pH-sensitive FC mutants

Abstract

The present invention relates to Fc variants with extended half-lives due to pH-dependent binding and dissociation to FcRn. The Fc variants of the present invention exhibit superior pH-selective FcRn binding and dissociation ability compared to conventional Fc variants with extended blood half-lives, thereby maximizing blood half-lives. Therefore, they can bind to numerous peptide pharmaceutical therapeutic agents with short half-lives and retention times in the body, and exert their therapeutic effects over a prolonged period due to their extended blood half-lives. This can also dramatically reduce the administration dose and frequency of antibodies and biopharmaceuticals, thereby reducing the cost of new drug development and significantly improving the feasibility of new drug development.

Description

[Technical field]

[0001] The present invention relates to Fc mutants whose half-lives are extended by binding to and dissociating from FcRn in a pH-dependent manner. [Background technology]

[0002] Protein therapeutics are highly specific to disease targets and have few side effects and toxicity, so they have quickly replaced non-specific small molecular weight compound therapeutics and are widely used in clinical practice. Among the protein therapeutics currently used in clinical practice, Fc fusion protein therapeutics, which are a fusion of an antibody therapeutic agent and an antibody Fc region, are the mainstream.

[0003] Therapeutic antibodies are considered to be one of the most effective cancer treatment methods because they have a very high target specificity, low biotoxicity, and few side effects compared to conventional small molecule drugs, and also have an excellent half-life in the blood of about three weeks. In fact, large pharmaceutical companies and research institutes around the world are actively researching and developing therapeutic antibodies that specifically bind to cancer cells, including the causative factors of cancer, and effectively remove them. The main companies developing therapeutic antibody drugs are Roche, Amgen, Johnson & Johnson, Abbott, and BMS, and in particular, Roche's representative products for anti-cancer treatments include Herceptin, Avastin, and Rituxan, and these three therapeutic antibodies achieved sales of about 19.5 billion dollars in the global market in 2012, generating large profits and leading the global antibody drug market. Johnson & Johnson, which developed Remicade, is also experiencing rapid growth in the global antibody market due to increased sales, and pharmaceutical companies such as Abbott and BMS are also known to have a large number of therapeutic antibodies in the final stages of development. As a result, biopharmaceuticals, including therapeutic antibodies that are specific to disease targets and have fewer side effects, are rapidly replacing small molecule drugs in the global pharmaceutical market, which had previously been dominant.

[0004] However, when administered orally, antibody and protein therapeutic agents have a very low absorption rate in the digestive tract, are easily denatured inside the digestive tract, are easily decomposed by proteolytic enzymes, and have very low bioavailability, so they must be administered by intravenous or subcutaneous injection, and frequent administration is required to obtain a significant therapeutic effect. Even for antibodies, which are one of the protein therapeutic agents with excellent stability in blood, a high dose of therapeutic antibody (2 to 8 mg / kg body weight) must be administered once every 2 to 3 weeks to obtain a therapeutic effect. Such frequent drug administration by injection causes great pain and inconvenience to patients, and has the problem of causing local and systemic side effects such as edema and infection. In order to solve the above problem, the half-life of the therapeutic antibody and protein therapeutic agent in the blood must be dramatically improved.

[0005] It has been reported that the in vivo half-life of immunoglobulins (antibodies) is brought about by Fc binding to FcRn. The Fc fragment of immunoglobulin is uptaken by endothelial cells using non-specific cellular absorption 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 retrieves immunoglobulins from the lysosomal degradation pathway. When serum immunoglobulin levels decrease, more FcRn molecules are available for immunoglobulin binding, and the amount of immunoglobulin can be increased. Conversely, when serum immunoglobulin levels increase, FcRn becomes saturated, and the proportion of immunoglobulins that degrade and are absorbed by cells can be increased (Non-Patent Document 1). That is, the half-life and persistence of an antibody in blood largely depend on the binding of the Fc portion of the antibody to FcRn (neonatal Fc receptor), which is one of the IgG-binding ligands. The Fc portion of an antibody, which acts to collect immune leukocytes or serum complement molecules and remove damaged cells such as cancer cells and infected cells, is located between the Cγ2 and Cγ3 domains and mediates the interaction with the neonatal receptor FcRn, and the binding recirculates the antibody transferred from the endosome to the bloodstream (Non-Patent Documents 1, 2). This process is associated with the prevention of kidney filtration due to the huge size of the full-length molecule, and has an advantageous antibody serum half-life in the range of 1 to 3 weeks. The binding of Fc to FcRn also plays an important role in antibody transport. Thus, the Fc portion plays an essential role in circulating antibodies and maintaining their prolonged serum persistence through intracellular trafficking and recycling mechanisms. [Prior art documents] [Patent documents]

[0006]

Patent Document 1

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0008] An object of the present invention is to develop antibodies into which mutations have been introduced that increase their binding ability to FcRn in intracellular endosomes (weakly acidic pH conditions) and decrease their binding ability to FcRn in blood (neutral pH conditions) in order to extend the half-life of the antibody in blood. [Means for solving the problem]

[0009] To solve the above problems, the present invention provides pH-sensitive Fc variants.

[0010] The present invention also provides polypeptides comprising pH-sensitive Fc variants.

[0011] Additionally, the present invention provides antibodies comprising pH-sensitive Fc variants.

[0012] Additionally, the present invention provides nucleic acid molecules encoding the pH sensitive Fc variants, polypeptides or antibodies.

[0013] Furthermore, the present invention provides a vector comprising the nucleic acid molecule.

[0014] Furthermore, the present invention provides a host cell comprising the vector.

[0015] Additionally, the present invention provides protein conjugates with extended in vivo half-lives. Effect of the Invention

