions allowing expression was run after mixing with an equal volume of 2X protein sample buffer.

FIG. 10 is a photograph of a 15% SDS-PAGE gel on which each product expressed and purified in example 3 was run after being mixed with a protein sample buffer without a reducing agent (e.g., DTT or. beta. -mercaptoethanol).

Detailed Description

In one aspect, the present invention relates to a method for mass-producing an immunoglobulin Fc region, comprising constructing a vector comprising a nucleotide sequence encoding a recombinant immunoglobulin Fc region comprising an immunoglobulin hinge region; transforming a prokaryotic cell with the vector; culturing the obtained transformant; and isolating and purifying the immunoglobulin Fc region expressed in the form of inclusion bodies from the transformant.

The present invention relates to a method for mass-producing immunoglobulin Fc region used as a carrier for protein drugs. When an immunoglobulin Fc region is fused at its N-terminus to a hinge region, it is found that the resulting recombinant immunoglobulin Fc region is expressed as an inclusion body, which is then solubilized and refolded into a dimer or monomer in an active form without the initial methionine residue encoded by the initiation codon. It is of importance to the present invention that the hinge region, when fused to an immunoglobulin Fc region, was found to play a key role in processing and refolding this recombinant Fc region into the native sequence form without the initial methionine residue encoded by the initiation codon.

The hinge region capable of allowing the immunoglobulin Fc region to be produced in a recombinant form in a large amount may be a derivative of IgG, IgA, IgM, IgE or IgD, preferably a derivative of IgG such as IgG1, IgG2, IgG3 or IgG4(SEQ ID Nos. 14 to 17), from humans and other animals including goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. The hinge region useful in the present invention may be a full-length hinge region or a fragment thereof. Preferably a hinge region fragment having two or more contiguous amino acid sequences, more preferably containing at least one cysteine residue in the amino acid sequence. Fragments for practical use according to the invention are fragments derived from the hinge region of Ig4 of SEQ ID NO.17, represented by the sequences SEQ ID NO.18, 19, 20 and 21. When the hinge regions of SEQ ID Nos. 18, 19 and 20 are used, the immunoglobulin Fc region may be prepared in a dimeric or monomeric form. The hinge region of SEQ ID NO.21 effectively supports the preparation of immunoglobulin Fc region monomers. In other embodiments of the invention, fragments of the hinge region of IgG1 derived from SEQ ID NO.14, represented by SEQ ID NO.48-55, and fragments of the hinge region of IgG2 derived from SEQ ID NO.15, represented by SEQ ID NO.56-60, are used to produce dimers of the immunoglobulin Fc region.

The immunoglobulin Fc region producible by the present invention may be in a native form isolated from humans and other animals (including goats, pigs, mice, rabbits, hamsters, rats and guinea pigs), or may be a recombinant or derivative thereof obtained from transformed animal cells or microorganisms. Preferably the Fc region of IgG, IgA, IgM, IgE and IgD from humans, or combinations or hybrids thereof. The term "combination" as used herein means that a polypeptide encoding a single chain immunoglobulin Fc fragment of the same origin is linked to a single chain polypeptide of a different origin to form a dimer or multimer. The term "hybrid" as used herein means that within a single chain immunoglobulin Fc fragment there are sequences encoding two or more immunoglobulin Fc fragments of different origin. The immunoglobulin is preferably an Fc region of IgG1, IgG2, IgG3, or IgG4, or a combination or hybrid thereof. The nucleotide sequences encoding the human immunoglobulin Fc region and the amino acid sequences limited to said Fc region that can be used in the present invention may be those encoded by nucleotide sequences from GenBank and/or EMBL databases.

The immunoglobulin Fc region of the present invention includes amino acid sequence derivatives. The term "amino acid sequence derivative" means a sequence in which one or more amino acid residues differ from the wild-type amino acid sequence, which may be naturally occurring or may be artificially generated. Immunoglobulin Fc regions include derivatives resulting from deletions, insertions, non-conservative or conservative substitutions or combinations of these forms. Typically, insertions are made by adding a contiguous amino acid sequence of about 1 to 20 amino acids, or insertions can also be made with longer sequences. Typically, a deletion is in the range of about 1 to 30 amino acid residues. Amino acid exchanges in Proteins and peptides that do not generally alter The activity of The protein or peptide are known in The art (h.neurath, r.l Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are the bidirectional exchanges of 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. These derivatives can be prepared by chemical peptide synthesis methods or recombinant methods based on DNA sequences known in the art (Sambrook et al, Molecular Cloning, Gold spring harbor Laboratory Press, New York, USA, 2d Ed., 1989).

In addition, the immunoglobulin Fc region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like, if necessary.

The immunoglobulin derivatives of the invention are preferably functional equivalents of their native form and thus have similar biological activity; or, if desired, the immunoglobulin derivatives of the invention may be prepared by altering the properties of the native form. Preferably, the derivatives of the immunoglobulin Fc region are proteins with increased structural stability or solubility against heat, pH, etc., or proteins with improved properties with respect to disulfide bond formation, compatibility with the expression host, complement fixation, Fc receptor binding, and antibody-dependent cell-mediated cytotoxicity (ADCC), provided that the resulting derivative does not differ from the native form of human in such a way that the derivative induces an unwanted immune response in humans and animals. A preferred derivative is the IgG1Fc region, in which a particular residue has been altered such that the affinity for Fc receptors that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is reduced. The resulting derivative may contain a deletion of the leucine residue at position 234 of the IgG1 CH2 sequence or a substitution of another amino acid (numbering of amino acid residues is seen in the sequences from the Kobat database). Most preferably, Leu234 is replaced with the amino acid residue phenylalanine at the corresponding position of IgG 4.

According to the present invention, a nucleotide sequence encoding a recombinant immunoglobulin Fc region, wherein the immunoglobulin Fc region is fused to an immunoglobulin hinge region, is prepared. The term "recombinant immunoglobulin Fc region" as used herein means an immunoglobulin Fc region linked at its N-terminus to a hinge region via a peptide bond.

The hinge region to be fused may be selected according to the immunoglobulin Fc region. Preferably a hinge region of the same origin as the immunoglobulin Fc region. In the practical practice of the invention, a nucleotide sequence encoding a recombinant immunoglobulin Fc region consisting of the amino acid sequence shown in SEQ ID NO.7, 9, 11 or 13 was prepared in which the Fc region derived from IgG4 was fused to the hinge region consisting of the amino acid sequence shown in SEQ ID NO.18, 19, 20 or 21. The nucleotide sequences encoding the Fc region of the recombinant immunoglobulins are represented by SEQ ID Nos. 6, 8, 10 and 12, respectively.

In another embodiment, a nucleotide sequence encoding a recombinant immunoglobulin Fc region consisting of the amino acid sequence shown in SEQ ID No.23, 25, 27, 29, 31, 33, 35 or 37 is prepared, wherein the Fc region derived from IgG1 is fused to a hinge region consisting of any of the amino acid sequences shown in SEQ ID nos. 48-55. The nucleotide sequence encoding the Fc region of the recombinant immunoglobulin formed is represented by SEQ ID Nos. 22, 24, 26, 28, 30, 32, 34 and 36.

In another embodiment, a nucleotide sequence encoding a recombinant immunoglobulin Fc region consisting of the amino acid sequence shown in SEQ ID No.39, 41, 43, 45, or 47 is prepared in which the Fc region derived from IgG2 is fused to a hinge region consisting of any of the amino acid sequences shown in SEQ ID nos. 56-60. The nucleotide sequences formed encoding the Fc region of the recombinant immunoglobulins are represented by SEQ ID Nos. 38, 40, 42, 44 and 46.

According to the present invention, there is provided a recombinant expression vector operably linked to a nucleotide sequence encoding a recombinant immunoglobulin Fc region.

The term "recombinant expression vector" as used herein, describing a vector capable of expressing a target protein in a suitable host cell, refers to a genetic construct comprising the necessary regulatory elements to which a gene insert is operably linked in such a way that it can be expressed in the host cell.

The term "operably linked" as used herein refers to a functional linkage between a nucleic acid expression control sequence and a second nucleic acid sequence encoding a target protein in a manner that allows for general function. Operable linkage to the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes widely known in the art. Suitable expression vectors include expression regulatory elements such as promoters, initiation codons, stop codons, polyadenylation signals, and enhancers. The start codon and stop codon are essential for functionality in the individual to whom the genetic construct has been administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible. In addition, expression vectors include selectable markers that allow for selection of host cells containing the vector and replication competent expression vectors that include an origin of replication. In a specific embodiment of the present invention, the following recombinant expression vectors were prepared: pmSCPFc, pmPSCFc, pmCPSFc, pmCPFc, pMEPKFC1, pMSCKFc1, pMDKTFC1, pMCPACF 1, pMPKSFc1, pMCPPFc1, pMPPCFc, pMPCPFc, pmPPCG2Fc, pmPCPG2Fc, pmCPG2Fc, pmCCVG2Fc, and pmCVE2 Fc.

The recombinant expression vector expressing the protein is transformed into a host cell.

For the purposes of the present invention, a host cell is a prokaryotic cell in which no glycosylation takes place. Examples of such prokaryotic cells include Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus (Proteus mirabilis) and Staphylococcus (Staphylococcus), preferably Escherichia coli. Illustrative, non-limiting examples of E.coli strains include BL21(DE3), JM109, DH series, TOP10 and HB 101. More preferably BL21(DE3) strain. Since escherichia coli lacks a system for glycosylation of proteins, it can be used as a host cell in which an immunoglobulin Fc region is produced in a form free of sugar moieties present in the CH2 domain of a native immunoglobulin. The carbohydrate moiety of the immunoglobulin CH2 domain does not affect the structural stability of the immunoglobulin, but causes the immunoglobulin to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) upon binding to Fc receptor-expressing cells and immune cells to secrete cytokines that induce inflammation. The sugar moiety also binds to the C1q moiety of the first complement component C1, resulting in complement fixation. Thus, when the immunoglobulin Fc region is produced in an unglycosylated form and linked to a therapeutic protein, the therapeutic protein can be present in serum for a longer period of time without the unwanted effector functions of immunoglobulins.

Transformation of a recombinant expression vector into a prokaryotic cell can be accomplished by any method known in the art that allows for the introduction of nucleic acids into the cell, and can be performed by selecting an appropriate standard technique depending on the host cell. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO)₄) Precipitate, calcium chloride (CaCl)₂) Precipitation, agitation with silicon carbide fibers, and transformation mediated by PEG-, dextran sulfate, and cationic liposomes.

In a specific embodiment of the present invention, the recombinant expression vectors are individually introduced into E.coli, thereby producing the following transformants: BL21/pmSCPFc (HM11200), BL21/pmPSCFc (HM11201), BL21/pmCPSFc (HM11204), BL 21/pmCPACF (HM11205), BL21/pMEPKFc1(HM11206), BL21/pMSCDFc1(HM11207), BL 21/pMDCTFc 1(HM11208), BL 1/pMFc 1(HM11209), BL 1/pMPKSFc1(HM11210), BL 1/CPPFCfc 1(HM11211), BL 1/pMPPCFc1(HM11212), BL 1/pMPCPFCfc 1(HM11213), BL 1/pmCPCPCPG 21 (pM 11214), BL 1/pmPGG 2 (HM11215), HM 1/pmPMPGG 2 (HM 11272/PMPGM 11272 (HM 11272), and PMCPVG 11272/PMVG 11272 (HM 11272/PMVG) and PMBCG 2 (HM 11272).

The transformant anchoring the recombinant expression vector is cultured by a general method.

