Novel glycosylated fc variant with effector function deleted

Abstract

The present invention relates to novel Fc variants in which the binding affinity for FcγRs and C1q is eliminated, thereby reducing effector functions. The human antibody Fc domain variants of the present invention are novel variants distinct from conventional Fab-arm exchange blocking variants, do not bind to any human FcγRs and C1q, and also do not bind to mouse or monkey FcγRs, have excellent serum half-life and thermal stability, and exhibit the same effect even when the variants are applied to IgGs of other subclasses. Therefore, the antibody variants to which the human antibody Fc domain variants are applied can be used for preventing immune cell / normal cell death (toxicity) mediated by the antibody Fc region in therapeutic antibodies or Fc-fusion protein pharmaceuticals.

Description

Novel glycosylated FC variants with removed functional group functions

[0001] The present invention relates to novel Fc variants having reduced effector functions due to abolished binding affinity for FcγRs and C1q.

[0002] Protein therapeutics are rapidly replacing non-specific small-molecule compound therapeutics due to their high specificity for disease targets and low side effects and toxicity, and are widely used in clinical practice. Currently, antibody therapeutics and Fc-fusion protein therapeutics that are fused with antibody Fc domains are the main types of protein therapeutics used clinically. Therapeutic antibodies are considered one of the most effective cancer treatments because they show much higher specificity for their targets than existing small-molecule drugs, have low biotoxicity and side effects, and have an excellent blood half-life of approximately three weeks. In fact, major pharmaceutical companies and research institutes around the world are accelerating the research and development of therapeutic antibodies that specifically bind to and effectively eliminate cancer cells, including carcinogens. Companies developing therapeutic antibody drugs include pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS. Roche, in particular, is leading the global antibody drug market with Herceptin, Avastin, and Rituxan, which are representative products for cancer treatment. These three therapeutic antibodies generated approximately $19.5 billion in global sales in 2012, generating significant profits and leading the global antibody drug market. Johnson & Johnson, which developed Remicade, is also rapidly growing in the global antibody market due to increasing sales. Pharmaceutical companies such as Abbott and BMS are also known to have a number of therapeutic antibodies in the final stages of development. As a result, biopharmaceuticals, including therapeutic antibodies that are specific to disease targets and have fewer side effects, are quickly replacing small molecule drugs in the global pharmaceutical market, which had been dominated by small molecule drugs.Antibodies provide a link between the humoral and cellular immune systems. While the Fab region of an antibody recognizes antigens, the Fc domain portion binds to receptors for antibodies (immunoglobulins) on cells (Fc receptors or FcRs) that are differentially expressed by all immunocompetent cells, and have different mechanisms depending on the type of FcγR expressed on the surface of the immune cell to which it binds. Binding of antibodies to Fc receptors on the cell surface via the Fc region triggers a variety of important biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, control of placental transport, and immunoglobulin production (Deo, YM et al., Immunol. Today 18(3):127-135 (1997)). Thus, the Fc domain plays a crucial role in the recruitment of immune cells and antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). In particular, the effector functions of antibodies, namely ADCC and ADCP, depend on the interaction with Fc receptors present on the surface of many cells. There are five types of Fc receptors in humans, and the type of immune cell recruited is determined by which Fc receptor the antibody binds to.For example, the Fc domain of an antibody is responsible for the major therapeutic effect of a therapeutic antibody by binding to FcγRⅢa to induce ADCC, binding to FcγRI or FcγRⅡa to induce ADCP, and binding to C1q to induce CDC (complement dependent cytotoxicity), thereby causing toxicity to the target antigen bound to the Fab region.

[0003] However, in a therapeutic context, the effector functions of antibodies are often undesirable, and can cause safety concerns and unwanted side effects by activating host immune defenses. For example, some therapeutic antibodies, such as immune checkpoint inhibitors and bispecific immune cell engagers, have been problematic in that their immune effectors activate the targeted immune cells, resulting in their destruction. Immune checkpoint inhibitors, which target immune checkpoint proteins expressed on the surface of immune cells, such as T cells, have the disadvantage of reducing the antibody's original efficacy due to the side effect of destroying immune cells that should be eliminating cancer cells by activating the immune response due to Fc-mediated immune effectors. In addition, among FcγRs, the immune cell activation inhibitory receptor (FcγRⅡb) is expressed on T cells and has been reported to reduce the efficacy of immune checkpoint inhibitory antibodies by interacting with antibody Fc (Bennion et al., Sci Transl Med., 2023), so it is known that the interaction of the immune cell surface and FcγR with antibody Fc is involved not only in the occurrence of side effects of immune checkpoint antibodies but also in the reduction of efficacy. In addition, immune cell-directing bispecific antibodies, which are antibody therapeutics that bind to antigens on the surface of cancer cells on one side and bind to immune cells on the other side, guide immune cells to cancer cells so that they can be eliminated more effectively, show side effects when the antibody has an Fc-mediated immune action mechanism, as the immune cells are destroyed and the cancer cells cannot be effectively eliminated. In addition, agonist antibodies that bind to target cells and induce cell activation or antagonist antibodies that block the interaction of the target antigen and the ligand have the problem of lowering the original effect of the antibody because they are toxic to the target cells and antigen due to the Fc-mediated immune action mechanism.In addition, when developing an Fc-fusion protein that fuses the Fc region to increase the half-life of an active substance such as a protein or chemical for therapeutic, diagnostic, or research purposes, there is a problem of toxicity due to the Fc-mediated immune mechanism.

[0004] Therefore, in order to prevent off-target toxicity caused by the antibody's immune action mechanism and to have an effective antibody therapeutic effect, it is essential to eliminate the Fc-mediated immune action mechanism. To this end, when developing antibodies, IgG2 antibodies, which have the lowest binding affinity to FcγR among human IgG subclasses and thus a very low immune action mechanism, are considered. However, IgG2 antibodies have multiple isotypes due to disulfide bond exchange in the hinge region, and there is a physical problem that aggregation occurs due to decreased stability. Therefore, IgG4 antibodies with the next lowest binding affinity are considered and are currently being used in clinical development. In this regard, anti-PD-1 antibodies (pembrolizumab (Keytruda) from Merck & Co., nivolumab (Opdivo) from Bristol-Myers Squibb, and cemiplimab (Libtayo) from Regeneron) that target the immune checkpoint protein PD-1 (Programmed cell death-1) expressed on T cells are all human IgG4 antibodies that have received FDA approval and are being used in high demand in clinical trials. Pembrolizumab has received clinical approval for various types of cancer and ranked second in global pharmaceutical sales in 2020, with sales of $14.3 billion, and nivolumab ranked eighth, with sales of $7.9 billion. However, IgG4 antibodies also have binding affinity for all FcγRs, and in particular, have a strong binding affinity of several nM for FcγRI, which causes the activation of various immune action mechanisms. Therefore, active research is being conducted to produce Fc with no binding affinity for FcγRs to prevent target cells from being destroyed by the immune action mechanism of the antibody.In addition, wild-type human IgG4 has a different amino acid at the 228th amino acid in the CH2 region as serine than human IgG1, and an intrachain disulfide bond is formed through a flexible core hinge, resulting in the production of a half-antibody due to a non-covalent linkage. Since IgG4 existing in this half-antibody form exhibits a Fab-arm exchange (FAE) phenomenon in which two half-antibody forms of IgG4 targeting different antigens combine, to prevent this, when the 228th amino acid of IgG4 is substituted with proline, which is the 228th amino acid of human IgG1, the hinge region is stabilized and Fab-arm exchange does not occur, so the corresponding S228P mutant is being developed for general application to IgG4 clinical antibodies. However, this conventional S228P variant has the same FcγR binding affinity as wild-type IgG4, and still has the problem of causing immune cell death side effects / off-target toxicity to normal cells.

[0005] An object of the present invention is to provide a novel human antibody Fc domain variant.

[0006] In addition, it is an object of the present invention to provide an antibody or a fragment thereof or a fragment thereof having immunological activity with reduced functional group function.

[0007] In addition, it is an object of the present invention to provide an Fc-fusion protein.

[0008] In addition, it is an object of the present invention to provide an antibody therapeutic agent.

[0009] In addition, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer.

[0010] In addition, it is an object of the present invention to provide a method for producing a human antibody Fc domain variant.

[0011] In addition, it is an object of the present invention to provide a method for producing an antibody or fragment thereof with reduced functional group function.

[0012] It is also an object of the present invention to provide a method for reducing the functional group function of an antibody.

[0013] It is also an object of the present invention to provide a use for the manufacture of antibody therapeutics.

[0014] In addition, it is an object of the present invention to provide a use for preventing or treating cancer.

[0015] In addition, it is an object of the present invention to provide a method for treating cancer.

[0016] To solve the above problem, the present invention provides a novel human antibody Fc domain variant with reduced functional group function.

[0017] In addition, the present invention provides an antibody comprising the novel human antibody Fc domain variant or a fragment thereof having immunological activity.

[0018] In addition, the present invention provides an Fc-fusion protein in which the human antibody Fc domain variant is fused with a protein therapeutic agent.

[0019] In addition, the present invention provides an antibody therapeutic agent comprising the antibody or a fragment thereof having immunological activity, and a drug moiety.

[0020] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the human antibody Fc domain variant, the antibody or a fragment thereof having immunological activity, or the antibody therapeutic agent as an active ingredient.

[0021] In addition, the present invention provides a method for producing the human antibody Fc domain variant.

[0022] In addition, the present invention provides a method for producing an antibody or fragment thereof with reduced functional group function.

[0023] The present invention also provides a method for reducing the functional group function of an antibody.

