pH sensitive Fc variants

Abstract

The present invention relates to an Fc variant with increased half-life that binds to and dissociates from FcRn in a pH-dependent manner. The Fc variant of the present invention, as a variant with maximized serum half-life, exhibits more excellent pH-selective FcRn binding and dissociation capabilities than existing Fc variants with increased serum half-life. Therefore, it is possible to exert a long-term drug effect with an increased serum half-life by combining with a variety of peptide drug therapeutic agents with low half-life and low maintenance time in vivo. In addition, the dosage and frequency of administration of antibodies and biopharmaceuticals can be greatly reduced, thereby not only reducing the cost of new drug development, but also having the effect of greatly increasing the possibility of new drug development.

Description

Technical Field

[0001] The present invention relates to Fc variants with improved half-life through pH-dependent binding to and dissociation from FcRn. Background Art

[0002] Since protein therapeutics show very high specificity for disease targets and less side effects and toxicity, they can be widely used in clinical practice by quickly replacing non-specific low-molecular compound therapeutics. Among the protein therapeutics used in clinical practice, antibody therapeutics and Fc fusion protein therapeutics that fused the Fc region of antibodies are currently the main ones used.

[0003] Compared with existing low-molecular drugs, therapeutic antibodies show very high specificity to targets, not only have the advantages of low biological toxicity and less side effects, but also have an excellent serum half-life of about 3 weeks and are used as one of the most effective cancer treatment methods. In fact, large pharmaceutical companies and research institutions around the world are committed to researching and developing therapeutic antibodies that specifically bind to cancer cells including cancer pathogens to effectively remove them. Pharmaceutical companies that develop therapeutic antibodies include Roche, Amgen, Johnson & Johnson, Abbott, Bristol-Myers Squibb, etc. In particular, in 2012, as Roche's representative products, three therapeutic antibodies such as Herceptin, Avastin and Rituxan for anti-cancer treatment achieved sales of approximately US$19.5 billion in the global market, not only creating huge profits, but also leading the global antibody drug market. Johnson & Johnson, which is known to have developed Remicade, has also grown rapidly in the global antibody market due to increased sales, and pharmaceutical companies such as Abbott and BMS also retain a variety of therapeutic antibodies in the final stages of development. As a result, in the global pharmaceutical market dominated by low-molecular-weight drugs, biopharmaceuticals including therapeutic antibodies that are specific to disease targets and have fewer side effects are rapidly taking their place.

[0004] However, when administered orally, antibodies and protein therapeutics have very low absorption rates through the digestive organs and are easily denatured or easily degraded by proteolytic enzymes inside the digestive organs, resulting in very low bioavailability. Therefore, they need to be administered by intravenous or subcutaneous injection. In order to achieve a significant therapeutic effect, frequent inoculations are required. Even as an antibody among protein therapeutics with excellent stability in the blood, in order to achieve a successful therapeutic effect, a high dose of therapeutic antibody (2mg to 8mg / kg body weight) needs to be administered once every 2 to 3 weeks. Frequent administration by the above-mentioned injection method not only causes pain and discomfort to the patient, but also has the problem of causing local and systemic edema, infection and other side effects. In order to solve the above problems, the half-life of therapeutic antibodies and protein therapeutics in the blood should be significantly improved.

[0005] It is 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 nonspecific cell uptake and then introduced into acidic endosomes. Under acidic pH (<6.5), FcRn binds to immunoglobulin in endosomes, and under alkaline pH (>7.4), FcRn releases immunoglobulin in the bloodstream. Thus, FcRn recovers immunoglobulin from the lysosomal degradation pathway. When the serum immunoglobulin level decreases, more FcRn molecules can be used for immunoglobulin binding to increase the amount of immunoglobulin. Conversely, when the serum immunoglobulin level increases, the proportion of immunoglobulin absorbed by cells degraded due to FcRn saturation can be increased (Ghetie and Ward, Annu. Rev. Immunol. 18: 739-766, 2000). That is, the serum half-life and persistence of antibodies are largely determined by the position of the Fc region of the antibody and the binding to FcRn (neonatal Fc receptor), one of the IgG binding ligands. The Fc region of antibodies that can collect immune leukocytes or serum complement molecules and remove damaged cells such as cancer cells or infected cells refers to the region between the Cγ2 and Cγ3 binding domains, mediating the interaction with the neonatal receptor FcRn, and its binding recirculates the antibody from the endosome to the bloodstream and then enters the cell (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766). In this process, the large size of the full-length molecule is associated with the inhibition of kidney filtration, and the antibody serum half-life is favorable in the range of 1 to 3 weeks. Furthermore, the binding of Fc to FcRn also plays an important role in antibody transport. Therefore, the Fc site plays an important role in maintaining prolonged serum persistence by circulating antibodies through intracellular trafficking and recycling mechanisms. Summary of the invention

[0006] Technical issues

[0007] The present invention aims to develop antibodies into which mutations are introduced so as to increase the binding affinity to FcRn in endosomes (under weakly acidic pH conditions) and to reduce the binding affinity to FcRn in blood (under neutral pH conditions).

[0008] Technical Solution

[0009] In order to achieve the above objectives, the present invention provides a pH-sensitive Fc variant.

[0010] Furthermore, the present invention provides polypeptides comprising pH-sensitive Fc variants.

[0011] Furthermore, the present invention provides antibodies comprising a pH-sensitive Fc variant.

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

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

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

[0015] Furthermore, the present invention provides a protein conjugate having an increased half-life in vivo.

[0016] Effects of the Invention

[0017] As a variant with maximized serum half-life, the Fc variant of the present invention exhibits more excellent pH-selective FcRn binding and dissociation ability than the existing Fc variant with improved serum half-life. Therefore, it can be combined with a variety of peptide drug therapeutic agents with low half-life and low maintenance time in vivo to exert a long-term drug effect with increased serum half-life. In addition, the dosage and frequency of antibody and biopharmaceutical administration can be greatly reduced, thereby not only reducing the cost of new drug development, but also having the effect of greatly increasing the possibility of new drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the binding ability of trastuzumab comprising Q311M and M428L Fc variants to FcRn confirmed by enzyme-linked immunosorbent assay (ELISA) under the conditions of pH 6.0 and pH 7.4:

[0019] PFc29: trastuzumab comprising the Q311R and M428L Fc variants; and

[0020] M: Trastuzumab comprising Q311M and M428L Fc variants.

[0021] Figure 2The figure shows a schematic diagram of the preparation of amino acid variants including the L309 position (saturation mutagenesis) and a photograph of an SDS-PAGE gel after expression and purification of trastuzumab containing each Fc variant.

[0022] Figure 3 This is a diagram showing the binding ability of trastuzumab containing Fc variants (Q311M, M428L and amino acid mutations at position 309) to FcRn confirmed by enzyme-linked immunosorbent assay under pH 6.0:

[0023] PFc29: trastuzumab containing Q311R and M428L Fc variants;

[0024] FM: trastuzumab containing L309F, Q311M, and M428L Fc variants;

[0025] IM: trastuzumab containing L309I, Q311M, and M428L Fc variants;

[0026] MM: trastuzumab containing L309M, Q311M and M428L Fc variants;

[0027] VM: trastuzumab containing L309V, Q311M and M428L Fc variants;

[0028] TM: trastuzumab containing L309T, Q311M and M428L Fc variants;

[0029] AM: trastuzumab containing L309A, Q311M, and M428L Fc variants;

[0030] YM: trastuzumab containing L309Y, Q311M, and M428L Fc variants;

[0031] HM: trastuzumab containing L309H, Q311M and M428L Fc variants;

[0032] QM: trastuzumab containing L309Q, Q311M, and M428L Fc variants;

[0033] NM: trastuzumab containing L309N, Q311M, and M428L Fc variants;

[0034] KM: trastuzumab containing L309K, Q311M, and M428L Fc variants;

[0035] EM: trastuzumab containing L309E, Q311M, and M428L Fc variants;

[0036] WM: trastuzumab containing L309W, Q311M, and M428L Fc variants;

[0037] RM: trastuzumab comprising L309R, Q311M and M428L Fc variants; and

[0038] SM: Trastuzumab comprising L309S, Q311M and M428L Fc variants.

