KR102382593B1 - Ph-sensitive fc variants - Google Patents

Abstract

The present invention relates to an Fc variant having improved half-life by binding and dissociating to FcRn in a pH-dependent manner. Since it is a maximized variant, it can bind to numerous peptide drug therapeutics with low half-life and retention time in the body, enabling long-term drug efficacy with increased blood half-life, thereby dramatically reducing the dosage and frequency of antibody and biopharmaceutical administration This has the effect of reducing the cost of new drug development and greatly improving the possibility of new drug development.

Description

pH-sensitive Fc variants {PH-SENSITIVE FC VARIANTS}

The present invention relates to an Fc variant having improved half-life by binding to and dissociating FcRn in a pH-dependent manner.

Protein therapeutics are widely used in clinical practice by rapidly replacing non-specific low-molecular compound therapeutics because they show very high specificity, low side effects, and toxicity to disease targets. Fc-fusion protein therapeutics fused with an antibody Fc region are the mainstays.

Therapeutic antibodies are considered as one of the most effective cancer treatment methods because they show very high specificity to the target compared to existing low molecular weight drugs, have low biotoxicity and few side effects, and have an excellent blood half-life of about 3 weeks. In fact, large pharmaceutical companies and research institutes around the world are spurring research and development of therapeutic antibodies that specifically bind to and effectively remove cancer cells, including cancer-causing factors. Pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS are mainly developing therapeutic antibody drugs. ) is a representative product, and these three therapeutic antibodies not only generated huge profits, such as achieving about $19.5 billion in sales in the global market in 2012, but also lead the global antibody drug market. Johnson & Johnson, which developed Remicade, is also growing rapidly in the global antibody market with increased sales, and pharmaceutical companies such as Abbott and BMS are also known to have many therapeutic antibodies in the final stage of development. As a result of this, biopharmaceuticals, including therapeutic antibodies that are specific to disease targets and have low side effects, are rapidly replacing the place in the global pharmaceutical market, where small-molecule drugs took the lead.

However, antibody and protein therapeutics have very low absorption through the digestive tract when administered orally, and their bioavailability is very low because they are easily denatured in the digestive tract or easily degraded by proteolytic enzymes. , frequent inoculation is required to achieve a visible therapeutic effect. It should be administered once every 3 weeks. Frequent drug administration through such injections causes considerable pain and discomfort in the patient, and there are problems that may lead to local and systemic side effects such as edema and infection. To solve this problem, it is necessary to dramatically improve the half-life of the therapeutic antibody and protein therapeutics in the blood.

It has been reported that the in vivo half-life of immunoglobulins (antibodies) is mediated by the binding of Fc to FcRn. The Fc fragment of immunoglobulin is taken up by endothelial cells through non-specific cellular uptake, and then introduced into acidic endosomes. FcRn binds to immunoglobulins under acidic pH (<6.5) in endosomes and releases immunoglobulins under basic pH (>7.4) in the bloodstream. Thus, FcRn recovers immunoglobulins from the lysosomal degradation pathway. When serum immunoglobulin levels decrease, more FcRn molecules can be used for immunoglobulin binding to increase the amount of immunoglobulins. Conversely, when serum immunoglobulin levels are elevated, FcRn may saturate and increase the proportion of cellular uptake immunoglobulins that are degenerate (Ghetie and Ward, Annu. Rev. Immunol. 18: 739-766, 2000). That is, the blood half-life and persistence of the antibody largely depend on the binding of the Fc region of the antibody to one of the IgG-binding ligands, FcRn (neonatal Fc receptor). The Fc region of an antibody, which serves to recruit immune leukocytes or serum complement molecules so that damaged cells such as cancer cells or infected cells can be eliminated, is a region between the Cγ2 and Cγ3 domains and interacts with the neonatal receptor FcRn mediates, and its binding recycles endocytosed antibodies from the endosome to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766). This process, due to the large size of the full-length molecule, has a favorable antibody serum half-life in the range of 1 to 3 weeks, associated with inhibition of kidney filtration. In addition, the binding of Fc to FcRn plays an important role in antibody transport. Therefore, the Fc region plays an essential role in maintaining prolonged serum persistence by circulating antibodies through intracellular trafficking and recycling mechanisms.

An object of the present invention is to improve the binding rate to FcRn in intracellular endosomes (weakly acidic pH conditions) in order to improve the blood half-life of antibodies, and introduce mutations that can dissociate rapidly from FcRn in blood (neutral pH conditions) developing an antibody.

In order to solve the above problems, the present invention provides a pH-sensitive Fc variant.

In addition, the present invention provides a polypeptide comprising a pH-sensitive Fc variant.

In addition, the present invention provides an antibody comprising a pH-sensitive Fc variant.