[0024] The present invention also provides the use of an antibody or an immunologically active fragment thereof comprising an Fc domain variant of the present invention for use in the manufacture of an antibody therapeutic agent.

[0025] Additionally, the present invention provides a use of the human antibody Fc domain variant of the present invention, the antibody of the present invention or a fragment thereof having immunological activity, or the antibody therapeutic agent of the present invention for the prevention or treatment of cancer.

[0026] In addition, the present invention provides a method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of a human antibody Fc domain variant of the present invention, an antibody of the present invention or a fragment thereof having immunological activity, or an antibody therapeutic agent of the present invention.

[0027] The human antibody Fc domain variants of the present invention are novel variants that are different from conventional Fab-arm exchange-prevention variants, and do not bind to all human FcγRs and C1q, nor to mouse and monkey FcγRs, and have excellent blood half-life and thermal stability. Since the variants exhibit the same effect even when applied to other subclasses of IgG, the antibody variants applied thereto can be utilized for the purpose of preventing immune cell / normal cell death (toxicity) by the antibody Fc region of a therapeutic antibody or Fc-fusion protein drug.

[0028] Figure 1 is a diagram showing the results of SDS-PAGE analysis after purification of glycosylated IgG4 Fc variants (Stapled Fc-1, Stapled Fc-2, Stapled Fc-3, Stapled Fc-4, Stapled Fc-5) that prevent Fab-arm exchange, abolish FcγRs and C1q binding.

[0029] Figure 2 is a diagram showing the results of SDS-PAGE analysis after expression and purification of purified FcγRI-GST, FcγRⅡa-131H-GST, FcγRⅡa-131R-GST, FcγRⅡb-GST, FcγRⅢa-158V-GST, and FcγRⅢa-158F-GST.

[0030] Figure 3 is a diagram showing the results of ELISA analysis of the binding affinity of the glycosylated pembrolizumab IgG4 Fc variants of the present invention to FcγRI, FcγRⅡa-131H, FcγRⅡa-131R, FcγRⅡb, FcγRⅢa-158V, and FcγRⅢa-158F.

[0031] Figure 4 is a diagram showing the results of SDS-PAGE analysis after expression and purification of purified mouse FcγRI-GST, mouse FcγRⅡb-GST, mouse FcγRⅢ-GST, and mouse FcγRⅣ-GST.

[0032] Figure 5 is a diagram showing the binding affinity of the glycosylated pembrolizumab IgG4 Fc variants of the present invention to mouse FcγRI, mouse FcγRⅡb, mouse FcγRⅢ, and mouse FcγRⅣ analyzed by ELISA.

[0033] Figure 6 is a diagram showing the results of SDS-PAGE analysis after expression and purification of purified cynomolgus monkey FcγRI-GST, cynomolgus monkey FcγRⅡa-GST, cynomolgus monkey FcγRⅡb-GST, and cynomolgus monkey FcγRⅢ-GST.

[0034] Figure 7 is a diagram showing the binding affinity of the glycosylated pembrolizumab IgG4 Fc variants of the present invention to cynomolgus monkey FcγRI, cynomolgus monkey FcγRⅡa, cynomolgus monkey FcγRⅡb, and cynomolgus monkey FcγRⅢ analyzed by ELISA.

[0035] Figure 8 is a diagram showing the C1q binding ability of the glycosylated pembrolizumab IgG4 Fc variants of the present invention analyzed by ELISA.

[0036] Figure 9 is a diagram showing the results of SDS-PAGE analysis after expression and purification of purified FcRn-GST.

[0037] Figure 10 is a diagram showing the pH-dependent FcRn binding ability of the glycosylated pembrolizumab IgG4 Fc variants of the present invention analyzed by ELISA.

[0038] Figure 11 is a diagram showing the in vivo analysis of the blood half-lives of the glycosylated pembrolizumab IgG4 Fc variants of the present invention in human FcRn expressing mice.

[0039] Figure 12 is a diagram showing the thermal stability of the glycosylated pembrolizumab IgG4 Fc variants of the present invention analyzed by DSF.

[0040] Figure 13 is a diagram showing the results of SDS-PAGE analysis after purification of the glycosylated trastuzumab IgG1 L235C variant (IgG1 L235C) of the present invention.

[0041] Figure 14 is a diagram showing the binding ability of the glycosylated trastuzumab IgG1 L235C variant (IgG1 L235C) of the present invention to FcγRI, FcγRⅡa-131H, FcγRⅡa-131R, FcγRⅡb, FcγRⅢa-158V, and FcγRⅢa-158F analyzed by ELISA.

[0042] Figure 15 is a diagram showing the pH-dependent FcRn binding ability of the glycosylated trastuzumab IgG1 L235C variant (IgG1 L235C) of the present invention analyzed by ELISA.

[0043] Figure 16 is a diagram showing the thermal stability of the glycosylated trastuzumab IgG1 L235C variant (IgG1 L235C) of the present invention analyzed by DSF.

[0044] Figure 17 is a diagram showing the results of SDS-PAGE analysis after purifying a comparative glycosylated Fc variant introduced into a trastuzumab model antibody.

[0045] Figure 18 is a diagram analyzing the binding affinity of the glycosylated trastuzumab IgG1 L235C variant (IgG1 LC) of the present invention to FcγRI, FcγRIIa-131H, FcγRIIa-131R, FcγRIIb, FcγRIIIa-158V, and FcγRIIIa-158F.

[0046] Figure 19 is a diagram analyzing real-time cell killing ADCC of the glycosylated trastuzumab IgG1 L235C variant (IgG1 LC) of the present invention.

[0047] Figure 20 is a diagram showing the results of SDS-PAGE analysis after purification of the IgG2 V235C variant (IgG2 VC) and IgG3 L235C variant (IgG3 LC) introduced into the trastuzumab model antibody.

[0048] Figure 21 is a diagram showing the results of ELISA analysis of the binding affinity of the glycosylated trastuzumab IgG2 V235C variant (IgG2 VC) and the glycosylated trastuzumab IgG3 L235C variant (IgG3 LC) of the present invention to FcγRI, FcγRIIa-131H, FcγRIIa-131R, FcγRIIb, FcγRIIIa-158V, and FcγRIIIa-158F.

[0049] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0050] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0051] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited herein as references are incorporated herein by reference.

[0052] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as generally accepted three-letter codes for other amino acids, such as Aib (α-aminoisobutyric acid) and Sar (N-methylglycine). Furthermore, amino acids referred to herein by abbreviations are described according to the IUPAC-IUB nomenclature as follows:

[0053] 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.

[0054] In one aspect, the present invention relates to a human antibody Fc domain variant, wherein an amino acid at any one or more positions selected from the group consisting of amino acids at positions 231, 232, 234 and 235 numbered according to the EU Index in Kabat et al. in a wild type human antibody Fc domain is replaced with a sequence different from that of the wild type.

[0055] In one embodiment, the human antibody Fc domain variant of the invention may comprise one or more amino acid substitutions selected from the group consisting of 231C, 232C, 234C, and 235C.

[0056] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-1 comprising an amino acid substitution of A231C, wherein the variant may comprise the amino acid sequence of SEQ ID NO: 1 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 2.

[0057] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-2 comprising an amino acid substitution of P232C, wherein the variant may comprise the amino acid sequence of SEQ ID NO: 3 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 4.

[0058] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-4 comprising an amino acid substitution of F234C, wherein the variant comprises the amino acid sequence of SEQ ID NO: 5 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 6.

[0059] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant Stapled Fc-5 comprising an amino acid substitution of L235C, wherein the variant may comprise the amino acid sequence of SEQ ID NO: 7 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 8.

[0060] In one embodiment, the human antibody (immunoglobulin) may be IgA, IgM, IgE, IgD or IgG, or a variant thereof, and may be IgG1, IgG2, IgG3 or IgG4.

[0061] In one embodiment, the human antibody (immunoglobulin) may be IgG1, IgG2, IgG3 or IgG4, or a variant thereof, may be a humanized antibody, and may be Trastuzumab, Pembrolizumab or Atezolizumab.

[0062] In one embodiment, the human antibody (immunoglobulin) may be IgG4 or a variant thereof, and the Fc domain of wild-type IgG4 comprising hinge, CH2 and CH3 may comprise the amino acid sequence of SEQ ID NO: 9 and may be encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 10.

[0063] In the present invention, the human antibody IgG4 of SEQ ID NO: 9 comprises the Fc domain among the entire sequence of wild-type IgG4, i.e., hinge (amino acids 1 to 12 of SEQ ID NO: 9), CH2 (amino acids 13 to 122 of SEQ ID NO: 9), and CH3 (amino acids 123 to 229 of SEQ ID NO: 9), and the variants are variants in which the amino acid of CH2 of the wild-type IgG4 is substituted. For example, the 17th amino acid L in the amino acid sequence of SEQ ID NO: 9 is the 235th amino acid when numbered according to the EU Index in Kabat et al., and this is substituted with C in the Stapled Fc-5 (L235C) variant.

[0064] In one embodiment, the human antibody (immunoglobulin) may be IgG1 or a variant thereof, and the Fc domain of wild-type IgG1 comprising hinge, CH2 and CH3 may comprise the amino acid sequence of SEQ ID NO: 11 and may be encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 12.

[0065] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant IgG1 L235C comprising an amino acid substitution of L235C, wherein the variant may comprise the amino acid sequence of SEQ ID NO: 13 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 14.

[0066] In one embodiment, the human antibody (immunoglobulin) may be an IgG2 or a variant thereof, and the Fc domain of a wild-type IgG2 comprising hinge, CH2 and CH3 may comprise the amino acid sequence of SEQ ID NO: 15 and may be encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 16.