[0016] The Fc variants of the present invention are variants that exhibit superior pH-selective FcRn binding and dissociation ability and maximize blood half-life compared to conventional blood half-life extended Fc variants. They bind to numerous peptide pharmaceutical therapeutic agents that have short half-lives and retention times in the body, and exert their pharmacological effects over a long period of time due to their extended blood half-life. This also has the effect of dramatically reducing the administration dose and frequency of antibodies and biopharmaceuticals, reducing the cost of new drug development, and significantly improving the possibility of new drug development. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the FcRn binding ability of trastuzumab containing Q311M and M428L Fc mutants at pH 6.0 and 7.4 as determined by ELISA. PFc29: Trastuzumab containing Q311R and M428L Fc mutants M: Trastuzumab containing Q311M and M428L Fc mutants [Diagram 2] 1 shows a schematic diagram of the preparation of amino acid mutants at L309 (saturation mutagenesis) and an SDS-PAGE gel photograph after expression and purification of trastuzumab containing each Fc mutant. [Diagram 3]FIG. 1 shows the FcRn-binding ability of trastuzumab containing Fc mutants (Q311M, M428L, and amino acid mutation at position 309) at pH 6.0, confirmed by ELISA. PFc29: trastuzumab containing the Q311R and M428L Fc variants FM: trastuzumab containing the L309F, Q311M and M428L Fc variants IM: trastuzumab containing the L309I, Q311M and M428L Fc variants MM: trastuzumab containing the L309M, Q311M and M428L Fc variants VM: trastuzumab containing the L309V, Q311M and M428L Fc variants TM: trastuzumab containing the L309T, Q311M and M428L Fc variants AM: trastuzumab containing the L309A, Q311M and M428L Fc variants YM: trastuzumab containing the L309Y, Q311M and M428L Fc variant HM: trastuzumab containing the L309H, Q311M and M428L Fc variants QM: trastuzumab containing the L309Q, Q311M and M428L Fc variants NM: trastuzumab containing the L309N, Q311M and M428L Fc variants KM: trastuzumab containing the L309K, Q311M and M428L Fc variants EM: trastuzumab containing the L309E, Q311M and M428L Fc variants WM: trastuzumab containing the L309W, Q311M and M428L Fc variants RM: trastuzumab containing the L309R, Q311M and M428L Fc variants SM: trastuzumab containing the L309S, Q311M and M428L Fc variants [Figure 4a] FIG. 1 shows the FcRn binding ability of trastuzumab containing Fc mutants at pH 6.0 confirmed by ELISA. PFc29: trastuzumab containing Q311R and M428L Fc mutants ML: trastuzumab containing Q311M and M428L Fc mutants YML: trastuzumab containing L309Y, Q311M, and M428L Fc mutants [Figure 4b] FIG. 13 shows the FcRn-binding ability of trastuzumab containing Fc mutants at pH 7.4, confirmed by ELISA. [Figure 4c]FIG. 13 shows the FcRn-binding ability of trastuzumab containing an Fc mutant at pH 6.0 and pH 7.4, confirmed and quantified by ELISA. [Diagram 5] FIG. 1 is a schematic diagram showing the binding rate (on rate) in a weakly acidic environment and the dissociation rate (off rate) in a neutral environment. [Figure 6a] Figure 2 shows the results of analyzing the binding rate of Fc variants at pH 6.0. PFc29: Trastuzumab with Q311R and M428L Fc variants PFc41: Trastuzumab with L309G and M428L Fc variants ML: Trastuzumab with Q311M and M428L Fc variants YML: Trastuzumab with L309Y, Q311M and M428L Fc variants [Figure 6b] FIG. 1 shows the results of an analysis of the dissociation rates of Fc mutants at pH 7.4. [Figure 6c] FIG. 1 shows the results of an analysis of the association and dissociation rates of Fc mutants. [Figure 7] 1 shows a schematic diagram of saturation mutagenesis at position L309 and an SDS-PAGE gel photograph after expression and purification of trastuzumab containing an Fc mutant. [Figure 8] Fig. 13 shows the FcRn-binding ability of trastuzumab containing Fc mutants at pH 6.0, confirmed by ELISA. [Figure 9] FIG. 13 shows the FcRn-binding ability of trastuzumab containing an Fc mutant at pH 6.0 and pH 7.4, confirmed by ELISA. [Figure 10a] FIG. 1 shows the FcFn-binding ability of trastuzumab containing EML mutants at pH 6.0, as determined by ELISA. [Figure 10b] FIG. 1 shows the FcFn-binding ability of trastuzumab containing EML mutants at pH 7.4, as determined by ELISA. [Figure 11] FIG. 1 shows the results of FACS analysis performed to confirm the binding ability of Fc mutants to FcRn at pH 6.0. [Figure 12]Photographs of SDS-PAGE gels after expression and purification of nine Trastuzumab Fc mutants (M428L, L309G mutants plus Q311L, Q311I, Q311V, Q311T, Q311A, Q311Y, Q311H, Q311K, or Q311W). [Figure 13] FIG. 1 shows the results of ELISA analysis of the binding affinity of nine trastuzumab Fc mutants to FcRn at pH 6.0 (M428L, L309G mutants with Q311L, Q311, Q311V, Q311T, Q311A, Q311Y, Q311H, Q311K or Q311W). [Figure 14] FIG. 1 shows the results of ELISA analysis of the FcRn-binding ability of three types of trastuzumab Fc mutants at pH 6.0 and pH 7.4. [Figure 15] Photograph of an SDS-PAGE gel after expression and purification of 30 trastuzumab Fc variants. [Figure 16] FIG. 1 shows the results of ELISA analysis of the FcRn-binding ability at pH 6.0 of 30 types of trastuzumab Fc mutants. [Figure 17a] FIG. 1 shows the results of ELISA analysis of the FcRn-binding ability of the EWL (L309E / Q311W / M428L) mutant at pH 6.0 and pH 7.4. [Figure 17b] FIG. 1 shows the results of ELISA analysis of the FcRn-binding ability of the EWL (L309E / Q311W / M428L) mutant at pH 6.0. [Figure 17c] FIG. 1 shows the results of ELISA analysis of the FcRn-binding ability of the EWL (L309E / Q311W / M428L) mutant at pH 7.4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention will be described in detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the present invention. The present invention can be modified and applied in various ways within the scope of the claims and their equivalents.

[0019] Unless otherwise defined, 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 or testing of the present invention, the preferred materials and methods are described herein.

[0020] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (Nmethylglycine), etc. In addition, the amino acids referred to in this invention by their abbreviations are written 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, Valine: V

[0021] In one aspect, the present invention relates to an Fc variant comprising an alteration of the amino acid residue L309Y according to the Kabat numbering system in the Fc region of a wild-type immunoglobulin.

[0022] In one example, the Fc variants of the invention may further comprise the following amino acid residue substitutions: M428L, Q311M, or M428L and Q311M.

[0023] In one aspect, the present invention relates to an Fc variant comprising an alteration of the amino acid residue Q311M according to the Kabat numbering system in the Fc region of a wild-type immunoglobulin.

[0024] In one example, Fc variants of the invention may further comprise the following amino acid residue substitutions: M428L, L309E, L309Y, M428L and L309E, or M428L and L309Y.

[0025] In one aspect, the present invention relates to an Fc variant comprising an amino acid residue modification of Q311W according to the Kabat numbering system in the Fc region of a wild-type immunoglobulin.

[0026] In one example, Fc variants of the invention may further comprise the following amino acid residue substitutions: M428L, L309E, or M428L and L309E.

[0027] In one embodiment, the immunoglobulin may be IgA, IgM, IgE, IgD or IgG, or a variant thereof, may be IgG1, IgG2, IgG3 or IgG4, and is preferably an anti-HER2 antibody, more preferably trastuzumab. Papain digestion of an antibody produces two Fab fragments and one Fc fragment, and in human IgG molecules, the Fc region is generated by papain digestion of the N-terminus of Cys226 (Non-Patent Document 3).

[0028] In one example, the Fc region of a wild-type immunoglobulin may comprise the amino acid sequence of SEQ ID NO:1.

[0029] In one example, the Fc variant may comprise the amino acid sequence of SEQ ID NO:2 (Q311M and M428L) or may comprise the amino acid sequence of SEQ ID NO:3 (L309Y, Q311M and M428L).

[0030] In one example, the Fc variant may comprise the amino acid sequence of SEQ ID NO:4 (L309E / Q311M / M428L).

[0031] In one example, the Fc variant may comprise the amino acid sequence of SEQ ID NO:5 (L309E / Q311W / M428L).

[0032] In one example, the Fc region of a wild-type immunoglobulin may comprise the nucleic acid sequence of SEQ ID NO:6.

[0033] In one example, the Fc variant may comprise the nucleotide sequence of SEQ ID NO: 7 (Q311M and M428L), or may comprise the nucleotide sequence of SEQ ID NO: 8 (L309Y, Q311M and M428L).

[0034] In one example, the Fc variant may comprise the nucleic acid sequence of SEQ ID NO:9 (L309E / Q311M / M428L).

[0035] In one example, the Fc variant may comprise the nucleic acid sequence of SEQ ID NO: 10 (L309E / Q311W / M428L).

