WO2017052324A1 - Procédé de production de région fc d'immunoglobuline comprenant un résidu méthionine initial - Google Patents

Procédé de production de région fc d'immunoglobuline comprenant un résidu méthionine initial Download PDF

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WO2017052324A1
WO2017052324A1 PCT/KR2016/010752 KR2016010752W WO2017052324A1 WO 2017052324 A1 WO2017052324 A1 WO 2017052324A1 KR 2016010752 W KR2016010752 W KR 2016010752W WO 2017052324 A1 WO2017052324 A1 WO 2017052324A1
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immunoglobulin
region
recombinant
protein
amino acid
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PCT/KR2016/010752
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English (en)
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Eui Joon Jeong
Yong Ho Heo
Jong Min Lee
Sung Hee Park
Sung Youb Jung
Se Chang Kwon
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Hanmi Pharm. Co., Ltd.
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Publication of WO2017052324A1 publication Critical patent/WO2017052324A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a method of producing an immunoglobulin Fc region including the initial methionine residue using a vector including a nucleotide sequence encoding a recombinant immunoglobulin Fc region including an immunoglobulin hinge region, and to a monomeric or dimeric immunoglobulin Fc region including the initial methionine residue prepared by the method.
  • the Fc region mediates effector functions such as complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), as well as antigen binding capacity, which is the unique function of immunoglobulins.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • antigen binding capacity which is the unique function of immunoglobulins.
  • the FcRn sequence present in Fc region plays a role in regulating serum IgG levels by increasing the transport of IgG to neonates and the half-life of the IgG (Ghetie and Ward, Immunology Today 18: 592-598, 1997), and the sequence regulates the interaction between protein A and protein G. Through the fusion of this Fc region with a therapeutic protein, many studies have been performed to enhance the stability of the therapeutic protein.
  • Korean Patent No. 249572 discloses a fusion protein which is prepared by linking an IgG1 heavy chain constant region (Fc) at an amino terminal end thereof to a carboxyl terminal end of a protein, such as an IL4 receptor, an IL7 receptor, a G-CSF receptor, or an EPO receptor, and producing the resulting fusion protein in mammalian cells.
  • a protein such as an IL4 receptor, an IL7 receptor, a G-CSF receptor, or an EPO receptor
  • US Patent No. 5,605,690 describes a fusion protein including a tumor necrosis factor receptor fused at its carboxyl terminal end to a human IgG1 Fc derivative, the fusion protein being produced in animal cells.
  • Tanox Inc. reports, in US Patent Nos.
  • 20030082679 discloses a fusion protein with an extended serum half-life, which is prepared by linking human G-CSF linked at its carboxyl terminal end to the amino terminal end of IgG1 Fc via a peptide linker and is produced in animal cells.
  • US Patent Publication No. 20010053539 US Patent No. 6,030,613, International PCT Application Publication Nos. WO 99/02709 and WO 01/03737, and European Patent No.
  • 0464533Bl disclose an Fc fusion protein with a longer serum half-life than a native protein, which includes an IgG1 Fc or Fc derivative linked at its amino terminal end through a peptide linker or without a peptide linker to the carboxyl terminal end of human EPO, TPO, human growth hormone, or human interferon beta, the Fc fusion protein being produced in animal cells.
  • Fc fusion proteins as described above, increase the serum half-life of target proteins, but have problems related to the mediation of effector functions by the Fc region (US Patent No. 5,349,053).
  • the effector functions of the Fc region fix complements or bind to cells expressing Fc ⁇ Rs, leading to lysis of specific cells, and induce the production and secretion of several cytokines inducing inflammation, leading to unwanted inflammation.
  • the protein sequence of the fusion region is a new amino acid sequence which does not exist in the human body, which could potentially induce immune responses if administered for a long time.
  • an aglycosylated antibody derivative as an anti-CD3 antibody can be prepared by replacing a glycosylated residue of antibodies, the asparagine residue at position 297 of the CH2 domain, with another amino acid.
  • This derivative exhibits reduced effector functions, but still retains its binding affinity to an FcRn receptor, with no change in serum half-life.
  • this derivative is also problematic in terms of being potentially recognized as a foreign material and rejected by the immune system due to the production of a novel recombinant construct having an abnormal sequence.
  • US Patent Publication No. 20030073164 discloses a method of producing an antibody by fusing a heavy chain and a light chain to a secretory sequence using E. coli cells devoid of glycosylation ability so as to prepare a therapeutic antibody deficient in effector functions.
  • the present inventors previously prepared an Fc region and a protein drug as separate polypeptides, not using a fusion method based on genetic recombination, but using the best expression systems, and covalently linking the two polypeptides together to use Fc as a drug carrier.
  • Fc a drug carrier
  • a prokaryotic expression system such as E. coli is used.
  • Protein production methods using an E. coli expression system have several advantages over conventional methods using animal cells, as follows.
  • An E. coli expression vector can be easily constructed, thus allowing rapid evaluation of protein expression. Due to its rapid growth rate, E. coli allows mass production of a protein of interest at low cost. Also, a relatively simple expression process can be used. Thus, E. coli is more useful for commercial production than other host cells.
  • European Patent No. 0227110 discloses the production of the immunoglobulin G1 Fc region using only the product (the cell lysate) which is expressed in the water soluble form upon the overexpression of the immunoglobulin G1 Fc region.