[0067] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant IgG2 V235C comprising an amino acid substitution of 235C, wherein the variant may comprise the amino acid sequence of SEQ ID NO: 17 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 18.

[0068] In one embodiment, the human antibody (immunoglobulin) may be IgG3 or a variant thereof, and the Fc domain of wild-type IgG3 comprising hinge, CH2 and CH3 may comprise the amino acid sequence of SEQ ID NO: 19 and may be encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 20.

[0069] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant IgG3 L235C comprising an amino acid substitution of L235C, wherein the variant may comprise the amino acid sequence of SEQ ID NO: 21 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 22.

[0070] In one embodiment, the human antibody Fc domain variant of the present invention may have reduced binding affinity to Fc gamma receptors (FcγRs) compared to a wild-type human antibody Fc domain, wherein the Fc gamma receptors may be human, mouse or monkey Fc gamma receptors (FcγRs), and may be FcγRI, FcγRⅡa, FcγRⅡb, FcγRⅢ, FcγRⅢa or FcγRIV.

[0071] In one embodiment, the human FcγR can be FcγRI, FcγRⅡa, FcγRⅡb or FcγRⅢa, wherein the FcγRⅡa can be FcγRⅡa-131H or FcγRⅡa-131R, and the FcγRⅢa can be FcγRⅢa-158V or FcγRⅢa-158F; the mouse FcγR can be mouse FcγRI, mouse FcγRⅡb, mouse FcγRⅢ or mouse FcγⅣ; and the monkey FcγR can be cynomolgus monkey FcγRⅡa, cynomolgus monkey FcγRⅡb or monkey FcγRⅢ.

[0072] In one embodiment, the human antibody Fc domain variant of the invention may have reduced binding affinity to C1q compared to a wild-type human antibody Fc domain.

[0073] In one embodiment, the human antibody Fc domain variant of the invention may have reduced effector function compared to a wild-type human antibody Fc domain.

[0074] In one embodiment, the effector function can be an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cell-mediated phagocytosis (ADCP), complement dependent cell-mediated cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof, preferably ADCC or CDC.

[0075] In one embodiment, the human antibody Fc domain variant of the invention may have increased thermal stability compared to a wild-type human antibody Fc domain.

[0076] In one embodiment, the human antibody Fc domain variant of the present invention may have an increased in vivo half-life compared to a wild-type human antibody Fc domain.

[0077] In one embodiment, the half-life of the human antibody Fc domain variant of the invention may be increased by at least 3%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a wild-type human antibody Fc domain, or may be increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to a wild-type Fc domain.

[0078] In one embodiment, the human antibody Fc domain variant of the present invention may have a higher binding affinity to FcRn at pH 5.6 to 6.5 compared to a wild-type human antibody Fc domain, which may be a slightly acidic condition within the endosome, and may be pH 5.8 to 6.0. In an embodiment, the pH-sensitive Fc variant of the present invention has a binding affinity for FcRn increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a wild-type Fc domain in the above pH range, or increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to a wild-type Fc domain, the Fc variant of the present invention has lower binding to FcRn compared to a wild-type immunoglobulin Fc region at pH 7.0 to 7.8. It can exhibit affinity, and can be in the normal pH range of blood, and can be pH 7.2 to 7.6. The degree of dissociation from FcRn of the Fc variant of the present invention in the above pH range can be the same or substantially unchanged compared to the wild-type Fc domain.

[0079] In one embodiment, the Fc domain variants of the present invention can be used for the purpose of not killing cells to which they bind.

[0080] In one embodiment, the Fc domain variants of the present invention can be applied to antibodies targeting immune cells or normal cells.

[0081] In one embodiment, the Fc domain variants of the present invention can be used in an immune checkpoint inhibitor antibody or a bispecific immune cell engaging bispecific antibody.

[0082] In the present invention, a variant comprising a mutation in an amino acid in the human antibody Fc region of the present invention is defined according to the amino acid mutation constituting the parent antibody Fc region, and the conventional antibody numbering is according to the EU Index described in the literature [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)].

[0083] As used in the present invention, the term “Fc domain variant” may be used interchangeably with “Fc variant”.

[0084] As used herein, the term "wild-type polypeptide" refers to an unmodified polypeptide that is later modified to produce a derivative. A wild-type polypeptide may be a polypeptide found in nature, a derivative of a naturally occurring polypeptide, or an engineered polypeptide. A wild-type polypeptide may refer to the polypeptide itself, a composition comprising the wild-type polypeptide, or an amino acid sequence encoding the same. Accordingly, the term "wild-type antibody" as used herein refers to an unmodified antibody polypeptide into which an amino acid residue has been modified to produce a derivative. Interchangeably with this term, the term "parent antibody" may be used to refer to an unmodified antibody polypeptide into which an amino acid modification has been introduced to produce a derivative.

[0085] As used herein, the term "amino acid modification / variation" refers to the substitution, insertion, and / or deletion, preferably substitution, of an amino acid in a polypeptide sequence. As used herein, the term "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a specific position in the polypeptide sequence of a wild-type human antibody Fc domain with another amino acid. For example, an Fc variant comprising an A231C substitution refers to a replacement of an alanine at position 231 in the amino acid sequence of the Fc domain of a wild-type antibody with a cysteine.

[0086] The term “Fc variant” as used herein means one that comprises modifications of one or more amino acid residues compared to a wild-type antibody Fc domain.

[0087] The Fc variants of the present invention comprise one or more amino acid modifications compared to a wild-type antibody Fc domain (region or fragment), thereby resulting in differences in amino acid sequence. The amino acid sequence of the Fc variants according to the present invention is substantially homologous to the amino acid sequence of the wild-type antibody Fc domain. For example, the amino acid sequence of the Fc variants according to the present invention will have at least about 80% homology, preferably at least about 90% homology, and most preferably at least about 95% homology to the amino acid sequence of the wild-type antibody Fc domain. The amino acid modifications can be performed genetically using molecular biological methods, or can be performed using enzymatic or chemical methods.

[0088] The Fc variants of the present invention can be prepared by any method known in the art. In one embodiment, the Fc variants of human antibodies according to the present invention encode a polypeptide sequence comprising a specific amino acid modification, which is then cloned into a host cell, if desired, and used to form a nucleic acid for expression and assay. Various methods for this are described in 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).

[0089] The nucleic acid encoding the Fc variant according to the present invention can be inserted into an expression vector for protein expression. The expression vector typically includes the protein operably linked, i.e., in a functional relationship, with a regulatory sequence, a selectable marker, an optional fusion partner, and / or additional elements. The Fc variant according to the present invention can be produced by a method in which a host cell transformed with the nucleic acid, preferably an expression vector containing the nucleic acid encoding the Fc variant according to the present invention, is cultured under appropriate conditions to induce protein expression. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are well known in the art and will vary depending on the host cell used. Preferably, the Fc variant according to the present invention is produced using Escherichia coli, which has low production costs and high industrial utility, as a host cell.

[0090] Accordingly, the scope of the present invention includes a method for producing an Fc variant, comprising the steps of culturing a host cell into which a nucleic acid encoding an Fc variant has been introduced under conditions suitable for protein expression; and purifying or isolating the Fc variant expressed from the host cell.

[0091] As used herein, the term "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is encoded at least in part by the FcRn gene. The FcRn may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein often comprises two polypeptides, referred to as a light chain and a heavy chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise stated herein, reference to FcRn or an FcRn protein refers to a complex of the FcRn heavy chain and beta-2-microglobulin.

[0092] In one aspect, the present invention relates to an antibody or an immunologically active fragment thereof comprising an Fc domain variant of the present invention.

[0093] In one embodiment, the antibody or immunologically active fragment thereof may have reduced binding to Fc gamma receptors (FcγRs) or C1q compared to a wild-type human antibody, such that binding to FcγRs is eliminated.

[0094] In one embodiment, the Fc gamma receptor can be a human, mouse or monkey Fc gamma receptor (FcγR), and can be FcγRI, FcγRⅡa, FcγRⅡb, FcγRⅢ, FcγRⅢa or FcγRIV.

[0095] In one embodiment, the human FcγR can be FcγRI, FcγRⅡa, FcγRⅡb or FcγRⅢa, wherein the FcγRⅡa can be FcγRⅡa-131H or FcγRⅡa-131R, and the FcγRⅢa can be FcγRⅢa-158V or FcγRⅢa-158F; the mouse FcγR can be mouse FcγRI, mouse FcγRⅡb, mouse FcγRⅢ or mouse FcγⅣ; and the monkey FcγR can be cynomolgus monkey FcγRⅡa, cynomolgus monkey FcγRⅡb or monkey FcγRⅢ.

[0096] In one embodiment, the antibody or immunologically active fragment thereof may have reduced effector function compared to a wild-type human antibody.

[0097] In one embodiment, the antibody can be a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an IgG-like bispecific antibody, a bispecific immune cell engager, an antibody mimetic, a chimeric antibody, an antibody conjugate, a human antibody, a humanized antibody, a bivalent antibody or a bispecific molecule, and the immunologically active fragment can be a Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, a single chain antibody, an Fv dimer, a complementarity determining region fragment or a diabody of the antibody.

[0098] Antibodies can be isolated or purified using a variety of methods known in the art. Standard purification methods include chromatography, electrophoresis, immunoassays, precipitation, dialysis, filtration, concentration, and chromatofocusing. As is known in the art, various natural proteins, such as bacterial proteins A, G, and L, bind to antibodies and can be used for purification. Often, purification using specific fusion partners may be possible.