[0036] In one embodiment, the amino acid sequence of SEQ ID NO: 11 may include a wild-type trastuzumab heavy chain, and the sequences GFNIKDTY, IYPTNGYT, and SRWGGDGFYAMDY represent CDR regions, respectively. The Fc region of wild-type trastuzumab in the amino acid sequence of SEQ ID NO: 11 corresponds to the sequence starting from amino acid 225.

[0037] In one example, the amino acid sequence of SEQ ID NO: 12 may comprise the wild-type trastuzumab light chain, with the sequences QDVNTA, SAS, and QQHYTTPPT representing the CDR regions, respectively.

[0038] In one example, the Fc variant of the present invention may be one that undergoes binding / dissociation reactions with FcRn in a pH-selective (dependent) manner.

[0039] In one example, the Fc variants of the present invention may have a lower binding affinity to FcRn at pH 7.0 to 7.8 compared to the wild-type immunoglobulin Fc region, which may be in the normal pH range of blood, or may be at pH 7.2 to 7.6. The Fc variants of the present invention may be such that the degree of dissociation from FcRn in the above pH range is the same or substantially unchanged compared to the wild-type Fc domain.

[0040] In one embodiment, the Fc variant of the present invention may have a higher binding affinity to FcRn at pH 5.6 to 6.5 compared to the wild-type immunoglobulin Fc region, or may be in the weakly acidic condition in the endosome, or may be at pH 5.8 to 6.0. The pH-sensitive Fc variant of the present invention may have a binding affinity to FcRn that is increased by 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, or 100% or more compared to the wild-type Fc domain in the above pH range, or may have a binding affinity that is increased by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-fold or more compared to the wild-type Fc domain.

[0041] In the present invention, the mutants containing amino acid mutations in the immunoglobulin Fc region of the present invention are defined according to the modification of the amino acids constituting the parent immunoglobulin Fc region, and the conventional immunoglobulin numbering follows the EU index by Kabat (Non-Patent Document 4).

[0042] The amino acids of SEQ ID NO:1 to SEQ ID NO:5 of the present invention start from position 221 of the Kabat numbering. For example, Asp (D, 1D), which is the first amino acid of SEQ ID NO:1 of the present invention, is identical to 221D of the Kabat numbering, Met (M, 91M), which is the 91st amino acid of SEQ ID NO:2, is identical to 311M of the Kabat numbering, and Tyr (Y, 89Y), which is the 89th amino acid of SEQ ID NO:3, is identical to 309Y of the Kabat numbering.

[0043] The "trastuzumab Fc variant" of the present invention refers to a variant in which the Fc variant of the present invention has been introduced in place of the Fc region of a wild-type trastuzumab heavy chain comprising the amino acid sequence of SEQ ID NO: 11. It also includes a wild-type trastuzumab light chain comprising the amino acid sequence of SEQ ID NO: 12. Thus, the "trastuzumab Fc variant" is produced by preparing an expression vector in which the Fc variant of the present invention has been introduced in place of the Fc region of a wild-type trastuzumab heavy chain, preparing an expression vector in which a wild-type trastuzumab light chain comprising the amino acid sequence of SEQ ID NO: 12 has been introduced, and transfecting them into animal cells to express them.

[0044] In the present invention, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody, which is at least partially encoded by the FcRn gene. The FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein contains two polypeptides, often referred to as a light chain and a heavy chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. In this specification, unless otherwise specified, FcRn or FcRn protein refers to a complex of FcRn heavy chain and β2-microglobulin.

[0045] By "wild-type polypeptide" in the present invention is meant an unmodified polypeptide which, when subsequently modified, produces a derivative. A wild-type polypeptide may be a polypeptide found in nature, or a derivative or engineered version of a polypeptide found in nature. A wild-type polypeptide may refer to the polypeptide itself, a composition comprising said wild-type polypeptide, or the amino acid sequence encoding it. Thus, by "wild-type immunoglobulin" in the present invention is meant an unmodified immunoglobulin polypeptide which, when amino acid residues are modified, produces a derivative. The term "parent immunoglobulin" is used interchangeably to mean an unmodified immunoglobulin polypeptide which, when amino acid modifications are introduced, produces a derivative.

[0046] In the present invention, "amino acid modification" refers to the substitution, insertion and / or deletion, preferably substitution, of an amino acid in a polypeptide sequence. In the present invention, "amino acid substitution" or "substitution" refers to the replacement of an amino acid with another amino acid at a specific position in a wild-type polypeptide sequence. For example, an Fc variant containing an L309Y substitution refers to an Fc variant in which the 309th amino acid residue in the amino acid sequence of a wild-type immunoglobulin Fc fragment, leucine, is replaced with tyrosine.

[0047] As used herein, the term "Fc variant" refers to one that contains at least one amino acid residue modification compared to a wild-type immunoglobulin Fc fragment. In the present invention, the Fc variant preferably contains at least one amino acid residue modification selected from the group consisting of L309Y, Q311M and M428L (said numbering is according to EU index of Kabat), L309E, Q311M and M428L (said numbering is according to EU index of Kabat), and L309E, Q311W and M428L (said numbering is according to EU index of Kabat), and has increased binding affinity to FcRn under weakly acidic conditions, decreased binding affinity to FcRn under neutral conditions, improved binding rate to FcRn in weakly acidic intracellular endosomes, and rapid dissociation in neutral blood compared to a wild-type immunoglobulin Fc fragment (region).

[0048] The present invention provides Fc variants with increased binding affinity to FcRn and / or extended serum half-life compared to the corresponding wild-type immunoglobulin Fc fragment. The in vivo half-life (i.e., persistence in the serum or other tissues of a subject) of antibodies and other bioactive molecules is an important clinical variable that determines the dosage and frequency of administration of an antibody (or any other pharmaceutical molecule). Thus, such bioactive molecules, including antibodies, with extended in vivo half-life are of great pharmaceutical importance. The half-life of the substituted Fc variants of the present invention may be increased by 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, or 100% or more compared to the wild-type Fc domain, and may be increased by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more compared to the wild-type Fc domain.

[0049] The Fc variants of the present invention contain at least one amino acid modification, and therefore have a different amino acid sequence, compared to the wild-type immunoglobulin Fc fragment (region or domain). The amino acid sequence of the Fc variants of the present invention is substantially homologous to the amino acid sequence of the wild-type immunoglobulin Fc fragment. For example, the amino acid sequence of the pH-sensitive Fc variants of the present invention has about 80% or more, preferably about 90% or more, and most preferably about 95% or more homology to the amino acid sequence of the Fc fragment of the wild-type immunoglobulin. The amino acid modification may be performed genetically using molecular biology methods, or may be performed using enzymatic or chemical methods.

[0050] In the present invention, the production and purification of trastuzumab can be performed with reference to Patent Document 1 (Korean Patent Application No. 10-2017-0045142).

[0051] The Fc variants of the present invention can be produced by any method known in the art. In one embodiment, the Fc variants of immunoglobulins according to the present invention are used to form nucleic acids that encode polypeptide sequences containing specific amino acid modifications, which can then be cloned into host cells, expressed, and assayed, if desired. Various methods for this purpose are described in the literature (Non-Patent Document 5).

[0052] Nucleic acids encoding Fc variants according to the invention may be inserted into an expression vector for protein expression. Expression vectors generally contain a protein operably linked, i.e. in functional relationship, with regulatory or control sequences, selection markers, any fusion partners, and / or additional elements. Fc variants according to the invention are produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector containing a nucleic acid encoding an Fc variant according to the invention, under appropriate conditions to induce protein expression. A variety of suitable host cells may be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and vary depending on the host cell used. It is preferred to produce the Fc variants according to the invention using E. coli as a host cell, which has low production costs and is highly industrially useful.