  • the immunoglobulin expressed in the water soluble form is as low as 15 mg/L in yield, which has no value in terms of industrial usefulness.
  • the Fc regions are overexpressed in E. coli, they are mostly expressed as inclusion bodies.
  • the present inventors have made many efforts to develop a method of efficiently producing an active aglycosylated immunoglobulin Fc region with no risk of immune response induction, and have found that when an immunoglobulin Fc region is expressed in a form fused at the N-terminus to a specific hinge region, the immunoglobulin Fc region is expressed as inclusion bodies which are ultimately a dimer or a monomer of an immunoglobulin Fc region including initial methionine residues through solubilization and refolding processes, thereby producing an immunoglobulin Fc region including the methionine initiation codon.
  • the present inventors have expressed an immunoglobulin Fc region with the initial methionine residue as inclusion bodies by linking a specific hinge region to the N-terminus of the immunoglobulin Fc region to produce a dimeric or monomeric immunoglobulin Fc region including the initial methionine residue through solubilization and refolding processes, in addition to expressing the immunoglobulin Fc region devoid of the initial methionine residue by linking a specific hinge region to the N-terminus of the immunoglobulin Fc region.
  • An object of the present invention is to provide a method of producing an immunoglobulin Fc region including the initial methionine residue, the method including preparing a vector including a nucleotide sequence encoding a recombinant immunoglobulin Fc region including an immunoglobulin hinge region; transforming a prokaryotic cell with the vector; culturing the transformant; and isolating and purifying the immunoglobulin Fc region expressed in the form of an inclusion body from the transformant.
  • Another object of the present invention is to provide an immunoglobulin Fc region in the form of a monomer or a dimer including the initial methionine residue, which is prepared by the above method.
  • a recombinant immunoglobulin Fc region including an immunoglobulin hinge region of the present invention may be used to produce a recombinant immunoglobulin Fc region as inclusion bodies in E. coli, and methionine is included in the expressed recombinant immunoglobulin Fc region.
  • the produced immunoglobulin Fc region is linked to a physiologically active polypeptide, and is thereby used for increasing serum half-life of the physiologically active polypeptide.
  • FIG. 1 shows the result of protein electrophoresis after expression of an Fc analogue including initial methionine (Met).
  • FIG. 2 shows the result of chromatography to examine purity of the Fc analogue including initial methionine.
  • FIG. 3 shows the result of examining serum half-life of a CA-Exendin-PEG-Fc analogue conjugate which is prepared by using the Fc analogue including initial methionine as a carrier.
  • an aspect of the present invention provides a method of producing an immunoglobulin Fc region including the initial methionine residue, the method comprising preparing a vector including a nucleotide sequence encoding a recombinant immunoglobulin Fc region including an immunoglobulin hinge region; transforming a prokaryotic cell with the vector; culturing the transformant; and isolating and purifying the immunoglobulin Fc region expressed in the form of an inclusion body from the transformant.
  • the immunoglobulin Fc domain is in the form of a monomer or a dimer.
  • the immunoglobulin Fc region is aglycosylated.
  • the hinge region has two or more consecutive amino acid sequences derived from the hinge region of IgG, IgA, IgM, IgE, or IgD
  • the IgG is IgG1, IgG2, IgG3, or IgG4.
  • the hinge region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, or 20.
  • the immunoglobulin Fc region consists of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 domains.
  • the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE, or IgM.
  • the immunoglobulin Fc region is a hybrid of domains, in which each domain has a different origin derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.
  • the immunoglobulin Fc region is a dimer or a multimer consisting of single chain immunoglobulins comprising the same origin.
  • the immunoglobulin Fc region is an IgG4 Fc fragment.
  • the immunoglobulin Fc region is a human aglycosylated IgG4 Fc fragment.
  • the recombinant immunoglobulin Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 12, 14, or 16.
  • the nucleotide sequence encoding the recombinant immunoglobulin Fc region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 11, 13, or 15.
  • the vector is pMCPSFc, pMYGPFc, pMKYGFc, or pMESKFc.
  • the prokaryotic cell is E. coli.
  • the transformant is BL21/pMCPSFc, BL21/pMYGPFc, BL21/pMKYGFc, or BL21/pMESKFc.
  • Another aspect provides an immunoglobulin Fc region in the form of a monomer or a dimer including the initial methionine residue, which is prepared by the method.
  • the present invention relates to a method of producing an immunoglobulin Fc region including the initial methionine residue, the method comprising preparing a vector including a nucleotide sequence encoding a recombinant immunoglobulin Fc region including an immunoglobulin hinge region; transforming a prokaryotic cell with the vector; culturing the transformant; and isolating and purifying the immunoglobulin Fc region expressed in the form of an inclusion body from the transformant.
  • the present invention pertains to a method of producing an immunoglobulin Fc region useful as a carrier for drugs.
  • an immunoglobulin Fc region is fused at the N-terminus to a hinge region, the resulting recombinant immunoglobulin Fc region is found to be expressed as an inclusion body and then solubilized and refolded into a dimer or monomer in an active form with the initial methionine residue encoded by the initiation codon.
  • the present invention is of great significance in terms of the finding that, when a hinge region is fused to an immunoglobulin Fc region, the expressed Fc region is processed and refolded into a form including the initial methionine residue without loss of activity, and its serum half-life is similar to that of the active monomeric or dimeric Fc region devoid of the initial methionine residue in terms of pharmacokinetics.