[0039] Figure 4a This is a graph showing the binding ability of trastuzumab containing Fc variants to FcRn confirmed by enzyme-linked immunosorbent assay under the condition of pH 6.0:

[0040] PFc29: trastuzumab containing the Q311R and M428L Fc variants;

[0041] ML: trastuzumab comprising Q311M and M428L Fc variants; and

[0042] YML: trastuzumab comprising L309Y, Q311M and M428L Fc variants.

[0043] Figure 4b This is a graph showing the binding ability of trastuzumab containing an Fc variant to FcRn confirmed by enzyme-linked immunosorbent assay under the condition of pH 7.4.

[0044] Figure 4c The bar graph shows the binding ability of trastuzumab containing Fc variants to FcRn confirmed by enzyme-linked immunosorbent assay under the conditions of pH 6.0 and pH 7.4.

[0045] Figure 5 It 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.

[0046] Figure 6a This is a graph showing the results of analyzing the binding speed of Fc variants under pH 6.0 conditions:

[0047] PFc29: trastuzumab containing the Q311R and M428L Fc variants;

[0048] PFc41: trastuzumab containing the L309G and M428L Fc variants;

[0049] ML: trastuzumab comprising Q311M and M428L Fc variants; and

[0050] YML: contains trastuzumab containing L309Y, Q311M and M428L Fc.

[0051] Figure 6b The figure shows the results of analyzing the dissociation rate of Fc variants under the condition of pH 7.4.

[0052] Figure 6c The graph shows the results of analyzing the association rate and dissociation rate of Fc variants.

[0053] Figure 7 The figure shows a schematic diagram of saturation mutation at the L309 position and a photograph of an SDS-PAGE gel after expression and purification of trastuzumab containing an Fc variant.

[0054] Figure 8 The graph shows the binding ability of trastuzumab containing Fc variants to FcRn confirmed by enzyme-linked immunosorbent assay under the condition of pH 6.0.

[0055] Fig. 9 This is a graph showing the binding ability of trastuzumab containing Fc variants to FcRn confirmed by enzyme-linked immunosorbent assay under the conditions of pH 6.0 and pH 7.4.

[0056] Fig.10a This is a graph showing the binding ability of trastuzumab containing an EML variant to FcRn confirmed by enzyme-linked immunosorbent assay under the condition of pH 6.0.

[0057] Fig.10b This is a graph showing the binding ability of trastuzumab containing an EML variant to FcRn confirmed by enzyme-linked immunosorbent assay under the condition of pH 7.4.

[0058] Fig.11 This is a graph showing the results of FACS analysis for confirming the binding ability of Fc variants to FcRn under the condition of pH 6.0.

[0059] Fig.12 A diagram showing photographs of SDS-PAGE gels after expression and purification of nine trastuzumab-Fc variants (Q311L, Q311I, Q311V, Q311T, Q311A, Q311Y, Q311H, Q311K, or Q311W in the M428L and L309G variants).

[0060] Fig.13 A graph showing the results of analyzing the binding ability of 9 trastuzumab-Fc variants to FcRn by enzyme-linked immunosorbent assay at pH 6.0 (M428L, L309G variants, Q311L, Q311I, Q311V, Q311T, Q311A, Q311Y, Q311H, Q311K or Q311W).

[0061] Fig.14 The graph shows the results of analyzing the binding ability of three trastuzumab-Fc variants to FcRn by enzyme-linked immunosorbent assay under the conditions of pH 6.0 and pH 7.4.

[0062] Fig.15 The figure shows a photograph of SDS-PAGE gel after expression and purification of 30 trastuzumab-Fc variants.

[0063] Fig.16 The figure shows the results of analyzing the binding ability of 30 trastuzumab-Fc variants to FcRn by enzyme-linked immunosorbent assay at pH 6.0.

[0064] Fig.17a The graph shows the results of analyzing the binding ability of the EWL (L309E / Q311W / M428L) variant to FcRn by enzyme-linked immunosorbent assay under the conditions of pH 6.0 and pH 7.4.

[0065] Fig.17b The figure shows the result of analyzing the binding ability of the EWL (L309E / Q311W / M428L) variant to FcRn by enzyme-linked immunosorbent assay at pH 6.0.

[0066] Fig.17c The figure shows the result of analyzing the binding ability of the EWL (L309E / Q311W / M428L) variant to FcRn by enzyme-linked immunosorbent assay at pH 7.4. DETAILED DESCRIPTION

[0067] Best Mode for Carrying Out the Invention

[0068] The present invention is described in detail below through examples of the present invention. However, the following examples are only examples of the present invention, and the present invention is not limited thereto. The present invention can be modified and applied in various ways within the scope of the invention and the equivalent scope explained thereby.

[0069] Unless otherwise defined, all technical terms and scientific terms used in this application have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described in this application can be used to perform the tests of the present invention, this application only describes preferred materials and methods.

[0070] Throughout the present specification, for naturally occurring amino acids, in addition to the commonly used single-letter and three-letter codes, other three-letter codes commonly used for amino acids are also used, for example, Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. In addition, in the present invention, amino acids referred to by abbreviations may also be recorded by the IUPAC-IUB nomenclature:

[0071] 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.

[0072] In one embodiment, the present invention relates to an Fc variant comprising a modification of the amino acid residue L309Y based on the Kabat numbering system in the Fc region of a wild-type immunoglobulin.

[0073] In one example, the Fc variant of the present invention may further comprise the substitution of the amino acid residues: M428L, Q311M, or M428L and Q311M.

[0074] In one embodiment, the present invention relates to an Fc variant comprising a modification of the amino acid residue Q311M based on the Kabat numbering system in the Fc region of a wild-type immunoglobulin.

[0075] In one example, the Fc variant of the present invention may further comprise substitutions of the amino acid residues: M428L; L309E; L309Y; M428L and L309E or M428L and L309Y.

[0076] In one embodiment, the present invention relates to an Fc variant comprising a modification of the amino acid residue Q311M based on the Kabat numbering system in the Fc region of a wild-type immunoglobulin.

[0077] In one example, the Fc variant of the present invention may further comprise the substitution of the amino acid residues: M428L, L309E, or M428L and L309E.

[0078] In one example, the immunoglobulin can be IgA, IgM, IgE, IgD, IgG or a modification thereof, and can be IgG1, IgG2, IgG3 or IgG4, preferably an anti-HER2 antibody, and more preferably trastuzumab. Papain cleavage of the antibody forms two Fab fragments and an Fc fragment. In human IgG molecules, the Fc region is generated by papain cleavage of the N-terminus of Cys 226 (Deisenhofer, Biochemistry 20: 2361-2370, 1981).

[0079] In one example, the Fc region of a wild-type immunoglobulin may comprise the amino acid sequence of SEQ ID NO: 1.

[0080] In one example, 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).

[0081] In one example, the Fc variant may comprise the amino acid sequence of SEQ ID NO: 4 (L309E / Q311M / M428L).

[0082] In one example, the Fc variant may comprise the amino acid sequence of SEQ ID NO: 5 (L309E / Q311W / M428L).

[0083] In one example, the Fc region of a wild-type immunoglobulin may include the base sequence of SEQ ID NO: 6.

[0084] In one example, the Fc variant may include the base sequence of SEQ ID NO: 7 (Q311M and M428L), and may include the base sequence of SEQ ID NO: 8 (L309Y, Q311M and M428L).

[0085] In one example, the Fc variant may comprise the base sequence of SEQ ID NO: 9 (L309E / Q311M / M428L).

[0086] In one example, the Fc variant may comprise the base sequence of SEQ ID NO: 10 (L309E / Q311W / M428L).

[0087] In one example, 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. In the amino acid sequence of SEQ ID NO: 11, the Fc region of wild-type trastuzumab corresponds to the sequence starting from position 225.