The culture conditions can be easily adjusted by those skilled in the art according to the bacterial strain. Typically, the medium used for culturing should contain all the nutrients necessary for cell growth and survival. The medium should contain various carbon sources, nitrogen sources and trace elements. Examples of the carbon source that can be used include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates (such as cellulose), fats (such as soybean oil, sunflower oil, castor oil, and 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). These carbon sources may be used singly or in combination of two or more. Examples of the nitrogen source that can be used include organic nitrogen sources such as peptone, yeast extract, meat extract, malt extract, Corn Steep Liquor (CSL) and soybean whey, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used singly or in combination of two or more thereof. The culture medium may contain a source of phosphorus, including potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts. In addition, the medium may contain a metal salt such as magnesium sulfate or ferrous sulfate. The medium may also include amino acids, vitamins, suitable precursors, and the like. The pH of the culture can be adjusted by adding a compound (e.g., ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid) to the culture by an appropriate method. During the incubation period, antifoams such as polyethylene glycol fatty acid esters may also be used to prevent bubble formation. In order to maintain the culture in a desired state, oxygen or an oxygen-containing gas (e.g., air) may be introduced into the culture. The temperature of the culture is generally 20 ℃ to 45 ℃, preferably 25 ℃ to 45 ℃. Large scale protein production can also be performed using fermentors. Protein production using a fermentor should take into account several factors including the growth rate of the host cell and the level of protein expression. Protein expression can be induced by adding, for example, IPTG to the culture medium under suitable culture conditions.

The immunoglobulin Fc region overexpressed as inclusion bodies can be purified by a general technique. By disrupting the cells with a French press, sonicator or the like; collecting only water-insoluble inclusion bodies containing an immunoglobulin Fc region by centrifugation; solubilizing and denaturing the collected fractions into their refolded form with a refolding agent such as urea, guanidine, arginine, cysteine (cysteine), β -mercaptoethanol, etc.; and purification of the refolded fusion protein by dialysis, various chromatographies (e.g., gel filtration, ion exchange, and reverse phase column chromatography), and ultrafiltration (alone or in combination of the above methods). In general, this refolding process is very complex and is known to produce very low refolding yields, and it is believed that refolded proteins can only have lower activity than water-soluble proteins.

However, the method of the present invention can overcome the above problems and produce an active immunoglobulin Fc region without an initial methionine residue on a large scale. In general, when a foreign protein is expressed and produced in E.coli, the protein has an initiation methionine residue encoded by an initiation codon. Repeated or excessive administration of a protein product having an initial methionine to a human may elicit an immune response sufficient to reduce its therapeutic effect or cause toxicity. However, when the recombinant immunoglobulin Fc region of the present invention is expressed in E.coli, it was found that the initial methionine residue is cleaved off by aminopeptidase, an endogenous cytoplasmic enzyme, as determined by N-terminal sequencing analysis (Adams et al, J.mol.biol.33: 571-589, 1968; Takeda, Proc.Natl.Acad.Sci.USA 60: 1487-1494, 1968). The activity of such aminopeptidases is known to depend on the sequence and structure of the Protein of interest (Morschell et al, J.biol.chem.265: 19638-. When the hinge region is fused to an immunoglobulin Fc region, the hinge region is affected by aminopeptidase so that the initial methionine is processed to an extent depending on the amino acid sequence of the hinge region.

Since the nature of the hinge region determines the post-translational modification of the protease, the ratio of dimer to monomer can be effectively controlled by selecting the appropriate hinge region. In addition, mismatches through cysteines in disulfide bonds can prevent the formation of precise dimers when inclusion bodies are refolded. However, the method of the present invention ensures the formation of precise disulfide bonds, resulting in the formation of active dimers.

In addition, the present invention can produce an immunoglobulin Fc region on a larger scale than conventional methods. For example, according to the method of European patent EP0227110, an immunoglobulin Fc region in which the G1Fc region is overexpressed can be produced at a yield of 15mg/L, and the G1Fc region is purified only from a cell lysate containing its water-soluble form; the immunoglobulin Fc region can be produced in a yield of 50-600mg/L according to the method of KR 0092783, wherein the immunoglobulin Fc region fused with the E.coli signal sequence is expressed in a water-soluble form rather than as inclusion bodies. However, the present invention can produce the immunoglobulin Fc region in a yield of up to 3-6g/L by purifying inclusion bodies of the recombinant immunoglobulin Fc region containing the hinge region. Thus, the method of the present invention ensures a highly useful system for the production of immunoglobulin Fc regions on an industrial scale with higher yields than conventional methods.

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

The immunoglobulin Fc region produced in prokaryotic cells such as E.coli according to the method of the present invention is not specifically limited to industrial applications. One exemplary application is as a carrier for forming conjugates with certain drugs. The construction of a conjugate comprising an immunoglobulin Fc region linked to a drug is not particularly limited. For example, different proportions of the immunoglobulin Fc region and the drug may be linked together, and the linkage may be mediated, for example, by a linker.

The drugs include polypeptides, compounds, extracts, and nucleic acids. Polypeptide (generally having the same meaning as "protein") drugs are preferred. Examples of linkers useful in the present invention include peptide linkers and non-peptide linkers, preferably non-peptide linkers, more preferably non-peptide polymers. A preferred example of an immunoglobulin heavy chain is Fc.

If it is desired to increase the serum half-life, any physiologically active polypeptide may be used as a chaperone for the immunoglobulin Fc region prepared according to the present invention to form a conjugate without particular limitation. Such physiologically active polypeptides include those useful for treating or preventing human diseases, including cytokines, interleukins, interleukin binding proteins, enzymes, antibodies, growth factors, transcription regulatory factors, clotting factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists, and derivatives and analogs thereof.

Specifically, non-limiting examples of such physiologically active polypeptides include human growth hormone, growth hormone releasing peptide, interferons and interferon receptors (e.g., interferon- α, - β and- γ, water 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-1-receptor, IL-receptor, IL-4 receptor, etc.), enzymes (e.g., glucocerebrosidase, iduronate-2-sulfatase), alpha-galactosidase-A, agalsidase alpha and beta, alpha-L-iduronidase (alpha-L-iduronase), butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, etc.), interleukins and cytokine binding proteins (e.g., IL-18bp, TNF binding proteins, etc.), macrophage activating factor, macrophage peptide, B cytokine, T cytokine, protein A, allergy inhibitor, cytonecrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor inhibitor, metastatic growth factor, alpha-1 trypsin inhibitor, etc, Albumin, alpha lactalbumin, apolipoprotein E, erythropoietin, hyperglycosylated erythropoietin, angiogenin, hemoglobin, thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX and factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C response protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, atriopeptide, cartilage inducing factor, elcatonin, connective tissue activating factor, tissue factor channel inhibitor, follicle stimulating hormone, luteinizing hormone releasing hormone, luteinizing hormone, Nerve growth factor (e.g., nerve growth factor, ciliary neurotrophic factor, axogenesis factor-1, brain natriuretic peptide, glial cell derived neurotrophic factor, nerve growth factor, neurotropic inhibitor factor, neurotrophic factor, neuturin, etc.), parathyroid hormone, relaxin, secretin, growth regulators, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autocrine motonectin, lactoferrin, tubocurarine, receptors (e.g., TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, B cell activator receptor, etc.), receptor antagonists (e.g., IL1-Ra, etc.), cell surface antigens (e.g., CD2, 3, 4, 5,7, 11a, 11B, CD2, 3, 4, 5,7, 11B, beta, etc.), cell surface antigens, 18. 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., scFv, Fab ', F (ab') 2, and Fd), and virus-derived vaccine antigens. Physiologically active polypeptides useful in the invention can be in the native form, can be produced by genetic recombination using prokaryotic cells (e.g., E.coli) or eukaryotic cells (e.g., yeast cells, insect cells, and animal cells), or can be derivatives having one or more amino acid mutations but exhibiting the same biological activity as the native form.

In a preferred embodiment of the present invention, the immunoglobulin Fc region fragment produced using HM11201 transformant and human Erythropoietin (EPO) are linked with polyethylene glycol to provide an EPO-PEG-immunoglobulin Fc region protein conjugate. The protein conjugate was found to exhibit a longer serum half-life than native EPO and aranesp (amgen), a second generation EPO known to have an increased serum half-life. Thus, the immunoglobulin Fc region without an initial methionine residue obtained from inclusion bodies using a hinge region according to the present invention can be used to increase the serum half-life and physiological activity of a physiologically active polypeptide linked thereto without increasing the risk of inducing an immune response.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.

Example 1: construction of expression vector for human immunoglobulin IgG4Fc region, expression and purification of IgG4Fc region, and N-terminal sequence analysis

<1-1> construction of expression vector for IgG4Fc region

To clone the heavy chain Fc region comprising the hinge region of human immunoglobulin IgG4, RT-PCR was performed using RNA from human blood cells as a template, as follows: first, total RNA was isolated from approximately 6ml of blood using the Qiamp RNA blood kit (Qiagen) and gene amplification was carried out using total RNA as a template with the aid of a one-step RT-PCR kit (Qiagen). Genes having different N-terminal sequences were amplified using primer pairs represented by SEQ ID NO.1 and 2, 3 and 2, 4 and 2, and 5 and 2. To facilitate the subsequent gene cloning operation, NdeI recognition site and the initiation codon ATG, which are necessary for protein expression, were introduced into the 5 'primers of SEQ ID Nos. 1, 3, 4 and 5, and a BamHI recognition site containing a stop codon was introduced into the 3' primer of SEQ ID No. 2. The amplified Fc region product was digested with Ndel and Hind III and inserted into pET22b (Novagen) treated with the same restriction enzymes to give the corresponding recombinant plasmid. These plasmids were designed to have a portion of the entire amino acid sequence Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro of the hinge region of IgG4, as described below.

The plasmid containing the gene amplified with SEQ ID NO.1 and 2, which was determined to have the sequence of SEQ ID NO.6 (corresponding to the amino acid sequence of SEQ ID NO. 7) by base sequencing, was called pmSCPFc and anchored to a DNA sequence encoding the N-terminal amino acid sequence whose starting sequence was Met-Ser-Cys-Pro. The plasmid containing the gene amplified by SEQ ID NO.3 and 2, which was determined to have the sequence of SEQ ID NO.8 (corresponding to the amino acid sequence of SEQ ID NO. 9) by base sequencing, was called pmPSCFc and was anchored to a DNA sequence encoding the N-terminal amino acid sequence whose starting sequence was Met-Pro-Ser-Cys-Pro. The plasmid containing the gene amplified by SEQ ID NO.4 and 2, which was determined to have the sequence of SEQ ID NO.10 (corresponding to the amino acid sequence of SEQ ID NO. 11) by base sequencing, was called pmCPSFc and was anchored to a DNA sequence encoding the N-terminal amino acid sequence whose starting sequence was Met-Cys-Pro-Ser-Cys-Pro. A plasmid containing the gene amplified by SEQ ID NO.5 and 2, which was determined to have the sequence of SEQ ID NO.12 (corresponding to the amino acid sequence of SEQ ID NO. 13) by base sequencing, was called pmCPFc and was anchored to a DNA sequence encoding the N-terminal amino acid sequence whose starting sequence was Met-Cys-Pro.

The expression vectors were transformed into E.coli BL21(DE3) to prepare transformants designated as BL21/pmSCPFc (HM11200), BL21/pmPSCFc (HM11201), BL21/pmCPSFc (HM11204), and BL21/pmCPAFC (HM11205), respectively. The transformants BL21/pmSCPFc (HM11200) and BL21/pmPSCFc (HM11201) were deposited at the Korean Collection of microorganisms (KCCM) at 2005, 6/20, under accession numbers KCCM-10659P and KCCM-10660P, respectively; the transformants BL21/pmCPSFc (HM11204) and BL21/pmCPAFC (HM11205) were deposited on KCCM at 7/28/2005 with accession numbers KCCM-10665P and KCCM-10666P, respectively.