  • the hinge region capable of allowing an immunoglobulin Fc region to be mass produced in a recombinant form therewith may be a hinge region derived from IgG, IgA, IgM, IgE, or IgD of humans and other animals, including goats, swine, mice, rabbits, hamsters, rats and guinea pigs, with preference for a hinge region derived from IgG, e.g., IgG1, IgG2, IgG3, or IgG4.
  • the hinge region may be a full-length hinge region or a fragment thereof. Preferably it is a hinge region fragment having two or more consecutive amino acid sequences, which more preferably contain at least one cysteine residue therein.
  • a fragment of the hinge region derived from IgG4 may be used, and the immunoglobulin Fc region may be prepared in an active dimer or monomer form including methionine.
  • a fragment of the hinge region derived from IgG4 was used, which is represented by SEQ ID NO: 17, 18, 19, or 20.
  • the immunoglobulin Fc region may be prepared in an active dimer or monomer form including methionine.
  • the immunoglobulin Fc region is linked to the immunoglobulin hinge region to prepare a nucleotide sequence encoding the recombinant immunoglobulin Fc region.
  • the immunoglobulin Fc region to be produced by the present invention refers to a protein that contains the heavy-chain constant region 2 (CH2) and the heavy-chain constant region 3 (CH3) (or including the heavy-chain constant region 4 (CH4)) of an immunoglobulin, excluding the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CH1), and the light-chain constant region (CL) of the immunoglobulin. It may further include a hinge region at the heavy-chain constant region.
  • the immunoglobulin Fc region of the present invention may be an extended immunoglobulin Fc region including a part or all of the Fc region including the heavy-chain constant region 1 (CH1) and/or the light-chain constant region (CL), except for the variable regions of the heavy and light chains, as long as it has an effect substantially similar to or better than the native protein. Also, it may be a region having a deletion in a relatively long portion of the amino acid sequence of CH2 and/or CH3.
  • the immunoglobulin Fc region of the present invention may include (1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, (2) a CH1 domain and a CH2 domain, (3) a CH1 domain and a CH3 domain, (4) a CH2 domain and a CH3 domain, (5) a combination of one or more domains of the constant region and an immunoglobulin hinge region (or a portion of the hinge region), and (6) a dimer of each domain of the heavy-chain constant regions and the light-chain constant region.
  • An immunoglobulin constant region including an Fc region is a biodegradable polypeptide which can be metabolized in vivo, so that it can safely be used as a drug carrier.
  • an immunoglobulin Fc fragment is more advantageous in terms of production, purification, and production yield of a complex than an entire immunoglobulin molecule owing to its relatively low molecular weight. Further, since it is devoid of Fab, which exhibits high non-homogeneity due to the difference in amino acid sequence from one antibody to another, it is expected to significantly enhance homogeneity and to reduce the possibility of inducing blood antigenicity.
  • the immunoglobulin Fc region may originate from humans or animals, such as cows, goats, swine, mice, rabbits, hamsters, rats, guinea pigs, etc., and may preferably be of human origin.
  • the immunoglobulin Fc region may be selected from constant regions derived from IgG, IgA, IgD, IgE, IgM, or combinations or hybrids thereof, preferably, derived from IgG or IgM, which are the most abundant in human blood, and most preferably, derived from IgG, which is known to improve the half-life of ligand-binding proteins.
  • the immunoglobulin Fc region may be a dimer or multimer consisting of single-chain immunoglobulins of domains of the same origin.
  • nucleotide sequences encoding human immunoglobulin Fc regions and amino acid sequences limited to the same may be those encoded by nucleotide sequences from the GenBank and/or EMBL databases.
  • the term "combination” means that polypeptides encoding single-chain immunoglobulin Fc regions (preferably Fc regions) of the same origin are linked to a single-chain polypeptide of a different origin to form a dimer or multimer. That is, a dimer or a multimer may be prepared from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc.
  • hybrid means that sequences encoding two or more immunoglobulin Fc regions of different origins are present in a single-chain of an immunoglobulin Fc region (preferably, an Fc region).
  • the hybrid domain can be composed of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc.
  • IgG is divided into the IgG1, IgG2, IgG3, and IgG4 subclasses, and the present invention may include combinations or hybrids thereof. Preferred are the IgG2 and IgG4 subclasses, and most preferred is the Fc region of IgG4 rarely having effector functions such as Complement Dependent Cytotoxicity (CDC).
  • CDC Complement Dependent Cytotoxicity
  • the immunoglobulin Fc region may have the glycosylated form to the same extent as, or to a higher or lesser extent than, the native form, or may be the deglycosylated form. Increased or decreased glycosylation or deglycosylation of the immunoglobulin Fc region may be achieved by typical methods, for example, by using a chemical method, an enzymatic method, or a genetic engineering method using microorganisms.
  • the complement (C1q) binding to an immunoglobulin Fc region becomes significantly decreased and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or removed, thereby not inducing unnecessary immune responses in vivo.
  • deglycosylated or aglycosylated immunoglobulin Fc regions are more consistent with the purpose of drug carriers. Accordingly, the immunoglobulin Fc region may be more specifically an aglycosylated Fc region derived from human IgG4, that is, a human IgG4-derived aglycosylated Fc region.