[0099] The above antibody is not only in the form of a whole antibody, but also includes functional fragments of antibody molecules. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule means a fragment that possesses an antigen-binding function, and examples of antibody fragments include (i) a Fab fragment consisting of a variable region (VL) of a light chain and a variable region (VH) of a heavy chain and a constant region (CL) of a light chain and a first constant region (CH1) of a heavy chain; (ii) a Fd fragment consisting of a VH and CH1 domain; (iii) an Fv fragment consisting of a VL and VH domain of a single antibody; (iv) dAb fragments consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) isolated CDR regions; (vi) F(ab')2 fragments, which are bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv) in which a VH domain and a VL domain are joined by a peptide linker that joins them to form an antigen-binding site; (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965); and (ix) diabodies, which are multivalent or multispecific fragments produced by gene fusion (WO94 / 13804).

[0100] The antibody of the present invention or a fragment thereof having immunological activity may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof having immunological activity. The antibody may be produced recombinantly or synthetically.

[0101] The above antibody or fragment having immunological activity may be isolated from a living organism (not existing in a living organism) or non-naturally occurring, for example, may be synthetically or recombinantly produced.

[0102] In the present invention, "antibody" refers to a substance produced by antigen stimulation within the immune system, and its type is not particularly limited, and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly). Antibodies are highly stable both in vitro and in vivo and have a long half-life, making them advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, and thus have very high avidity. A complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The constant region of antibodies is divided into the heavy chain constant region and the light chain constant region. The heavy chain constant region has the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and the subclasses are gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has the kappa (κ) and lambda (λ) types.

[0103] In the present invention, the term "heavy chain" refers to a variable region domain V comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three invariant region domains CH 1 , C H 2 and C H 3 and is interpreted to mean both full-length heavy chains and fragments thereof, including hinges. In addition, the term "light chain" is interpreted to mean a variable region domain V comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and invariant region domain C L It is interpreted to mean including both full-length light chains and fragments thereof.

[0104] In the present invention, the term "Fc domain", "Fc fragment" or "Fc region" constitutes an antibody together with a Fab domain / fragment, and the Fab domain / fragment comprises a variable region (V) of a light chain. L ) and variable region of heavy chain (V H ) and the constant region of the light chain (C L ) and the first constant region of the heavy chain (C H 1) and the Fc domain / fragment is the second constant region of the heavy chain (C H 2) and the third invariant region (C H 3) It consists of:

[0105] In one aspect, the present invention relates to a nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same, or a fragment having immunological activity thereof.

[0106] In one aspect, the present invention relates to a vector comprising the nucleic acid molecule and a host cell comprising the vector.

[0107] The nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from the surrounding genetic sequence present in the genome of the organism from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a procedure may be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a pre-sequence or secretory leader is operably linked to the DNA of a polypeptide if the polypeptide is expressed as a preprotein, i.e., the form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Operably linked generally means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and within the same reading frame. However, enhancers need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in a conventional manner.

[0108] The isolated nucleic acid molecule encoding the Fc domain variant of the present invention, or the antibody comprising the same, or the fragment having immunological activity thereof, may undergo various modifications in the coding region within a range that does not change the amino acid sequence of the Fc domain variant expressed from the coding region, or the antibody comprising the same, or the fragment having immunological activity thereof, due to codon degeneracy or in consideration of the codons preferred in the organism to be expressed, and various modifications or alterations may also be made in a portion other than the coding region within a range that does not affect the expression of the gene, and it will be well understood by those skilled in the art that such modified genes are also included in the scope of the present invention. That is, the nucleic acid molecule of the present invention may undergo mutations by substitution, deletion, insertion, or a combination thereof of one or more nucleic acid bases, as long as it encodes a protein having an activity equivalent thereto, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

[0109] An isolated nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same, or a fragment thereof having immunological activity, may be inserted into an expression vector for protein expression. The expression vector typically comprises the protein operably linked, i.e., in a functional relationship, with a regulatory sequence, a selectable marker, an optional fusion partner, and / or additional elements. Under appropriate conditions, a host cell transformed with the nucleic acid, preferably an expression vector containing an isolated nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same, or a fragment thereof having immunological activity, may be cultured to induce protein expression, thereby producing an Fc domain variant of the present invention, or an antibody comprising the same, or a fragment thereof having immunological activity. Various suitable host cells may be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are well known in the art and will vary depending on the host cell used. Preferably, E. coli, which has a low production cost and high industrial utility value, can be produced as a host cell.

[0110] The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like. A suitable vector may include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and may be manufactured in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence may include, but is not limited to, a PhoA signal sequence, an OmpA signal sequence, etc. when the host is an Escherichia sp. fungus; an α-amylase signal sequence, a subtilisin signal sequence, etc. when the host is a Bacillus sp. fungus; an MFα signal sequence, a SUC2 signal sequence, etc. when the host is a yeast; and an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc. when the host is an animal cell. Additionally, the vector may include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, an origin of replication.

[0111] As used herein, the term "vector" refers to a carrier 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 may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art can construct vectors by standard recombinant techniques (see, e.g., 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).

[0112] In one embodiment, when producing the vector, depending on the type of host cell to be produced, the Fc domain variant, or the antibody containing the Fc domain variant, or the fragment having immunological activity thereof, an expression control sequence such as a promoter, terminator, enhancer, etc., a sequence for membrane targeting or secretion, etc. may be appropriately selected and variously combined according to the purpose.

[0113] As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide additional functions.

[0114] In the present invention, the term "host cell" includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. The host cell may be transfected or transformed by the vector, which refers to the process by which an exogenous nucleic acid molecule is transferred or introduced into the host cell.

[0115] In one embodiment, the host cell may be a bacterial or an animal cell, the animal cell line may be a CHO cell, a HEK cell, or a NSO cell, and the bacteria may be E. coli.

[0116] In one aspect, the present invention relates to a fusion protein comprising a human Fc domain variant of the present invention, or an antibody or fragment thereof having immunological activity, linked to a cargo molecule.

[0117] In one embodiment, the cargo molecule can be a detector, a therapeutic agent, a drug, a peptide, a growth factor, a cytokine, a receptor trap, a chemical compound, a carbohydrate moiety, an enzyme, an antibody or a fragment thereof, a DNA-based molecule, a viral vector, or a cytotoxic agent; one or more liposomes or nanocarriers loaded with a detector, a therapeutic agent, a drug, a peptide, an enzyme, an antibody or a fragment thereof, a DNA-based molecule, a viral vector, or a cytotoxic agent; or one or more nanoparticles, nanowires, nanotubes, or quantum dots.

[0118] In one embodiment, the fusion protein can be an agonist antibody, an antagonist antibody or an antibody therapeutic.

[0119] In one aspect, the present invention relates to an Fc-fusion protein comprising a human antibody Fc domain variant of the present invention and a protein therapeutic agent.

[0120] In one embodiment, the protein therapeutic agent may be a T-cell modulatory polypeptide (TMP), an immune checkpoint protein or immune effector cell-specific targeting molecule, an immune checkpoint inhibitor antibody, a bispecific immune cell engaging bispecific antibody, an agonist antibody, or an antagonist antibody.

[0121] In one embodiment, the immune effector cell may be an effector T cell, a regulatory T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a dendritic cell, or a B cell.

[0122] In one embodiment, the immune checkpoint protein can be CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, or PD-L2.

[0123] In one embodiment, the immune checkpoint inhibitor antibody can be atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0124] In one aspect, the present invention provides an antibody therapeutic agent comprising an antibody of the present invention or a fragment thereof having immunological activity, and a drug moiety.

[0125] In one embodiment, the drug moiety is selected from the group consisting of an immunomodulatory drug (IMiD), an immunogenic apoptosis inducer, an inhibitor of microtubulin structure formation, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, a chimeric antigen receptor (CAR) cell therapy, an oncolytic drug, an immunotherapy agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, an anti-cytokine agent, an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1BB agent, an anti-GITR agent, an anti-OX40 agent, Anti-TRAILR1 agents, anti-TRAILR2 agents, tagretin, interferon-alpha, clobetasol, peginterferon, prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, anti-chemokines, vorinostat, gabapentin, cyclosporine, rapamycin, FK506, detectable markers or reporters, TNF antagonists, antirheumatic drugs, muscle relaxants, narcotics, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials, psoriasis medications, corticosteroids, anabolic steroids, erythropoietin, immunization, immunoglobulins, immunosuppressants, growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma medications, beta agonists, inhaled steroids, Epinephrine or its analogues, cytokines, cytokine antagonists, PD-1 antagonists, adenosine A2AR antagonists, CD73 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors,anthracyclines or any combination thereof;

[0126] In one embodiment, the antibody therapeutic may have reduced effector function.

[0127] In one embodiment, the antibody therapeutic may be an immune checkpoint inhibitor or a bispecific immune cell engager.

[0128] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a human antibody Fc domain variant of the present invention, an antibody comprising the same or a fragment having immunological activity thereof, an Fc-fusion protein or an antibody therapeutic agent.

[0129] In one embodiment, the cancer can be any one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

[0130] In one embodiment, the composition of the present invention may further comprise an immunogenic apoptosis inducer, wherein the immunogenic apoptosis inducer may be at least one selected from the group consisting of an anthracycline-based anticancer agent, a taxane-based anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide-based anticancer agent, a GADD34 / PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin, and the anthracycline-based anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or It could be valrubicin, and the taxane family of anticancer drugs could be paclitaxel or docetaxel.

[0131] The pharmaceutical composition of the present invention can be used as a single therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy, and when such combination therapy is performed, cancer can be treated more effectively.