[0053] Thus, the scope of the present invention includes a method for producing an Fc variant, comprising culturing a host cell into which a nucleic acid encoding an Fc variant has been introduced under conditions suitable for expression of the protein, and purifying or isolating the expressed Fc variant from the host cell.

[0054] Antibodies can be separated or purified by various methods known in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunoprecipitation, dialysis, filtration, concentration and chromatofocusing techniques. As known in the art, various natural proteins, such as bacterial proteins A, G, L, can be bound to antibodies and used for purification. Sometimes purification by specific fusion partners is also possible.

[0055] In one aspect, the invention relates to polypeptides comprising the Fc variants of the invention.

[0056] In one embodiment, the polypeptide may have an increased in vivo half-life compared to the wild-type polypeptide.

[0057] In one aspect, the invention relates to an Fc variant or an antibody comprising said polypeptide.

[0058] In one example, an antibody of the present invention may have an extended in vivo half-life compared to an antibody comprising a wild-type Fc region.

[0059] In one example, the antibody may be a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a chimeric antibody, an antibody conjugate, a human antibody or a humanized antibody, or a fragment thereof.

[0060] The antibodies of the present invention can maximize the half-life of the Fc domain (region) or a polypeptide comprising same by optimizing the Fc region (L309Y, Q311M, and M428L; L309E, Q311M, and M428L; L309E, Q311W, and M428L).

[0061] In one aspect, the present invention relates to a protein conjugate having an extended in vivo half-life, in which an Fc variant of the present invention, a non-peptidyl polymer, and a physiologically active polypeptide are covalently linked.

[0062] In one example, the Fc variants of the present invention may be useful as carriers for extending the in vivo half-life of biologically active polypeptides such as protein drugs.

[0063] In one example, protein conjugates comprising the Fc variants of the invention may be used as long-acting drug formulations with significantly extended in vivo half-lives.

[0064] The non-peptidic polymer usable in the present invention is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitin, hyaluronic acid, and combinations thereof, and is preferably polyethylene glycol. Derivatives thereof known in the art and derivatives that can be easily prepared at the technical level of the art are also included in the present invention.

[0065] The physiologically active polypeptide to be bound to the Fc variant of the present invention may be any polypeptide whose half-life in blood needs to be extended, including, for example, various physiologically active polypeptides, such as cytokines, interleukins, interleukin-binding proteins, enzymes, antibodies, growth factors, transcriptional regulators, blood factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, and receptor antagonists, and derivatives and analogs thereof, that are used for the purpose of treating or preventing human diseases.

[0066] Examples of physiologically active polypeptides that can be used in the present invention include human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons and interferon receptors (e.g., interferon α, β, and γ, soluble type 1 interferon receptor), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), glucagon-like peptides (GLP-1), G-protein-coupled receptors, interleukins (e.g., IL-1 receptor, IL-4 receptor), enzymes (e.g., glucocerebrosidase, iduronate-2-sulfatase, α-galactosidase A, agalsidase α), and the like. alpha), beta, alpha-L-iduronidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidaseendopeptidase, myeloperoxidase, interleukin and cytokine binding proteins (e.g., IL-18bp, TNF binding protein), macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitory factor, cytotoxic glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha1-antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, erythropoietin, hyperglycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin ombomodulin), blood factor VII, blood factor VIIa, blood factor VIII, blood factor IX, blood factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, endostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, insulin, atriopeptin, cartilage-inducing factor, elcatonin, connective tissue activating factor, tissue factor pathway inhibitor factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factors (e.g., nerve growth factor, cilliary neurotrophic factor, axogenesis factor-1, brain-natriuretic peptide, glial derived neurotrophic factor, netrin, neutrophil inhibitory factorfactor, neurotrophic factor, neuturin, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenal cortex hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, receptors (e.g., TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, B cell activating factor receptor), receptor antagonists (e.g., IL1-Ra), cell surface antigens (e.g., CD2, 3, 4, 5, 7, 11a, 11b, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69), monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., scFv, Fab, Fab', F(ab')2, and Fd), virus-derived vaccine antigens, and the like, but are not limited thereto. The antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, and scFv, which have the ability to bind to a specific antigen.

[0067] In one example, an antibody drug may be conjugated to the protein conjugate comprising the Fc variant, and the antibody drug for treating cancer may be Trastuzumab, Cetuximab, Bevacizumab, Rituximab, Basiliximab, Infliximab, Ipilimumab, Pembrolizumab, Nivolumab, Atezolizumab, or Avelumab.

[0068] The invention includes methods of producing a sustained release drug formulation by covalently linking the Fc variants to a biologically active polypeptide via a non-peptidyl polymer.

[0069] The production method according to the present invention may comprise the steps of covalently linking a physiologically active polypeptide and an Fc variant via a non-peptidyl polymer having a reactive group at its terminus, and separating the conjugate in which the physiologically active polypeptide, the non-peptidyl polymer and the Fc variant are covalently linked.

[0070] In one aspect, the invention relates to nucleic acid molecules encoding the Fc variants, polypeptides or antibodies of the invention.

[0071] The nucleic acid molecules of the present invention may be isolated or recombinant and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. An isolated nucleic acid is one that is isolated from a naturally occurring source and is separated from surrounding genetic sequences present in the genome of the individual from whom the nucleic acid is isolated. In the case of a nucleic acid that is enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid produced during such processes is considered to be an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide when it is expressed as a preprotein, the form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence when it is positioned to facilitate translation. Generally, operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in the same reading frame. Enhancers, however, do not have to be contiguous. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0072] In one aspect, the present invention relates to a vector comprising said nucleic acid molecule.

[0073] In the present invention, a "vector" refers to a carrier into which a nucleic acid sequence is inserted for introduction into a cell where it will be replicated. The nucleic acid sequence may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art can construct vectors by standard recombinant techniques (e.g., Non-Patent Documents 6 and 7).

[0074] In the present invention, an "expression vector" refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide or peptide. Expression vectors contain various regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors also contain nucleic acid sequences that provide other functions.

[0075] In one aspect, the invention relates to a host cell comprising said vector.

[0076] In the present invention, the term "host cell" refers to any transformable organism, including eukaryotes and prokaryotes, that is capable of replicating the vector or expressing a gene encoded by the vector. A host cell is transfected or transformed by the vector, which refers to the process by which an exogenous nucleic acid molecule is delivered or introduced into the host cell.

[0077] Preferred host cells of the present invention include, but are not limited to, bacterial cells, CHO cells, HeLa cells, HEK293 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, and NIH3T3 cells.

[0078] In one aspect, the invention relates to a method for extending the in vivo half-life of a biologically active polypeptide comprising the step of covalently linking an Fc variant of the invention to the biologically active polypeptide via a non-peptidyl polymer.

[0079] In one aspect, the invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an Fc variant, polypeptide or antibody of the invention.

[0080] In one embodiment, the composition may further comprise an immunogenic cell death inducer.

[0081] In one embodiment, the immunogenic cell death inducer is an anthracycline anticancer drug, a taxane anticancer drug, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, The anticancer drug may be at least one selected from the group consisting of cyclophosphamide glycoside, a cyclophosphamide anticancer drug, a GADD34 / PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, and oxaliplatin, the anthracycline anticancer drug may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin, and the taxane anticancer drug may be paclitaxel or docetaxel.