<1-2> expression and purification of IgG4Fc

The bacterial transformants prepared in example <1-2> were inoculated into fermentors (Marubishi Co.), respectively, and allowed to grow, followed by determining whether they expressed an immunoglobulin Fc region fragment.

First, each transformant was grown in 100ml of LB medium, stirred overnight, and inoculated into a fermenter for large-scale culture. The fermentor was maintained at either 28 ℃ or 35 ℃. To avoid the transition from aerobic to anaerobic conditions, the cultures were aerated with 20vvm of air and stirred at 500 rpm. To compensate for nutrient deficiencies for cell growth during fermentation, the culture is supplemented with glucose and yeast extract depending on the fermentation state of the bacteria. When OD of culture₆₀₀When the value reached 80, the inducer IPTG was added to the culture to induce protein expression. The culture was further cultured for 40 to 45 hours, so that the OD at 600nm was increased to 100 to 120.

Expression of immunoglobulin Fc, formation of inclusion bodies, and formation of dimers of the expressed Ig Fc in the e.coli transformants were examined as follows. To study the overall intracellular expression of the immunoglobulin Fc region, a portion of the fermentation broth was mixed with an equal volume of 2x protein sample buffer and electrophoresed on a 15% SDS-PAGE gel (Criterion gel, Bio-Rad). As a result, it was observed that the immunoglobulin Fc was overexpressed in all transformants formed. Then, the cells were disrupted by an ultrasonicator (Misonix Co.). The cell lysate thus obtained is centrifuged to separate the water-soluble substance from the water-insoluble substance. Most of the overexpressed material was found to be present as inclusion bodies, as determined by electrophoresis on 15% SDS-PAGE. Inclusion bodies were subjected to the following refolding process to examine the extent of Fc refolding and whether and to what extent the Fc region of the dimer was formed. 10g of the fermentation broth was sonicated in 100mL of lysis buffer (10mM Tris, pH9.0, 1mM EDTA, 0.5% Triton X-100, 0.2M NaCl) to disrupt the cells. The cell lysate was separated into a water-soluble fraction and a water-insoluble fraction in the form of inclusion bodies by centrifugation at 10,000rpm for 20 minutes. 2g of this inclusion body was dissolved in a mixture of 20mL of 1M Tris (pH9.0) and 20mL of a solubilization buffer (6M guanidine, 50mM Tris), and allowed to react at 4 ℃ for 30 minutes with gentle stirring. After completion of the reaction, the inclusion body solution was mixed with 10 volumes of refolding buffer (2M urea, 50mM Tris, 0.25M arginine, 3mM cysteine, pH9.0) overnight with gentle stirring. To this mixture was added a protein sample buffer without any reducing agent such as DTT or β -mercaptoethanol, followed by electrophoresis on 15% SDS-PAGE (Criterion Gel, Bio-Rad). Protein bands are visualized with dyes such as coomassie blue. FIG. 1 is a photograph of a gel in which proteins refolded from inclusion bodies expressed by the transformant HM11201 at 32 deg.C (lane 1) and 28 deg.C (lane 2), HM11200 at 28 deg.C (lane 3) and 32 deg.C (lane 4), HM11204 at 28 deg.C (lane 5) and 32 deg.C (lane 6), and HM11205 at 32 deg.C (lane 7) and 28 deg.C (lane 8) were run in an electric field together with Fc protein purified by a conventional method from E.coli (lane C) as a control. As can be seen from fig. 1, the major part of the total protein is the Fc protein, most of which is present as a dimer after refolding. However, the ratio of dimers and monomers of the Fc protein in each transformant varies depending on the N-terminal amino acid sequence expressed by the transformant. For example, most of the HM11201 Fc protein (starting with Met-Pro-Ser-Cys-Pro-CH2-CH 3) is present in dimeric form. Almost all of the Fc protein of HM11205 (starting with Met-Cys-Pro-CH2-CH 3) exists as a monomer, but not as a dimer. This is believed to be due to the fact that the processing specificity of aminopeptidases in E.coli host cells differs depending on the N-terminal sequence of Fc.

<1-3> N-terminal sequence analysis

The Fc region fragment of the dimer resulting from the refolding of the inclusion bodies differs in amino acid sequence from the wild type due to the presence of the initiating methionine residue. To determine whether methionine residues were processed by protease of E.coli, the N-terminal amino acid sequence of the protein was analyzed by Korea's basic scientific research institute. Samples for N-terminal amino acid sequence analysis were prepared as follows.

First, a PVDF membrane (Bio-Rad) was soaked in methanol for about 2 to 3 seconds to activate the membrane, and the membrane was sufficiently humidified with a blocking buffer (170mM glycine, 25mM Tris-HCl (pH 8.0), 20% methanol). Protein samples separated on non-reducing SDS-PAGE gels prepared in examples <1-2> were transferred to PVDF membrane for about 1 hour using a blotting kit (Hoefer Semi-Dry Transfer unit, Amersham). The proteins transferred to the PVDF membrane were stained with the protein dye Coomassie blue R-250(Amnesco) for a short period (3-4 seconds) and washed with destaining solution (water: acetic acid: methanol: 5: 1: 4). Then, the protein-containing membrane fragments were cut with scissors and subjected to N-terminal sequence analysis.

As a result, it was found that the N-terminal sequence of IgG4Fc protein having a hinge region was Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys Pro-Ser-Cys-Pro-CH2-CH 3. The amino acid sequence and N-terminal sequence of the protein expressed in the transformants are given in Table 1 below.

TABLE 1

Transformant	N-terminal sequence	Results of sequence analysis
				IIPolymer	Monomer
HM11200	Met-Ser-Cys-Pro-CH2-	Ser-Cys-Pro-CH2	Pro-CH2
				HMl1201	Met-Pro-Ser-Cys-Pro-CH2-	Pro-Ser-Cys-Pro- CH2	Pro-Ser-Cys- Pro-CH2
HM11204	Met-Cys-Pro-Ser-Cys-Pro-CH2	Pro-Ser-Cys-Pro- CH2	mixed
				HM11205	Met-Cys-Pro-CH2-CH3		Pro-CH2

[0074] Data from amino acid sequencing analysis revealed that the Fc fragment refolded from inclusion bodies produced by the e.coli transformants of the invention was processed to have the correct N-terminal sequence without initiating methionine. Protein products that remain monomeric even after refolding lose cysteine residues and therefore are unable to form dimers. In addition, it is apparent from FIG. 1 thatThe monomeric moiety in the refolded Fc fragment differs from transformant to transformant, and no dimer is present in HM 11205. These results indicate that the amino acid sequence of the N-terminal site has a great influence on the processing of the N-terminal, and thus a protein having a desired N-terminal sequence can be obtained by controlling the N-terminal sequence. As revealed by the following experiments, even if proteins have the same amino acid sequence, they can be processed differently depending on the culture conditions, particularly the culture temperature, of E.coli host cells. When HM11200 was grown at low temperature (28 ℃ C. to 32 ℃ C.), it expressed the same amount of Fc fusion protein in soluble form as in inclusion body form. The soluble form of the Fc fusion protein exists as a monomer without the N-terminal amino acid sequence Met-Ser-Cys. Thus, the present inventors recognized that a controlled ratio of monomeric and dimeric immunoglobulin Fc fragments can be obtained by regulating the N-terminal amino acid sequence of the Fc fusion protein and the culture conditions of the host cell.

To quantitatively determine the expression of the immunoglobulin Fc region in e.coli transformants, the immunoglobulin Fc region of the refolding solution was purified using a protein a affinity column known to have strong immunoglobulin affinity as described below.

The inclusion bodies collected by centrifugation were refolded and then purified by column chromatography. 5ml of protein A affinity column (Pharmacia) was equilibrated with PBS, and then the cell lysate was loaded onto the column at a flow rate of 5 ml/min. Unbound protein was washed off with PBS and bound protein was eluted with 100mM citric acid (pH 3.0). The collected fractions were desalted using a HiPrep26/10 desalting column (Pharmacia) with 10mM Tris buffer (pH 8.0). Then, a second anion exchange column chromatography was performed using 50ml Q HP 26/10 column (Pharmacia). The preliminarily purified recombinant immunoglobulin Fc region was loaded on a Q-Sepharose HP 26/10 column (Pharmacia), and the column was eluted with a linear gradient (0-0.2M NaCl) in 10mM Tris buffer (pH 8.0) to obtain a fraction of high purity. The expression level of the recombinant Ig Fc region was determined after partial purification with a protein A affinity column, and the results are given in Table 2 below.

TABLE 2

Plasmids	Transformant	Expression yield after protein A purification
			pmSCPFc	HM11200	5-6gL
pmPSCFc	HM11201	4-5gL
			pmCPSFc	HM11204	4-5gL
pmCPAFc	HM11205	3-4gL

Example 2 construction of expression vector for human immunoglobulin IgG1Fc region, expression and purification of IgG1Fc region, and N-terminal sequence analysis

Construction of <2-1> IgG1Fc region expression vector

In order to clone the heavy chain Fc region comprising the hinge region of human immunoglobulin IgG1, RT-PCR was performed in the same manner as in example <1-1 >. To amplify genes with different N-terminal sequences, the following primers were used.

TABLE 3

5' primer sequences used

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

For the 3 ' primer, it has the sequence 5'-CGC GGA TCC TCA TTT ACC CGGAGA CAG GGA GAG GCT CTT C-3' and is used to amplify all genes with different N-terminal sequences. To facilitate the subsequent gene cloning process, Nde I recognition sites were introduced into each 5 'primer and BamHI recognition sites were introduced into the 3' primers. The Fc region product amplified by the primer pair was inserted into a vector to obtain recombinant plasmids each having a part of the entire amino acid sequence Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys-Pro of the hinge region of IgG1 as follows. A plasmid containing the gene amplified with the MEPK primer, designated pMEPKFc1, anchored to the DNA sequences of CH2 and CH3 encoding IgG1 with the starting sequence Met-Glu-Pro-Lys, was determined to have the sequence of SEQ ID No.22 (corresponding to the amino acid sequence of SEQ ID No. 23) by base sequencing analysis. The plasmid containing the gene amplified with the MSCD primer, which was determined to have the sequence of SEQ ID No.24 (corresponding to the amino acid sequence of SEQ ID No. 25) by base sequencing analysis, was designated pMSCKFc1 and was anchored to the DNA sequences of CH2 and CH3 encoding IgG1 with the starting sequence Met-Ser-Cys-Asp. A plasmid containing the gene amplified by the MDKT primer, designated pMDKTFC1, anchored to the DNA sequences encoding CH2 and CH3 of IgG1 with the starting sequence Met-Asp-Lys-Thr, was determined by base sequencing analysis to have the sequence of SEQ ID No.26 (corresponding to the amino acid sequence of SEQ ID No. 27). The plasmid containing the gene amplified with the MCPA primer, designated pMCPACF 1, anchored to the DNA sequences of CH2 and CH3 encoding IgG1 with the starting sequence Met-Cys-Pro, was determined by base sequencing analysis to have the sequence of SEQ ID No.28 (corresponding to the amino acid sequence of SEQ ID No. 29). The plasmid containing the gene amplified with the MPKS primer, which was determined to have the sequence of SEQ ID No.30 (corresponding to the amino acid sequence of SEQ ID No. 31), was designated pMPKSFc1, and was anchored to the DNA sequences of CH2 and CH3 encoding IgG1 with the starting sequence Met-Pro-Lys-Ser. The plasmid containing the MCPP-amplified gene, designated pMCPPFc1, anchored to the DNA sequences of CH2 and CH3 encoding IgG1 with the starting sequence Met-Cys-Pro-Pro, was determined by base sequencing analysis to have the sequence of SEQ ID No.32 (corresponding to the amino acid sequence of SEQ ID No. 33). The plasmid containing the MPPC primer amplified gene, designated pMPPCFc, anchored to the DNA sequences of CH2 and CH3 encoding IgG1 with the starting sequence Met-Pro-Pro-Cys, was base sequenced to determine the sequence of SEQ ID No.34 (corresponding to the amino acid sequence of SEQ ID No. 35). The plasmid containing the gene amplified with the MPCP primer, which was determined to have the sequence of SEQ ID No.36 (corresponding to the amino acid sequence of SEQ ID No. 37) by base sequencing analysis, was designated pMPCPFc, anchored to the DNA sequences of CH2 and CH3 of IgG1 whose starting sequence was Met-Pro-Cys-Pro. The expression vectors were transformed into E.coli BL21(DE3) to prepare transformants designated BL21/pMEPKFc1(HM11206), BL21/pMSCDFc1(HM11207), BL 21/pMDCTFC 1(HM11208), BL 21/pMCPFc 1(HM11209), BL21/pMPKSFc1(HM11210), BL21/pMCPPFc1(HM11211), BL21/pMPPCFc1(HM11212) and BL21/pMPCPFc1(HM11213), respectively.