[0132] The pharmaceutical composition for preventing or treating cancer of the present invention can increase the cancer treatment effect of conventional anticancer drugs through the cancer cell killing effect by administering it together with a chemical anticancer drug (anticancer agent), etc. The combined administration can be performed simultaneously with or sequentially with the anticancer agent. Examples of the anticancer agent include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; Anti-cancer antibiotics include, but are not limited to, dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids include, but are not limited to, vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan.

[0133] In the present invention, the term “prevention” means any act of inhibiting or delaying the occurrence, spread, and recurrence of cancer by administering a pharmaceutical composition according to the present invention.

[0134] The term "treatment" as used herein refers to any action that kills cancer cells or improves or beneficially alters the symptoms of cancer through administration of the composition of the present invention. Those skilled in the art to which the present invention pertains will be able to accurately determine the criteria for diseases for which the composition of the present invention is effective and determine the degree of improvement, enhancement, and treatment by referencing materials provided by the Korean Medical Association and other sources.

[0135] The term "therapeutically effective amount" used in combination with the active ingredient in the present invention means the amount of a pharmaceutically acceptable salt of the composition effective in preventing or treating the target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage for use in humans should be determined as an appropriate amount by taking both safety and efficacy into consideration. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

[0136] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, cancer type and severity, drug activity and sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0137] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0138] The composition of the present invention may also include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and examples thereof include compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main-use dosage form such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0139] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer it once or several times a day.

[0140] Liquid preparations for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0141] In one aspect, the present invention relates to a method for producing a human antibody Fc domain variant, comprising the steps of: a) culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the human antibody Fc domain variant of the present invention; and b) recovering a polypeptide expressed by the host cell.

[0142] In one aspect, the present invention relates to a method for producing an antibody or fragment thereof with reduced functional group function, comprising the steps of: a) culturing a host cell comprising a vector comprising a nucleic acid molecule encoding an antibody of the present invention or a fragment thereof having immunological activity; and b) purifying the antibody expressed from the host cell.

[0143] In one embodiment, purification of the antibody may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably affinity chromatography using Protein A.

[0144] In one aspect, the present invention relates to a method for reducing the functional group function of an antibody, comprising the step of replacing an amino acid at any one or more positions selected from the group consisting of amino acids at positions 231, 232, 234 and 235 numbered according to the EU Index in Kabat et al in the Fc domain of the antibody with a sequence different from that of the wild type amino acid.

[0145] In one embodiment, the substitution may be any one substitution selected from the group consisting of 231C, 232C, 234C and 235C.

[0146] In one embodiment, the human antibody may be IgG1, IgG2, IgG3 or IgG4.

[0147] In one embodiment, the substitution may reduce effector function by reducing binding to Fc gamma receptors (FcγRs) compared to a wild-type human antibody.

[0148] In one embodiment, the Fc gamma receptors (FcγRs) can be human, mouse or monkey Fc gamma receptors (FcγRs), and can be FcγRI, FcγRⅡa, FcγRⅡb, FcγRⅢ, FcγRⅢa or FcγRIV.

[0149] In one embodiment, the human FcγR can be FcγRI, FcγRⅡa, FcγRⅡb or FcγRⅢa, wherein the FcγRⅡa can be FcγRⅡa-131H or FcγRⅡa-131R, and the FcγRⅢa can be FcγRⅢa-158V or FcγRⅢa-158F; the mouse FcγR can be mouse FcγRI, mouse FcγRⅡb, mouse FcγRⅢ or mouse FcγⅣ; and the monkey FcγR can be cynomolgus monkey FcγRⅡa, cynomolgus monkey FcγRⅡb or monkey FcγRⅢ.

[0150] In one embodiment, the substitution may reduce binding to C1q compared to a wild-type human antibody Fc domain, thereby reducing effector function.

[0151] In one embodiment, the effector function can be an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0152] In one aspect, the present invention relates to a method for producing an antibody or fragment thereof with reduced functional group function, comprising the step of replacing an amino acid at any one or more positions selected from the group consisting of amino acids at positions 231, 232, 234 and 235 (numbered according to the EU Index in Kabat et al) in the Fc domain of the antibody with a sequence different from that of the wild type amino acid.

[0153] In one embodiment, the substitution may be any one substitution selected from the group consisting of 231C, 232C, 234C and 235C.

[0154] In one embodiment, the human antibody may be IgG1, IgG2, IgG3 or IgG4.

[0155] In one embodiment, the substitution may result in decreased binding affinity to Fc gamma receptors (FcγRs) compared to a wild-type human antibody, thereby reducing effector function.

[0156] In one embodiment, the Fc gamma receptors (FcγRs) can be human, mouse or monkey Fc gamma receptors (FcγRs), and can be FcγRI, FcγRⅡa, FcγRⅡb, FcγRⅢ, FcγRⅢa or FcγRIV.

[0157] In one embodiment, the human FcγR can be FcγRI, FcγRⅡa, FcγRⅡb or FcγRⅢa, wherein the FcγRⅡa can be FcγRⅡa-131H or FcγRⅡa-131R, and the FcγRⅢa can be FcγRⅢa-158V or FcγRⅢa-158F; the mouse FcγR can be mouse FcγRI, mouse FcγRⅡb, mouse FcγRⅢ or mouse FcγⅣ; and the monkey FcγR can be cynomolgus monkey FcγRⅡa, cynomolgus monkey FcγRⅡb or monkey FcγRⅢ.

[0158] In one embodiment, the substitution may result in decreased binding affinity to C1q compared to a wild-type human antibody Fc domain, thereby reducing effector function.

[0159] In one embodiment, the effector function can be an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

[0160] In one aspect, the present invention relates to the use of an antibody or an immunologically active fragment thereof comprising an Fc domain variant of the present invention for use in the manufacture of an antibody therapeutic.

[0161] In one aspect, the present invention relates to the use of a human antibody Fc domain variant of the present invention, an antibody of the present invention or a fragment thereof having immunological activity, or an antibody therapeutic agent of the present invention for the prevention or treatment of cancer.

[0162] In one aspect, the present invention relates to a method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of a human antibody Fc domain variant of the present invention, an antibody of the present invention or a fragment thereof having immunological activity, or an antibody therapeutic agent of the present invention.

[0163] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0164] Example 1. Production of an IgG4 Fc variant that prevents Fab-arm exchange and eliminates FcγR binding.

[0165] In order to prevent the Fab-arm exchange phenomenon in which half-antibody forms of wild-type human IgG4 are combined and to produce FcγRs and C1q-binding ablative Fc variants to avoid the toxicity problem of the conventional S228P Fc variant, various Fab-arm exchange-preventing variants were produced by introducing cysteine ​​into the lower hinge of the FcγRs-binding ablative glycosylated Fc variant SL001 (E233C) from the inventors' previous study (patent application number 10-2023-0077425) (Table 1).

[0166]

[0167] Example 2. Expression and purification of glycosylated pembrolizumab IgG4 Fc variants.

[0168] The glycosylated Fc variants produced in Example 1 were cloned into the heavy chain gene of pembrolizumab, a model antibody that does not have a Fab-arm exchange variant, to produce an expression vector. After that, the heavy chain gene and light chain gene of the variants were first mixed in a 1:1 ratio in 3 ml of Freestyle 293 expression medium (Gibco, 12338-018), and then mixed in a ratio of PEI:variant gene = 4:1, left at room temperature for 20 minutes, and then incubated with 2 × 10 6After mixing with Expi293F cells subcultured at a density of 10 cells / ml, the mixture was cultured for 7 days in a CO2 shaking incubator at 37°C, 125 rpm, and 8% CO2, centrifuged, and only the supernatant was collected. The supernatant was equilibrated with 25× PBS and filtered through a 0.2 μm syringe filter. After that, Protein A resin was added to the culture medium containing pembrolizumab Fc variants, stirred at 4°C for 16 hours, spun down, and the resin was recovered, washed with 2 ml PBS, and eluted with 600 μl of 100 mM glycine (pH 2.7) buffer. The eluate was neutralized using 200 μl of 1 M Tris-HCl (pH 8.0) and the buffer was exchanged using Amicon Ultra-4 centrifugal filter units 30K (Merck Millipore, UFC503096). Then, it was confirmed through SDS-PAGE gel analysis that the glycosylated antibody pembrolizumab Fc variants were purified with high purity. In particular, while the wild-type IgG4 antibody produced half-antibodies (75 kDa), it was confirmed that the glycosylated pembrolizumab Fc variants of the present invention did not produce half-antibodies, thereby preventing Fab-arm exchange (Fig. 1).

[0169] Example 3. Binding Ability Analysis of Glycosylated Pembrolizumab IgG4 Fc Variants to Human FcγRs

[0170] 3-1. Expression and purification of human FcγRs

[0171] To analyze the binding affinity of the glycosylated Fc variants of the present invention to FcγRs using ELISA, FcγRI-GST, FcγRⅡa-131H-GST, FcγRⅡa-131R-GST, FcγRⅡb-GST, FcγRⅢa-158V-GST, and FcγRⅢa-158F-GST were each cloned into an animal cell expression vector, transfected into Expi293F cells using PEI, and cultured for 7 days under conditions of 37°C, 125 rpm, and 8% CO2. After the culture was completed, the supernatant was collected, equilibrated with 25× PBS, and each receptor protein was purified using anti-GST affinity chromatography. Afterwards, SDS-PAGE analysis was performed to confirm that FcγRI-GST, FcγRⅡa-131H-GST, FcγRⅡa-131R-GST, FcγRⅡb-GST, FcγRⅢa-158V-GST, and FcγRⅢa-158F-GST were purified with high purity (Fig. 2).