[0088] In one example, the amino acid sequence of SEQ ID NO: 12 may include a wild-type trastuzumab light chain, and the QDVNTA, SAS, and QQHYTTPPT sequences represent CDR regions, respectively.

[0089] In one example, the Fc variants of the present invention can produce a pH-selective (dependent) binding / dissociation reaction with FcRn.

[0090] In one example, compared to the wild-type immunoglobulin Fc region, the Fc variant of the present invention may exhibit a lower binding affinity to FcRn under conditions of pH 7.0 to 7.8, which may be within the normal pH range of blood, which may be pH 7.2 to 7.6. Compared to the wild-type Fc domain, the Fc variant of the present invention may have the same or substantially unchanged degree of dissociation with FcRn within the above pH range.

[0091] In one example, compared to the wild-type immunoglobulin Fc region, the Fc variant of the present invention can exhibit a higher binding affinity to FcRn under conditions of pH 5.6 to 6.5, which can be a weakly acidic condition in the endosome, or pH 5.8 to 6.0. The pH-sensitive Fc variant of the present invention can increase the binding affinity of FcRn to FcRn by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 100% compared to the wild-type Fc domain within the above pH range, or can increase by more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 20 times, more than 30 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times or more than 100 times compared to the wild-type Fc domain.

[0092] In the present invention, variants comprising amino acid variations in the Fc region of the immunoglobulin of the present invention are defined based on the amino acid modifications constituting the Fc region of the parent immunoglobulin, and the usual immunoglobulin numbering is based on the EU index of Kabat (Kabat et al., Sequence of proteins of immunological interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, 1991).

[0093] The amino acids of sequence numbers 1 to 5 of the present invention start from the 221st position of the Kabat numbering. For example, the amino acid Asp (D, 1D) at position 1 of sequence number 1 of the present application is the same as the Kabat numbering 221D, the amino acid Met (M, 91M) at position 91 of sequence number 2 is the same as the Kabat numbering 311M, and the amino acid Tyr (Y, 89Y) at position 89 of sequence number 3 is the same as the Kabat numbering 309Y.

[0094] In the present invention, the term "trastuzumab-Fc variant" used refers to the Fc variant of the present application introduced by replacing the Fc region of the wild-type trastuzumab heavy chain containing the amino acid of sequence number 11. And, it contains the wild-type trastuzumab light chain containing the amino acid of sequence number 12. Therefore, the preparation of the above-mentioned "trastuzumab-Fc variant" replaces the Fc region of the wild-type trastuzumab heavy chain to prepare an expression vector introducing the Fc variant of the present application, and can be expressed by preparing an expression vector introducing the wild-type trastuzumab light chain containing the amino acid of sequence number 12 and transfecting it into animal cells.

[0095] In the present invention, the term "FcRn" or "neonatal Fc receptor" used refers to a protein that binds to the Fc region of an IgG antibody, at least a portion of which is encoded by an FcRn gene. However, the above-mentioned FcRn is not limited thereto and can be derived from any organism including humans, mice, rats, rabbits and monkeys. As is well known in the art to which the present invention belongs, a functional FcRn protein comprises two polypeptides commonly 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 an FcRn protein refers to a complex of an FcRn heavy chain and β-2-microglobulin.

[0096] In the present invention, the term "wild-type polypeptide" used refers to an unmodified polypeptide that is subsequently modified to produce a derivative. A wild-type polypeptide may be a polypeptide found in nature, a derivative of a polypeptide found in nature, or a modified polypeptide. A wild-type polypeptide may be the polypeptide itself, a composition comprising the above-mentioned wild-type polypeptide, or an amino acid sequence encoding it. Therefore, the term "wild-type immunoglobulin" used in the present invention refers to an unmodified immunoglobulin polypeptide that produces a derivative due to the modification of amino acid residues. Interchangeably with the above terms, "parent immunoglobulin" may be used as an unmodified immunoglobulin polypeptide that produces a derivative due to the introduction of amino acid modifications.

[0097] In the present invention, the term "amino acid modification" used refers to the substitution, insertion and / or deletion of the amino acids in the polypeptide sequence, preferably the meaning of substitution. The term "amino acid substitution" or "substitution" used in the present invention means that the amino acid in a specific position of the wild-type polypeptide sequence is replaced by another amino acid. For example, in the Fc variant comprising the L309Y substitution, the leucine at the amino acid residue at position 309 in the amino acid sequence of the wild-type immunoglobulin Fc fragment is replaced by tyrosine.

[0098] In this specification, the term "Fc variant" used refers to a modification comprising one or more amino acid residues compared to the wild-type immunoglobulin Fc fragment. Preferably, in the present invention, the Fc variant comprises a modification of one or more amino acid residues selected from the group consisting of: L309Y, Q311M and M428L (the above numbering is based on the EU index recorded by Kabat); L309E, Q311M and M428L (the above numbering is based on the EU index recorded by Kabat); L309E, Q311W and M428L (the above numbering is based on the EU index recorded by Kabat), compared to the wild-type immunoglobulin Fc fragment (region), due to the increased binding affinity to FcRn under weakly acidic conditions and the reduced binding affinity to FcRn under neutral conditions, the binding speed to FcRn in weakly acidic intracellular endosomes is increased, and it will be rapidly dissociated in neutral blood.

[0099] The present invention provides Fc variants having increased binding affinity for FcRn and / or serum half-life compared to wild-type immunoglobulin Fc fragments. The in vivo half-life of antibodies and other physiologically active molecules (i.e., persistence in the serum or other tissues of the subject) is an important clinical variable for determining the dosage and frequency of the antibody (or any other pharmaceutical molecule). Therefore, the above-mentioned physiologically active molecules, including antibodies with increased in vivo half-life, are very important in pharmacology. The half-life of the substituted Fc variants of the present invention 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, or can be increased by more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, or more than 10 times compared to the wild-type Fc domain.

[0100] Compared to the wild-type immunoglobulin Fc fragment (region or binding domain), the Fc variant of the present invention comprises one or more amino acid modifications, thereby, the amino acid sequence will be different. The amino acid sequence of the Fc variant of the present invention is substantially the same as the amino acid sequence of the wild-type immunoglobulin Fc fragment. For example, the amino acid sequence of the pH-sensitive Fc variant of the present invention has a homology of about 80% or more to the amino acid sequence of the Fc fragment of the wild-type immunoglobulin, preferably more than 90%, and most preferably more than 95%. Amino acid modification can be performed on a genetic level by molecular biological methods, or it can also be performed by enzymatic or chemical methods.

[0101] In the present invention, the production and purification of trastuzumab can be prepared with reference to Korean patent application 10-2017-0045142.

[0102] The Fc variants of the present invention can be prepared by any method disclosed in the corresponding technical field. In one embodiment, the Fc variants of the immunoglobulin of the present invention can be prepared by the following method, that is, after encoding a polypeptide sequence containing a specific amino acid modification, cloning into a host cell under desired conditions, and forming a nucleic acid for expression and verification. For various methods thereof, reference can be made to the literature (Molecular Cloning-A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).

[0103] The nucleic acid encoding the Fc variant of the present invention can be inserted into an expression vector for protein expression. The expression vector contains a common regulatory sequence or a regulatory sequence, a selection marker, an arbitrary fusion partner and / or a protein that is operably connected to an additional element, that is, a protein placed in a functional relationship. Under appropriate conditions, the Fc variant of the present invention can be produced by a host cell converted into a nucleic acid. Preferably, a method of inducing protein expression by culturing an expression vector containing a nucleic acid encoding the Fc variant of the present invention can be used. A variety of appropriate host cells including mammalian cells, bacteria, insect cells and enzymes can be used, but are not limited thereto. The method for introducing exogenous nucleic acids into host cells is a method known in the art to which the present invention belongs, and changes are produced based on the host cell used. Preferably, Escherichia coli with low production cost and high industrial utilization value can be used as a host cell to produce the Fc variant of the present invention.

[0104] Therefore, within the scope of the present invention, the method for preparing Fc variants comprises the steps of: culturing host cells introduced with nucleic acid encoding Fc variants under conditions suitable for protein expression; and purifying or isolating the expressed Fc variants from the host cells.