[0082] When the pharmaceutical composition for preventing or treating cancer of the present invention is administered together with a chemical anticancer drug (anticancer agent), it can increase the cancer treatment effect of the conventional anticancer agent by killing cancer cells. The co-administration may be performed simultaneously or sequentially with the anticancer agent. Examples of the anticancer drugs include DNA alkylating agents such as mechlorethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin, and anti-cancer antibiotics such as actinomycin D, plicamycin, and mitomycin C, and plant alkaloids. Examples of alkaloids include, but are not limited to, vincristine, vinblastine, etoposide, teniposide, topotecan, and irinotecan.

[0083] In one embodiment, the cancers include leukemias, acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, lymphomas such as Hodgkin's Disease, non-Hodgkin's lymphomas, multiple myeloma, brain tumors, childhood solid tumors such as neuroblastoma, retinoblastoma, Wilms Tumor, bone tumors, soft-tissue sarcomas, lung cancer, breast cancer, Examples of cancers that can be prevented or treated in the present invention include common solid tumors in adults, such as pancreatic cancer, prostate cancer, urinary cancer, uterine cancer, oral cancer, pancreatic cancer, melanoma and other skin cancer, stomach cancer, ovarian cancer, brain tumor, liver cancer, laryngeal cancer, thyroid cancer, esophageal cancer, and testicular cancer, and the types of cancers that can be prevented or treated in the present invention are not limited.

[0084] In the present invention, "prevention" refers to any action that inhibits or delays the onset, spread and recurrence of cancer by administering the pharmaceutical composition according to the present invention.

[0085] In the present invention, "treatment" refers to any action of killing cancer cells or improving or favorably changing the symptoms of cancer by administering the composition of the present invention. A person having ordinary skill in the art to which the present invention pertains would be able to grasp the exact criteria for diseases for which the composition of the present invention is effective and to judge the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association, etc.

[0086] In the present invention, the "therapeutically effective amount" used in combination with the active ingredient means the amount of a pharma- ceutically acceptable salt of the composition that is effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention varies depending on various factors, such as the administration method, the target site, and the condition of the patient. Therefore, when used in the human body, the dosage should be determined appropriately taking into consideration both safety and efficacy. The amount used in humans can also be estimated from the effective amount determined by animal experiments. These matters to be considered when determining the effective amount are described, for example, in Non-Patent Documents 8 and 9.

[0087] The pharmaceutical composition of the present invention is administered in a pharmacologic effective amount. In the present invention, the term "pharmacologic effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects, and the effective dose level is determined by factors including the patient's health condition, type of cancer, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, co-administered or co-used drugs, and other factors known in the medical field. The composition of the present invention may be administered alone 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. It is important to administer an amount that provides maximum effect with minimum amount without side effects, taking into account all of the above factors, and this can be easily determined by a person skilled in the art.

[0088] The pharmaceutical composition of the present invention may further contain a pharma- ceutically acceptable additive, and examples of the pharma- ceutically acceptable additive include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, glucose, sorbitol, talc, etc. The pharma- ceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

[0089] The composition of the present invention may also include a carrier, diluent, excipient, or a combination of two or more thereof that is commonly used in biological preparations. The pharma- ceutical acceptable carrier is not particularly limited as long as it is suitable for delivering the composition into the body, and may be, for example, a mixture of one or more of the compounds described in Non-Patent Document 10, saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerin, ethanol, and their components, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be further added to formulate the composition into a weekly dosage form such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets. Furthermore, it is preferable to formulate the composition according to each disease or component by a suitable method in the art or the method disclosed in Non-Patent Document 11.

[0090] The composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or locally in the form of an injection) or orally depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / mL, preferably 0.0001 to 5 mg / mL. It is more preferable to administer it once a day or in several divided doses.

[0091] Liquid preparations for oral administration of the composition of the present invention include suspensions, liquid preparations for internal use, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water and liquid paraffin, which are commonly used diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc.

[0092] In one aspect, the present invention relates to a method for producing an Fc variant having an in vivo half-life extended compared to the wild-type, comprising the steps of culturing a host cell comprising a vector comprising a nucleic acid molecule encoding an Fc variant of the present invention, and recovering the Fc variant expressed by the host cell.

[0093] In one aspect, the present invention relates to a method for producing a polypeptide comprising an Fc variant having an in vivo half-life extended compared to the wild-type, the method comprising the steps of culturing a host cell comprising a vector comprising a nucleic acid molecule encoding a polypeptide of the present invention, and recovering the polypeptide expressed by the host cell.

[0094] In one aspect, the invention relates to a method for producing an antibody comprising a pH-sensitive Fc mutation having an extended in vivo half-life relative to the wild-type antibody, comprising the steps of culturing a host cell comprising a vector comprising a nucleic acid molecule encoding an antibody of the invention, and purifying the antibody expressed by the host cell.

[0095] In one embodiment, antibodies are purified using filtration, HPLC, anion or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, with affinity chromatography using Protein A being preferred. EXAMPLES

[0096] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the present invention. EXAMPLES

[0097] Fc variant with altered amino acid residue L309Y <1-1> Production and purification of Fc mutant (Q311M / M428L) As seen in M252Y of YTE and M428L of LS, which are known to have a half-life extension effect in previous studies, it was confirmed that Met was replaced with other amino acids in mutants with improved FcRn binding ability (amino acid sequence of wild-type Fc domain: SEQ ID NO: 1). For these reasons, it was determined that Met in the FcRn binding site of antibody Fc is an amino acid that inhibits pH-dependent binding ability to FcRn. To confirm whether or not the introduction of PFc29 (Q311R and M428L Fc mutants) actually inhibits FcRn binding ability, trastuzumab-ML was produced by introducing Fc mutant (Q311M / M428L) mutant (SEQ ID NO: 2) into the trastuzumab heavy chain (SEQ ID NO: 11) and transfected into Expi293F animal cells. Specifically, first, the heavy chain gene and the light chain gene of the Fc mutant (Q311M / M428L) in which Met was introduced into Q311 were mixed in a ratio of 1:1 with 30 mL of Freestyle 293 expression culture medium (Gibco, 12338-018). Next, PEI (Polyethylenimine) (Polysciences, 23966): mutant gene were mixed in a ratio of 4:1 and left to stand at room temperature for 20 minutes. Then, 2 × 10 6The cells were mixed with the Expi293F cell line, which had been cultured at a density of 10 ...

[0098] <1-2> Confirmation of pH-dependent binding ability of Fc mutant (Q311M / M428L) The pH-dependent binding ability of Trastuzumab-ML purified in Example 1-1 to FcRn was confirmed by ELISA. Specifically, 50 μL of the IgG Fc mutant diluted to 4 μg / mL in 0.05M Na2CO3 (pH 9.6) was immobilized on a flat bottom polystyrene High Bind 96-well microplate (costar) at 4° C. for 16 hours. Then, blocking was performed with 100 μL of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0) at room temperature for 2 hours. After washing four 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 allowed to react at room temperature for 1 hour. After washing, 50 μL of anti-GST-HRP conjugate (GE Healthcare) was used to react with the antibody at room temperature for 1 hour, followed by washing. Then, 50 μL of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to each well to develop color, and 50 μL of 2M H2SO4 was added to each well to terminate the reaction, and the results were analyzed using an Epoch Microplate Spectrophotometer (BioTek).

[0099] As a result, it was confirmed that Met, which was expected to inhibit the binding strength to FcRn, actually binds better than PFc29 at pH 6.0 and shows the same binding strength as PFc29 at pH 7.4. That is, the Q311M mutant binds better than PFc29 at pH 6.0 and shows the same binding strength as PFc29 at pH 7.4 (Figure 1).