[0172] 3-2. Binding analysis for FcγRs

[0173] To confirm the FcγR binding ability of the purified glycated pembrolizumab Fc variants from Example 2, ELISA analysis was performed. Specifically, the FcγRs-GST (FcγRI-GST, FcγRⅡa-131H-GST, FcγRⅡa-131R-GST, FcγRⅡb-GST, FcγRⅢa-158V-GST, and FcγRⅢa-158F-GST) purified in Example 3-1 above were diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6, and 50 μl of each was immobilized in a flat-bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, 50 μl of glycated pembrolizumab Fc variants serially diluted with 1% skim milk were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed for 1 hour at room temperature with 50 μl of HRP-Protein L (GenScript, M00098) and washed again. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl of 2 M H2SO4 at a time. The absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0174] As a result, the stapled Fc-1 (A231C) variant showed reduced binding affinity to all FcγRs except FcγRI compared to the wild-type IgG4 antibody, the stapled Fc-2 (P232C) variant showed FcγR binding affinity similar to the wild-type IgG4 antibody, and the stapled Fc-4 (F234C) variant showed eliminated binding affinity to all FcγRs except FcγRI (Fig. 3). In particular, the stapled Fc-5 (L235C) variant showed eliminated binding affinity to all FcγRs, and showed significantly lower FcγR binding affinity than the preceding S228P / L235E variant (SPLE), which was previously used clinically and prevented Fab-arm exchange and reduced FcγR binding affinity (Fig. 3).

[0175] Example 4. Binding Ability of Glycosylated Pembrolizumab IgG4 Fc Variants to Mouse FcγRs

[0176] 4-1. Expression and purification of mouse FcγRs

[0177] In order to analyze the binding affinity of the glycosylated Fc variants of the present invention to mouse FcγRs, which are widely used as preclinical animal models, using ELISA, mouse FcγRI-GST, mouse FcγRⅡb-GST, mouse FcγRⅢ-GST, and mouse FcγRⅣ-GST were each cloned into an animal cell expression vector, transfected into Expi293F cells using PEI, and cultured for 7 days under conditions of 37°C, 125 rpm, and 8% CO2. After the culture was completed, the supernatant was collected, equilibrated with 1× PBS, and each receptor protein was purified using anti-GST affinity chromatography. Thereafter, SDS-PAGE analysis was performed to confirm that mouse FcγRI-GST, mouse FcγRⅡb-GST, mouse FcγRⅢ-GST, and mouse FcγRⅣ-GST were purified with high purity (Fig. 4).

[0178] 4-2. Binding analysis for mouse FcγRs

[0179] To confirm the binding affinity of the purified glycated pembrolizumab Fc variants in Example 2 to mouse FcγRs, an ELISA analysis was performed. Specifically, mouse FcγRI-GST, mouse FcγRⅡb-GST, mouse FcγRⅢ-GST, and mouse FcγRⅣ-GST purified in Example 4-1 were diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6, and 50 μl of each was immobilized in a flat-bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, followed by blocking with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, 50 μl of glycated pembrolizumab Fc variants serially diluted with 1% skim milk were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed for 1 hour at room temperature with 50 μl of HRP-Protein L (GenScript, M00098) and washed again. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl of 2 M H2SO4 at a time. The absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0180] As a result, the stapled Fc-5 (L235C) variant was found to have significantly lower FcγR binding affinity than the wild-type IgG4 antibody and the conventional precursor S228P / L235E variant (SPLE), and in particular, the binding affinity to all mouse FcγRs was completely eliminated (Fig. 5).

[0181] Example 5. Binding Ability of Glycosylated Pembrolizumab IgG4 Fc Variants to Monkey FcγRs

[0182] 5-1. Expression and purification of cynomolgus monkey FcγRs

[0183] To analyze the binding affinity of the glycosylated Fc variants of the present invention to the FcγRs of cynomolgus monkeys, which are widely used as preclinical animal models, using ELISA, cynomolgus monkey FcγRI-GST, cynomolgus monkey FcγRⅡa-GST, cynomolgus monkey FcγRⅡb-GST, and cynomolgus monkey FcγRⅢ-GST were each cloned into an animal cell expression vector, transfected into Expi293F cells using PEI, and cultured for 7 days under conditions of 37°C, 125 rpm, and 8% CO2. After the culture was completed, the supernatant was collected, equilibrated with 25× PBS, and each receptor protein was purified using anti-GST affinity chromatography. Afterwards, SDS-PAGE analysis was performed to confirm that cynomolgus monkey FcγRI-GST, cynomolgus monkey FcγRⅡa-GST, cynomolgus monkey FcγRⅡb-GST, and cynomolgus monkey FcγRⅢ-GST were purified with high purity (Fig. 6).

[0184] 5-2. Binding analysis to cynomolgus monkey FcγRs

[0185] To confirm the binding affinity of the purified glycated pembrolizumab Fc variants in Example 2 to cynomolgus monkey FcγRs, an ELISA analysis was performed. Specifically, cynomolgus monkey FcγRI-GST, cynomolgus monkey FcγRⅡa-GST, cynomolgus monkey FcγRⅡb-GST, and cynomolgus monkey FcγRⅢ-GST purified in Example 5-1 were diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6, and 50 μl of each was immobilized in a flat-bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, followed by blocking with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, 50 μl of glycated pembrolizumab Fc variants serially diluted with 1% skim milk were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed for 1 hour at room temperature with 50 μl of HRP-Protein L (GenScript, M00098) and washed again. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl of 2 M H2SO4 at a time. The absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0186] As a result, the stapled Fc-5 (L235C) variant was found to have significantly lower FcγR binding affinity than the wild-type IgG4 antibody and the conventional precursor S228P / L235E variant (SPLE), and in particular, the binding affinity to all cynomolgus monkey FcγRs was completely eliminated (Fig. 7).

[0187] Example 6. Analysis of binding affinity of glycosylated pembrolizumab IgG4 Fc variants to human C1q.

[0188] To confirm the C1q binding affinity of the purified glycated pembrolizumab Fc variants in Example 2, an ELISA assay was performed. 50 μl of each glycated pembrolizumab Fc variant, diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6), was immobilized in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) at 4°C for 16 h, and then blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 h. After washing four times with 180 μl of 0.05% PBST, 50 μl of C1q (Quidel, A400) protein serially diluted with 1% skim milk was dispensed into each well and reacted at room temperature for 1 h. After washing, antibody reaction was performed at room temperature for 1 hour using 50 μl of anti-C1q-HRP (Invitrogen, PA1-84324), and washed again. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl each of 2 M H2SO4. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0189] As a result, it was found that all of the glycosylated pembrolizumab Fc variants of the present invention had no binding affinity to C1q (Fig. 8).

[0190] Example 7. In vivo half-life analysis of glycosylated pembrolizumab IgG4 Fc variants.

[0191] 7-1. Binding analysis for human FcRn

[0192] To confirm the binding affinity of the glycosylated Fc variants of the present invention to human FcRn, which is involved in the in vivo half-life, an ELISA analysis according to pH was performed. First, FcRn-GST was cloned into an animal cell expression vector and prepared, and then transfected into Expi293F cells using PEI, and cultured for 7 days under conditions of 37°C, 125 rpm, and 8% CO2. After the culture was completed, the supernatant was collected, equilibrated with 25× PBS, and FcRn-GST was purified using anti-GST affinity chromatography, and its high purity was confirmed by SDS-PAGE analysis (Fig. 9). In addition, the purified glycated pembrolizumab Fc variants from Example 2 were diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6 and 50 μl each were immobilized in a flat-bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, the purified FcRn-GST was serially diluted in 1% skim milk (pH 6.0 / pH 7.4) and 50 μl each was dispensed into each well and reacted at room temperature for 1 hour. After washing, 50 μl of anti-GST-HRP conjugate (GE Healthcare, RPN1236V) was added to each well, and antibody reaction was performed at room temperature for 1 hour, followed by washing again. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to develop color, and 50 μl of 2 M H2SO4 was added to terminate the reaction. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0193] As a result, all of the glycosylated pembrolizumab Fc variants of the present invention were found to maintain the FcRn properties of the wild-type IgG4 antibody, and in particular, the stapled Fc-5 (L235C) variant was found to have a significantly improved binding affinity for FcRn at pH 6.0 than the wild-type IgG4 antibody (Fig. 10), confirming that its half-life was increased compared to the wild-type.

[0194] 7-2. In vivo half-life analysis in mice expressing human FcRn

[0195] To compare the in vivo half-lives of the stapled Fc-5 (L235C) variant, which was confirmed to have increased binding affinity to human FcRn in a pH-dependent manner, with the conventional variant S228P, a pharmacokinetic analysis was performed using Tg276 female mice harboring human FcRn. Specifically, 2 or 3 mice per variant were injected intravenously with 5 mg / kg of pembrolizumab containing each variant, and blood samples were collected from each mouse after 0.5, 24, 168, 336, 504, and 696 hours, and serum was obtained by centrifugation at 1000 × g for 15 minutes. To measure serum antibody concentrations using ELISA, 50 μl of HER2-His diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6 was immobilized in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) at 4°C for 16 h, and then blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 h. After washing four times with 180 μl of 0.05% PBST, pembrolizumab containing each variant was serially diluted from 1 μg / ml with 1% skim milk to create a standard curve, and 50 μl of serum was dispensed into each well and reacted at room temperature for 1 h. After washing, antibody reaction was performed for 1 hour at room temperature using 50 μl of goat anti-human IgG H+L (Jackson Immunoresearch, 109-036-003) and washed again.1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl each of 2 M H2SO4. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek). Antibody concentration was analyzed using a standard curve.