The present invention also provides a nucleic acid molecule encoding a pH sensitive Fc variant, polypeptide or antibody.

In addition, the present invention provides a vector comprising the nucleic acid molecule.

In addition, the present invention provides a host cell comprising the vector.

In addition, the present invention provides a protein conjugate having an increased half-life in vivo.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer.

In addition, the present invention provides a method for preparing a pH-sensitive Fc variant.

In addition, the present invention provides a method for producing a polypeptide comprising a pH-sensitive Fc variant.

In addition, the present invention provides a method for producing an antibody comprising a pH-sensitive Fc variant.

In addition, the present invention provides a method for increasing the in vivo half-life of a bioactive polypeptide.

Since the Fc variant of the present invention is a variant with a maximized blood half-life that shows superior pH-selective FcRn binding and dissociation ability than conventional blood half-life-enhancing Fc variants, it binds to numerous peptide drug therapeutics with low half-life and retention time in the body This can enable long-term drug efficacy with increased half-life in the blood, which can dramatically reduce the dose and frequency of administration of antibodies and biopharmaceuticals, and have the effect of reducing new drug development costs and greatly improving new drug development possibilities. there is.

1 is a diagram confirming the binding affinity of trastuzumab containing Q311M and M428L Fc variants with FcRn at pH 6.0 and 7.4 by ELISA:
PFc29: trastuzumab with Q311R and M428L Fc variants; and
M: Trastzumab with Q311M and M428L Fc variants.
2 is a diagram showing a SDS-PAGE gel photograph after expression purification of trastuzumab containing each of the Fc variants and a schematic diagram of amino acid variant production at the L309 position (saturation mutagenesis).
3 is a diagram confirming the binding affinity of Trastuzumab containing Fc variants (amino acid mutations at positions Q311M, M428L and 309) with FcRn at pH 6.0 by ELISA:
PFc29: trastuzumab with Q311R and M428L Fc variants;
FM: Trastzumab with L309F, Q311M and M428L Fc variants;
IM: Trastzumab with L309I, Q311M and M428LFc Fc variants;
MM: trastuzumab with L309M, Q311M and M428LFc Fc variants;
VM: Trastuzumab with L309V, Q311M and M428LFc Fc variants;
TM: Trastzumab with L309T, Q311M and M428LFc Fc variants;
AM: Trastzumab with L309A, Q311M and M428LFc Fc variants;
YM: Trastzumab with L309Y, Q311M and M428LFc Fc variants;
HM: Trastzumab with L309H, Q311M and M428LFc Fc variants;
QM: trastuzumab with L309Q, Q311M and M428LFc Fc variants;
NM: trastuzumab with L309N, Q311M and M428LFc Fc variants;
KM: trastuzumab with L309K, Q311M and M428LFc Fc variants;
EM: Trastzumab with L309E, Q311M and M428LFc Fc variants;
WM: Trastzumab with L309W, Q311M and M428LFc Fc variants;
RM: Trastzumab with L309R, Q311M and M428LFc Fc variants; and
SM: Trastuzumab with L309S, Q311M and M428LFc Fc variants.
Figure 4 is a diagram confirming the binding affinity of trastuzumab containing Fc variants with FcRn at pH 6.0 and 7.4 by ELISA:
PFc29: trastuzumab with Q311R and M428L Fc variants;
ML: Trastzumab with Q311M and M428L Fc variants; and
YML: Trastzumab with L309Y, Q311M and M428L Fc variants.
5 is a schematic diagram of the association rate (on rate) in a weakly acidic environment and the dissociation rate (off rate) in a neutral environment.
6 is a diagram showing the results of analysis of the association rate and dissociation rate of Fc variants:
PFc29: trastuzumab with Q311R and M428L Fc variants;
PFc41: Trastzumab with L309G and M428L Fc variants;
ML: Trastzumab with Q311M and M428L Fc variants; and
YML: Trastzumab with L309Y, Q311M and M428L Fc variants.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention. However, the following embodiments are presented as examples for the present invention, and the present invention is not limited thereto, and the present invention is capable of various modifications and applications within the scope of equivalents interpreted and interpreted from the following claims. .

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice to test the present invention, the preferred materials and methods are described herein.

Throughout this specification, common one-letter and three-letter codes for naturally occurring amino acids are used as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Amino acids referred to by abbreviation in the present invention are also described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

In one aspect, the present invention relates to an Fc variant comprising modification of amino acid residues of L309Y according to the Kabat numbering system in the Fc region of wild-type immunoglobulin.

In one embodiment, the Fc variant of the present invention may further comprise a substitution of amino acid residues of M428L, Q311M, or M428L and Q311M.