  • the human-derived Fc region is more preferable than a non-human derived Fc region, which may act as an antigen in the human body and cause undesirable immune responses such as the production of a new antibody against the antigen.
  • the immunoglobulin Fc region of the present invention includes not only the immunoglobulin Fc region with the native amino acid sequence, but also a recombinant immunoglobulin Fc region, immunoglobulin Fc analogue, or sequence derivative (mutant).
  • the amino acid sequence derivative means that it has an amino acid sequence different from the wild-type amino acid sequence as a result of deletion, insertion, conserved or non-conserved substitution of one or more amino acid residues, or a combination thereof.
  • the recombinant immunoglobulin Fc region and immunoglobulin Fc analogue may have the same meaning as above.
  • the recombinant immunoglobulin Fc region, immunoglobulin Fc analogue, or sequence derivative, as used herein may refer to an immunoglobulin Fc region including an immunoglobulin hinge region.
  • it may be an IgG Fc analogue which is prepared to include a hinge region at the N-terminus, and additionally, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 in IgG Fc, known to be important for linkage, may be used as the sites suitable for modification.
  • Various derivatives such as those prepared by removing the sites capable of forming disulfide bonds, removing several N-terminal amino acids from native Fc, or adding methionine to the N-terminus of native Fc, may be used.
  • complement fixation sites e.g., C1q fixation sites, or ADCC sites may be eliminated to remove the effector function.
  • the techniques of preparing the sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478.
  • the recombinant immunoglobulin Fc region or the immunoglobulin Fc analogue of the present invention may be a recombinant immunoglobulin Fc analogue having an amino acid sequence of SEQ ID NO: 10, 12, 14, or 16.
  • a recombinant immunoglobulin Fc analogue having an amino acid sequence of SEQ ID NO: 10, 12, 14, or 16 was prepared, the recombinant immunoglobulin Fc analogue including a hinge region at the N-terminus and the initial methionine based on an IgG4-derived Fc region.
  • amino acids may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.
  • the immunoglobulin derivative is preferably a functional equivalent to its natural form, thus having a similar biological activity, or, if desired, may be made by altering the properties of the natural form.
  • the derivatives of the immunoglobulin Fc region are derivatives that have increased structural stability against heat, pH, etc., or solubility, or that have improved characteristics in terms of disulfide bond formation, compatibility with an expression host, complement binding, Fc receptor binding, and antibody-dependent cell-mediated cytotoxicity (ADCC), so long as the derivatives produced are not so different from natural forms of humans that they induce unwanted immune responses in humans and animals.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the altered region in the Fc region derivative should not be so different from those of humans that they induce immune responses when administered into humans.
  • Preferred derivatives are IgG1 Fc regions which are altered in such a specific residue as to have reduced affinity to Fc receptors mediating antibody-dependent cell-mediated cytotoxicity.
  • the derivative may include a deletion or a substitution with another amino acid in the leucine residue at position 234 of an IgG1 CH2 sequence (see the sequence from the Kobat database for the numbering of the amino acid residues), and most preferably, may be replaced by phenylalanine, an amino acid residue at a corresponding position in IgG4.
  • the immunoglobulin Fc region may be obtained from a native type isolated from humans or animals such as cow, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or may be their recombinants or derivatives obtained from transformed animal cells or microorganisms.
  • native Fc regions may be obtained by protease digestion of the entire gamut of immunoglobulins isolated from human or animal samples. Immunoglobulins are cleaved into Fab and Fc by papain and into pF'c and F(ab) 2 by pepsin, followed by size-exclusion chromatography to separate Fc or pF'c therefrom.
  • a nucleotide sequence encoding the recombinant immunoglobulin Fc region may be prepared by fusing the immunoglobulin Fc region to the immunoglobulin hinge region.
  • the recombinant immunoglobulin Fc region means the immunoglobulin Fc region linked at the N-terminus to a hinge region via a peptide bond.
  • the hinge region to be fused may be chosen.
  • a hinge region which is the same in origin as the immunoglobulin Fc region.
  • the nucleotide sequence encoding the recombinant immunoglobulin Fc region of the present invention means a nucleotide sequence encoding the above recombinant immunoglobulin Fc region, and for example, it may be a nucleotide sequence encoding the recombinant immunoglobulin Fc analogue having an amino acid sequence of SEQ ID NO: 10, 12, 14, or 16, which includes the hinge region and the initial methionine based on IgG4-based Fc region, and in particular, it may be a nucleotide sequence of SEQ ID NO: 9, 11, 13, or 15.
  • the recombinant immunoglobulin Fc analogue having the amino acid sequence of SEQ ID NO: 10, 12, 14 or 16, which includes the hinge region and the initial methionine based on IgG4-based Fc region, and also a nucleotide sequence of SEQ ID NO: 9, 11, 13, or 15 encoding the recombinant immunoglobulin Fc region, were prepared.
  • a vector to which the nucleotide sequence encoding the recombinant immunoglobulin Fc region is operably linked may be provided.
  • vector which describes a recombinant vector capable of expressing a target protein in a suitable host cell, refers to a genetic construct that includes essential regulatory elements to which a gene insert is operably linked in such a manner as to be expressed.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein in such a manner as to allow general functions.
  • the operable linkage to a vector may be prepared using a genetic recombinant technique well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes generally known in the art.