[0100] <1-3> Search for Fc variants with improved FcRn binding ability We attempted to find further mutants with improved FcRn-binding ability than the Fc mutant having the Q311M and M428L mutations selected in Example 1-1. Specifically, 15 mutants in which 15 amino acids excluding L, G, P, C, and D were introduced at position L309 were prepared in the same manner as in Example 1-1, and cultured in animal cells and purified (Figure 2).

[0101] <1-4> Confirmation of pH-dependent binding ability of antibodies containing Fc mutants In order to confirm the binding ability of the mutants purified in Example 1-3 to FcRn at pH 6.0, ELISA analysis was performed in the same manner as in Example 1-2. As a result, the L309Y, Q311M and M428L (trastuzumab-YML) (YM in FIG. 3, hereinafter referred to as YML, SEQ ID NO: 3) mutants with the highest binding ability at pH 6.0 were obtained (FIG. 3). Therefore, the pH-dependent binding ability of the selected YML mutants to FcRn was measured by ELISA. As a result, it was found that the binding ability to FcRn at pH 6.0 was higher than that of the conventional PFc29 (Q311R and M428L Fc mutants) and the ML mutants (Q311M and M428L Fc mutants) selected in Example 1-1. In addition, the same degree of dissociation was shown at pH 7.4 (FIGS. 4a to 4c).

[0102] <1-5> Confirmation of the effect of Fc mutants on extending the blood half-life of FcRn Since the pH-dependent binding strength of Fc to FcRn is important for extending the half-life in blood (see FIG. 5), the binding instantaneous rate (on rate) in a weakly acidic environment and the dissociation instantaneous rate (off rate) in a neutral environment were confirmed for trastuzumab-ML (Q311M and M428L Fc mutants) and trastuzumab-YML (L309Y, Q311M and M428L Fc mutants) selected in Examples 1-1 and 1-4, with reference to Souders et al. 2015. As control groups, conventional PFc29 (Q311R and M428L Fc mutants) and PFc41 (L309G and M428L Fc mutants) were used. Specifically, 40 μg / mL of His-tagged human FcRn was immobilized on an NI-NTA biosensor (Pall Fortebio) for 3 minutes. Then, PBS buffer at pH 6.0 was used as a baseline, and then 700 nM of each antibody Fc mutant (in PBS pH 6.0) was bound for 20 seconds. In a state where the antibody Fc mutant was bound to FcRn in a weakly acidic environment (pH 6.0), the buffer was changed to PBS at pH 7.4 and dissociated for 5 seconds, and the data was measured by biolayer interferometry assay (BLItz, Pall Fortebio). In the data (sensorgram) measured in this way, the instantaneous binding rate and the instantaneous dissociation rate were confirmed by the slope of the linear section of each pH (pH 6.0, association: 2 seconds / pH 7.4, dissociation: 1 second). Then, to compare the Fc mutants, the slope value of each Fc mutant was divided to obtain a ratio. The average of the binding rate ratio and the dissociation rate ratio were scored to confirm the Fc mutant with the fastest binding rate and dissociation rate. As a result, an antibody Fc mutant (YML, SEQ ID NO: 3) that was expected to have the greatest half-life extension effect was selected (FIGS. 6a to 6c). EXAMPLES

[0103] Fc variant with altered amino acid residue Q311M <2-1> Production and purification of trastuzumab Fc mutants carrying the Q311M / M428L mutations Previous studies have shown that mutants with improved FcRn-binding ability have Met substituted with other amino acids (amino acid sequence of wild-type Fc domain: SEQ ID NO: 1). For these reasons, it was determined that Met in the FcRn-binding site of antibody Fc is an amino acid that inhibits the pH-dependent binding ability to FcRn. Therefore, this experiment was performed to confirm whether introduction of Met into PFc29 (Q311R / M428L), which the present inventors discovered, actually inhibits FcRn-binding ability.

[0104] More specifically, a heavy chain expression vector was prepared by introducing an Fc variant (Q311M / M428L, SEQ ID NO: 2) into a wild-type trastuzumab heavy chain (SEQ ID NO: 11), and a wild-type trastuzumab light chain (SEQ ID NO: 12) expression vector was prepared. The two vectors were then transfected into Expi293F animal cells. One day before transfection, Expi293F cells were cultured at 2 × 10 6 The cells were subcultured in 300 mL to a density of 1000 cells / mL, and the next day, transfection was performed using PEI (Polyethylenimine, Polysciences, 23966). First, the mutant heavy chain gene and light chain gene were mixed in a ratio of 1:1 in 30 mL of freestyle 293 expression culture medium (Gibco, 12338-018). Next, PEI: mutant gene = 4:1 was mixed and left to stand at room temperature for 20 minutes, mixed with cells that had been subcultured the day before, and cultured in a shaking CO2 incubator at 37 ° C, 125 rpm, and 8% CO2 for 7 days, after which the cells were centrifuged and only the supernatant was collected. Then, equilibration was performed using 25 × PBS. Filtration was performed using a bottle top filter and a 0.2 μm filter (Merck Millipore). 500 μL of Protein A resin was poured into the filtered culture medium, stirred at 4°C for 16 hours, and then the column was run. The resin was collected, washed with 5 mL of PBS, and eluted in 3 mL of 100 mM glycine pH 2.7 buffer, then neutralized with 1 M Tris-HCl pH 8.0. A centrifugal filter unit 3K (Merck Millipore) was used to change the buffer.

[0105] <2-2> ELISA analysis of purified trastuzumab Fc mutant (Q311M / M428L) To measure the pH-dependent binding ability of trastuzumab-ML purified in Example 2-1 to FcRn, ELISA was performed.

[0106] More specifically, to confirm the pH-dependent binding strength to FcRn, 50μL of each IgG Fc mutant diluted to 4μg / mL in 0.05M Na2CO3 pH9.6 was immobilized on a flat-bottom polystyrene high bind 96-well microplate (Costar) at 4℃ for 16 hours, and then blocked with 100μL of 4% skim milk (genome-based) (in 0.05% PBST pH6.0 / pH7.4) at room temperature for 2 hours. After washing four times with 180μL of 0.05% PBST (pH6.0 / pH7.4), 50μL of FcRn serially diluted with 1% skim milk (in 0.05% PBST pH6.0 / pH7.4) was dispensed into each well and allowed to react at room temperature for 1 hour. After washing, 50 μL of anti-GST-HRP conjugate (GE Healthcare) was added to each well to allow antibody reaction at room temperature for 1 hour, followed by washing. 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well to develop color, and 50 μL of 2M H2SO4 was then added to each well to terminate the reaction, followed by analysis using an Epoch microplate spectrophotometer (BioTek). As a result, it was confirmed that Met, which was expected to inhibit the binding strength to FcRn, bound better than PFc29 at pH 6.0, and showed the same binding strength as PFc29 at pH 7.4 (Figure 1).

[0107] That is, contrary to the prediction before the experiment, it was confirmed that the Q311M mutation improves the binding strength at pH 6.0.

[0108] <2-3> Further exploration of Fc variants with improved FcRn binding ability and expression and purification in animal cells We attempted to find mutants with improved binding ability compared to the previously selected Q311M / M428L. Specifically, 15 mutants in which 15 amino acids excluding L, G, P, C, and D were introduced at position L309, which is known to be important for FcRn binding, were cultured in animal cells in the same manner as in Example 2-2 and purified (FIG. 7).

[0109] <2-4> ELISA analysis of purified trastuzumab Fc mutants As described above, ELISA was performed to confirm the binding ability of the mutants prepared in Example 2-3 to FcRn at pH 6.0.