In one embodiment, the immunoglobulin may be IgA, IgM, IgE, IgD or IgG, or a variant thereof, may be IgG1, IgG2, IgG3 or IgG4, preferably an anti-HER2 antibody, preferably trastuzumab more preferably. Papain digestion of antibodies forms two Fab fragments and one Fc fragment, and in human IgG molecules, the Fc region is produced by papain digestion of the N-terminus of Cys 226 (Deisenhofer, Biochemistry 20: 2361-2370, 1981). .

In one embodiment, the Fc region of wild-type immunoglobulin may include the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the Fc variant may include the amino acid sequence of SEQ ID NO: 2 (Q311M and M428L) and may include the amino acid sequence of SEQ ID NO: 3 (L309Y, Q311M and M428L).

In one embodiment, the Fc variant of the present invention is capable of pH-selective (dependently) binding/dissociation reaction with FcRn.

In one embodiment, the Fc variant of the present invention may be a pH-sensitive Fc variant.

In one embodiment, the Fc variant of the present invention may exhibit a lower binding affinity to FcRn compared to the wild-type immunoglobulin Fc region at pH 7.0 to 7.8, and may be in the normal pH range of blood, and may be at pH 7.2 to 7.6. In the Fc variant of the present invention, the degree of dissociation from FcRn in the above pH range may be the same or substantially unchanged compared to that of the wild-type Fc domain.

In one embodiment, the Fc variant of the present invention may exhibit a higher binding affinity to FcRn compared to the wild-type immunoglobulin Fc region at pH 5.6 to 6.5, and may be in weakly acidic conditions in endosomes, and may be at pH 5.8 to 6.0. The pH-sensitive Fc variant of the present invention has a binding affinity for FcRn in the above pH range of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more compared to the wild-type Fc domain. % or more, 80% or more, 90% or more, or 100% or more, or at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, 9 It may be increased by at least fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least 100 fold.

In the present invention, a variant comprising an amino acid mutation in the immunoglobulin Fc region of the present invention is defined according to the amino acid modification constituting the parent immunoglobulin Fc region, and conventional immunoglobulin numbering follows the EU index by Kabat (Kabat). et al., Sequence of proteins of immunological interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, 1991).

Amino acids of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 of the present invention start from number 221 of Kabat numbering. For example, amino acid 208M of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 is identical to 428M by Kabat numbering, amino acid 91M of SEQ ID NO: 2 and SEQ ID NO: 3 is identical to 311M by Kabat numbering, and in SEQ ID NO: 3 Amino acid 89Y is identical to 309Y by Kabat numbering.

As used herein, the term “FcRn” or “neonatal Fc receptor” refers to a protein that binds to an IgG antibody Fc region, which is encoded, at least in part, by the FcRn gene. The FcRn may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit and monkey. As is known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a light chain and a heavy chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise stated herein, FcRn or one FcRn protein refers to a complex of FcRn heavy chain and beta-2-microglobulin.

As used herein, the term "wild-type polypeptide" refers to an unmodified polypeptide that is later modified to produce a derivative. The wild-type polypeptide may be a polypeptide found in nature or a derivative or engineered one of a polypeptide found in nature. The wild-type polypeptide may refer to the polypeptide itself, a composition comprising the wild-type polypeptide, or an amino acid sequence encoding the same. Accordingly, as used herein, the term “wild-type immunoglobulin” refers to an unmodified immunoglobulin polypeptide in which amino acid residues are modified to produce derivatives. Interchangeable with the above term, "parent immunoglobulin" may be used, which refers to an unmodified immunoglobulin polypeptide into which amino acid modifications have been introduced to yield derivatives.

As used herein, the term "amino acid modification" refers to a substitution, insertion and/or deletion, preferably substitution, of an amino acid in a polypeptide sequence. As used herein, the term "amino acid substitution" or "substitution" means that an amino acid at a specific position in a wild-type polypeptide sequence is replaced with another amino acid. For example, an Fc variant containing the L309Y substitution means that leucine, which is the 309th amino acid residue, is replaced with tyrosine in the amino acid sequence of a wild-type immunoglobulin Fc fragment.

As used herein, the term “Fc variant” is meant to include modifications of one or more amino acid residues compared to a wild-type immunoglobulin Fc fragment. Preferably, the Fc variant in the present invention comprises a wild-type immunoglobulin Fc fragment (region) comprising a modification of one or more amino acid residues selected from the group consisting of L309Y, M428L and Q311M (the numbering is according to the EU index as described in Kabat). ), the binding affinity to FcRn is increased in weakly acidic conditions and the binding affinity to FcRn is decreased in neutral conditions, so that the binding rate to FcRn is improved in weakly acidic intracellular endosomes, and it is rapidly dissociated in neutral blood.