[0105] Antibodies can be separated or purified by various methods known in the art to which the present invention belongs. Standard purification methods may include chromatography, electrophoresis, immunization, sedimentation, dialysis, filtration, concentration and chromatofocusing techniques. As is known in the art to which the present invention belongs, for example, various natural proteins such as bacterial proteins A, G and L bind to antibodies, and the above proteins can be used for purification. Generally, it can be used for purification by specific fusion partners.

[0106] In one embodiment, the invention relates to a polypeptide comprising an Fc variant of the invention.

[0107] In one embodiment, the polypeptide may have an increased half-life in vivo compared to the wild type.

[0108] In one embodiment, the present invention relates to an antibody comprising an Fc variant or the above-mentioned polypeptide.

[0109] In one embodiment, the antibodies of the present invention may have an increased half-life in vivo compared to antibodies comprising a wild-type Fc region.

[0110] In one example, the antibody can be a polyclonal antibody, a monoclonal antibody, a minibody, a binding 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.

[0111] The antibodies of the present invention can maximize the half-life of the Fc domain (region) or a polypeptide comprising the same by optimizing the Fc region (L309Y, Q311M and M428L; L309E, Q311M and M428L; L309E, Q311W and M428L).

[0112] In one embodiment, the present invention relates to a protein conjugate having an increased in vivo half-life, which is linked to the Fc variant of the present invention, a non-peptidyl polymer, and a physiologically active polypeptide by a covalent bond.

[0113] In one embodiment, the Fc variant of the present invention can be effectively used as a carrier for increasing the in vivo half-life of physiologically active polypeptides such as protein drugs.

[0114] In one example, a protein conjugate comprising an Fc variant of the present invention can be used as a long-lasting pharmaceutical formulation with a significantly longer half-life in vivo.

[0115] The non-peptidyl polymer that can be used in the present invention can be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers such as polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof, preferably polyethylene glycol. Their derivatives known in the technical field to which the present invention belongs and derivatives that can be easily prepared at the level of the technical field to which the present invention belongs all belong to the scope of the present invention.

[0116] There is no particular limitation on the physiologically active polypeptides that can bind to the Fc variants of the present invention, as long as the serum half-life needs to be increased. For example, various physiologically active polypeptides or their derivatives and analogs used for treating or preventing human diseases, such as cytokines, interleukins, interleukin-binding proteins, enzymes, antibodies, growth factors, transcriptional regulatory factors, blood factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, and receptor antagonists, can be used.

[0117] Physiologically active polypeptides that can be used in the present invention include hepatic growth hormone, growth hormone-releasing hormone, growth hormone-releasing peptide, interferons and interferon receptors (e.g., interferon-α, interferon-β, interferon-γ, soluble type 1 interferon receptor), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CS), glucagon-like peptide-1 (GLP-1), G protein-coupled receptor (G-protein-coupled receptor), interleukins (e.g., IL-1 receptor, IL-4 receptor), enzymes (e.g., glucocerebrosidase), iduronate-2-sulfatase, α-galactosidase-A, agalsidase α ( alpha), beta-L-iduronidase, alpha-L-iduronidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, interleukin- and cytokine-binding proteins (e.g.,IL-18bp, TNF-binding protein), macrophage active factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitory factor, necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor factor, transfer growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor active peptide, thrombomodulin, blood factor 7, blood factor 7a, blood Factor 8, blood factor 9, blood factor 13, plasminogen activating factor, fibrin binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial cell growth factor, epidermal cell growth factor, angiostatin, endostatin, angiotensin, bone formation growth factor, bone morphogenetic protein, calcitonin, insulin, atrial natriuretic peptide, chondroitin, elcatonin, connective tissue activator, 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 factor inhibitorfactor), neurotrophic factors, neutrinos (neutrin)), parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, adrenocorticotropin-releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors (e.g., TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, B cell activation 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, etc., but not limited thereto. The antibody fragment can be selected from Fab, Fab', F(ab')2, Fd or scFv that can bind to a specific antigen.

[0118] In one example, an antibody drug can be conjugated to a protein conjugate comprising an Fc variant, and the antibody drug for cancer treatment can be trastuzumab, cetuximab, bevacizumab, rituximab, basiliximab, infliximab, ipilimumab, pembrolizumab, nivolumab, atezolizumab or avelumab.

[0119] The present invention includes a method for preparing a durable pharmaceutical preparation by covalently linking the above-mentioned Fc variant and a physiologically active polypeptide through a non-peptidyl polymer.

[0120] The preparation method of the present invention may include the following steps: covalently linking a physiologically active polypeptide and an Fc variant via a non-peptidyl polymer having a reactive group at the end; and separating a conjugate linked to the physiologically active polypeptide, the non-peptidyl polymer and the Fc variant via a covalent bond.

[0121] In one embodiment, the invention relates to a nucleic acid molecule encoding an Fc variant, polypeptide or antibody of the invention.

[0122] The nucleic acid molecules of the present invention can be separated or recombinant, not only including DNA and RNA in single-stranded and double-stranded forms, but also including corresponding complementary sequences. In the case of separation from natural sources, the isolated nucleic acid is a nucleic acid separated from the surrounding genetic sequence already present in the individual of the isolated nucleic acid. In the case of a PCR product, a cDNA molecule or an oligonucleotide synthesized by an enzyme or chemistry from a model, the nucleic acid generated by the above steps can be understood as a separated nucleic acid molecule. The isolated nucleic acid molecule refers to a nucleic acid molecule as a component component of a separated fragment or a larger nucleic acid. When configured according to a functional relationship with other nucleic acid sequences, the nucleic acid is operably connected. For example, when the DNA of the current sequence or secretion leader is expressed as a preprotein (preprotein) in a state before secretion of a polypeptide, it is operably connected to the DNA of the polypeptide, therefore, if a promoter or enhancer affects the transcription of the polypeptide sequence, the coding sequence is operably connected, or, when the ribosome binding site is configured to promote translation, the coding sequence is operably connected. Usually, operably connected refers to the adjacent fixed position of a DNA sequence, and in the case of a secretion leader, it refers to the adjacent presence in the same leader frame. However, enhancers need not be located adjacent to each other. Linking is accomplished by ligation at convenient restriction endonucleases. When no such endonucleases are present, synthetic oligonucleotides are used for linkage or peptide linkage in the usual manner.

[0123] In one embodiment, the present invention relates to a vector comprising the above-mentioned nucleic acid molecule.

[0124] In the present invention, the term "vector" used refers to a vector into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence can be exogenous or heterologous. The vector can be a plasmid, a cosmid, and a virus (e.g., a bacteriophage), but is not limited thereto. A person of ordinary skill in the art to which the present invention belongs can construct a vector by standard recombinant techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988: and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

[0125] In the present invention, the term "expression vector" refers to a vector containing a nucleic acid sequence that encodes at least a portion of a transcribed gene product. In some cases, the RNA molecule can then be translated into a protein, polypeptide or peptide. The expression vector may contain a variety of regulatory sequences. In addition to regulatory sequences for regulating transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide other functions.

[0126] In one embodiment, the present invention relates to a host cell comprising the above-mentioned vector.

[0127] In the present invention, the term "host cell" used includes eukaryotic organisms and prokaryotes, and refers to any transformable organism that can replicate the above-mentioned vector or can express the gene encoded by the vector. The host cell can be transfected (transfected) or transformed (transformed) by the above-mentioned vector, which means the process of delivering or introducing exogenous nucleic acid molecules into the host cell.

[0128] Preferably, the host cell of the present invention can be a bacterial cell, a CHO cell, a HeLa cell, a HEK293 cell, a BHK-21 cell, a COS7 cell, a COP5 cell, an A549 cell, a NIH3T3 cell, etc., but is not limited thereto.

[0129] In one embodiment, the present invention relates to a method for increasing the in vivo half-life of a physiologically active polypeptide, comprising the step of covalently linking the Fc variant of the present invention to the physiologically active polypeptide via a non-peptidyl polymer.