<2-2> expression and purification of IgG1Fc

The bacterial transformants prepared in example <2-1> were inoculated into fermentors (Marubishi Co.) respectively, and allowed to grow, as with IgG4, followed by determining whether they expressed immunoglobulin Fc region fragments.

First, each transformant was grown in 100ml of LB medium, stirred overnight, and inoculated into a fermenter for large-scale culture. The fermentor was maintained at either 28 ℃ or 35 ℃. To avoid the conversion of aerobic to anaerobic environment, the cultures were aerated with 20vvm air and operated at 500rpmAnd (4) stirring. To compensate for nutrient deficiencies for cell growth during fermentation, the culture is supplemented with glucose and yeast extract depending on the fermentation state of the bacteria. When OD of culture₆₀₀When the value reached 80, the inducer IPTG was added to the culture to induce protein expression. The culture was further cultured for 40 to 45 hours, so that the OD at 600nm was increased to 100 to 120.

Expression of immunoglobulin Fc, formation of inclusion bodies, and formation of expressed Ig Fc dimers in e.coli transformants were examined as follows. To study the overall intracellular expression of the immunoglobulin Fc region, the fermentation broth was aliquoted before and after induction.

A portion of the fermentation broth was mixed with an equal volume of 2 Xprotein sample buffer under the following reducing conditions and electrophoresed on a 15% SDS-PAGE gel (Criterion gel, Bio-Rad). The results of the electrophoresis are given in FIG. 7. Lane 1 is a control of IgG4Fc, while lanes 2 to 4 show the expression level of HM11208 transformant over time, and lanes 5 to 7 are the expression level of HM11206 transformant over time. Lanes 8 to 13 show the expression levels of the HM11207, HM11212, HM11209, HM11210, HM11213, and HM11211 transformants, respectively. As shown in figure 7, a single 30kDa band (Fc region) was very clearly shown in all the samples subjected to IPTG induction, which was not observed before IPTG induction, indicating that recombinant IgG1Fc region was expressed, unlike the G4Fc control. The Fc region is also overexpressed, at least in an amount of about 30% of the total amount of protein expressed.

To quantitatively determine the expression of the immunoglobulin Fc region in e.coli transformants, the immunoglobulin Fc region of the refolding solution was purified using a protein a affinity column known to have strong immunoglobulin affinity in the same manner as for IgG4 Fc.

Among the transformants, the pMSCDFc plasmid transformant was determined to have the highest expression rate, reaching 340mg per 10g of inclusion body, while the pMDTFc, pMEPKFc, pMPPCFc and pMPCPFc transformants showed expression rates of 133.3mg, 159mg, 110mg and 120mg, respectively.

The amount of dimeric IgG1Fc in the expression product was determined in the same manner as used for determining the amount of dimeric IgF4 Fc. Cells in the fermentation broth were disrupted with an sonicator (Misonix Co.). The obtained cell lysate is centrifuged to separate the water-soluble substance from the water-insoluble substance. Most of the overexpressed material was found to be present as inclusion bodies, as determined by 15% SDS-PAGE gel electrophoresis. Inclusion bodies were refolded to examine the extent of Fc refolding and whether a dimeric Fc region was formed and to a corresponding extent. The refolded Fc protein was purified using a protein A affinity column and mixed with a protein sample buffer without reducing agents such as DTT or β -mercaptoethanol, followed by electrophoresis on 15% SDS-PAGE (Criterion Gel, Bio-Rad). The protein bands are visualized with a dye such as coomassie blue.

FIG. 8 is a photograph of a gel running in an electric field under non-reducing conditions from protein isolates of inclusion body weight-folded protein A columns expressed by the transformant HM11208 (lane 1), the transformant HM11206 (lane 2), and the transformant HM11207 (lane 4), the transformant HM11212 (lane 5), and the transformant HM11213 (lane 7), and IgG4Fc protein as a control (lanes 3, 6, and 8). As shown in fig. 8, all IgG1Fc fragments used in this assay were found to form dimers, although the amount of dimers varied to some extent.

<2-3> N-terminal sequence analysis

Like IgG4Fc, the N-terminal amino acid sequence determines post-translational processing, such as whether the initiating methionine residue is retained or whether the initiating methionine residue is processed correctly, or whether the initiating methionine together with other amino acid residues results in an amino acid sequence that differs from the desired amino acid sequence. To examine whether methionine residues were processed by e.coli protease, different N-terminal amino acid sequences of IgG1Fc region were analyzed by seoul basic scientific research institute in korea. The results of the analysis are summarized in table 4 below.

TABLE 4

Transformant	N-terminal sequencing results (dimer)
		HM11208	Met
HM11206	Met
		HM11207	Ser
HM11212	Pro
		HM11213	Pro

As shown in table 4, the initial methionine residues in transformants HM11208 and HM11206 were still unprocessed, in which the IgG1Fc region in dimeric form was overexpressed, whereas the fermentation products of HM11207, HM11212 and HM11213 did not have the initial methionine residues due to correct post-translational processing.

In summary, the data obtained by the above experiments indicate that when IgG1Fc region is expressed in E.coli, its N-terminal sequence determines expression, expression level, dimer proportion, and its N-terminal processing; and the Fc region free of the initial methionine residue can be produced on a large scale by using the N-terminal sequence. The IgG1Fc region obtained by the present invention can be used for increasing the serum half-life and physiological activity of the physiologically active polypeptide connected with the IgG1Fc region, and has no immune response induced by the addition of exogenous amino acid residues.

Example 3: construction of expression vector for human immunoglobulin IgG2Fc region

<3-1> construction of expression vector for IgG2Fc region

To clone the heavy chain Fc region comprising the hinge region of IgG2, RT-PCR was performed in the same manner as for the IgG4Fc region. In order to amplify genes having different N-terminal sequences, the following primers were used.

TABLE 5

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

The 3 ' primer has the sequence 5'-CGC GGA TCC TCA TTT ACC CGG AGA CAGGGA GAG GCT CTT C-3' and is used to amplify all genes with different N-terminal sequences. To facilitate the subsequent gene cloning process, Nde I recognition sites were introduced into each 5 'primer and BamHI recognition sites were introduced into the 3' primers. The Fc region product amplified by the primer set was inserted into a vector to obtain recombinant plasmids each having a part of the entire amino acid sequence G1u-Arg-Lys-Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys-Pro of the IgG1 hinge region. The plasmid containing the gene amplified with the G2MPPCS primer, designated pmPPCG2Fc, anchored to the DNA sequences of CH2 and CH3 encoding IgG2 with the starting sequence Met-Pro-Pro-Cys, was determined by base sequencing analysis to have the sequence of SEQ ID No.38 (corresponding to the amino acid sequence of SEQ ID No. 39). The plasmid containing the gene amplified with the G2MPCPSS primer, designated pmPCPG2Fc, anchored to the DNA sequences of CH2 and CH3 encoding IgG2 with the starting sequence Met-Pro-Cys-Pro, was determined by base sequencing analysis to have the sequence of SEQ ID No.40 (corresponding to the amino acid sequence of SEQ ID No. 41). The plasmid containing the gene amplified by the G2MCPSS primer, which was determined to have the sequence of SEQ ID NO.42 (corresponding to the amino acid sequence of SEQ ID NO. 43) by base sequencing analysis, was designated pmCPG2Fc, and was anchored to the DNA sequences of CH2 and CH3 encoding IgG2 with the starting sequence Met-Cys-Pro. The plasmid containing the gene amplified by the G2MCCVSS primer, which was determined to have the sequence of SEQ ID No.44 (corresponding to the amino acid sequence of SEQ ID No. 45) by base sequencing analysis, was designated pmCCVG2Fc, and was anchored to the DNA sequence of CH2 and CH3 encoding IgG2 with the starting sequence Met-Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys-Pro. The plasmid containing the gene amplified by the G2MCVESS primer, designated pmCVEG2Fc, anchored to the DNA sequence encoding CH2 and CH3 of IgG2 with the starting sequence Met-Cys-Val-Glu-Cys-Pro-Pro-Cys-Pro, was determined by base sequencing analysis to have the sequence of SEQ ID No.46 (corresponding to the amino acid sequence of SEQ ID No. 47). The expression vectors were transformed into E.coli BL21(DE3) to prepare transformants designated as BL21/pmPPCPG2Fc (HM11214), BL21/pmPCPG2Fc (HM11215), BL21/pmCPG2Fc (HM11216), BL21/pmCCVG2Fc (HM11217) and BL21/pmCVEG2Fc (HM11218), respectively.

<3-2> expression, purification and N-terminal sequence analysis of IgG2Fc

The bacterial transformants prepared in example <3-1> were inoculated into fermentors (Marubishi Co.) respectively, and allowed to grow, as with IgG4, followed by determining whether they expressed immunoglobulin Fc region fragments. The culture conditions were not significantly different from those set for IgG4 Fc. The IgG2Fc region fragment was found to be overexpressed under various conditions (including temperature, medium composition, inducer concentration, etc.) as determined by SDS-PAGE under reducing conditions. FIG. 9 shows the results of a 15% SDS-PAGE electrophoresis of fermentation broth mixed with an equal volume of 2X protein sample buffer. The IgG4Fc fragment of lane 1 was used as a control, while the fragments expressed by HM11214, HM1215, HM11216, HM11217 and HM11218 were run in lanes 2 to 6, respectively. As shown in fig. 9, all 5 transformants used in this experiment over-expressed the Fc fragment.

The content of dimer IgG4Fc in the expression product was measured in the same manner as described above. The cells in the fermentation broth are disrupted and the water-insoluble material of the cell lysate is refolded, after which only the Fc region fragment is purified using a protein a affinity column. The purified expression products were mixed with protein sample buffer without reducing agent (e.g., DTT or. beta. -mercaptoethanol) and the expression products were separated on 15% SDS-PAGE (criterion gel, Bio-Rad). The protein bands are visualized with a dye such as Coomassie Brilliant blue. FIG. 10 shows the electrophoresis results. The IgG4Fc fragment of lanes 1 and 7 was used as a control, and dimers of the fragments from HM11214, HM11215, HM11216, HM11217 and HM11218 were observed in lanes 2 to 6. As can be seen from the data of FIG. 10, although the expression products of the transformants differ from each other in N-terminal sequence or expression conditions, they can form dimers.

To examine whether methionine residues were processed by e.coli protease, different N-terminal amino acid sequences of dimeric IgG4Fc region were analyzed by seoul basic scientific research institute in korea. The starting methionine was removed from the products of the HM11214 and HM11215 transformants, both of which were proline residues at position 2.