[0110] More specifically, to confirm the pH-dependent binding strength to FcRn, 50 μL of each IgG Fc mutant diluted to 4 μg / mL in 0.05M Na2CO3 pH 9.6 was immobilized on a flat-bottom polystyrene high bind 96-well microplate (Costar) for 16 hours at 4°C, and then blocked with 100 μL of 4% skim milk (genome base) (in 0.05% PBST pH 6.0) for 2 hours at room temperature. The plate was washed four times with 180 μL of 0.05% PBST (pH 6.0), and then 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 washing, 50 μL of anti-GST-HRP antibody complex (GE Healthcare) was used to perform antibody reaction at room temperature for 1 hour, and then washed. 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well to develop color, and then 50 μL of 2 M H2SO4 was added to each well to terminate the reaction, which was then analyzed using an Epoch microplate spectrophotometer (BioTek). As a result, two mutants were obtained: trastuzumab-YML, in which a heavy chain formed by introducing Fc mutants (L309Y / Q311M / M428L, sequence number 3) into a wild-type trastuzumab heavy chain (sequence number 11), which has a higher binding strength at pH 6.0 than PFc29, is linked to a wild-type trastuzumab light chain (sequence number 12); and trastuzumab-EML (sequence number 4), in which a heavy chain formed by introducing Fc mutants (L309E / Q311M / M428L, sequence number 3) into a wild-type trastuzumab heavy chain (sequence number 11) is linked to a wild-type trastuzumab light chain (sequence number 12) (Figure 8).

[0111] <2-5>Analysis of pH-dependent FcRn binding ability of Trastuzumab Fc mutants (L309Y / Q311M / M428L(YML), L309E / Q311M / M428L(EML)) ELISA was performed to confirm the pH-dependent binding ability of the two mutants, trastuzumab-YML and trastuzumab-EML (SEQ ID NO: 4), purified in Example 2-4 to FcRn.

[0112] More specifically, to confirm the pH-dependent binding strength to FcRn, 50 μL of each IgG Fc mutant diluted to 4 μg / mL in 0.05M Na2CO3 pH 9.6 was immobilized on a flat-bottom polystyrene High Bind 96-well microplate (Costar) at 4 °C for 16 hours. Then, 100 μL of 4% skim milk (genome-based) (in 0.05% PBST pH 6.0 / pH 7.4) was blocked at room temperature for 2 hours. After washing four times with 180 μL of 0.05% PBST (pH 6.0 / pH 7.4), 50 μL of FcRn serially diluted with 1% skim milk (in 0.05% PBST pH 6.0 / pH 7.4) was dispensed into each well and allowed to react at room temperature for 1 hour. After washing, 50 μL of anti-GST-HRP antibody conjugate (GE Healthcare) was added to each well to allow antibody reaction at room temperature for 1 hour. After washing, 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well to develop color, and 50 μL of 2M H2SO4 was added to each well to terminate the reaction. The plate was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0113] As a result, it was confirmed that YML showed higher binding strength than PFc29 at pH 6.0 than EML, but that the binding strength of YML was also improved at pH 7.4 (Figure 9).

[0114] Therefore, we concluded that the EML mutant, which shows binding affinity equivalent to that of PFc29 at pH 7.4 and improved binding affinity at pH 6.0, has improved pH-dependent binding affinity, thereby improving the antibody recycling process and extending the blood half-life (Figures 10a to 10b). EXAMPLES

[0115] Fc variant with altered amino acid residue Q311W <3-1> Construction and analysis of 17 amino acid substitution mutants at Q311 of PFc41 (L309G, M428L) Based on PFc41 (L309G, M428L) and PFc29 (Q311R and M428L Fc mutants) whose half-lives were extended in previous studies, we fixed L309G and constructed pMopac12-NlpA-Fc mutant plasmids (variants plasmid) to analyze mutants with 17 amino acid substitutions at Q311, excluding Q, C, and R, by bacterial display. FACS analysis was performed on the constructed plasmids to select mutants with improved binding to human FcRn at pH 6.0 using FACS. As a result, nine mutants with substitutions of L, I, V, T, A, Y, H, K, or W at position Q311 were selected, which have improved binding strength at pH 6.0 compared to PFc29 found in previous studies (M428L, L309G Fc mutant with Q311L, Q311I, Q311V, Q311T, Q311A, Q311Y, Q311H, Q311K, or Q311W) (Figure 11).

[0116] <3-2> Production and purification of trastuzumab Fc mutants containing L, I, V, T, A, Y, H, K, and W mutations at position Q311 in PFc41 (L309G, M428L) To confirm the characteristics of the nine mutants selected in Example 3-1 in the antibody format, a heavy chain expression vector in which an Fc mutant was introduced into a wild-type trastuzumab heavy chain (SEQ ID NO: 11) and a wild-type trastuzumab light chain expression vector (SEQ ID NO: 12) were prepared. Then, they were transfected into Expi293F animal cells.

[0117] More specifically, 1 day before transfection, Expi293F cells were cultured at 2 × 10 6The cells were subcultured in 30 mL to a density of 10000 cells / mL, and the next day, transfection was performed using PEI (Polyethylenimine, Polysciences, 23966). First, the mutant heavy chain gene and light chain gene were mixed in a ratio of 1:1 in 3 mL of freestyle 293 expression culture medium (Gibco, 12338-018). Next, PEI: mutant gene were mixed in a ratio of 4:1 and left to stand at room temperature for 20 minutes, mixed with cells that had been subcultured the day before, and cultured in a shaking CO2 incubator at 37°C, 125 rpm, and 8% CO2 for 7 days, after which the cells were centrifuged and only the supernatant was collected. Then, equilibration was performed using 25×PBS. Filtration was performed using a bottle top filter and a 0.2 μm filter (Merck Millipore). 100 μL of Protein A resin was poured into the filtered culture medium, stirred at 4°C for 16 hours, and then the column was run to collect the resin. It was then washed twice with 1 mL of PBS, and then eluted in 1 mL of 100 mM glycine pH 2.7 buffer, and then neutralized with 1 M Tris-HCl pH 8.0. A centrifugal filter unit 30K (Merck Millipore) was used to change the buffer. The finished sample was confirmed by SDS-PAGE gel (Figure 12).

[0118] <3-3> ELISA analysis of purified trastuzumab Fc mutants ELISA was performed to measure the pH-dependent binding strength of trastuzumab mutants, which are heavy chains obtained by introducing Fc mutants into wild-type trastuzumab heavy chains (SEQ ID NO: 11) and wild-type trastuzumab light chains (SEQ ID NO: 12), to FcRn. First, to confirm the binding strength to FcRn under pH 6.0 conditions, 50 μL of IgG Fc mutants diluted to 4 μg / mL in 0.05 M Na2CO3 pH 9.6 were immobilized on a flat-bottom polystyrene high bind 96-well microplate (Costar) at 4° C. for 16 hours. Then, the plates were blocked with 100 μL of 4% skim milk (genome-based) (in 0.05% PBST pH 6.0) at room temperature for 2 hours. The wells were washed four times with 180 μL of 0.05% PBST (pH 6.0), and then 50 μL of FcRn serially diluted in 1% skimmed milk (in 0.05% PBST pH 6.0) was dispensed into each well and reacted at room temperature for 1 hour. After washing, 50 μL of anti-GST-HRP antibody conjugate (GE Healthcare) was used for antibody reaction at room temperature for 1 hour, and then washed. 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well to develop color, and then 50 μL of 2M H2SO4 was injected to terminate the reaction. The wells were then analyzed using an Epoch microplate spectrophotometer (BioTek). As a result, the top three mutants (L309G / Q311Y / M428L, L309G / Q311H / M428L, and L309G / Q311W / M428L) with improved binding affinity to FcRn at pH 6.0 were selected (Figure 13).