The present invention provides Fc variants having increased binding affinity for FcRn and/or serum half-life compared to the corresponding wild-type immunoglobulin Fc fragment. The in vivo half-life (ie, persistence in a subject's serum or other tissues) of antibodies and other bioactive molecules is an important clinical variable that determines the dosage and frequency of the antibody (or any other pharmaceutical molecule). Therefore, such bioactive molecules, including antibodies with increased in vivo half-life, are of great pharmaceutical importance. The half-life of the substituted Fc variants of the present invention is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more compared to a wild-type Fc domain. may be increased by at least 100% or at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold over the wild-type Fc domain. there is.

The Fc variants of the present invention contain one or more amino acid modifications compared to the wild-type immunoglobulin Fc fragment (region or domain), thereby having differences in amino acid sequence. The amino acid sequence of the Fc variant according to the present invention is substantially identical to the amino acid sequence of the wild-type immunoglobulin Fc fragment. For example, the amino acid sequence of the pH-sensitive Fc variant according to the present invention is about 80% or more, preferably about 90% or more, and most preferably about 95% compared to the amino acid sequence of the Fc fragment of wild sheep immunoglobulin. will have more homology. Amino acid modifications may be performed genetically using molecular biological methods, or may be performed using enzymatic or chemical methods.

The Fc variants of the present invention may be prepared by any method known in the art. In one embodiment, the Fc variant of an immunoglobulin according to the present invention is used to form a nucleic acid that is cloned into a host cell, expressed and assayed, if desired, after encoding a polypeptide sequence comprising a specific amino acid modification. Various methods for this are described in Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons.

The nucleic acid encoding the Fc variant according to the present invention may be inserted into an expression vector for protein expression. Expression vectors usually comprise a protein operably linked, ie, placed in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements. In an appropriate state, the Fc variant according to the present invention can be produced by a method of inducing protein expression by culturing a host cell transformed with a nucleic acid, preferably, an expression vector containing a nucleic acid encoding the Fc variant according to the present invention. there is. A variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing an exogenous nucleic acid into a host cell are known in the art and will vary depending on the host cell used. Preferably, the Fc variant according to the present invention is produced by using Escherichia coli, which has high industrial use value due to low production cost, as a host cell.

Accordingly, the scope of the present invention includes the steps of culturing a host cell into which a nucleic acid encoding an Fc variant is introduced under conditions suitable for protein expression; And it includes a method for producing an Fc variant comprising the step of purifying or isolating the Fc variant expressed from the host cell.

Antibodies can be isolated or purified by various methods known in the art. Standard purification methods include chromatography techniques, electrophoresis, immunization, sedimentation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, various native proteins, such as, for example, bacterial proteins A, G, and L, bind antibodies and these proteins can be used for purification. Often, purification by a specific fusion partner may be possible.

In one aspect, the present invention relates to a polypeptide comprising an Fc variant of the present invention.

In one embodiment, the polypeptide may have an increased half-life in vivo compared to the wild-type.

In one aspect, the present invention relates to an antibody comprising an Fc variant or said polypeptide.

In one embodiment, the antibody of the present invention may have an increased half-life in vivo compared to an antibody comprising a wild-type Fc region.

In one embodiment, the antibody is a polyclonal antibody, monoclonal antibody, minibody, domain antibody, bispecific antibody, antibody mimic, chimeric antibody, antibody conjugate, human antibody or humanized antibody , or a fragment thereof.

The antibody of the present invention can maximize the half-life of the Fc domain (region) or a polypeptide comprising the same through optimization of the Fc region (L309Y, M428L and Q311M).

In one aspect, the present invention relates to a protein conjugate having an increased in vivo half-life in which the Fc variant of the present invention, a non-peptidyl polymer, and a physiologically active polypeptide are covalently linked.

In one embodiment, the Fc variant according to the present invention can be usefully used as a carrier for increasing the in vivo half-life of a bioactive polypeptide such as a protein drug.

In one embodiment, the protein conjugate comprising the Fc variant of the present invention can be used as a long-acting drug formulation with significantly increased in vivo half-life.

Non-peptidyl polymers usable in the present invention include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA ( It may be selected from the group consisting of biodegradable polymers such as polylactic acid, polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitin, hyaluronic acid, and combinations thereof, preferably polyethylene glycol am. Derivatives thereof already known in the art and derivatives that can be easily prepared at the level of the art are also included in the scope of the present invention.