[0196] As a result, the stapled Fc-5 (L235C) variant was found to have a longer blood half-life than the S228P variant, which is a Fab-arm exchange-prevention variant that has been used clinically (Fig. 11).

[0197] Example 8. Thermal stability analysis of glycosylated pembrolizumab IgG4 Fc variants

[0198] To confirm the thermal stability of the purified glycated pembrolizumab IgG4 Fc variants according to temperature, differential scanning fluorimetry (DSF) analysis was performed. Specifically, 45 μl of the glycated pembrolizumab Fc variants diluted to 5 μM in 1× PBS and 5 μl of SYPRO Orange (Invitrogen, S6651) dye diluted to 200× were mixed and dispensed onto a PCR plate (Thermo Scientific, AB0900W). 1× PBS was also prepared as a control using the same method (all samples were performed in triplicate). An optically clear sealing film (Thermo Scientific, AB1170) was attached to the plate containing the samples, and the fluorescence intensity was measured while increasing the temperature by 0.03°C per second from 25°C to 99.9°C using a QuantStudio 3 Real-Time PCR System (Applied Biosystems, A28567). The fluorescence values ​​at each temperature were fitted to the Boltzmann model using OriginPro software, and then the midpoint of the sigmoidal transition curve was calculated.

[0199] As a result, the glycosylated pembrolizumab Fc variants of the present invention were found to have higher decomposition temperatures than the wild-type IgG4 antibody, the conventional Fab-arm exchange prevention variant (S228P), and the conventional leading variant SPLE (S228P / L235E), and in particular, the stapled Fc-5 (L235C) variant was found to have the highest decomposition temperature (Fig. 12), confirming excellent thermal stability.

[0200] Example 9. Expression and purification of L235C-introduced IgG1 Fc variant (glycosylated trastuzumab Fc variant)

[0201] In order to apply the stapled Fc-5 (L235C), an IgG4 Fc variant with all FcγR binding affinity eliminated, not only to IgG4 antibodies but also to the IgG1 subclass with high FcγR binding affinity, an Fc variant was prepared by introducing the mutation (L235C) into a trastuzumab model antibody as in Example 1. The trastuzumab L235C variant (IgG1 L235C), wild-type IgG1, and the conventional FcγR binding affinity reduced Fc variant LALA prepared in this way were expressed and purified in animal cells using the same method as in Example 2, and it was confirmed through SDS-PAGE gel analysis that the glycosylated trastuzumab Fc variant was purified with high purity (Fig. 13).

[0202] Example 10. Analysis of FcγR binding affinity of glycosylated trastuzumab IgG1 Fc variants

[0203] The binding ability of the purified glycosylated trastuzumab Fc variant (IgG1 L235C) in Example 9 to FcγRs was confirmed by ELISA analysis using FcγRs-GST (FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, FcγRIIIa-158F-GST) as in Example 3-2.

[0204] As a result, the IgG1 L235C variant was found to have abolished binding affinity to all FcγRs, and was found to have a significantly lower FcγR binding affinity than the LALA variant previously used in clinical practice (Fig. 14).

[0205] Example 11. Half-life analysis of glycosylated trastuzumab IgG1 Fc variants

[0206] To confirm the FcRn binding affinity involved in the in vivo half-life of the purified glycosylated trastuzumab Fc variant (IgG1 L235C) in Example 9, an ELISA assay was performed according to pH. Specifically, HER2-His diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was immobilized in 50 μl of a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. After washing each well four times with 180 μl of 0.05% PBST (pH 6.0 / pH 7.4), 50 μl of glycosylated trastuzumab antibody diluted in 1% skim milk (pH 6.0 / pH 7.4) was dispensed into each well and incubated for 1 hour at room temperature. After washing each well four times with 180 μl of 0.05% PBST (pH 6.0 / pH 7.4), 50 μl of FcRn-GST serially diluted in 1% skim milk (pH 6.0 / pH 7.4) was dispensed into each well and incubated for 1 hour at room temperature. After washing, 50 μl of anti-GST-HRP conjugate (GE Healthcare, RPN1236V) was treated to each well and antibody reaction was performed for 1 hour at room temperature, followed by washing. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at 50 μl each to develop color, and then 2 M H2SO4 was added at 50 μl each to terminate the reaction. The absorbance was then analyzed using an Epoch microplate spectrophotometer (BioTek).

[0207] As a result, it was confirmed that the glycosylated IgG1 L235C variant, which applied the mutation of the IgG4 Fc variant to IgG1, had a significantly increased binding affinity at pH 6.0 compared to the wild-type IgG1 antibody and LALA, and thus the half-life was increased (Fig. 15).

[0208] Example 12. Thermal stability analysis of glycosylated trastuzumab IgG1 Fc variants

[0209] The thermal stability of the glycosylated trastuzumab Fc variant according to temperature was confirmed using the same method as in Example 8.

[0210] As a result, it was confirmed that the glycosylated trastuzumab IgG1 Fc variant (IgG1 L235C) had a higher decomposition temperature than wild-type IgG1 and LALA (Fig. 16), indicating excellent thermal stability.

[0211] Example 13. Measurement of binding constants of glycosylated trastuzumab IgG1 Fc variants

[0212] 13-1. Expression and Purification of Glycosylated Trastuzumab IgG1 L234A / L235A / P329G Fc Variants

[0213] To compare the binding affinity and ADCC activity with IgG1 L235C, the conventional Fc variant, L234A / L235A / P329G (LALAPG) Fc variant, was introduced into the trastuzumab model antibody as in the above example, produced, expressed and purified in animal cells, and confirmed. As a result, it was confirmed that a highly pure glycosylated trastuzumab Fc variant was purified (Fig. 17).

[0214] 13-2. Measurement of coupling constants

[0215] For quantitative binding analysis of glycosylated trastuzumab IgG1 Fc variants (IgG1 LC), binding constant measurements were performed. Deionized water was dispensed into a black 96-well plate (Greiner, 655209), and a FAB2G biosensor (Satorius, 18-5125) was inserted and hydrated for 10 minutes. Each antibody (IgG1 LC, IgG1 WT, IgG1 LALA, and IgG1 LALAPG) was diluted in 1× PBS (pH 7.4) and dispensed into each well. The remaining FcγRs, except FcγRI, were serially diluted 2-fold from 5,000 nM and dispensed into each well, and 1× PBS (pH 7.4) was added to the reference well. The biosensor and sample plate were placed in an Octet ® The binding strength was measured by placing it in R8 (Satorius) and reacting it for 60 sec (baseline) with deionized water, 300 sec (loading) with antibody, 60 sec (baseline) with 1× PBS (pH 7.4), 30 sec (association) with FcγR, and 30 sec (dissociation) with 1× PBS (pH 7.4). When measuring FcγRI, it was replaced with 1× kinetic buffer, and the measurement was made from 500 nM, and the binding strength was measured for 300 sec with dissociation.

[0216] As a result, the conventional mutant LALA was found to have binding affinity to all FcγRs, LALAPG was found to have binding affinity to FcγRI with high affinity and non-specific binding affinity to FcγRIIIa, whereas IgG1 L235C was found to have lower binding affinity than the conventional mutants LALA and LALAPG and to have completely lost binding affinity (Fig. 18).

[0217] Example 14. Real-time ADCC analysis of glycosylated trastuzumab IgG1 Fc variants

[0218] To confirm the ability of glycosylated trastuzumab IgG1 Fc variants to eliminate Fc effector function according to the ablated FcγR binding affinity, a real-time cell death ADCC assay was performed using SKBR-3 expressing the target antigen HER2. SKBR-3 (10,000 cells / well) were diluted in RPMI medium (Gibco, 11875-093) containing 10% FBS (Gibco, 16000-044) and cultured in E-Plate (Agilent, 300600900). PBMCs were isolated from whole blood obtained from healthy donors using Histopaque 1077 (Sigma Aldrich, 10771), washed with 1× PBS, and diluted in RPMI medium containing 10% FBS. PBMCs and each antibody (IgG1 LC, IgG1 WT, IgG1 LALA, IgG1 LALAPG, and serum IgG) were treated on the E-plate, and the E-plate was placed in the RTCA equipment and observed in real time using RTCA Software 1.2 (Agilent) at 37°C and 5% CO2 over time, and cell lysis (cytolysis) was analyzed according to the time point.

[0219] As a result, LALA and LALAPG, which were confirmed to have retained FcγR binding affinity in Example 14, showed high ADCC, whereas IgG1 L235C of the present invention was confirmed to have ADCC eliminated (Fig. 19).

[0220] Example 15. Expression and purification of L235C-introduced IgG2 Fc variants and IgG3 Fc variants.

[0221] Since the above examples confirmed that all FcγR binding affinity was eliminated by the L235C mutation introduced into IgG4 Fc and IgG1 Fc, in order to confirm whether the mutant exhibits the ability to eliminate FcγR binding affinity in other human IgG subclasses, an IgG2 V235C Fc mutant and an IgG3 L235C Fc mutant were produced by introducing cysteine ​​into the V235 position of IgG2 and the L235 position of IgG3, which are identical to the 235 position of IgG1, by aligning the IgG subclasses according to the EU numbering (EU Index in Kabat et al) with the trastuzumab model antibody (Table 2). The IgG2 V235C mutant and the IgG3 L235C mutant introduced into the trastuzumab model antibody, and the wild-type IgG2 and IgG3 were expressed and purified using a Protein G resin, and confirmed.