[0130] In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the Fc variant, polypeptide or antibody of the present invention.

[0131] In one example, an immunogenic apoptosis-inducing agent may also be included.

[0132] In one example, the immunogenic apoptosis inducer may include one or more selected from the group consisting of anthracycline anticancer drugs, taxane anticancer drugs, anti-EGFR antibodies, BK channel agonists, bortezomib, cardiac glycosides, cyclophosphamide anticancer drugs, GADD34 / PP1 inhibitors, LV-tSMAC, measles virus, bleomycin, mitoxantrone or oxaliplatin, the anthracycline anticancer drugs may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin or valrubicin, and the taxane anticancer drugs may be paclitaxel or docetaxel.

[0133] The pharmaceutical composition for preventing or treating cancer of the present invention can be administered together with chemical anticancer drugs (anticancer agents) to enhance the apoptosis effect of cancer cells and increase the cancer treatment effect of existing anticancer agents. The combined administration can be carried out simultaneously or sequentially with the above anticancer agents. Examples of the anticancer agent include DNA alkylating agents such as mechlorethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; and anticancer antibiotics such as actinomycin (actinomycin D), plicamycin, and mitomycin C. and plant alkaloids such as vincristine, vinblastine, etoposide, teniposide, topotecan and iridotecan, but are not limited thereto.

[0134] In one example, cancer includes: leukemias and lymphomas such as acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's Disease, non-Hodgkin's lymphomas, and multiple myeloma; childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms Tumor, bone tumors, and soft-tissue sarcomas; and lung cancer, breast cancer, and The present invention can prevent or treat common solid tumors in adults, such as ovarian 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. However, the types of cancers to be prevented or treated by the present invention are not limited thereto.

[0135] In the present invention, the term "prevention" used refers to all effects of inhibiting or delaying the occurrence, spread and recurrence of cancer by administering the pharmaceutical composition of the present invention.

[0136] In the present invention, the term "treatment" used refers to all effects of effectively improving apoptosis of cancer cells or cancer symptoms by administering the composition of the present invention. A person skilled in the art of the present invention can refer to the public information of the Korean Medical Association and the like to accurately understand the disease standards for which the composition of the present application is effective, and can judge the degree of improvement, enhancement and treatment.

[0137] In the present invention, the term "therapeutically effective amount" used in combination with an active ingredient refers to a pharmaceutically acceptable amount of the composition required to effectively prevent or treat the target disease. The therapeutically effective amount of the composition of the present invention may vary based on a variety of factors, such as the method of administration, the location of the target part, the patient's condition, etc. Therefore, the dosage for human body should be determined in consideration of safety and efficiency. The amount for human body can also be estimated based on the effective amount determined by animal experiments. Alternatively, when determining the effective amount, the precautions can also be considered with reference to the literature, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10 th ed. (2001), PergamonPress; and EWMartin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0138] The pharmaceutical composition of the present invention should be administered in an effective amount that is pharmaceutically acceptable. In the present invention, the term "pharmaceutically effective amount" used refers to an amount that can be used to treat a disease in a reasonable benefit / risk ratio for medical treatment without causing side effects. The standard of the effective dose should be determined based on factors including the patient's health status, cancer type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment time, mixed or simultaneously used drugs, and other well-known elements in the medical field. The composition of the present invention can be used as a single therapeutic agent for administration, or it can be administered in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered alone or multiple times. Taking all the above factors into consideration, it is particularly important to administer the amount that can obtain the maximum effect with the minimum amount and without side effects, which can be easily determined by a person of ordinary skill in the art to which the present invention belongs.

[0139] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives. In this case, the pharmaceutically acceptable additives include 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, calcium stearate, white sugar, glucose, sorbitol, and talc. Preferably, the pharmaceutically acceptable additives of the present invention may include 0.1 to 90 parts by weight relative to the above composition, but are not limited thereto.

[0140] The composition of the present invention may also include a carrier, a diluent, an excipient or a combination of two or more thereof commonly used in biological preparations. There is no particular restriction on pharmaceutically acceptable carriers, as long as they are suitable for delivering the composition to the organism. For example, compounds recorded in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol or a mixture of the above components may be used, and commonly used additives such as antioxidants, buffers, and antibacterial agents may be added as required. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form injections, pills, capsules, granules or tablets such as aqueous solutions, suspensions, and emulsions. Furthermore, preferably, preparations may be made according to the appropriate methods in the technical field of the present invention or the methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) according to each disease or component.

[0141] The composition of the present invention can be administered parenterally (e.g., for intravenous, subcutaneous, intraperitoneal or local injection preparations) or orally according to the target method, and the range of the dosage varies based on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the disease. For the composition of the present invention, the daily dosage is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and more preferably, it should be administered once or multiple times a day.

[0142] Liquid preparations for oral administration of the composition of the present invention include suspensions, liquid solutions, internal solutions, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.

[0143] In one embodiment, the present invention relates to a method for preparing an Fc variant having an increased half-life in vivo compared to the wild type, comprising the following steps: culturing a host cell containing a vector containing a nucleic acid molecule encoding the Fc variant of the present invention; and recovering the Fc variant expressed by the above host cell.

[0144] In one embodiment, the present invention relates to a method for preparing an Fc variant having an increased half-life in vivo compared to the wild type, comprising the following steps: culturing a host cell containing a vector containing a nucleic acid molecule encoding the polypeptide of the present invention; and recovering the polypeptide expressed by the above host cell.

[0145] In one embodiment, the present invention relates to a method for preparing an antibody comprising a pH-sensitive Fc variant having an increased in vivo half-life compared to the wild type, comprising the steps of: culturing a host cell comprising a vector containing a nucleic acid molecule encoding the antibody of the present invention; and purifying the antibody expressed by the above host cell.

[0146] In one example, purification of the antibody may include filtration, high performance liquid chromatography, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof. Preferably, affinity chromatography using Protein A may be used.

[0147] Embodiments of the present invention

[0148] The present invention is further described in detail below by way of examples. However, the following examples are only used to specifically implement the content of the present invention, and the present invention is not limited thereto.

[0149] Example 1. Amino acid residue modified Fc variant of L309Y

[0150] 1-1. Production and purification of Fc variant (Q311M / M428L)

[0151] In the previous study, for M252Y of YTE and M428L of LS, which are known to have the effect of increasing half-life, Met was confirmed to be substituted with other amino acids in variants that improve FcRn binding ability (wild-type Fc domain amino acid sequence: sequence number 1). For this reason, Met is judged to be an amino acid that inhibits FcRn pH-dependent binding ability in the FcRn binding site of antibody Fc. Therefore, in order to confirm whether the FcRn binding ability is actually inhibited when introduced into PFc29 (Q311R and M428L Fc variants), trastuzumab-ML was prepared in which the Fc variant (Q311M / M428L) variant (sequence number 2) was introduced into the trastuzumab heavy chain (sequence number 11) and transfected into Expi293F animal cells. Specifically, first, the heavy chain gene and light chain gene of the Fc variant (Q311M / M428L) with Met introduced into the Q311 were mixed in a ratio of 1:1 with 30 ml of Freestyle293expression culture medium (Gibco, 12338-018). Then, polyethyleneimine (PEI, Polyethylenimine) (Polyscience, 23966) and the variant gene were mixed in a ratio of 4:1 at room temperature and left for 20 minutes, and then mixed with 2x10 6 The Expi293F cell line was cultured at a density of cells / ml. After culturing for 7 days in a shaking culture incubator at 37°C, 125rpm and 8% CO2, only the supernatant was obtained by centrifugation. Subsequently, 25×PBS was used to maintain equilibrium and filtered using a 0.2μm filter (Merck Millipore) and a bottle top filter. 500μl of protein A resin was added to the filtered culture solution and stirred at a temperature of 4°C for 16 hours. Then, the resin was recovered by column flow and washed with 5ml of PBS, eluted with 3ml of 100mM glycine (pH2.7) buffer, and neutralized with 1M Tris-HCl pH8.0. A centrifugal filter device 3K (Merck Millipore) was used to replace the buffer.