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

Bioactive polypeptides usable in the present invention include liver growth hormone, growth hormone-releasing hormone, growth hormone-releasing peptide, interferons and interferon receptors (eg, interferon-alpha, -beta and -gamma, water-soluble type I interferon receptor); granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), glucacon-like peptides (GLP-1), G-protein-coupled receptor, Interleukins (eg IL-1 receptor, IL-4 receptor), enzymes (eg glucocerebrosidase, iduronate-2-sulfatase, alpha-galactosidase) Agent-A, agalsidase alpha, beta, alpha-L-iduronidase, butyrylcholinesterase, chitinase, glutamate dicar glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activatingfactor acetylhydrolase, neutral endopeptidase , myeloperoxidase), interleukin and cytokine binding protein (eg IL-18bp, TNF-binding protein), macrophage activator, macrophage peptide, B cell factor, T cell factor, protein A, allergy suppression Factor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, red blood cells Generating factor, hyperglycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin Lombin receptor activator peptide, thrombomodulin, blood factor VII, blood factor VIIa, blood factor VIII, blood factor IX, blood factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hist hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, endostatin (endostatin) angiotensin, bone growth factor, bone growth promoting protein, calcitonin, insulin, atriopeptin, cartilage inducer, elcatonin, connective tissue activator, tissue factor pathway inhibitor ), follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors (eg, nerve growth factor, cilliary neurotrophic factor, axogenesis factor-1, brain- natriuretic peptide, glial derived neurotrophic factor, netrin, neutrophil inhibitor factor, neurotrophic factor, neuturin), parathyroid hormone, Lil Raxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin , myostatin, receptors (eg TNFR(P75), TNFR(P55), IL-1 receptor, VEGF receptor, B cell activator receptor), receptor antagonist (eg IL1-Ra), cell surface antigen (e.g. CD 2, 3, 4, 5, 7, 11a, 11b, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69), monoclonal antibody, polyclonal antibody, antibody fragments (eg scFv, Fab , Fab', F(ab') 2 and Fd), and various types of virus-derived vaccine antigens, but are not limited thereto. Antibody fragments may be selected from Fab, Fab', F(ab') 2 , Fd or scFv having the ability to bind to a particular antigen.

In one embodiment, an antibody drug may be bound to a protein conjugate comprising an Fc variant, and the antibody drug for cancer treatment is trastzumab, cetuximab, bevacizumab, rituximab. rituximab, basiliximab, infliximab, Ipilimumab, Pembrolizumab, Nivolumab, Atezolizumab or Abelumab (Avelumab).

The present invention includes a method for preparing a long-acting drug formulation by covalently linking the Fc variant to a physiologically active polypeptide through a non-peptidyl polymer.

The manufacturing method according to the present invention comprises the steps of covalently linking a bioactive polypeptide and an Fc variant through a non-peptidyl polymer having a reactive group at the end; And it may include the step of separating the conjugate in which the bioactive polypeptide, the non-peptidyl polymer, and the Fc variant are covalently linked.

In one aspect, the invention relates to a nucleic acid molecule encoding an Fc variant, polypeptide or antibody of the invention.

The nucleic acid molecules of the present invention may be isolated or recombinant, and include single-stranded and double-stranded forms of DNA and RNA as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, a nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of separate fragments or as a component of a larger nucleic acid construct. Nucleic acids are operably linked when placed into a functional relationship with another nucleic acid sequence. For example, the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when expressed as a preprotein in the form before the polypeptide is secreted, and the promoter or enhancer is the polypeptide sequence is operably linked to a coding sequence when it affects the transcription of, or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, operably linked means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, they are contiguous and in the same reading frame. However, enhancers need not be located adjacent to each other. Linkage is achieved by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

In one aspect, the present invention relates to a vector comprising said nucleic acid molecule.

As used herein, the term "vector" refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994 et al.).

As used herein, the term "expression vector" refers to a vector comprising a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. The expression vector may include various regulatory sequences. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions.

In one aspect, the present invention relates to a host cell comprising the vector.

In the present invention, the term "host cell" includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. A host cell may be transfected or transformed by the vector, which refers to a process in which an exogenous nucleic acid molecule is delivered or introduced into a host cell.

The host cell of the present invention preferably includes bacterial cells, CHO cells, HeLa cells, HEK293 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, NIH3T3 cells, etc., but is not limited thereto. not.

In one aspect, the present invention relates to a method for increasing the half-life of a bioactive polypeptide in vivo, comprising the step of covalently linking the Fc variant of the present invention to the bioactive polypeptide through a non-peptidyl polymer. .

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the Fc variant, polypeptide or antibody of the present invention.

In one embodiment, it may further comprise an immunogenic apoptosis inducer.

In one embodiment, the immunogenic apoptosis inducer is an anthracycline-based anticancer agent, a taxane-based anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosmide-based anticancer agent, GADD34 It may be any one or more selected from the group consisting of /PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin, and the anthracycline-based anticancer agent is daunorubicin ), doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin or valrubicin may be used, and the taxane-based anticancer agent is paclitaxel ) or docetaxel (docetaxel).