  • a suitable expression vector includes expression regulatory elements, such as a promoter, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer. The initiation and stop codons are necessary for functionality in an individual to whom a genetic construct has been administered, and must be in frame with the coding sequence.
  • a general promoter may be constitutive or inducible.
  • expression vectors include a selectable marker that allows selection of host cells containing the vector, and replicable expression vectors include a replication origin.
  • pMCPSFc, pMYGPFc, pMKYGFc, and pMESKFc were prepared.
  • the recombinant expression vector expressing the recombinant immunoglobulin Fc region of the present invention is transformed into host cells.
  • the host cells are prokaryotic cells in which glycosylation does not occur.
  • these prokaryotic cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, and Staphylococcus, with preference for E. coli.
  • E. coli includes include E. coli XL-1 blue, E. coli BL21(DE3), E. coli JM109, E. coli DH series, E. coli TOP10, and E. coli HB101, and more preferably, E. coli BL21(DE3), but is not limited thereto. Because it lacks a system, for protein glycosylation, E.
  • an immunoglobulin Fc region is produced in the form of being devoid of sugar moieties that are present in a CH2 domain of a native immunoglobulin.
  • Sugar moieties of the immunoglobulin CH2 domain do not affect the structural stability of immunoglobulins, but cause immunoglobulins to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) upon binding to cells expressing Fc receptors, and immune cells to secrete cytokines inducing inflammation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the sugar moieties bind to the C1q part of the complement component, leading to complement fixation.
  • the transformation of the vector into prokaryotic cells may be achieved by any method that allows nucleic acids to be introduced into cells and, as known in the art, may be performed by selecting suitable standard techniques according to host cells. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation with silicon carbide fiber, and PEG-, dextran sulfate-, and lipofectamine-mediated transformation.
  • BL21/pMCPSFc BL21/pMYGPFc, BL21/pMKYGFc, and BL21/pMESKFc were prepared.
  • the transformants transformed with the recombinant expression vectors are cultured through a general method.
  • the medium used for the culture should contain all nutrients essential for the growth and survival of cells.
  • the medium should contain a variety of carbon sources, nitrogen sources and trace elements.
  • available carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and 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.
  • nitrogen sources examples 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.
  • a phosphorus source may be contained in the medium, which includes potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts.
  • the medium may contain a metal salt, such as magnesium sulfate or iron sulfate.
  • the medium may further include amino acids, vitamins, suitable precursors, and the like.
  • the pH of the culture may be adjusted by adding a compound, such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid, to the culture using a suitable method. Also, during the culture, antifoaming agents such as polyglycol fatty acid esters may be used to prevent bubble formation.
  • oxygen or an oxygen-containing gas e.g., air
  • the temperature of the culture is generally 20°C to 45°C, and preferably 25°C to 40°C.
  • a fermentor may be used. Protein production using a fermentor should be carried out taking into consideration several factors, including the growth rate of host cells and protein expression levels. Protein expression may be induced through adding, for example, IPTG to the medium under suitable culture conditions.
  • An immunoglobulin Fc region overexpressed as inclusion bodies may be purified through a general technique.
  • the immunoglobulin Fc regions produced in the transformants may be obtained by disrupting cells using a French press, an ultrasonicator, etc., collecting only water-insoluble inclusion bodies containing the immunoglobulin Fc region through centrifugation, solubilizing, and denaturing the collected fraction with refolding agents, such as urea, guanidine, arginine, cysteine, beta-mercaptoethanol, etc., to the refolding thereof, and purifying the refolded fusion protein through dialysis, gel filtration, various chromatographie, such as ion exchange and reverse phase column chromatography, and ultrafiltration, alone or in combination.
  • this refolding process is very complicated and is known to produce a very low refolding yield and assure the refolded protein only of lower activity than that of the water soluble protein.
  • the method of the present invention can overcome the above-mentioned problems and produce an active immunoglobulin Fc region including the initial methionine residue from inclusion bodies.
  • an exogenous protein has an initial methionine residue encoded by the initiation codon.
  • properties of the hinge region to be added determine the post-translational modification of proteases, the ratio of dimers to monomers can be effectively controlled by selecting proper hinge regions.
  • inclusion bodies are refolded, the formation of accurate dimers is hindered by the mismatching of cysteines in disulfide bonds.
  • the method of the present invention ensures the formation of disulfide bonds, thereby leading to the efficient formation of active dimers.
  • an immunoglobulin G1 Fc region is produced at a yield of 15 mg/L according to the method of European Patent No. EP0227110, in which a G1 Fc region is overexpressed and purified only from a cell lysate containing the water soluble form thereof, and at a yield of 50 mg/L to 600 mg/L according to the method of Korean Patent application No. 0092783, in which an immunoglobulin Fc region fused to an E. coli signal sequence is expressed in a water soluble form, but not as an inclusion body.
  • an immunoglobulin Fc region may be produced at a yield of 5 g/L or higher from inclusion bodies of the recombinant immunoglobulin Fc region including the hinge region of the present invention. Accordingly, the method of producing the immunoglobulin Fc region of the present invention ensures a highly useful system for producing the immunoglobulin Fc region at a very high yield compared to the previous method.
  • the present invention relates to an immunoglobulin Fc region prepared by the method of the present invention, and specifically, to a monomeric or dimeric immunoglobulin Fc region including the initial methionine residue which is prepared by the above method.
  • the immunoglobulin Fc region prepared by the method of the present invention includes the hinge region, and methionine as the initiation amino acid at the N-terminus.