[0152] 1-2. Confirmation of pH-dependent binding ability of Fc variant (Q311M / M428L)

[0153] The pH-dependent binding ability of the trastuzumab-ML purified in Example 1-1 above to FcRn was confirmed by enzyme-linked immunosorbent assay (ELISA). Specifically, 50 μl of the above IgG Fc variant diluted to 4 μg / ml in 0.05 M Na2CO3 pH9.6 was injected into a Flat Bottom Polystyrene High Bind 96-well microplate (costar) and fixed at 4°C for 16 hours. Then, at room temperature, it was blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH6.0) for 2 hours. After washing 4 times with 180 μl of 0.05% PBST (pH6.0), 50 μl of FcRn diluted in sequence with 1% skim milk (in 0.05% PBST, pH6.0) was dispensed into each well and reacted for 1 hour at room temperature. After washing, 50 μl of anti-GST-HRP conjugate (GE Healthcare) was used for antibody reaction for 1 hour at room temperature and then washed. Subsequently, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and 50 μl of 2M H2SO4 was added to terminate the reaction, and then analyzed using an Epoch Microplate Spectrophotometer (Epoch Microplate Spectrophotometer, BioTek).

[0154] As a result, the binding of Met, which is expected to inhibit binding to FcRn, was superior to that of PFc29 at pH 6.0, and it was confirmed that the binding was similar to that of PFc29 at pH 7.4. That is, the binding of the Q311M variant was superior to that of PFc29 at pH 6.0, and the binding was similar to that of PFc29 at pH 7.4 ( Figure 1 ).

[0155] 1-3. Exploration of Fc variants with enhanced FcRn binding ability

[0156] In order to find additional variants with increased FcRn binding ability compared with the Fc variants having the mutations of Q311M and M428L screened in Example 1-1 above, 15 variants having 15 amino acids other than L, G, P, C, and D at the L309 position were prepared in the same manner as in Example 1-1 above, and purified after culturing in animal cells ( Figure 2 ).

[0157] 1-4. Confirmation of pH-dependent binding ability of antibodies containing Fc variants

[0158] In order to confirm the binding ability of the variants 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, L309Y, Q311M, and M428L (trastuzumab-YML) with the highest binding ability at pH 6.0 were confirmed. Figure 3 YM, hereinafter, represented by YML, sequence number 3) variant ( Figure 3 ). In this regard, the pH-dependent binding ability of the selected YML variants to FcRn was measured by enzyme-linked immunosorbent assay. As a result, the binding ability to FcRn at pH 6.0 was higher than that of the existing PFc29 (Q311R and M428L Fc variants) and the ML variants (Q311M and M428L Fc variants) selected in Example 1-1 above, and a similar degree of dissociation was shown at pH 7.4 ( Figures 4a to 4c ).

[0159] 1-5. Confirmation of the effect of Fc variants on increasing the serum half-life of FcRn

[0160] In order to increase the serum half-life, the pH-dependent binding of Fc to FcRn is particularly important (refer to Figure 5), referring to Souders et al 2015, the instantaneous binding rate (on rate) in a weakly acidic environment and the instantaneous dissociation rate (off rate) in a neutral environment were confirmed in the trastuzumab-ML (Q311M and M428L Fc variants) and trastuzumab-YML (L309Y, Q311M and M428L Fc variants) screened in the above Examples 1-1 and 1-4. As a control group, the existing Fc29 (Q311R and M428L Fc variants) and PFc41 (L309G and M428L Fc variants) were used. Specifically, 40 μg / ml of His-tagged human FcRn was fixed to the NI-NTA biosensor (Pall Fortebio) for 3 minutes. Subsequently, after maintaining the baseline with a PBS buffer at pH 6.0, 700 nM of each antibody Fc variant (in PBS pH 6.0) was bound for 20 seconds. In a weakly acidic environment (pH 6.0), the antibody Fc antibody was bound to FcRn, and the buffer was replaced with PBS at pH 7.4 for 5 seconds and measured by biolayer interferometry (BLItz, Pall Fortebio). In the measurement data (sensorgram), the instantaneous association rate and instantaneous dissociation rate were confirmed based on the slope of the linear interval (pH 6.0, association: 2 seconds / pH 7.4, dissociation: 1 second) at each pH. Then, in order to compare the Fc variants, the slope value of each Fc variant was divided and the ratio was calculated. The Fc variant with the highest association rate and dissociation rate was confirmed by evaluating (scoring) according to the average of the association rate ratio and the dissociation rate ratio. As a result, the antibody Fc variant (YML, sequence number 3) with the greatest expected half-life increase effect was screened out ( Figures 6a to 6c ).

[0161] Example 2. Amino Acid Residue Modification of Q311M Fc Variants

[0162] 2-1. Production and purification of trastuzumab-Fc variants with Q311M / M428L mutations

[0163] It is known from existing studies that in variants with increased FcRn binding ability, Met is substituted with other amino acids (wild-type Fc domain amino acid sequence: SEQ ID NO: 1). For this reason, Met is judged to be an amino acid that inhibits FcRn pH-dependent binding ability in the FcRn binding site of antibody Fc. Therefore, when Met is introduced into PFc29 (Q311R / M428L) discovered by the present inventors, this experiment was conducted to confirm whether the FcRn binding ability is actually inhibited.

[0164] More specifically, a heavy chain expression vector for introducing an Fc variant (Q311M / M428L, sequence number 2) into a wild-type trastuzumab heavy chain (sequence number 11) and a wild-type trastuzumab light chain (sequence number 12) expression vector were prepared. Next, the two vectors were transfected into Expi293F animal cells. One day before transfection, 2×10 6 300ml of Expi293F cells were subcultured at a density of cells / ml, and the next day, polyethyleneimine (PEI, Polyethylenimine, Polyscience, 23966) was used for transfection. First, the heavy chain gene and light chain gene of the variant were mixed in a ratio of 1:1 to 30ml of Freestyle 293expression culture medium (Gibco, 12338-018). Subsequently, polyethyleneimine and variant genes were mixed in a ratio of 4:1 and placed for 20 minutes at room temperature, and the cells subcultured the day before were mixed and cultured in a CO2 shaking incubator at 37°C, 125rpm and 8% CO2 for 7 days, and only the supernatant was obtained by centrifugation. Afterwards, 25×PBS was used to maintain equilibrium and filtered through a 0.2μm filter (MerckMillipore) using a bottle top filter. 500μl of protein A resin was added to the filtered culture solution and stirred for 16 hours at a temperature of 4°C. Then, the resin was recovered by column flow and washed with 5 ml of PBS, eluted with 3 ml of 100 mM glycine buffer (pH 2.7), and neutralized with 1 M Tris-HCl pH 8.0. A centrifugal filter device 3K (Merck Millipore) was used for buffer replacement.

[0165] 2-2. ELISA analysis of purified trastuzumab-Fc variant (Q311M / M428L)

[0166] The pH-dependent binding ability of the trastuzumab-ML purified in Example 2-1 to FcRn was confirmed by enzyme-linked immunosorbent assay (ELISA).

[0167] More specifically, 50 μl of IgG Fc variant diluted to 4 μg / ml in 0.05 M Na2CO3 pH9.6 was injected into each well of a Flat Bottom Polystyrene High Bind 96-well microplate (costar) and fixed at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH6.0 / pH7.4) for 2 hours at room temperature. After washing four times with 180 μl of 0.05% PBST (pH6.0 / pH7.4), 50 μl of FcRn diluted in sequence with 1% skim milk (in 0.05% PBST, pH6.0 / pH7.4) was dispensed into each well and reacted at room temperature for 1 hour. After washing, the antibody reaction was carried out for 1 hour at room temperature with 50μl of anti-GST-HRP conjugate (GE Healthcare) and then washed. Subsequently, 50μl of 1-StepUltra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and 50μl of 2M H2SO4 was added to terminate the reaction, and then analyzed using an Epoch microplate spectrophotometer (BioTek). As a result, it was expected that the binding of Met to FcRn would be better than that of PFc29 at pH 6.0, and it was confirmed that it showed a binding force similar to that of PFC29 at pH 7.4 ( Figure 1 ).