By administering the pharmaceutical composition for preventing or treating cancer of the present invention together with a chemical anticancer drug (anticancer agent), it is possible to increase the cancer treatment effect of the conventional anticancer agent through the apoptosis effect of cancer cells. Co-administration may be performed simultaneously or sequentially with the anticancer agent. Examples of the anticancer agent include mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide as DNA alkylating agents. ifosfamide), carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin and carboplatin ; dactinomycin (actinomycin D), plicamycin and mitomycin C as anti-cancer antibiotics; and plant alkaloids including vincristine, vinblastine, etoposide, teniposide, topotecan and iridotecan, etc. , but is not limited thereto.

In one embodiment, the cancer is leukemias and acute lymphocytic leukemia, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia, Lymphomas such as Hodgkin's Disease, non-Hodgkin's lymphomas and multiple myeloma, brain tumors, neuroblastoma, retinoblastoma, Wilms Childhood solid tumors such as Wilms Tumor, bone tumors and soft-tissue sarcomas, lung cancer, breast cancer, prostate cancer , urinary cancers, uterine cancers, oral cancers, pancreatic cancer, melanoma and other skin cancers, stomach cancer, ovarian cancer ), brain tumors, liver cancer, laryngeal cancer, thyroid cancer, esophageal cancer and testicular cancer common solid tumors in adults may include, and the type of cancer to be prevented or treated in the present invention is not limited.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action that improves or advantageously changes the apoptosis of cancer cells or symptoms of cancer by administering the composition of the present invention. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented by the Korean Medical Association, etc., know the exact standard of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount of a pharmaceutically acceptable salt of a composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention is It may vary depending on several factors, for example, administration method, target site, patient's condition, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, type of cancer, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or locally as an injection formulation) or orally according to a desired method, and the dosage may vary depending on the patient's weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide and administer once to several times a day.

Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweetening agents, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

In one aspect, the present invention provides a method comprising: culturing a host cell comprising a vector comprising a nucleic acid molecule encoding an Fc variant of the present invention; And it relates to a method for producing an Fc variant having an increased in vivo half-life compared to the wild-type, comprising the step of recovering the Fc variant expressed by the host cell.

In one aspect, the present invention provides a method comprising: culturing a host cell comprising a vector comprising a nucleic acid molecule encoding a polypeptide of the present invention; And it relates to a method for producing a polypeptide comprising an Fc variant having an increased in vivo half-life compared to the wild-type, comprising the step of recovering the polypeptide expressed by the host cell.

In one aspect, the present invention provides a method comprising: culturing a host cell comprising a vector comprising a nucleic acid molecule encoding an antibody of the present invention; And it relates to a method for producing an antibody comprising a pH-sensitive Fc mutation * having an increased in vivo half-life compared to the wild-type comprising the step of purifying the antibody expressed by the host cell.

In one embodiment, the purification of the antibody may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably using Protein A. affinity chromatography can be used.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Production and purification of Fc variants (Q311M/M428L)

As seen in M252Y of YTE and M428L of LS, which are known to have a half-life improvement effect in previous studies, it was confirmed that Met was substituted with another amino acid in the mutant with improved FcRn binding ability. For this reason, Met in the FcRn binding site of antibody Fc is judged to be an amino acid that inhibits FcRn pH-dependent avidity, and when introduced into PFc29 (Q311R and M428L Fc variants), to find out whether the FcRn binding force actually inhibits the avidity, Q311 To prepare an Fc variant (Q311M/M428L) into which Met was introduced, it was transfected into Expi293F animal cells. Specifically, in 30 ml of Freestyle 293 expression culture medium (Gibco, 12338-018), the heavy chain gene and light chain gene of the Fc variant (Q311M/M428L) in which Met was introduced into Q311 were first mixed at a ratio of 1:1, and then PEI (Polyethylenimine) (Polyscience, 23966): Mutant gene = 4:1 ratio, mixed and left at room temperature for 20 minutes. The day before, it was dispensed at a density of 2x10 ⁶ cells/ml and mixed with the cultured Expi293F cell line. After culturing for 7 days under 8% CO ₂ conditions, centrifugation was performed to obtain only the supernatant. Thereafter, the mixture was equilibrated with 25x PBS and filtered using a 0.2 μm filter (Merck Millipore) and a bottle top filter. 500 μl of Protein A resin is added to the filtered culture, stirred at 4°C for 16 hours, and then flowed through the column to recover the resin, washed with 5 ml PBS, eluted with 3 ml 100 mM glycine (pH 2.7) buffer, and then eluted with 1M Neutralized using Tris-HCl pH 8.0. To change the buffer, centrifugal filter units 3K (Merck Millipore) were used.