It is apparent from these experiments that the IgG2Fc region can be expressed on a large scale in E.coli. In addition, the data obtained in the above experiments indicate that the N-terminal sequence of the IgG1Fc region determines expression, expression level, dimer proportion, and N-terminal processing thereof; the Fc region free of the initial methionine residue can be produced on a large scale by using the N-terminal sequence. The IgG1Fc region obtained by the present invention can be used for increasing the serum half-life and physiological activity of the physiologically active polypeptide connected with the IgG1Fc region, and has no immune response induced by the addition of exogenous amino acid residues.

Example 4: c1q binding detection by ELISA

To determine in the embodiment<1-2>Whether the derivatives produced in (1) and the protein corresponding to the Fc region of immunoglobulin expressed in E.coli transformants and purified bind to human C1q was measured by enzyme-linked immunosorbent assay (ELISA) as follows. Immunoglobulin Fc regions produced by the HM11200 and HM11201 transformants prepared in the above examples were used as a test group. Glycosylated immunoglobulin (IVIGG-globulin S, Green Cross PBM) was used as standard. Test and standard samples were prepared at a concentration of 1. mu.g/ml in 10mM carbonate buffer (pH 9.6). The samples were dispensed into 96-well plates ((Nunc) at 200 ng/well, the plates were coated overnight at 4 ℃ and then PBS-T (137mM NaCl, 2mM KCl, 10mM Na)₂HPO₄、2mM KH₂PO₄0.05% Tween 20) was washed 3 times per well, blocked with 250. mu.l blocking buffer (1% bovine serum albumin in PBS-T) for 1 hour at room temperature, and then washed 3 times with the same PBS-T. The standard and test samples were diluted in PBS-T to predetermined concentrationsAnd added to antibody-coated wells, incubated the plate at room temperature for 1 hour, and washed 3 times with PBS-T. Thereafter, 2. mu.g/ml C1q (R) was added to the plate&D Systems) and reacted at room temperature for 2 hours, and the plate was washed 6 times with PBS-T. Mu.l of a 1: 1000 dilution of human anti-human C1q antibody peroxidase conjugate (Biogenesis, USA) diluted in blocking buffer was added to each well and reacted at room temperature for 1 hour. After washing each well 3 times with PBS-T, equal volumes of color reagent A and B were mixed (color reagent A: stabilized peroxide; color reagent B: stabilized chromogen; DY999, R&D Systems) and 200 μ l of the mixture was added to each well, followed by incubation for 30 minutes. Then 50. mu.l of a reaction stop solution (2M sulfuric acid) was added to each well. The plate was read with a microplate reader (Molecular Device). The absorbance of the standard sample and the test sample was measured at 450nm, and the results are shown in FIG. 2.

As shown in fig. 2, the immunoglobulin Fc region protein of the present invention produced in e.coli exhibits significantly reduced C1q binding affinity. These results indicate that the immunoglobulin Fc region protein of the present invention has little risk of inducing immune responses (e.g., cytotoxicity and inflammation) in the body when it is used as a carrier for physiologically active polypeptides in the form of conjugates.

Example 5: detection of Fc gamma RI, Fc gamma RIII and FcRn alpha beta by ELISA₂In combination with

Immunoglobulin Fc is known to bind to the blood cell receptors Fc γ RI and Fc γ RIII to mediate effector functions such as antibody-dependent cellular cytotoxicity. To determine whether the immunoglobulin Fc produced in e.coli could mediate this effector function, each receptor was obtained and tested for its binding capacity by ELISA. The ability of immunoglobulin Fc to bind to the receptor FcRn, which is known to affect the in vivo metabolism of immunoglobulins, was also examined in the same manner.

<5-1>Human Fc γ RI, Fc γ RIII and FcRn α β₂Construction of expression Strain

Isolation of Total RN from human peripheral blood mononuclear cells Using a kit (Qiagen, Cat. No.)A, and the total RNA was used to fishe encoding human Fc γ RI, Fc γ RIII and FcRn α β by RT-PCR and PCT₂The extracellular ligand-binding domain of (a). The gene was fused with GST (glutathione S-transferase) and cloned into the corresponding mammalian cell expression vector anchored to the dihydrofolate reductase gene. The prepared recombinant pHM000 plasmid was transfected into CHO cells. According to 1x10 per 6cm culture dish⁶Individual cell amounts were seeded into the CHO cells and incubated at 37 ℃ in 5% CO₂Incubate in the incubator for 24 hours, wash 2 times with Opti-MEM (Gibco., Cat. No. 31985-070). 1ml of Opti-MEM containing 10. mu.g pHM000 and 1ml of Lipofectamine^TMReagents (Invitrogen, Cat. No.18324-020) were mixed. After 20 minutes of holding, the resulting mixture was added to the prepared CHO cells. These cells were incubated in a 5% CO2 incubator at 37 ℃ for 18 hours, changed to DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and then incubated for another 48 hours. To select transformants, the cells were treated with 0.5% trypsin (Gibco., Cat. No.15400-054) in selection medium α -MEM (Welgene, Cat. No. 61-234 RG) containing 10% dialyzed fetal calf serum, 1% penicillin-streptomycin and 800. mu.g/ml geneticin (Mediatech, Cat. No. 61-234 RG), followed by centrifugation. The cells thus transformed were transferred to T25 petri dishes (Nunc) and incubated at 5% CO₂Incubate in an incubator at 37 ℃ to 90% or greater confluency. To determine Fc γ RI, Fc γ RIII and FcRn α β₂At increasing concentrations of MTX (Sigma, Cat. No. M-8407) (starting at 20nM and increasing at 20nM every 2 weeks) in 5% CO₂The selected strains were incubated in an incubator at 37 ℃.

<5-2>Human Fc γ RI, Fc γ RIII and FcRn α β₂Production and purification of

Fc γ RI, Fc γ RIII and FcRn α β were purified as follows₂. The selected cell lines were processed at 3.5X 10 cells per factory⁸The amount of individual cells was seeded in a cell factory (Nunc, Cat. No.170009) and in 5% CO₂The cells were grown in an incubator at 37 ℃ for 48 hours and then washed 2 times with 1 liter PBS per plant. Cells were supplemented with 1 literProduction Medium CHO-A-SFM containing 0.3mM sodium butyrate (SigmA, Cat. No. B-5887) and 5% CO₂The culture was carried out at 33 ℃ in an incubator, during which the expression supernatant was collected every other day for a total of 7 times. The collected supernatant was centrifuged, filtered through a 0.22 μ M filtration system (corning) and concentrated with a concentration system (PALL, cat No. pn OS010C70), and loaded on chelating sepharose FF resin (amersham pharmacia, cat No.17-0575-02) loaded with 0.1M nickel sulfide (Sigma, cat No. n4887) to allow Fc γ RI, Fc γ RIII and FcRn α β to₂GST of (a) binds to nickel. Bound FcyRI, FcyRIII and FcRn α β were analyzed and purified from the column with 50mM NaPi (pH 8.0), 300mM NaCl and 250mM imidazole₂。

<5-3> detection of binding to Fc γ RI

The Fc γ RI purified in example <5-2> was diluted in PBS (pH7.4) to a concentration of 0.75 μ g/ml with PBS, and then dispensed into 96-well plates (Nunc, Maxisorp) in an amount of 100 μ l/well, and incubated at 4 ℃ for 18 hours to adsorb the receptor to the bottom of the 96-well plates. Each well of the 96-well plate was washed 3 times with 300. mu.l of washing buffer PBS (pH7.4) containing 0.05% Tween-20(Amresco, Cat. No. 0777). Then, 300. mu.l of PBS (pH7.4) containing 0.1% Tween-20 and 3% BSA (bovine serum albumin, Amresco, Cat. No.0332) was added to each well to avoid undesired adsorption of other substances to the bottom of the well, and incubated at 37 ℃ for 1 hour, after which the reaction solution was completely removed therefrom. Human serum IgG as a control, Fc isolated from human serum IgG treated with papain, and HM11200 and HM11201 products purified in example 2 were diluted with detection buffer to a concentration of 9 μ g/ml, respectively, and then the 1: 3 serial dilution was repeated 7 times with detection buffer. 100. mu.l of the dilution was added to each well of the 96-well plate and allowed to react for 2 hours at 25 ℃ under constant shaking, and the wells were washed 6 times with the washing buffer. To check whether all of the HM11200, HM11201 products and controls anchored to the bottom of the well plate bound Fc γ RI, a volume of 100 μ l of HRP-conjugated goat anti-human heavy chain antibody (Chemicon, AP309P) diluted 1: 100000 in detection buffer was added to each well and allowed to react for 2 hours at 25 ℃ under constant shaking conditions. After washing 6 times with the washing buffer, 100. mu.l of a substrate capable of reacting with HRP conjugated to an antibody (BD bioscience, Cat. No.555214) was added to each well and reacted at 25 ℃ for 20 minutes. The reaction was stopped with 2N sulfuric acid and the color intensity was measured at 450nm with an ELISA microplate reader (Molecular Devices). As shown in fig. 3, almost none of the Fc proteins produced in e.coli bound Fc γ RI, while both human IgG and Fc (both glycosylated) bound strongly to Fc γ RI.

<5-4> detection of binding to Fc γ RIII

Human serum IgG as a control and the Fc separated from the human serum IgG treated with papain, respectively, the HM11200 and HM11201 products purified in examples <1-2> were diluted with carbonate buffer (pH9.0) to a concentration of 9. mu.g/ml, followed by repeated 1: 3 serial dilutions 7 times with carbonate buffer. To each well of the 96-well plate, 100. mu.l of the dilution was added and incubated at 4 ℃ for 18 hours so that they were adsorbed to the bottom of the 96-well plate. Each well of the 96-well plate was washed 3 times with 300. mu.l of a washing buffer consisting of PBS (pH7.4) containing 0.05% Tween-20(Amresco, Cat. No. 0777). Then, 300. mu.l of detection buffer consisting of PBS (pH7.4) containing 0.1% Tween-20 and 5% skim dry milk powder (Difco, Cat. No.232100) was added to each well to avoid undesired adsorption of other substances to the bottom of the well, and incubated at 37 ℃ for 1 hour, after which the reaction solution was completely removed therefrom. The Fc γ RIII purified in example <4-2> was diluted to a concentration of 1 μ g/ml with a detection solution. To each well of the 96-well plate, 100. mu.l of the dilution was added, and allowed to react at 25 ℃ for 2 hours with constant shaking. Wells were washed 6 times with wash buffer. Rabbit anti-GST antibody (Chemicon, AB3282) capable of binding to the products HM11200, HM11201 and control-linked GST (glutathione S-transferase) of Fc γ RIII was diluted 1: 10000 with assay buffer, 100. mu.l of the dilution was added to each well and allowed to react for 2 hours at 25 ℃ under constant shaking. Subsequently, after washing the wells 6 times with wash buffer, 100 μ l of a 1: 7500 dilution of the antibody against rabbit antibody in detection buffer was added to each well.

After 2 hours of reaction at 25 ℃ under constant shaking, the 96-well plate was washed 6 times with washing buffer. The substrate was added in the same manner as in example <5-3> and the color intensity was measured with an ELISA plate reader. As shown in fig. 4, almost none of the Fc γ RIII proteins produced in e.coli bound Fc γ RI, whereas both human IgG and Fc (both glycosylated) bound Fc γ RIII strongly.