[0168] That is, contrary to the prediction before the experiment, in the case of the Q311M mutation, an increase in binding force at pH 6.0 was confirmed.

[0169] 2-3. Additional exploration of Fc variants with increased FcRn binding ability and animal cell expression and purification

[0170] In order to find variants with stronger binding ability than the Q311M / M428L screened above, 15 variants with 15 amino acids other than L, G, P, C, and D at the L309 position, which is particularly important for FcRn binding, were cultured in animal cells by the same method as in Example 2-2 and purified ( Figure 7 ).

[0171] 2-4. ELISA analysis of purified trastuzumab-Fc variants

[0172] In order to confirm the FcRn binding ability of the variants prepared in the above Example 2-3 at pH 6.0, an enzyme-linked immunosorbent assay was performed.

[0173] More specifically, in order to confirm the pH-dependent binding ability to FcRn, 50 μl of IgG Fc variant diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6 was injected into a Flat Bottom Polystyrene High Bind 96-well microplate (costar) and fixed at 4°C for 16 hours. Then, it was blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0) for 2 hours at room temperature. After washing four times with 180 μl of 0.05% PBST (pH 6.0), 50 μl of FcRn diluted in sequence 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, antibody reaction was carried out with 50 μl of anti-GST-HRP conjugate (GE Healthcare) at room temperature for 1 hour and washed. Subsequently, 50 μl of 1-Step UltraTMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to each sample for color development, and 50 μl of 2M H2SO4 was added to each sample to terminate the reaction. The samples were then analyzed using an Epoch microplate spectrophotometer (BioTek). Figure 8 ), i.e., trastuzumab-YML in which the heavy chain of the wild-type trastuzumab heavy chain (sequence number 11) having a higher binding force than PFc29 at pH 6.0 is introduced into the heavy chain of the Fc variant (L309Y / Q311M / M428L, sequence number 3) and the wild-type trastuzumab light chain (sequence number 12) is combined; and trastuzumab-EML (sequence number 4) in which the heavy chain of the wild-type trastuzumab heavy chain (sequence number 11) is introduced into the heavy chain of the Fc variant (L309E / Q311M / M428L, sequence number 3) and the wild-type trastuzumab light chain (sequence number 12) is combined.

[0174] 2-5. pH-dependent FcRn binding analysis of trastuzumab Fc variants (L309Y / Q311M / M428L (YML), L309E / Q311M / M428L (EML))

[0175] In order to confirm the pH-dependent binding ability of the two variants of trastuzumab-YML and trastuzumab-EML (SEQ ID NO: 4) purified in Example 2-4 to FcRn, an enzyme-linked immunosorbent assay was performed.

[0176] More specifically, in order to confirm the pH-dependent binding ability to FcRn, 50 μl of IgG Fc variants diluted to 4 μg / ml in 0.05 M Na2CO3 pH9.6 were injected into Flat Bottom Polystyrene High Bind 96-well microplate (costar) and fixed at 4°C for 16 hours. Subsequently, blocking was performed for 2 hours at room temperature with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH6.0). After washing 4 times with 180 μl of 0.05% PBST (pH6.0 / pH7.4), 50 μl of FcRn diluted in sequence with 1% skim milk (in 0.05% PBST, pH6.0 / pH7.4) was dispensed into each well and reacted for 1 hour at room temperature. After washing, an antibody reaction was performed with 50 μl of anti-GST-HRP conjugate (GE Healthcare) at room temperature for 1 hour. After washing, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and then 50 μl of 2 M H2SO4 was added to terminate the reaction. Then, the samples were analyzed using an Epoch microplate spectrophotometer (BioTek).

[0177] As a result, YML showed higher binding ability than EML at pH 6.0 than PFc29, and in the case of YML, the binding ability was also increased at pH 7.4 ( Fig. 9 ).

[0178] Since the pH-dependent binding of the EML variants, which showed similar binding to PFc29 at pH 7.4 and increased binding at pH 6.0, was improved, it can be judged that this may increase the serum half-life by improving the recycling process of the antibody ( Figure 10a to Figure 10b ).

[0179] Example 3. Amino Acid Residue Modification of Q311W Fc Variants

[0180] 3-1. Preparation and analysis of 17 amino acid substitution variants at position Q311 of PFc41 (L309G, M428L)

[0181] pMopac12-NlpA-Fc variant plasmid was prepared. In the previous study, L309G was fixed based on PFc41 (L309G, M428L) and PFc29 (Q311R and M428L Fc variants) with increased half-life, and variants substituted with 17 amino acids other than Q and CR at the Q311 position were analyzed by bacterial display. In order to screen variants with increased binding to human FcRn by FACS at pH 6.0, FACS analysis was performed on the prepared plasmid. As a result, 9 variants were screened out in which the Q311 position was replaced by L, I, V, T, A, Y, H, K, and W (Q311L, Q311I, Q311V, Q311T, Q311A, Q311Y, Q311H, Q311K, or Q311W in the M428L and L309G Fc variants) with stronger binding ability at pH 6.0 than those found in the prior studies. Fig.11 ).

[0182] 3-2. Production and purification of trastuzumab Fc variants containing L, I, V, T, A, Y, H, K, W variants at the Q311 position of PFc41 (L309G, M428L)

[0183] In order to confirm the characteristics of the 9 variants screened in Example 3-1 above in the antibody format, a heavy chain expression vector (SEQ ID NO. 11) in which the wild-type trastuzumab heavy chain (heavy chain) Fc variants were introduced and a wild-type trastuzumab light chain (SEQ ID NO. 12) expression vector was prepared. Subsequently, the expression vectors were transfected into Expi293F animal cells.

[0184] More specifically, one day before transfection, 2 × 10 6300 ml of Expi293F cells were subcultured at a density of 1:1 cells / ml, and the next day, polyethylenimine (Polyscience, 23966) was used for transfection. First, the heavy chain gene and light chain gene of the variant were mixed in a ratio of 1:1 to 3 ml of Freestyle 293expression culture medium (Gibco, 12338-018). Subsequently, polyethylenimine and variant genes were mixed in a ratio of 4:1 and left for 20 minutes at room temperature, and then the cells subcultured the day before were mixed and cultured in a CO2 shaking incubator at 37°C, 125rpm and 8% CO2 for 7 days, and only the supernatant was obtained by centrifugation. Afterwards, 25×PBS was used to maintain equilibrium. Filtered using a bottle top filter to a 0.2μm filter (Merck Millipore). 100 μl of protein A resin was added to the filtered culture solution and stirred at 4°C for 16 hours, and then the resin was recovered by passing through the column. Then, after washing twice with 1 ml of PBS, elution was performed with 1 ml of 100 mM glycine (pH 2.7) buffer, and neutralization was performed with 1 M Tris-HCl pH 8.0. A centrifugal filter device 30K (Merck Millipore) was used to replace the buffer. The completed sample ( Fig.12 ).

[0185] 3-3. Enzyme-linked immunosorbent assay (ELISA) analysis of purified trastuzumab Fc variants

[0186] In order to determine the pH-dependent binding of the heavy chain of the wild-type trastuzumab heavy chain (SEQ ID NO. 11) to the wild-type trastuzumab light chain (SEQ ID NO. 12) to FcRn, an enzyme-linked immunosorbent assay was performed. In order to confirm the binding ability to FcRn under pH 6.0 conditions, first, IgG Fc variants diluted to 4 μg / ml in 0.05M Na2CO3 pH9.6 were injected into Flat Bottom Polystyrene High Bind 96-well microplates (costar) at 50 μl each and fixed at 4°C for 16 hours. Then, at room temperature, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH6.0) was used to block 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 for 1 hour at room temperature. After washing, 50 μl of anti-GST-HRP conjugate (GE Healthcare) was used for antibody reaction at room temperature for 1 hour and washed. Subsequently, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and 50 μl of 2M H2SO4 was added to terminate the reaction. Then, the analysis was performed using an Epoch microplate spectrophotometer (BioTek). As a result, the top three variants with increased binding ability to FcRn at pH 6.0 were screened (L309G / Q311Y / M428L, L309G / Q311H / M428L, L309G / Q311W / M428L) ( Fig.13 ).