Example 2. Confirmation of pH-dependent binding affinity of Fc variants (Q311M/M428L)

The pH-dependent binding ability of the Fc variant (Q311M/M428L) (SEQ ID NO: 2) purified in Example 1 to FcRn was confirmed by ELISA. Specifically, 50 μl of the IgG Fc variant diluted to 4 μg/ml in 0.05 M Na ₂ CO ₃ (pH 9.6) was immobilized on a 96-well microplate (costar) with Flat Bottom Polystyrene High Bind at 4° C. for 16 hours. Then, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0) was blocked at room temperature for 2 hours. After washing 4 times with 180 μl of 0.05% PBST (pH 6.0), 50 μl of FcRn serially diluted with 1% skim milk (in 0.05% PBST, pH 6.0) was dispensed into each well and reacted at room temperature for 1 hour. After the washing process, the antibody reaction was performed for 1 hour at room temperature using 50 μl of anti-GST-HRP conjugate (GE Healthcare) and washed. After that, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, and 50 μl of 2M H ₂ SO ₄ was added to terminate the reaction, and then analyzed using an Epoch Microplate Spectrophotometer (BioTek). did. As a result, it was confirmed that Met, which was expected to inhibit the binding affinity to FcRn, rather binds better than PFc29 at pH 6.0 and exhibits similar binding affinity to PFc29 at pH 7.4. That is, the Q311M mutant bound better than PFc29 at pH 6.0 and showed a similar binding affinity to PFc29 at pH 7.4 (Fig. 1).

Example 3. Searching for Fc variants having improved FcRn binding affinity

In order to further discover variants having improved FcRn binding ability than the Fc variants having Q311M and M428L mutations selected in the above example, L, G, P, C, and D variants containing 15 amino acids except for L309 position 15 species were prepared as in the above example, cultured in animal cells, and then purified (FIG. 2).

Example 4. Confirmation of pH-dependent binding affinity of antibodies containing Fc variants

In order to confirm the binding affinity to FcRn at pH 6.0 of the mutants purified in Example 3, ELISA analysis was performed as in Example 2. As a result, L309Y, Q311M, and M428L (indicated by YML) variants having the highest binding affinity at pH 6.0 were obtained ( FIG. 3 ). Accordingly, as a result of measuring the pH-dependent binding affinity of the selected YML mutant to FcRn by ELISA, the pH 6.0 than the conventional PFc29 (Q311R and M428L Fc mutant) and the ML mutant (Q311M and M428L Fc mutant) selected in the above Examples. It was found that the binding affinity to FcRn was high, and a similar degree of dissociation was shown at pH 7.4 (FIG. 4).

Example 5. Confirmation of pH-dependent binding affinity to FcRn of Fc variants

Since the pH-dependent binding ability of Fc to FcRn is important for blood half-life improvement (see FIG. 5), referring to Souders et al 2015, the on rate of binding in a weakly acidic environment and the instantaneous rate of dissociation in a neutral environment ( off rate) was confirmed in the ML variants (Q311M and M428L Fc variants) and YML variants (L309Y, Q311M and M428L) selected in the above Examples. As a control thereof, conventional PFc29 (Q311R and M428L Fc variants) and PFc41 (L309G and M428L Fc variants) were used. Specifically, 40 μg/ml of His-tagged human FcRn was immobilized on an NI-NTA biosensor (Pall Fortebio) for 3 minutes and the baseline was set with a PBS buffer of pH 6.0, and 700 nM of each antibody for 20 seconds Fc variants (in PBS pH 6.0) were bound. In a weakly acidic environment (pH 6.0), in a state in which the antibody Fc variant was bound to FcRn, the buffer was changed to PBS at pH 7.4, dissociated for 5 seconds, and measured by biolayer interferometry assay (BLItz, Pall Fortebio). In the measured data (sensorgram), the instantaneous rate of association and instantaneous dissociation were confirmed through the slope of the linearity section (pH6.0, association: 2 sec / pH 7.4, dissociation: 1 sec) at each pH, and Fc variants For comparison between the two, it was calculated as a ratio by dividing the slope value of each Fc variant. By scoring the average of the association rate ratio and the dissociation rate ratio, the Fc variant having the fastest association rate and dissociation rate was identified. As a result, the antibody Fc variant (YML) (SEQ ID NO: 3), which is expected to have the greatest half-life increasing effect, was selected ( FIG. 6 ).