<5-5>Binding to FcRn alpha beta₂Detection of (2)

Fc separated from control human serum IgG and papain-treated human serum IgG in carbonate buffer (pH9.0) was used in the examples<1-2>The purified HM11200 and HM11201 products of (1) were diluted to a concentration of 20. mu.g/ml, followed by 7 repeated 1: 3 serial dilutions with carbonate buffer. 100 μ l of the dilution was added to each well of the 96-well plate and incubated at 4 ℃ for 18 hours so that they were adsorbed to the bottom of the 96-well plate. Each well of the 96-well plate was washed 3 times with 300. mu.l of a washing buffer consisting of PBS (pH7.4) containing 0.05% Tween-20(Amresco, Cat. No. 0777). Then, 300. mu.l of detection buffer consisting of PBS (pH7.4) containing 0.1% Tween-20 and 5% BSA (Amresco, Cat. No.0332) was added to each well to avoid undesired adsorption of other substances to the bottom of the well, and incubated at 37 ℃ for 1 hour, after which the reaction solution was completely removed therefrom. Will be described in the examples with the detection solution<5-2>In the purification of FcRn alpha beta₂Diluted to a concentration of 3. mu.g/ml. To each well of the 96-well plate, 100. mu.l of the dilution was added, and allowed to react for 2 hours at 25 ℃ under constant shaking. Wells were washed 6 times with wash buffer. FcRnAlpha.beta capable of linking to HM11200, HM11201 products and controls were diluted 1: 10000 with assay buffer₂GST (glutathione S-transferase) conjugated rabbit anti-GST antibody (Chemicon, AB3282), 100. mu.l of a dilution was added to each well and allowed to react for 2 hours at 25 ℃ under constant shaking. Subsequently, after washing the wells 6 times with wash buffer, 100 μ l of a 1: 7500 antibody dilution against rabbit antibody in detection buffer was added to each well.

After 2 hours of reaction at 25 ℃ under constant shaking, the 96-well plate was washed 6 times with washing buffer. According to the following embodiments<5-2>Substrate was added in the same manner and color intensity was measured using an ELISA plate reader. As shown in FIG. 5, the Fc protein produced in E.coli was found to be similar to that of human IgG and glycosylated Fc, and to that of FcRn. alpha. beta.₂Strongly bound.

Example 6: preparation and pharmacokinetic analysis of human EPO conjugates

<6-1> preparation of human EPO

To prepare a human EPO (erythropoietin) conjugate, EPO gene was first amplified by RT-PCR using total RNA isolated from blood cells and cloned into pBluescript II (Stratagen) vector to generate pBlueEP vector. In order to transfer the cloned EPO gene into the dhfr gene-containing animal cell expression vector pCMV/dhfr- (pCDNA3.1 (Invitrogen)), pBlueEP was digested with HindIII and BamHI, and the fragment containing the EPO gene thus obtained was inserted into the same restriction enzyme-treated animal cell expression vector, thereby obtaining pcmvEP. The expression vector carrying the EPO gene was transfected into CHO cells (a protein-expressing strain) using Lipofectamine reagent (Gibco). The cells were treated with increasing concentrations of MTX (maximum concentration of 120nM) to increase their expression levels. EPO is expressed at high levels, exceeding 100 mg/l.

<6-2> preparation of human EPO-PEG complexes

3.4kDa polyethylene glycol ALD-PEG-ALD (shearwater) having aldehyde-reactive groups at both ends thereof was mixed with 100mM solution containing 5mg/ml of the aldehyde-reactive groups<6-1>The phosphate buffer solutions of EPO prepared in (1: 1), 1: 2.5, 1: 5, 1: 10 and 1: 20 EPO: PEG molar ratios are mixed. To this mixture was added the reducing agent sodium cyanoborohydride (NaCNBH)₃Sigma), final concentration 20mM, and reacted at 4 ℃ for 2 hours with gentle stirring to allow selective attachment of PEG to the amino terminus of EPO. To obtain a 1: 1 complexation of PEG with EPOComposition of Superdex^RThe reaction mixture was subjected to size exclusion chromatography using a column (Pharmacia). The EPO-PEG complex was eluted from the column using 10mM potassium phosphate buffer (pH 6.0) as an elution buffer, while EPO not bound to PEG, unreacted PEG and dimer by-products (in which PEG is linked to two EPO molecules) were removed. The purified EPO-PEG complex was concentrated to 5 mg/ml. It was found by this experiment that the optimum reaction molar ratio of EPO to PEG was 1: 2.5 to 1: 5 in order to obtain the highest reactivity and to produce the least amount of by-products (e.g., dimers).

<6-3> preparation of conjugate of human EPO-PEG Complex and recombinant immunoglobulin Fc region

Will be in the examples<6-2>Prepared EPO-PEG complexes were attached in the examples<1-3>The immunoglobulin Fc region prepared using HM 11201. In particular, it will be in the examples<1-3>The immunoglobulin Fc region fragment (about 53kDa) prepared in (1) was dissolved in 10mM phosphate buffer and mixed with EPO-PEG complex at a molar ratio of EPO-PEG complex: Fc region of 1: 1, 1: 2, 1: 4 and 1: 8. After the phosphate buffer concentration of the reaction solution was adjusted to 100mM, a reducing agent NaCNBH was added to the reaction solution₃(final concentration: 20mM) and reacted at 4 ℃ for 20 hours under a condition of slight stirring. It was found by this experiment that the optimum reaction molar ratio of EPO-PEG complex to Fc region fragment was 1: 2 in order to obtain the highest reactivity and to produce the least by-products (e.g., dimers).

After the coupling reaction, the reaction mixture was subjected to high pressure liquid chromatography to remove unreacted materials and by-products. The coupling reaction solution was desalted using a HiPrep26/10 desalting column (Pharmacia) with 10mM Tris buffer (pH 8.0). Then, the reaction solution was loaded on a 50ml Q HP 26/10 column (Pharmacia) at a flow rate of 8ml/min, and the column was eluted with a linear gradient of NaCl from 0M to 0.2M to obtain the desired fraction. The collected fractions were loaded again on a polycat21.5 × 250 column equilibrated with 10mM acetate buffer (pH 5.2) at a flow rate of 15ml/min, and the column was eluted with a linear gradient of 0.1M to 0.3M NaCl, thereby obtaining high purity fractions.

<6-4> pharmacokinetic analysis

Each group of 5 SD rats was subcutaneously injected with the native EPO prepared in example <5-1>, Aranesp (Amgen) having a higher sialic acid content to increase its half-life, and the EPO-PEG-Fc conjugate prepared in example <5-3> (test group) at a dose of 100. mu.g/kg. After subcutaneous injection, blood samples were collected at 0.5, 1, 2, 4, 6, 12, 24 and 48 hours for the control group, and at 1, 12, 24, 30, 48, 72, 96, 120, 144, 168, 192, 240, 288, 336 and 384 hours for the test group. Blood samples were collected in 1.5ml tubes, coagulated, and centrifuged in an Eppendorf high speed microcentrifuge for 10 minutes to remove blood cells. Serum protein levels were determined by ELISA using antibodies specific for EPO.

Table 6 below, as well as figure 6, shows the serum half-lives of the native proteins and protein conjugates. The EPO-PEG-Fc (E.coli) protein conjugates prepared using the immunoglobulin Fc region generated by the present invention as a carrier exhibit a longer serum half-life than native EPO. This extended half-life was found to be longer than that of Aranesp, which is known to be a second generation EPO with a long serum half-life.

TABLE 6

	EPO	EPO-PEG-Fc conjugates	Aranesp
				C_max ¹(ng/ml)	30.4	192.8	96.8
T_max ²(hr)	12.0	48.0	12.0
				T_1/2 ³(hr)	6.1	47.0	16.4
AUC⁴ (ng.hr/ml)	713	20436	4064
				MRT⁵(hr)	15.1	88	32
¹Maximum serum concentration²Time required to reach maximum drug concentration³Serum half-life of drug⁴Area under the curve of serum concentration versus time curve⁵Mean time of residence of drug molecules in the body

[0142] Industrial applicability

As described above, the present invention allows the production of immunoglobulin Fc regions in the form of inclusion bodies on a large scale in Escherichia coli using recombinant immunoglobulin Fc regions comprising a hinge region. When linked to a physiologically active protein, the immunoglobulin Fc region produced can be used to effectively increase the serum half-life and physiological activity of the physiologically active protein without the risk of inducing an immune response.

Sequence listing

<110> Korea medicine Industrial Co., Ltd

<120> method for mass-producing immunoglobulin Fc region deleted initial methionine residue

<160>60

<170>KopatentIn 1.71

<210>1

<211>39

<212>DNA

<213>Artificial Sequence

<220>

<223>primer for amplification of IgG4 Fc

<400>1

gggcatatgt catgcccagc acctgagttc ctgggggga

39

<210>2

<211>32

<212>DNA

<213>Artificial Sequence

<220>

<223>primer for amplification of IgG4 Fc

<400>2

gggggatccc tatttaccca gagacaggga ga

32

<210>3

<211>39

<212>DNA

<213>Artificial Sequence

<220>

<223>primer for amplification of IgG4 Fc

<400>3

gggcatatgc catcatgccc agcacctgag ttcctgggg

39

<210>4

<211>40

<212>DNA

<213>Artificial Sequence

<220>

<223>primer for amplification of IgG4 Fc

<400>4

gggcatatgt gcccatcatg cccagcacct gagttcctgg

40

<210>5

<211>36

<212>DNA

<213>Artificial Sequence

<220>

<223>primer for amplificaiton of IgG 4Fc

<400>5

gggcatatgt gcccagcacc tgagttcctg ggggga

36

<210>6

<211>663

<212>DNA

<213>homo sapiens

<220>

<221>CDS

<222>(1)..(663)

<223>Met-Ser-Cys-Pro-Fc

<400>6

atg tca tgc cca gca cct gag ttc ctg ggg gga cca tca gtc ttc ctg

48

Met Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

1 5 10 15

ttc ccc cca aaa ccc aag gac act ctc atg atc tcc cgg acc cct gag

96

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

20 25 30

gtc acg tgc gtg gtg gtg gac gtg agc cag gaa gac ccc gag gtc cag

144

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

35 40 45

ttc aac tgg tac gtg gat ggc gtg gag gtg cat aat gcc aag aca aag

192

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

50 55 60

ccg cgg gag gag cag ttc aac agc acg tac cgt gtg gtc agc gtc ctc

240

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

65 70 75 80

acc gtc ctg cac cag gac tgg ctg aac ggc aag gag tac aag tgc aag

288

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

85 90 95

gtc tcc aac aaa ggc ctc ccg tcc tcc atc gag aaa acc atc tcc aaa

336

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

100 105 110

gcc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc

384

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

115 120 125

cag gag gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa

432

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

130 135 140

ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag

480

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

145 150 155 160

ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc

528

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

165 170 175

tcc ttc ttc ctc tac agc agg cta acc gtg gac aag agc agg tgg cag

576

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

180 185 190

gag ggg aat gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac

624

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

195 200 205

cac tac aca cag aag agc ctc tcc ctg tct ctg ggt aaa

663

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210>7

<211>221

<212>PRT

<213>homo sapiens

<400>7

Met 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>8

<211>666

<212>DNA

<213>homo sapiens

<220>

<221>CDS

<222>(1)..(666)