  • the immunoglobulin Fc region produced in prokaryotic cells such as E. coli according to the method does not have specifically limited industrial applications.
  • One exemplary application is use as a carrier for the formation of a conjugate with a certain drug. Construction of the conjugate including the immunoglobulin Fc region linked to a drug is not specifically limited.
  • the immunoglobulin Fc region and the drug may be linked together at various ratios, and the linkage may be mediated, for example, through a linker.
  • the drug includes polypeptides, compounds, extracts, and nucleic acids. Preferred is a polypeptide drug (used to have a meaning identical to the word "protein").
  • the linker includes peptide and non-peptide linkers, with preference for a non-peptide linker, and higher preference for a non-peptide polymer.
  • a preferred example of the immunoglobulin heavy chain is Fc.
  • any physiologically active polypeptide may be used without specific limitation to be linked to the immunoglobulin Fc region prepared according to the method of the present invention.
  • physiologically active polypeptides include those used for treating or preventing human diseases, which include cytokines, interleukins, interleukin binding protein, enzymes, antibodies, growth factors, transcription regulatory factors, coagulation factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists, and derivatives and analogues thereof.
  • the physiologically active polypeptide may be exemplified by human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons and interferon receptors (e.g., interferon- ⁇ , - ⁇ , and - ⁇ , 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-I receptor, IL-4 receptor, etc.), enzymes (e.g., glucocerebrosidase, iduronate-2-sulfatase
  • the physiologically active polypeptide useful in the present invention may be a native form, may be produced by genetic recombination using prokaryotic cells such as E. coli or eukaryotic cells, such as yeast cells, insect cells and animal cells, or may be a derivative having one or more amino acid mutations but showing biological activity identical to that of the native form.
  • an immunoglobulin Fc region produced from the transformant was linked to Exendin using polyethylene glycol, thus providing an Exendin-PEG-immunoglobulin Fc region protein conjugate.
  • This protein conjugate was found to exhibit serum half-life similar to an Exendin-PEG-Fc protein conjugate prepared by using an immunoglobulin Fc region devoid of Met as a carrier.
  • the immunoglobulin Fc region including the initial methionine residue obtained from inclusion bodies using the hinge region in accordance with the present invention, is used to enhance the serum half-life of the physiologically active polypeptide.
  • Example 1 Construction of vector expressing Fc region analogue including methionine (Met)
  • RNA from human blood cells serving as a template, as follows.
  • total RNA was isolated from about 6 mL of blood using a Qiamp RNA blood kit (Qiagen), and gene amplification was performed using the total RNA as a template with the aid of a One-Step RT-PCR kit (Qiagen).
  • Qiagen Qiamp RNA blood kit
  • gene amplification was performed using the total RNA as a template with the aid of a One-Step RT-PCR kit (Qiagen).
  • Qiagen Qiamp RNA blood kit
  • PCR was performed to amplify individual genes.
  • Fc analogues including methionine For amplification of Fc analogues including methionine, PCR was performed under the following conditions: 18 cycles consisting of 95°C for 10 seconds, 55°C for 5 seconds, and 68°C for 6 minutes.
  • the Fc analogue fragments including methionine obtained under the conditions were inserted into pET22b vector to express them as inclusion bodies in cells, respectively.
  • the expression vectors thus obtained were designated as pET22b-MetFc analogues 1 to 4, respectively.
  • the expression vectors included nucleic acids encoding amino acid sequences of Fc analogues 1 to 4 including methionine under control of t7 promoter, and expressed Fc analogue proteins including methionine in the form of inclusion bodies in host cells.
  • Fc analogues including Met were performed under control of T7 promoter.
  • each of the Fc analogue expression vectors including methionine prepared in Example 1 was transformed into E. coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB-mB-) gal ⁇ DE3); Novagen). The transformation was performed as recommended by Novagen. Single colonies transformed with each recombinant expression vector were taken and inoculated in 2X Luria Broth (LB) medium containing ampicillin (50 ⁇ g/mL), followed by incubation at 37°C for 15 hours.
  • LB 2X Luria Broth
  • the recombinant cell culture broth and 2X LB medium containing 30% glycerol were mixed at a ratio of 1:1 (v/v). Each 1 mL thereof was aliquoted in a cryotube and stored at -140°C, and used as a cell stock for production of recombinant fusion proteins.
  • each 1 vial of the cell stocks was thawed and inoculated in 500 mL of 2X Luria Broth (LB), and followed by shaking incubation at 37°C for 14 to 16 hours. When OD600 values reached 4.0 or higher, cultivation was terminated. These culture broths were used as seed culture broths. Each seed culture broth was inoculated in 1.7 L of a fermentation medium using a 5 L fermentor (MDL-8C, B.E.MARUBISHI, Japan), and initial bath fermentation was started.
  • LB 2X Luria Broth
  • the culture conditions were maintained at a temperature of 37°C, an airflow rate of 2 L/min (1 vvm), and an agitation speed of 500 rpm at pH 6.70 by using a 20% ammonia solution. Fermentation was performed in a fed-batch mode by adding a feeding solution when nutrients in the culture broth were depleted. Cell growth was monitored by OD values, and IPTG was introduced at a final concentration of 0.5 mM at an OD value of 120 or higher. After introduction, the cultivation was further performed for about 23 to 25 hours. After completing the cultivation, recombinant strains were harvested using a centrifuge, and stored at -80°C until use. After cultivation, expression was examined by electrophoresis on SDS-PAGE (FIG. 1).