<110> Korea University Research and Business Foundation <120> PH-SENSITIVE FC VARIANTS <130> DP-2019-0911 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 227 <212> PRT <213> Homo sapiens <400> 1 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 2 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> ML (Q311M/M428L) <400> 2 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Met Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 3 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> YML (L309Y/Q311M/M428L) <400> 3 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Tyr His Met Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225

Claims (26)

An Fc variant comprising modification of amino acid residues of L309Y, Q311M and M428L according to the Kabat numbering system in the Fc region of wild-type immunoglobulin. delete The Fc variant of claim 1 , wherein the immunoglobulin is selected from the group consisting of IgA, IgM, IgE, IgD and IgG. The Fc variant according to claim 1, wherein the Fc region of wild-type immunoglobulin comprises the amino acid sequence of SEQ ID NO: 1. delete The Fc variant according to claim 1, wherein the Fc variant comprises the amino acid sequence of SEQ ID NO:3. The Fc variant according to claim 1, which exhibits a lower binding affinity to FcRn compared to the wild-type immunoglobulin Fc region at pH 7.0 to 7.8. The Fc variant according to claim 1, which exhibits a high binding affinity to FcRn compared to the wild-type immunoglobulin Fc region at pH 5.6 to 6.5. The Fc variant of claim 1 , which is pH sensitive. A polypeptide comprising the Fc variant of claim 1 . The polypeptide according to claim 10, wherein the half-life in vivo is increased as compared to the wild-type. An antibody comprising the Fc variant of claim 1 . 13. The antibody of claim 12, which has an increased half-life in vivo compared to wild-type. 13. The method of claim 12, wherein the polyclonal antibody, monoclonal antibody, minibody, domain antibody, bispecific antibody, antibody mimic, chimeric antibody, antibody conjugate, human or humanized antibody, or Antibodies that are fragments thereof. A nucleic acid molecule encoding the Fc variant of claim 1 , the polypeptide of claim 10 , or the antibody of claim 12 . A vector comprising the nucleic acid molecule of claim 15 . A host cell comprising the vector of claim 16 . The protein conjugate of claim 1, wherein the Fc variant, the non-peptidyl polymer, and the physiologically active polypeptide are covalently linked to have an increased half-life in vivo. 19. The method of claim 18, wherein the bioactive polypeptide is human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, colony stimulating factor, interleukin, interleukin soluble receptor, TNF soluble receptor, glucocerebrosidase, macrophage activator , macrophage peptide, B cell factor, T cell factor, protein A, allergy suppressor factor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor factor, metastatic growth factor, alpha-1 antitrypsin, albumin, Apolipoprotein-E, erythropoietin, high glycosylated erythropoietin, blood factor VII, blood factor VIII, blood factor IX, plasminogen activator, urokinase, streptokinase, protein C, C-reactive protein, renin inhibitor , Collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, osteogenic growth factor, osteogenic protein, calcitonin, insulin, insulin derivative, glucagon, glucagon analog peptide-1 (Glucagon Like Peptide-1) atriopeptin, cartilage inducing factor, connective tissue activator, follicle-stimulating hormone, luteinizing hormone, follicle-stimulating hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin- Growth factor, corticosteroid hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid stimulating hormone, receptors, receptor antagonists, cell surface antigens, monoclonal antibodies, polyclonal antibodies, antibody fragments , and a protein conjugate selected from the group consisting of a virus-derived vaccine antigen. A pharmaceutical composition for preventing or treating cancer comprising the Fc variant of claim 1, the polypeptide of claim 10, and the antibody of claim 12. The pharmaceutical composition for preventing or treating cancer according to claim 20, further comprising an immunogenic apoptosis inducing agent. The method of claim 21, wherein the immunogenic apoptosis inducer is an anthracycline-based anticancer agent, a taxane-based anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclofosmide-based anticancer agent, GADD34 / PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin (bleomycin), mitoxantrone (mitoxantrone) or any one or more selected from the group consisting of oxaliplatin (oxaliplatin), a pharmaceutical composition for preventing or treating cancer. 1) culturing a host cell containing a vector containing a nucleic acid molecule encoding the Fc variant of claim 1; and
2) A method for producing an Fc variant having an increased in vivo half-life compared to the wild-type, comprising the step of recovering the Fc variant expressed by the host cell. 1) culturing a host cell containing a vector containing a nucleic acid molecule encoding the polypeptide of claim 10; and
2) A method for producing a polypeptide comprising an Fc variant having an increased in vivo half-life compared to the wild-type, comprising recovering the polypeptide expressed by the host cell. 1) culturing a host cell containing the vector containing the nucleic acid molecule encoding the antibody of claim 13; and
2) A method for producing an antibody comprising an Fc variant having an increased in vivo half-life compared to the wild-type, comprising the step of purifying the antibody expressed by the host cell. A method for increasing the half-life of a bioactive polypeptide in vivo, comprising covalently linking the Fc variant of claim 1 to a bioactive polypeptide through a non-peptidyl polymer.

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