[0119] ELISA was performed to measure the pH-dependent binding ability of the three selected trastuzumab mutants to FcRn. First, to confirm the pH-dependent binding ability to FcRn, 50 μL of each IgG Fc mutant diluted to 4 μg / mL in 0.05 M Na2CO3 pH 9.6 was immobilized on a flat-bottom polystyrene high bind 96-well microplate (Costar) at 4°C for 16 hours. Then, the plate was blocked with 100 μL of 4% skim milk (genome-based) (in 0.05% PBST pH 6.0 / pH 7.4) at room temperature for 2 hours. The wells were washed four times with 180 μL of 0.05% PBST (pH 6.0 / pH 7.4), and then 50 μL of FcRn serially diluted in 1% skim milk (in 0.05% PBST pH 6.0 / pH 7.4) was dispensed into each well and reacted at room temperature for 1 hour. After washing, 50 μL of anti-GST-HRP antibody conjugate (GE Healthcare) was used to perform antibody reaction at room temperature for 1 hour, and a washing process was performed. 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well to develop color, and then 50 μL of 2M H2SO4 was injected to each well to terminate the reaction, and then the results were analyzed using an Epoch microplate spectrophotometer (BioTek).

[0120] As a result, the binding ability to FcRn was improved compared to PFc29 at pH 6.0, but was also improved compared to PFc29 at pH 7.4, and there was no pH-dependent increase in binding ability (Figure 14).

[0121] <3-4>Saturation mutagenesis and expression and purification in animal cells Of the three mutants (L309G / Q311Y / M428L, L309G / Q311H / M428L, L309G / Q311W / M428L) selected in Example 3-3, 15 trastuzumab mutants were prepared, including Q311Y or W and substituted at L309 with F, I, M, V, T, A, Y, H, Q, N, K, E, W, R, or S, except for Q311H, which was already known in a previous study. The 30 trastuzumab mutants were cultured in animal cells using the expression vectors prepared in the same manner as described above, and then purified and confirmed by SDS-PAGE gel (FIG. 15).

[0122] <3-5> ELISA analysis of trastuzumab Fc mutants against FcRn at pH 6.0 ELISA was performed to measure the binding strength of the 30 trastuzumab mutants of Example 3-4 to FcRn at pH 6.0. First, in order to confirm the pH-dependent binding strength to FcRn, 50 μL of each IgG Fc mutant diluted to 4 μg / mL in 0.05M Na2CO3 pH 9.6 was immobilized on a flat-bottom polystyrene high bind 96-well microplate (costar) at 4°C for 16 hours, and then blocked with 100 μL of 4% skim milk (genome-based) (in 0.05% PBST pH 6.0) at room temperature for 2 hours. After washing four 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 washing, 50 μL of anti-GST-HRP antibody conjugate (GE Healthcare) was used to perform antibody reaction at room temperature for 1 hour, followed by washing. 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well to develop color, and then 50 μL of 2M H2SO4 was added to each well to terminate the reaction, and then the mixture was analyzed using an Epoch microplate spectrophotometer (BioTek). As a result, the L309E / Q311W / M428L (EWL, SEQ ID NO: 8) mutant was found to have improved binding strength at pH 6.0 compared to PFc29 (Figure 16).

[0123] <3-6>Analysis of pH-dependent FcRn binding of trastuzumab Fc mutants with L309E / Q311W / M428L (EWL) mutations ELISA was performed to measure the pH-dependent binding strength of trastuzumab-EWL, which is a heavy chain containing an EWL Fc mutant (SEQ ID NO: 5) and a trastuzumab light chain (SEQ ID NO: 12) bound to a wild-type trastuzumab heavy chain (SEQ ID NO: 11), to FcRn. First, in order to confirm the pH-dependent binding strength to FcRn, 50 μL of each of the IgG Fc mutants diluted to 4 μg / mL in 0.05 M Na2CO3 pH 9.6 was immobilized on a flat-bottom polystyrene High Bind 96-well microplate (Costar) at 4°C for 16 hours, and then blocked with 100 μL of 4% skim milk (genome-based) (in 0.05% PBST pH 6.0 / pH 7.4) at room temperature for 2 hours. The wells were washed four times with 180 μL of 0.05% PBST (pH 6.0 / pH 7.4), and then 50 μL of FcRn serially diluted with 1% skimmed milk (in 0.05% PBST pH 6.0 / pH 7.4) was dispensed into each well and allowed to react at room temperature for 1 hour.

[0124] After washing, 50 μL of anti-GST-HRP antibody conjugate (GE Healthcare) was used to perform antibody reaction at room temperature for 1 hour, followed by washing. 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) was added to each well to develop color, and then 50 μL of 2M H2SO4 was injected to each well to terminate the reaction, and then the results were analyzed using an Epoch microplate spectrophotometer (BioTek). As a result, it bound better to human FcRn at pH 6.0 than PFc29, and showed lower binding strength than PFc29 at pH 7.4 (Figures 17a to 17c). Therefore, it is predicted that the mutant EWL improves pH-dependent binding strength, thereby maximizing the antibody recycling process and extending the blood half-life.

Claims

1. An Fc variant comprising an amino acid residue modification selected from the group consisting of L309Y, Q311M and Q311W according to the Kabat numbering system in the Fc region of a wild-type immunoglobulin, wherein the Fc variant consists of the amino acid residue modifications 1) L309E, Q311M and M428L, 2) L309Y, Q311M and M428L, or 3) L309E, Q311W and M428L.

2. The Fc variant of claim 1 , wherein the immunoglobulin is selected from the group consisting of IgA, IgM, IgE, IgD and IgG.

3. The Fc variant of claim 1, which has a lower binding affinity to FcRn at pH 7.0 to 7.8 compared to a wild-type immunoglobulin Fc region.

4. The Fc variant of claim 1, which has a higher binding affinity to FcRn than a wild-type immunoglobulin Fc region at pH 5.6 to 6.

5.

5. A polypeptide comprising the Fc variant of claim 1.

6. The polypeptide according to claim 5, which has an extended in vivo half-life compared to the wild type.

7. An antibody comprising the Fc variant of claim 1.

8. The antibody of claim 7, having an extended in vivo half-life compared to the wild-type antibody.

9. A nucleic acid molecule encoding the Fc variant of claim 1, the polypeptide of claim 5, or the antibody of claim 7.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the vector of claim 10.

12. A protein conjugate having an extended in vivo half-life, comprising the Fc variant of claim 1, a non-peptidyl polymer and a physiologically active polypeptide covalently linked thereto.

13. The biologically active polypeptide may be human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, colony stimulating factor, interleukin, interleukin soluble receptor, TNF soluble receptor, glucocerebrosidase, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitory factor, cytotoxic glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, α1-antitrypsin, albumin, apo Lipoprotein E, erythropoietin, hyperglycosylated 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 derivatives, glucagon, glucagon-like peptide-1 13. The protein conjugate of claim 12, wherein the protein conjugate is selected from the group consisting of: IL-1, IL-2, IL-1-Like Peptide-1, atriopeptin, chondroinductive factor, connective tissue activating factor, follicle stimulating hormone, luteinizing hormone, follicle stimulating hormone releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical 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 virus-derived vaccine antigens.

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