  • each cell pellet corresponding to 1 L of the culture broth was resuspended in 1 L lysis buffer (50 mM Tris (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M sodium chloride and 0.5% triton X-100).
  • the cells were disrupted using a microfluidizer M-110EH (AC Technology Corp. Model M1475C) at a pressure of 15,000 psi.
  • the disrupted cell lysates were centrifuged at 7,000 rpm and 4°C for 30 minutes and the supernatant was discarded.
  • Each cell pellet was resuspended in 1 L of a washing buffer (0.5% triton X-100 and 20 mM Tris (pH 7.0)). Centrifugation was performed at 7,000 rpm and 4°C for 30 minutes, and the resulting pellet was resuspended in 1 L of washing buffer (20 mM Tris (pH 7.0)), followed by centrifugation in the same manner. Each pellet was resuspended in 1 L of buffer (8 M urea, 1.5 mM EDTA, 20 mM Tris (pH 7.0)), and agitated at room temperature for 4 hours. Centrifugation was performed for 4 hours in the same manner. The supernatants were collected, and 0.67 mM cysteine was added thereto, followed by agitation at 4°C for 1 hour.
  • buffer was replaced at a flow rate of 1000 mL/hr using a Sartorius Sartocon cassette (3021405907E-SG) and a peristaltic pump.
  • a primary buffer (2 M urea, 10 mM Tris (pH 8.5), 0.125 M arginine), a secondary buffer (1 M urea, 10 mM Tris (pH 8.5)), and a tertiary buffer (10 mM Tris (pH 8.5)
  • a primary buffer (2 M urea, 10 mM Tris (pH 8.5), 0.125 M arginine
  • secondary buffer (1 M urea, 10 mM Tris (pH 8.5)
  • a tertiary buffer (10 mM Tris (pH 8.5)
  • the refolded samples were loaded onto a DEAE (GE healthcare) column which had been equilibrated with 10 mM Tris (pH 8.0) buffer, and then Fc analogue proteins including methionine were eluted with a linear gradient from 0% to 45% over 4 column volumes using 10 mM Tris (pH 8.0) and 0.5 M sodium chloride buffer.
  • DEAE GE healthcare
  • Salts were removed from the samples eluted in Example 6 using a Sartorius Sartocon cassette (3021405907E-SG) and a peristaltic pump at a rate of 1000 mL/hr, followed by replacement of a buffer (10 mM Tris, pH 7.5).
  • Fc expressed as a dimer by refolding of inclusion body in the above Example includes the initiation codon, methionine.
  • methionine residue was processed by E. coli proteases, N-terminal sequences of the proteins were analyzed by the Basic Science Research Institute, Seoul, Korea. The samples for analysis were prepared as follows.
  • a PVDF membrane (Bio Rad) was activated in methanol for about 2 to 3 seconds, and then fully soaked in a blocking buffer (170 mM glycine, 25 mM Tris-HCl (pH 8), 20% methanol).
  • the samples on a non-reduced SDS-PAGE gel, prepared in Example ⁇ 1-2>, were blotted onto the PVDF membrane for about 1 hour using a blotting kit (Hoefer Semi-Dry Transfer unit, Amersham). Proteins transferred onto the PVDF membrane were stained with a protein dye, Coomassie Blue R-250 (Amnesco), for a moment (3 to 4 seconds), and washed with a destaining solution (water:acetic acid:methanol 5:1:4). Then, fragments containing proteins (each of dimer and monomer) were cut out from the washed membrane using scissors and subjected to N-terminal sequence analysis. Ultimately, it was confirmed that Met was included.
  • ALD-PEG-ALD Shearwater
  • a 3.4 kDa polyethylene glycol having an aldehyde reactive group at both ends was mixed with amounts of a 100 mM HEPES (pH 7.5) buffer containing CA-Exendin at a concentration of 12 mg/mL appropriate to form a molar ratio of CA-Exendin:PEG of 1:7.5.
  • a reducing agent sodium cyanoborohydride (NaCNBH 3 , Sigma) was added at a final concentration of 30 mM and was allowed to react at 4°C for 4 hours with gentle agitation.
  • NaCNBH 3 sodium cyanoborohydride
  • the refolded samples were loaded onto a Source S (GE healthcare) column which had been equilibrated with 20 mM sodium citrate (pH 2.0) and 45% ethanol buffer, and CA-Exendin-PEG complex was purified with a linear gradient from 0% to 10% over 0.0625 column volumes for 0.5 minutes and over 7 column volumes for 65 minutes using 20 mM sodium citrate (pH 2.0) and 0.25 M potassium chloride buffer.
  • Source S GE healthcare
  • the CA-Exendin-PEG complex prepared in Example 9 was linked to the Fc analogue including methionine produced in Example 7.
  • the immunoglobulin Fc region fragment prepared in Example 7 was mixed with the CA-Exendin-PEG complex at a molar ratio of CA-Exendin-PEG complex:Fc analogue including methionine of 1:2.
  • HEPES pH 8.2
  • buffer concentration of the reaction solution was adjusted to 100 mM, and 3%(v/v) Triton X-100 was added.