[0222]

[0223] As a result, it was confirmed that wild-type IgG2, wild-type IgG3, glycosylated trastuzumab IgG2 V235C Fc variant (IgG2 VC), and glycosylated trastuzumab IgG3 L235C Fc variant (IgG3 LC) were purified with high purity (Fig. 20).

[0224] Example 16. ELISA analysis of FcγR binding affinity of glycosylated trastuzumab IgG2 and IgG3 Fc variants

[0225] The FcγR binding affinity of the glycosylated trastuzumab IgG2 V235C Fc variant (IgG2 VC) and the glycosylated trastuzumab IgG3 L235C Fc variant (IgG3 LC) prepared after purification in Example 15 above was confirmed by ELISA analysis.

[0226] FcγRs-GST (FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, FcγRIIIa-158F-GST) were diluted to 4 μg / ml in 0.05 M Na2CO3, pH 9.6, and 50 μl of each was immobilized in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) at 4°C for 16 h, followed by blocking with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 h. After washing four times with 180 μl of 0.05% PBST, 50 μl of glycated trastuzumab antibody serially diluted with 1% skimmed milk was dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed for 1 hour at room temperature with 50 μl of HRP-Protein L (GenScript, M00098) and washed again. After color development, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added at each time, and the reaction was terminated by adding 50 μl of 2 M H2SO4 at each time. The absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0227] As a result, it was confirmed that the IgG2 V235C mutant and the IgG3 L235C mutant had significantly lower FcγR binding affinity than the wild-type IgG1, IgG2, and IgG3, and that all FcγR binding affinities were eliminated (Fig. 21).

Claims

1. A human antibody Fc domain variant, wherein at least one amino acid at a position selected from the group consisting of amino acids at positions 231, 232, 234, and 235 numbered according to the EU Index in Kabat et al. in a wild type human antibody Fc domain is substituted with a sequence different from that of the wild type amino acid.

2. A human antibody Fc domain variant comprising at least one amino acid substitution selected from the group consisting of 231C, 232C, 234C, and 235C, according to claim 1.

3. In claim 1, the human antibody is a human antibody Fc domain variant that is IgG1, IgG2, IgG3 or IgG4.

4. A human antibody Fc domain variant having reduced binding affinity to Fc gamma receptors (FcγRs) compared to a wild-type human antibody Fc domain in claim 1.

5. A human antibody Fc domain variant according to claim 4, which is a human, mouse or monkey Fc gamma receptor (FcγR).

6. A human antibody Fc domain variant having reduced binding affinity to C1q compared to a wild-type human antibody Fc domain in claim 1.

7. A human antibody Fc domain variant having a reduced effector function compared to a wild-type human antibody Fc domain according to claim 1.

8. A human antibody Fc domain variant according to claim 7, wherein the effector function is an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

9. A human antibody Fc domain variant having increased thermal stability compared to a wild-type human antibody Fc domain according to claim 1.

10. A human antibody Fc domain variant having an increased in vivo half-life compared to a wild-type human antibody Fc domain according to claim 1.

11. An antibody or a fragment thereof having immunological activity, comprising a human antibody Fc domain variant of paragraph 1.

12. An antibody or a fragment thereof having immunological activity, wherein the binding affinity to Fc gamma receptors (FcγRs) is reduced compared to a wild-type human antibody in claim 11.

13. An antibody or a fragment thereof having immunological activity, wherein the binding affinity to C1q is reduced compared to a wild-type human antibody in claim 11.

14. An antibody or a fragment thereof having immunological activity, wherein the functional group function is reduced compared to a wild-type human antibody in claim 11.

15. In claim 11, the antibody is a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an IgG-like bispecific antibody, a bispecific immune cell engager, an antibody mimetic, a chimeric antibody, an antibody conjugate, a human antibody, a humanized antibody, a bivalent antibody, or a bispecific molecule, or an antibody or a fragment thereof having immunological activity.

16. A nucleic acid molecule encoding a human antibody Fc domain variant of paragraph 1, an antibody of paragraph 11, or a fragment thereof having immunological activity.

17. An Fc-fusion protein comprising a human antibody Fc domain variant of paragraph 1 and a protein therapeutic agent fused thereto.

18. In claim 17, the protein therapeutic agent is an Fc-fusion protein that is a T-cell modulatory polypeptide (TMP), an immune checkpoint protein or an immune effector cell-specific targeting molecule, an immune checkpoint inhibitor antibody, a bispecific immune cell engaging bispecific antibody, an agonist antibody, or an antagonist antibody.

19. In claim 18, the immune checkpoint protein is CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, or PD-L2, an Fc-fusion protein.

20. In claim 18, the immune checkpoint inhibitor antibody is atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, an Fc-fusion protein.

21. An antibody therapeutic agent comprising an antibody of claim 11 or a fragment thereof having immunological activity, and a drug moiety.

22. In claim 21, the drug moiety is selected from the group consisting of an immunomodulatory drug (IMiD), an immunogenic apoptosis inducer, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, a chimeric antigen receptor (CAR) cell therapy, an oncolytic drug, an immunotherapy agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, an anti-cytokine agent, an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1BB agent, an anti-GITR agent, Anti-OX40 agents, anti-TRAILR1 agents, anti-TRAILR2 agents, tagretin, interferon-alpha, clobetasol, peginterferon, prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, anti-chemokines, vorinostat, gabapentin, cyclosporine, rapamycin, FK506, detectable markers or reporters, TNF antagonists, antirheumatic agents, muscle relaxants, narcotics, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterials, psoriasis medications, corticosteroids, anabolic steroids, erythropoietin, immunization, immunoglobulins, immunosuppressants, growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma medications, beta Agonists, inhaled steroids, epinephrine or its analogues, cytokines, cytokine antagonists, PD-1 antagonists, adenosine A2AR antagonists, CD73 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors,Antibody therapy, an anthracycline or any combination thereof; 23. An antibody therapeutic agent having a reduced functional group function in claim 21.

24. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient the human antibody Fc domain variant of claim 1, the antibody of claim 11 or a fragment thereof having immunological activity, the Fc-fusion protein of claim 17, or the antibody therapeutic agent of claim 21. 25.a) A step of culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the human antibody Fc domain variant of paragraph 1; and b) A method for producing a human antibody Fc domain variant, comprising the step of recovering a polypeptide expressed by a host cell. 26.a) A step of culturing a host cell comprising a vector containing a nucleic acid molecule encoding the antibody of claim 11 or a fragment thereof having immunological activity; and b) A method for producing an antibody or fragment thereof with reduced functional group function, comprising a step of purifying an antibody expressed from a host cell.

27. A method for reducing the functional group function of an antibody, comprising the step of replacing an amino acid at any one or more positions selected from the group consisting of amino acids at positions 231, 232, 234, and 235 numbered according to the Kabat numbering system in the Fc domain of the antibody with a sequence different from that of the wild type amino acid.

28. A method for reducing the functional group function of an antibody, wherein the substitution in claim 27 is any one substitution selected from the group consisting of 231C, 232C, 234C, and 235C.

29. A method for reducing the functional group function of an antibody according to claim 27, wherein the human antibody is IgG1, IgG2, IgG3 or IgG4.

30. A method for reducing the effector function of an antibody, wherein the effector function is reduced by reducing the binding affinity to Fc gamma receptors (FcγRs) compared to a wild-type human antibody in claim 27.

31. A method for reducing the functional function of an antibody, wherein the binding affinity to C1q is reduced compared to a wild-type human antibody Fc domain, thereby reducing the functional function.

32. A method for reducing an effector function of an antibody according to claim 27, wherein the effector function is an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

33. A method for producing an antibody or fragment thereof or a fragment thereof with reduced functional group function, comprising the step of replacing an amino acid at any one or more positions selected from the group consisting of amino acids at positions 231, 232, 234 and 235 numbered according to the Kabat numbering system in the Fc domain of an antibody with a sequence different from that of the wild type amino acid.

34. A method for producing an antibody or fragment thereof or fragment thereof with reduced functional group function, wherein the substitution in claim 33 is any one substitution selected from the group consisting of 231C, 232C, 234C and 235C.

35. A method for producing an antibody or fragment thereof or fragment thereof with reduced functional group function, wherein the human antibody is IgG1, IgG2, IgG3 or IgG4, in claim 33.

36. A method for producing an antibody or fragment thereof or fragment thereof having a reduced effector function, wherein the antibody or fragment thereof has a reduced binding affinity to Fc gamma receptors (FcγRs) compared to a wild-type human antibody, thereby reducing the effector function, in accordance with claim 33.

37. A method for producing an antibody or fragment thereof or fragment thereof having a reduced functional group function, wherein the antibody or fragment thereof has a reduced binding affinity to C1q compared to a wild-type human antibody Fc domain, and thus has a reduced functional group function, in accordance with claim 33.

38. A method for producing an antibody or fragment thereof or fragment thereof with reduced effector function, according to claim 33, wherein the effector function is an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cell-mediated phagocytosis (ADCP), complement dependent cell-mediated cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.

39. Use of an antibody or a fragment thereof having immunological activity comprising the Fc domain variant of claim 1 for use in the manufacture of an antibody therapeutic agent.

40. Use of the human antibody Fc domain variant of paragraph 1, the antibody of paragraph 11 or a fragment thereof having immunological activity, or the antibody therapeutic agent of paragraph 21 for the prevention or treatment of cancer.

41. A method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of the human antibody Fc domain variant of claim 1, the antibody of claim 11 or a fragment thereof having immunological activity, or the antibody therapeutic agent of claim 21.

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