[0187] In order to determine the pH-dependent binding of the three trastuzumab variants screened to FcRn, an enzyme-linked immunosorbent assay was performed. First, to confirm the pH-dependent binding to FcRn, 50 μl of IgG Fc variants diluted to 4 μg / ml in 0.05 M Na2CO3 pH9.6 were injected into Flat Bottom Polystyrene High Bind 96-well microplates (costar) and fixed at 4°C for 16 hours. Then, at room temperature, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH6.0 / pH7.4) was used for blocking 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 reacted for 1 hour at room temperature. After washing, an antibody reaction was performed at room temperature for 1 hour with 50 μl of anti-GST-HRP conjugate (GE Healthcare) and a washing process was performed. Subsequently, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and 50 μl of 2M H2SO4 was added to terminate the reaction, and then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0188] As a result, the binding ability to FcRn was improved compared to PFc29 at pH 6.0, and the binding ability was higher than that of PFc29 even at pH 7.4, so the pH-dependent binding ability was not improved ( Fig.14 ).

[0189] 3-4. Saturation mutagenesis, animal cell expression and purification

[0190] Among the three variants screened in Example 3-3 (L309G / Q311Y / M428L, L309G / Q311H / M428L, L309G / Q311W / M428L), Q311H known in prior studies was excluded and Q311Y or W was included to express 15 trastuzumab variants in which the L309 position was substituted with F, I, M, V, T, A, Y, H, Q, N, K, E, W, R, and S into 30 plasmids. The prepared expression vectors were used to culture the 30 trastuzumab variants in animal cells by the same method as above, and then purified, and then confirmed by SDS-PAGE gel ( Fig.15 ).

[0191] 3-5. Analysis of trastuzumab-Fc variants by enzyme-linked immunosorbent assay for FcRn at pH 6.0

[0192] In order to determine the binding capacity of the 30 trastuzumab variants of Examples 3-4 to FcRn at pH 6.0, an enzyme-linked immunosorbent assay was performed. First, in order to confirm the pH-dependent binding capacity to FcRn, 50 μl of IgG Fc variants diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6 were injected into a Flat Bottom Polystyrene High Bind 96-well microplate (costar) and fixed at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0) for 2 hours at room temperature. After washing four times with 180μl of 0.05% PBST (pH6.0), 50μl of FcRn serially diluted with 1% skim milk (in 0.05% PBST, pH6.0) was dispensed into each well and reacted at room temperature for 1 hour. After washing, an antibody reaction was performed with 50μl of anti-GST-HRP conjugate (GE Healthcare) at room temperature for 1 hour and then washed. Subsequently, 50μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and 50μl of 2M H2SO4 was added to terminate the reaction, and then analyzed using an Epoch microplate spectrophotometer (BioTek). As a result, it was found that the binding force at pH6.0 was higher than that of the L309E / Q311W / M428L (EWL, sequence number 8) variant of PFc29 ( Fig.16 ).

[0193] 3-6. pH-dependent FcRn binding analysis of trastuzumab-Fc variants with L309E / Q311W / M428L (EWL) mutations

[0194] In order to determine the pH-dependent binding affinity of trastuzumab-EWL to FcRn, which contains the heavy chain of the wild-type trastuzumab (SEQ ID NO. 11) and the heavy chain of the EWL Fc variant and the trastuzumab light chain (SEQ ID NO. 12), an enzyme-linked immunosorbent assay was performed. First, in order to confirm the pH-dependent binding affinity to FcRn, 50 μl of the IgG Fc variant diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6 was injected into a Flat Bottom Polystyrene High Bind 96-well microplate (costar) and fixed at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0 / pH 7.4) for 2 hours at room temperature. 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 reacted at room temperature for 1 hour.

[0195] After washing, the antibody reaction was carried out for 1 hour at room temperature with 50 μl of anti-GST-HRP conjugate (GE Healthcare) and then washed. Subsequently, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for color development, and 50 μl of 2M H2SO4 was added to terminate the reaction, and then analyzed using an Epoch microplate spectrophotometer (BioTek). The results showed that the binding affinity to human FcRn was higher than that to PFc29 at pH 6.0, and the binding affinity was lower than that to PFc29 at pH 7.4. Figures 17a to 17c ). Therefore, with the improved pH-dependent binding of the variant EWL, the serum half-life can be increased by maximizing the recycling process of the antibody. Sequence Listing <110> Korea University Industry-Academic Cooperation Group <120> pH sensitive Fc variants <130> P22115167WP <150> KR 10-2020-0010336 <151> 2020-01-29 <150> KR 10-2020-0148372 <151> 2020-11-09 <150> KR 10-2020-0148373 <151> 2020-11-09 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 227 <212> PRT <213> Unknown <220> <223> Wild Fc <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> Unknown <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> Unknown <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 <210> 4 <211> 227 <212> PRT <213> Unknown <220> <223> EML (L309E / Q311M / M428L) <400> 4 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 Glu 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> 5 <211> 227 <212> PRT <213> Unknown <220> <223> EWL (L309E / Q311W / M428L) <400> 5 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 Glu His Trp 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> 6 <211> 681 <212> DNA <213> Unknown <220> <223> Wild - type Fc DNA sequence <400> 6 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggacga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ccccgggtaa a 681 <210> 7 <211> 681 <212> DNA <213> Unknown <220> <223> ML DNA sequence <400> 7 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac atggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggacga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gctgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ccccgggtaa a 681 <210> 8 <211> 681 <212> DNA <213> Unknown <220> <223> YML DNA sequence <400> 8 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg aacagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtctatcac atggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cacctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gctgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ccccgggtaa a 681 <210> 9 <211> 681 <212> DNA <213> Unknown <220> <223> EML DNA sequence <400> 9 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcgaacac atggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cacctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gctgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ccccgggtaa a 681 <210> 10 <211> 681 <212> DNA <213> Unknown <220> <223> EWL DNA sequence <400> 10 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcgaacac tgggactggc tgaatggcaa ggatcaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc gggacaac tacaagacca cacctcccgt gctggactcc 540 gacggctcct tcttctctca cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gctgcatgag gctctgcaca accactacac gcaagaagagc 660 ctctccctgt ccccgggtaa a 681 <210> 11 <211> 451 <212> PRT <213> Unknown <220> <223> Trastuzumab-WT (heavy chain) <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 12 <211> 214 <212> PRT <213> Unknown <220> <223> Trastuzumab-WT (light chain) <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An Fc variant, characterized in that The invention relates to a modification of an amino acid residue selected from the group consisting of L309Y, Q311M and Q311W based on the Kabat numbering system in the Fc region of wild-type IgG1, wherein the Fc variant consists of 1) L309E, Q311M and M428L, 2) L309Y, Q311M and M428L or 3) L309E, Q311W and M428L.

2. The Fc variant according to claim 1, characterized in that Under conditions of pH 7.0 to pH 7.8, it exhibits lower binding affinity to FcRn compared to the wild-type immunoglobulin Fc region.

3. The Fc variant according to claim 1, characterized in that Under conditions of pH 5.6 to pH 6.5, it exhibits higher binding affinity to FcRn than the wild-type immunoglobulin Fc region.

4. A polypeptide, characterized in that Comprising the Fc variant of claim 1.

5. The polypeptide according to claim 4, characterized in that Compared to the wild type, it has an increased half-life in vivo.

6. An antibody, characterized in that Comprising the Fc variant of claim 1.

7. The antibody according to claim 6, characterized in that Compared to the wild type, it has an increased half-life in vivo.

8. A nucleic acid molecule, characterized in that Encodes the Fc variant of claim 1, the polypeptide of claim 4, and the antibody of claim 6.

9. A carrier, characterized in that Comprising the nucleic acid molecule of claim 8.

10. A host cell, characterized in that Comprising the vector of claim 9.

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