  • a reducing agent, NaCNBH 3 was added thereto at a final concentration of 50 mM and was allowed to react at RT for 15 hours with gentle agitation.
  • the reaction mixture was subjected to anion exchange chromatography and hydrophobic interaction chromatography so as to eliminate unreacted substances and byproducts and to purify a conjugate of CA-Exendin-PEG-Fc analogue protein containing Met.
  • the coupling reaction solution was loaded onto a Source Q (GE healthcare) column equilibrated with 20 mM bis Tris (pH 6.0) buffer, and then the conjugate of the CA-Exendin-PEG-Fc analogue protein containing Met was purified with a linear gradient from 0% to 100% over 16 column volumes for 143.2 minutes using 20 mM bis Tris (pH 6.0) and 0.25 M sodium chloride buffer.
  • the purified fraction was diluted 3.3 times for a final concentration of 50 mM potassium phosphate (pH 6.5), and 5 M sodium chloride was added to the diluted sample for a final concentration of 1.2 M sodium chloride.
  • the prepared sample was loaded onto the Source Phenyl (GE healthcare) column which had been equilibrated with 20 mM potassium phosphate (pH 6.5) and 1.2 M sodium chloride buffer, and step elution was performed from 0% to 100% at a flow rate of 2 mL/min using 20 mM potassium phosphate (pH 6.5) buffer to purify the conjugate of the CA-Exendin-PEG-Fc analogue protein containing Met.
  • the conjugate of CA-Exendin-PEG-Fc devoid of Met (control group) and the conjugate of the CA-Exendin-PEG-Fc analogue containing Met (test group) prepared in Example 10 were subcutaneously injected at a dose of 5 nmol/kg into three SD rats per group. After the subcutaneous injection, blood samples were collected at 1, 4, 8, 10, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336 hours from the control and test groups. The blood samples were collected in 1.5 mL tubes, coagulated, and centrifuged for 10 minutes using an Eppendorf high-speed micro centrifugator to remove blood cells. Serum protein levels were measured by ELISA using an antibody specific to Exendin.
  • FIG. 3 shows serum half-lives of the samples.
  • the CA-Exendin-PEG-Fc conjugate prepared by using the Fc analogue containing Met produced according to the present invention as a carrier, exhibited a similar serum half-life to that of the control group, CA-Exendin-PEG-Fc devoid of Met.

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Abstract

La présente invention concerne un procédé de production d'une région Fc d'immunoglobuline monomère ou dimère comprenant le résidu méthionine initial au moyen d'un vecteur comprenant une séquence nucléotidique codant pour une région Fc d'immunoglobuline recombinée comprenant une région charnière d'immunoglobuline, et une région Fc d'immunoglobuline monomère ou dimère comprenant le résidu méthionine initial préparée à l'aide du procédé. Une région Fc d'immunoglobuline recombinée comprenant une région charnière d'immunoglobuline de la présente invention peut être utilisée pour produire une région Fc d'immunoglobuline recombinée en tant que corps d'inclusion dans E coli, et de la méthionine est incluse dans la région Fc d'immunoglobuline recombinée exprimée. La région Fc d'immunoglobuline produite est liée à un polypeptide physiologiquement actif, et est ainsi utilisée pour augmenter la demi-vie sérique du polypeptide physiologiquement actif.
PCT/KR2016/010752 2015-09-25 2016-09-26 Procédé de production de région fc d'immunoglobuline comprenant un résidu méthionine initial WO2017052324A1 (fr)

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US20080293106A1 (en) * 2005-08-16 2008-11-27 Jung Sung Youb Method For the Mass Production of Immunoglobulin Fc Region Deleted Initial Methionine Residues
US8124094B2 (en) * 2005-04-08 2012-02-28 Hanmi Holdings Co., Ltd. Immunoglobulin Fc fragment modified by non-peptide polymer and pharmaceutical composition comprising the same
US20140113370A1 (en) * 2011-04-13 2014-04-24 Bristol-Myers Squibb Company Fc fusion proteins comprising novel linkers or arrangements
WO2014119956A1 (fr) * 2013-01-31 2014-08-07 Hanmi Pharm. Co., Ltd. Transformé de levure recombinée et procédé de préparation de fragment fc d'immunoglobuline faisant appel à celui-ci

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Publication number Priority date Publication date Assignee Title
US8124094B2 (en) * 2005-04-08 2012-02-28 Hanmi Holdings Co., Ltd. Immunoglobulin Fc fragment modified by non-peptide polymer and pharmaceutical composition comprising the same
US20080293106A1 (en) * 2005-08-16 2008-11-27 Jung Sung Youb Method For the Mass Production of Immunoglobulin Fc Region Deleted Initial Methionine Residues
US20140113370A1 (en) * 2011-04-13 2014-04-24 Bristol-Myers Squibb Company Fc fusion proteins comprising novel linkers or arrangements
WO2014119956A1 (fr) * 2013-01-31 2014-08-07 Hanmi Pharm. Co., Ltd. Transformé de levure recombinée et procédé de préparation de fragment fc d'immunoglobuline faisant appel à celui-ci

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WANG ET AL.: "Impact of Methionine Oxidation in Human IgG1 Fc on Serum Half-life of Monoclonal Antibodies", MOLECULAR IMMUNOLOGY, vol. 48, no. 6, 21 January 2011 (2011-01-21), pages 860 - 866, XP028143840 *

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