<223>Met-Pro-Ser-Cys-Pro-Fc

<400>8

atg cca tca tgc cca gca cct gag ttc ctg ggg gga cca tca gtc ttc

48

Met Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

1 5 10 15

ctg ttc ccc cca aaa ccc aag gac act ctc atg atc tcc cgg acc cct

96

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

20 25 30

gag gtc acg tgc gtg gtg gtg gac gtg agc cag gaa gac ccc gag gtc

144

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

35 40 45

cag ttc aac tgg tac gtg gat ggc gtg gag gtg cat aat gcc aag aca

192

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

50 55 60

aag ccg cgg gag gag cag ttc aac agc acg tac cgt gtg gtc agc gtc

240

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

65 70 75 80

ctc acc gtc ctg cac cag gac tgg ctg aac ggc aag gag tac aag tgc

88

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

85 90 95

aag gtc tcc aac aaa ggc ctc ccg tcc tcc atc gag aaa acc atc tcc

336

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

100 105 110

aaa gcc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca

384

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

115 120 125

tcc cag gag gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc

432

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

130 135 140

aaa ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag agc aat ggg

480

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

145 150 155 160

cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac

528

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

165 170 175

ggc tcc ttc ttc ctc tac agc agg cta acc gtg gac aag agc agg tgg

576

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

180 185 190

cag gag ggg aat gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac

624

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

195 200 205

aac cac tac aca cag aag agc ctc tcc ctg tct ctg ggt aaa

666

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210>9

<211>222

<212>PRT

<213>homo sapiens

<400>9

Met Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

1 5 10 15

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

20 25 30

Glu Val Thr Cys Val Val Val Asp Val ser Gln Glu Asp Pro Glu Val

35 40 45

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

50 55 60

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

65 70 75 80

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

85 90 95

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

100 105 110

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

115 120 125

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

130 135 140

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

145 150 155 160

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

165 170 175

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

180 185 190

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

195 200 205

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210>10

<211>669

<212>DNA

<213>homo sapiens

<220>

<221>CDS

<222>(1)..(669)

<223>Met-Cys-Pro-Ser-Cys-Pro-Fc

<400>10

atg tgc cca tca tgc cca gca cct gag ttc ctg ggg gga cca tca gtc

48

Met Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

1 5 10 15

ttc ctg ttc ccc cca aaa ccc aag gac act ctc atg atc tcc cgg acc

96

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

cct gag gtc acg tgc gtg gtg gtg gac gtg agc cag gaa gac ccc gag

144

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

35 40 45

gtc cag ttc aac tgg tac gtg gat ggc gtg gag gtg cat aat gcc aag

192

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

aca aag ccg cgg gag gag cag ttc aac agc acg tac cgt gtg gtc agc

240

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

gtc ctc acc gtc ctg cac cag gac tgg ctg aac ggc aag gag tac aag

288

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

tgc aag gtc tcc aac aaa ggc ctc ccg tcc tcc atc gag aaa acc atc

336

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

100 105 110

tcc aaa gcc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc

384

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

cca tcc cag gag gag atg acc aag aac cag gtc agc ctg acc tgc ctg

432

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

130 135 140

gtc aaa ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag agc aat

480

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

145 150 155 160

ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc

528

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

gac ggc tcc ttc ttc ctc tac agc agg cta acc gtg gac aag agc agg

576

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

180 185 190

tgg cag gag ggg aat gtc ttc tca tgc tcc gtg atg cat gag gct ctg

624

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

gac aac cac tac aca cag aag agc ctc tcc ctg tct ctg ggt aaa

769

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210>11

<211>223

<212>PRT

<213>homo sapiens

<400>11

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>12

<211>660

<212>DNA

<213>homo sapiens

<220>

<221>CDS

<222>(1)..(660)

<223>Met-Cys-Pro-Fc

<400>12

atg tgc cca gca cct gag ttc ctg ggg gga cca tca gtc ttc ctg ttc

48

Met Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

1 5 10 15

ccc cca aaa ccc aag gac act ctc atg atc tcc cgg acc cct gag gtc

96

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

acg tgc gtg gtg gtg gac gtg agc cag gaa gac ccc gag gtc cag ttc

144

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

35 40 45

aac tgg tac gtg gat ggc gtg gag gtg cat aat gcc aag aca aag ccg

192

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

cgg gag gag cag ttc aac agc acg tac cgt gtg gtc agc gtc ctc acc

240

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

65 70 75 80

gtc ctg cac cag gac tgg ctg aac ggc aag gag tac aag tgc aag gtc

288

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

tcc aac aaa ggc ctc ccg tcc tcc atc gag aaa acc atc tcc aaa gcc

336

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

100 105 110

aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cag

384

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

115 120 125

gag gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc

432

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

130 135 140

ttc tac ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg

480

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

145 150 155 160

gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc

528

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

165 170 175

ttc ttc ctc tac agc agg cta acc gtg gac aag agc agg tgg cag gag

576

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

180 185 190

ggg aat gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac

624

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

195 200 205

tac aca cag aag agc ctc tcc ctg tct ctg ggt aaa

660

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210>13

<211>220

<212>PRT

<213>homo sapuens

<400>13

Met Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

1 5 10 15

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

35 40 45

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

65 70 75 80

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

100 105 110

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

115 120 125

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

130 135 140

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

145 150 155 160

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

165 170 175

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

180 185 190

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

195 200 205

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210>14

<211>15

<212>PRT

<213>homo sapiens

<220>

<221>SITE

<222>(1)..(15)

<223>Ig G1 hinge

<400>14

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210>15

<211>12

<212>PRT

<213>homo sapiens

<220>

<221>SITE

<222>(1)..(12)

<223>Ig G2 hinge

<400>15

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210>16

<211>62

<212>PRT

<213>homo sapiens

<220>

<221>SITE

<222>(1)..(62)

<223>Ig G3hinge

<400>16

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

35 40 45

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

50 55 60

<210>17

<211>12

<212>PRT

<213>homo sapiens

<220>

<221>SITE

<222>(1)..(12)

<223>Ig G4 hinge

<400>17

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro

1 5 10

<210>18

<211>3

<212>PRT

<213>homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG4 hinge

<400>18

Ser Cys Pro

1

<210>19

<211>4

<212>PRT

<213>homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(4)

<223>fragment of IgG4 hinge

<400>19

Pro Ser Cys Pro

1

<210>20

<211>5

<212>PRT

<213>homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(5)

<223>fragment of IgG4 hinge

<400>20

Cys Pro Ser Cys Pro

1 5

<210>21

<211>2

<212>PRT

<213>homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(2)

<223>fragment of IgG4 hinge

<400>21

Cys Pro

1

<210>22

<211>699

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(699)

<223>Met-Glu-Pro-Lys-Fc

<400>22

atg gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca

48

Met Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa

96

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg

144

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac

192

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag

240

Val Asp Gly VaL Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac

288

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa

336

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag

384

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg

432

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc

480

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac

528

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc

576

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc

624

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag

672

The Ser Cys Ser Val Met His GLu Ala Leu His Asn His Tyr Thr Gln

210 215 220

aag agc ctc tcc ctg tct ccg ggt aaa

699

Lys Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210>23

<211>233

<212>PRT

<213>Homo sapiens

<400>23

Met Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 7 5 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210>24

<211>690

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(690)

<223>Met-Ser-Cys-Asp-Fc

<400>24

atg tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa

48

Met Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac

96

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac

144

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc

192

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

50 55 60

gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac

240

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

65 70 75 80

agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg

288

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

85 90 95

ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca

336

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

100 105 110

gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gag

384

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac

432

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

130 135 140

cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc

480

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

145 150 155 160

gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc

528

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag

576

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

180 185 190

ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc

624

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

195 200 205

tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc

672

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

tcc ctg tct ccg ggt aaa

690

Ser Leu Ser Pro Gly Lys

225 230

<210>25

<211>230

<212>PRT

<213>Homo sapiens

<400>25

Met Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

Leu 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 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 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 Ala Leu Pro

100 105 110

Ala Pro 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 Arg Asp Glu Leu 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 Lys

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys

225 230

<210>26

<211>684

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(684)

<223>Met-Asp-Lys-Thr-Fc

<400>26

atg gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg

48

Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

1 5 10 15

ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc

96

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc

144

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag

192

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg

240

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

65 70 75 80

tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat

288

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc

336

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

100 105 110

atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gag cca cag

384

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc

432

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

130 135 140

agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg

480

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct

528

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc

576

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg

624

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg

672

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

tct ccg ggt aaa

684

Ser Pro Gly Lys

225

<210>27

<211>228

<212>PRT

<213>Homo sapiens

<400>27

Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 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

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 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 Ala Leu Pro Ala Pro

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 Pro Ser Arg Asp Glu Leu 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 Lys Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys

225

<210>28

<211>660

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(660)

<223>Met-Cys-Pro-Fc

<400>28

atg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc

48

Met Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

1 5 10 15

ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc

96

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc

144

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

35 40 45

aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg

192

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc

240

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

65 70 75 80

gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc

288

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc

336

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

100 105 110

aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cgg

384

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

115 120 125

gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc

432

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

130 135 140

ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg

480

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

145 150 155 160

gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc

528

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

165 170 175

ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag

576

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

180 185 190

ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac

624

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

195 200 205

tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa

660

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>29

<211>220

<212>PRT

<213>Homo sapiens

<400>29

Met Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

1 5 10 15

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

35 40 45

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

65 70 75 80

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

Ser Asn Lys A1a Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

100 105 110

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

115 120 125

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

130 135 140

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

145 150 155 160

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

165 170 175

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

180 185 190

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

195 200 205

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>30

<211>696

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(696)

<223>Met-Pro-Lys-Ser-Fc

<400>30

atg ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca

48

Met Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc

96

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg

144

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg

192

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag

240

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag

288

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc

336

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc

384

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

cga gag cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc

432

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc

480

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac

528

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac

576

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc

624

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag

672

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

agc ctc tcc ctg tct ccg ggt aaa

696

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210>31

<211>232

<212>PRT

<213>Homo sapiens

<400>31

Met Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210>32

<211>669

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(669)

<223>Met-Cys-Pro-Pro-Fc

<400>32

atg tgc cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc

48

Met Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

1 5 10 15

ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc

96

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag

144

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag

192

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

aca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc

240

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag

288

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc

336

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

100 105 110

tcc aaa gcc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc

384

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg

432

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

130 135 140

gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat

480

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

145 150 155 160

ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc

528

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg

576

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

180 185 190

tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg

624

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa

669

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>33

<211>223

<212>PRT

<213>Homo sapiens

<400>33

Met Cys Pro Pro Cys Pro Ala Pro Glu Leu 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 His Glu Asp Pro Glu

35 40 45

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Tyr 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 Ala Leu Pro Ala Pro 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 Arg Asp Glu Leu 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 Asr Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

180 185 190

Trp Gln Gln 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 Pro Gly Lys

210 215 220

<210>34

<211>666

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(666)

<223>Met-Pro-Pro-Cys-Fc

<400>34

atg cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc

48

Met Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

1 5 10 15

ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct

96

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

20 25 30

gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc

144

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

35 40 45

aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca

192

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

50 55 60

aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc

240

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

65 70 75 80

ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc

288

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

85 90 95

aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc

336

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

100 105 110

aaa gcc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca

384

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

115 120 125

tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc

432

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

130 135 140

aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg

480

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

145 150 155 160

cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac

528

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

165 170 175

ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg

576

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

180 185 190

cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac

624

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

195 200 205

aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa

666

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>35

<211>222

<212>PRT

<213>Homo sapiens

<400>35

Met Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

1 5 10 15

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

20 25 30

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

35 40 45

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

50 55 60

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

65 70 75 80

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

85 90 95

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

100 105 110

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

115 120 125

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

130 135 140

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

145 150 155 160

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

165 170 175

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

180 185 190

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

195 200 205

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>36

<211>663

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(663)

<223>Met-Pro-Cys-Pro-Fc

<400>36

atg ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc

48

Met Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

1 5 10 15

ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag

96

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

20 25 30

gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag

144

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

35 40 45

ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag

192

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

50 55 60

ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc

240

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

65 70 75 80

acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag

288

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

85 90 95

gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa

336

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

100 105 110

gcc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc

384

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

115 120 125

cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa

432

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

130 135 140

ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag

480

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

145 150 155 160

ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc

528

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

165 170 175

tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag

576

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

180 185 190

cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac

624

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

195 200 205

cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa

663

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>37

<211>221

<212>PRT

<213>Homo sapiens

<400>37

Met Pro Cys Pro Ala Pro Glu Leu 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 His Glu Asp Pro Glu Val Lys

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 Tyr 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 Ala Leu Pro Ala Pro 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

Arg Asp Glu Leu 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 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

180 185 190

Gln 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 Pro Gly Lys

210 215 220

<210>38

<211>663

<212>DNA

<213>Homto sapiens

<220>

<221>CDS

<222>(1)..(663)

<223>Met-Pro-Pro-Cys-Fc

<400>38

atg cca ccg tgc cca gca cct ccg gtg gcg gga ccg tca gtc ttc ctc

48

Met Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

1 5 10 15

ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag

96

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

20 25 30

gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc cag

144

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

35 40 45

ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag

192

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

50 55 60

ccg cgg gag gag cag ttt aac agc acg ttt cgt gtg gtc agc gtc ctc

240

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

65 70 75 80

acc gtc gtg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag

288

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

85 90 95

gtc tcc aac aaa ggc ctc cca gcc ccc atc gag aaa acc atc tcc aaa

336

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

100 105 110

acc aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc

384

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

115 120 125

cgg gaa gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa

432

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

130 135 140

ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag

480

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

145 150 155 160

ccg gag aac aac tac aag acc acg cct ccc atg ctg gac tcc gac ggc

528

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

165 170 175

tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag

576

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

180 185 190

cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac

624

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

195 200 205

cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa

663

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>39

<211>221

<212>PRT

<213>Homo sapiens

<400>39

Met Pro Pro Cys Pro Ala Pro Pro Val Ala 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 His 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 Phe Arg Val Val Ser Val Leu

65 70 75 80

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

85 90 95

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

100 105 110

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

115 120 125

Arg 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 Met Leu Asp Ser Asp Gly

165 170 175

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

180 185 190

Gln 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 Pro Gly Lys

210 215 220

<210>40

<211>660

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(660)

<223>Met-Pro-Cys-Pro-Fc

<400>40

atg ccg tgc cca gca cct ccg gtg gcg gga ccg tca gtc ttc ctc ttc

48

Met Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

1 5 10 15

ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc

96

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc cag ttc

144

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

35 40 45

aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg

192

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

cgg gag gag cag ttt aac agc acg ttt cgt gtg gtc agc gtc ctc acc

240

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

65 70 75 80

gtc gtg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc

288

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

tcc aac aaa ggc ctc cca gcc ccc atc gag aaa acc atc tcc aaa acc

336

Ser Asr Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

100 105 110

aaa ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cgg

384

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

115 120 125

gaa gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc

432

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

130 135 140

ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg

480

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

145 150 155 160

gag aac aac tac aag acc acg cct ccc atg ctg gac tcc gac ggc tcc

528

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

165 170 175

ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag

576

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

180 185 190

ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac

624

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

195 200 205

tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa

660

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>41

<211>220

<212>PRT

<213>Homo sapiens

<400>41

Met Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

1 5 10 15

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

35 40 45

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

65 70 75 80

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

100 105 110

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

115 120 125

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

130 135 140

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

145 150 155 160

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

165 170 175

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

180 185 190

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

195 200 205

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>42

<211>657

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(657)

<223>Met-Cys-Pro-Fc

<400>42

atg tgc cca gca cct ccg gtg gcg gga ccg tca gtc ttc ctc ttc ccc

48

Met Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

1 5 10 15

cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca

96

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

20 25 30

tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc cag ttc aac

144

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

35 40 45

tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg

192

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

50 55 60

gag gag cag ttt aac agc acg ttt cgt gtg gtc agc gtc ctc acc gtc

240

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

65 70 75 80

gtg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc

288

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

85 90 95

aac aaa ggc ctc cca gcc ccc atc gag aaa acc atc tcc aaa acc aaa

336

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

100 105 110

ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cgg gaa

384

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

115 120 125

aag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc

432

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

130 135 140

tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag

480

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

145 150 155 160

aac aac tac aag acc acg cct ccc atg ctg gac tcc gac ggc tcc ttc

528

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

165 170 175

ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg

576

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

180 185 190

aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac

624

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

195 200 205

acg cag aag agc ctc tcc ctg tct ccg ggt aaa

657

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210>43

<211>219

<212>PRT

<213>Homo sapiens

<400>43

Met Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

1 5 10 15

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

20 25 30

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

35 40 45

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

50 55 60

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

65 70 75 80

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

85 90 95

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

100 105 110

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

115 120 125

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

130 135 140

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

145 150 155 160

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

165 170 175

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

180 185 190

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

195 200 205

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210>44

<211>678

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(678)

<223>Met-Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys-Pro-Fc

<400>44

atg tgt tgt gtc gag tgc cca ccg tgc cca gca cct ccg gtg gcg gga

48

Met Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

1 5 10 15

ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc

96

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

20 25 30

tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa

144

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

35 40 45

gac cct gag gtc cag ttc aac tgg tac gtg gac ggc gtg gag gtg cat

192

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

50 55 60

aat gcc aag aca aag ccg cgg gag gag cag ttt aac agc acg ttt cgt

240

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

65 70 75 80

gtg gtc agc gtc ctc acc gtc gtg cac cag gac tgg ctg aat ggc aag

288

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

85 90 95

gag tac aag tgc aag gtc tcc aac aaa ggc ctc cca gcc ccc atc gag

336

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

100 105 110

aaa acc atc tcc aaa acc aaa ggg cag ccc cga gag cca cag gtg tac

384

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

115 120 125

acc ctg ccc cca tcc cgg gaa gag atg acc aag aac cag gtc agc ctg

432

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

130 135 140

acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg

480

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

145 150 155 160

gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc atg

528

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

165 170 175

ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac

576

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

180 185 190

aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat

624

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

195 200 205

gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg

672

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

210 215 220

ggt aaa

678

Gly Lys

225

<210>45

<211>226

<212>PRT

<213>Homo sapiens

<400>45

Met Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

1 5 10 15

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

20 25 30

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

35 40 45

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

50 55 60

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

65 70 75 80

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

85 90 95

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

100 105 110

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

115 120 125

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

130 135 140

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

145 150 155 160

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

165 170 175

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

180 185 190

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

195 200 205

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

210 215 220

Gly Lys

225

<210>46

<211>675

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(675)

<223>Met-Cys-Val-Glu-Cys-Pro-Pro-Cys-Pro-Fc

<400>46

atg tgt gtc gag tgc cca ccg tgc cca gca cct ccg gtg gcg gga ccg

48

Met Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

1 5 10 15

tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc

96

Ser Val Phe Leu Phe Pro pro Lys Pro Lys Asp Thr Leu Met Ile Ser

20 25 30

cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac

144

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

35 40 45

cct gag gtc cag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat

192

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

50 55 60

gcc aag aca aag ccg cgg gag gag cag ttt aac agc acg ttt cgt gtg

240

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

65 70 75 80

gtc agc gtc ctc acc gtc gtg cac cag gac tgg ctg aat ggc aag gag

288

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

85 90 95

tac aag tgc aag gtc tcc aac aaa ggc ctc cca gcc ccc atc gag aaa

336

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

100 105 110

acc atc tcc aaa acc aaa ggg cag ccc cga gag cca cag gtg tac acc

384

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

115 120 125

ctg ccc cca tcc cgg gaa gag atg acc aag aac cag gtc agc ctg acc

432

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

130 135 140

tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag

480

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

145 150 155 160

agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc atg ctg

528

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

165 170 175

gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag

576

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

180 185 190

agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag

624

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

195 200 205

gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt

672

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

aaa

675

Lys

225

<210>47

<211>225

<212>PRT

<213>Homo sapiens

<400>47

Met Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

1 5 10 15

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

20 25 30

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

35 40 45

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

50 55 60

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

65 70 75 80

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

85 90 95

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

100 105 110

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

115 120 125

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

130 135 140

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

145 150 155 160

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

165 170 175

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

180 185 190

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

195 200 205

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

Lys

225

<210>48

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgGl hinge

<400>48

Glu Pro Lys

1

<210>49

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgGl hinge

<400>49

Ser Cys Asp

1

<210>50

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG1 hinge

<400>50

Asp Lys Thr

1

<210>51

<211>2

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(2)

<223>fragment of IgG1 hinge

<400>51

Cys Pro

1

<210>52

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG1 hinge

<400>52

Pro Lys Ser

1

<210>53

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG1 hinge

<400>53

Cys Pro Pro

1

<210>54

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG1 hinge

<400>54

Pro Pro Cys

1

<210>55

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG1 hinge

<400>55

Pro Cys Pro

1

<210>56

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG2 hinge

<400>56

Pro Pro Cys

1

<210>57

<211>3

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(3)

<223>fragment of IgG2 hinge

<400>57

Pro Cys Pro

1

<210>58

<211>2

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(2)

<223>fragment of IgG2 hinge

<400>58

Cys Pro

1

<210>59

<211>9

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(9)

<223>fragment of IgG2 hinge

<400>59

Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5

<210>60

<211>8

<212>PRT

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(8)

<223>fragment of IgG2 hinge

<400>60

Cys Val Glu Cys Pro Pro Cys Pro

1 5

Claims

1. A method of producing an immunoglobulin Fc region free of an initiating methionine residue, the method comprising:

preparing a recombinant expression vector comprising a nucleotide sequence encoding a recombinant immunoglobulin Fc region consisting of an immunoglobulin Fc region linked at its N-terminus to an immunoglobulin hinge region via a peptide bond;

transforming prokaryotic cells with the recombinant expression vector to generate a transformant;

culturing the transformant to express an immunoglobulin Fc region in the form of inclusion bodies; and

isolating and purifying the immunoglobulin Fc region,

wherein the immunoglobulin hinge region has cysteine, serine or proline as an N-terminal starting amino acid and includes at least one cysteine in its sequence, and

wherein the prokaryotic cell is a prokaryotic cell in which no glycosylation takes place and which has aminopeptidase activity.

2. The method of claim 1, wherein the hinge region has two or more contiguous amino acid sequences derived from a hinge region of IgG, IgA, IgM, IgE, or IgD.

3. The method of claim 2, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG 4.

4. The method of claim 3, wherein the amino acid sequence of the hinge region is SEQ ID No.18, 19, 20, 21, 49, 51, 53, 54, 55, 56, 57, 58, 59, or 60.

5. The method of claim 1, wherein the immunoglobulin Fc region is selected from the group consisting of Fc regions from IgG, IgA, IgM, IgE, IgD, and combinations and hybrids thereof.

6. The method of claim 5, wherein the immunoglobulin Fc region is an Fc region of an IgG selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

7. The method of claim 1, wherein the immunoglobulin Fc region is formed from a peptide selected from the group consisting of C_H1、C_H2、C_H3、C_H4 and C_L1 to 4 domains of a domain.

8. The method of claim 1, wherein the amino acid sequence of the recombinant immunoglobulin Fc region is SEQ ID No.7, 9, 11, 13, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47.

9. The method of claim 1, wherein the recombinant expression vector comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID No.7, 9, 11, 13, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47.

10. The method of claim 1, wherein the prokaryotic cell is E.

11. The method of claim 1, wherein the transformant is transformed with a recombinant expression vector comprising a nucleotide sequence encoding a recombinant immunoglobulin Fc region selected from the group consisting of SEQ ID No.7, 9, 11, 13, 25, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47.

12. A monomeric or dimeric immunoglobulin Fc region devoid of initiating methionine residues produced by the method of claim 1, wherein said immunoglobulin Fc region is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO.6, 8, 10, 12, 24, 28, 30, 32, 34, 36, 40, 44 and 46.

13. The process of claim 11, wherein the transformant is selected from the group consisting of the transformants deposited under accession numbers KCCM-10659P, KCCM-10660P, KCCM-10665P and KCCM-10666P.